Oral presentation
1st INTERNATIONAL MULTIDISCIPLINARY
CANCER RESEARCH CONGRESS
18-22 September, 2019
Culture and Congress Centre
Dicle University, Diyarbakır, Turkey
CONGRESS ABSTRACT BOOK
The congress is a bilingual congress in Turkish and English.
www.mokad2019.com
Dear Cancer Researchers,
You are cordially invited to participate in the 1st International Multidisciplinary Cancer
Research Congress to be held in Diyarbakır, Turkey on 18 to 22 September 2019.
The Congress provides a platform for young and renowned researches and clinicians come
together to discuss, present and showcase high-quality research and exchange of ideas on a
range of fundamental and applied topics in the area of current cancer research.
As a member of EACR -European Association for Cancer Research, MOKAD committee has
decided to organize a meeting at the international level. 1st International Multidisciplinary
Cancer Research Congress is the internationally boardened version of the national MOKAD
congress series called “Multidisciplinary Cancer Reasearh Congress”.
Young researchers will have ample opportunity to present their own research during open slots
for short talks and poster sessions. Interactions with cancer field scientists, during coffee breaks
and social events will give chance for young scientist candidates to improve their research
subjects and broaden their horizon. Among the young applicants, the ones who bring excellent
research with excellent presentation skills during short talks and poster sessions, will be
awarded!
1st International Multidisciplanary Cancer Research Congress involves two days practical
course which is intended to train students on basic cell culture and cell death detection
techniques. Students attending the course will have interactive environment with the well
experienced professional trainers who can answer the raised questions about the details of
experimental process.
The congress is held in Diyarbakır, a city hosted 26 different civilisations over 5000 years, a
city the cradle of cultures. Diyarbakır has unique standing with the strong multicultural
inhabitants and spectacular landscape. We believe that Diyarbakır’s fantastic atmosphere with
rich history will inspire young and experienced researchers and create an interactive, and
fruitful platform to discuss cancer research.
We are looking forward to an exciting and memorable scientific meeting and welcome you in
Diyarbakır, Turkey.
On Behalf of Organising Committee
Local head of organizing committee Head of Congress
Prof. Dr. Beran Yokuş Prof. Dr. Engin Ulukaya
2
1st INTERNATIONAL MULTIDISCIPLINARY CANCER RESEARCH CONGRESS
18-22 September, 2019
18 September 2019 (Course in Turkish)
Kurs 1.Gün: Kursiyerler iki gruba ayrılacaklar: Grup A ve Grup B
Kurs, sitotoksisite ve hücre ölüm modu (apoptozis ve nekrozis) değerlendirme yöntemlerini
kapsamaktadır.
Grup A: 09:00 – 13:00
09:00-09:30 Dr. Öğr. Üyesi Didem Karakaş
• Genel Giriş
09:30-13:00 Dr. Öğr. Üyesi Didem Karakaş
• Dondurulmuş hücrenin aktivasyonu ve flaska ekilmesi
• Total ve canlı hücre sayımları, hücre sayılarının hesaplaması ve pasajlama
• İlaç hazırlanması, konsantrasyon hesabı ve hücrelere ilaç uygulaması
12:00-14:00 Öğle Yemeği (Lunch box)
Grup B: 13:00 – 17:00
13:00-13:30 Dr. Öğr. Üyesi Nazlıhan Aztopal
• Genel Giriş
13:30-17:00 Dr. Öğr. Üyesi Nazlıhan Aztopal
• Dondurulmuş hücrenin aktivasyonu ve flaska ekilmesi
• Total ve canlı hücre sayımları, hücre sayılarının hesaplaması ve pasajlama
• İlaç hazırlanması, konsantrasyon hesabı ve hücrelere ilaç uygulaması
19 September 2019 (Course in Turkish)
Kurs 2.Gün: Grup A+B: Dr. Öğr. Üyesi Nazlıhan Aztopal, Dr. Öğr. Üyesi Didem Karakaş
09:00-09:30
• Apoptozisin M30 (kaspazla kırılmış sitokeratin 18) ELISA testi ile tayini
09:30-11:30
• SRB canlılık testi
11:30-12:30 Öğle Yemeği (Lunch box)
12:30-13:00
• M30 ELISA testi sonuç alma
3
13:00-16:00
• Hücre canlılığı, büyüme hızı hesaplamaları, ilgili parametrelerin tanımlanmaları*
• M30 elisa testi sonuç analizi (Grafik çizimi ve değerlendirme*)
• Hücre ölüm modunun floresan boyama ile tayini (erken ve geç apoptozis ile birlikte
nekrozisin nükleus ve hücre membranı temelinde ayırt edilmesi)
16:00-17:00 Prof. Dr. Engin Ulukaya
Sitotoksisite/Hücre ölüm modu çalışmalarında önemli detaylar, yapılan hatalar!
* Öğrencilerin kişisel bilgisayarları ile katılımı önerilmektedir.
20 September 2019 Social Programme
21 September 2019 (1st Day of the Congress)
8:30-9:00 Registration
9:00 Opening Ceremony
9:15 Opening Lecture / Keynote Lecture
Chairs: Hana Alguel,
Bülent Özpolat / Development of targeted therapies in cancer – A historical perspective –
past present and future
10:00-11:00 Special Session: Munich Technical University
Chairs: Hatice Mehtap Kutlu, Engin Ulukaya
Hana Alguel / From bench to bed: novel concepts in mutant KRAS driven cancers
Proffered Paper:
Kıvanç Görgülü / Autophagy and its role in pancreatic cancer
*the speaking sessions of the invited speakers and the oral presentations will be carried out
parallelly in Congress Hall D and Congress Hall E respectively. (Davetli konuşmacı sunumları
(Salon D) ve sözlü sunumlar (Salon E) paralel olarak gerçekleşecektir.)
11:00-11:15 Coffee Break
11:15-12:15 Session 1:
Chairs: Burçin Güngör, Gamze Tanrıöver
Maryam Moghaddam-Matin / Targeted therapy in colorectal cancer
Burçin Güngör / Protein lipidation on cancer metabolism
Sema Yılmaz / Molecular diagnosis in childhood cancers, from molecular side to bed side
4
12:15-13:15 Lunch Break
13:15-14:15 Session 2:
Chairs: Süreyya Bozkurt, Konstantinos Dimas
Nihal Karakaş / Glioblastoma multiforme beyin tümörünün erken tanı ve tedavisine yönelik
otoantikor çalışmaları
Halil Kavaklı / A molecule that desablize Cryptochrome 1 enhances apoptosis in cancer cells
Ceyda Açılan Ayhan / New therapeutic approaches targeting MLL-AF9 leukemia through
epigenetic reprogramming
14:15-14:30 Coffee Break
14:30-15:50 Session 3:
Chairs: Beran Yokuş, Ebru İnce Bostancı
Alexandre Tavartkiladze / Personalized vaccines and CAR-T cell for cancer immunotherapy
Güneş Esendağlı / Cancer cells adaptation to immune responses
Konstantinos Dimas / Mouse models for immunotherapeutic approaches
Süreyya Bozkurt / Diagnostic and prognostic importance of cytogenetics in hematologic
cancers
15:50-16:05 Coffee Break
16:05-17:25 Session 4:
Chairs: Mehmet Küçüköner, Dilek Ülker Çakır
Abdurrahman Şengül / Antikanser Çalışmalarında Platin Koordinasyon Bileşiklerinin Rolü
Gülperi Öktem / Kanser kök hücrede güncel tedavilere yaklaşım
Ata Özçimen / Aurora Kinases and Cancer
Şeyma Demirsoy / A Parkinson-related gene in melanoma: Exploringthe role of the P5
typeATPase ATP13A2, a Parkinson disease’sassociated gene, in melanoma cell proteostasis
17:30-18:30 Poster Session
20:00 Gala Dinner (Traditional Diyarbakır Night Event)
5
22 September 2019 (2nd Day of the Congress)
10:00-11:15 Session 5 Young Stars are Talking
Chairs: Engin Ulukaya, Ceyda Açılan Ayhan
Seçil Demirkol Canlı / A novel transcriptomic based gene panel for the prognostication of
pancreatic cancer
Burcu Şengez / The Transcription Factor Elf3 is Essential for a Successful Mesenchymal to
Epithelial Transition
Nazlıhan Aztopal / Kanser kök hücre ve epigenetik yaklaşımlar
Didem Karakaş / Pankreatik Kanserde Tümör Mikroçevresinin Hedeflenmesi
Şeyma Aydınlık / Hedefe yönelik tedavide kansere özgü moleküler mekanizmaların
aydınlatılması
11.15-11.30 Coffee Break
11.30-12.50 Session 6:
Chairs: Ceyhun Bozkurt, Hasan İçen
Ebru İnce Bostancı / Yeni antikanser ilaç keşfinde genomik ve metabolomik: fırsatlar ve
zorluklar
Dilek Odacı Demirkol / Akıllı İlaç taşıyıcı sistemlerin kanser hücrelerinin hedeflenmesinde
kullanımına genel bakış
Dilek Ülker Çakır / Nanopartiküllerin biyokimyasal ve hematolojik etkileri
Hasan İçen / Hayvanlarda görülen multiple myelomalar
12.50-14.00 Lunch time
14.00-15.00 Session 7:
Chairs: Elif İkitimur Armutak, İlhan Yaylım
Mehmet Küçüköner / Kanserde çığır açan moleküler hedef tedaviler
Ferda Arı / Kanser Tedavisinde Histon Deasetilaz İnhibitorleri
Closing Lecture: Tuba Günel / Over kanserinde liquid biyopsi ile elde edilen moleküllerle
biyobelirteç çalışmaları
15.00-16.00 Award ceremony and closing
6
Congress Chair
Engin Ulukaya
Congress Co-Chair
Beran Yokus
Course Instructors
Didem Karakaş
Nazlihan Aztopal
Scientific Secreteriat
Burçin Güngör
Melda Sarıman
Selim Karahan
Organising Committee
Alexander Tavartkiladze (Georgia)
Beran Yokuş
Bircan Çeken Toptancı
Bülent Özpolat (USA)
Engin Ulukaya
Hana Alguel (Germany)
Kıvanç Görgülü (Germany)
Konstantinos Dimas (Greece)
Maryam Moghaddam-Matin (Iran)
Local Organising Committee
Beran Yokuş
Ebru İnce Bostancı
İlhan Sabancılar
Mehmet Hüseyin Alkan
Neval Berrin Arserim
Selçuk Tunik
Selim Karahan
Sevcan İzgi
Scientific Committee
Abdurrahman Şengül
Alexandre Tavartkiladze
Ata Özçimen
Azmi Yerlikaya
7
Batu Erman
Burcu Şengez
Burçin Güngör
Buse Cevatemre
Bülent Özpolat
Ceyda Açılan Ayhan
Ceyhun Bozkurt
Devrim Gözüaçık
Didem Karakaş
Dilek Ülker Çakır
Dilek Odacı Demirkol
Ebru İnce Bostancı
Egemen Dere
Elif İkitimur Armutak
Erdal Karaöz
Ferda Arı
Funda Acar Yağcı
Gülperi Öktem
Güneş Esendağlı
Halil Kavaklı
Hana Alguel
Hasan İçen
İlhan Yaylım
Kıvanç Görgülü
Konstantinos Dimas
Maryam Moghaddam-Matin
Mehmet Hüseyin Alkan
Mehmet Küçüköner
Mehmet Sarımahmut
Meryem Alagöz
Milica Pesic
Nazlıhan Aztopal
Neval Berrin Arserim
Nihal Karakaş
Nuray Erin
Seçil Demirkol Canlı
Selçuk Tunik
Sema Yılmaz
Serap Çelikler
Sonja Levenat
Süreyya Bozkurt
Şeyma Aydınlık
Tolga Sütlü
Tuba Günel
Ümit Zeybek
Veysel Turan Yılmaz
Yasemin Delen Akçay
8
1st International Multidisciplanary Cancer Research Congress covers a wide rage of cancer field
topics including:
Biosimilars
Cancer immunotherapy
Cancer stem cells
CAR-NK Cells
Cell death (apoptosis/necrosis and
autophagy) in cancer
DNA damage
Epigenetics and cancer
Epithelial-mesenchymal transition
Exosomes in cancer
Kinases in cancer
KRAS in oncology
Lipids in cancer, lipidomics
Liquid biopsy: Future of oncology
Nanoparticles against cancer
Novel anticancer small molecules
Precision oncology
SPEAKER UNIVERSITY TITLE PAGE
Bülent Özpolat MD Anderson
Cancer Center, USA
Development of targeted
therapies in cancer – A historical
perspective – past present and
future
15
Hana Alguel Technical University
of Munich, Germany
From bench to bed: novel
concepts in mutant KRAS
driven cancers
16
Kıvanç Görgülü Technical University
of Munich, Germany
Autophagy and its role in
pancreatic cancer 17
Maryam
Moghaddam-Matin
Ferdowsi University,
Iran
Targeted therapy in colorectal
cancer 18
Burçin Güngör İstinye University,
Istanbul
Protein lipidation on cancer
metabolism 19
Sema Yılmaz Yeditepe University,
Istanbul
Molecular diagnosis in
childhood cancers, from
molecular side to bed side
20
Nihal Karakaş Medipol University,
Istanbul
Glioblastoma multiforme beyin
tümörünün erken tanı ve
tedavisine yönelik otoantikor
çalışmaları
22
Halil Kavaklı Koç University,
Istanbul
A molecule that destabilize
Cryptochrome 1 enhances
apoptosis in cancer cells
24
Ceyda Açılan Ayhan Koç University,
Istanbul
New therapeutic approaches
targeting MLL-AF9 leukemia
through epigenetic
reprogramming
25
9
Alexandre
Tavartkiladze
Tblisi State Medical
University, Georgia
Personalized vaccines and CAR-
T cell for cancer immunotherapy 27
Güneş Esendağlı Hacettepe
University, Ankara
Cancer cells adaptation to
immune responses 28
Konstantinos Dimas University of
Thessaly, Greece
Mouse models for
immunotherapeutic approaches 29
Süreyya Bozkurt İstinye University,
Istanbul
Diagnostic and prognostic
importance of cytogenetics in
hematologic cancers
30
Abdurrahman
Şengül
Bülent Ecevit
University,
Zonguldak
Antikanser Çalışmalarında
Platin Koordinasyon
Bileşiklerinin Rolü
31
Gülperi Öktem Ege University,
Izmir
Kanser kök hücrede güncel
tedavilere yaklaşım 32
Ata Özçimen Mersin University,
Mersin Aurora Kinases and Cancer 34
Şeyma Demirsoy Hacettepe
University, Ankara
A Parkinson-related gene in
melanoma: Exploringthe role of
the P5 typeATPase ATP13A2, a
Parkinson disease’sassociated
gene, in melanoma cell
proteostasis
37
Seçil Demirkol Canlı Hacettepe
University, Ankara
A novel transcriptomic based
gene panel for the
prognostication of pancreatic
cancer
38
Burcu Şengez
International
Biomedicine and
Genom Institute
(IBG), Izmir
The Transcription Factor Elf3 is
Essential for a
Successful Mesenchymal to
Epithelial Transition
39
Nazlıhan Aztopal Istinye University,
Istanbul
Kanser kök hücre ve epigenetik
yaklaşımlar 40
Didem Karakaş İstinye University,
Istanbul
Pankreatik Kanserde Tümör
Mikroçevresinin Hedeflenmesi 41
Şeyma Aydınlık Tübitak, Gebze
Hedefe yönelik tedavide kansere
özgü moleküler mekanizmaların
aydınlatılması
43
Ebru İnce Bostancı Dicle University,
Diyarbakır
Yeni antikanser ilaç keşfinde
genomik ve metabolomik:
fırsatlar ve zorluklar
44
Dilek Odacı
Demirkol
Ege University,
Izmir
Akıllı İlaç taşıyıcı sistemlerin
kanser hücrelerinin 45
10
hedeflenmesinde kullanımına
genel bakış
Dilek Ülker Çakır
Çanakkale Onsekiz
Mart University,
Çanakkale
Nanopartiküllerin biyokimyasal
ve hematolojik etkileri 46
Hasan İçen Dicle University,
Diyarbakır
Hayvanlarda görülen multiple
myelomalar 48
Mehmet Küçüköner Dicle University,
Diyarbakır
Kanserde çığır açan moleküler
hedef tedaviler 49
Ferda Arı Uludağ University,
Bursa
Kanser Tedavisinde Histon
Deasetilaz İnhibitorleri 51
Tuba Günel İstanbul University,
Istanbul
Over kanserinde liquid biyopsi
ile elde edilen moleküllerle
biyobelirteç çalışmaları
53
Oral Presentations
SPEAKER TITLE PAGE
Adnan Ayhanci
Stone Alkaline Water Induces Apoptosis of
Prostate Cancer Cells and Inhibits Tumor Cell
Induced Angiogenesis In Vitro
55
Asli Kurden Pekmezci MALT1 Paracaspase Inhibition Reduces
Hepatocellular Carcinoma Cell Survival 56
Ayca Üvez
In Vitro and In Vivo Investigation of Mangiferin
and Paclitaxel combination on Ehrlich Ascites
Carcinoma
57
Aydın Demiray Akciğer Kanseri ve KOAH’da Biyobelirteç Olarak
Aday miRNA Araştırması 59
Baran Erman Underlying Primary İmmunodeficiency in Patients
with Lymphoma 60
Berkcan Doğan
Determination of MiRNAs and Xeno-miRs
Targeting Most of the Genes Associated with
Colorectal Neoplasms and Central Obesity by "in
silico" Analysis
62
Burcu Salman Investigation of DHRS2 Gene Effects on Breast
Cancer Cell Line 63
Canan Vejselova Sezer
Bir Seramidaz İnhibitörü olan N-
Oleoylethanolamine Bileşiğinin A549 ve Beas-2B
Hücreleri Üzerindeki Sitotoksik ve Morfolojik
Değişimlerinin Araştırılması
65
Çevik Gürel
Doksorubisin İle İndüklenen Sıçan Nefrotoksisite
Modelinde Fluvastatinin Olası Koruyucu ve
Tedavi Edici Etkilerinin Histokimyasal ve
İmmünohistokimyasal Olarak Değerlendirilmesi
67
11
Cihangir Yandım Abnormal Expression Patterns of Repetitive DNA
Elements Uncovered in Breast Cancer 68
Damla Uludağ
Hedefe Yönelik Tedaviler İçin Çeşitli Kanser
Hücrelerinde IL13Rα2 Ekspresyonunun
Belirlenmesi
69
Dehan Çömez
Fluidic Shear Stress Regulates Biological
Behaviours of Hepatocellular Carcinoma Cells
Through c-Met Signalling
70
Derya Akçiçek
Investigation of the Anticancer Effect of
Haplophyllum Buxbaumii Plant on HCT-116
Colon Cancer Cells
71
Dilek Pirim
Identification of Shared Candidate Key Genes and
Pathways Associated with Cancer and Heart
Failure Pathogenesis Using Bioinformatic
Analyses
72
Ebru Temiz Investigation of Anti-cancer Properties of
Haplophyllum Ptilostylum Plant 73
Ece Gümüşoğlu The İmportance of Dysregulated miRNAs on
Ovarian Cysts and Tumors 74
Elif Merve Aydın
Investigation of Helicobacter Pylori Virulence
Factors and İmmune Response in Precancerous
Lesions
75
Esra Arslan Ateş
A Useful Tool for Distinguishing
Gene/Pseudogene Variants Detected via NGS:
Haplotype Analysis
76
Esra Bozgeyik
Identification of lncRNA Molecules İnvolved in
the Regulation of EGF-İnduced of Cellular
Proliferation
77
Feryal Akay
Morus Nigra (Karadut) Yaprağı Etanol
Ekstraktının Doğal bir DNA İleri Glikasyon Son
Ürünleri (DNA-AGE'ler) Bileşik Kaynağı Olarak
Değerlendirilmesi
78
Gamze Tan
The Antiproliferative and Apoptotic Effects of
Gold Nanoparticles Synthesized by Allium Cepa
Extract on Leukemia Cell Lines
80
Gülsün Bağcı The Role of c-Met Signaling in Organ Specific
Extravasation of HCC cells 81
Gürcan Günaydın The Effects of Cancer Associated Fibroblasts on
Tumor Associated Macrophages 82
Gülşah Torkay Cryptonin An Antimicrobial Peptide from Cicada
and its Effects on Melanoma Cell Lines 84
İbrahim Halil Kılıç
Güneydoğu Anadolu Bölgesinde Yetişen Patlıcan
Çeşitlerinin Kabuklarının Sitotoksik Etkilerinin
Saptanması
85
Hande Özkan
How Dose and Duration of Exposure Affect the
Rate of Acquired Resistance to Doxorubicin in
Gastric Cancer?
86
Hatice Mehtap Kutlu İnsan Meme Kanseri Hücrelerinde D-erythro-
MAPP’ın Antikanser Özelliklerinin Araştrılması 87
Hüseyin Özevren Experimental Traumatic Brain Injury Models 88
12
Işık Didem Karagöz In vitro Self-renewal Properties in A549 and
H1299 Non-Small Cell Lung Cancer Cells 89
Işıl Yıldırım
Immunohistochemical Determination of
Glutathione-S-Transferase Isoenzym Family
Expression in Invasive Lobular Breast Cancer
Tissue
90
İsmail Korkmaz Karadutun (Morus nigra) Anti Kanser Etkilerinin
Araştırılması 91
Kadir Eği The Investigation of Anticancer Effect of CEDAR
TAR Prepared by Traditional Methods 92
Lütfiye Kadıoğlu Dalkılıç
Mide Kanserinin Teşhisinde Kullanılabilecek
Biyobelirteç Adayı Genlerin Bİyoinformatik
Araçlar İle Belirlenmesi
93
Mehmet Tahir Hüsunet
L-Askorbik Asit’in (Vitamin C) İnsan
Promiyelösitik Kanser Hücre Hattında (HL-60)
MTT Testi ile Araştırılması
95
Melda Sarıman
Investigation of TMED9-ERAP1 Candidate Gene
Expressions Obtained from Multiple Myeloma
Transcriptome Data by RT-PCR
96
Melike Bayındır Bilgiç The Effects of The LIF Antagonist Molecule
(EC359) on the Chordoma Cells 97
Merve Nur Ataş Investigation of Apoptotic Effect of Betulinic
Acid in Renal Cancer Cells 98
Mümin Alper Erdoğan
Selective Cannabinoid-1/2 Receptor (CB1 and
CB2) Agonists Suppress Cell Proliferation and
Clonogenicity in Pancreatic and Breast Cancer
Cells
99
Murat Ferhat Ferhatoğlu
Usefulness of Simple Prognostic Markers of
Complete Blood Count to Predict Lymph Node
Metastasis in Colon Carcinoma
100
Necla Birgül İyison Characterization of BRI3 as a Novel Wnt/β-
catenin Pathway Target 101
Nur Ekimci Gürcan Sub-Culturing Tumorspheres Reduced the
Pluripotency of a Chordoma Cell Line 102
Onur Dirican Beyin Tümörü Dokularında CYP1A1, CYP1B1,
GST-M, GST-P ve MDR Proteinlerinin Rolü 103
Özen Leylek
Defining the Synergistic Sequential Application
Schedules for Chemotherapeutic–Molecular
Targeted Agent Dual Combinations in Gastric
Adenocarcinoma Cells
105
Özge Akbulut Targeting TACC3 as a Novel Therapeutic
Strategy for Breast Cancer Treatment 107
Özlem Demirci DNA Damaging Activities of Terbuthylazine 109
Pınar Kaygın hBD-1 ve hBD-3 Antimikrobiyal Peptidlerin
Ürotelyal Kanser Dokularındaki Ekspresyonları 110
Seda Baykal-Köse
Investigation of the Relationship Between Cellular
Adaptation and Therapeutic Resistance in Chronic
Myeloid Leukemia Cells under İmatinib Pressure
111
13
Selim Yalçın
Neutrophil-to-Lymphocyte Ratio and Platelet
Distribution Width Predicts Invasiveness of
Bladder Carcinoma
113
Semih Dalkılıç PİCOA VE TERFEZİA Türlerinin Anti-Kanser
Etkilerinin Araştırılması 115
Serap Sancar-Baş
The Effects of Mackia Amurensis
Leukoagglutinin on the Expressions of Some
Extracellular Matrix Proteins in Anaplastic
Thyroid Carcinoma Cell Line, 8505C
116
Sibel Bayıl Oğuzkan
An Investigation of the Antitumoral Activity of
Phycocyanin Derived from Spirulina (Arthrospira)
Platensis on Mice
117
Suna Bektaş
Effects of Maackia Amurensis Leukoaglutinin
Treatment on the Transcriptome of Human
Anaplastic Thyroid Cancer Cell Line 8505C
118
Süreyya Bozkurt Cytogenetic Anomalies in Lymphomas: A single
center study 119
Tevriz Dilan Demir
The Role of Nuclear P-glycoproteins in Nuclear
Sparing and Chemoresistance to Doxorubicin in
Gastric Adenocarcinoma Cell Models
120
Tuğba Güngör Synthesis, Antiinflammatory and Anticancer
Effects of Novel Amide Containing Compounds 122
Utku Özbey As a Potential Overall-Biomarker of Cancer Stem
Cells CD90: on Drug Resistance Perspective 124
Yeliz Yılmaz The Role of c-Met Neddylation in Hepatocellular
Carcinoma 125
Zelal Zuhal Kaya Keton Cisimlerinin in vitro İnsan Meme Kanseri
Hücrelerinin Canlılığını Azaltıcı Etkisi 126
Zeliha Emrence
Effect of Doxorubicin on Expression of RNA
m6A Methylation Enzymes in Leukemia Cell
Lines
127
Poster Presentations
Meltem Güleç Plants Used Against Cancer in Folk Medicine in
Turkey 129
Ayten Kılınçlı Targetıng Trem2 Reduces Prolıferatıon, Induces
Cell Death On Breast Cancer Cells 130
Bircan Çeken Toptancı
DNA Degradation by Water and Ethanol extracts
of Allium tuncelianum in the Presence of Copper
Ions: Implications for Anticancer Properties
131
Selahattin Can Özcan Role of NEK2A in PLK4 Induced Multipolar
Cell Divisions 132
Batuhan M. Kalkan Investigation of Nek2 Kinase Targets with Focus
on Centrosomal Unclustering 134
İpek Bulut
New Therapeutıc Approaches Targetıng Mll-Af9 Leukemıa Through Epıgenetıc Reprogrammıng
136
14
İsmail Koyuncu
Investigation of the free amino acid profile of
different breast cancer cell lines (MCF-7, MDA-
MB-231, and CRL-4010)
137
Elif Bozbulut
Hepatoselüler Karsinoma, Mide ve Kolorektal
Kanser Hastalarında Tedavi Öncesi ve Sonrası
Oksidatif Stresin Araştırılması
138
Gamze Tanriover
Cross-talk between tumor and stem cells:
determination of conditioned media cytokine
levels
139
Hatice DİNÇER
Yeni Sentezlenen Naftakinon Türevli
Bileşiklerin Üçlü Negatif Meme Kanseri
Hücreleri Üzerindeki Sitotoksik Etkilerinin
İncelenmesi
140
İlhan Yaylım
The Importance of FOXP3 C/A (rs:3761548) Genetic Variants and Protein Expressions in tumoral tissues of patients with Non-Small Cell Lung Cancer
141
Mehmet Emin Köse
Investigation of the Cell Cycle Specifity of Zn(II)
5,5-Diethylbarbiturate Complex on A-549 Human
Lung Cancer Cell Line
143
Ozyurt R Is Notch1 Associated with LeptinR, IL-6 and
TNF-α in the CRC Tumorigenesis? 144
Sayra Dilmac Effects of phosphorylation changes of E2F1 from
S235 on proliferation and migration 145
Selin Ulukaya The Role of p62 in Pancreatic Cancer
Development and Metastasis 146
Sedat Aydin
Expression levels of glutathione S transferase
isoenzymes in pleomorphic adenoma of the
parotid gland and normal gland
147
Sibel Gökşen New Orthotopic Mouse Colorectal Cancer Model 149
Invited speaker talk
15
Development of targeted therapies in cancer – A historical perspective – past present
and future
Bülent Özpolat
MD Anderson Cancer Center, USA
Invited speaker talk
16
From Bench to Bed: Novel Concepts in Mutant KRAS Driven Cancers
Hana Algül
Comprehensive Cancer Center Munich, Technical University of Munich, Germany
e-mail: [email protected]
Mutations in the KRAS gene drive several common and deadly cancers. Unfortunately, most
mutant KRAS proteins cannot be targeted therapeutically. Among the mutant KRAS driven
cancers pancreatic ductal adenocarcinoma (PDAC) poses the most devastating malignancy.
Due to the late diagnosis, aggressive disease, and a lack of effective treatment options PDAC
is projected to become the second leading cause of cancer-related death by 2030. So far,
conventional oncology has failed to improve the outcome of patients with PDAC thus urging
for novel approaches. Fortunately, with the deeper understanding of the molecular basis of
PDAC new avenues and opportunities emerge at the horizon. Here, we will shed light on these
new opportunities and discuss new challenges to the implementation of an individualized
therapy approach in the context of precision medicine.
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Levels of the Autophagy Related Gene 5 (Atg5) Determine Pancreatic Tumor Formation
and Metastasis in Mice
Kivanc Görgülü1*, Kalliope N. Diakopoulos1*, Marija Stevanovic1, Angeliki-Faidra
Karpathaki1, Jiaoyu Ai1, Derya Kabacaoglu1, Katrin J. Ciecielski1, Ezgi Kaya-Aksoy1,
Dietrich A. Ruess1, Sonja M. Wörmann1, Thomas Wartmann2, Yue Zhao2, Walter Halangk2,
Svetlana Voronina3, Alexey Tepikin3, Anna Artati4, Jerzy Adamski4 7, Michaela Aichler5,
Axel Walch5, Roland M. Schmid1, Martin Jastroch6, Götz Hartleben7, Stephan Herzig7, Bruno
Sainz Jr8, Christos S. Mantzoros9, Marina Lesina1 and Hana Algül1
1 II. Medizinische Klinik, Klinikum rechts der Isar, Technische Universität München, Munich,
Germany
2 Klinik für Chirurgie Bereich Experimentelle Operative Medizin, Universitätsklinikum Magdeburg,
Magdeburg, Germany
3 Institute of Translational Medicine, University of Liverpool, Liverpool, UK
4 Institute of Experimental Genetics, Genome Analysis Centre, Helmholtz Zentrum München,
Neuherberg, Germany
5 Research Unit Analytical Pathology, Helmholtz Zentrum München, Neuherberg, Germany
6 Helmholtz Diabetes Center & German Diabetes Center (DZD), Helmholtz Zentrum München,
Neuherberg, Germany
7 Institute for Diabetes and Cancer, German Center for Diabetes Research (DZD), Neuherberg,
Germany
8 Department of Biochemistry, School of Medicine, Autónoma University of Madrid, Madrid, Spain
9 Division of Endocrinology, Diabetes, andMetabolism, Beth Israel Deaconess Medical Centre,
Harvard Medical School, Boston, Massachusetts
Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease characterized by late-stage
diagnosis and metastasis. A multitude of underlying mechanisms has been suggested e.g.,
alterations in cell homeostasis and metabolism. Autophagy is a central regulator of homeostasis
and metabolism. Here we show for the first time that varying levels of the Autophagy related
gene 5 (Atg5) determine pancreatic carcinogenesis. Using an in vivo model of PDAC, we
generated mice with pancreas specific homozygous or heterozygous deletion of Atg5.
Homozygous deletion of Atg5 elevated tumor initiation but blocked progression; heterozygous
deletion of Atg5 increased tumor aggressiveness and metastasis. In vitro with in vivo analysis
reveal that Atg5-Heterozygosity not only influences cell homeostasis but also affects the tumor
microenvironment enhancing cell migration. Pro-tumorigenic inflammation, changes in
calcium homeostasis, and increased extracellular cathepsin activities collectively shape the
microenvironment enhancing metastasis. Thus, tumor stage and genetic background have to be
considered when targeting autophagy in PDAC.
Invited speaker talk
18
Targeted Therapy in Colorectal Cancer
Maryam M. Matin1,2
1Department of Biology and Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad,
Iran 2Stem Cells and Regenerative Medicine Research Group, Academic Center for Education, Culture,
and Research (ACECR), Khorasan Razavi Branch, Mashhad, Iran
Colorectal cancer is the third most common cancer worldwide and its incidence has been
increasing in Iran in the past decade. Depending on the stage of colon cancer, various treatments
including surgery, chemotherapy, immunotherapy or targeted therapy might be considered in
the clinic. Despite many advances in these fields, patients still suffer from poor prognosis and
further studies are required on early detection and targeted therapy of this cancer. In the past
few years my lab has focused on different approaches to improve the efficacy of colorectal
cancer treatment. While use of natural products like ferutinin was beneficial, further research
indicated that safer and more efficient treatments can be achieved by targeted therapy. Some
examples include downregulation of certain long noncoding RNAs and also specific approaches
like "bacterial directed enzyme prodrug therapy" for which both in vitro and preclinical results
in mouse models with colorectal cancer will be discussed. However, despite all these promising
results, I think a combinatorial approach targeting multiple mechanisms would be more
appropriate in colorectal cancer therapy in the clinic.
Invited speaker talk
19
Protein lipidation on cancer metabolism
Burçin Güngör
Department of Medical Biochemistry, Faculty of Medicine, Istinye University, İstanbul, Turkey
The importance of protein lipidation and lipid metabolism in cancer is well-recognized, but the
full spectrum of dysregulation of lipid post-translational modifications (PTMs) in cancer
remains largely unexplored. Till now, it has been known that a wide range of proteins, involved
in cancer, are modified by covalent linkage of fatty acids and/or isoprenoid groups. Attachment
of these hydrophobic groups plays a major role in regulating protein structure and function such
as correct subcellular localization of the protein. The saturated nature of the fatty acid promotes
insertion of palmitoylated proteins into liquid ordered domains of the membrane, known as
lipid rafts. Multiple signaling proteins are enriched in lipid rafts, and raft association promotes
intracellular signal transduction. Proteins actively functioning in malignant cell growth and
movement require lipidation. Therefore the action of lipid linkage and dissociation have
emerged as potential drug targets in a wide variety of disease states. Moreover, our
understanding of the dynamics of intracellular trafficking of lipid modified proteins has
dramatically expanded over the past few years. Ras proteins (K-, H, and N-Ras) are among the
best-known oncoproteins. H- and N-Ras are prenylated and then palmitoylated. Another group
of proteins possessing prominent lipidation features is Rho GTPases. Among these, Rac1 is one
of the key proteins for signal transduction for transcriptional regulation, ROS production, cell
motility, etc. Rac1 has double lipidation; prenylation and palmitoylation. Lipidation of Rac1
has been reported for protein localization to lipid rafts to regulate actin cytoskeleton thus cell
movement. Targeting Rac1 lipidation in cancer cells can be one of the important focus on the
therapeutic development for cancer cell invasion.
Invited speaker talk
20
Molecular Diagnosis in Childhood Cancers
Sema Yilmaz
Health Science University Kartal Lutfi Kirdar Education and Research Hospital, Pediatric
Hematology/Oncology, Istanbul/Turkey
Cancer is a Genetic Disease (Theodor Boveri, 1914). Gene and chromosome abnormalities
observed in cancer[Gene mutations (oncogenes, tumor suppressor genes), Chromosome
structural, abnormalities (translocations, deletions, insertions) and Chromosome number
abnormalities (aneuploidy, polysomy)].
Molecular oncology (Personalized approach) provides, risk assessment, differential diagnosis,
prognosis prediction of treatment response pharmacokinetics, monitoring treatment response,
monitoring recurrence in patients without symptoms of cancer.
Diagnostic/Prognostic information
Molecular assays are currently available for testing on tumors including
• Fluorescent in situ hybridization (FISH)
• Polymerase chain reaction (PCR)
• Single nucleotide polymorphism (SNP)-based microarrays
• Next-generation sequencing (NGS) [Whole-genome sequencing (WGS), Whole
exome sequencing (WES) and RNA sequencing (RNAseq)
Next-generation sequencing (NGS), allows for massively parallel sequencing of genomic
fragments generating thousands to millions of short “reads” in a single run. Point mutations
including single nucleotide polymorphisms as well as small (generally, less than 20–30 base
pairs) insertions/deletions (indels) in large numbers of genes at once can be detected. NGS can
also identify unanticipated targetable mutations, copy number alterations, differentially
expressed genes, and gene fusions.
Pediatric cancers:
Pediatric cancers are significantly different from adult cancers because of mutation frequency
and type of altered genes, exhibit a lower mutational burden, relatively high prevalence of
specific structural variations (e.g., gene fusions and chromosomal rearrangements). Most of the
Invited speaker talk
21
genetic alterations in pediatric leukemias and solid tumors involve well-known genes and
oncogenic pathways such as:
• Receptor tyrosine kinases,
• Phosphoinositide 3-kinase-AKT and related pathways, TP53, CDK12,
NOTCH1, ARID1 and
• Include amplification of other genes such as MYCN, MCL1, MDM2.
Precision cancer treatment
Precision cancer treatment is largely based on matching the patient’s tumor mutations with the
appropriate targeted therapy. The use of novel targeted therapy has greatly revolutionized
cancer treatment and is largely based on blocking actionable gene mutations or over-expressed
signaling transduction pathways.
Invited speaker talk
22
Glioblastoma Multiforme Beyin Tümörünün Erken Tanı ve Tedavisine Yönelik
Otoantikor Çalışmaları
Nihal Karakaş
İstanbul Medipol Üniversitesi Tıp Fakültesi, Tıbbi Biyoloji ABD, Rejeneratif ve Restoratif Tıp
Araştırmaları Merkezi (REMER)
Primer beyin tümörlerinin %60 kadarını gliomalar oluşturur ve gliomalar içerisinde ise Dünya
Sağlık Örgütü’nün sınıflandırmasına göre 4.evre (grade IV) olarak sınıflandırılan ileri grade’li
gliomalardan “Glioblastoma multiforme (GBM)” yetişkinlerde en sık görülen, agresif ve
malign bir beyin tümörüdür. GBM’in tedavisindeki bugünkü yöntemler, cerrahi tedavi ve bunu
takip eden radyoterapi ile kemoterapi uygulamalarıdır. Ancak hastalara GBM tanısı
konulduktan sonra yaşam süresi maalesef 1 yılı geçmemektedir. Dolayısıyla GBM ile
mücadelede, hastalığa özgü biyobelirteçlerin saptanması, erken tanı ve tedavi yöntemlerinin
geliştirilmesine öncülük edebilecektir.
Kanser oluşumu ile ilişkili olduğu düşünülerek vücut sıvılarında sirküle olan otoantikorların
araştırılması, özellikle kanserin erken tanısında yeni biyoişaretleyicilerin belirlenmesi açısından
etkin bir çalışma alanı oluşturmuştur. Akciğer ve meme kanseri gibi olgularda tespit edilen
otoantikorların, patolojik ve klinik olarak kanser tipine özgünlük gösterdiği tespit edilmiştir.
Buna karşılık, literatürde otoantikorların, beyin tümörleri ile ilişkisine yönelik, hastalığın
patolojisiyle ilgili olabilecek bilinen antijenlere özgü antikorlara ait tarama yapılmasına karşılık
olası yeni otoantikorlar ve ilişkili antijenlerin tespitine yönelik bir araştırma bildirilmemiştir.
Dolayısıyla, gliomalar içinde tedavisi en güç olan GBM’e özgü anti-GBM otoantikorların
araştırılması ve daha önce tanımlanmamış, yeni hedef antijenlerin keşfi GBM teşhis ve
tedavisinde bir dizi çalışmanın önünü açabilecek niteliktedir. Çalışmamızda, GBM ve non-
GBM glioma olarak gliomalar iki gruba ayrılmıştır ve hastaların tümör doku ve serumları
toplanmıştır. Kontrol olgu olarak ise tümör gelişimi oluşmamış epilepsi hastalarının örnekleri
kullanılmıştır. Elde edilen doku numunelerinde western blot analizleri yapılmış, primer antikor
yerine hasta ve kontrol serumları kullanılmıştır. Anlamlılık gösteren hedef proteinlerin tespit
edilebilmesi için kütle spektrometrisi analizleri ile aday proteinler tanımlanmıştır. Bu aday
proteinlere yönelik otoantikorların GBM serumlarında varlığı immünopresipitasyon ve
immünohistokimya yöntemleri uygulanarak çalışılmıştır.
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Western blot analizleri ile serum örneklerinin, doku örneklerinden elde edilen proteinlerle
etkileştiği ve kontrol olgularına kıyas ile GBM olgularında anlamlı farklılıklar olduğu
saptanmıştır. Bu sonuçlara dayanarak 5 kontrol, 5 düşük grad ve 5 GBM olgularından elde
edilen proteinler kütle spekrometrisi yöntemi ile analiz edilmiştir. Biyoinformatik
değerlendirmeler sonucuda GBM olgularında düşük grad glioma ve kontrol olgularına kıyas ile
fazla eksprese edilen proteinler tespit edilmiştir. Bu aday proteinlerden birinin otoantijen
olduğu ve GBM hasta olgularında bu otoantijene güçlü oranda bağlanan özgün otoantikorların
varlığı immünopresipitasyon yöntemi ile belirlenmiştir. İmmünhistokimya çalışmaları
neticesinde GBM doku preparatlarında anlamlı derecede artmış otoantijen boyaması
gözlemlenmiştir. Bu bulgular kütle spektrometrisi ve immünopresipitasyon çalışmalarından
elde edilen bulgular ile uyumluluk göstermiştir. Sonuç olarak, bu çalışmamızda, GBM’ lerde
fazla ekspese edilen proteinlerden birini tanıyan bir otoantikor tespit edilmiştir. Ayrıca bu
otoantikorun, GBM için prognostik ve/veya diagnostik biyobelirteç adayı olduğu gösterilmiştir.
Böylece GBM’ye özgü bir otoantikor tanımlanmış ve bu yönüyle GBM hastalığının hasta
serumları kullanılarak erken dönem tanısına imkan sağlayabilecek bir buluş ortaya
konulmuştur.
Bu çalışma TÜBİTAK 216S nolu proje desteği ile gerçekleştirimiş ve patent başvuruları
başlatılmıştır.
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Discovery of A Molecule That Destabilize Cryptochrome 1
Şeref Gül1, Kübra Akyel2, Tuğba Korkmaz3, Ibarhim Daniş4,5, Özgecan Şavlug İpek6,7, Fatih
Aygenli3, Ali Cihan Taskin8, Nuri Ozturk3, Narin Öztürk2, Durişehvar Özer Ünal4,5, Mustafa
Güzel6,9, Metin Turkay10, Alper Okyar2, Ibrahim Halil Kavakli1,11
1 Department of Chemical and Biological Engineering, Koc University, İstanbul
2 Faculty of Pharmacy Department of Pharmacology, Istanbul University, İstanbul, Turkey, 3 Department of Molecular Biology and Genetics, Gebze Technical University, Kocaeli, Turkey, 4 İstanbul University Faculty of Pharmacy Department of Analytical Chemistry Beyazit-İstanbul,
5 İstanbul University Drug Research and Application Center (ILAM) Beyazıt İstanbul, 6 İstanbul Medipol University, Regenerative and Restorative Medicine Research Center (REMER),
Kavacik Campus, Kavacik-Beykoz/İstanbul, 7 Yildiz Technical University, Graduate School Of Natural And Applied Sciences, Department of
Chemistry, Besiktas/İstanbul, Turkey, 8 Animal Research Facility, Research Center For Translational Medicine, Koç University,
Rumelifeneri yolu, Sariyer, İstanbul, 9 İstanbul Medipol University, International School of Medicine, Department of Medical
Pharmacology, Kavacik Campus, Kavacik- Beykoz-İstanbul, 10 Department of Industrial Engineering, Koc University, İstanbul,
11 Department of Molecular Biology and Genetics, Koc University, İstanbul, Turkey.
We discovered a molecule that destabilizes Cryptochrome1 (CRY1) both in vitro and in vivo
using a structure-based drug design approach. The molecule, M47, enhanced the degradation
rate of CRY1 and modulated the period of U2-OS Bmal1-dLuc cells. In addition,
pharmacological studies with mice indicated that M47 had no toxic effect with half-life of ~6
h in blood. In addition, subcellular fractionation studies from mice liver indicated that M47
selectively decrease CRY1 level in the nucleus. Furthermore, M47 mediated CRY1 reduction
enhanced cisplatin-induced apoptosis in Ras-transformed p53-null fibroblast cells. Effect of
M47 on the oxaliplatin-induced apoptosis makes it a very promising agent to treat p53 mutant
dependent forms of cancer.
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REWIRING THE EPIGENETIC NETWORKS AS A THERAPEUTIC APPROACH
IN MLL-REARRANGED LEUKEMIAS
Ipek Bulut1, Buse Cevatemre2, Adam Lee3, Arasu Ganesan3, Ceyda Acılan Ayhan2,4*
1Koc University, Graduate School of Science and Engineering, Istanbul, Turkey, 2Koc University, Research Center for Translational Medicine, Istanbul, Turkey,
3University of East Anglia, School of Pharmacy, Norwich, England, 4Koc University, School of Medicine, Istanbul, Turkey,
*To whom correspondence should be addressed
Defects in epigenetic pathways involving increased expression levels or abnormal patterns of
activity are one of the key drivers of cell proliferation in cancer. A particular example of these
defects is the MLL-AF9 fusion gene, seen in mixed-lineage leukemia (MLL). While the normal
function of the MLL gene regulates expression of developmental genes, chimeric MLL shows
abnormal activity playing an important role in the process of malignant transformation. The
main purpose of our studies is to reverse this transformation and to develop a personalized
medicine approach for the treatment of leukemia by targeting epigenetic processes dysregulated
in patients bearing the MLL-AF9 fusion protein.
Dual targeting in drug discovery is particularly important, as drugs with two targets reduce the
possibility of intrinsic or acquired drug resistance in tumors. While the normal function of MLL
gene has methyltransferase activity, the MLL-AF9 fusion protein recruits Lysine-Specific
Demethylase (LSD1) leading to activation of several genes that inhibit differentiation and cause
uncontrolled cell proliferation. Among the Histone De-Acetylases (HDACs), HDAC6 is
particularly unique, as it has targeted both in the nucleus and cytoplasm. Additionally, it is the
only HDAC, whose knockout mice is viable, making it an attractive target with potentially less
toxicity. Therefore, our project involves the use of LSD1 and HDAC6 inhibitors for dual
targeting.
The activity and the targeting efficiency of the inhibitors were verified by enzyme-based
profiling. The selectivity was measured through inhibition of other enzymes with similar
structure through in vitro enzyme inhibition and cellular thermal shift assays. Cell viability was
assessed using CTG assay, and the IC50 values were lower in cells containing MLL-AF9
compared to no MLL fusion. The epigenetic changes in response to these inhibitors were tested
via western blotting with epitope-specific antibodies. The differential expression of target genes
was evaluated using RT-qPCR, and a dramatic increase was observed CD11b and CD86
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expression indicating that the drugs exhibited the expected changes. The synergy of these
molecules with the drugs used in the clinic was examined using both CTG and flow cytometry.
A significant synergy was observed with doxorubicin, which did not appear to be a result of
topoisomerase II inhibition. The mechanism of action for this synergistic effect is still under
examination.
In summary, the molecules under investigation showed promising activity in MLL-AF9
leukemic cells and will be characterized further as potential drugs in the clinic.
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Treatment of refractory recurrent Gastrointestinal Stromal Tumors with adoptive
cellular immunotherapy (TILs) and Personalized Vaccine: a case report
A Tavartkiladze., R. Khutsishvili., P.Revazishvili., M.Maisuradze., L.Tavartkiladze
Institute for Personalized Medicine, Tbilisi State Medical University, Tbilisi, Georgia
We report the successful treatment of a patient with recurrent Gastrointestinal Stromal Tumors
with adoptive cellular immunotherapy and personalized vaccine. The patient is a young adult
with recurrent progressive disease refractory to aggressive multi-modality therapy including
repetitive surgical resection, radiation, and chemotherapy and all lines targeted therapy. He
received multiple courses of systemic intravenous administration of allogenous
phytohemagglutinin (PHA) activated T-lymphocytes in combination with a low dose of
interleukin-2 (IL-2) and personalized vaccine, which was adopted in laboratory from donors B-
lymphocytes and Patient’s Circulated Tumor Cells. The side-effects of this treatment were
limited and manageable. The patient achieved complete remission, as demonstrated by MRI
after 2 months from initiation immune therapy and confirmed by glucose-positron emission
tomography (PET) imaging 7 months after initiation of immune therapy. 14 months later, the
patient is still in remission with improving performance status. Adoptive cellular
immunotherapy utilizing allogenous phytohemagglutinin (PHA) activated T-lymphocytes and
personalized vaccine with low dose IL-2 appears to be a safe and effective therapy for a subset
of patients with primary, recurrent or progressive Gastrointestinal Stromal Tumors following
conventional therapy.
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Cancer Cells Adaptation to Immune Responses
Güneş Esendağlı
Department of Basic Oncology, Cancer Institute, Hacettepe University, Ankara-Turkey
The straightforward perspective that tumor cells are immunosuppressive has been contradicted
by the expression of potent costimulatory molecules on certain cancer cells. Indeed, the
stimulatory support offered by certain cancer cells such as myeloid leukemia or basal-like breast
cancer cells can provoke helper T cell responses. Unfavorably, this interaction can lead these
malignant cells to acquire an immune-suppressive capacity. Strong immune reactions are
required for the elimination of tumor cells. Tumor cells found in the circulation are considered
to be more susceptible to immune attack; the likelihood of these cells to come across with
immune cells is enhanced, and they are devoid of a protective (immunosuppressive)
microenvironment. On the other hand, anti-tumor immunity does not always correlate with
reduction in tumor growth and increase in patient survival. Moreover, immune-stimulatory
interventions have been shown to enhance the ability of immune cells to eradicate some, but
not all, tumor cells. Essentially, reduction in immune-provoking signals derived from the tumor
can diminish the effector phase of immune responses. The dogma in tumor immunology is that
the tumor cells express inhibitory molecules and anti-inflammatory cytokines to escape from
anti-tumor responses. However, it becomes more intriguing since the costimulatory molecules
can also be found on the tumor cells. Tumor cells hide from immune recognition and/or cope
with the immune attack. In other words, tumor cells which can successfully evade the anti-
tumor immunity may emerge as a consequence of adaptation to the selective pressure applied
by the immune system. Our results demonstrate the rapid adaptation capacity of leukemia,
breast cancer and lung cancer cells in response to anti-tumor immune responses. In our
experience, only a small subpopulation of cancer cells was able to provoke immunity, especially
through costimulation, adopts this de novo immunosuppressive character. Thus, during immune
evasion, tumor cells may benefit from being composed of heterogeneous subpopulations.
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Mouse Models for Immunotherapeutic Approaches
Konstantinos Dimas
Department of Pharmacology, Faculty of Medicine, Health Science School, University of Thessaly,
Larissa, Greece
Any preclinical model system should mimic human cancer development. This includes models
that reproduce the genomic heterogeneity of human cancer, as well as develop a milieu that
incorporates the multitude of immune and stromal cell populations that make up the complex
tumor microenvironment. The gold standard for the development of standard cytotoxic cancer
therapies is to utilize xenograft models of human cancer cell lines engrafted into
immunocompromised mice to evaluate pharmacology, efficacy, and safety profiles of these
agents. However, the development of immunotherapy requires a model system with a
functionally intact immune system. Additionally, the immune system shows an inherent
heterogeneity and adaptability that to some extent explains the relative success of
immunotherapy, in that it is able to constantly adapt and evolve along with the tumor. However,
finding preclinical models that can recapitulate this adaptability in the face of tumor
heterogeneity remains a major impediment in the development of cancer immunotherapies. In
this brief talk, we discuss the current landscape of preclinical models available for preclinical
immunotherapeutic evaluation and the pros and cons associated with each model in this context.
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Diagnostic and Prognostic Importance of Leukemia Cytogenetics
Süreyya Bozkurt
Department of Medical Biology, Faculty of Medicine, İstinye University, Istanbul Turkey
Cytogenetic Anomalies have a very important role in the prognosis and diagnosis of
hematologic cancers. Some cytogenetic abnormalities predict good prognosis while others
indicate poor prognosis. Cytogenetic is the gold standard for detecting to Ph chromosome and
monitoring cytogenetic response and is one of the most important prognostic markers in CML.
The emergence of additional chromosomal anomalies (ACA) besides the Philadelphia
chromosome in CML is thought to be a sign of disease progression. The frequencies of ACA s
in higher in CML-BC (Blastic crisis phase) patients than in CML-CP (Chronic Phase) patients
or AP patients. ACAs can be classified as major and minor route of abnormalities. The minor
route abnormalities are loss of Y chromosome, trisomy 21, hypodiploidy, hyperdiploidy, and
polyploidy. The treatment of the chronic phase Ph + CML is the oral administration of tyrosine
kinase inhibitors (TKI). Cytogenetic and molecular monitoring help selecting the most
convenient TKI drug and to optimize TKI treatment. The emergence of ACAs in CML
karyotype is an alarm for the development of AML or MDS. Cytogenetic findings play a central
role in diagnostic classification in AML as in all myeloid malignancies. In the development of
AML, some cytogenetic abnormalities function specifically as prognostic markers. Therefore
cytogenetic is another important diagnostic marker after induction therapy in AML. While del
(11q) is a marker of good prognosis, inv
(3) / t (3q) / del (3q) is a marker of poor prognosis in Myelodysplastic syndromes (MDS).
Despite highly advanced molecular techniques, conventional cytogenetic hematological
cancers remain the gold standard. Because cytogenetic anomalies shed light on new treatment
options as a guide in disease pathogenesis. It is also a guide in predicting prognosis and helps
in the selection of treatments to be applied.
References
Bennour A, Saad A, Sennana H. Chronic myeloid leukemia: Relevance of cytogenetic and molecular
assays. Crit Rev Oncol Hematol. 2016;97:263-74
François Guilhot. Cytogenetics in CML: more important than you think. Blood 2016 127:2661-2662
Ravandi F, Walter RB, Freeman SD. Evaluating measurable residual disease in acute myeloid
leukemia. Blood Adv. 2018 Jun 12;2(11):1356-1366
Hosono N. Genetic abnormalities and pathophysiology of MDS. Int J Clin Oncol. 2019;24(8):885-892
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ANTİKANSER ÇALIŞMALARINDA PLATİN KOORDİNASYON BİLEŞİKLERİNİN
ROLÜ
Abdurrahman Şengül
Zonguldak Bülent Ecevit Üniversitesi, Fen-Edebiyat Fakültesi, Kimya Bölümü 67100 İncivez,
Zonguldak
E-mail: [email protected]
Kanser, günümüzde dünyanın en önemli sağlık problemidir. Kanser, vücudumuzun herhangi
bir yerinde anormal hücrelerin kontrol edilemez bir şekilde büyümesi olarak tanımlanmaktadır.
WHO, kanserin genetik faktörlerin ve çevresel faktörlerin (UV, radyasyon, kimyasal
kanserojenler ve biyolojik kanserojenler) kişiyle etkileşimi sonucu meydana geldiğini rapor
etmiştir. Kanserin tipine, safhasına ve hastanın durumuna göre birçok farklı tedavi yöntemi
mevcuttur. Bu sunumda, 1965 yılında Barnett (Barney) Rosenberg’in Cisplatin’in (cis-
[Pt(NH3)2Cl2]) antitümör aktivitesini keşfetmesinden sonra kemoterapi alanında klinik
kullanımı onaylanmış olan platin(II)-koordinasyon bileşiklerinin özellikleri ve kanser
tedavisindeki rolleri vurgulanacaktır.
Cisplatin’in birçok kanser türüne karşı çok aktif olmasına rağmen ciddi yan etkilere sahip
olması ve bazı kanser tiplerinin Cisplatin’e direnç göstermesinden dolayı binlerce platin-esaslı
antitümör ilaç sentezlenmiştir. Bunlardan sadece birkaç tanesi (örneğin: Oxaliplatin) Cisplatin
direncini yenmesi ve daha az toksik özellik göstermesine rağmen günümüzde Cisplatin’den
daha etkili bir kemoterapi ajanı henüz keşfedilmemiştir. Günümüzde kanser hastalarının yarısı
Cisplatin ile tedavi edilmektedir. Yan etkileri daha az ve geniş spektruma sahip ilaç geliştirmek
için birinci nesil, ikinci nesil, üçüncü nesil ve yeni nesil antikanser platin ilaçları
sentezlenmiştir. Klasik kovalent bağlanma yapan ilaçların yerine daha sonra interkalasyon ve
groove bağlanma özelliği olan ilaçlar geliştirilmiştir. Günümüzde kinetik olarak daha kararlı
olan ve hücre içinde indirgenerek aktif hale geçen platin(IV) esaslı kompleksler büyük aşama
kaydetmiştir. Bunların yanında hidrofobik özellikleri olan dinükleer platin(II) bileşikleri de
umut verici sonuçlar göstermektedir. Bu çalışmada tüm bu bileşiklerin, in vitro ve in vivo
deneyleri sonucunda belirlenen antitümör aktiviteleri ve ilaçların moleküler yapıları ile olan
ilişkileri irdelenecektir.
Literatür ışığında, daha büyük antikanser özelliğe sahip fakat daha az yan etki gösteren
platin(II)-koordinasyon bileşikleri sentezlenmiş ve bunların DNA etkileşimleri ve sitotoksisite
çalışmaları grubumuz tarafından yapılmıştır.
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Kanser Kök Hücresinde Güncel Tedavilere Yaklaşım
Gülperi Öktem
Ege Üniversitesi, Tıp Fakültesi Histoloji ve Embriyoloji ABD, İzmir
Ege Üniversitesi, Sağlık Bilimleri Enstitüsü Kök Hücre ABD, İzmir
Tümör kitlesindeki heterojen hücre popülasyonlarının çok küçük bir kısmını oluşturan Kanser
Kök Hücreleri (CSC) veya Tümör Başlangıç Hücreleri (TICs) malign progresyona sebep olan,
tedaviye yanıtı değiştiren hücrelerdir. Pek çok araştırma gurubu tarafından incelenen bu hücre
gurubu hedef tedavilerin de gözdesi haline gelmiştir. CSC’nin eradike edilmesininin kanseri
ortadan kaldıracağı yaygın olarak kabul görmektedir. Bu hücre gurubuna ait pek çok biobelirteç
araştırma gurupları tarafından identifiye edilmiş ve akademik makalelerde yayınlanmıştır.
Özellikle yüzey belirteçlerini hedefleyen spesifik monoklonal antikor tedavileri kanser tedavi
protokollerini değiştirmeye başlamıştır. Ancak görünen odur ki; günümüzde CSC’ni identifiye
etmek için kullanılan yüzey belirteçlerinin %73 ü insan embriyonik kök hücrelerinde (hESCs),
erişkin kök hücrelerinde ve somatik hücrelerde de bulunmaktadır. Bu da CSC’in epigenetik
veya genetik değişiklikler sonunda normal kök hücrelerden farklılaştığını göstermektedir. Bu
sebeple yüzey belirteçlerine göre kanser kök hücrelerinin izole edilmesi veya hedef tedavilerde
kullanılabilmesi için ancak spesifik bir beliteçin bulunması veya birden fazla yüzey belirtecinin
kullanılarak hedef tedavilerin yapılması uygun seçenek gibi görünmektedir. Bununla beraber,
kanser araştırmalarında değişen gündem son yıllarda egzozomların stratejik hedef olarak
karşımıza çıktığını göstermektedir. Hücresel sinyallemenin ana düzenleyicisi olarak, hem
normal hem de kanser kök hücreleri, tümör mikro-ortamını, büyümesini, ilerlemesini ve
varsayılan immünolojik fonksiyonunu değiştiren çeşitli otokrin ve parakrin sinayalleri orkestra
etmek için eksozomlar salgılanmaktadır. Bu endozomal yolak orijinli nano kesecikler
orijinlendiği hücrenin parmak izini taşıması yönü ile özellikle kanserin tanı ve tedavisinde
potansiyel belirteçler olma özelliği taşır, mRNA ve mikroRNA(miRNA)‘lar gibi taşıdıkları
kargo içerikleri ile kanser ilerleyişine, immün sistemden ve apoptozisten kaçış, anjiyogenez,
invazyon, metastaz gibi onkojenik hedefleri uyararak, amtitümör immün yanıtı değiştirerek
katkı sağlarlar. Hücreler arası iletişiminde eksozomların ve eksozomal miRNA vasıtası ile bilgi
aktarımı kanser gelişimindeki en önemli mekanizmalardan biridir. CSC’nin onkojenik fenotipi
teşvik etmek için tümör mikro ortamları ile nasıl etkileşime girdiği konusunda bilgilerimiz hala
eksiktir. Bu nedenle tanı ve tedavide tüm vücut sıvılarından elde edilebilen eksozomlar serum
ve plazmada korunmuş halde bulunan hücre tipine göre spesifik olan ve kökenlendiği tümör ile
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ilgili biyobelirteçler içeren eksozomal miRNA’lar kullanılması çok daha büyük bir potansiyel
taşımaktadır.
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AURORA KINASES AND CANCER
Ata Özçimen
University of Mersin, Department of Biology, Mersin-Turkey
Cancer is a disease caused by uncontrolled cell proliferation of bad tumors in the body. Human
cancer cells are often characterized by changes in the amount or organization of DNA resulting
from errors in mitosis, resulting in chromosome instability and aneuploidy. Auroras are the
serine/threonine kinase family, which are highly conserved throughout evolution in cell
division processes. As the main regulators of mitotic chromosome degradation, condensation
and orientation in the metaphase plate, spindle assembly and completion of cytokinesis play an
important role in cell division. They also play an important role in the proliferation of cancer
cells. Three mammalian subtypes were identified from these kinases as Aurora -A, -B and -C.
All the Aurora genes are characterized by 78–84% of identity between human and rodent
orthologs [3]. Phylogenetic trees show that the Aurora genes have originally evolved from a
single gene from urochordata ancestor (Tunicata). This gene was first called Increases in
Ploidy-1 (Ipl1). It has previously been identified in yeast genomes such as Saccharomyces
cerevisiae and Saccharomyces pombe, suggest that the functions of Aurora vary throughout
evolution. These only encode an Aurora–like homolog (Ipl1) [1,4]. The ancestral AURK gene
shares 41% of identity with the human AURKA gene. Aurora-A family is ubiquitous among
all vertebrates. In invertebrates and non-mammalian vertebrates, Ipl1 gene gave rise to AURKA
and AURKB/AURKC. The invertebrate Aurora-A and Aurora-B kinases have protein
families that are quite different from their vertebrate counterparts. The Aurora-B and Aurora-
C families in humans have emerged from a gene duplication event in mammals. These all three
were highly conserved in evolution processes [3]. Three mammalian Aurora kinases occur at
certain locations during mitosis. Aurora-A, which is the “polar kinases”, combines mainly
with separating centrosomes. The gene is encoded in human Chromosome 20q13 locus. It’s
overexpression and oncogenic transformation have inhibited the p53 activity [5]. Aurora-B,
which is the “equatorial kinase”, is a chromosomal transition protein. This gene is encoded in
human chromosome 17q13 locus. Aurora-B is a specific protein of Chromosomal Passenger
Complex (CPC), which manages kinase activity and localization. This complex is combined
with Inner Centromere Protein (INCENP), survivin (BIRC5), borealin (CDCA8) and non-
enzymatic sub-units [5,6]. Aurora-C, the least studied, appears to be localized in anaphase to
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telophase centrosome, highly expressed in germ and testis. This gene is encoded in human
chromosome 19q13 locus.
Beside the increased expression in normal cell division and meiosis, all Aurora kinases have
differentially expressed in tumor cells. Aurora-A is highly amplified, overexpressed and
hyperactivated in several aneuploidy tumors such as gastrointestinal, nasopharyngeal, prostate
and breast cancers [3]. Aurora-B is also overexpressed in most cancer cells, which were
transformed from over proliferated non-cancerous cells. This gene is mainly active in stemness,
invasion and proliferation of tumors. Due to the actions of Aurora-B is a poor diagnostic factor
for several tumors such as hepatocellular carcinoma, non-small cell lung carcinoma and oral
squamous cell carcinoma [7]. Aurora-C is expressed in meiotically-active germ cells and in
human thyroid carcinomas, cervical and colorectal tumors. Aurora-C protein level is specially
correlated with the malignancies of colorectal carcinoma. However, Aurora-C overexpression
in tumors was not yet well described [8]. In the shed of light, molecular approach of the cancer
treatment is way to block proteins cause irregularity and their irregular important mediators in
cell division. One of the best approaches is suitable for Chromosomal Passenger Complex that
regulates kinetochore-microtubule associations in mitosis [2]. The therapeutic inhibition of
these kinases has probably become an attractive target for the development of potential anti-
cancer therapies due to their fundamental role during cell division. In fact, several small
molecule Aurora kinase inhibitors have already been developed, and some have shown
promising clinical efficacy in some human tumors in Phase-I and-II clinical trials [7].
As a result of the studies, one of the most advanced clinical compounds showing strong activity
against aurora kinases is Tozasertib (VX680), Alisertib (MLN 8237) and Danusertib
(formerly known as PHA-739358). Danusertib is named as Pan-Aurora Kinase inhibitor
because of its’ effective on all three Aurora kinases group. Further pharmaco-active studies of
pan-aurora kinase inhibitors might be potential anti-tumor compounds for cancer patients [7].
References
[1]. Brown, J. R., Koretke, K. K., Birkeland, M. L., Sanseau, P., Patrick, D. R. (2004). Evolutionary
relationships of Aurora kinases: Implications for model organism studies and the development of anti-
cancer drugs. BMC Evolutionary Biology, 10.1186/1471-2148-4-39. 10.
[2]. Bolanos-Garcia, V.M. (2005). Aurora kinases, The International Journal of Biochemistry & Cell
Biology 37. 1572–1577.
[3]. Willems, E., Dedobbeleer, M., Digregorio, M., Lombard, A., Lumapat, P. N., Rogister, B. (2018).
The functional diversity of Aurora kinases: a comprehensive review. Cell Div 13:7. 17.
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[4]. Sasai, K., Katayama, H., Stenoien, D. L., Fujii, S., Honda, R., Kimura, M., Okano, Y., Tatsuka, M.,
Suzuki, F., Nigg, E. A., Earnshaw, W. C., Brinkley, W. R., Sen, S. (2004). Aurora-C kinase is a novel
chromosomal passenger protein that can complement Aurora-B kinase function in mitotic cells. Cell
Motility and the Cytoskeleton, 59(4), 249–263.
[5]. Seeling, J. M., Farmer, A. A., Mansfield, A., Cho, H., Choudhary, M. (2018). Differential
selective pressures experienced by the aurora kinase gene family. International Journal of Molecular
Sciences, 19(1), 1-16.
[6]. Adams, R. R., Carmena, M., Earnshaw, W. C. (2001). Chromosomal passengers and the (aurora)
ABCs of mitosis. TRENDS in Cell Biology 11(2). 49-54.
[7]. Kitzen, J.J.E.M., de Jonge, M.J.A., Verweij. J. (2010). Aurora kinase inhibitors. Critical Reviews
in Oncology/Hematology 73, 99–110.
[8]. Gavriilidis, P., Poutahidis, T., Giakoustidis, A., Makedou, K., Angelopoulou, K., Hardas, A.,
Andreani, P., Zacharioudaki, A., Saridis, G., Gargavanis, A., Louri, E., Antoniadis, N., Karampela, E.,
Psychalakis, N., Michalopoulos, A., Papalois, A., Iliadis, S., Mudan, S., Azoulay, D., Giakoustidis, D.
(2018). Targeting hepatocarcinogenesis model in C56BL6 mice with pan-aurora kinase inhibitor
Danusertib. Journal of Cancer, 9(5), 914-922.
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A Parkinson-related Gene in Melanoma: Exploring The Role of The P5 Type ATPase
ATP13A2, A Parkinson Disease’s Associated Gene, in Melanoma Cell Proteostasis
Seyma Demirsoy 1,3, Peter Vangheluwe2 and Patrizia Agostinis1
1Laboratory of Cell Death Research & Therapy, Department of Cellular and Molecular Medicine, KU
Leuven, Belgium, 2Laboratory of Cellular Transport Systems, Department of Cellular and Molecular Medicine, KU
Leuven, Belgium, 3Laboratory of Molecular Microbiology, Department of Medical Microbiology, Hacettepe University,
Ankara, Turkey
ATP13A2 (also called PARK9), is a transmembrane endo-/lysosomal-associated P5 type
transport ATPase. Loss-of-function mutations in ATP13A2 result in the Kufor-Rakeb
Syndrome (KRS), a form of autosomal Parkinson’s disease (PD). In spite of a growing interest
in ATP13A2, very little is known about its physiological role in stressed cells. Recent studies
suggest that the N-terminal domain of ATP13A2 may hold key regulatory functions, but their
nature remains incompletely understood. To this end, we generated a set of melanoma and
neuroblastoma cell lines stably overexpressing wild-type (WT), catalytically inactive (D508N)
and N-terminal mutants, or shRNA against ATP13A2. We found that under proteotoxic stress
conditions, evoked by the proteasome inhibitör Bortezomib, endo-/lysosomal associated full-
length ATP13A2 WT, catalytically-inactive or N-terminal fragment mutants, reduced the
intracellular accumulation of ubiquitin-conjugated (Ub) proteins, independent of autophagic
degradation. In contrast, ATP13A2 silencing increased the intracellular accumulation of Ub-
proteins, a pattern also observed in patient-derived fibroblasts harboring ATP13A2 loss-of-
function mutations. In treated cells, ATP13A2 evoked endocytic vesicle relocation and
increased cargo export through nanovesicles. Expression of an ATP13A2 mutant abrogating
PI(3,5)P2 binding or chemical inhibition of the PI(3,5)P2-generating enzyme PIKfyve,
compromised vesicular trafficking/nanovesicles export and rescued intracellular accumulation
of Ub-proteins in response to proteasomal inhibition. Hence, our study unravels a novel activity
–independent scaffolding role of ATP13A2 in trafficking/export of intracellular cargo in
response to proteotoxic stress.
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A Novel Transcriptomic Based Gene Panel for The Prognostication of Pancreatic
Cancer
Seçil Demirkol Canlı1, Ali Osmay Güre2
1Molecular Pathology Application and Research Center, Hacettepe University, Ankara, Turkey
2Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey
PDAC is amongst the most lethal cancer types in the world with records of 5 year survival less
than 5%. A projection of cancer incidence and death rates to 2030 in US showed that pancreas
cancer will become the second cause of cancer-related death by 2030. A comprehensive study
of death rate trends in pancreatic cancer since 1970 indicated complex trends that are largely
unexplainable by known risk factors. For the evaluation of risk, currently the only FDA
approved biomarker for PDAC patients is CA19-9 with its limitations, and along with that
AJCC TNM staging and performance status are considered important prognosis indicators in
clinical practice. In order to identify a novel panel of prognostic genes, we utilized publically
available microarray and RNAseq data of PDAC tumors from GEO and TCGA. Expression of
20 genes was significantly associated with overall survival and recurrence free survival in four
datasets and a score generated based on the expression matrix of these genes have been
validated in two independent cohorts. The 20 gene score is dramatically elevated in metastatic
tissue, compared to primary tumor, and is higher in primary tumor compared to normal. Further
analyses of transcriptomic data from the prognostic sub-groups showed that the ‘low risk’
tumors have overall more immune cell infiltration, a higher CD8 T cell/Treg ratio. Analyses of
proteomic data from TCGA PAAD indicated higher levels of Cyclin B1, RAD51, EGFR and a
lower E-cadherin-Fibronectin ratio in ‘high risk’ group. In summary, our score defines both
prognostic and biological sub-groups among PDAC tumors, and has potential to have a
predictive role in terms of response to targeted therapy options.
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The Transcription Factor Elf3 Is Essential for a Successful Mesenchymal to Epithelial
Transition
Burcu Şengez
Izmir Biomedicine and Genome Center, Izmir, Turkey
The epithelial to mesenchymal transition (EMT) and the mesenchymal to epithelial transition
(MET) are two critical biological processes that are involved in both physiological events such
as embryogenesis and development and also pathological events such as tumorigenesis. They
present with dramatic changes in cellular morphology and gene expression exhibiting acute
changes in E-cadherin expression. Despite the comprehensive understanding of EMT, the
regulation of MET is far from being understood.
To find novel regulators of MET, we hypothesized that such factors would correlate with Cdh1
expression. Bioinformatics examination of several expression profiles suggested Elf3 as a
strong candidate. Depletion of Elf3 at the onset of MET severely impaired the progression to
the epithelial state. This MET defect was explained, in part, by the absence of E-cadherin at the
plasma membrane. Moreover, during MET, ELF3 interacts with the Grhl3 promoter and
activates its expression.
Our findings present novel insights into the regulation of MET and reveal ELF3 as an
indispensable guardian of the epithelial state. A better understanding of MET will, eventually,
lead to better management of metastatic cancers.
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Kanser Kök Hücre ve Epigenetik Yaklaşımlar
Nazlıhan Aztopal
İstinye Üniversitesi, Fen Edebiyat Fakültesi, Moleküler Biyoloji ve Genetik Bölümü, İstanbul
Kanser tedavisi, son yıllarda yeni ilaçların da kullanıma girmesine rağmen, tatmin edici başarı
seviyesine ulaşmamıştır. Son yıllarda giderek artan kanıtlar, tedavi başarısızlığındaki en önemli
nedenlerden biri olarak tümör içerisinde kök hücrelerle benzer şekilde kendini yenileme ve
farklılaşma kabiliyetine sahip özel bir hücre alt grubunun, kanser kök hücrelerin (KKH),
varlığını işaret etmektedir. Tümörün çok küçük bir kısmını (%0,1-2) oluşturan bu hücre
grubunun; tümör oluşumu ve gelişimi, artmış invazif özellik, radyasyon ve kemorezistans ile
ilişkili oldukları düşünülmektedir. Bu nedenle, çoğunluğu oluşturan kanser hücrelerinin yanı
sıra KKH’lerini de hedefleyen tedavi modaliteleri önem kazanmaktadır.
Son yıllarda giderek artan kanıtlar, epigenetik değişikliklerin KKH özelliklerinin
düzenlenmesinde önemli bir role sahip olduğunu göstermektedir. KKH’lerinde, tümörün büyük
çoğunluğunu oluşturan ve kök hücre özelliği taşımayan kanser hücrelerinden farklı olarak;
hayatta kalma, kendini yenileme ve proliferasyon sürecinde kritik rol oynayan Wnt/ β-katenin
sinyalizasyonunun yeniden aktive olduğu ve tedavi direncine katkı sağladığı bilinmektedir. Her
ne kadar Wnt/ β-katenin sinyal yolağının aşırı aktivasyonu yolak bileşenlerinde genetik
mutasyonlar ile ilişkilendirilse de epigenetik mekanizmaların da bu düzenlemede oldukça
belirgin bir rol oynadığı ve terapötik olarak hedeflenebildiği gösterilmiştir. Özellikle, histon
asetilasyon programının değişimi farklılaşma süreci ile ilişkilendirilmekte ve bundan dolayı
farklılaşmayı arttırmak için histon deasetilazları (HDAC) hedefleyen ilaçlar kullanılmaktadır.
Epigenetik belirteçlerin terapötik birer hedef olarak önemi dikkate alındığında, KKH’lerini
hedefleyen ve bu hücreleri KKH özelliği taşımayan hücrelere dönüştürebilen bu
mekanizmaların anlaşılması ve aydınlatılması yeni tedavi seçenekleri/yaklaşımları sunabilir.
Dolayısıyla, tedaviye dirençten hatta tedavi sonrası nükslerin oluşumundan (kanser kök
hücreleri üzerinden) sorumlu tutulan Wnt/ β-katenin sinyal yolağını düzenleyen epigenetik
modifikasyonları daha iyi anlamak, terapötik bir hedef olarak rolünü tanımlamak ve yeni
epigenetik terapi yaklaşımları geliştirmek son yıllarda yaptığımız çalışmaların temelini
oluşturmaktadır.
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Pankreatik Kanserde Tümör Mikroçevresinin Hedeflenmesi
Didem Karakaş
İstinye Üniversitesi, Fen-Edebiyat Fakültesi, Moleküler Biyoloji ve Genetik, İstanbul
Pankreatik kanser semptomlarının azlığı, erken deteksiyon ve efektif tedavi seçeneklerinin
bulunmaması nedeniyle en öldürücü kanser türleri arasında yer almaktadır. 5 yıllık sağ kalım
oranı %5’ten daha düşüktür ve hastaların %50’si ilk 6 ay içinde hayatını kaybetmektedir [1].
Günümüzde pankreatik kanserin tedavisi için sitotoksik ya da hedefli tedaviler gibi pek çok
yaklaşım denenmesine rağmen etkin bir sonuç alınamamaktadır. Son yıllarda yapılan
çalışmalarla, yalnızca karsinoma hücrelerinin değil, tümör mikroçevresinin de pankreatik
kanserin bu agresif doğasına katkı sağladığı ortaya konmuştur [2,3]. Bu nedenle sadece tümör
hücrelerini hedefleyen tedaviler başarısız olmaktadır ve etkin tedavi için karsinoma hücreleriyle
birlikte tümör mikroçevresinin de hedeflenmesi gerektiği düşünülmektedir.
Pankreatik kanser stroma bakımından en zengin kanserlerden biridir [4]. Tümör kütlesinin
yaklaşık %90’ı dezmoplastik stromadan oluşurken, tümör hücreleri tümörün sadece %10’lık
kısmını oluşturmaktadır [5]. Pankreatik kanseri eşsiz yapan bu yoğun stroma tedavi
başarısızlığındaki en büyük nedenlerden biri olarak görülmektedir. Çünkü son yıllarda yapılan
çalışmalar ile “tümör mikroçevresi” olarak da adlandırılan stromal hücrelerin tümör gelişimi,
invazyon, kemoresistans, apoptozisten ve immün sistemden kaçış ve metastaz süreçlerinde aktif
rol oynadığı bilinmektedir [6-8]. Bu nedenle günümüzde sadece tümör hücrelerini hedefleyen
tedavilerin başarısız olduğu ve tümör hücreleriyle birlikte stromal hücrelerin de hedeflenmesi
gerektiği bilinmektedir.
Bu kapsamda ilk olarak, pankreatik kanser ve mikroçevre hücreleri arasındaki etkileşim
mekanizmalarının aydınlatılması gerekmektedir. Pankreatik kanser hücreleri ile makrofajlar,
nöronlar ve pankreatik stellat hücreleri arasındaki interaksiyonların moleküler
mekanizmalarının incelenmesi ve bu mekanizmaların biyomarker olarak kullanılabilme
potansiyellerinin araştırılması ve bu interaksiyonları hedef alacak tedavi yaklaşımlarının
geliştirebilmesi son yıllardaki çalışmalarımızın temelini oluşturmaktadır.
Referanslar:
1. Michaud, D.S. 2017. “Pancreatic Cancer: Reference Module in Biomedical Sciences”, ABD.
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2. Fujita, H. et al. 2010. “α-Smooth muscle actin expressing stroma promotes an aggressive tumor biology in pancreatic ductal adenocarcinoma”, Pancreas, 39, 1254–1262.
3. Miyamoto, H. et al. 2004. “Tumor–stroma interaction of human pancreatic cancer: acquired resistance
to anticancer drugs and proliferation regulation is dependent on extracellular matrix proteins”, Pancreas,
28, 38–44.
4. Feig, C. et al. 2012. “The pancreas cancer microenvironment”, Clin Cancer Res, 15,18(16),4266-76.
5. Li, J., Wientjes, M. G., Au, J.L. 2010. “Pancreatic cancer: pathobiology, treatment options, and drug delivery”, AAPS J., 2, 223–232.
6. Farrow, B., Albo, D., Berger, D.H. 2008. “The role of the tumor microenvironment in the progression of pancreatic cancer”, J. Surg. Res, 149,319–328.
7. Hanahan, D., Weinberg, R.A. 2000. “The hallmarks of cancer”, Cell, 100: 57–70.
8. Liotta, L.A., Kohn, E.C. 2001. “The microenvironment of the tumour-host interface”, Nature, 411,
375–379.
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HEDEFE YÖNELİK TEDAVİDE KANSERE ÖZGÜ MOLEKÜLER MEKANİZMALARIN
AYDINLATILMASI
Şeyma Aydınlık1, Egemen Dere1, Engin Ulukaya2
1Biyoloji Bölümü, Fen Edebiyat Fakültesi, Uludağ Üniversitesi, Bursa, Türkiye
2Tibbi Biyokimya Bölümü, Tıp Fakültesi, İstinye Üniversitesi, İstanbul, Türkiye
Metastatik kolon kanseri agresif kemoterapi rejimlerinin kullanılmasına rağmen, % 5'den az bir
hayatta kalma oranına sahiptir. Kolon kanserinde epidermal büyüme faktörü reseptörünün
(EGFR) prognostik ve prediktif önemi hala tartışmalıdır. Bu nedenle EGFR reseptörünü bloke
etmek amacıyla çalışmada tirozin kinaz inhibitörü canertinib kullanılmıştır. Bu amaçla Uludağ
üniversitesi kimya bölümü tarafından sentezlenenen Palladyum(Pd) (II) bileşiği
[PdCl(terpy)](sac).2H2O] ve bu bileşiğin canertinib ile kombinasyonunun kansere özgü
moleküler mekanizmalar üzerine etkileri araştırılmıştır. Ayrıca kemoterapötik ilaç 5-
Fluorourasil (5-FU) ve bu ajanın canertinib ile kombinasyonu ise pozitif kontrol olarak
kullanılmıştır.Dolayısıyla Palladyum (II) bileşiği ile canertinib tek başına ve kombinasyon
şeklinde, aynı şekilde 5- Fluorourasil tek başına ve canertinib ile kombinasyonunun insan kolon
kanseri hücre soyları (HCT-15, HT-29) üzerine sitotoksik etkileri SRB canlılık testi ile
belirlenmiştir. Hücrede gerçekleşen apoptozis, asidik vezikülasyon, mitokondriyal
depolarizasyon, flow sitometri, Anneksin-V-Cy3/SYTOX ikili boyama, akridin ve JC-1
boyama yöntemi belirlenmiştir. EGFR ilişkili yaşam yolaklarında, bu reseptörün inhibe
edilmesi ile gerçekleşen değişimler, epidermal mezenkimal dönüşüm (EMT) ve apoptoz/otofaji
ilişkili proteinlerdeki değişimler ise western blot yöntemi ile değerlendirildi. Ayrıca bu yaşam
yolağının inhibisyonunun kombinasyon tedavileri ile birlikte invazyon, yara iyileşmesi ve
koloni oluşturma yetenekleri üzerine etkileri belirlendi. Son olarak HUVEC endotel
hücrelerinde EGFR inhibisyonun kombinasyon tedavileri ile birlikte tüp oluşturma yeteneği in
vitro matrigel testi ile ve damar oluşumu üzerine etkileri ayrıca in vivo Chorio-allantoik
membran (CAM) testi ile değerlendirildi. Sonuç olarak, Pd (II) bileşiği ve canertinib
kombinasyonunun kolon kanseri hücre soylarında apoptozis ve sitotoksik etkilerinde artışa,
invazyon, yara iyileşmesi yeteneklerinde ve damar oluşumunda azalmaya neden olduğu
bulunmuştur.
*Bu çalışma Uludağ Üniversitesi Bilimsel Araştırmalar Projeler Birimi tarafından
desteklenmiştir (DDP(F)-2017).
Anahtar Kelimeler: Trozin kinaz inhibitörleri, Anjiyogenez, Otofaji, EMT, İnvazyon ve migrasyon
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Yeni Antikanser İlaç Keşfinde Genomik ve Metabolomik: Fırsatlar ve Zorluklar
Ekrem Kum, Süleyman Özakın, Ebru İnce*
Dicle Üniversitesi Fen Fakültesi Biyoloji Bölümü 21280 Diyarbakır
Elli yıldan uzun bir süredir, mikroorganizmalardan ve bitkilerden elde edilen küçük organik
moleküller çok sayıda kullanışlı kanser kemoterapötik ilacına kaynak teşkil etmiştir. Doğal
ürünlerin bu tip öncü bileşiklerinin tarama çalışmaları son yıllarda ivme kazanmış, karasal bitki
ve mikroorganizmalar kadar, deniz fauna ve florasını oluşturan bileşenlerinin de anti-kanser
aktiviteleri araştırılmaya başlanmıştır. Doğal ürünlerin biyosentezlerinin daha iyi anlaşılması
ile beraber, geleneksel ilaç veya ilaç öncülü tarama çalışmaları yerini genomik bilginin
kimyasal verilerle kombine edilmesi prensibe dayalı farklı stratejilere bırakmıştır. Anti-kanser
dahil olmak üzere farklı biyolojik aktivite ve emsalsiz kimyasal çeşitliliğe sahip mikrobiyal
doğal ürünlerin üreticisi olan Aktinomisetler kompleks bir yaşam döngüsüne ve diğer bakteriler
ile kıyaslandığında büyük genomlara sahiptirler. Yeni nesil dizileme teknolojileri sayesinde
genomlarında biyoaktif doğal ürün sentezinden sorumlu yüksek miktarda biyosentetik gen
kümesine sahip oldukları bulunmuştur. Bu makalede; mikrobiyal kaynaklı yeni biyoaktif
bileşik keşfinde genomik ve metabolomik tarama stratejileri, kullanılan biyoinformatik
programlar, avantaj veya zorluklarına değinilerek, son yıllarda bu teknoloji ile elde edilmiş yeni
anti-kanser ilaçların altı çizilmiştir.
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Kanser Hücrelerini Tanımak İçin Akıllı Biyofonksiyonel Yüzeylerin Tasarımı
Dilek Odacı Demirkol
Ege Üniversitesi Fen Fakültesi Biyokimya Bölümü 35100 Bornova-İzmir
Kanser, kendi dokularımızdan ortaya çıkar ve hastalıklı doku ve sağlıklı doku arasındaki
benzerlikler nedeniyle hem tespiti hem de tedavi yöntemlerinin seçimini zorlaştırır. Erken tanı
kanser hastasının prognozunun iyileştirilmesinde oldukça önemlidir. Şu anda, kanserin tespiti,
öncelikle görüntüleme tekniklerine veya hastalıktan şüphelenilen hücrelerin veya dokuların
morfolojik analizi ile yapılmaktadır. Nanoteknoloji alanındaki son araştırmalar kanser tanısı ve
tedavisi için şu anda mevcut uygulamaların dezavantajlarını bertaraf etmeye odaklanmıştır.
Kanserin erken teşhisi ve tedavisi amacıyla gerçekleştirilen nanomalzeme temelli araştırmaları
dört kategoriye ayırabiliriz [1]:
(1) Ekstraselüler kanser biyobelirteçlerinin belirlenmesi,
(2) Kanser hücrelerinin dedeksiyonu,
(3) in vivo dokularda kanserli hücrelerin tespiti ve
(4) Hedefe yönelik akıllı ilaç taşıyıcı sistemlerinin geliştirilmesidir.
Bazı nanoyapılı problar; avantaj sağlayan çeşitli benzersiz özellikleri ile kanserin erken teşhis
edilmesinde ve hedeflenmiş tedavisinde kullanılmaktadırlar [2]. Karbon yapılı
nanomalzemeler, polimerik yapılı malzemeler, metalik nanopartiküller, nanolifler ve veziküler
bu bağlamda kullanım potansiyeline sahip moleküllerdir [2,3]. Burada, nanomalzemeler ve
bunların biyolojik moleküllerle konjugasyonu sonrasında elde edilen yapıların kanserin teşhis
ve tedavisindeki kullanımı hakkında güncel yaklaşımlar tartışılacaktır.
Kaynaklar
[1]Fulya Ekiz Kanik, Alexandra Sneider, Prakash Rai, Dilek Odaci Demirkol, Suna Timur, Nanomaterials in Cell Imaging (Chapter 4). Innovations in Nanomaterials (Eds: Al-Nakib Chowdhury, Joe Shapter, and Abu Bin Imran) Nova Science Publishers New York, 2015.
[2]Bahar Güler, Bilal Demir, Emine Güler, Kadri Güleç, Ozan Yeşiltepe, Dilek Odacı Demirkol, Suna
Timur, Targeting and Imaging of cancer cells using nanomaterials (Chapter 8), Applications of
Nanobiomaterials (Ed: Alexandru Mihai Grumezescu), Elsevier, 2015.
[3]Emine Guler, Bilal Demir, Bahar Guler, Dilek Odaci Demirkol, Suna Timur, Biofuctionalized Nanomaterials for Targeting Cancer Cells, Therapeutic Nanostructures; Volume II: Drug Delivery (Ed: Alexandru Mihai Grumezescu), Elsevier, 2016.
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Nanopartiküllerin biyokimyasal ve hematolojik etkileri
Dilek U. Çakır
Tıbbi Biyokimya Anabilim Dalı, Tıp Fakültesi, Çanakkale Onsekiz Mart Üniversitesi, Terzioğlu
Kampüsü
Günümüzde yine devrim niteliğinde olabilecek maddeyi atomik boyutlarda inceleyip işlemek
fikri ve uygulamaları, bilim ve teknolojiye yeni bir boyut kazandırmıştır. Yeni devrim ve
gelecek yüzyılın teknolojisi diye adlandırılan bu gelişmeler dönemi: nanobilim ve
nanoteknoloji dönemidir. Çözünmüş, hapsedilmiş veya adsorbe haldeki etkin maddeyi
kontrollü olarak açığa çıkaran, doğal ya da sentetik yapıdaki polimerlerle hazırlanan katı
kolloidal polimerik partiküler sistemlere nanopartiküller denilmektedir.
Hedeflendirilmiş nanopartikül taşıyıcıların kullanılması nano tıpta en önemli alanlardan biridir.
İlaç hedefleme; ilaç etken maddesinin hedef doku veya organda seçici ve kantitatif toplanma
yeteneği olarak tanımlanır. Nanoteknoloji temelli ilaç salınımı; ilacın geçirgenliğini, kontrollü
salınımını ve ilacın hedefe yönelik olması özelliklerini arttırmayı hedefler. Nanokapsülleri ya
da nanopartikülleri kullanan geleneksel ilaç salınım sistemi hedefe yönelik olmadığı için,
vücuttaki normal ve hassas hücreler üzerinde yan etki ve toksik etki oluşturabilir. Son
zamanlarda geliştirilen hedefli terapilerle nanopartiküllerin hedef bölgeye ulaşma şansı
artmıştır ve toksik etkileri ve ilaç dozu azalmıştır. Nanopartiküller, normal hücrelerde toksik
etki yaratmaktan kaçınarak, tümör hücrelerindeki ilaç konsantrasyonunu arttırmak için pasif ve
aktif hedefleme stratejilerini kullanır. Bu durum özellikle tümör kemoterapisinde, sadece
çevresindeki normal hücrelere dokunulmadan seçici şekilde tümör hücrelerine ilaç verilmesi
açısından önemlidir. Elde edilen mikro/nanopartiküllerin canlı vücudunda kullanılabilir
olabilmesi için fiziksel ve kimyasal özelliklerinin iyi bilinmesi ve hücreler ile allerjik, toksik,
karsinojenik reaksiyon vermemesi istenir. Nanoteknoloji alanındaki heyecan verici gelişmelere
rağmen, nano ölçekli materyallerin toksik etkilerinin ve sağlık açısından oluşturduğu riskin
değerlendirilmesi gerekir.Bu nedenle nanotıp alanındaki araştırma trendi, nano parçacıkların
toksik özelliklerinin daha iyi anlaşılması ve azaltılması üzerinedir.
İlaç taşıyıcı olarak kullanılan yavaş bozunan ya da bozunmayan nanopartiküllerde ilaç salımı
uzun sürer ve etki bölgesinde ilacın toplanmasına neden olur bu da sonuçta kronik inflamatuar
cevaba yol açar. Nanopartiküllerin yapısında bulundurduğu maddelerden gelen fizikokimyasal
özellikler nanopartiküllerin vücutta dağılımını, kan beyin bariyerini geçişi ve kan pıhtılaşma
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yolaklarını tetikleyebilir. Altın ve polistiren içeren nanopartiküllerin hemoliz ve kan
pıhtılaşmasına neden olduğu gözlenmiştir. Karaciğer, dalak, böbrek, lenf nodları, kalp,
akciğerler ve kemik iliğinde nanoparçacıkların toksik etkisinin olduğu rapor edilmiştir.
Raporlarda nanoparçacıkların etkisi kısa dönem için gösterilmiştir ama uzun dönemdeki etkileri
ile ilgili çalışmalara da ihtiyaç vardır. Bu açıdan, nanomateryallerin ne kadar güvenli olduğu ve
ne derece toksik etkiye sahip oldukları henüz cevaplanmamış önemli sorulardır.
Nanoparçacıkların çevre, doğa ve insan sağlığına etkilerinin incelenmesi için sağlık otoriteleri
yeni çerçeve kurallar belirlemeye çalışmaktadır. Bu doğrultuda, klasik farmasötik kimya ve
biyoteknolojinin kullandığı ve biyomateryal ruhsatlandırılması için gerekli ve geçerli olan
testlerin yanında yeni bazı deneylerin de yapılması ve standart hale getirilmesi bir ihtiyaçtır.
Mevcut gelişmeleri göz önünde bulundurarak, hedefe odaklı kanser ve diğer hastalıkların
nanoparçacıklarla tedavi edilebilmesi için, nanomateryaller ve toksisite ile ilgili ileri düzeyde
çalışmaların yapılmasına ihtiyaç duyulmaktadır.
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Hayvanlarda Multiple Myeloma (Plazmasitom, Plasma Cell Myelloma)
Hasan İçen
Veterinerlik Fakültesi, Dicle Üniversitesi, Diyarbakır
Multipl miyelom (MM), kemik iliğinde üretilen B lenfositlerinden köken alan plazma
hücrelerinin (plazma B hücreleri ya da plazmositler) proliferasyonu ile karakterize bir plazma
hücresi malignitesi olarak tanımlanan ve paraprotein olarak bilinen homojen bir
immünoglobulin salgılarlar. Çoğunlukla köpek, kedi ve insanlarda görülmesi ile birlikte fare,
at, inek, domuz ve tavşanlarda tespit edilmiştir. Tüm malign tümörlerin <%1'ini, malign
hematopoetik tümörlerin ise ~%8'ini, biyopsi ile teşhis edilen tüm primer ve sekonder kemik
tümörlerinin ise %3,6'sını oluştururlar.
Multipl miyelomun klinik belirtileri oldukça değişkendir ve çoklu organ yetmezliklerine yol
açabilir. Etkilenen hayvanlarda; uyuşukluk, halsizlik, topallık, kemik ağrısı, kanama (örn.,
Mukoza zarındaki peteşi, dişeti kanaması ve burun kanaması), poliüri / polidipsi ve nörolojik
bozukluklar gibi klinik belirtilerle beraber rejeneratif anemi, patolojik kemik kırıkları, böbrek
yetmezliği, immün yetmezlik sonucu sekonder enfeksiyonlar, pıhtılaşma bozuklukları, kronik
anemi, kalp yetmezliği gibi belirtilere de neden olabilir.
Hayvanlarda multipl miyelom teşhisi, kemik iliği aspirasyonunda veya biyopsisinde %20 den
fazla plazma hücrelerine rastlanılması, serum ya da idrarda Bence-Jones proteinlerinin tespit
edilmesi, ile radyografik bulgularla tanı konulabilir. Biyokimyasal olarak hiperproteinemi,
hiperviskozite ve hiperkalsemi görülebilir. Multipl miyelom için genelde palyatif tedavi ile
birlikte melfalan veya siklofosfamid ile prednizondan oluşur.
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Kanserde Çığır Açan Moleküler Hedefler ve Tedavileri
Mehmet Küçüköner
Dicle Üniversitesi Tıbbi Onkoloji Bilimdalı, Diyarbakır
Kanser, çeşitli faktörlerin genetik materyal üzerinde yaptığı değişiklikler sonucu gelişen çok
basamaklı klonal bir hastalıktır. Kanser nedeni olan genetik değişikliklerin, onkolojik tanı
uygulamalarında önemle gözönünde bulundurulması gerekir. Kanser dokusunda bu tür
mutasyonların varlığı, tanı ve tedavi seçiminde yanıltıcı olabilir. Bu mutasyonların tek başına
varlığı kanser gelişiminin nedeni olarak değerlendirilmemelidir. Bu mutasyonlar, gelişen
kanser için yalnızca kolaylaştırıcı faktörler olarak bilinmektedir. Bu nedenle saptanan genetik
değişikliklerin tedavi hedefi olarak değerlendirilebilmesi için, o mutasyonun kanser dokusuna
özgü somatik mutasyon olduğunun ileri çalışmalarla gösterilmesi gerekir. Bu sebeplerle birçok
karsinom ve sarkomlar için moleküler testler kullanılmaktadır. Klinik uygulamalarda genel
olarak kullanılan moleküler yöntemler, dizi analizi, hibridizasyon veya PCR yöntemleridir.
Kanserde moleküler hedef tedavilerin başarılı örneklerinden biri akciğer kanseridir. Üzerinde
en çok çalışılan kanser tiplerinden biri olan akciğer kanserinde son yıllarda hem patogenezini
anlama hem de tedavi yaklaşımlarını belirlemede büyük gelişmeler yaşanmıştır. Pek çok
moleküler değişiklik driver mutasyonlar/moleküler değişiklik olarak tanımlanmıştır. Driver
mutasyonlar/moleküler değişiklikler kanserin başlangıcından ve gelişiminden sorumludur.
Özellikle hedefe yönelik tedavi sürecinde son derece etkili farmakolojik formları da bulunan
epidermal büyüme faktörü reseptörü (EGFR), anaplastik lenfoma kinaz (ALK) ve proto-
onkojen tirozin-protein kinaz 1 ROS (ROS1) gibi moleküler değişiklikler, akciğer kanseri
tedavisinde yeni bir çığır açmıştır. Büyüme faktör reseptörlerine karşı geliştirilen monoklonal
antikorlar ve hücre içi sinyal iletiminde rol alan tirozin kinaz reseptörlerini inhibe eden ilaçlar
kanser tedavisinde kullanılmaktadır. Son yıllarda T lenfosit ilişkili protein 4 (CTLA-4),
programlanmış ölüm-1 (PD-1) ve programlanmış ölüm ligandı 1’e (PD-L1) yönelik geliştirilen
antikorlar kanser tedavisinde ön plana çıkmışlardır. Ayrıca siklin bağımlı kinaz inhibitörleri,
Poli (ADP-Riboz) Polimeraz-1 (PARP) inhibitörleri bazı kanser türlerinde tedavi seçeneği
olmuştur.
Her geçen gün yeni bir hedefe yönelik tedavi ajanının ortaya atıldığı bu dönemde doğru hastaya
doğru tedavinin uygulanabilmesi için moleküler testler önem kazanmaktadır. Moleküler testler
sadece tedavi sürecinin belirlenmesinde değil, aynı zamanda tedavi direnç mekanizmalarının
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anlaşılmasında ve çözümünde, hastalıkların prognozunun tahmininde ve daha da önemlisi
kanserin patogenezinin anlaşılmasında rol almaktadır.
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KANSER TEDAVİSİNDE HİSTON DEASETİLAZ İNHİBİTÖRLERİ
Ferda Arı
Bursa Uludağ Üniversitesi, Fen Edebiyat Fakültesi, Biyoloji Bölümü, Moleküler Biyoloji Anabilim
Dalı 16059 Nilüfer/BURSA
Epigenetik terimi DNA dizisinde değişiklik oluşturmadan gen ekspresyonunda gelişen
kalıtımsal değişikliklerdir. Epigenetik mekanizmalar; DNA metilasyonu, histon
modifikasyonu, kromatinin yeniden modellenmesi olarak tanımlanmaktadır. Bu süreçler DNA
yapısına, DNA-bağlanma proteinlerinin yapısına, RNA ve protein degredasyonuna etki ederek
gen fonksiyonunun kontrolünü sağlamaktadır. Epigenetik mekanizmalarda ki işleyiş
bozuklukları özellikle kanser gibi çeşitli hastalıkların oluşumunda önemlidir.
Histon modifikasyonları histon proteinlerinin posttranslasyonel modifikasyonları olup; N-
terminal kuyruğunda gerçekleşir. Gen fonkiyonuna, kromatin yapısına, gen ekspresyonuna etki
edebilen histon modifikasyonları aynı zamanda DNA tamirinde ve replikasyonunda işlev
görmektedirler. Histon modifikasyonları çoğunlukla asetilasyon, metilasyon, fosforilasyon,
glikolizasyon şeklinde olmaktadır. Histonların asetilasyonunun, pozitif yüklerini nötralize
ettiği ve negatif yüklü DNA ile olan etkileşimlerini gevşettiği düşünülmektedir. Histonların
histon deasetilazlar (HDAC) tarafından deasetilasyonu, DNA ile olan etkileşimini sıkılaştırır
ve kapalı bir kromatin yapısına ve gen transkripsiyonunun engellenmesine neden olmaktadır.
Histon modifikasyonunun düzenlenmesinin yanı sıra HDAC'lar, transkripsiyon faktörleri,
şaperonlar ve sinyal molekülleri de dahil olmak üzere birçok histon proteininin post-
translasyonel asetilasyon durumunu düzenleyerek protein stabilitesinde, protein protein
etkileşimlerinde ve protein-DNA etkileşimlerinde değişikliğe neden olabilmektedirler.
HDAC’lar genetik olarak yüksek oranda korunmuş olsalar da kanserdeki rollerine odaklanan
mevcut veriler birçok tümör yapısında farklı ekspresyon düzeylerine sahip olduklarını
göstermiştir. HDAC’ların tümörijenezdeki birçok moleküler yolakta etkilerinden dolayı
hematolojik malignitelerde ve solid tümörlerde uzun süredir etkin hedef olarak
görülmektedirler. Bu amaçla çok sayıda HDAC inhibitörü (HDACi) doğal kaynaklardan
saflaştırılmış veya sentezlenmiştir. HDACi’leri hidroksamatlar, siklik peptidler, alifatik asitler
ve benzamitler olarak sınıflandırılmaktadır. HDACi, özellikle çeşitli kemoterapi ve/veya
radyoterapiyle kombinasyon halinde etkin bir anti-kanser tedavi seçeneği sunmaktadır. Ayrıca
birçok in-vitro çalışmada antitümör etkinliği farklı kanser modellerinde gösterilmiştir.
Vorinostat, romidepsin ve belinostat HDAC inhibitörleri T-hücreli lenfoma ve multipl miyelom
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için FDA tarafından onaylanmıştır. Solid malignitelerin tedavisi için ise Vorinostat, Valproik
asit, CHR-3996 ve Practinostat’ın devam eden veya tamamlanan klinik faz çalışmaları
bulunmaktadır. Bunlara ilaveten, son yıllarda bazı HDACi’lerinin kanser kök hücreleri ve
epitelyal-mezenkimal geçiş üzerine olan etkileri araştırılmakta ve umut verici sonuçlar
alınmaktadır.
Sonuç olarak, HDACi’leri kanserde epigenetik tedavide kullanılabilecek potansiyel moleküller
olup daha ileri klinik analizlerin yapılması gerekmektedir. Araştırmalar ilerledikçe şüphesiz ki
kanser tedavisinde HDACi’leri önemli bir yer alacaktır.
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Over Kanserinde Likit Biyopsi ile Elde Edilen Moleküllerle Biyobelirteç Çalışmaları
Tuba Günel
İstanbul Üniversitesi Fen Fakültesi Moleküler Biyoloji ve Genetik Bölümü
Biyoteknoloji ve Genetik Mühendisliği Araştırma ve Uygulama Merkezi (BİYOGEM)
Tumor biomarkers are molecules that are produced by cancer cells or cells around them, which
can be measured in body fluids or in the blood during the diagnosis, screening or treatment of
cancer so-called “liquid biopsy”. Molecules that can be used as tumor biomarkers can be
counted as cytoplasmic proteins, enzymes, hormones, surface antigens, receptors, oncofetal
antigens (reemerging proteins in cancer that is normally lost after birth), oncogenes or their
products. An ideal tumor biomarker should be sensitive enough for early detection of small
tumors while retaining the specificity of the identified cancer type. Recent studies have
discovered a great number of non-invasive tumor diagnosis and screening has become an
important area of study
Contrary to tissue biopsy, through detection of circulating tumor cells (CTCs), tumor nucleic
acids (“circulating tumor DNA/RNA”), and exosomes, predictive and prognostic markers may
potentially be developed which is far less invasive. Hence early and multiple evaluations of the
disease can be made, including retrospective follow-up, identification of treatment effects and
investigation of clonal development. Isolation and characterization of CTCs, exosomes, and
circulating tumor DNA (ctDNA) will improve cancer diagnosis, treatment, and imaging. Liquid
biopsy can be performed “real-time” and at every stage of cancer. The asymptomatic patients,
it can be used to identify to improve early diagnosis and better intervention. Although, it has
some potential disadvantages such as; still is not certain to use in cancer diagnosis, difficulties
in analysis of data obtaining from high-throughput screening and lack of data verification
through clinical trials; it has significant potential for clinical cancer diagnosis in future
Ovarian cancer is one of the most important and fatal gynecological cancer types worldwide.
The biomarkers with high sensitivity and specificity obtained from the studies performed with
liquid biopsy will increase the five-year survival rate.
54
ORAL PRESENTATIONS
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Stone Alkaline Water Induces Apoptosis of Prostate Cancer Cells and Inhibits Tumor
Cell Induced Angiogenesis In Vitro
Sila Appak-Baskoy1, lknur Kulcanay Sahin2, Ozgun Teksoy3, Mustafa Cengiz4, Asuman
Deveci Ozkan5, Gamze Guney Eskiler5, Namık Bilici6, Pinar Oztopcu Vatan3, Adnan
Ayhanci3
1Ryerson University, Faculty of Science, Toronto, Ontario, Canada
2Kırıkkale University, Vocational School of Health Services, Kırıkkale, Turkey
3Eskisehir Osmangazi University, Faculty of Science and Arts, Department of Biology, Eskisehir, Turkey
4Siirt University, Faculty of Education, Department of Elementary Education, Siirt, Turkey
5Sakarya University, Department of Medical Biology, Faculty of Medicine, Sakarya, Turkey
6Karabük University Faculty of Medicine Department of Medical Pharmacology, Karabuk, Turkey.
Prostate cancer is the second most common cancer in men. Treatment options to improve
overall survival rate of patients are limited since tumor cells acquire resistance to the
chemotherapeutic drugs. Thus, new alternative therapeutic strategies are urgently needed to
improve the overall life quality and expectancy of the patients. Stone alkaline water (SAW) is
a high alkaline mineral water that is obtained by breaking certain stone and it may have
anticancer effects due to its rich mineral content. Among the most noteworthy minerals in SAW
are sodium (Na) and magnesium (Mg). In this study, we aimed to determine anticancer effects
of SAW on PC-3 and DU-145 prostate adenocarcinoma cell lines. Our findings indicated that
SAW treatment significantly decreased cell viability and induced apoptotic cell death through
accumulation of cells at G0/G1 phase. Additionally, SAW treatment was found to be more
effective in PC-3 cells that have more metastatic activity than DU-145 cells that have a
moderate metastatic activity. Furthermore, treatment of the prostate cancer cells with SAW
inhibited tumor cell induced HUVEC tube formation and migration stating that SAW inhibits
tumor cell derived angiogenesis in vitro.
Key Words: Prostate cancer, Alkaline Water, Apoptosis, Angiogenesis
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MALT1 PARACASPASE INHIBITION REDUCES HEPATOCELLULAR
CARCINOMA CELL SURVIVAL
Asli Kurden Pekmezci1,2, Ozlem Silan Coskun1,2, Ece Cakiroglu1,2, Dilara Demirci2, Serif
Senturk1,2
1 Dokuz Eylul University Izmir International Biomedicine and Genome Institute,
2 Izmir Biomedicine and Genome Center
Hepatocellular carcinoma (HCC) is a leading cause of cancer related deaths around the world
and is associated with several etiological factors including infections with hepatitis B and C
viruses, heavy alcohol consumption and chronic aflatoxin B1 exposure. Treatment strategies
for HCC are currently limited. Hepatocarcinogenesis process involves alterations in several
molecular pathways including activation of NF-κB pathway. MALT1 (Mucosa-Associated
Lymphoid Tissue Lymphoma Translocation Protein 1) is a cytosolic signaling molecule which
acts both as a scaffold protein and as a protease for NF-κB pathway activation. To date, the
contribution of MALT1 to HCC cell survival has not been studied. In the present study, we
investigated the effects of MALT1 inhibition on HCC cell survival. For this purpose, we
depleted the MALT1 levels in HCC cell lines via RNAi system and small molecule inhibitor
and we analyzed the cell survival with different methods. Based on the preliminary results,
shRNA-mediated gene silencing or pharmacological inhibition with MI-2 reduced cell growth
in vitro, and this decrease in cell growth is associated with inhibition of cell proliferation as
well as reduced expression of well-known NF-kB downstream targets, an indirect indication of
NF-κB pathway inactivation. Conclusively, our results suggest that MALT1 plays an important
role in HCC cell growth. Further studies focusing on delineating the molecular mechanisms
underlying the growth regulating effects of MALT1 are currently underway. By all means, we
think that pharmacological inhibition of MALT1 in HCC is a promising avenue to be explored
further.
Key Words: Hepatocellular carcinoma, NF-κB, MALT1, RNAi
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In vitro and In vivo Investigation of Mangiferin and Paclitaxel combination on Ehrlich
Ascites Carcinoma.
Ayca Uvez1, Engin Ulukaya2, Elif Ilkay Armutak1
1Istanbul University-Cerrahpasa, Faculty of Veterinary Medicine, Department of Histology and
Embryology, Istanbul-Turkey
2Istinye University, Faculty of Medicine, Department of Clinical Biochemistry, Istanbul-Turkey
Aims: Recent years, combinatory usage of various natural compounds in cancer therapy is
aimed to increase activate of chemotherapeutic agents Mangiferin is a polyphenol that has been
reported may have an important role in cancer. The aim of this study was to investigate in vitro
and in vivo effects of Mangiferin and Paclitaxel alone and in their combinations on cell
proliferation, apoptosis, autophagy and survival pathway In addition, the effect on angiogenesis
was evaluated by HUVEC tube formation method and in vivo CAM model.
Methods: The effect of Mangiferin (6.25-300 µM) and Paclitaxel (0.25-15.9 µM) alone and in
combination on (Ehrlich Ascites Carcinoma) EAC and HUVEC cells were assessed by ATP
cytotoxicity test. In combination with Mangiferin and Paclitaxel, apoptosis, autophagy, and
survival markers were determined by western blot. The effect of Mangiferin (12.5-50 µM) and
Paclitaxel (1.99-15.9 µM) alone and in combination on HUVEC cells were evaluated at 0-16 h
by tube formation test. Mangiferin (12.5-5 mg/ml) and Paclitaxel (0.3-0.0375 mg/ml µM) alone
and in combination were treated in CAM assay. Seventy eight female Balb/c mice with EAC
solid tumors were administered 12.5 mg/kg Paclitaxel by subcutaneously and 25-50-100 mg/kg
Mangiferin by gavage. Tumor volumes determined by caliper then proliferation, apoptosis,
autophagy, and survival markers were evaluated by immunohistochemistry.
Results: The ATP test results showed that the cytotoxic effect increased synergistically in a
dose and cell type dependent in the combination group. According to evaluation of western blot
results, the increase of LC3B with the decrease of p62 along with the increase of FAS with
cleavage of PARP1 was shown that apoptosis occurred through the external pathway in the
presence of autophagy. Furthermore, the survival parameters were suppressed. The in vitro
results were parallel to in vivo. The most effective dose on tumor size, tumor weight, apoptosis,
autophagy, and survival were determined as Mangiferin 50 mg/kg alone and combination. In
vitro tube formation test was observed that Mangiferin 50 µM dose was effective alone. On the
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in vivo CAM assay, mangiferin was shown more effective antiangiogenic effect in combination
groups.
Conclusions: According to results, Mangiferin and Paclitaxel combinations selectively
increased apoptosis in the presence of autophagy, decreased proliferation and survival
parameters, suppressed tumor size and observed strong antiangiogenic effect. In vivo, low doses
of Mangiferin combination with Paclitaxel were shown stronger synergistic effect than alone.
Mangiferin could be a promising potential for the development of new strategies in the
treatment of cancer.
Keywords: Mangiferin, Breast Cancer, Paclitaxel, Apoptosis, Autophagy.
The present work was supported by the Research Fund of Istanbul University-Cerrahpasa.
Project No. TDK-2017-26107.
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Akciğer Kanseri ve KOAH’da Biyobelirteç olarak aday miRNA Araştırması
Aydın Demiray
Pamukkale Üniversitesi Tıbbi Genetik Anabilim Dalı, Denizli, Türkiye ([email protected])
Genel bilgiler: Akciğer kanserleri, kanser nedenli ölüm sebepleri arasında ilk sırada, insidansı
üçüncü sırada yer almaktadır (1). Akciğer kanserlerinin %85’i Küçük Hücre Dışı Akciğer
Kanserleri (KHDAK) oluşturmaktadır (1). Kronik Obstrüktif Akciğer Hastalığı (KOAH),
genellikle zararlı partikül veya gazlara ciddi maruziyetin neden olduğu havayolu ve/veya
alveoler anormalliklere bağlı kalıcı hava akımı kısıtlanması ve solunumsal semptomlarla
karakterize, yaygın, önlenebilir ve tedavi edilebilir bir hastalıktır (2). Yapılan çalışmalarda
KOAH akciğer kanseri gelişimi açısından artmış risk ile ilişkilendirilmiştir. Akciğer kanseri
hastalarının % 40-70'ini KOAH hastaları oluşturmaktadır (3). Bu durumun ana etmeni sigara
kullanımı öngörülmektedir. Biz bu çalışmamızda sigara kullanan 30 KHDAK ve 30 KOAH
hastalarında serum düzeyinde prediktif bir faktör olarak miRNA araştırması yaptık.
Materyal-Metod: Pamukkale Üniversitesine başvuran 30 KHDAK ve KOAH hasta
serumlarından miRNA izolasyonu elde edilerek cDNA (ABM) çevirimi gerçekleştirilmiş. Bu
cDNA’lar literatürde belirlenen 20 miRNA (ABM) ile panel oluşturularak incelenmiştir.
Sonuçlar SPSS 17. Paket programında analiz edilmiştir.
Sonuç: Elde edilen miRNA profili normal akciğer hücre hattı MRC-5 normalizasyonu
sağlanmıştır. Analiz sonucunda KHDAK kanseri hastalarında hsa-miR-26a ve hsa-miR-93
KOAH hastalarına oranla daha yüksek ifade edildiği ve hsa-miR-20b ifadesi de her iki grupta
yüksek olarak saptanmıştır.
Tartışma: KOAH hastalarının sigara içimine bağlı olarak KHDAK oluşumu rapor edilmiştir
(3). Buna bağlı olarak sigara içen KOAH hastalarında KHDAK oluşumu prediktif miRNA
taraması yapılmıştır. Pinelo ve arkadaşlarının ve Leidinger ve arkadaşlarının yapmış oldukları
çalışmalar ile sonuçlarımız örtüşmektedir (4,5). Bizim çalışmamızda KHDAK hastalarında hsa-
miR-26a ve hsa-miR-93 yüksek ifade edilirken her iki grupta hsa-miR-20b yüksek ifade
edilmiştir. Ayrıca hsa-miR-26a ve hsa-miR-93 ise PI3K/AKT yolağında aktifleyen
miRNA’lardır. hsa-miR-26a PTPN13 hedefleyerek src aktivasyonu sonucu EGFR alt yolağını
aktiflediği hsa-miR-93 ise PTEN baskılanması sonucu PI3K/AKT yolağını aktiflediği rapor
edilmiştir. hsa-miR-26a ve hsa-miR-93 ikisi de KHDAK’lerinde onkomiR olarak rapor
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edilmiştir (6,7). Ayrıca hsa-miR-20b literatürde akciğer kanserlerinde BTG3 geni üzerinden
p53 baskılanmasını ortadan kaldırdığı rapor edilmiştir (8). hsa-miR-20b her iki grupta artmış
olmasının moleküler yolakların aydınlatılması KOAH’ın KHDAK dönüşümünü
aydınlatabileceği öngörülmektedir.
Referans
(1) Siegel R, DeSantis C, Virgo K, Stein K, Mariotto A. Cancer Treatment and Survivorship Statistics, 2012. CA-Cancer J Clin 2012; 62: 220-241.
(2) Global Strategy for the Diagnosis, Management and Prevention of COPD, Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2017. Available from: http://goldcopd.org.
(3) Wu AH, Fontham ET, Reynolds P, Greenberg RS, Buffler P, Liff J, et al., Previous lung disease and risk of lung cancer among lifetime nonsmoking women in the United States. American journal of epidemiology,1995:141(11);1023-1032.
(4) Petra Leidinger, Andreas Keller, Anne Borries, Hanno Huwer, Mareike Rohling, Junko Huebers, Hans-Peter Lenhof, Eckart Meese Specific peripheral miRNA profiles for distinguishing lung cancer from COPD. Lung Cancer 2011:74;41-47
(5) Sonia Molina-Pinelo, M. Dolores Pastor, Rocı´o Suarez, Beatriz Romero-Romero, Miriam Gonza´lez et al. MicroRNA clusters: dysregulation in lung adenocarcinoma and COPD. Eur Respir J 2014; 43: 1740–1749
(6) Shudi Xu, Tao Wang, Zhiwei Yang, Ying Li, Weijie Li et al. miR-26a desensitizes non-small cell lung cancer cells to tyrosine kinase inhibitors by targeting PTPN13. Oncotarget 2016:29(7);45687-45701
(7) Wei Yang, Jinquan Bai, Dayong Liu, Shuwei Wang, Nan Zhao et al. MiR-93-5p up-regulation is involved in non-small cell lung cancer cells proliferation and migration and poor prognosis. Gene 2018:647;13-20
(8) LIJUN PENG, SHAOBIN LI, YUCHAN LI, MINGHUI WAN, XISHENG FANG et al. Regulation of BTG3 by microRNA-20b-5p in non-small cell lung cancer. ONCOLOGY LETTERS 2019:18;137-144
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Underlying primary immunodeficiency in patients with lymphoma
Baran Erman
Department of Molecular Biology and Genetics, Istinye University, Istanbul, Turkey
Primary immunodeficiencies (PIDs) are rare and heterogeneous congenital diseases of immune
system. The most common clinical symptoms in PID patients are high susceptibility to
infections, inflammation, allergy, autoimmunity and malignancies. The vast majority of
primary immunodeficiency diseases shows monogenic inheritance and almost 400 genetic
defects have been identified so far.2 The overall prevalence of PIDs is approximately 1:10.000.
However, the high consanguinity and fertility rates lead to a higher prevalence in some countries
like Turkey.
After severe infections, malignancy is the most prevalent cause of death in both children and
adults with PIDs. The possible explanation is impaired immunosurveillance mechanisms leads
cancer development in the patients with PID. PIDs more often associated with cancer include
common variable immunodeficiency (CVID), Wiskott–Aldrich syndrome, ataxia-
telangiectasia, and severe combined immunodeficiency. Hematologic cancers, especially
lymphoma, are the most common type of malignancies in PID patients.
We identified PID mutations in two patents following with lymphoma by next generation
sequencing (NGS). In last decade, NGS technologies have been facilitated rapid genetic
diagnosis in complex congenital diseases like PIDs. We applied whole exome sequencing and
determined two distinct PIDs due to mutations in CARD9 and CD70 genes in the patients. The
first patient is a 8-year-old boy received hematopoietic stem cell transplantation from HLA-
matched mother due to EBV-associated lymphoma. NGS analysis revealed homozygous
missense, c.332C>T mutation in CD70 gene. Other two siblings of the patient had the same
mutation, as well. In the other patient, a 14-year-old boy, disseminated lymphadenopathies were
detected. He had also weight loss and lymphoma was considered first. However, we found
homozygous, nonsense c.883C>T mutation in CARD9 gene.
In conclusion, underlying PID should be considered in the patients presented with lymphoma.
Moreover, NGS based approaches may be the first option for a rapid and accurate genetic
diagnosis in PID patients.
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Determination of MiRNAs and Xeno-miRs Targeting Most of the Genes Associated
with Colorectal Neoplasms and Central Obesity by "in silico" Analysis
Berkcan Dogan1,2
1Uludag University, Faculty of Medicine, Department of Medical Genetics, Bursa, Turkey, 2Istanbul University, Institute of Graduate Studies in Science, Department of Biology and Genetics,
Istanbul, Turkey
Scope: Recent studies have found a close connection between obesity and the development of
colorectal neoplasia. It is known that central obesity is associated with an increased risk of
colorectal cancer and that these diseases have common genes associated with pathogenesis.
Hsa-miR-335-5p, targeting the highest number of identified mutual genes, regulated by DNA
methylation that has been reported to possess both tumor suppressor and tumor promoter
activities. Also, upregulation of hsa-miR-335-5p correlated with obesity in adipose tissue in
mice. The purpose of this study specifies common genes for colorectal neoplasms and central
obesity (CN&CO) and microRNAs (miRNAs) and Xeno-miRs targeting these genes that may
involve in the pathogenesis of CN&CO diseases’ via “in silico” analysis.
Methods and Results: Biomolecules that were reported to be associated with both conditions
have been searched by using Ingenuity Pathways Knowledge Base (IPKB) in Ingenuity
Pathway Analysis (IPA) software (QIAGEN Inc., Germany). Nineteen common genes (ACE,
ACE2, ADRA1A, ADRA1B, ADRA1D, CA1, CA12, CA14, CA2, CA5A, CA7, CYP2E1, ESR1,
GCG, GH1, GHR, NISCH, REN and SLC5A2) are involved in the pathogenesis of CN&CO by
IPKB. Using miRNet (www.mirnet.ca), six genes (ACE, CA12, CA14, CA5A, CYP2E1, REN)
targeted by hsa-miR-335-5p in nineteen genes have been found. Hsa-miR-335-5p is upregulated
in CN&CO and suppresses its target genes as reported by IPA software and it is correlated with
other experimental studies. Subsequently, analysis conducted with Dietary MicroRNA
Database showed that Xeno-miR bta-miR-335 was found only in Bos taurus, and this matched
%100 sequence similarity.
Conclusion: Hsa-miR-335-5p targets the highest number of colorectal cancer- and obesity-
related genes and shows same sequence of bta-miR-335 in bovine. MiR-335-5p can have a
therapeutic value for cancer- and obesity-related disorders. Consequently, personalized dietary
interventions can improve obesity-colorectal cancer management if these results can be
validated by using human cell lines and model organisms experimentally.
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Investigation of DHRS2 Gene Effects on Breast Cancer Cell Line
Burcu Salman1, Sema Sırma Ekmekci1, Suzan Çınar2, Neslihan Abacı1
1Department of Genetics, Aziz Sancar Institute of Experimental Medicine, İstanbul University, Turkey
2Department of Immunology, Aziz Sancar Institute of Experimental Medicine, İstanbul University,
Turkey
Breast cancer is the most common malignancy in women and takes the second place between
all cancers. Even though recent research increased life quality of patients and therapy
succession, our knowledge on molecular mechanisms and the tumor behavior on this cancer is
limited. Interactions between p53 and Mdm2 molecules are widely researched but the
regulatory mechanisms of these proteins are still not fully known. Mdm2 and p53 proteins affect
cell metabolism and regulate each other. The dehydrogenase/reductase gene we selected to
study, DHRS2, regulates p53 via suppressing Mdm2. We aimed to investigate the relationship
between p53 and DHRS2. To better understand this regulation, DHRS2 gene expression on
MCF7 cell line was suppressed via RNAi. On the control group and DHSR2 siRNA applied
experiment group, apoptosis percentage was measured on the flow cytometry. In addition, cell
proliferation and viability were evaluated with MTT assay and cell staining with trypan blue.
Last of all migration test were performed using scratch assay method which is very commonly
used nowadays. 24- and 48-hours groups were compared. Apoptotic cell count was lower on
DHRS2 suppressed cells according to the control group. Again, results on DHRS2 gene
suppressed cells showed us decelerate wound recovery and decreased cell viability and
proliferation. Decreased apoptotic cell count on experiment group supports increased protein
amount of Mdm2 and the decreased p53 because of active Mdm2. Our findings on the cell
migration could use as a lead on this research area. This thesis research shows the specialty of
being the first research on our country that specifically investigates DHRS2 gene on breast
cancer.
This work was supported by Scientific Research Projects Coordination Unit of Istanbul
University (grant number 30936).
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Bir seramidaz inhibitörü olan N-Oleoylethanolamine bileşiğinin A549 ve Beas-2B
hücreleri üzerindeki sitotoksik ve morfolojik değişimlerinin araştırılması
Emre Çömlekçi1, Hüseyin İzgördü1, Canan Vejselova Sezer1*, O. Tansel Korkmaz2, Hatice
Mehtap Kutlu1
1Eskişehir Teknik Üniversitesi, Fen Fakültesi, Biyoloji Bölümü, Eskişehir, Türkiye 2Osmangazi Üniversitesi, Tıp Fakültesi, Fizyoloji Anabilimdalı, Eskişehir, Türkiye
*Sorumlu yazar: [email protected]
Sfingolipid metabolizmasında bulunan enzimler hücrenin sağ kalım ve ölümünde önemli roller
oynamaktadır ve bu metabolizmanın merkez molekülü olan seramidin kanserli hücreleri
apoptoza teşvik ettiği ortaya konmuştur. Yeni nesil kanser çalışmalarında sfingolipidler önemli
bir yer tutmaktadır. Seramid ve sfingozin katabolizmasından sorumlu bir enzim olan
seramidazların inhibitörü olan N-Oleoylethanolamine (NOE) seramidin sfingosine
dönüşümünü engellemektedir ve bununla birlikte farklı kanser hücrelerinde apoptozu
tetiklediği bildirilmiştir. Yaptığımız çalışmada, N-Oleoylethanolamine (NOE) bileşiğinin
akciğer kanseri hücreleri ve sağlıklı akciğer hücreleri üzerindeki sitotoksisitesinin ölçülmesi ve
hücreler üzerindeki morfolojik değişiklerin saptanması amaçlanmıştır. Sitotoksisiteyi tespit
etmek için MTT testi kullanılmıştır. A549 ve Beas-2B hücreleri, 96 kuyucuklu plakalarda 24
saat boyunca farklı konsantrasyonlarda muamele edilmiştir. İnkübe edilen hücreler, 570 nm
dalga boyunda bir plaka okuyucu kullanılarak spektrofotometrik olarak ölçülmüştür. Elde
edilen absorbanslardan canlılık yüzdeleri hesaplanmış ve IC50 değeri belirlenmiştir. A549
hücrelerine farklı konsantrasyonlarda N-Oleoylethanolamine (NOE) uygulanması ile 24 saat
içinde hücre ölümünde önemli bir artışa neden olduğu görülmüştür. Bu çalışmada NOE ile
tedavi edilen A549 ve Beas-2B hücreleri, morfolojik değişikliklerin tespit edilmesi amacıyla
konfokal mikroskop altında incelenmiştir. A549 ve Beas-2B hücreleri, sitotoksisite analizi ile
hesaplanan IC50 değeri ile uygulanan NOE konsantrasyonları ile inkübe edilmiştir. İşlem gören
hücreler Alexa fluor-488 phalloidin ve akridin oranj ile tespit edilerek boyanmış ve daha sonra
konfokal mikroskop altında gözlemlenmiştir. Konfokal mikroskopi ile NOE'nin insan akciğer
kanseri hücreleri üzerindeki apoptotik etkileri gösterilmiştir. Çalışmamızdan elde edilen
sonuçlara göre, N-Oleoylethanolamine (NOE) bileşiğinin insan akciğer kanseri tedavisinde bir
anti kanser ajan olma potansiyeline sahip olduğu gösterilmiştir ve daha ileri deneylerin
gerçekleştirilmesi tarafımızdan önerilmektedir.
Anahtar Kelimeler: N-Oleoylethanolamine, NOE, akciğer kanseri, apoptoz.
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Doksorubisin İle İndüklenen Sıçan Nefrotoksisite Modelinde Fluvastatinin Olası
Koruyucu ve Tedavi Edici Etkilerinin Histokimyasal ve İmmünohistokimyasal Olarak
Değerlendirilmesi
Fatih Oltulu1, Çevik Gürel1,2
1Ege Üniversitesi Tıp Fakültesi, Histoloji ve Embriyoloji Anabilin Dalı, İzmir, Türkiye 2Harran Üniversitesi Tıp Fakültesi, Histoloji ve Embriyoloji Anabilim Dalı, Şanlıurfa, Türkiye
Amaç: Doksorubisin, solid tümörlerin ve hematolojik malignitelerin tedavisinde sıklıkla
kullanılan fakat başta kalp ve böbrek olmak üzere birçok organda ortaya çıkan akut ve kronik
yan etkiler nedeniyle kullanımı önemli ölçüde kısıtlanan antrasiklin türevi bir antibiyotiktir. Bu
çalışmada amaç, doksorubisin ile indüklenen nefrotoksisite modelinde fluvastatinin olası
koruyucu ve tedavi edici etkilerinin ve bu etkilerin altında yatan moleküler mekanizmalarının
araştırılmasıdır.
Gereç ve Yöntemler: Bu çalışma için 35 adet yetişkin erkek Sprague Dawley türü sıçan
kullanılmıştır. Sıçanlar rastgele; Kontrol (n=7), Fluvastatin (n=7), Doksorubisin (n=7),
Profilaksi (n=7) ve Tedavi (n=7) gruplarına ayrılmıştır. Kontrol grubuna deneyin 1.,4. ve 7.
günlerinde serum fizyolojik uygulaması yapılmış başka herhangi bir uygulama veya girişim
yapılmamıştır. Fluvastatin grubundaki hayvanlara ise 7 gün boyunca, gavaj ile 6 mg/kg/gün
fluvastatin uygulaması yapılmıştır. Doksorubisin, Proflaksi ve Tedavi gruplarında, deneyin 1.,
4. ve 7. günlerinde, 7,5 mg/kg/gün doksorubisin uygulaması yapılarak nefrotoksisite modeli
indüklenmiştir. Proflaksi grubuna, 1., 4. ve 7. günlerde doksorubisin uygulamasından bir saat
önce olacak şekilde, deneyin ilk gününden başlanarak 7 gün boyunca gavaj ile 6 mg/kg/gün
fluvastatin uygulanmıştır. Tedavi grubundaki hayvanlara ise 8. günden itibaren 7 gün boyunca
gavaj ile 6 mg/kg/gün fluvastatin uygulanmıştır. Deney protokolünün sonunda, sakrifiye edilen
sıçanlardan alınan örnekler histokimyasal, immünohistokimyasal, biyokimyasal ve Real-Time
PCR analizleri ile değerlendirilmiştir.
Bulgular: Histopatolojik ve biyokimyasal değerlendirmede doksorubisin grubunda
vakualizasyon, kollajen miktarında artış, Bowman kapsülünde kalınlaşma ve Bowman
boşluğunda daralma, intersitisiyel ödem, infiltrasyon, tübüler dilatasyon, tübül epitel
hücrelerinde dökülmeler, MDA seviyesinde artış ve SOD aktivitesinde azalma gibi patolojik
değişiklikler saptanmıştır. Bunanla birlikte, proflaksi ve tedavi gruplarında, doksorubisin
grubunda gözlemlenen patolojik bulguların çoğunun bulunmadığı görülmüştür. Yapılan
immünohistokimyasal ve Real-Time PCR analizlerinde elde edilen sonuçlar ise doksorubisin
uygulamasının böbreklerde mTOR mRNA ve protein ekspresyonlarını baskıladığını buna
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karşın LC3 mRNA ve protein ekspresyonunda ise anlamlı bir artışa neden olduğunu
gözlemlenmiştir. Öte yandan, proflaksi ve tedavi gruplarında doksorubisin grubuna oranla,
mTOR ekspresyonlarında artış ve LC3 ekspresyon seviyelerinde ise anlamlı bir azalma olduğu
tespit edilmiştir.
Sonuç ve Tartışma: Yapılan analizlerden elde edilen sonuçlar, doksorubisin kaynaklı
nefrotoksisitede fluvastatinin, antioksidan sistemin aktivasyonu ile lipit peroksidasyonunun
inhibisyonu ve otofaji yolağının regülasyonu yoluyla bu kemoterapötik ajanın kullanımından
kaynaklanan patolojileri önleyebileceğini göstermektedir. Ek olarak, fluvastatinin profilaktik
olarak kullanımının, tedavi amaçlı kullanımına kıyasla daha olumlu sonuçlar verdiği
gözlemlenmiştir.
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67
Abnormal Expression Patterns of Repetitive DNA Elements Uncovered in Breast Cancer
Cihangir Yandım1, 2,, Gökhan Karakülah2, 3*
1İzmir University of Economics, Faculty of Engineering, Department of Genetics and Bioengineering, 35330, Balçova, İzmir, Turkey
2İzmir Biomedicine and Genome Center (IBG), Dokuz Eylül University Health Campus,
35340, İnciraltı, İzmir, Turkey 3İzmir International Biomedicine and Genome Institute (iBG-İzmir), Dokuz Eylül University,
35340, İnciraltı, İzmir, Turkey
e-mails: [email protected] ; [email protected]
*corresponding
Repetitive DNA elements, which constitute more than half of the human genome with more
than a thousand types of distinct motifs 1,2, are normally hardly expressed in healthy cells with
a stable genome3. Notably, a common feature of breast cancer is genomic instability, which is
associated with heterogeneity and resistance to treatment 4. The contribution of repeats to this
pathogenic phenomenon largely remained unexplored mostly due to difficulties in mapping the
repeats using sequencing data. Only a handful of repeats (i.e. HERVK, HSATI and II) were
previously reported in breast cancer literature 5-7, and a genome-wide analysis was needed to
get a global picture. Hence, we analysed an RNA sequencing dataset comprising the
transcriptomes of ER+/HER2- breast tumours obtained from 22 patients along with their
healthy breast tissues 8. We used ultimate bioinformatics tools that allow proper repeat
quantification 9,10 and identified 56 repeat motifs being significantly dysregulated in tumours;
many of them being novel for cancer literature. Among these, D20S16 and REP522 satellites
globally correlated well with breast cancer driver genes such as AKT, mTOR, KRAS, BRCA1
and BRCA2. We also studied how the repeat motifs could interfere with the expressions of
proximal genes through regression analysis, and determined transposons such as L2b, L2c,
MER5B or LTR5_Hs to be potentially involved in the dysregulation of breast cancer genes.
Our results not only open a new avenue of research for breast cancer but also provide a guiding
bioinformatics pipeline on how repetitive DNA, which is often overlooked, could be studied in
the context of cancer genomics. The repeats identified in this study may serve as biomarkers
for assessing genomic instability or prognosis in breast cancer patients, and warrant further
research in terms of understanding complex events that take place in the axis of genomic
distortion underlying this devastating disease.
Oral Presentation
68
References
1 de Koning, A. P., Gu, W., Castoe, T. A., Batzer, M. A. & Pollock, D. D. Repetitive elements
may comprise over two-thirds of the human genome. PLoS Genet 7, e1002384,
doi:10.1371/journal.pgen.1002384 (2011).
2 Lander, E. S. et al. Initial sequencing and analysis of the human genome. Nature
409, 860-921, doi:10.1038/35057062 (2001).
3 Saksouk, N., Simboeck, E. & Dejardin, J. Constitutive heterochromatin formation and
transcription in mammals. Epigenetics & chromatin 8, 3, doi:10.1186/1756-8935-8-3
(2015).
4 Kwei, K. A., Kung, Y., Salari, K., Holcomb, I. N. & Pollack, J. R. Genomic instability in
breast cancer: pathogenesis and clinical implications. Molecular oncology 4, 255-266,
doi:10.1016/j.molonc.2010.04.001 (2010).
5 Ichida, K. et al. Overexpression of satellite alpha transcripts leads to chromosomal
instability via segregation errors at specific chromosomes. International journal of
oncology, doi:10.3892/ijo.2018.4321 (2018).
6 Johanning, G. L. et al. Expression of human endogenous retrovirus-K is strongly associated
with the basal-like breast cancer phenotype. Scientific reports 7, 41960,
doi:10.1038/srep41960 (2017).
7 Zhu, Q. et al. Heterochromatin-Encoded Satellite RNAs Induce Breast Cancer. Molecular
cell 70, 842-853 e847, doi:10.1016/j.molcel.2018.04.023 (2018).
8 Solovyov, A. et al. Global Cancer Transcriptome Quantifies Repeat Element
Polarization between Immunotherapy Responsive and T Cell Suppressive Classes.
Cell reports 23, 512-521, doi:10.1016/j.celrep.2018.03.042 (2018).
9 Papin, C. et al. Combinatorial DNA methylation codes at repetitive elements. Genome
research 27, 934-946, doi:10.1101/gr.213983.116 (2017).
10 Yandim, C. & Karakulah, G. Expression dynamics of repetitive DNA in early
human embryonic development. BMC genomics 20, 439, doi:10.1186/s12864-019-
5803-1 (2019).
Oral Presentation
69
Hedefe Yönelik Tedaviler İçin Çeşitli Kanser Hücrelerinde IL13Rα2 Ekspresyonunun
Belirlenmesi
Damla Uludağ1, Ozan Topcu1, Nihal Karakaş1,2
1İstanbul Medipol University, Regenerative and Restorative Medicine Research Center (REMER), Kavacik Campus, Kavacik-Beykoz/ISTANBUL 34810
2Istanbul Medipol University, School of Medicine, Department of Medical Biology, Kavacik Campus, Kavacik-Beykoz/ISTANBUL 34810
Çeşitli kanser türlerinde uygulanan standart tedaviler, hastalığın prognozunun olumlu yönde
seyri için yetersiz kalmakla birlikte yalnızca kanser hücrelerini hedeflememektedir. Bu durum
uygulanan tedavilerin sağlıklı hücreler üzerine olumsuz etkilerinin varlığı ile sonuçlanmaktadır.
Hedefe yönelik tedavilerde ise esas amaç kanser hücrelerinin selektif olarak hedeflenerek
ortadan kaldırılmasıdır. Bu kapsamda hedefe yönelik reseptörlerden biri, kanser hücrelerine
özgü olan interlökin-13 (IL-13) reseptörüdür. IL-13, normal hücrelerde ya hiç ya da az düzeyde
eksprese edilen fakat çeşitli kanser hücre hatlarında yoğun olarak eksprese edilen IL13Rα2
reseptörlerine yüksek afinite ile bağlanır. Böylece IL-13 ve yüksek afinite gösterdiği IL13Rα2
reseptörü hedefe yönelik terapötiklerin oluşturulmasında kanser seçiciliği yönüyle oldukça
önemlidir. Bununla birlikte, kanser hastalığı ve prognozuna dair klinik öncesi araştırmalarda
tümördeki gerilemenin takibi için kullanılan yöntemler de sınırlıdır. Diğer taraftan
görüntülenebilir tümör çalışmaları, hedefe yönelik tedavilerin etkinliğini en hassas şekilde
ortaya koymaktadır.
Bu doğrultuda çalışmamızda; yeşil floresan proteini (GFP) ile biyolüminesan görüntüleme
ajanlarından Firefly lusiferazı (Fluc) birlikte içeren lentiviral vektörler kullanılarak, bu ajanları
eksprese eden kanser hücre hatları oluşturulmuştur. Ardından terapötik hedefleme için IL13Rα2
yüzey reseptörünün çeşitli kanser hücrelerindeki ekspresyon seviyeleri belirlenmiştir. Terapötik
hedefleme sonucunda hücre canlılığının in vitro analizi, bu hücrelerde canlılığın tespit
edilmesini sağlayan luminesan sinyal ölçümü için Fluc’a özgü D-luciferin kullanılarak
gerçekleştirilmiştir. Hücre canlılık analizi sonrasında kontrol hücrelerine kıyasla IL13Rα2
eksprese eden hücrelerde hedefe yönelik terapötik kullanımının hücre ölümüne yol açtığı tespit
edilmiştir.
Bu proje, TUBİTAK 117S421 nolu 1001 projesi kapsamında desteklenmektedir.
Oral Presentation
70
Fluidic Shear Stress Regulates Biological Behaviours of Hepatocellular Carcinoma
Cells Through c-Met Signalling
Dehan Comez1,3*, Gulsun Bagci1,2*, Hande Topel1,2*, Ezgi Bagirsakci1,2, Yeliz Yilmaz1,3,
Aysim Gunes1, Nese Atabey1
• Equal contribution 1Izmir Biomedicine and Genome Center, Balcova, Izmir, Turkey
2Department of Medical Biology and Genetics, Health Sciences Institute, Dokuz Eylul University,
Balcova, Izmir, Turkey 3Izmir International Biomedicine and Genome Institute, Balcova,Izmir, Turkey
Tumour metastasis is the most common reason of the cancer related death. Metastasis process
requires cancer cells to detach primer tumour and intravasation to blood circulation and survive
the shear stress in blood stream, homing and proliferation in the secondary organ. Fluidic shear
stress (FSS) in blood circulation effects tumour cell’s survival, apoptosis, invasion, metastasis
and proliferation. Several clinic-pathological studies underline that in HCC intravascular
invasion is poor prognosis factor and it is significantly important in metastasis. When tumour
cells intravasate to sinusoidal capillaries, shear stress stimulates apoptosis. Frequency of
intrahepatic metastasis is high in HCC and it occurs as invasion dependent manner indicating
survival of tumour cells under FSS is crucial step for intrahepatic and extrahepatic metastasis.
Nevertheless, mechanisms behind process hasn’t been illuminated adequately yet. Our previous
studies showed that HGF/c-Met pathway plays crucial role in cell survival, invasion, metastasis
during hepatocarcinogenesis. In this study we aimed to examine the role of HGF/c-Met pathway
on survival and proliferation of HCC cells under FSS. In order to do this, we used peristaltic
pump to apply FSS to HCC cells which have low (HuH-7 and HepG2) and high (SNU-449) c-
Met levels. c-Met expression and activation levels were determined by RT-qPCR and Western
blotting, cell survival and proliferation analysis by trypan blue and (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide) (MTT) in HCC cells. The effect of FSS on colony
formation, epithelial-mesenchymal transition (EMT) and invasion were determined by 3D
microfluidic system. Our data showed that, FSS treatment increased the expression and
activation of c-Met stimulating EMT phenotype, cell survival, proliferation and invasion in
HCC cells and c-Met inhibitor (SU11274) reversed these effects suggesting HGF/c-Met
pathway has critical role on the survival of HCC cells under FSS and induces aggressive
phenotype and c-Met inhibitors could be used to inhibit survival of circulating tumour cells’
metastasis in HCC.
Keywords: HCC metastasis, fluidic shear stress, circulating tumour cell, cancer
Oral Presentation
71
Investigation of the Anticancer Effect of Haplophyllum Buxbaumii Plant on HCT-116
Colon Cancer Cells
Ataman Gönel, Derya Akçiçek, İsmail Koyuncu, Nihayet Bayraktar, Ebru Temız
Department of Medical Biochemistry, Harran University, Sanliurfa, Turkey
Colon cancer is the second leading cause of cancer-related deaths in the world. Mostly, the main
treatment option is postoperative chemotherapy. Although the cytotoxic effects of
chemotherapeutic drugs are to ensure the complete destruction of cancer cells, healthy cells are
damaged through these drugs. The discovery of phytotherapeutic agents in which the toxic
effect for healthy cells is minimized can be promising for cancer patients. The aim of this work
was to investigate the anticancer feature of Haplophyllum buxbaumii plant, which is grown
endemically in Şanlıurfa.
In our study, MTT method was used to investigate the cytotoxic effect of HCT-116 colon cancer
cell line and healthy (HUVEC) cells from extracts obtained from Haplophyllum buxbaumii
plant. Flow cytometric annexin V analysis, cell cycle analysis (BD Cycletest ™ Plus DNA
Reagent), intracellular ROS effect analysis (flow cytometry analysis) and apoptotic
morphological change analysis (fluorescence Acridine orange / ethidium bromide staining)
were performed to detect the apoptotic activity of the extract material. At the end of the study,
the IC50 value of the cytotoxicity found by MTT method in HCT-116 cells was determined at
14.17 μg / ml. For the normal cells, IC50 dose was found to be 70 µg/ml. It was concluded that
the cytotoxic effect was caused by apoptosis in HCT-116 cells and there was no apoptotic effect
in HUVEC cell.
This study showed that Haplophyllum buxbaumii plant extract may have the potential of
chemotherapeutic effect for the HCT-116 colon cancer cells.
Keywords: HCT-116, HUVEC, Haplophyllum Buxbaumii, Cytotoxicity, Annexin V
Oral Presentation
72
Identification of shared candidate key genes and pathways associated with cancer
and heart failure pathogenesis using bioinformatic analyses
Dilek Pirim
Uludag University, Faculty of Arts and Sciences, Department of Molecular Biology and Genetics,
Bursa, Turkey
Background: Emerging evidence suggests that heart failure (HF) may contribute to the
oncogenic process and tumor development. High incidence of cancer in patients with HF
highlights the link between cancer and HF which needs to be elucidated to alleviate the
worldwide burden of the two diseases. The objective of our study is to identify the potential
common key genes and molecular pathways associated with cancer and HF.
Methods: Microarray data sets (GSE9128, GSE103512) were downloaded from Gene
Expression Omnibus and integrated analyses were performed by using bioinformatic tools.
Differentially expressed genes (DEGs) were analyzed in GEO2R using Limma R package.
Functional and pathway enrichment analyses of DEGs were performed by NetworkAnalyst
online analytics tool. STRING database was used for construction of protein-protein
interaction network. Network was visualized by Cytoscape software; hub genes were
determined using plug in Cytohubba. The expression levels of hub genes were evaluated in
independent datasets through C/VD and ENCORI databases for validation purposes.
Results: A total of 54 genes were identified to be dysregulated in HF and 4 types of cancers
(Breast, colorectal, non small cell lung and Pancreatic cancer). Top most enriched Kyoto
Encyclopedia of Genes Genomes (KEGG) categories was related immune system and most
significant Gene Ontology (GO) term related biological process was the response to abiotic
stimuli. Network analyses revealed top ten hub genes with the highest scores in Maximal
Clique Centrality ranking as VEGFA, JUN, DUSP1, FOS, CXCL8, EGR1, MYC, CXCL2,
NR4A2, FOSL2 and five hub genes including VEGFA, MYC, CXCL8, CXCL2, FOSL2 were
found to be significantly different expressed in independent datasets related both HF and
cancer.
Conclusion: This study shed light on the shared critical pathways and potential genes
regarding the common molecular mechanism of the pathogenesis of HF and cancer. Our
results pinpoint candidate molecular targets for further investigation and management of
cancer.
Oral Presentation
73
Investigation of Anti-cancer Properties of Haplophyllum Ptilostylum Plant
Ebru Temız, İsmail Koyuncu Ataman Gönel, M.Orhan Tunçel
Department of Medical Biochemistry, Harran University, Sanliurfa, Turkey
Characterizing by uncontrolled cell growth, cancer is one of the most common diseases. Since
the current methods damage to the immune system of the body during the treatment process, a
new treatment method, which has less side effect and high specificity, have been searched.
Phytotherapy, which has become widespread as one of the cancer treatment methods in recent
years, has many advantages. The present study aims to investigate the antiproliferative effects
of Haplophyllum Ptilostylum extract on liver cancer. The cytotoxic effects of the extract
obtained from Haplophyllum Ptilostylum on liver cancer cell line (SNU-423) and control cell
line (HUVEC) were examined by MTT method. In addition, flow cytometric; annexin V
analysis, cell cycle analysis, mitochondrial membrane potential analysis and apoptotic
morphological change analysis (fluorescence Acridine orange/ethidium bromide staining) were
performed to detect the apoptotic activity of the extracted material. At the end of the study, the
cytotoxic effect (IC50) was measured as 5μg/ml in liver cancer cells and 102μg/ml in control
cells. The apoptotic activities in SNU-423 and HUVEC cells were measured as %25.6 and
%7.9, respectively; the effects of the extract on the cell cycle were found as follows: Sub-G1:
%2,3 and G0/G1: %55,0 in SNU-423; and Sub-G1: %0 and G0/G1: %58,2 in HUVEC. The
mitochondria membrane potentials in SNU-432 and HUVEC were recorded as %33.2 and %
15.7, respectively. According to the obtained results, Haplophyllum Ptilostylum plant creates
an apoptotic activity by disrupting the mitochondria membrane potential in the liver cancer cell.
Based on these grounds, we suggest that Haplophyllum Ptilostylum plant can be tested as a
pharmacologic agent in different cancer types.
Keywords: Haplophyllum Ptilostylum, SNU-423, Liver Cancer, Cell Death, Cytotoxicity
Oral Presentation
74
The importance of dysregulated miRNAs on ovarian cysts and tumors
Ece Gumusoglu1, Tuba Gunel1, Mohammad Kazem Hosseini1, Nogayhan Seymen2, Taylan Senol3, Uğur Sezerman2, Samet Topuz4, Kılıç Aydınlı5
1Istanbul University, Faculty of Science, Department of Molecular Biology and Genetics, Istanbul, Turkey.
2Acıbadem University, School of Medicine, Department of Basic Sciences, Biostatistics, Istanbul Turkey.
3Bağcılar Training and Research Hospital, Department of Obstetrics and Gynecology, İstanbul,
Turkey. 4Istanbul University, Department of Obstetrics and Gynecology, Istanbul Medical Faculty, Istanbul,
Turkey. 5Medicus Healthcare Centre, Istanbul, Turkey
Ovarian cancer is the fifth most important cause of cancer-related deaths among women and
the most lethal gynecologic malignancy. Epithelial ovarian cancer (EOC) is asymptomatic and
few screening tests are available. In our study, we aimed to find the importance of dysregulated
miRNAs in ovarian cyst and their expression profile difference between ovarian cancer cases.
Another goal of our study is to identify novel circulating miRNAs to be used as therapeutic
prediction of EOC. In this study, we studied three different samples: serums of EOC patients,
healthy individuals (HI) and benign ovarian cysts (BOC). Their miRNA expressions have been
compared by microarray. Microarray data were analyzed according to miRNA expressions the
relation between miRNAs target genes and EOC were examined by bioinformatic tools. 75 and
66 significantly dysregulated miRNAs were identified by microarray in BOC vs. EOC and BOC
vs. HI comparison, respectively. Then, we focused on common miRNAs that found in both
comparison and finally 46 important miRNAs were detected which can represent the only
common sample group, BOC. After these findings, we also considered miRNA profiling in
EOC and HI, and surprisingly any common miRNAs were found with these 46 miRNAs. Thus,
we analyzed them depending on their potential importance on BOC pathogenesis. After
bioinformatic analysis, our findings indicated that there are several biological processes and
pathways which can be considered to be related BOC development.
Oral Presentation
75
Investigation of Helicobacter Pylori virulence factors and immune response in
precancerous lesions
Elif Merve Aydın1†‡, Tevriz Dilan Demir2‡, Ayça Sayı Yazgan3* Arzu Tiftikçi 4*
1Molecular Biology- Genetics and Biotechnology Research Center, Istanbul Technical University,
Istanbul, Turkey
2Molecular Biology- Genetics and Biotechnology Research Center, Istanbul Technical University, Istanbul, Turkey
3Molecular Biology- Genetics and Biotechnology Research Center, Istanbul Technical University, Istanbul, Turkey
4Internal Medicine Gastroenterology Department, Acibadem University, Istanbul, Turkey
†Presenting Author, ‡Equal contribution to the study
The developmental stages of gastric cancer could be classified as chronic gastritis, atrophic
gastritis, intestinal metaplasia and dysplasia. It is not possible to determine whether patients
with gastric pathology will develop gastric cancer, as the genetic and immunological aspects of
gastric pathogenesis are not fully known. At the same time, the relation between the
characteristic of the virulence factors of H. pylori and the impact on the host's immune response
is not fully understood. Therefore, we aimed to investigate the ten H. pylori virulence factors
and adaptive immune response in patients with gastric pathology.
This study included in total 32 patients; 20 with active chronic gastritis, 3 with inactive chronic
gastritis, and 9 with intestinal metaplasia. 15 subjects who had no signs of inflammation and
did not carry infection were included to this study as control. Three tissue samples were taken
from the antrum of the stomach during the endoscopy. DNA and RNA was extracted from two
of the received tissue samples and third biopsy specimen was used to determine the
pathogenesis of gastric tissue and H.pylori presence using histological staining. DNA samples
were used to detect ten H. pylori virulence factor genes; ureA, ureB, hpaA, cagA, napA, dupA,
oipA, vacA m1/m2, vacA s1/s2, babA using conventional PCR. qRT-PCR was used to
investigate the expression of regulatory T cell specific transcription factor, FOXP-3; Th-1
specific cytokine IFN-γ, and immune checkpoint ligand PD-L1 at mRNA level. The most
common virulence factors in patients with active chronic gastritis were shown to be napA, hpaA
and cagA. The expression level of IFN -ɣ was increased and FOXP-3 was decreased in gastric
precancerous lesions. Conversely, PD-L1 expression level was higher in precancerous lesions.
Overall, we investigated virulence factors of Helicobacter pylori and immune response of the
host in gastric precancerous lesions.
Oral Presentation
76
A Useful Tool for Distinguishing Gene/Pseudogene Variants Detected via NGS:
Haplotype Analysis
Esra Arslan Ateş1,2, Ahmet İlter Güney2, Tuba Günel1 1Istanbul University, Faculty of Science, Department of Molecular Biology and Genetics, Istanbul.
2Marmara University Pendik Training and Research Hospital, Department of Medical Genetics,
Istanbul.
e-mail: [email protected]
Introduction: Lynch Syndrome is an inherited cancer prone disease mostly associated with
colon, endometrium, ovarian and gastrointestinal tract cancers. Mutations affecting missmatch
repair genes (MLH1, PMS2, MSH2, MSH6) are responsible from Lynch Syndrome. PMS2 has
a psedudogene called PMS2CL showing a high sequence similarity to PMS2 gene. Here we
report a novel mutation in PMS2 gene associated with Lynch Syndrome which was proven to
be on the active gene using haplotype analysis.
Case Report: A 69 year-old woman referred to us because of endometrium cancer. She had a
history of colon cancer diagnosed at the age of 66. Endometrial tumor showed high
microsatellite instability and MLH1 and PMS2 expression loss were detected in
immunohistochemical analysis. There were no other cancer history in her family. BRAF V600E
mutation and MLH1 promoter hypermethyation were not detected in tumor tissue. On the basis
of immunohystochemical findings MLH1 and PMS2 genes were screened for germline
mutations from peripheral blood lymphocytes via next generation sequencing. We detected a
novel 47bp insertion (c.1362_1407dup; p.Pro470Valfs*3) in exon 11 of PMS2 gene with a
variant fraction of 26%. The false positive result due to pseudogene was excluded by haplotype
analysis using the SNPs that allow us to distinguish PMS2 and PMS2CL.
Conclusion: PMS2 gene is a MMR system component which has an important role in DNA
replication and homologous recombination, located on chromosome 7p and consist of 15 exons.
There is a pseudogene, PMS2CL, which shows 98% sequence similarity to exons 9 and 11–15
of PMS2 and it is important to distinguish the variations between gene and pseudogene
sequences. NGS data enable us to assess the haplotypes in an amplicon using the distinct SNPs
on gene and psedudogene.
Oral Presentation
77
Identification of lncRNA molecules involved in the regulation of EGF-induced of cellular
proliferation
Esra Bozgeyik
Tekirdag Namik Kemal University, Faculty of Medicine, Department of Medical Biology, Tekirdag, Turkey
Lung cancer is one of the leading causes of cancer-related deaths worldwide. According to 2019
cancer statistics, it is estimated that over 230000 new lung cancer cases will occur and 143000
people will die due to lung cancer. Moreover, lncRNAs (long non-coding RNA) are functional
RNA molecules longer than 200 nucleotides that do not have protein coding capacity and play
important roles in the vital cellular processes such as proliferation, growth, division,
differentiation, survival, and migration of cells. Due to their wide range of biological functions,
deregulation of these transcripts highly contributes to molecular pathogenesis of cancers
including lung cancer. EGF is key factor that is involved in the regulation of cell proliferation.
Since EGF is a very chief protein for the maintenance of normal physiological processes,
deregulation of EGF-induced signaling significantly contributes to the several hallmark
capabilities of human cancers. Thus, determination of lncRNA molecules involved in the
regulation of EGF-induced promotion of cellular proliferation is of great interest to illuminate
the molecular pathology of human cancers. Accordingly, in this study we aimed to identify
differentially expressed lncRNAs involved in the regulation of EGF-induced stimulation of
cellular proliferation. As a result, expression levels of MALAT1, ANRIL and MCM3AP-AS1
lncRNAs was found to be increased whereas expression levels of LOC105372161 and MEG3
were found to be decreased. However, only changes in the expression of LOC105372161 were
found to be statistically significant, suggesting that LOC105372161 is involved in the regulation
of EGF-induced cell proliferation and BCL2 might be the putative target of LOC105372161.
Keywords: EGF, LOC105372161, lncRNA, lung cancer.
Oral Presentation
78
Morus nigra (Karadut) yaprağı etanol ekstraktının doğal bir DNA ileri glikasyon son
ürünleri (DNA-AGE'ler) bileşik kaynağı olarak değerlendirilmesi
Feryal Akay, Bircan Çeken Toptancı, Nesrin İnceören, Göksel Kızıl, Murat Kızıl
University of Dicle, Faculty of Science, Chemistry Department, Diyarbakır, Turkey
Gelişmekte olan ülkelerde nişasta bazlı gıdaların tüketiminin artması ile metabolik hastalıklarda
hızlı bir artış meydana gelmiştir. Glikoz kanda insulin tarafından tanınırken fruktoz tanınmaz
ve lipogenezise katılarak hücre içi lipit birikimini arttırır. İndirgen şekerler; biyolojik makro
moleküllerde bazı reaktif ürünler ve AGE’leri (İleri Glikasyon Son ürünleri) oluşumuna neden
olur. Diyabetin sürekliliği dokularda protein birikimine ve Maillard reaksiyonuna sebep
olabilir. İleri glikasyon son ürün reseptörü (RAGE) ekspresyonuna neden olabilir ve karşılıklı
olarak AGE formasyonuda hiperglisemiyi hızlandırır. Bu ürünler çeşitli mekanizmalarla bir dizi
hastalığın patojenezinde birikerek doku hasarına neden olabilir.
AGEs’ler hücrelerin çekirdek ve sitoplazmalarında birikerek DNA hasarı oluşturur. Böylece
kanserli hücre oluşabilir. AGEs reaktif oksijen türlerini arttırarak antioksidan sistemlere hasar
verebilirler.
AGEs ciltteki elastikliği zayıflatarak yaşlanmayı da aktif hale getirir. Eğer deriye yakınsa deri
kanserine sebep olur. UV ile aktifleşerek proteinleri modifiye edip yine DNA hasarına neden
olur. Derideki glikolizlenmiş proteinler foto-aktif olarak, foto-yaşlanma ve foto-kanserde eşit
derecede işlev görürler.
Bazı doğal gıda bileşenleri AGE'lerin oluşumuna ve birikmesine karşı koruyucu bir etki
gösterebilir. Oksidasyon birçok AGE'nin oluşumunda rol oynayan çok önemli bir adım
olduğundan, başlangıçta antioksidan, radikal temizleme veya metal şelatlama özelliklerine
sahip polifenoller AGE inhibitörleri olarak araştırılmaktadır.
Çalışmanın amacı farklı ekstraksiyon teknikleri kullanılarak elde edilen Morus nigra (Karadut)
yaprağı etanol ekstraktlarının; toplam fenolik, flavonoid, DPPH ve hidroksi radikalini
söndürme aktivitelerini araştırlaması ve bu parametrelerde en yüksek aktivite gösteren ekstrakt
ve M. nigra yaprağının etken maddelerinden Quersetinin; DNA-fruktoz ve DNA-aspartam
sisteminde in vitro antiglikasyon kapasitesini Floresans spektrometresi, UV spektrometresi,
fruktozamin (NBT) testi ve agarose jel elektroforez tekniklerini kullanarak araştırmaktır.
Sonuç olarak, fruktozun ve aspartamın DNA da oksidatif hasara neden olduğu ve M. nigra
yaprak ekstraktının ve quersetinin, antioksidant aktivitesinin olduğu, hidroksi radikali kaynaklı
Oral Presentation
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DNA oksidasyonunu inhibe ettiği tespit edildi, ancak fruktoz kaynaklı DNA-AGE oluşumunu
etkin bir şekilde inhibe ettmediği tespit edildi. M. nigra yapraklarında ana etken madde olarak
bulunan Quersetinin fruktoz kaynaklı DNA kesimini konsantrasyona bağlı olarak önlediği
belirlendi, ancak M. nigra yaprağı etanol ekstraktı DNA kesimini önlemeyip, DNA kesimini
artırdığı tespit edildi.
Oral Presentation
80
The antiproliferative and apoptotic effects of gold nanoparticles synthesized by Allium
cepa extract on leukemia cell lines
Gamze Tan1*, Kamile Öztürk1, Mehtap Tarhan1
1Department of Biology, Faculty of Science and Letters, Aksaray University, Aksaray, Turkey
*[email protected]; [email protected]
In biomedicine, the use of metallic nanoparticles prepared by using plant extracts is increasing.
It is preferred because the extract of the plants contains many different metabolites, which are
especially useful in the reduction of metal ions. They are also low-cost, easily available, and
environmentally friendly alternatives to chemical reducing agents. In addition, plants have been
used in the treatment of many diseases and obtaining medicines in modern medicine. Allium
cepa (Onion) with a large number of functional compounds has been used to protect human
health, thanks to their therapeutically useful properties, such as antibacterial, anti-inflammatory
and antioxidant activities in particular. In this study, it is aimed to show the synthesis of gold
nanoparticles using the aqueous extract of onion (O-AuNPs), and evaluate their antiproliferative
and apoptotic effects on four leukemia cell lines.
The chemical structure and physical properties of bio-fabricated AuNPs were confirmed with
TEM, zeta potential, and ICP-OES. Furthermore, cellular responses of O-AuNPs was
investigated on K562, HL-60, CEM and Raji leukemia cell lines by using MTT test for 48 and
72 hours. Pro-apoptotic Bax, anti-apoptotic Bcl-2 and Caspase-3 gene expression levels were
determined by using real time quantitative RT-PCR method.
According to TEM images, O-AuNPs had spherical in shape with approximately 13.5 nm in
size. The zeta potential result showed that surface charge of the particles was about -14.4 mV.
O-AuNPs decreased cell viability in all cell lines in both dose and time dependent manner.
Moreover, O-AuNPs induced apoptosis via changing the expression Bax, Bcl-2 and caspase 3
gene in all cell line.
In conclusion, our results suggest that O-AuNPs have antiproliferative and apoptotic effects on
leukemia cell line through by up-regulating Bax/Bcl-2 gene expression ration and increased
caspase-3 levels.
Keywords: Allium cepa, antiproliferative effect, leukemia, apoptosis, gold nanoparticles
Oral Presentation
81
The Role of c-Met Signaling in Organ Specific Extravasation of HCC cells
Gulsun Bagci1,2*, Dehan Comez1,2 *,Hande Topel1,3*, Ezgi Bagirsakci1,2, Yeliz Yilmaz1,3, Aysim Gunes1, Gizem Batı Ayaz4, Devrim Pesen Okvur4, Nese Atabey1
*Equal contribution 1Izmir Biomedicine and Genome Center, Balcova, Izmir, Turkey
2Department of Molecular Biology and Genetics, Izmir International Biomedicine and Genome
Institute, Balcova,Izmir, Turkey 3Department of Medical Biology and Genetics, Health Sciences Institute, Dokuz Eylul University,
Balcova, Izmir, Turkey 4Izmir Institute of Technology, Department of Molecular Biology and Genetics, Izmir, Turkey
HCC (Hepatocellular Carcinoma) is the fifth most common cancer worldwide and the third
cause of cancer deaths. Due to difficulties in diagnosis, many patients are diagnosed at late
stages. HCC is associated with high mortality rate due to high recurrence and metastasis risk in
patients after surgical resection. Metastasis consists of multiple steps as dissemination of cancer
cells from primary tumor, intravasation into blood stream, survival in circulation, extravasation
to secondary organs and colonization. c-Met signaling pathway mediates embryogenesis and
organ regeneration, its aberrant activation can promote invasion, motility, proliferation,
angiogenesis and evading apoptosis in several cancers including HCC. Recent data suggest that
activation of c-Met signaling might be an important factor in the regulation of tumor metastasis.
However, role of c-Met signaling in the organ specific extravasation is not defined in literature
yet. Lab-on-a-chip is integrated 3D cell culturing to study for tumor-microenvironment
interactions, drug screening, studying metastatic patterns of cancer cells to distant organs. In
this study, a Lab-on-chip device was used to mimic the extravasation capacities of SNU-449,
SNU-398, SNU-398-Met HCC cells to investigate role of c-Met on the organ specific
extravasation. Homing cells were THLE-2, BEAS-2B and HS-27A to represent liver epithelial,
lung epithelial and bone marrow/stroma cells respectively. HUVEC cells were used to mimic
endothelial barrier. HCC cells were exposed to 0.5 dyn/cm2 FSS for 4 hours to simulate FSS in
hepatic sinusoid. As a result, activation of c-Met signaling is significantly increased organ
specific extravasation of HCC cells and c-Met inhibitor SU11274 decreased extravasation rate
of HCC cells approximately 70% in total. Our data support that c-Met signaling could play role
in organ specific extravasation especially in intrahepatic metastasis and bone metastasis and
SU11274 could be used as anti-cancer drug to prevent metastasis especially to prevent HCC
and can be used for therapeutic purposes.
Keywords: HCC, c-Met, metastasis, extravasation
Oral Presentation
82
The Effects of Cancer Associated Fibroblasts on Tumor Associated Macrophages
Gurcan Gunaydin
Department of Basic Oncology, Cancer Institute, Hacettepe University, Ankara, Turkey
Introduction: Monocytes polarize into either M1 or M2 macrophages. Most macrophages in
tumor microenvironment display M2-like-features. Monocytes/macrophages demonstrated
plasticity and investigating the mechanisms underlying this plasticity will pave the way for
more effective and successful diagnostic/therapeutic approaches cancer treatment. Therefore,
the current study aims to shed light on the effects of cancer-associated-fibroblasts(CAFs) on
macrophage-differentiation in breast-cancer patients.
Materials/Methods: An-enzymatic-protocol utilizing Collagenase-I/Hyaluronidase was used
to obtain CAFs and normal-fibroblasts(NFs) from patients undergoing total-
mastectomy/reduction-mammoplasty. Surface-expressions of markers (e.g. α-smooth-muscle-
actin[α-SMA],vimentin), which differentiate CAFs/NFs, were analyzed with
immunocytochemistry. CAF/NF cultures were used to acquire conditionedmediums(CMs).
Peripheral-blood-mononuclear-cells were isolated from healthy-volunteers’ peripheralblood
via Histopaque-1077. CD14+ monocytes, CD4+ T-cells were isolated from PBMCs via
magneticseparation. Surface-expressions of CD206,CD163 were analyzed with flow-
cytometry. Functional-effects of CAF/NF-educated-monocytes on CD4+ T-lymphocytes were
demonstrated via CFSE proliferation assays with CD4+ T-cells (activated-with-CD3/CD28-
magnetic-beads). Transwell-migration-assays were utilized to demonstrate the alterations in
monocyte-recruitment due to CAFs/NFs. E-cadherin,vimentin protein expressions were
determined by Western-blot. In addition, alterations in MDA-MB-231 breastcancer- cell-
invasion due to CAF/NF-educated-monocytes were investigated with Transwell-inserts.
Results: Although CAFs expressed α-SMA;NFs did not. CAFs were able to potently recruit
monocytes. This recruitment may be mediated through Monocyte-chemotactic-protein-1(MCP-
1) or stromal-cellderived- factor-1(SDF-1) cytokines, since monocyte migration was
significantly reduced as a result of MCP- 1 or SDF-1 inhibition through MCP-1 or CXCR4(a-
chemokine-receptor-specific for SDF-1) blockingantibodies. CD163, CD206 (associated-with
M2-macrophages) expressions were higher in CAF-educatedcells than in NF-educated- cells. T
cell-mediated immune-responses were shown to be suppressed by CAFeducated- monocytes.
While CAF-educated-monocytes augmented breast-cancer-cell-invasion, NFeducated-
Oral Presentation
83
monocytes did not. Moreover, CAF-educated-monocytes increased vimentin
expression,decreased E-cadherin-expression in breast-cancer- cells. Last but not least, CAFs
differentiated M1-macrophages to M2-like-macrophages, as CD163-expression significantly
increased in M1-macrophages due to CAFs.
Discussion: CAFs polarized monocytes to M2-like pro-tumoral macrophages phenotypically
and functionally; whereas NFs did not. CAFs were shown to be very potent in recruiting
monocytes. MCP-1 and SDF-1, which are secreted from stromal-cells, may provide evidence
to be pivotal monocytechemotactic- cytokines.
Keywords: breast cancer, CAFs, monocytes, macrophages, oncoimmunology, tumor
microenvironment
Authors declare no conflict of interest.
Oral Presentation
84
Cryptonin: An Antimicrobial Peptide from Cicada and its Effects on Melanoma Cell
Lines
Gülşah Torkay, Serap Sancar-Baş
İstanbul University, Science Faculty, Biology Department, Molecular Biology Section, Vezneciler,
34134, Fatih, ISTANBUL
Introduction: Antimicrobial peptides are important part of the innate immune response and
synthesized by various organisms ranging from bacteria to mammals. These peptides
generally have 12-50 amino acids and most of them are positively charged. Accumulating
data have shown that antimicrobial peptides not only have effects on microorganisms but
also may have toxic effects on tumor cells. So, we can say that some of these peptides are
not only “antimicrobial” anymore. Since they have relatively high positive charge, they can
interact with tumor cell membranes which have anionic character like bacterial membranes.
Aim: In this study, we aimed to investigate the effects of cryptonin which is an antimicrobial
peptide defined in the hemolymph of Cicada, Cryptotympana dubia (Park et al., 2007) and
the anticancer activity has not studied before. Cryptonin has an alpha-helix structure in
aqueous solution, 24 amino acids in length, 50% hydrophobicity and +8 net charge. For these
reasons, cryptonin can bind to membrane bilayers to form pores and cause tumor cells to
death.
Methods: The anti-proliferative effects of cryptonin was studied with MTT test on metastatic
and non-metastatic melanoma cell lines and possible lytic effects were studied with lactate
dehydrogenase and HMGB1 release. The morphology of cell death type was studied with
acridine orange/ethidium bromide staining. Besides, reactive oxygen levels were measured.
Results: We detected that cryptonin decreased the cell viability in the melanoma cell lines in
time and dose dependent manner via damaging to cell membranes. This lytic effect leads to
releasing of lactate dehydrogenase and HMGB1 protein, which are the marks of necrotic cell
death. The ethidium bromide stained nucleus number is also increased in higher peptide
concentrations.
Discussion: Taken together, the antimicrobial peptide cryptonin may have therapeutic
potential for treatment of melanoma, especially in metastatic cell lines leading immediate
cell death.
Key-words: Cryptonin, Antimicrobial peptide, Melanoma, Necrotic cell death
Oral Presentation
85
Güneydoğu Anadolu Bölgesinde Yetişen Patlican Çeşitlerinin Kabuklarının Sitotoksik
Etkilerinin Saptanması
İbrahim Halil Kılıç, Işık Didem Karagöz, Başak Simitçioğlu, Sami Serhat Tonus, Bekir
Sıddık Kurt
Biyoloji Bölümü, Gaziantep Üniversitesi, Gaziantep
Günümüzde bazı bitki özütleri gıda sanayisinde kullanılmak üzere marketlerde görülmeye
başlamıştır. Bazı bileşiklerin antioksidan kapasitesinin, sentetik olanlardan daha yüksek olduğu
yine dünya nüfusunun çoğunluğu için bitkiler, ilaç hammaddesi olarak yaygın olarak
kullanmaktadırlar. Özellikle gelişmekte olan ülkelerde nüfusun % 80’i sağlık gereksinimlerini
ilk etapta geleneksel tıbbi bitkilerden sağlamaktadır. Tıbbi ilaç dışında bitkiler endüstri
sektöründe de oldukça önemli yere sahiptir. Gelişen ilaç sektörüne rağmen birçok hastalığa
çözüm bulunmamakla birlikte yeni etkin bileşik keşfi için araştırmalar hızla devam etmektedir.
Ülkemizin önemli sebzelerinden olan patlıcanın özellikle Güneydoğu Anadoluda bölgesinde
patlıcanın farklı çeşitleri üretilmekte ve tüketilmekte olup özellikle dolmalık çeşidi (kuru
dolmalık) tüm ülkemize bu bölgeden gönderilmektedir. Gaziantep’te yetişen Dolmalık patlıcan,
Kemer patlıcan, Nizip patlıcan ve Birecik patlıcanı olmak üzere dört çeşit patlıcan kabukları
soyulup kurutulmuş ve su,metanol,hekzan ve diklormetan özütleri elde edilmiştir. Özüt
verimleri hesaplanmıştır.
Sitotoksisitenin değerlendirilmesi amacıyla kulanılan enzimatik test yöntemlerinden biri olan
MTT testi ile belirlenmiştir. H1299 ve HUVEC hücre hatları kullanılmıştır. Özellikle Dolmalık
ve Kemer patlıcan çeşitlerinin Metanaol özütleri H1299 kanser hücreleri üzerine toksik etki
gösterirken sağlıklı HUVEC hücre hatları üzerine sitotoksik etkisi göstermemiştir. Seçici
sitotoksik etki saptanmış olması etken bileşiklerin saflaştırılmasına temel veriler
oluşturmaktadır.
Anahtar kelimeler: Dolmalık patlıcan, Nizip patlıcanı, Sitotoksite
Oral Presentation
86
How dose and duration of exposure affect the rate of acquired resistance to doxorubicin
in gastric cancer?
Hande Özkan1,2†, Gülnihal Özcan2,3*
1 Koc University Graduate School of Health Sciences, Istanbul, Turkey 2 Koc University Research Center for Translational Medicine (KUTTAM), Istanbul, Turkey
3 Koc University School of Medicine, Istanbul, Turkey
†Presenting Author *Corresponding author, e-mail: [email protected]
Chemotherapy is the mainstay for the treatment of numerous cancers. However, patients mostly
develop chemoresistance due to the presence of naturally resistant cancer cells within the tumor
mass or acquired resistance after chemotherapy. Therefore, there is an urgent need to develop
new therapeutic strategies that can overcome chemoresistance. Today’s chemotherapy
protocols are based on the administration of chemotherapeutics at maximum tolerated doses
with intermittent chemotherapy sessions. Although these protocols decrease the size of the
tumor at the short-term, remaining cancer cells adapt themselves to the presence of
chemotherapeutics, escape from their anti-cancer action and develop chemoresistant clones
which cause the regrowth of the tumor at later stages. Therefore, an increase in long-term
survival could not be achieved with this strategy. Recently it was suggested that doses below
maximum tolerated dose applied over a longer period of time may decrease the rate of acquired
resistance to chemotherapeutics and increase overall survival, despite a lower anti-cancer
efficacy at the short-term. To test this hypothesis in gastric cancer, we exposed gastric
adenocarcinoma cells to doxorubicin, one of the most potent and most commonly used
chemotherapeutics in cancer, with a high dose-intermittent protocol or low dose-continuous
protocol. Our preliminary studies suggest that, high doses of doxorubicin applied over a short-
term may induce a higher increase in resistance index compared to the application of lower
doses over a longer period of time. We also observed that a specific group of multi-nucleated
giant cells were selected by high dose-intermittent application and these cells replenished the
original cell population during the drug-free interval. On the other hand, the topology of the
initial cell population was mostly preserved with low dose-continuous exposure. These results
suggest that, low dose-continuous administration of doxorubicin might be tested as a strategy
to decrease rate of acquired resistance in gastric cancer.
Funding/Support: “The authors gratefully acknowledge use of the services and facilities of the Koç University Research Center for Translational Medicine (KUTTAM), funded by the Presidency of Turkey, Presidency of Strategy and Budget. The content is solely the responsibility of the authors and does not
necessarily represent the official views of the Presidency of Strategy and Budget.”
Oral Presentation
87
İNSAN MEME KANSERİ HÜCRELERİNDE D-erythro-MAPP’ın ANTİKANSER
ÖZELLİKLERİNİN ARAŞTIRILMASI
Hüseyin İzgördü1, Canan Vejselova Sezer1, Emre Çömlekçi1, Hatice Mehtap Kutlu1*
1Eskişehir Teknik Üniversitesi, Fen Fakültesi, Biyoloji Bölümü, Eskişehir
*Sorumlu yazar:[email protected]
Giriş ve Amaç: Kanser günümüzde öldürücü hastalıklar arasında ilk sıralarda yer almaktadır.
Kanser, tedavi sürecinde hastalara büyük zorluklar yaşatan ve tedavisi çok pahalı olan bir
hastalıktır. Meme kanseri erkeklerde ve kadınlarda görülen bir kanser çeşidir. Meme kanseri,
hem gelişmiş hem de gelişmekte olan ülkelerde tüm kanserler içinde üçüncü sırada yer
almaktadır. Kanser tanısı konduktan sonra hastaların hayatta kalmaları ortalama olarak 5 yıl ile
sınırlıdır. Bu sebeple ileriye dönük daha etkin tedavi biçimlerinin geliştirilmesine ihtiyaç
duyulmaktadır. Seramid, sfingozin ve sfingozin-1-fosfat; hücre proliferasyonunu ve apoptozu
kontrol etmekte görev alan sfingolipid metabolitleridir. Seramid ve sfingozin hücre ölümüne,
yaşlanmasına ve hücre döngüsünün durdurulmasına aracılık etmektedirler. Bu çalışmanın
amacı bir seramidaz inhibitörü olan D-erythro-MAPP’ın insan meme kanseri (MCF-7) hücreleri
üzerindeki potansiyel antiproliferatif, proapoptotik ve sitotoksik etkilerinin araştırılmasıdır.
Gereç ve Yöntemler: Bu çalışmada D-erythro-MAPP’ın insan meme kanseri hücreleri
üzerindeki sitotoksisitesi MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium
Bromide) kolorimetrik testi ile incelenmiştir. MTT deney sonuçlarından D-erythro-MAPP’ın
insan meme kanseri hücrelerin yarısını öldüren IC50 konsantrasyonu belirlenmiştir. D-erythro-
MAPP’ın neden olduğu hücre ince yapısal ve morfolojik değişiklikler geçirimli elektron
mikroskobu (TEM) ve konfokal mikroskopi yöntemi ile araştırılmıştır.
Sonuçlar: MTT sonuçlarından elde edilen verilere göre D-erythro-MAPP’ın MCF-7
hücrelerinin canlılığını 24 saatlik bir uygulama sonucunda doza bağımlı olarak düşürmüştür.
D-erythro-MAPP’ın MTT sonucuna göre IC50 değeri 4,4µM olarak bulunmuştur. Konfokal
mikroskopi ve TEM bulguları ise çekirdekte yapısal anomali, membranlarda tomurcuklanma,
kromatin yoğunlaşması, çekirdek ve hücre iskeletinde parçalanmalar gibi proapoptotik etkiler
ile bağlantılı olarak değerlendirilmiştir. Sonuç olarak D-erythro-MAPP’ın bu çalışma
sonucunda ciddi bir antikanser ajanı olma potansiyele sahip olduğu ortaya konulmuştur ve daha
ileri ve detaylı kanser araştırmalarına ihtiyaç vardır.
Anahtar Kelimeler: D-erythro-MAPP, MCF-7, TEM, Konfokal
Bu çalışma TÜBİTAK 118Z943 no’lu 1002 projesi ile desteklenmiştir.
Oral Presentation
88
Experimental traumatic brain injury models
Hüseyin Özevren
Departments of Neurosurgery, Dicle University School of Medicine, Diyarbakir, Turkey.
Head trauma is still a serious cause of morbidity and mortality. It has physical, psychological,
social and economic effects on societies. In spite of many diagnostic and therapeutic
approaches, the point reached is unfortunately still not at the desired level. Therefore, new
treatment approaches that can shed light on the treatment are very important for both the
problem and the head trauma patient. Experimental studies for head trauma are the most
appropriate experimental methods to investigate neuroprotective methods
The experimental model of traumatic brain injury was first made in the mid-20th century.
Denny-Brown and Russell divided experimental brain injury into acceleration and compression
in 1941; have used the term acceleration for blunt closed head trauma and the term compression
for penetrating injury of the skull. Over the years, several improvements have been made.
In the 1980s, there was a huge explosion in traumatic brain injury experimental research. In
parallel with the development of instruments used in cell and molecular biology, 3 new models
have been developed. These models; fluid percussion damage, controlled cortical effect, weight
reduction from high
It was accepted that the main animal used in experimental studies was rodents. They are used
because of their similarity to the human brain, easy to grow and cost-effective.
Therefore, new treatment approaches that can shed light on the treatment are very important for
both the problem and the head trauma patient. Experimental studies on head trauma are the most
appropriate experimental methods to investigate neuroprotective methods.
Keywords: Experimental, Neuroprotective. Traumatic brain injury
Oral Presentation
89
In vitro Self-renewal Properties in A549 and H1299 Non-Small Cell Lung Cancer Cells
Hiba H. A. Khair and Isik Didem Karagoz,
Department of Biology, Faculty of Art and Science, Gaziantep University, Gaziantep, Turkey
Lung cancer remains at the top of the list of cancers with the highest incidence and mortality
rates worldwide. The cancer stem cell paradigm proposes that a small subpopulation of cancer
cells called cancer stem cells (CSCs) can self-renew, recapitulate tumor heterogeneity by
differentiating into different lineages of cells that constitute the bulk of the tumor and
substantially lead to tumor recurrence. Accumulating evidence suggests that a small population
of stem-like cells are responsible for the initiation, progression, and metastasis of lung cancer.
This study aims to assess cancer stem-like properties in A549 and H1299 non-small cell lung
cancer cells. The spheroid formation assay was used to assess self-renewal ability indicated by
tumorspheres formation and to calculate sphere-forming efficiency in vitro. The limiting
dilution assay was used to calculate the minimum number of cells needed to be cultured to see
at least one spheroid in vitro. Cells derived from both cell lines could form tumor spheroids.
The sphere-forming efficiencies of A549 and H1299 cells were 4.14 ± 1.06% and 5.81 ± 1.28%,
respectively. However, H1299 cells produced more and larger tumorspheres when compared to
A549 cells. The mean diameter of tumor spheroids in H1299 and A549 cells were 95.25 ± 41.03
and 80.70 ± 31.72 μm, respectively (p=0.00003). In limiting dilution assay, A549 and H1299
single cells produced spheroids at 23.5 ± 43.7% and 62.5 ± 49.4%, respectively. The frequency
of sphere-forming cells (stem-like cells) was found to be 6.06-fold higher for H1299 cells than
those for A549 cells. These results suggest that the self-renewal ability of H1299 cells is higher
and more pronounced compared to A549 cells. As a conclusion, both A549 and H1299 cells
comprise stem-like cells with self-renewal ability.
Key words: Lung cancer, Cancer stem-like properties, Self-renewal.
Oral Presentation
90
IMMUNOHISTOCHEMICAL DETERMINATION OF GLUTATHIONE-S-
TRANSFERASE ISOENZYM FAMILY EXPRESSION IN INVASIVE LOBULAR
BREAST CANCER TISSUE
Işıl Yıldırım1, Bilge Aydın Türk2, Pınar Kaygın3, Serpil Oğuztzüzün3
1Beykent University, Vocational School, Pharmacy Services Program, Biochemistry Area, 34500
İstanbul, Turkey,
2Adıyaman University, Faculty of Medicine, Department of Pathology, 02100, Adıyaman, Turkey
3Kırıkkale University, Faculty of Science, Department of Biology, 71450, Kırıkkale, Turkey
Corresponding author email: [email protected]; [email protected]
Aim: GST isozymes are present in a variety of normal and malignant human tissues including
breast carcinomas. The aim of the present study was to evaluate the association between GSTM,
GSTA1, GSTP1 of invasive lobular breast cancers tissues.
Method: Breast biopsy specimens from at least 14 invasive breast lobular carcinoma women
whose diagnosis and treatment protocols were determined between 09.07.2007 - 01.12.2018
were used at Adiyaman University Medical Pathology Department by Ethics Committee with
resolution of 2019 / 1-28. The protein expression of GSTM, GSTA1, GST1 in breast carcinoma
cells, determined by immunohistochemistry. GST was correlated with clinical like age and
several pathological parameters of prognostic significance including tumor size, nodal status,
estrogen receptor protein positivity, or progesterone receptor protein positivity.
Results: When pathologic diagnosis and histological gland conditions were considered for
GSTM1 protein, invasive lobular carcinoma was found protein staining grade +1 for right breast
tissue, in normal tissue and in tumor tissue was 16.6 %, 50 %, respectively. In both invasive
lobular carcinoma breast right and left, +3 staining was 33.3 %. GSTA1 protein was not
expressed for both histological glands. GSTPi protein expression invasive lobular carcinoma
for both right and left was found 66.66 % and 33.3 % for staining +1 degree. Since P > 0.05
right and left breast carcinoma, GSTM1, GSTA1 and GSTPi were not statistically different in
the degree of staining. Mean age for invasive lobular breast carcinoma right gland 59.85±17.61,
while mean age for invasive lobular carcinoma left breast 49±5.38. Tumor seize for invasive
lobular carcinoma right gland varies between 0.1 cm and 2.5 cm according to biopsy material,
but tumor size of left glands was found 0.2 cm and 5.4 cm. Estrogen receptor 62 % ± 0.36 for
invasive lobular carcinoma left glands, but for left glands was 68%±0.45.
Oral Presentation
91
KARADUTUN (Morus nigra) ANTİ KANSER ETKİLERİNİN ARAŞTIRILMASI
Semih Dalkılıç, Sevda Kırbağ, Lütfiye Kadıoğlu Dalkılıç, İsmail Korkmaz, Şule İnci
Fırat Üniversitesi, Fen Fakültesi, Biyoloji Bölümü, Moleküler Biyoloji ve Genetik Programı, ELAZIĞ
Kanser tüm dünyada oldukça yaygın olarak görülen bir hastalıktır. Dünya Sağlık Örgütü’nün
verilerine göre kanserin görülme sıklığı her yıl daha da artmaktadır. Yapılan tahminlere göre
2030 yılına gelindiğinde 22 milyon yeni kanser vakasının ortaya çıkması beklenmektedir.
Günümüzde özellikle tıp alanında, diagnostik ve tedavi yöntemlerinde elde edilen ilerlemeler
sayesinde kanser tedavisinde önemli başarılar elde edilmiştir. Ancak tüm bu gelişmelere
rağmen özellikle bazı kanser türlerinde elde edilen başarı tatmin edici değildir. Yeni ilaç hedefi
moleküllerin tanımlanması ve daha etkili ilaçların geliştirilmesi için yoğun çalışmalar
yapılmaktadır.
Bunlara ilave olarak bazı doğal maddelerin kanser tedavisinde etkili olabileceği ileri
sürülmektedir. Bu tür uygulamalar neredeyse insanlık tarihi kadar eskidir ve günümüzde bile
uygulanmaktadır ve alternatif tıp veya tamamlayıcı tıp olarak adlandırılmaktadır.
Halk arasında da özellikle doğal yoldan elde edilen kırmızı meyveler şifalı olarak
görülmektedir. Bu çalışmada amacımız kırmızı meyvelerden biri olan karadutun (Morus nigra)
anti kanser özelliğinin incelenmesidir.
Doğal yollardan elde edilen karadut (Morus nigra) meyvesinin, anti kanser etkisi, meme kanseri
(MDA MB 231) ve prostat kanseri (PC3) hücre hatları kullanılarak, MTT (3- (4,5-dimetiltiazol-
2-il) -2,5-difeniltetrazolyum bromür) Assay ile test edildi. Karadut (Morus nigra) meyvesinin
ekstresi kanser hücre hatları üzerine 1/10, 1/25, 1/50, 1/75 ve 1/100 oranlarında 72 saat süreyle
uygulandı.
Karadutun (Morus nigra) farklı konsantrasyonlardaki ekstresinin, prostat ve meme kanser
hücrelerinin canlılığını farklı oranlarda etkilediği gözlenmiştir.
Anahtar Kelimeler: Karadut, Ekstrakt, Meme kanseri, Prostat kanseri, MTT
Oral Presentation
92
The Investigation Of Anticancer Effect Of Cedar Tar Prepared By Traditional Methods
İsmail Koyuncu, Kadir Eği, Ebru Temiz, Ataman Gönel
Department of Medical Biochemistry, Harran University, Sanliurfa, Turkey
Cancer is one of the leading causes of morbidity and mortality worldwide and it ranks first after
cardiovascular diseases. Colon adenocarcinoma is the most common cancer of gastrointestinal
system, although it is seen in different populations in different populations. Various methods
such as chemotherapy, radiotherapy, surgical methods, immunotherapy, gene therapy and
alternative therapy are applied together or separately in cancer treatment. Chemotherapy is the
most commonly used method. The biggest handicap of chemotherapy is the destruction of
normal cells while killing cancerous cells. Therefore, there has now been a trend towards plant-
based drugs to provide a more effective treatment for cancer with minimal toxicity. In this study;
the Toros Mountains in Turkey (Tarsus) is grown and used for centuries among the people,
from the cedar trees have been viewed etnofarmosötik feature anticancer activity of tar obtained
by conventional methods. In this study, the cytotoxic effect of cedar tar HCT-116 (colon cancer
cell line) and HUVEC (human normal cell line) cells was determined using MTT Assay method.
At the end of 24 hours, the cytotoxic effect (IC50) on colon cancer was calculated to be 30
µg/ml, whereas the effect on normal cells was approximately twice as high.
Apoptotic effect was examined by Annexin-V, Cell Cycle, JC-1, ROS method. Cedar tar HCT-
116 cells showed apoptotic activity by disrupting mitochondral membrane potential.
Morphological images were also determined by Acridine orange / Ethidium bromide staining
methods. The effect of cedar tar on cell cycle was observed in 70% of G0-G1 phase. In addition,
apoptotic effect correlated with ROS increase.
In this study, as a result of the data obtained by examining some chemical and biological activity
properties of tar, it will be determined whether there will be a new herbal medicine candidate
for cancer treatment.
Keywords: Tar, Cytotoxicity, Cell Death, Colon Cancer, HCT-116
Oral Presentation
93
MİDE KANSERİNİN TEŞHİSİNDE KULLANILABİLECEK BİYOBELİRTEÇ
ADAYI GENLERİN BİYOİNFORMATİK ARAÇLAR İLE BELİRLENMESİ
Semih Dalkılıç, Lütfiye Kadıoğlu Dalkılıç
Fırat Üniversitesi Fen Fakültesi Biyoloji Bölümü Moleküler Biyoloji ve Genetik Programı
Geçtiğimiz yüzyıl süresince insidansında bir azalma olsa da mide kanseri, kanserle ilişkili
ölümlerde ikinci sırada yer almaktadır. Özellikle doğu Asya’da en yaygın kanser türüdür (1).
Yapılan bu çalışmada National Center for Biotechnology Information (NCBI) Gene Expression
Omnibus (GEO) veri tabanında bulunan GSE54129 kodlu veri seti kullanılmıştır(2). Bu veri
seti içerisinde 111 adet Mide kanseri tümör dokusu örneği, 21 adet normal mide dokusu
kullanılarak genom boyu gen ifade analizi yapılmıştır.İfade analizi Affymetrix Human
GeneChip 133 Plus 2.0 (HG-U133 Plus 2.0) mikrodizinlerine hibridize edilmiştir. İşlenmemiş
gen ekspresyon verisi (CEL dosyaları) bu veritabanından alınarak biyoinformatik analizler
yapılmıştır. Tüm analizler Applied Biosystem’e ait olan Transcriptome Analysis Console 4.0
(TAC) ile yapılmıştır.
Analiz sırasında öncelikle verinin kalite kontrolü ve önişlem (preprocessing) analizleri
yapılmıştır. Önişlemler, görüntü analizi (Image Analysis), arkaplan düzeltmesi (Background
Correction), normalizasyon (Normalization) ve özetleme (Summarization) adımlarından
oluşmaktadır. Bu işlemlerden sonra bu veri üzerine varyans filtreleme uygulanmıştır. Varyans
filtreleme işleminden sonra ise kanser ve kontrol grubu arasında karşılaştırma yapılarak bu iki
grup arasında ifade düzeyi farklılık gösteren genler (DEG) belirlenmiştir. Tüm bu analizler
RMA (Robust Multi-Array Average) metodu ile gerçekleştirilmiştir. Yalancı pozitif sonuçların
benzerliğini düşürmek için ham p değerleri üzerinden Benjamini-Hochberg düzeltmesi
yapılmıştır. Daha sonra p<0.05 için kat değişimi (log2 fold change, FC > 5) olacak şekilde bir
cut-off değeri belirlenmiştir.
Elde edilen farklı ifade edilen gen listesi daha sonra DAVID (The Database for Annotation,
Visualization and Integrated Discovery v6.8) veritabanına yüklenerek gen zenginleştirme
(GeneSet Enrichment) ve fonksiyonel kümeleme analizleri (Functional Annotation Clustering)
yapılmıştır. Bu veritabanına entegre olan KEGG (Kyoto Encyclopedia of Genes and Genomes)
veritabanı üzerinden de yolak analizleri yapılmıştır.
Elde edilen sonuçlara göre mide kanserinde neoplastik transformasyon sırasında bir çok genin
ifade düzeyi sağlıklı kontrol bireylerle kıyaslandığı zaman anlamlı düzeyde değişmektedir. Elde
Oral Presentation
94
edilen bu sonuçlara göre bu genler mide kanserinin teşhis edilmesinde veya moleküler
tiplendirmesinde kullanılabilecek biyobelirteç adayı olabilecek genlerdir.
Anahtar Kelimeler: Mide kanseri, Gen ifadesi, Biyoinformatik, Biyobelirteç
Oral Presentation
95
L-Askorbik Asit’in (Vitamin C) İnsan Promiyelösitik Kanser Hücre Hattında (HL-60)
MTT Testi ile Araştırılması
Mehmet Tahir Hüsunet1, Mehmet Bertan Yılmaz2, Hasan Basri İla1
1Çukurova Üniversitesi Fen Edebiyat Fakültesi Biyoloji Bölümü Adana/Türkiye;
2Çukurova Üniversitesi Tıp Fakültesi Tıbbi Biyoloji Bölümü Adana/Türkiye
Amaç: L-Askorbik Asit (LAA) Proliferatif Etkisinin İnsan Promiyelösitik Kanser Hücre
Hattında (HL-60) MTT Testi ile Araştırılması
Giriş: L-Askorbik Asit insanlar tarafından sentezlenmeyen, takviye diyet ile dışardan
alınan ve antioksidan özellik gösteren bir maddedir. LAA, birçok meyve ve sebzenin
yapısında bulunmaktadır. LAA içeren gıdalar aynı zamanda antioksidan özellikler de
gösterebilmektedir. Çalışmamızda LAA’nın HL-60 hücre hattı üzerindeki proliferatif
etkisine bakıldı. Bu amaçla LAA konsantrasyonları 0,5 mg/mL, 1 mg/mL ve 2 mg/mL
olarak belirlendi ve MTT testi yapıldı. Buna göre; LAA’nın, HL-60 insan promiyelositik
lösemi hücre hattında 570 nm’deki absorbans sonuçları incelendiğinde doza bağlı bir
proliferasyon artışı görüldü. 0,5 mg/mL ve 1 mg/mL dozları kontrol düzeyinde artışa neden
olurken en yüksek doz (2 mg/mL) hücre canlılığını kontrole göre çok önemli düzeyde
(p<0,001) artığı görüldü.
Materyal-Metod: HL-60 hücre hattının %5 CO2, 37 oC sıcaklık ve nemli ortamda hücre
kültürü yapılmıştır. RPMI-1640 besi ortamı kullanılarak 24 saatlik kültür yapılmıştır.
Kültürün 20. saatinde MTT maddesi verilerek kültür 24. saatte sonlandırılmıştır. Daha sonra
spektrofotometrede 570 nm absorbans değerleri alınarak sonuçlar değerlendirilmiştir.
Sonuç: En yüksek dozun (2 mg/mL) HL-60 insan promiyelositik lösemi hücre hattını ciddi
oranda bölünmeye teşvik ettiği görüldü (p<0,001). Bu durumun daha iyi anlaşılabilmesi için
ileri düzey moleküler çalışmalara ihtiyaç duyulmaktadır. Moleküler çalışmaların MTT testi
ile paralel sonuçlar vermesi durumunda kanser tedavisi gören insanlara antioksidan
içermeyen diyetler tavsiye edilebilecektir.
Oral Presentation
96
Investigation of TMED9-ERAP1 Candidate Gene Expressions Obtained from Multiple
Myeloma Transcriptome Data by RT-PCR
Melda Sarıman1,2, Büşra Karaçam1, Mesut Ayer3, Sema Sırma Ekmekci1, İlknur Suer4, Kıvanç Çefle4, Şükrü Palanduz4, Şükrü Öztürk4, Neslihan Abacı1
1Department of Genetics, Istanbul University, Aziz Sancar Institute of Experimental Medicine,
Istanbul, Turkey 2Molecular Cancer Research Center, Istinye University, Istanbul, Turkey
3Clinic of Hematology, İstanbul Haseki Training and Research Hospital, İstanbul, Turkey 4Department of Internal Medicine, Division of Medical Genetics, İstanbul University İstanbul School
of Medicine, İstanbul, Turkey
Multiple Myeloma (MM) in the plasma cell dyscrasias group is characterized by transformation
of B cells into malignant plasma cells originating from bone marrow. Monoclonal Gammopathy
of Undetermined Significance (MGUS) is an asymptomatic premalignant plasma cell disorder
to be characterized by the presence of serum M-protein. The expression levels of genes were
investigated in our previous study by RNA-Sequencing from cell pools of MM patients and
healthy bone marrow controls. MM group was compared with healthy bone marrow donor
group over RPKM values and candidate genes with the highest expression in MM group were
determined by filtering with control group. These genes were analyzed by molecular properties
of the genes determined by using bioinformatics tools such as Gene Set Enrichment Analysis
(GSEA), MSGDB (Broad Institute), Panther, and String databases, and biological pathways
within the cell, and the pathways and protein-protein interactions were analyzed in silico.
Candidate genes obtained from transcriptome data are very valuable and specific, and their
direct or indirect relationships of these genes have been revealed and this study was aimed to
contribute to the elucidation of MM pathogenesis.
In our study, bone marrow materials from 30 newly diagnosed, untreated MM patients and from
11 MGUS and 9 healthy bone marrow donor groups were used. Expression levels of the
candidate genes (TMED9 and ERAP1) that we identified previously were examined by qRT-
PCR method. The results of MM group, the expression of TMED9 and ERAP1 genes were
found to be higher than that of MGUS and healthy bone marrow donor group.
According to these results, we think that ERAP1 and TMED9 genes in MM will directly or
indirectly contribute to the elucidation of the pathogenesis of the disease.
Keywords: Multiple Myeloma, Bioinformatic, qRT-PCR, TMED9, ERAP1
This work was supported by the Scientific Research Projects Coordination Unit of Istanbul University
(grant number 30846).
Oral Presentation
97
The Effects Of The Lıf Antagonıst Molecule (Ec359) On The Chordoma Cells
Melike Bayindir Bilgic, Nur Ekimci Gurcan, Utku Ozbey, Aysegul Kuskucu, Omer Faruk
Bayrak Yeditepe University, Institute of Biotechnology, Istanbul
Introduction: Chordoma is a type of malignant bone cancer observed on the skull base, axial
skeleton and around the rump-bone, which is thought to originate from ectopic-notochord
residues in the embryonic period (1). Chordomas are resistant to conventional chemotherapy
and radiotherapies as well as slow growth. Total block resection is usually used as the first
choice in the treatment of chordoma. In addition, radiotherapy is among the treatment methods
used (2, 3). Resistance to chemotherapy, recurrence and metastasis capacity make it difficult to
treat chordoma. Therefore, new treatment foci and strategies targeted for development are of
great importance for chordoma. Our study group has previously demonstrated that Leukemia
Inhibitory Factor (LIF) molecule increases chordoma aggressiveness (4). EC359 is a LIF
antagonist molecule. The EC359, LIF antagonist has been shown to inhibit a wide variety of
tumor types, including tumors resistant to standard therapies, including NSCLC, breast, ovary,
colon and pancreas. In this study, the efficacy of EC359 on chordoma cells was investigated.
Materials and Methods: The EC359 molecule was dissolved with DMSO. The EC359
solution prepared at different concentration values was treated with 6 different chordoma cell
lines (U-CH1, U-CH2, Um-CHOR1, Mug-CHOR1, JHC7, YU-CHOR1). After 8 days of study,
viability was determined by 8th day MTS assay.
Results: During the eight-day experimental period, viability of chordoma cells treated with
EC359 decreased compared to the control group. This significant decrease in viability of EC359
treatment in chemotherapy / radiotherapy resistant chordoma was found to be worth further
research. Therefore, Westernblot and qPCR investigations of the samples will also be carried
out to clarify the mechanism in downstream pathways.
(1) Chugh R, Tawbi H, Lucas DR, Biermann JS, Schuetze SM, Baker LH. Chordoma: The
Nonsarcoma Primary Bone Tumor. The Oncologist. 2007 Jan 11;12(11):1344–50.
(2) Bailey CS, Fisher CG, Boyd MC, Dvorak MFS. En bloc marginal excision of a multilevel cervical
chordoma: Case report. Journal of Neurosurgery: Spine. 2006 May 1;4(5):409–14.
(3) Carrabba G, Dehdashti AR, Gentili F. Surgery for clival lesions: open resection versus the expanded
endoscopic endonasal approach. Neurosurgical Focus. 2008 Dec 1;25(6):E7.
(4) Gulluoglu, S., Sahin, M., Tuysuz, E. C., Yaltirik, C. K., Kuskucu, A., Ozkan, F., ... & Bayrak, O. F.
(2017). Leukemia inhibitory factor promotes aggressiveness of chordoma. Oncology Research
Featuring Preclinical and Clinical Cancer Therapeutics, 25(7), 1177-1188.
Oral Presentation
98
Investigation of Apoptotic Effect of Betulinic Acid in Renal Cancer Cells
Merve Nur Ataş1, Barış Ertuğrul1, Elif Sinem İplik2, Bedia Çakmakoğlu1, Arzu Ergen1
1Istanbul University, Aziz Sancar Institute of Experimental Medical, Department of Molecular
Medicine, Istanbul, Turkey 2Istanbul Yeni Yuzyil University, Faculty of Pharmacy, Istanbul, Turkey
E-mail: [email protected]
Kidney cancer or renal cancer is the fourteenth most frequent cancer in the world. With its
increasing incidence in the last few years, the renal cell carcinoma is the most common kidney
cancer type. Apart from the environmental factors, increasing activities of expressions of genes
and/or their pathways make cells become cancerous. AKT-1 and mTOR promote cell growth,
proliferation and survival, participate in cellular homeostasis and have ability to suppress
apoptosis in various ways.
Betulinic acid which is a lupane-type pentacyclic triterpenoid is derived from various plants.
Betulinic acid exhibits wide range biological activities such as anti-inflammatory, anti-viral,
anti-bacterial and particularly anti-cancer activities. It is known that betulinic acid promotes
apoptosis in cancer cells along with no toxic effect in normal cells. This effect makes betulinic
acid a potential anti-cancer drug. In our study, clear cell renal carcinoma cell line CAKI-2 and
healthy cell line MRC-5 are used to research the effect of betulinic acid on apoptosis and gene
expression levels. In order to identify the toxic effect of betulinic acid, WST-1 and to detect
apoptosis Annexin-V were conducted. Expression analysis of AKT-1 and mTOR genes were
conducted with Real-Time PCR to measure the effect of betulinic acid in gene expressions
levels.
As a result of this study, the apoptotic activity of betulinic acid on kidney cancer cell line was
detected with Annexin-V. In gene expression analysis, there was statistically significant
decrease in AKT-1 expression level while mTOR expression level was increased. Betulinic acid
with its apoptotic effect on renal cancer cell line and non-toxic effect on normal cell line is a
potential anti-cancer drug promising for future studies.
Keywords: Betulinic acid, renal cancer, apoptosis
Acknowledgment: The present work was supported by the Research Fund of Istanbul University.
Project No: 31565
Oral Presentation
99
Selective cannabinoid-1/2 receptor (CB1 and CB2) agonists suppress cell proliferation and clonogenicity in pancreatic and breast cancer cells
Nergiz Hacer Turgut1, Mumin Alper Erdogan1
1Izmir Katip Celebi University, Faculty of Pharmacy, Department of Pharmacology, Izmir,TURKEY 2Izmir Katip Celebi University, Faculty of Medicine, Department of Physiology, Izmir,TURKEY
Introduction/Aim: Cancer ranks first among the causes of morbidity and mortality all over the
world and is expected to continue to be the main cause of death in the coming years. Therefore,
novel molecular targets and therapeutics strategies are urgently needed. In many cases, some
reports show that levels of endocannabinoids and their receptors are increased in cancer, a
situation that frequently correlates with tumor aggressiveness. Recent studies have suggested
that cannabinoid-1/2 receptors contribute to the tumor growth in a variety of cancers including
pancreas, colon, prostate and breast cancer. Understanding how cannabinoids are able to
regulate essential cellular processes involved in tumorigenesis, such as cell proliferation and
cell death are crucial for improving existing and developing new therapeutic approaches for
cancer patients. In this study, we investigated the impact of CB1-2 receptor agonists on cell
proliferation and clonogenicity in pancreatic(PANC1) and breast(MDA-MB-231) cancer cells.
Material/Methods: The impact of CB1/2 receptors on cell proliferation and clonogenicity was
investigated using selective CB2-agonist-L-759633, CB1-agonist-ACEA and CB1-agonist-
ACP A in our cancer cells, utilizing MTS and colony formation assay, respectively.
Mechanisms of these cell behaviours are also under investigating by Western blot.
Results: CB1/2 receptor agonists led to a significant decrease in proliferation and colony
formation in all cells with the doses of 1, 10, 50, 100, 250µM for 72 h and 14 days, respectively.
Based on our findings, these agonists led to the inhibition of both cell viability and clonogenic
growth as a dose dependent manner. Our first western blot data is also suggesting that CB1/2
agonists may inhibit proliferation and tumorigenesis through the upregulation of apoptotic
proteins in these cells.
Conclusion: Our data suggests that CB1/2 agonists have the therapeutic potential through the
inhibition of survival of these cancer cells and also might be linked with further cellular
mechanisms for the prevention.
Keywords: Cannabinoids, Cancer, CB1/2 Receptor, Agonist, Proliferation, Clonogenicity, Pancreatic
Cancer, Breast Cancer, Neuroblastoma
Oral Presentation
100
Usefulness of simple prognostic markers of complete blood count to Predict lymph node
metastasis in colon carcinoma
Murat Ferhat Ferhatoğlu
Istanbul Okan University, Faculty of Medicine, Department of General Surgery
Background: According to estimates from the World Health Organization (WHO) in 2015,
cancer is the first or second leading cause of death before age 70 years in 91 of 172 countries.
Cancer incidence and mortality are rapidly growing worldwide. With rapid population growth
and aging worldwide, the rising prominence of cancer as a leading cause of death partly reflects
marked declines in mortality rates of stroke and coronary heart disease, relative to cancer, in
many countries. According to GLOBOCAN 2018 data, colon cancer is the fourth most
commonly diagnosed type of cancer and the sixth most common cause of cancer deaths. Lymph
node metastasis is common in patients with colorectal cancer. And, it is a known fact that lymph
node metastasis in patients with colon cancer is an indicator of a poor prognosis. Complete
blood count is a laboratory test frequently used in clinical practice and comprises white blood
cell, red blood cell and platelet counts, and their morphological indices. In this study, we tried
to asses the association between the parameters of complete blood count and lymph node
metastasis in colon cancer.
Materials and Methods: This is a retrospective cohort study conducted in a state hospital from
January 2010 to December 2015. One hundred and five colon carcinoma patients following an
elective colon surgery were enrolled in the study. Pathology results and preoperative complete
blood count parameters were collected.
Results: There were significant positive correlations between leukocyte, lymphocyte,
neutrophil measurements and metastatic lymph node positive cases (0.001, 0.02, 0.014;
respectively).
Conclusion: We found a positive correlation between leukocyte, lymphocyte, neutrophil
measurements and metastatic lymph node positive cases.
Oral Presentation
101
Characterization of BRI3 as a Novel Wnt/β-catenin Pathway Target
İzzet Akiva, Necla Birgül İyison Department of Molecular Biology and Genetics, Boğaziçi University North Campus Kpark Building,
Bebek, Istanbul, Turkey
Background: The Wnt/β-catenin signaling is an evolutionary conserved pathway which has
important functions in vertebrate early development, axis formation, cellular proliferation and
morphogenesis. The activation of this pathway leads to translocation of the transcriptional
activator β -catenin into the nucleus where it activates T-cell factor/Lymphoid enhancer factor
(Tcf/Lef) family of transcription factors, which regulate expression of developmental and cell
cycle-related genes. Apart from its roles in various cellular processes, Wnt/β-catenin signaling
pathway is also one of the most important intracellular pathways for cancer progression. A
significant number of identified target molecules of this pathway, are known to have
tumorigenic characters.
Objective: Previous studies confirmed BRI3 gene to be one of the transcriptional target genes
of this pathway, but it has not been associated with cancer up to now and its function remained
largely unknown. Further characterization of this gene in order to elucidate its biological roles
and eventual implications in cancer, is one of the main objectives of this study.
Methods: Overexpression studies in Huh7 cells, Luciferase Reporter Assay, Western Blotting,
Quantitative Real-Time PCR Analysis, Cell Proliferation and Cell Migration Assay, Xenograft
Assay in SCID mice, RNA Isolation from tumor tissues and RNA-Sequencing.
Results: Functional characterization of this novel Wnt/β-catenin pathway target has been
carried out by using both in vitro and in vivo techniques. BRI3 is found to be upregulated in
response to TNF-α treatment and overexpression of BRI3 results in an increase in NFkB
promoter activity. Cell proliferation and migration assays show that, Huh7 cells stably
expressing BRI3 gene have greater proliferative and invasive capabilities compared to control.
Furthermore, in vivo xenograft experiments show that stable overexpression of this gene in
Huh7 cell lines results in significantly larger tumor sizes in SCID mice. In order to determine
the possible interacting pathways in this tumorigenesis process, RNA-Seq analysis was carried
out from BRI3 expressing tumors and control tumors from SCID mice.
Acknowledgements: This study is supported by funding from TUBITAK - 108T183 and BAP (Boğaziçi
University Research Projects) 08B101.
Keywords: Wnt/β-catenin pathway, BRI3, Xenograft, RNA-Sequencing.
Oral Presentation
102
Sub-Culturing Tumorspheres Reduced the Pluripotency of a Chordoma Cell Line
Nur Ekımcı Gurcan1,2, Melike Bayındır Bılgıc1,2, Utku Ozbey1,2, Aysegul Kuskucu2, Omer
Faruk Bayrak2
1 Yeditepe University, Institute of Natural and Applied Sciences, Department of Biotechnology
2 Yeditepe University, School of Medicine, Department of Medical Genetics
Cancer is a result of accumulated mutations altering pathways that control cellular growth,
division and apoptosis. Cancer stem cells (CSCs) are a minor subpopulation within tumors that
have self-renewal and differentiation potential which provides the cancer enhanced resistance
capacity to cellular stress, therapeutic agents and radiation. Studies suggest that invasion and
metastasis capabilities of cancer cells are driven by increased epithelial mesenchymal transition
(EMT) status and associated with CSC phenotype. CH22 is a Chordoma cell line, which is a
rare, malignant bone cancer with poor prognosis. Chordoma cells are highly resistant to
chemotherapeutic drugs. Tumorsphere formation model is a known CSC enrichment method
based on culturing and eliminating non-stem like cancer cells on an ultra-low attachment
surface with specific culture medium conditions. Surviving cancer cell subpopulations are the
ones that exhibit higher stem-like characteristics. However, tumorsphere formation protocols
differ whether they suggest a repetitive sub-culturing and sphere formation or termination of
the experiment with the initial spheres.
In this study, parental CH22 cells, first generation tumorspheres achieved from CH22 cells and
sub-cultured tumorspheres are compared based on pluripotency, EMT and MET related genes
expression changes via qPCR.
Results revealed that sphere formation increases pluripotency markers OCT4, SOX2, Nanog,
KLF4 and c-Myc comparing parental CH22 cell line, but sub-cultured sphere formation
decreases that elevated expression levels. MET markers E-Cad, Cytokeratin 19 and Muc1 are
not elevated in both sphere groups as expected. Surprisingly, some of the EMT related genes
N-Cad, Twist, Snail and Slug are not altered significantly with sphere formation assays.
However, ZEB2 and fibronectin increased with sphere formation and sub-culturing spheres lead
an additional increase in these genes.
This study suggests that sub-culturing tumorspheres may reduce stem-like properties, instead
of CSC enrichment.
Oral Presentation
103
Beyin Tümörü Dokularında CYP1A1, CYP1B1, GST-M, GST-P ve MDR Proteinlerinin
Rolü
Onur Dirican1, Pınar Kaygın1, Serpil Oğuztüzün1, Gülçin Şimşek3, Yusuf İzci4, Cahit Kural4, Tülay Çoban5, Sezen Yılmaz Sarıaltın5, Oğuz Kul2, Işıl Yıldırım6
1Kırıkkale Üniversitesi, Fen Fakültesi, Biyoloji Anabilim Dalı, Kırıkkale,Türkiye.
2 Kırıkkale Üniversitesi, Veteriner Fakültesi, Patoloji Anabilim Dalı, Kırıkkale,Türkiye.
3 Sağlık Bilimleri Üniversitesi, Keçiören Eğitim ve Araştırma Hastanesi, Tıbbi Patoloji Anabilim Dalı,
Ankara,Türkiye.
4Sağlık Bilimleri Üniversitesi, Gülhane Eğitim ve Araştırma Hastanesi, Beyin Cerrahisi Anabilim
Dalı, Ankara, Türkiye.
5 Ankara Üniversitesi, Eczacılık Fakültesi, Farmasötik Toksikoloji Anabili Dalı, Ankara, Türkiye.
6 Beykent Üniversitesi, Eczane Hizmetleri Meslek Yüksekokulu,Eczane Hizmetleri Bölümü, İstanbul,
Türkiye.
Beyin tümörü dokularında, immunohistokimyasal yöntemle yapılan bu çalışmada; CYP1A1,
CYP1B1, GST-M, GST-P ve MDR protein ekspresyonları açısından değerlendirilmiştir.
Dokuların elde edildiği hastaların yaş, cinsiyet, alkol kullanımı, sigara kullanımı, tümör
lokalizasyonu, metastatik durumları, kemoterapik veya radyoterapik tedavi alma durumları ve
sağ kalım oranları göz önünde bulundurularak, karşılaştırmalı olarak ortaya konulması
amaçlanmıştır. Çalışma 2017-2019 yılları arasında tümör teşhisi ile beyin cerrahisi bölümü ve
kliniğine başvuran 141 hastadan cerrahi müdahale ile elde edilen ve incelenmesi için patoloji
bölümüne iletilen taze dokuların, immunohistokimyasal metod ile ekspresyon durumları
skorlanarak (0=ekspresyon yok, 1=zayıf ekspresyon, 2=güçlü ekspresyon) değerlendirilmeye
alınmıştır. Hastalardan alınan verilerde, ilgili parametrelere bağlı olarak; 83'ü (%58,9) erkek,
58'i (%41,1) kadındı. Tüm yaşlar için yaş ortalaması 49.44 (6-83) idi; kadınlarda ortalama yaş
50.25 (6-77) ve erkeklerde ortalama yaş 48.91 (11-83) idi. Hastaların 44'ü (%31,2) sigara
içiyordu ve 15'i (%10,6) alkol kullanıyordu. Hastaların 52'sine (%36,9) radyoterapi, 30'una
(%21,3) kemoterapi uygulandı. Çalışmanın sonunda 94 (%66.7) hasta yaşıyordu.
İmmunohistokimyasal metod sonrası kullanılan protein belirleyicilere bağlı olarak boyama
yoğunluğu istatistiksel olarak değerlendirildiğinde; CYP1A1 için; tümör (%6,7) ve normal
(%5,4) dokuları arasında ekspresyon paternleri açısından benzer bulundu (p=0.627). CYP1B1
için ekspresyon oranı tümörlü dokularda 4 kat gibi yüksek bir oranda gözlenirken, tümör
(%41,6) ve normal (%9,4) dokuları arasında oldukça önemli ekspresyon seviyesi farkı görüldü
(p<0.0001). Tümör dokularındaki GST-M ve GST-P ekspresyonu, normal dokularda ki
ekspresyonundan neredeyse 3 kat daha yüksek olduğu belirlendi (GST-M ve GST-P
ekspresyonları için; p<0.0001). Tümör dokularında, normal dokularla karşılaştırıldığında daha
yüksek MDR ekspresyonu saptandı. Tümör dokularının 65'i (%43.6) zayıf ve 22'si (%14.8)
güçlü, normal dokuların 9'u (% 6.0) zayıf ve 1'i (%0.7) güçlü MDR ekspresyonuna sahip olduğu
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gözlenmiştir (p<0.0001). Yapılan bu çalışma da, beyin tümörlü dokularda, CYP1A1’e oranla
oldukça yoğun eksprese olduğu tespit edilen CYP1B1, GST-M, GST-P ve MDR proteinlerinin
dokudaki tespiti, yapılacak benzer çalışmalarda, metabolik sinyal yolaklarının anlaşılması ve
prognostik faktörler açısından müdahalesinin sorgulanması gerektiği düşünülmektedir.
Anahtar Kelimeler : Beyin tümörü, immunohistokimya, CYP, GST, MDR.
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Defining the synergistic sequential application schedules for chemotherapeutic–
molecular targeted agent dual combinations in gastric adenocarcinoma cells
Özen Leylek1,2,†, Gülnihal Özcan2,3,*
1Koc University Graduate School of Health Sciences, Istanbul, Turkey 2Koc University Research Center for Translational Medicine (KUTTAM), Istanbul, Turkey
3Koc University School of Medicine, Istanbul, Turkey
†Presenting Author, *Corresponding author, e-mail:[email protected]
Gastric cancer is the fifth most common cancer and the third leading cause of deaths from
cancer. It is diagnosed mostly at the metastatic stage where there is no curative strategy. Though
chemotherapeutics and molecular-targeted agents with different mechanisms of action are
combined to increase treatment efficacy, a significant increase in overall survival could not be
achieved. Therefore, there is an urgent need for new strategies to increase the success of
combination chemotherapy in gastric cancer.
In today’s combination protocols, anticancer drugs are being administered concomitantly to the
patients in chemotherapy sessions. Recent studies suggest that application of the anti-cancer
drugs with a time-interval can increase the degree of synergism provided that the appropriate
time-interval and order of application are determined. In this study, we aim to investigate how
the time-interval and order of application affect the efficacy of chemotherapeutic-molecular
targeted agent dual combinations, and determine the sequential application schedules that
achieve maximum synergistic effect in gastric cancer.
To achieve the goals of the study, dual combinations of five different chemotherapeutics from
anthracyclins, platinium derivatives, taxanes, fluoropyrimidines and topoisomerase inhibitors,
and three different molecular-targeted agents targeting EGFR, mTOR or c-Met are being tested
on four different gastric adenocarcinoma cell lines. In addition to concomitant application, the
drug pairs at each combination are being applied according to ten different schedules at which
the drugs are applied with two different orders and five different time-intervals. The degree of
synergism at each schedule is being measured with Chou-Talalay Method.
Our preliminary data suggests that the time-interval and order of application for
chemotherapeutic-molecular targeted agent dual combinations are important determinants of
the degree of synergism in gastric cancer cells. We believe that the treatment schedules that will
be defined in this project can increase the success of chemotherapeutic-molecular targeted agent
combinations in gastric cancer.
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Funding/Supports:“The authors gratefully acknowledge The Scientific and Technological Research
Council of Turkey (TÜBİTAK) for funding by 3501- Career Development Program (CAREER) Grant
(Grant No: KBAG-117Z460) and use of the services and facilities of the Koç University Research
Center for Translational Medicine (KUTTAM), funded by the Presidency of Turkey, Presidency of
Strategy and Budget. The content is solely the responsibility of the authors and does not necessarily
represent the official views of the TÜBİTAK and the Presidency of Strategy and Budget.”
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Targeting TACC3 as a novel therapeutic strategy for breast cancer treatment
Ö. Akbulut1*, D. Lengerli2, Ö. Saatci3, E. D. Ergül4, U. Ö. Ş. Şeker4, B. Çalışkan2, E.
Banoğlu2, Ö. Şahin1,3,4* 1Department of Molecular Biology and Genetics, Faculty of Science, Bilkent University,
Ankara, Turkey 2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, Ankara,
Turkey 3Department of Drug Discovery and Biomedical Sciences, University of South Carolina,
Colombia, SC, USA 4UNAM-National Nanotechnology Research Center, Institute of Material Science and
Nanotechnology, Bilkent University, Ankara, Turkey *Corresponding author; [email protected]
Transforming Acidic Coiled-Coil Containing Protein 3 (TACC3) is an essential microtubule-
associated protein which is concentrated at centrosomes where it ensures chromosomal
segregation and microtubule stability. Abnormal expression is frequently observed in a
variety of cancers highlighting its importance to be used as a therapeutic target. KHS101 and
SPL-B are two small molecule inhibitors targeting TACC3, shown to suppress tumor growth
of glioblastoma and ovarian cancer xenografts, respectively. However, the clinical arm of
these inhibitors is still missing.
Here, we combined rational drug design approaches with screening methods where some
chemical fragments of already available TACC3 inhibitors were changed with their isosteric
equivalents to improve potency as well as drug-like properties. We identified a novel TACC3
inhibitor, BO-264, which showed higher potency (in nanomolar range) in in vitro and in vivo
systems and can be used as a mitotic blocker in breast cancer. Inhibitor-TACC3 binding was
validated through target engagement assay, drug affinity responsive target stability and
isothermal titration calorimetry methods. Compared to other available inhibitors, it showed
superior effects on the cellular processes such as mitotic progression, DNA repair and cell
viability, recapitulating the siRNA effect used against TACC3. Notably, BO-264 had
remarkable cytotoxicity effect on several cancer cell lines in NCI-60 human tumor cell line
panel while it doesn’t affect non-cancerous cells. Importantly, oral administration of BO-264
significantly suppressed the tumor growth in BC xenografts both in immunocompromised
and immunocompetent mice models which also increased survival. Finally, kinome profiling
was performed to test the specificity of BO-264 for TACC3 and further validated by state-
of-art binding assays.
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High potency independent from subtype and tissue and minimum toxicity of BO-264
highlight its potential use as mitotic blocker and a novel TACC3 inhibitor not only in breast
cancer, but also in other cancers.
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DNA Damaging Activities of Terbuthylazine
Özlem Demirci
University of Dicle, Faculty of Science, Biology Department, Diyarbakır
Pesticides and their metabolites are important xenobiotics that cause environmental pollution
and lead to very important ecological and health problems. Pesticide residues are present in
water areas, soils, fruits, and vegetables. Although, their application is based on selective
toxicity for certain organisms yet it has resulted in serious effects on many non-target organisms
as well. The use of pesticides has created a type of chemical environment which is proving
harmful to the living systems. Investigation of the interaction of macromolecules with
compounds that cannot be eliminated by means of detoxification mechanisms in organisms
exposed to xenobiotics such as pesticides is a valuable tool for revealing toxic damage.
Persons are exposed to pesticides directly not only when they are applied to vegetables, but also
through the metabolites that are stored in different structures which may turn out to be more
dangerous. Plants can bio-concentrate these environmental agents and convert pro-mutagens
into toxic metabolites. This fact raises the concern that plant systems might also activate
agrochemicals and environmental agents, thereby introducing new mutagens into the human
food chain. For this reason, the present study designed to investigate DNA damaging activity
of terbuthylazine.
Terbutylazine is a herbicide that is widely used in agricultural fields that act as photosynthesis
II inhibitors in target plants. In this study, DNA damaging activity of terbuthylazine was
investigated by using Agarose Gel Electrophoresis.
It was observed that this insecticide have ability to damage DNA.
Keywords: DNA Damage, terbuthylazine, pesticide pollution
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hBD-1 ve hBD-3 Antimikrobiyal Peptidlerinin Ürotelyal Kanser Dokularındaki
Ekspresyonları
Pınar Kaygın1, Serpil Oğuztüzün1, Gülçin Güler Şimşek2, Şeyma Özkan2, Sezen Yılmaz Sarıaltın3, Arzu Kaya Koçdoğan4, Onur Dirican1, Sema Çetin1, Muharrem Atlı1
1Kırıkkale Üniversitesi, Fen Edebiyat Fakültesi, Biyoloji Bölümü, Kırıkkale, Türkiye 2Sağlık Bilimleri Üniversitesi, Keçiören Eğitim ve Araştırma Hastanesi, Patoloji Bölümü, Ankara,
Türkiye 3Ankara Üniversitesi, Eczacılık Fakültesi, Toksikoloji Ana Bilim Dalı, Ankara, Türkiye
Antimikrobiyal Peptidler (AMP), bakteri, virüs, mantar gibi patojenlere karşı antimikrobiyal
aktivite göstererek vücudu koruma görevi üstlenmiş, bağışıklık sisteminin doğal elemanlarıdır.
Antimikrobiyal peptidlerin iki geniş ailesini oluşturan Human Beta Defensin (hBD)’ler ve
katelisidin (LL-37/hCAP-18) peptidleri, memelilerde epitel hücreleri, makrofaj ve
nötrofillerden eksprese edilmektedir. Bu peptidlerin antimikrobiyal etkilerinin yanında kanser
biyolojisinde de çeşitli rollerinin olduğu bildirilmiştir. Katelisidin ve defensinler, memelilerde
eksprese edilen AMP’lerin iki büyük üyesidirler. Ürotel kanserinin etiyolojisinde yeni bulgular
immun sistemin bu hastalıktaki önemini vurgulayan niteliktedir. Ancak ürotel kanserlerinin
baskılanmasında immun sistemin rolü hakkında çok az şey bilinmektedir. Bu çalışmada; Ürotel
kanser gelişiminde, hastalardan alınan ürotel karsinomlu ve benign ürotel epiteli dokularda,
hBD-1 ve hBD-3 antimikrobiyal peptidlerinin ekspresyon farklılıklarının incelenmesi
amaçlanmıştır. Ankara Keçiören Eğitim Araştırma Hastanesi Patoloji Kliniği arşivinden 69
hastadan alınan ve bu kişilere ait olan ürotel karsinomlu ve benign ürotel epiteli dokuların
belirtilen antikorlar ile immunohistokimyasal yöntemle boyanmış ve sonra boyama şiddetine
göre incelenmiştir. Negatif boyanma (0), hafif şiddette boyanma (+1), orta şiddette boyanma
(+2) ve şiddetli boyanma (+3) şeklinde değerlendirilen boyama sonuçlarına göre dokularda
peptidlerin ekspresyonları arasındaki farklılıklar SPSS istatistik analiz programında Anova
Testleri kullanılarak; peptidlerle hastaların klinik ve demografik veriler arasındaki ilişkiler ise
aynı programda Pearson Correlation testi ile belirtilmiştir. Buna göre, ürotel karsinomlu
dokularda hBD-1 peptidinin, benign ürotel epiteli dokularında hBD-3 peptidinin daha fazla
eksprese olduğu istatistiksel olarak anlamlı bulundu (p<0.05). Sonuç olarak, belirtilen
antimikrobiyal peptidlerin ürotel kanser biyolojisinde rol oynayabileceği ilk olarak bu çalışma
ile gösterilmiştir. Buna ek olarak bu peptidlerin ürotel kanserindeki rollerinin, daha fazla
örneklem ile çalışılarak ve kanser yolağındaki diğer gen ve proteinlerle karşılaştırılarak
aydınlatılması gerektiği düşüncesindeyiz.
Anahtar Kelime: Ürotel Kanser, Antimikrobiyal Peptidler, hBD-1, hBD-3
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Investigation of the relationship between cellular adaptation and therapeutic resistance
in chronic myeloid leukemia cells under imatinib pressure
Seda Baykal-Köse1, Ahmet Sinan Yavuz2, Eda Açıkgöz3-4 ,Öykü Gönül-Geyik1, Halil Ateş5,
Uğur Sezerman6, Güner Hayri Özsan5 and Zeynep Yüce1,*
1Department of Medical Biology, Faculty of Medicine, Dokuz Eylul University, Izmir, Turkey
2Department of Molecular Biology, Genetics and Bioengineering, Faculty of Engineering and Natural
Sciences, Sabanci University, Istanbul, Turkey
3Department of Histology and Embryology, Faculty of Medicine, Ege University, Izmir, Turkey
4Department of Histology and Embryology, Faculty of Medicine, Yuzuncu Yil University, Van,
Turkey
5Department of Hematology, Faculty of Medicine, Dokuz Eylul University, Izmir, Turkey
6Department of Biostatistics and Medical Informatics, Faculty of Engineering, Acibadem University,
Istanbul, Turkey
Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease characterized by the
t(9;22)(q34;q11) translocation, resulting in the chimeric BCR-ABL oncogene. Despite the
significant success of tyrosine kinase inhibitors (such as imatinib) inhibiting Bcr-Abl
oncoprotein function in the treatment of CML, drug resistance is still a serious and maybe the
most important problem affecting 20-30% of patients.
Our in vitro studies are designed using a TKI resistant K562 subclone (K562-Ir) which does not
harbour any BCR-ABL amplification or kinase domain mutation. The K562-Ir subclone was
developed in our laboratory by natural selection under imatinib pressure. We performed
immunofluorescence, Western blot and mRNA microarray (transcriptome) techniques for gene
expression analyses; three-dimensional cell culture studies; cell differentiation experiments;
different TKIs applications with cell death analysis; flow cytometric cell membrane marker
screening; proliferation assays; senescence assays; Q-PCR for amplification and expression
confirmation studies.
We observed that unlike K562 wild type cells, K562-Ir cells were adherent and resistant to
nilotinib, dasatinib, bosutinib and ponatinib in addition to imatinib. K562-Ir cells are more
resistant to cell death and proliferate slower. They are capable of forming tumor spheroids in
three-dimensional cell culture studies. We also showed a significant differences in the
expression of genes that regulate cell-tissue-organ differentiation and development processes
in K562-Ir cells. There is an increase in the expression of embryonic and cancer stem cell
surface markers and reweal that these cells have drifted away from their hematological origin.
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In addition miRNAs which are responsible of cell differentiation, proliferation and cell death
resistance are differentially expressed in the K562-Ir cells.
In conclusion, we present a new TKI resistance model suggesting that under imatinib stress a
sub-clone of the leukemic cells develop transcriptional instability resulting in a Bcr-Abl-
independent/aggressive population with phenotypic adaptation capacity. We believe that the
new model will contribute to a better understanding of drug resistance and relapse problems in
CML biology and the emergence of new therapeutic approaches.
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Neutrophil-to-Lymphocyte Ratio and Platelet Distribution Width Predicts
Invasiveness of Bladder Carcinoma
Selim Yalçın1, Ercan Yuvanç2
1Kırıkkale University Faculty of Medicine, Department of Medical Oncology
2 Kırıkkale University Faculty of Medicine, Department of Urology
Aim: Bladder cancer (BC) is a common tumor in the urinary tract and 75-85% of urothelial
carcinomas (UC) are non-muscle-invasive while 15-25% are invasive. Intracavitary BCG
therapy is one of the prophylaxis and treatment options and one of the anti-tumor mechanisms
of BCG is related to local immunological responses on urothelial cells. Inflammatory cells
around the tumor cells play a significant role in the progress and prognosis of tumors.
Patients and Methods: For this retrospective study, we reviewed the surgical and
pathological reports of 250 patients with UC operated between 2013 and 2019. Samples for
full blood count analysis were collected preoperatively.
Statistics: Factors analyzed were patient age, gender, lifestyle, occupational environment,
tumor stage, tumor size, tumor multiplicity, MPV, neutrophil count, lymphocyte count, platelet
count, platelet distribution width (PDW) and neutrophil/lymphocyte ratio (NLR).
Results: The study population was divided into three groups, i.e. muscle-invasive urothelial
tumors (n=107) and non-muscle-invasive urothelial tumors (n = 143) and healty individuals
(n=150). NLR had the most significant p value in correlation with pathological stage (p=0.005)
while the platelet count had the most risk (OR= 4.2746). When we sorted out the hazard ratios
of these parameters from strongest to weakest, it could be summarized as platelet count > PDW
> female gender > NLR > age (4.2746 – 4.1254 – 3.9734 – 2.8787 – 2.5434, OR value,
respectively). In that study, increased lymphocyte counts were shown in 78% of the patients
with non-muscle-invasive bladder carcinoma but in only 45% of those with infiltrating tumor.
Healthy individuals with a high NLR ratio have a significantly lower NK activity than those
with a low NLR ratio. The platelet count was found to significantly correlate with tumor size,
tumor stage and surgical margin status (p= 0.038 – 0.042 – 0.031, respectively).
Conclusions: The degree of systemic inflammation reflects the local tumor burden and reported
a correlation between lymphocyte reactivity against bladder tumor cells and clinical stage. The
ability of tumor invasion and metastasis is dependent both on the intrinsic characteristics of the
tumor cells and on the environment around the tumor. Patients with high preoperative NLR
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should be considered candidates for additional therapies after resection. Increase of neutrophils
and decrease of lymphocytes is important for determination of the prognosis. In our study, we
found a correlation between pathological stage and age, PDW, NLR, female gender and platelet
count with logistic regression analysis. These parameters can be used to predict the pathological
stage of the tumor before the treatment. NLR is an easily measurable parameter of systemic
inflammation, which can predict pathological stage. The abnormal phenotype of the tumor may
stimulate an influx of inflammatory lymphocytes into tissues surrounding the tumor. The
systemic inflammatory response also features changes in the relative levels of circulating white
blood cells. Predicting the prognosis of patients with cancer by examination of peripheral blood
leukocytes would seem to be an easier and useful procedure. Thrombocytosis may adversely
affect survival by facilitating cell invasion and metastasis. There is evidence that platelets
protect tumor cells by shielding them from the host’s immune system.
Keywords: Urothelial carcinoma, Neutrophil-to-lymphocyte ratio, Platelet distribution width
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Picoa Ve Terfezia Türlerinin Anti-Kanser Etkilerinin Araştırılması
Semih Dalkılıç, Sevda Kırbağ, Lütfiye Kadıoğlu Dalkılıç, Şule İNCİ, İsmail Korkmaz
Fırat Üniversitesi Fen Fakültesi Biyoloji Bölümü ELAZIĞ
İnsanlık tarihi boyunca, bazı mantarlar hem beslenmek için hem de hastalıkları önlemek için
fonksiyonel gıdalar olarak kullanılmıştır. Tüm yenilebilir mantarlar arasında yer mantarı bir
dizi ayırt edici özelliğe sahiptir ve diğer mantarlardan daha fazla dikkat çekmiştir. Yer altında
yetişen bu mantarlar, lezzetli tadı ve misk aroması nedeniyle çok miktarda tüketilir.
Terfezia ve Picoa türleri geleneksel olarak hem beslenmek, hem de iyileştirici özellikleri,
afrodizyak özellikleri için Elazığ-Malatya bölgesi civarında toplanmaktadır. Bu çalışmada, yarı
kurak, kurak veya çöl türüflerinin antikanser aktivitelerinin belirlenmesi amaçlanmıştır.
Yapılan çalışmada Terfezia ve Picoa türleri (Terfezia boudieri, T. claveryi, T. olbiensis, Picoa
juniperi ve P. lefebvrei) gibi çöl mantarlarından elde edilen ekstreler kullanılmıştır.
Terfezia ve Picoa türlerinin su ve metanol ekstrelerinin antikanser etkisi, MDA-MB-231 meme
kanseri hücre hattı ve PC3 prostat kanseri hücre hatları kullanılarak MTT (3- (4,5-dimetiltiazol-
2-il) -2,5-difeniltetrazolyum bromür) Assay ile test edildi. Mantarların Metanol ve su ile elde
edilen ekstreleri kanser hücre hatları üzerine 50 -100-200 ve 400 mg/ml konsantrasyonda 72
saat süresince uygulandı.
Çöl trüflerinin metanol ve su ile elde edilen ekstreleri, kanser hücrelerinin canlılığını farklı
oranlarda etkilediği gözlendi. Özellikle T. claveryi ve T. olbiensis'in ekstrelerinin anlamlı
düzeyde antikanser etki gösterdiği belirlenmiştir.
Key words: Yer mantarı, Ekstre, Anti-kanser, MTT
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The effects of Mackia amurensis leukoagglutinin on the expressions of some extracellular
matrix proteins in anaplastic thyroid carcinoma cell line, 8505C
Serap Sancar-Baş, Engin Kaptan Istanbul University, Faculty of Science, Department of Biology, Section of Molecular Biology
Vezneciler 34134 İstanbul/Turkey
Introduction: Anaplastic thyroid carcinoma (ATC) is one of the most lethal malignancy and
most of the patients are diagnosed with advanced and metastatic disease. Therefore, the
preventative therapies could be important for metastatic progression of ATC. The extracellular
matrix proteins regulate the remodelling and deposition of ECM and also effect cell motility,
adhesion and survival. High stromal expression of secreted protein acidic and cysteine -rich
(SPARC) have been reported and it has been observed that lysyl-oxidase (LOX) activity leads
to cell invasion, migration and high metastatic activity in ATC. On the other hand, osteopontin
(OPN), an important glycoprotein related to cell-cell and cell-ECM interactions, has been
associated with invasiveness of papillary thyroid carcinoma. In this study we aimed to
investigate the effect of Maackia amurensis leukoagglutinin (MAL-II), which is spesific for α-
2,6 sialylated glycans, on the levels of these metastasis related proteins in ATC cell line, 8505C.
Material-Method: Anaplastic cells were cultured and treated with 0.25 µM MAL-II for 24 h
at the non-lethal dose and duration according to our previous experiments. MAL-II treated cells
were lysed and RNA and proteins were isolated. The relative mRNA expression levels of
SPARC, LOX and OPN genes were determined with quantitative Real-time PCR and the
measurement of protein levels were studied with Western blotting.
Results: MAL-II treatment decreased the expressions of SPARC, LOX and osteopontin genes.
The protein levels of LOX and SPARC were also decreased with MAL-II, but no changes
osteopontin levels was observed.
Discussion: These results have pointed out that MAL-II treatment alters the expressions of
some extracellular matrix proteins of ATC cells. When considering MAL-II glycan specifity,
targeting α-2,6 sialylated glycans in ATC can be important strategy for the prevention of its
metastatic properties. In conclusion, MAL-II may have a therapeutic potential for treatment of
metastatic ATC.
Keywords: Anaplastic thyroid carcinoma, Mackia amurensis-II lectin, Metastasis, LOX, SPARC,
Osteopontin.
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An Investigation of the Antitumoral Activity of Phycocyanin Derived from Spirulina
(Arthrospira) Platensis on Mice
Sibel Bayıl Oğuzkan, İzzettin Güler, Mehmet Özaslan, Kemal Bakır, Betül Kut Güroy
1Department of Medical Services and Techniques, Vocational School of Health Services, University of
Gaziantep, Turkey
2Department of Biology, Molecular Biology and Genetic, University of Gaziantep, Turkey
3Department of Pathology, Medicine Faculty, University of Sanko, Gaziantep,Turkey
4Central Research Laboratory, University of Yalova, Turkey
Spiruluna is the most important and economically important phycocyanin that is a blue
photosynthetic pigment that is water-soluble and strong fluorescent properties. In this current
study, following the administration of phycocyanin by gavage feeding to balb-c mice with
Ehrlich Ascites Tumor (EAT), its antitumoral effect was studied in vivo. Phycocyanin was
administered to three different experimental groups; i.e. treatment, control and prophylaxis
(n=6) in different concentrations and the antitumoral activity in each group in terms of dosage
effect was detected. At the end of the treatment, cardiac blood was collected from every animal
to determine the oxidant and antioxidant status and liver enzymes parameters (TAL, TOL, ALT
and AST). The subjects were sacrificed under ether anesthesia, the kidney, stomach, small
intestine, and large intestine tissue was removed and pathologically evaluated for tumor
development.. As a result, while the TAL value of the prophylaxis groups was found to be
statistically significant compared with the control group (p<0.05), no statistically significant
difference was found in the other parameters (p<0.05). When the prophylaxis groups were
statistically compared between each other, while a significant increase was observed in values
as the dose increased, a statistically significant difference was found in terms of antioxidant
activity between the prophylaxis group, which was administered the lowest phycocyanin dose.
When the TAL, TOL, ALT and AST values of the treatment groups were statistically compared
with the control group, it was detected that the treatment group (200 mg) had the highest TAL
value, with a statistical significance (p<0.05). Likewise, there was a statistically significant
difference between treatment group and control group in terms of the ALT values, and the same
results were also found to be similar in the comparison by dosage difference. The
histopathological evaluations demonstrated that dosage difference does not have a preventive
effect against tumor formation.
Keywords: phycocyanin, EAT, TAS, TOS, ALT, AST
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Effects of Maackia amurensis leukoaglutinin treatment on the transcriptome of human
anaplastic thyroid cancer cell line 8505C
Suna Bektas, Engin Kaptan
Istanbul University Faculty of Science Department of Biology, Section of Molecular Biology, 34134
Vezneciler, Istanbul, Turkey
Lectins are bioactive molecules and have highly important roles in various cellular process.
They can bind specifically and irreversibly to free sugars and sugar residues of glycoconjugates.
Effects of plant lectins on normal and malignant cells and the underlying molecular mechanisms
have been subject of many researches. Although it is known that Maackia amurensis
leukoagglutin-II (MAL) decreases the tumorigenic and malignant capabilities of human
anaplastic thyroid cancer cells, its molecular mechanism has not been known yet. In this study,
it was amed to investigate the possible molecular mechanisms playing role in suppressing
malignant and tumorigenic characteristics of anaplastic thyroid cancer cells through the
treatment of MAL. For this purpose, whole transcriptome analysis was performed using RNA-
Seq in total RNA isolates of 8505C human anaplastic thyroid cancer cells treated with 0.25μM
MAL during 24 hours. Gene expression profile of MAL treated 8505C cells exhibited
significant changes in the expression of various genes related to the tumorigenic and malignant
characteristics of cancer cells such as LGR5, GPC5, PTGFR, CHFR. Our findings indicated
that the expression pattern of these genes were found to be associated with Wnt/β-catenin
pathway, which was an important signal transduction pathway in proliferation, stem cell control
and carcinogenesis. Besides, MAL treatment were also affected to G-protein-mediated receptor
signalling processes. Our data also pointed out that MAL is an effective bioactive molecule for
the suppression of the tumorigenic and malignant characteristics of anaplastic thyroid cancer.
In addition, our findings indicate that MAL has a significant potential for the treatment of
anaplastic thyroid cancer. However, in order to elucidate specific mechanisms for the effect of
MAL on 8505C cells, the functionality of the related genes and pathways needs to be tested by
conventional methods.
Keywords: Lectins, MAL, Thyroid cancer, RNA sequencing
Funding information: This study was supported by Scientific Research Projects Coordination Unit
of Istanbul University with the project number 32144.
Oral Presentation
119
Cytogenetic Anomalies in Lymphomas: A single center study
Süreyya Bozkurt
Department of Medical Biology, Faculty of Medicine, Istinye University, Istanbul
Lymphomas are clonal tumors originating from T or B lymphocytes and natural killer (NK)
cells. Lymphomas show different morphological, immunological and clinical traits. Although
there are differences in distribution according to geographical regions and etiologic factors,
75% of all lymphomas are non-Hodgkin and 25% are Hodghkin lymphomas. Chromosomal
translocations is one of the etiologic factor in lymphomas (1,2,3).
This study included 150 lymphoma patients who came to cytogenetic laboratory for
conventional karyotyping between 2008-2016. Fifty-five patients were female and 95 were
male. Karyotyping could not be performed in 34 patients (%24) of the bone marrow samples
and 114 (%76) of them were karyotyped. Fifteen patients had Hodgkin lymphoma and 46 had
non-Hodgkin lymphoma. Of the non-Hodgkin's lymphomas, 7 were T-cell lymphoma, 16 were
B-cell lymphoma, 5 were Mantle cell lymphoma and 8 were Burkitt lymphoma. Bone marrow
of the remaining 89 patients came to the laboratory with the diagnosis of lymphoma alone
without any sub-classification information.When the karyotype results are examined, it is seen
that 33 of the patients with NHL have normal karyotype and 9 of them have abnormal
karyotype. Karyotyping could not be performed in 33 patients. In patients with Hodgkin
lymphoma, normal karyotype was found in 10 patients and no results were obtained in 5
patients. None of the patients had abnormal karyotype in Hodgkin lymphoma. Numerical and
structural abnormalities have been identified in NHL patients. As structural anomaly;
t(8;14)(q24;q32), del(1p), del(9p), dup(1q) and del(3q) were found. As numerical anomaly
monosomy of chromosome 6, 8, 11, 14 were detected.
Refences
1-Jiang M, Bennani N N and Feldman A L. Lymphoma classification update: T-cell lymphomas,
Hodgkin lymphmas, and histiocytic/dendritic cell neoplasm .Expert Rev Hematol.
2017.March;10(3):239-249.
2-Lynch R C, Gratzinger D, Advani R H.Clinical impact of the 2016 update to the WHO lymphoma
Classification. Curr.Treat.Options in Oncol.(2017) 18:45.
3-Jaffe E S.Diagnosis and classification of lymphoma: Impact of technical advances.Seminars in
Hematology.(2019) 56:30-36.
Oral Presentation
120
The Role of Nuclear P-glycoproteins in Nuclear Sparing and Chemoresistance to
Doxorubicin in Gastric Adenocarcinoma Cell Models
Tevriz Dilan Demir1,2 †‡, Hande Özkan1,2‡, Gülnihal Özcan2,3*
1Koc University Graduate School of Health Sciences, Istanbul, Turkey 2Koc University Research Center for Translational Medicine (KUTTAM), Istanbul, Turkey
3Koc University School of Medicine, Istanbul, Turkey
†Presenting Author, ‡Equal contribution to the study
*Corresponding author, [email protected]
Doxorubicin is one of the most potent and most widely used chemotherapeutics in cancer
treatment. Its’ primary mechanism of action is DNA damage through binding to DNA and
inhibition of topoisomerase II. Therefore, intra-nuclear concentration of doxorubicin is an
important determinant for its anti-cancer efficacy. In sensitive cancer cells doxorubicin
predominantly accumulates at the nucleus leading to cell death. On the other hand, nuclear
accumulation could not be achieved in resistant cells despite the presence of the drug at the
cytoplasm. This phenomenon is known as “nuclear sparing” in the literature. However, the
underlying mechanism for the nuclear sparing and its causal relationship with chemoresistance
is unknown yet. Here, we aim to investigate the role of nuclear p-glycoprotein in nuclear-
sparing and resistance to doxorubicin. P-glycoprotein (p-gp), is a drug efflux pump localized
on the cell membrane which decreases the intracytoplasmic concentration of various
chemotherapeutics including doxorubicin. Recent studies and our findings showed that p-gp’s
can also be localized on the nuclear membrane. It was also suggested that p-gp’s on the cell
membrane can translocate to the nuclear membrane after exposure to doxorubicin. We
hypothesized that increased nuclear expression of p-gp may lead to nuclear sparing and may be
an acquired mechanism of resistance to doxorubicin. To test our hypothesis, we established
doxorubicin resistant gastric adenocarcinoma cell lines by continuous exposure to doxorubicin.
In immunofluorescence and subcellular fractionation studies we observed that the nuclear
expression of p-gp increased with increasing degree of resistance. The nuclear accumulation of
doxorubicin decreased significantly in resistant cells compared to age-matched control cells
investigated with fluorescence imager-reader and confocal microscope. These findings suggest
that increased expression of nuclear p-gp’s may be an important mechanism for acquired
resistance to doxorubicin via decreasing the accumulation of the drug within the nucleus of
gastric adenocarcinoma cells.
Oral Presentation
121
Funding/Supports:
“The authors gratefully acknowledge The Scientific and Technological Research Council of Turkey
(TÜBİTAK) for funding by 3001 Starting R&D Projects Funding Program (Grant No: SBAG-217S342) and
use of the services and facilities of the Koç University Research Center for Translational Medicine
(KUTTAM), funded by the Presidency of Turkey, Presidency of Strategy and Budget. The content is
solely the responsibility of the authors and does not necessarily represent the official views of the
TÜBİTAK and the Presidency of Strategy and Budget.”
Oral Presentation
122
Synthesis, Antiinflammatory and Anticancer Effects of Novel Amide Containing
Compounds
Tuğba Güngör1, Adem Özleyen2, Y. Berkay Yılmaz2, Tuğba Taşkın Tok3, Mehmet Ay1, Tuğba Boyuneğmez Tümer4
1Department of Chemistry, Faculty of Arts and Science, Natural Products and Drug Research
Laboratory, Çanakkale Onsekiz Mart University, Çanakkale, Turkey 2Graduate Program of Biomolecular Sciences, Institute of Natural and Applied Sciences, Çanakkale
Onsekiz Mart University, Canakkale, Turkey 3Department of Chemistry, Faculty of Arts and Science, Gaziantep University, Gaziantep, Turkey 4Department of Molecular Biology and Genetics, Faculty of Arts and Science, Çanakkale Onsekiz
Mart University, Çanakkale, Turkey
Non-Steroidal Anti-inflammatory Drugs (NSAIDs) are the most widely used drug classes all
over the world. The best-known examples of NSAIDs are aspirin, celecoxib, nimesulide,
diclofenac, ibuprofen, etodolac, naproxen etc. Despite the widespread usage of these drugs on
multifunctional diseases such as inflammation, type II diabetes, metabolic syndrome and
cancer, their long term usage are associated with serious gastrointestinal, cardiac and renal side
effects. To overcome these problems, there is a great need to develop new superior anti-
inflammatory drugs.
In this study, nimesulide, N-(4-nitro-2-phenoxyphenyl)methanesulfonamide, which inhibits
COX-2 selectively and shows analgesic and antipyretic effects was selected as a reference
compound1-3. In recent years, it is reported that nimesulide can be used as an anticancer agent
since it suppresses the proliferation of cancer cells and induces apoptosis2,3. Herein, we
designed and synthesized a series of novel amide containing nimesulide derivatives with
sequential multistep organic reactions and characterized their structures by using melting point,
FT-IR, 1H NMR, 13C NMR and MS analysis. The compounds were screened for their selective
COX-2 inhibitory properties and secondary anticancer effects by using in vitro and cell based
assays. Among the synthesized compounds, five of them demonstrated better inhibitory COX-
2 activity as compared to nimesulide. Two compounds demonstrated moderate antiproliferative
effects against colon cancer cell line. Results showed that lead compounds can be further
developed by using in silico design and synthesis studies for in vivo experiments.
Keywords: Nimesulide, Anticancer, Antiinflammatory, Synthesis, In silico design
*This study was funded by TUBITAK, Grant No.117Z398.
Oral Presentation
123
References
1. Akarca U. S. 2005. “Gastrointestinal Effects of Selective and Non-Selective Non-Steroidal Anti-Inflammatory Drugs”, Current Pharmaceutical Design, 11, 1779-1793.
2. Hida T., Kozaki K., Muramatsu H., Masuda A., Shimizu S., Mitsudomi T., Sugiura T., Ogawa M., Takahashi T. 2000. “Cyclooxygenase-2 inhibitor induces apoptosis and enhances cytotoxicity of various anticancer agents in non-small cell lung cancer cell lines”, Clinical Cancer Research, 6 (5),
2006-2011.
3. Renard J. F., Julémont F., de Leval X., Pirotte B. 2006. “The use of nimesulide and its analogues in cancer chemoprevention”, Anti-Cancer Agents in Medicinal Chemistry, 6 (3), 233-237.
Oral Presentation
124
As a potential overall-biomarker of cancer stem cells CD90: on drug resistance
perspective
Utku Ozbey1,2, Nur Ekimci Gurcan1,2, Negar Taghavi Pourianazar1,2, Emre Can Tuysuz1,2, Melike Bayindir Bilgic1,2, Aysegul Kuskucu2, Omer Faruk Bayrak2
1Department of Genetics and Bioengineering, Yeditepe University, 34755, Istanbul, Turkey
2Department of Medical Genetics, Yeditepe University Medical School, 34755, Istanbul, Turkey
Cancer stem cells (CSCs) also known as tumor initiating cells (TICs) are like normal stem cells
and they show the same pattern in tumor. According to recent studies, CSCs are associated with
tumor progression, recurrence and prognosis. Therefore, targeting CSCs in cancer therapy is
important challenge in researches.
CD90 (THY-1) is a membrane glycoprotein which is founded in several cancers. Therefore,
CD90 expression is associated with stemness properties in cancer cells. As an example, small
populations of CD90 expressing HCC, GBM and gastric cancers Epithelial-Mesenchymal
Transition (EMT) profiles, ALDH1 activities and stemness biomarker expressions are
upregulated.
In this study, we perfomed CRISPR/Cas9 technology for silencing THY-1 gene in AGS,
HGC27, A549 and PANC1 cancer cell lines. CD90-/- populations are sorted via BD FACS Aria
III system and stable cell line of knockout cells has been generated and CSC related gene
expressions (Sox2, Oct4, CDH1, Twist, ABCG2) have been studied which are also associated
with drug resistance in cancers. After that, drug resistance studies with cisplatin, cytarabine,
etoposite and taxol on both knock-out and parental cells had been continued over a year. After
that, resistant cells CD90 expressions and profiling of molecular weight of CD90 protein via
western blot. Our findings suggest that, CD90 is potential CSC biomarker in these cancers and
CSC associated drug resistance. Additinaly, molecular weight differentiation of CD90 protein
in drug resistant cells is associated with glycosylation pattern of CD90 is related with drug
resistance in these cell lines.
Consequently, CD90 protein is associated with CSC properties in several cancers and targeting
of CD90 is effecting development of drug resistance in cancers. Therefore, glycosylation
pattern of CD90 might be effective on drug resistance in cancer cells. Finally, according to these
findings, targeting of CD90 in cancer could be new important therapy strategy in drug resistance
inhibition manner.
Oral Presentation
125
The Role of c-Met Neddylation in Hepatocellular Carcinoma
Yeliz Yılmaz1, 2, Bora Güloğlu1,3, Hande Topel1, Ezgi Karaca1, 4, Neşe Atabey1
1Izmir Biomedicine and Genome Center, Izmir, Turkey 2Department of Medical Biology and Genetics, Institute of Health Sciences, Dokuz Eylul University,
Izmir, Turkey 3Department of Biochemistry, University of Oxford, Oxford, United Kingdom
4Izmir International Biomedicine and Genome Institute, Izmir, Turkey
Neddylation is the covalent binding of NEDD8 protein to lysine residues of target proteins.
Neddylation causes alteration of activation, stabilization and localization of proteins. Receptor
tyrosine kinase c-Met has elevated expression and activation in cancer. Enlightening the
mechanisms of sustained expression and activation is of great importance. Only two receptor
kinases were shown to be neddylated. We hypothesize that c-Met might also be prone to
neddylation which might render the kinase more stable and active, resulting in more aggressive
hepatocellular carcinoma (HCC) clones. To conduct our hypothesis, we first investigated total
neddylation status in several HCC cell lines and tumor tissue microarrays, NEDD8 is
ubiquitously expressed and both c-Met and NEDD8 stainings are positively correlated.
Immunoprecipitation of c-Met from lysates treated with c-Met ligand hepatocyte growth factor
and/or c-Met inhibitor SU11274 revealed that c-Met is neddylated upon ligand activation and
neddylation is diminished with inhibitor treatment. c-Met activation is also decreased when
cells are treated with MLN4924 with/without SU11274. The use of SU11274 and MLN4924 in
combination also decreases HCC migration and proliferation. Next we aim to identify potential
neddylation sites through computational analysis. We focused on c-Met’s C-terminal tail and
identified two lysine residues as possible targets of E3 ligases. When we looked at the crystal
structure, we identified K1360 on the tail region as conformationally available. When we
focused on E3 ubiquitin ligase c-Cbl as a known interactor of c-Met, three possible
ubiquitination sites were identified via UbPred as K27, K104 and K1232. Since K27 and K104
belong to the extracellular moiety, K1232 was chosen as the other candidate for neddylation. In
conclusion, we identified a novel post-translational modification of c-Met which alters its
activation and biological responses that leads the cells more invasive and metastatic. NEDD8:c-
Met interaction might be a target for the treatment of hepatocellular carcinoma.
Oral Presentation
126
Keton Cisimlerinin In Vıtro İnsan Meme Kanseri Hücrelerinin Canlılığını Azaltıcı Etkisi
Zelal Zuhal Kaya1, Ayşe Mine Yılmaz2, Gökhan Biçim2, Seval Kaya3, A. Suha Yalçın2
1Tıbbi Biyokimya Bölümü, Tıp Fakültesi, Acıbadem Mehmet Ali Aydınlar Üniversitesi, 34752,
İstanbul, Türkiye 2Tıbbi Biyokimya Bölümü, Tıp Fakültesi, Marmara Üniversitesi, 34854, İstanbul, Türkiye
3Histoloji ve Embriyoloji Bölümü, Tıp Fakültesi, Dicle Üniversitesi, 21280, Diyarbakır, Türkiye
Glikoz katabolizmasının indüklenmesi ve pirüvatın laktik aside dönüştürülmesi, oksijen
varlığında bile kanser hücrelerinde başlıca metabolik modülasyonlardır. Bu fenomen Warburg
etkisi olarak bilinir. Önceki çalışmalar kanser hücrelerinin glikoz bağımlılığı nedeniyle keton
cisimlerini birincil enerji kaynağı olarak kullanamadıklarını göstermiştir. Bu çalışmada, keton
cisimlerininin farklı meme kanseri hücrelerinin canlılığını ve çoğalmasını in vitro olarak inhibe
edip etmeyeceğini araştırdık. İnsan meme kanseri hücreleri (MCF-7, MDA-MB-231, MDA-
MB-435) ve insan foreskin fibroblastları (kontrol), yüksek glikozlu standart ortam, glikozsuz
standart ortam ve glikozsuz ortama ek olarak 10 mM ve 20 mM asetoasetat veya 10 mM ve 20
mM β-hidroksibütirat içerisinde büyütüldü. Asetoasetat ve β-hidroksibütiratın hücre canlılığı
üzerindeki doza bağlı etkisi MTT testi ile belirlenirken, morfolojik değişiklikler ışık
mikroskobu ile gözlenmiştir. Apoptotik hücre miktarı, Annexin V-FITC / PI apoptoz saptama
kiti ile ölçülmüştür. Keton cisimlerinin uygulanması, glikoz varlığına kıyasla, tüm meme
kanseri hücrelerinde hücre çoğalmasını ve canlılığını önemli ölçüde azaltmıştır. Aksine, kontrol
hücrelerinin canlılığı glikoz varlığında / yokluğunda değişmedi. Ek olarak, keton cisimlerinin
hücre canlılığı üzerindeki etkisinin şiddeti, üç meme kanseri hücresinde farklıydı.
Sonuçlarımızın meme kanserinde yeni tedavi yaklaşımlarının gelişimine katkı sağlayacağına
inanıyoruz. Bununla birlikte, bu farkın moleküler mekanizmasını açıklığa kavuşturmak için
daha fazla çalışmaya ihtiyaç vardır.
Anahtar Sözcükler: Keton cisimleri, Asetoasetat, β-hidroksibütirat, Meme kanseri hücreleri
Bu çalışma, Marmara Üniversitesi Bilimsel Araştırma Projeleri Birimi (Proje no: SAG-C-YLP-
081117-0614) tarafından finansal olarak desteklenmiştir.
Oral Presentation
127
Effect of Doxorubicin on Expression of RNA m6A Methylation Enzymes in Leukemia
Cell Lines
Zeliha Emrence1, Melda Sarıman2, Burcu Salman1, Neslihan Abacı1, Sema Sırma Ekmekci1
1Department of Genetics, Istanbul University, Aziz Sancar Institute of Experimental Medicine,
Istanbul, Turkey. 2Molecular Cancer Research Center, Istinye University, Istanbul, Turkey
Up to date, more than 100 chemical modifications of RNA have been identified. N6-
metyladenosine (m6A) is the most commonly observed modification in mRNA, modulating
gene expression by altering features such as structure, maturation, stability, splicing, export,
translation, decay, and affecting cell fate decision, cell cycle regulation, cell differentiation, and
circadian rythim. RNA modification enzymes have been associated in many diseases, including
cancer. It has been shown to play a role in cancer development, diagnosis and treatment by
effects such as proliferation, migration and invasion, and its mechanisms are not fully
elucidated. We aimed to investigate the expression of m6A writers (WTAP, METTL3,
METTL14) and m6A eraser (ALKBH5) via treatment Doxorubicine, a chemotherapeutic agent,
in acute lymphoblastic cell line, REH, and acute promyelocytic cell line, NB4. For this purpose,
500nM doxorubicine was applied to REH and NB4 cells and incubated for 24 hours. At the end
of the incubation period, RNA was isolated from the cells, and cDNA was synthesized. Gene
expression was determined using quantitative real time PCR and analyzed using the 2^ ct
method. Demethylase ALKBH5 expression in NB4 and REH did not change compared to
control. In methylase enzymes, WTAP expression was decreased 1.6 fold in NB4, 1.3 fold in
REH and METTL3 expression was decreased 2.9 fold in REH while did not change in NB4;
METTL14 was decreased 1,8 fold in NB4 and 3 fold in REH. Our results releaved that
administration of doxorubicin ensue decreased expression of methylase enzymes in REH and
NB4 cell lines. These results suggest that doxorubicin may contributes to treatment by affecting
RNA m6A methylation, besides the previously known effects.
128
POSTER PRESENTATIONS
Poster Presentation
129
Plants Used Against Cancer in Folk Medicine in Turkey
Meltem Güleç1, Ayşegül Çalışkan2, Ayşe Şeyma Büyük3
1Istinye University Faculty of Pharmacy, Department of Pharmacognosy, Istanbul, Turkey
2Istinye University Faculty of Pharmacy, Department of Analytical Chemistry, Istanbul, Turkey
3Istinye University Faculty of Pharmacy, Department of Clinical Pharmacy, Istanbul, Turkey
Since time immemorial humankind benefits from plants as nutritient, medicine, cosmetic or to
make tools and shelters for itself (Hoppe 1958). Through history, traditional knowledge was
gathered by trial and error method, practices were developed and sustained (Sezik 1991). It has
been passed on from generation to generation to reach our day. While indigenous people
change their lifestyle, knowledge and traditional medicine based on plants was abandoned and
replaced mostly with conventional medicine (Walter 2003). This study aims to gather
information on medicinal plants used traditionally against cancer in Turkey before it is lost
forever. 124 taxa has been identified and given as a table including their scientific and local
names, used parts and usage against cancer.
More studies are needed to provide scientific evidences to folklore use. It may be helpful in the
development of future medicines or treatments.
Keywords: Cancer, Traditional medicine, Ethnobotany, Turkey, Medicinal plant
References
Hoppe, H.A. 1958. Drogenkunde: Handbuch der Pflanzlichen und Tierichen Rohstoffe, Cram, De
Gruyter und Co., Hamburg.
Sezik, E. 1991. Dünya’da bitkilerle tedavi yaygınlaşıyor, Bilim ve Teknik Dergisi 21:278.
Walter, H.L. 2003. Pharmaceutical Discoveries Based on Ethnomedicinal Plants: 1985 to 2000 and
Beyond, Economic Botany 57(1):126-134.
Poster Presentation
130
Targetıng Trem2 Reduces Prolıferatıon, Induces Cell Death On Breast Cancer Cells*
Ayten Kılınçlı, İbrahim Tekedereli
Department of Medical Biology and Genetics, Inonu University, Malatya
Triggering receptor expressed on myeloid cells-2 (TREM-2) is a cell surface receptor on human
monocyte-derived dentritic cells. TREM transmembrane proteins play vital roles in regulating
inflammation and immune response through their association with adaptor proteins. TREM2
is considered to be a protective negative regulator of inflammation. Besides playing a critical
role in immune responses, TREM2 was involved in a variety of other biological processes,
including osteoclastogenesis, brain homeostasis, and phagocytosis. To date, studies on whether
there is any relationship between TREM2 and cancer development are limited. The aim of this
study is to investigate the role of blocking TREM2 on breast cancer cell lines.
This study was conducted on MCF10A, MDA-MB-231 and MCF7 cells. Western Blot was
used for analysis the expression of proteins. After knocking down of TREM2 by siRNA, cell
proliferation, colony forming ability and cell death rates were tested by MTS, colony formation
assay, trypan blue exclusion assay, respectively.
TREM2 expression was higher in MCF10A than MDA-MB-231 and MCF7 cells. After
treatment with siRNA, cell proliferation was reduced by 60 % in MDA-MB-231, 50 % in
MCF7 cells. Colony forming ability was decreased by 90 % in MDA-MB-231, 80 % in MCF7
cells. Cell death rates were 45% in MDA-MB-231 and 40 % in MCF7.
In this study, for the first time TREM2 expression was analyzed in breast cancer cells and
immortalized human mammary epithelial cells in vitro. TREM2 suppression reduced cell
proliferation and increased cell death. This study suggested that targeting TREM2 could be a
new hope for the treatment of breast cancer.
This work is supported by The Scientific and Technological Research Council of Turkey (TUBITAK) project number 114S979
Poster Presentation
131
DNA Degradation by Water and Ethanol extracts of Allium tuncelianum in the
Presence of Copper Ions: Implications for Anticancer Properties
Selim Gün1, Bircan Çeken Toptancı2, Göksel Kızıl2, Murat Kızıl2, Süleyman Kızıl3
1University of Van Yüzüncü Yıl, Faculty of Science, Chemistry Department, Van, Turkey 2University of Dicle, Faculty of Science, Chemistry Department, Diyarbakır, Turkey
3University of Dicle, Faculty of Agriculture, Department of Field Crops, Diyarbakir, Turkey
There has been an increasing interest in the potential cancer chemopreventive properties of
diet-derived phytochemical agents. Therefore, dietary antioxidants derived plant have attracted
considerable interest for their ability to induce apoptosis and regression of tumors in animal
models. While it is believed that the antioxidant properties of these agents may contribute to
lowering the risk of cancer induction by impeding oxidative injury to DNA, it could not account
for apoptosis induction and chemotherapeutic observations.
Most studies on anticancer mechanisms of plant polyphenols invoke the induction of cell cycle
arrest and modulation of transcription factors that lead to anti-neoplastic effects [1]. Dietary
antioxidants can alternatively switch to a prooxidant action in the presence of transition metals
such as copper. Such a prooxidant action leads to strand breaks in cellular DNA and growth
inhibition in cancer cells. Copper is an essential metal found in chromatin, and observed to be
elevated in a number of malignancies [2]. Further, the cellular DNA breakage and anticancer
effects were found to be significantly enhanced in the presence of copper ions.
In the present study, we tested the ability of water and ethanol extracts of Allium tuncelianum
which is grown Ovacık district of Tunceli province, to cause DNA strand breaks on pBluescript
M13+ plasmid DNA system, both in the absence and the presence of Cu(II), as measured by
standard an agarose gel electrophoresis assay. Both extract tested caused some breakage of
cellular DNA, the degree of such breakage is enhanced in the presence of copper.
The results clearly indicate that extracts of Allium tuncelianum can alternately behave as
prooxidants in the presence of Cu(II) leading to cleavage of plasmid DNA that such DNA
breakage is physiologically feasible and could be of biological significance that have great
potential as putative chemopreventive or therapeutic agents.
1) Kuo, M. L., Huang, T. S., & Lin, J. K. (1996). Curcumin, an antioxidant and antitumor promoter, induces apoptosis in human leukemia cells. Biochimica et Biophysica Acta, 1317, 95-100.
2) Linder M.C (2012). The relationship of copper to DNA damage and damage prevention in humans. Mutation Research, 733(1-2):83-91.
Poster Presentation
132
Role of NEK2A in PLK4 Induced Multipolar Cell Divisions
Selahattin Can Özcan1, Batuhan Mert Kalkan2, Erkam Keskin1, Ceyda Açılan Ayhan1
1Koc University, School of Medicine, Istanbul, Turkey
2Koc University, Graduate School of Health Sciences, Istanbul, Turkey
Introduction: Centrosomes are the major microtubule organizing centers of animal cells, and
they play important roles in organizing the mitotic spindle poles. Centrosomes consist of two
centrioles; a mother and a daughter centriole, surrounded by pericentriolar matrix. Polo like
kinase 4 (PLK4) is important in initiation of centriole duplication and over-expression of PLK4
leads to multiple rounds of centriole duplication in a single cell cycle. Furthermore, PLK4 over-
expression produces “rosette” shaped centrosomes, which consist of several centriols
surrounding the mother centriole. NIMA like kinase (NEK2) is responsible from centrosome
seperation by phosphorylating centrosome cohesion proteins like C-Nap-1, Rootletin and
Cep68. Centriolar links in rosette structures is currently undefined and to understand the role
of Nek2 on PLK4 induced centrosomes, we hypothesised that co-expression of PLK4 and
NEK2 could synergically contribute to centrosome amplification and multipolar spindles
(MPS).
Material and Method: Protein expressions of Nek2 and PLK4-GFP was confirmed by
Western blotting. PLK4 mediated centrosome amplification was visualized by Centrin-2
staining with a confocal microscope. GFP positive metaphase and interphase cells were scored
manually using a fluorescent microscope.
Results: Overexpression of PLK4-GFP in HEK293T cells resulted in centrosome
amplification as determined by confocal microscopy. Metaphase cells were scored in PLK4
over-expressing HEK293T and HEK293T+NEK2 cells. We observed that PLK4
overexpression increased the rate of MPS, and NEK2 over-expression further increased
multipolarity. Also we scored interphase cells for centrosome numbers and observed a similar
phenomenon.
It is known that rosettes generated by PLK4 expression could contribute to multipolarity more
in second cell division (48H) due to disengagement of centriols from rosette centrosomes. But
the observation of increased multipolarity in NEK2+PLK4 cells in the first cell division (24H),
compared to WT+PLK4 cells (%14 to %22) may be attributed to the role of NEK2 on centriolar
links in rosettes.
Poster Presentation
133
Conclusion and future plans: PLK4 over-expression increases the frequency of MPS in
HEK293T cells and NEK2 further contributes to this increase. While the structure of PLK4
induced rosette centrosomes is defined, centriolar links in rosettes remain unknown. To
understand whether this links are similar with normal centrosomes, high-end microscopy
techniques like STED imaging is required. Our most important future plan is to characterize
centriolar links in rosettes and to find out whether NEK2 could phosphorylate these links and
seperates centriols from rosette centrosomes.
Poster Presentation
134
Investigation of Nek2 Kinase Targets with Focus on Centrosomal Unclustering
Batuhan M. Kalkan1, S. Can Ozcan2, Ceyda Açılan Ayhan2
1Koc University, Graduate School of Health Sciences, Istanbul, Turkey
2Koc University, School of Medicine, Istanbul, Turkey
Background and Purpose: The chemotherapeutics used today mostly suffer from the
destructive side effects on healthy tissues as a result of failure to selectively target tumor cells.
Unlike normal cells, tumor cells frequently exhibit extra centrosomes, which tend to form
multipolar spindles (MPS), leaving one/some of the cells with less genetic material triggering
death pathways. Nevertheless, cancer cells divide successfully by clustering their extra
centrosomes into two poles. Nek2A kinase is a key molecule regulating mitotic processes,
including centrosome separation, and we have shown that Nek2 overexpression leads to
centrosomal unclustering. In this project, we aim to investigate how and through which
molecules Nek2A accomplishes its unclustering activity. For this reason, we started with
known Nek2A targets with relevant function and assess their involvement in centrosomal
unclustering. The targets that can efficiently uncluster centrosomes and leads to cell death will
be put forward as novel molecules with a chemotherapeutic potential.
Method: A total of 4 candidate Nek2A targets, (C-NAP1, Rootletin, Centlein, Trf1) will be
tested in different cancer cell lines containing extra centrosomes and exhibiting efficient
centrosome clustering. For candidates, whose silencing can cluster multiple centrosomes and,
the effect of Nek2A upregulation will be tested to understand if Nek2A unclustering
mechanism depends on these targets.
Results: Our previous data showed that overexpression of Nek2A induces unclustering of
centrosomes and MPS, while its disruption provides clustering of extra centrosomes leading
bipolar divisions. Moreover, we have observed a significant reduction in relative cell viability
after overexpression of Nek2A in the cells with supernumerary centrosomes, which is probably
due to unclustering of extra centrosomes leading to MPS. We have previously designed three
guide RNAs for each Nek2A targets and successfully cloned into LentiCRISPR-v2 vectors,
followed by viral packaging.
Future Directions: Beside its known targets, novel Nek2A targets will be investigated using
a phosphoproteomic approach comparing Nek2A overexpressing cells and inactivated cells
using a dominant negative form of Nek2A, knock-out and knock-down separately. Proteins,
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which have a known function in mitosis, will be confirmed through western blotting,
coimmunoprecipitation, colocolization studies and kinase assays. Following characterization,
the newly established Nek2A targets will be tested for their ability to override Nek2A
reclustering phenotype when Nek2A is silenced. Lastly, cell death following inhibition of
centrosomal unclustering via silencing of candidate molecules will be determined.
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136
New Therapeutıc Approaches Targetıng Mll-Af9 Leukemıa Through Epıgenetıc
Reprogrammıng
İpek Bulut1, Buse Cevatemre2, Adam Lee3, Arasu Ganesan3, Ceyda Açılan Ayhan1, 4
1Koc University, Graduate School of Science and Engineering, Istanbul, Turkey
2Koc University, Research Center for Translational Medicine, Istanbul, Turkey
3University of East Anglia, School of Pharmacy, Norwich, England
4Koc University, School of Medicine, Istanbul, Turkey
Leukemia is a highly complex disorder of blood and bone marrow and characterized by
inhibition of differentiation during hematopoiesis and leading to an uncontrolled abnormal cell
proliferation. Mixed lineage leukemia (MLL) is a form of acute leukemia which represents
poor prognosis and due to chromosomal translocation, resulting in a hyperactive MLL fusion
protein. It has been indicated that MLL leukemias are largely based on epigenetic irregulations
rather than genomic instability. A transcription factor (AF9) that fuses with MLL plays a role
in the uncontrolled proliferation of MLL cell lines.
Chromatin modifying enzymes are aberrantly expressed in leukemias. Targeting these
regulators such as the Lysine-specific demethylase (LSD1) and Histone deacetylase (HDAC)
has been considered as novel treatment modality. In MLL-AF9 leukemias recruiting of these
enzymes due to translocation leads the activation of several genes that inhibit differentiation
and cause uncontrolled cell proliferation.
In this study, novel compounds which synthesized to inhibit LSD1, HDAC6 and both LSD1&
HDAC6 (Dual Inhibitor) are characterized. In our study, we showed that the compounds inhibit
target enzymes by in vitro enzyme activity tests, we also showed that the compounds inhibit
target enzymes in the cell by cellular thermal shift assay (CETSA). Toxic effects of the
compounds on leukemia cells were shown with ATP-dependent cell viability tests. Increased
histone methylation and acetylation levels after inhibition of target enzymes were indicated
with Western blot. Increased expression of downstream genes due to enzyme inhibiton were
showed using RT-qPCR. The synergistic effect of the inhibitors with the therapeutic drugs used
in the clinic was investigated. We showed that the death response could be increased with
combination of Doxorubicin and our compounds.
Defects in epigenetic pathways involving increased expression levels or abnormal patterns of
activity are one of the key drivers of cell proliferation in cancer. Targeting MLL-AF9 is an
attractive strategy for therapeutic intervention in patients with this genetic translocation. Since
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our inhibitors are more effective on MLL-AF9 leukemias, we hope that our study will result in
the characterization of targeted drugs for use in leukemia therapy.
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138
Investigation of the free amino acid profile of different breast cancer cell lines (MCF-7,
MDA-MB-231, and CRL-4010)
İsmail Koyuncu, Ebru Temız, Ataman Gönel, Mevlüt Bahçevan, Nihayet Bayraktar
Department of Medical Biochemistry, Harran University, Sanliurfa, Turkey
Cancer is one of the leading causes of death worldwide, and its early diagnosis improves the
chances of survival for patients. Affecting one out of every twenty women, breast cancer is the
most common cancer type. Due to the increasing incidence of cancer worldwide, genomic,
proteomic and metabolic methods are used to develop a new technology, which has high
specificity and is faster and more sensitive. Metabolomic is a powerful analytic tool based on
new biomarkers and therapeutic targets as well as has a capable of changing the production,
usage, and level of many metabolites. The present study aims to compare the free amino acid
profile of breast and normal cell lines, which have different characteristics, and to investigate
their biomarker potentials for the diagnosis and treatment of breast cancer. In this framework,
intracellular free amino acid profiles of MDA-MB-231 (triple-negative), MCF-7 (ER+) breast
cancer and CRL-4010 normal cells were investigated by the method of LC-MS/MS. According
to the obtained results, trans-4-OH-proline and 4-OH-proline (p<0,05*), ortophospo-serine,
citrulline, aspartate, 3-amino-isobutyric acid (p<0,01**), and GABA (p<0,001***) were found
to be statistically increased in MDA-MB-231 line compared to the CRL4010; however,
isoleucine, losin, valine, tyrosine, tryptophan, threonine, lysine, histidine, glutamine, aspartate,
1-MHIS (p<0,05*), norvaline, carnitine, serine, asparagine, arginine, alanine (p<0,01**), and
argininosuccinic acid, sarcosin, trans-4-OH-proline, cystathionine, 4-oh-proline (p <0.001 ***)
were found to be significantly increased in MCF-7 line compared to the CRL 4010.
Consequently, our data show that the levels of some amino acids are changed in the pathogenies
of the tumor. At this stage, we can speculate that these amino acids can play roles in tumor
progression and drug activity (sensitive/resistant) but detailed amino acid level manipulating
studies are required to elucidate the relationship between amino acid profile and cancer
pathogenesis.
Keywords: Breast cancer, MCF-7, MDA-MB-231, CRL-4010, metabolomics, LC-MS/MS
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Hepatoselüler Karsinoma, Mide ve Kolorektal Kanser Hastalarında Tedavi Öncesi ve
Sonrası Oksidatif Stresin Araştırılması
Elif Bozbulut1, Mustafa Gökçe2, Muhammed Yunus Bektay3, Murat Serilmez 4, Mustafa Uzun5, Turgut Aksoy5, Abdurrahim Kocyigit1, Eray Metin Guler1
1 Bezmialem Vakıf Üniversitesi Tıp Fakültesi Tıbbi Biyokimya A.D. Fatih – İstanbul 2 Bezmialem Vakıf Üniversitesi Eczacılık Fakültesi Farmakoloji A.D. Fatih – İstanbul
3 Bezmialem Vakıf Üniversitesi Eczacılık Fakültesi Klinik Eczacılık A.D. Fatih – İstanbul
4 İstanbul Üniversitesi Onkoloji Enstitüsü Fatih – İstanbul
5 Bezmialem Vakıf Üniversitesi Tıp Fakültesi Fatih – İstanbul
Dünya genelinde her sene 1 milyondan fazla yeni kolorektal kanser (KRK) vakası teşhis
edilmektedir. KRK dünya genelinde en sık görülen üçüncü kanser ve dördüncü en yaygın
kanserden ölüm sebebidir. KRK özellikle sanayi ülkelerinde yaygın ve kanser ölümlerinde
ikinci sıradadır. Mide kanseri dünya genelinde en sık görülen dördüncü kanser türü olup son
yıllarda görülme sıklığının azalmasına rağmen, kanserle ilişkili ölüm nedenlerinin başında
gelmektedir. Hepatoselüler karsinoma (HSK) karaciğerin sık rastlanan malin tümörüdür ve en
çok Afrika, Çin ve Avrupa’da gözlenmektedir. Son yılların verilerine göre kanserler arasında
sıklık açısından beşinci sırada yer alırken kanserden ölüm sıklıkları arasında üçüncü sırada
bulunmaktadır. HCC gelişiminde birçok faktörün önemli rol oynadığı bilinmekte; siroz, alkol
kullanımı, oksidatif stres ve hepatit B, C virüsleri risk faktörleri arasında değerlendirilmektedir.
Hepatokarsinogenez patogenezinde oksidatif stresin artışı, proto-onkogenler, growth faktörler
ve tümör baskılayıcı genlerde mutasyonların rol oynadığı düşünülmektedir. Kanserin ortaya
çıkmasında enfeksiyon ve enflamasyona bağlı oksidatif stresin rol oynadığı her ne kadar bilinse
de, bu ilişki şimdiye kadar tam olarak aydınlatılamamıştır.
Kanser tanısı konan 45-65 yaş arası 150 hasta ile demografik açıdan aynı özellikteki 50 sağlıklı
kontrol gruplarından alınan venöz kan örnekleri santrifüj edilerek serumları ayrıldı. Serum
örneklerinden total tiyol, natif tiyol, total antioksidan seviyeleri (TAS) ve total oksidan
seviyeleri (TOS) fotometrik yöntem ile ölçüldü. Oksidatif stres indeks (OSI) seviyeleri ve
disülfit ise hesapla bulundu.
Bu çalışmada amacımız, mide kanseri, kolorektal kanser ve hepatoselüler karsinoma teşhisi
konan hastaların tedavi öncesi ve ilk kemoterapi sonrası oksidatif stres durumunun
değerlendirilmesidir. Sonuç olarak, tüm kanser türlerinde tedavi öncesi kontrole göre yüksek
çıkan oksidatif stres seviyeleri tedavi sonrası da daha da arttığı gözlemlenmiştir.
Anahtar Kelimeler: Mide kanseri, kolorektal kanser, hepatoselüler karsinoma, oksidatif stres
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Cross-talk between tumor and stem cells: determination of conditioned media cytokine
levels
Gamze Tanriover*, Sayra Dilmac*, Mustafa Gokhan Ertosun**, Gulperi Oktem***, Altug
Yavasoglu***
*Akdeniz University, School of Medicine, Department of Histology and Embryology
**Akdeniz University, School of Medicine, Department of Plastic and Reconstructive Surgery
***Ege University, School of Medicine, Department of Histology and Embryology
Tumor and surrounding microenvironment cells are closely related and interact constantly. The
mutual communication between these cells may affect some factors such as cytokines that
involved in tumor progression. It is known that the interactions between stem cells and their
niche are reciprocal; since secrete some factors. We hypothesized that depending on
microenvironment cytokines can modulate to stem cells response.
DPSC (dental pulp stem cells) which is confirmed for co-culture experiments and MDA-MB-
231 cells were used in this study. Firstly, we established single and co-cultures using MDA-
MB-231 and DPSC cells and obtained conditioned media (CM). As a control conditioned
media were prepared from single cell line cultures. Secondly, TGF 1 and GDF15 levels were
determined in CM using ELISA.
Surprisingly, the CM from MDA-MB-231 incubate with DPSC cells and CM from DPSC
incubate with MDA-MB-231 cells have increased TGF 1 and GDF15 expressions but, the
mediums from individual cell lines did not identify any sensible change for the cytokines. It
was suggested that increasing these cytokines might be related with the cells cross talk’s.
An improved understanding of this microenvironment will help us to cross-talk the tumor and
stem cells or to develop better therapeutic strategies for inhibition of some cytokines. It could
be thought that this relation would improve the survival and quality of life of cancer patients.
Keywords: MDA-MB-231, DPSC, cross-talk, TGF 1, GDF15, ELISA
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Yeni Sentezlenen Naftakinon Türevli Bileşiklerin Üçlü Negatif Meme Kanseri
Hücreleri Üzerindeki Sitotoksik Etkilerinin İncelenmesi
Hatice Dinçer, Didem Karakaş, Zeliha Gökmen, Engin Ulukaya
Kanser Biyolojisi ve Farmakolojisi, Sağlık Bilimleri Enstitüsü, İstinye Üniversitesi Cevizlibağ,
İstanbul
Kanser ve kanser nedenli ölümlerin her geçen gün artış göstermesi günümüz dünyasının en
önemli problemlerinden biri olarak karşımıza çıkmaktadır. Meme kanseri kadınlarda en sık
görülen kanser türleri arasında ilk sırada yer almaktadır. Üçlü negatif meme kanseri (TNBC)
östrojen reseptörü, progesteron reseptörü ve insan epidermal büyüme faktörü reseptörü II
geninin eksprese olmadığı bir tümör grubudur. İlaçla hedeflenebilir reseptörlerin eksikliği
nedeniyle kemoterapi, hastalık sonuçlarını iyileştirmek için önerilen tek sistemik tedavidir.
Ancak klinikte kullanılan kemoterapötik ajanlar henüz TNBC hastalarına kalıcı bir çözüm
sunmamaktadır. Bunun sebebi kemoterapi sonrası hücrelerde gelişen direnç ve relapstır.
Metastatik relapstan sonra sağkalım diğer meme kanseri alt tiplerine göre daha kısa, tedavi
seçenekleri az, cevap oranları düşüktür. Bu nedenle TNBC'nin tedavi sonucunu iyileştirmek
için, güvenilir yeni ilaçlar ve tedaviler geliştirilmelidir.
Doğada bulunan kinonik bileşiklerin antibakteriyel, antifungal ve antitümorijenik özelliklere
sahip olduğu bilinmektedir. Naftokinonlar, kinon ailesinin üyeleridir ve umut verici özellikleri
nedeniyle araştırmalarda yaygın olarak kullanılırlar. Bu doğrultuda kinon çekirdeği içeren
klinik olarak önemli birçok antitümör ilaç temel alınarak yeni naftakinon türevli bileşikler
sentezlenmiştir.
Bu çalışmada yeni sentezlenmiş olan naftakinon türevli bileşiklerin (ZP-2, ZP-3, ZP-4, ZP-5,
ZP-11) belirli dozlarının (50-25-12,5-6,25-3,125-1,562 µM) MDA-MB-231 TNBC hücre
hattındaki 48. saatteki sitotoksik etkilerine bakılmıştır. Yapılan SRB sonuçlarına göre en etkili
bulunan 2 bileşiğin (ZP-2, ZP-4) IC90 değerleri belirlenerek 12 ve 24. saatlerdeki sitotoksik
etkileri MUSE Flow Sitometri cihazında Annexin-V/PI ve Caspase 3/7 testleri ile
gösterilmiştir.
Anahtar Kelimeler: Üçlü negatif meme kanseri, MDA-MB-231, naftakinon, sitotoksisite, apoptoz
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The Importance of FOXP3 C/A (rs:3761548) Genetic Variants and Protein Expressions
in tumoral tissues of patients with Non-Small Cell Lung Cancer
Dilara Sönmez1, Şeyda Demirkol1,2, Merve Uzunoğlu1, Cem Horozoğlu3, Akif Turna 4, Volkan Kara4, Ebru Nur Ay1,5, Mehmet Tolgahan Hakan1,6, Özlem Küçükhüseyin1, İlhan
Yaylım1
1Molecular Medicine Department, Aziz Sancar Institute of Experimental Medicine / Istanbul Medical Faculty at Istanbul University, Istanbul- TURKEY.
2Istanbul Biruni University, Vocational School of Health Services, Istanbul- TURKEY. 3Department of Pathology Laboratory Techniques, Vocational School of Health Services, Istanbul
Gelisim University, Istanbul, Turkey 4Department of Thoracic Surgery, İstanbul University-Cerrahpaşa, Cerrahpaşa School of Medicine,
Fatih, 34096, İstanbul-TURKEY. 5Istinye University, Vocational School of Health Services, İstanbul, Turkey
6Hitit University, Art and Science Faculty, Department of Biology, Çorum-TURKEY
Regulatory FOXP3 + T-cells are known to cause low lymphocyte infiltration by controlling
tumor stromal infiltration in non-small cell lung cancer (NSCLC) patients. Therefore, in
previous studies, the increase of FOXP3 + T-cells in tumor environment has been associated
with poor prognosis in NSCLC. A single nucleotide polymorphism (SNP) in the FOXP3 gene
(rs3761548 in the promoter region) has been found to be associated with susceptibility to
NSCLC.
This study was conducted on 48 patients and 124 healthy controls diagnosed as non-small-cell-
lung cancer in İstanbul Cerrahpaşa University Training and Research Hospital. In order to
determine the different gene variants of FOXP3 C/A(rs:3761548) molecule in the blood
samples of the laryngeal cancer and healthy controls, DNA isolation was performed and then
polymerase chain reaction-restriction fragment lenght polymorphism (PCR-RFLP) was
applied. And then also, proteins isolated from the control and tumor tissues of 22 patients with
trizol (Ambion) were measured by western blot protein determination method. Statistical
analysis of the study was determined by SPSS 13 program.
As a result of our study, FOXP3 rs3761548 (A/C) polymorphism was associated with a
significantly increased cancer risk for non-small-cell lung cancer (p<0.001). In addition, our
study demonstrated that the frequencies of CC and AA/CC genotypes significantly increased
in NSCLC patients (for both results; p<0.01) [ respectively, odds ratio (OR)=3.495; 95%
confidence interval (CI)=2.055-5.944, OR=2.087; 95% CI=1.653-2.634]. When
histopathological data of our lung cancer patients were evaluated, we found increased
frequencies of CA genotype (p=0,044) and A allele for our patients with the presence of
vascular invasion (p=0.024 OR=2.656; 95% CI=1.040-6.781) than those with absence of
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143
vascular invasion. We didn’t find any correlation between FOXP3 rs3761548 genotypes and
FOXP3 protein expressions in the tumoral tissues of the NSCLC patients.
According to our results, we have indicated the modulating effect of rs3761548 in the promoter
region may be an important parameter for the risk and tumor progression for NSCLC. Our data
also investigated possible correlation between FOXP3 SNP (C/A - rs:3761548) and FOXP3
protein expression on the etiopathogenesis of NSCLC.
The present work was supported by the Research Fund of Istanbul University. Project No. 24517
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144
Investigation of the Cell Cycle Specifity of Zn(II) 5,5-Diethylbarbiturate Complex on
A-549 Human Lung Cancer Cell Line
Mehmet Emin Köse1*, Buse Cevatemre2, Ferda Arı3
1Department of Biology, Faculty of Arts and Sciences, Uludag University, 16120, Bursa, Turkey
Current address: Cancer Biology and Pharmacology, Health Sciences Institute, Istinye University, 34010, İstanbul, Turkey
2Koç University Research Center for Translational Medicine, 34450, İstanbul, Turkey 3Department of Biology, Faculty of Arts and Sciences, Uludag University, 16120, Bursa, Turkey
Lung cancer is a significant health problem and the survival rate is still not satisfactory despite
the discoveries of new treatments. Survival time (5 years) fails by 60-70% and in advanced
stages to 5%. Many scientists are focused on the cell cycle because of the promising effect of
the combination of cytostatic and cytotoxic agents. Therefore, this study was conducted to
determine that at whether newly synthesized [Zn(barb-κN)2(phen-κN,N’)]·H2O complex was
sensitive and/or specific to the cell cycle in A549 lung cancer cells. Synchronization process
carried out by using the cell cycle specific agents [aphidicolin (2 µg/mL) and mimosine (0,5
mM), thymidine (2 mM), nocodazole (40 ng/mL) and colcemid (20 ng/mL) Using the cell cycle
specific agents, aphidicolin and mimosine, thymidine, nocodazole and colcemid which are
specific for G1 phase, S phase, G2/M phase, respectively.]. Following the verification of the
synchronization by Flow Cytometry (Muse) Cell analyzer, A-549 lung cancer cells were
treated with Zn(II) complex and then subjected to sulforhodamine B assay to determine cell
viability at 48h. After synchronization, no significant effect on viability was observed
compared to Zn (II) and its combinations. In conclusion, effect of Zn(II) complex was found
regardless of cell cycle status.
Keywords: Lung cancer, anti-cancer activity, cell cycle, synchronization, [Zn(barb-κN)2(phen-
κN,N’)]·H2O complex.
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Is Notch1 Associated with LeptinR, IL-6 and TNF-α in the CRC Tumorigenesis?
Erkasap N1, Ozyurt R1, Ozkurt M1, Erkasap S2, Yaşar F2, İhtiyar E2, Canaz F3, Yılmaz E3,
Çolak E4.
1Department of Physiology, 2General Surgery, 3Pathology, 4Statistics, Eskisehir Osmangazi
University Medical Faculty, Eskisehir / Turkey
The aim of this study is to investigate the interaction of Notch, leptinR, IL-6 and TNF-α in
cancerous and adjacent normal colorectal tissues and the effects of various related genes on
colorectal cancer (CRC) development mechanism. One of the important intracellular pathways
involved in CRC is the Notch signaling pathway. Notch has an important role in regulating the
balance between proliferation, differentiation and apoptosis. Recent studies in several cancers
have reported that one of the Notch regulators is leptin. Most cancer studies have shown
changes in blood leptin levels. Such as leptin another adipokines, IL-6 and TNF-α have been
reported to have proangiogenic activity and increase tumor neovascularization. All these data
indicate that adipokines play an important role in the molecular regulation of cancer
development. However, the role of these four molecules has not yet been fully understood in
the CRC. Notch1, leptinR, IL-6 and TNF-α expressions were detected by qRT-PCR in
cancerous (n:40) and adjacent normal (n:40) colorectal tissues at a distance of 10 cm.
According to qRT-PCR results, in cancerous tissue comparison with normal tissues, mRNA
levels of Notch1, leptinR, IL-6 and TNF-α increased respectively (p<0.05), (p<0.001),
(p<0.001) and (p<0.001). The results of this study give information about Notch1, leptinR, IL-
6 and TNF-α related intracellular pathways involved in the formation of colorectal cancer and
may provide new treatment methods to the literature.
This study is supported by a grant from The Scientific And Technological Research Council Of Turkey
(TUBITAK 1001, no: 116S628).
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146
Effects of phosphorylation changes of E2F1 from S235 on proliferation and migration
Sayra Dilmac*, Mustafa Gokhan Ertosun**, Osman Nidai Ozes***, Gamze Tanriover*
*Akdeniz University, School of Medicine, Department of Histology and Embryology
**Akdeniz University, School of Medicine, Department of Plastic and Reconstructive Surgery ***Akdeniz University, School of Medicine, Department of Medical Biology and Genetics
Breast cancer is one of the causes of death in women due to the lack of effective treatment
methods. The basic stage of tumor development is the uncontrolled proliferation of tumor cells.
Controlling of cell proliferation depends on the limited progression of the cell cycle. Many
factors are involved in the cell cycle regulation. One of these factors, E2F1 which is the
transcription factor, is the regulation of cell cycle and plays a role in apoptosis. E2F1 is
necessary the progression of the cell cycle. In addition to, E2F1 could be affect motility of the
tumor cells. We aimed to determine if, E2F1 phosphorylation effects in cell proliferation and
migration in tumor cells.
E2F1 S235 (Serine 235) phosphorylation changes was achieved by Site-Directed Mutagenesis
(SDM) methods. We changed serine aminoacid at position 235 to Alanine (A) (inhibition form
of phosphorylation) and Glutamic acid (E) (phosphomimetic form). We used NT-4T1 (Non-
Treated), WT (Wild-type E2F1) and E2F1 S235 mutant forms (S235A, S235E) 4T1 cells. 4T1
cells (1000/per well) were seeded 94-well plate for MTT assay. Cells were treated 24, 48 and
72 hours for MTT. 4T1 cells were seeded (9x105 cells/per well) 6-well plate for migration
assay. A straight scratch was made on the cell layer using a 200 μl sterile pipette tip. 4T1 cells
were photographed at 0 and 48 hours. Wound healing was measured with using ImageJ
Changed to from E2F1-Serin235 to Alanine cell viability was increase. On the contrary to, if
changed this Serine to Glutamic Acid cell viability was decreased. Similarly, 4T1 cells
migration have increased with E2F1-S235A. Migration ability of 4T1 cells have decreased with
E2F1-S235E.
Our results suggested that the phosphorylation of E2F1 on S235, could decrease proliferation
and migration on 4T1 cells. These results indicate that S235 phosphorylation of E2F1 is very
important for tumor cells progression.
Keywords: E2F1, S235 phosphorylation, 4T1, MTT, migration
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147
The Role of p62 in Pancreatic Cancer Development and Metastasis
Kivanc Görgülü1, Kalliope N. Diakopoulos1, Selin Ulukaya1, Jiaoyu Ai1, Derya Kabacaoglu1,
Katrin Ciecielski1, Ezgi Kaya-Aksoy1, Dietrich A. Ruess1, Götz Hartleben2, Stephan Herzig2,
Roland M. Schmid1, Bruno Sainz Jr3, Marina Lesina1 and Hana Algül1
1II. Medizinische Klinik, Klinikum rechts der Isar, Technische Universität München, Munich, Germany
2Institute for Diabetes and Cancer, German Center for Diabetes Research (DZD), Neuherberg, Germany
3Department of Biochemistry, School of Medicine, Autónoma University of Madrid, Madrid, Spain
Pancreatic ductal adenocarcinoma (PDAC) is the most common malignancy of the pancreas
with 5-year survival rate. Therapeutic approaches are still limited for patients due to the
cancer’s aggressive nature and late diagnosis.. Recently, inhibition of autophagy in PDAC has
shown promising results in pancreatic cancer patients. Autophagy is a pivotal player to
maintain cellular homeostasis in physiological and pathological conditions. Here we show that
p62/SQSTM1, is a degradation marker of autophagy, has multiple roles in pancreatic cancer
development and progression. In order to reveal the function of p62 in pancreatic cancer, we
generated mice that express oncogenic Kras in primary pancreatic cancer cells and have
homozygous disruption of p62 (p62;Kras) and compared them with mice with only oncogenic
Kras (controls). Pancreas specific deletion of p62 attenuated pancreatic cancer development.
However, once tumor developed homozygous deletion of p62 enhanced metastasis in mice. To
understand the mechanism of in vivo findings, primary tumor cells were isolated and used to
perform several analyses. We observed that Kras cells had more proliferative, organoid
forming and colony forming capacity compared to p62;Kras cells. Having morphological
differences in mitochondria via transmission electron microscopy analysis, metabolic profiling
of cancer cells has shown that p62;Kras cells had higher glycolytic flux. To confirm, metastatic
phenotype of p62;Kras mouse model, intra-tail vein injection of cancer cells is performed,
p62;Kras cells had more metastatic dissemination compared to Kras cells. RPPA analysis of
cancer cells depicted that epigenetic regulation and mitochondrial membrane organization
related pathways may be underlying mechanism of these changes in p62;Kras cells. Therefore,
particular functions of autophagy related proteins in pancreatic cancer should be conceived
when planning therapeutic strategies targeting autophagy in pancreatic cancer.
Poster Presentation
148
Expression levels of glutathione S transferase isoenzymes in pleomorphic adenoma of
the parotid gland and normal gland.
Sedat Aydin1, Arzu Kaya Koçdoğan2, Mehmet Gokhan Demir3, Serpil Oguztuzun4, Murat Kilic5, Nagehan Barışık6, Nuran Topal4, Pınar Kaygın4
1Department of Ear Nose Throat and Oral Maxillofacial Surgeon, Istanbul University, Istanbul,
Turkey 2Istanbul Gelisim University, Vocational School of Health Sevices, Patology Laboratory Techniques,
Istanbul, Turkey 3Department of Otorhinolaryngology, Kartal Dr. Lutfi Kirdar Kartal Education and Research
Hospital, Istanbul, Turkey 4Department of Biology, Kirikkale University, Faculty of Arts and Sciences, Kirikkale, Turkey 5Department of Pharmacy Services, Ankara University Vocational School of Health Services,
Ankara, Turkey 6Department of Patology, Kartal Dr. Lutfi Kirdar Kartal Education and Research Hospital,
Istanbul, Turkey
Pleomorphic adenoma is the most common salivary gland tumor that has detected on parotid
gland. Enzymatic and genetic markers are of considerable significance in understanding the
mechanisms of tumorigenesis and in the identification, diagnosis, prevention and possible
treatment of tumors and prognosis and follow-up. Glutathione S-transferases (GSTs) are a well
known enzyme family which role in detoxification, either by conjugating glutathione with
electrophilic compounds or by direct binding with carcinogens. GSTs have been divided into
several subgroups. GSTs enzymes might be used as tumor markers to diagnose neoplastic
lesions. We have known that parotid gland tumors are diagnosed via fine neeble aspiration
biopsy. Sometimes diagnostic difficulties has arisen and recurrent fine needle aspiration is
needed. The tumor markers might be used also in this situation. The normal distribution of the
GST in salivary gland was investigated and reported the higher GST activity in adenoma than
normal tissue. In this study, we investigated the distribution of GST in parotid gland
pleomorphic adenoma and normal parotid gland tissue . The sudy group is composed of 52
patients (25 female, 27 male) aged between 18-77 (mean 40.2) who were operated due
pleomorphic adenoma of the parotid gland. It was found that GSTP1 expression was higher in
the normal tissues of 14 patients (42.31 %) compared to tumor tissues and 22 (26.92%). GSTK1
expression was higher in the normal tissues of 12 patients (50%) compared to tumor tissues
and 26 (23%). Higher levels of GSTK1 expression in normal tissues in comparison to tumor
tissues was found to be statistically significant (p<0,05). There was no statistically significant
correlation between expression of GST isoenzymes and clinical information of patients. Our
findings showed the decreased GSTK1 staining in tumor cells which is significantly different
from normal parotid cells. GSTK1 might be useful tool to differentiate the normal cell from
Poster Presentation
149
pleomorphic adenoma. We think that this information will reduce the confusion that may be
encountered in the differentiation of malignant and benign tumors especially in fine needle
aspiration biopsy.
Keywords: Parotid gland pleomorphic adenoma, Glutathione-S-transferase, immunohistochemistry,
fine needle aspiration biopsy
Poster Presentation
150
New Orthotopic Mouse Colorectal Cancer Model
Sibel Gökşen1, Sefa Özdemir2, Süleyman Can Öztürk2, Güneş Esendağlı2
1Department of Medical and Surgical Research, Hacettepe University Institute of Health Sciences,
Ankara, Turkey. 2Department of Basic Oncology, Hacettepe University Cancer Institute, Ankara, Turkey.
Mouse cancer models are the important milestone for drug testing and screening tumor
development. Orthotopic models give an opportunity to observe tumor growth on its own tumor
microenviroment. In our study, we developed a new method for mouse colorectal cancer model
for orthotopic tumor engraftment from cell culture to animal.
CT26 murine colon carcinoma cells were grown until they reach to 70% confluency using full
RPMI medium (10% FBS, 1% penicilline/streptomycine antibiotics and 1% L-glutamine) at
37 °C and 5 % CO2. For harvest, after trypsinization the cells were washed with PBS and
centrifuged (400g x 5 minutes). CT26 cells (2x106/100μl) in PBS coated with Matrigel matrix
(1:10) and prepared for engraftment to 8 weeks old male BALB/c mouse. Anesthesia was
performed using ketamine/xylazine method. The cecum was delivered through 15 mm incision,
firmly scratched with a 28G needle and then returned back to abdomen. Prepared cells were
poured on the small wounds of scratching and incision was closed using 5/0 monofilament
absorbable suture. 0,5 ml sterile 0.9 % NaCl was injected subcutaneously to prevent
dehydration. On 10th day after surgery, mouse was euthanized and dissected. Primary tumors
were observed only on cecum and colon.
In our new methodology there is no need to do cecotomy or direct injection of the cells into
colon. This our new mouse colorectal cancer model can be used for drug treatment and
development, tumor screening and cancer biogenesis. Also, it will give new insights for cancer
studies due to its orthotopic usage.
151
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The importance of dysregulated miRNAs on ovarian cysts and tumors
Ece Gumusoglu1, Tuba Gunel1, Mohammad Kazem Hosseini1, Nogayhan Seymen2, Taylan Senol3, Uğur Sezerman2, Samet Topuz4, Kılıç Aydınlı5
1Istanbul University, Faculty of Science, Department of Molecular Biology and Genetics, Istanbul,
Turkey.
2Acıbadem University, School of Medicine, Department of Basic Sciences, Biostatistics, Istanbul
Turkey.
3Bağcılar Training and Research Hospital, Department of Obstetrics and Gynecology, İstanbul,
Turkey.
4Istanbul University, Department of Obstetrics and Gynecology, Istanbul Medical Faculty, Istanbul,
Turkey.
5Medicus Healthcare Centre, Istanbul, Turkey
Abstract
Ovarian cancer is the fifth most important cause of cancer-related deaths among women and
the most lethal gynecologic malignancy. Epithelial ovarian cancer (EOC) is asymptomatic and
few screening tests are available. In our study, we aimed to find the importance of dysregulated
miRNAs in ovarian cyst and their expression profile difference between ovarian cancer cases.
Another goal of our study is to identify novel circulating miRNAs to be used as therapeutic
prediction of EOC. In this study, we studied three different samples: serums of EOC patients,
healthy individuals (HI) and benign ovarian cysts (BOC). Their miRNA expressions have been
compared by microarray. Microarray data were analyzed according to miRNA expressions the
relation between miRNAs target genes and EOC were examined by bioinformatic tools. 75 and
66 significantly dysregulated miRNAs were identified by microarray in BOC vs. EOC and
BOC vs. HI comparison, respectively. Then, we focused on common miRNAs that found in
both comparison and finally 46 important miRNAs were detected which can represent the only
common sample group, BOC. After these findings, we also considered miRNA profiling in
EOC and HI, and surprisingly any common miRNAs were found with these 46 miRNAs. Thus,
we analyzed them depending on their potential importance on BOC pathogenesis. After
bioinformatic analysis, our findings indicated that there are several biological processes and
pathways which can be considered to be related BOC development.
Introduction
Ovarian cancer is the fifth most important cause of cancer-related deaths among women and
the most lethal gynecologic malignancy (Srivastava et al, 2017). Among the types of ovarian
cancer, epithelial ovarian cancer (EOC) is the most lethal ovarian cancer type (Srivastava et al,
2017). EOC is usually asymptomatic and few screening tests are available, almost 70% of
women present with advanced-stage (stage III or IV) disease, with 5-year survival rates of less
than 30% (Retamales et al, 2017). Patients who are diagnosed with stage I disease have a 5-
year survival rate of up to 90%, and patients with stage (stage III or IV) disease have a 5-year
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survival rate of up to 70%. Therefore, early detection of EOC has a great importance in clinic
(Retamales et al, 2017).
An ovarian cyst is a type of body surrounded by a very thin wall and filled with fluid secreted
from the local microenvironment, tumor cells, and stroma (Onur et al, 2013). Ovarian cysts can
be divided into 2 groups: functional cysts and non-functional cysts. Most ovarian cysts belongs
to non-functional class and they are asymptomatic (Lee et al, 2000), which includes polycystic
ovary syndrome (PCOS) (Woo et al, 2013).
MicroRNAs (miRNAs) are small regulatory RNA family found in plasma and used for
diagnosis of human diseases. miRNAs are non-coding RNAs that represses specific mRNAs
and regulates post-transcriptional gene expression (Lan et al, 2015). Although miRNA
expression is irregular in ovarian cancer, mutations are found rarely in cancer related miRNAs.
Several studies showed that different miRNA profile between normal ovarian epithel and
ovarian cancer (Lan et al, 2015).
In this study, miRNA expression profiles have been compared between patients suffering
ovarian cancer, simple ovarian cysts and healthy individuals. In this study, we aimed to detect
significantly dysregulated serum miRNAs with the comparison of their expression levels
between patients (BOC and EOC) and healthy individuals. Thus, we aim to illuminate
pathogenesis of BOC and to make contribution to the difference from ovarian cancer in terms
of molecular mechanisms. Furthermore, we can make a small step to develop novel therapeutic
approaches for cystic ovarian syndromes.
Material Methods
This study is ethical approved by Istanbul University Faculty of Medicine Clinical Researches
Ethics Committee (Permission no: 2014/1175) on 08/08/2014. Scientific Research Projects
Coordination Unit of Istanbul University also funded our study under the projects numbered as
47803 and 51472. Two main steps were followed in this study: serum miRNA profiling by
microarray approaches and bioinformatic analysis of target genes and pathways.
Sample collection
In this study, 10 epithelial ovarian cancer (EOC) serum samples which are valuable in common
clinical practice, from patients, 11 benign ovarian cyst (BOC) serum samples and 15 serum
samples from HI were collected to compare miRNA expression levels by microarray. The
samples were collected by Department of Obstetrics and Gynaecology, Istanbul Medical
Faculty in Istanbul University. All volunteers have been asked to confirm and sign the informed
consent approved by the ethical committee. Before cyst or tumor removal surgery, peripheral
blood samples were collected from each group in clot activator tube (10 ml) and centrifuged
immediately at 3500 rpm for 15 min at 4 °C. The supernatant fluid, serum, was transferred to
RNase-free tubes after centrifugation. Following this step, serum samples were immediately
stored at − 80 °C until studied.
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Total RNA extraction
Total RNA was extracted with the mirVana™ miRNA Isolation kit (Ambion, life technology,
USA) for microarray analysis following to the manufacturer’s protocols. In the final step of
extraction, total RNAs were eluted into 35 μl mirVana elution solution.
Quality Control of Extracted RNAs
The quantity control step was performed on extracted all RNAs by NanoDrop ND-2000
Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). 8 samples with the highest
quality were selected from each group (EOC, BOC and HI) for microarray study. All total RNA
samples were stored in -80°C until laboratory preparation.
MiRNA expression analysis with microarray
The miRNA expressions from all sample types were analyzed by Agilent miRNA microarray
chips (Agilent SurePrint G3 Human miRNA r21 8x60K). The miRNA Labeling and
Hybridization Kit (Agilent Technologies) was used according to the manufacturer’s
instructions. Cyanine 3-cytidine bisphosphate (pCp-Cy3) was used for each total RNA (which
included small RNAs) labeling. After this step, for hybridization was done for 24 h on Human
miRNA Microarray Version 16 (Agilent Technologies) slides which include 1368 miRNAs
encoded by genes located across all chromosome. These slides were scanned by an Agilent
SureScan Microarray Scanner (Model G2600D) and “AgilentFeature Extraction” software
(v.12.0) was used for image analysis. Tissue and serum derived RNAs from each group (EOC
and BOC) were analyzed in single microarray chip, then compared each other. The target genes
and pathways of these miRNAs were analyzed with KEGG (Kyoto Encyclopedia of Genes and
Genomes) and GO (Gene Ontology) databases with p value < 0,005.
Results
Sample characterization
The median age of EOC and BOC, the mean of CA-125 (cancer antigen-125) level, the
consumption of alcohol and tobacco profiles, histological types and stages, the number of
patients with metastasis or other diseases and their survival situations were shown in Table 1.
None of the patients had taken neoadjuvant chemotherapy, all patients had undergone primary
cytoreductive surgery.
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Table 1. Demographic and clinical characteristic of BOC and EOC.
BOC EOC
Number of Samples 11 10
The median of age 40 47
The mean of CA-125 (U/ml) 85,3 1520,2
Alcohol N N
Cigarette N N
Histological types and stages 4 cases are endometrioid cysts, 2 5 cases are stage 3C grade 3
cases are dermoid cysts, 2 cases serous cancers, 2 cases are stage
are mucinous cystadenoma and
2 3B grade 3 serous cancers, 1 case
cases are serous cystadenoma (No is 2B grade 1 serous cancer, 1
valid staging system for cyst case 2A endometrioid and 1 case
patients) is 1A grade 3 serous cancers
The number of patients with 1 patient developed borderline 9 patients developed metastasis
metastasis or other diseases ovarian cancer
occurred after sample
collection
Survival All still alive 8 patients alive, two patients
passed away (23 and 32
months)
*N: No usage
Microarray results
MiRNA expression profiling results has shown that there were several significantly changed
miRNAs in serum samples. All dysregulated miRNAs were selected based on p values (< 0.05)
and fold changes (> 2). 75 miRNAs were found as dysregulated in BOC compared to EOC
samples. Otherwise, from BOC vs HI comparison, 66 miRNAs were found as significantly
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dysregulated. After analysis of these two results, it was found that 46 miRNAs were common
in both group and their expression alteration also similar (up- or down- regulation). (Table 2)
According to these results, 4 miRNAs were down and 2 miRNAs were up regulated in BOC
tissue samples.
Table 2. Dysregulated common miRNAs in serum samples in BOC vs. EOC and BOC vs. HI
comparison
BOC vs. EOC BOC vs. HI
miRNA logFC adj.P.Val logFC adj.P.Val
hsa-miR-1207- -1.362 0.01307976 -1.864 0.003935395
5p
hsa-miR-1227- -2.742 2.43E-05 -2.484 0.000243633
5p
hsa-miR-1915- -3.134 2.29E-06 -2.374 0.000281523
3p
hsa-miR-2861 -2.127 0.000104813 -1.659 0.003935395
hsa-miR-3162- 1.479 0.004755918 1.515 0.012906392
3p
hsa-miR-328- -1.331 0.047903462 -2.06 0.010634825
5p
hsa-miR-34b- 2.099 1.23E-05 1.27 0.008383498
3p
hsa-miR-3591- 1.884 0.000136467 1.313 0.013365004
3p
hsa-miR-3656 -1.851 8.38E-05 -2.133 2.92E-05
hsa-miR-3663- -1.452 0.027134019 -1.621 0.0404849
3p
hsa-miR-3665 -2.544 4.85E-08 -1.792 4.21E-05
hsa-miR-371b- -3.304 1.79E-06 -3.006 1.15E-05
5p
hsa-miR-3940- -1.748 5.35E-06 -1.978 1.76E-06
5p
hsa-miR-3960 -1.402 0.005525347 -1.508 0.010634825
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hsa-miR-4254 1.594 5.80E-06 1.119 0.00155371
hsa-miR-4281 -2.097 0.000110739 -2.498 2.92E-05
hsa-miR- -1.074 0.018695511 -1.717 0.001791038
4433a-3p
hsa-miR-4446- 1.379 0.000149864 1.314 0.000851001
5p
hsa-miR-4466 -2.632 2.29E-06 -2.357 2.85E-05
hsa-miR-451a 2.521 0.016837998 2.95 0.01841656
hsa-miR-451b 2.183 0.002086641 2.283 0.004670585
hsa-miR-4530 -1.21 0.031159443 -1.639 0.013973395
hsa-miR-466 1.627 1.34E-05 1.006 0.007591278
hsa-miR-4730 2.335 0.005006931 2.411 0.012906392
hsa-miR-4763- -2.552 4.94E-05 -2.447 0.000228055
3p
hsa-miR-4787- -2.245 0.000129136 -2.117 0.000805023
5p
hsa-miR-5001- -3.01 1.54E-05 -2.602 0.000281987
5p
hsa-miR-5010- 1.729 0.000217645 1.771 0.000639126
3p
hsa-miR-572 -1.297 0.011149386 -1.377 0.022997867
hsa-miR-6068 -2.639 0.00010944 -2.249 0.002020182
hsa-miR-6087 -1.782 0.003970873 -1.823 0.010951788
hsa-miR-6088 -2.439 3.78E-06 -2.488 5.61E-06
hsa-miR-6090 -1.296 0.019690327 -2.38 0.000421357
hsa-miR-6125 -2.39 0.000110739 -2.218 0.000796256
hsa-miR-638 -1.564 0.00162682 -1.175 0.039770036
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hsa-miR-6514- 2.233 0.000109411 1.564 0.010301901
3p
hsa-miR-6716- 1.986 0.009854892 2.357 0.009626385
3p
hsa-miR-6724- -1.957 0.000297636 -2.356 0.000122162
5p
hsa-miR-6791- -3.662 1.16E-08 -3.163 5.42E-07
5p
hsa-miR-6800- -2.58 4.77E-06 -1.977 0.000454228
5p
hsa-miR-6821- -2.452 5.80E-06 -2.08 0.000184515
5p
hsa-miR-6834- 1.704 0.000804102 1.292 0.023323819
3p
hsa-miR-6850- -2.207 0.005629416 -2.264 0.014502295
5p
hsa-miR-7108- -2.25 0.000117511 -2.478 0.000122162
5p
hsa-miR-7704 -1.668 0.003856916 -1.687 0.011303091
hsa-miR-8485 2.08 0.001038117 1.65 0.021095792
According to target gene and pathway analysis of these 46 miRNAs, Table 3 showed that
3 pathways were detected with KEGG analysis.
Table 3: The pathways and target genes of common 46 miRNAs from KEGG database
Pathways miRNAs P value Target Genes
hsa-miR-1915-3p 2,46E-18 FUT3,FUT6
Glycosphingolipid biosynthesis
hsa-miR-34b-3p 0,0020494 FUT9
hsa-miR-3665 9,08E-27 FUT3,FUT6
- lacto and neolacto series
hsa-miR-4530 8,07E-08 FUT3,B4GALT3
hsa-miR-6514-3p 1,14E-08 B3GALT5,FUT9
GALNT13, GALNT1,
hsa-miR-34b-3p 1,02E-07 C1GALT1
hsa-miR-4466 8,44E-12 GALNT14
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Mucin type O-Glycan
POC1B-GALNT4, B4GALT5,
biosynthesis
hsa-miR-5010-3p 0,000405 GALNT4, GALNT1, GCNT1
hsa-miR-6088 0,0042684 GALNT7,WBSCR17
hsa-miR-6090 5,53E-07 GALNT8
hsa-miR-6724-5p 6,17E-11 GALNT6,GALNT8
COL27A1, COL3A1, COL1A1,
hsa-miR-3663-3p 1,34E-13 COL4A4, COL11A1
ECM-receptor interaction
COL24A1, COL27A1,
hsa-miR-4254 1,18E-11 COL6A3, COL4A6
hsa-miR-6088 0,0049984 LAMA3, COL24A1, COL1A1
hsa-miR-7704 3,77E-06 COL27A1
Discussion
The physiological and pathological roles of altered miRNAs have been demonstrated in many
tumor types and may play an important role in the diagnosis, prognosis, and treatment of cancer
(Wan et al, 2014). In our study, we aimed to find a new perspective to illuminate the pathogenesis
of ovarian cyst and cancer. Therefore, we focused on miRNA expressions in both group and also
HI group for control. After microarray and bioinformatics analysis of results, 46 miRNAs were
selected because of their common expression level alteration in both comparisons. When they were
evaluated due to their target genes and pathways, we have found 3 pathways which are also
important in some other cancer developments. For instance, Mucin Type O-Glycan Biosynthesis
pathway is an evolutionarily conserved protein modification present on membrane-bound and
secreted proteins (Tran et al, 2013). Aberrations in O-glycosylation causes some human diseases
and are related with disease risk factors (Tran et al, 2013). There are researches about this
pathway and cancer relation. Broukhausen indicated that O-Glycan structures are often
unusual or abnormal in cancer, and greatly contribute to the phenotype and biology of
cancer cells. Some of the mechanisms of changes in O-glycosylation pathways have been
determined in cancer model systems. Therefore, this pathway can be effective in cyst
formation. We suggested that these genes and pathways preferred to be analyzed with
further investigations to prove their certain role in cyst and cancer formation. Also, we
recommend also their role in malignant transformation whether it is effected in cyst to
cancer transformation. Besides all these, our miRNA findings derive the understanding of
ovarian cyst and cancer mechanism.
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big implications. Cancer Lett. 2017.
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2013;288(10):6921–6929. doi:10.1074/jbc.R112.418558
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In vitro Self-renewal Properties in A549 and H1299 Non-Small Cell Lung Cancer Cells
Hiba H. A. Khair, Isik Didem Karagoz
Department of Biology, Faculty of Art and Science, Gaziantep University, Gaziantep, Turkey
Abstract:
Lung cancer remains at the top of the list of cancers with the highest incidence and mortality
rates worldwide. The cancer stem cell paradigm proposes that a small subpopulation of cancer
cells called cancer stem cells (CSCs) can self-renew, recapitulate tumor heterogeneity by
differentiating into different lineages of cells that constitute the bulk of the tumor and
substantially lead to tumor recurrence. Accumulating evidence suggests that a small
population of stem-like cells are responsible for the initiation, progression, and metastasis of
lung cancer. This study aims to assess cancer stem-like properties in A549 and H1299 non-
small cell lung cancer cells. The spheroid formation assay was used to assess self-renewal
ability indicated by tumorspheres formation and to calculate sphere-forming efficiency in
vitro. The limiting dilution assay was used to calculate the minimum number of cells needed
to be cultured to see at least one spheroid in vitro. Cells derived from both cell lines could
form tumor spheroids. The sphere-forming efficiencies of A549 and H1299 cells were 4.14 ±
1.06% and 5.81 ± 1.28%, respectively. However, H1299 cells produced more and larger
tumorspheres when compared to A549 cells. The mean diameter of tumor spheroids in H1299
and A549 cells were 95.25 ± 41.03 and 80.70 ± 31.72 μm, respectively (p=0.00003). In
limiting dilution assay, A549 and H1299 single cells produced spheroids at 23.5 ± 43.7% and
62.5 ± 49.4%, respectively. The frequency of sphere-forming cells (stem-like cells) was found
to be 6.06-fold higher for H1299 cells than those for A549 cells. These results suggest that the
self-renewal ability of H1299 cells is higher and more pronounced compared to A549 cells.
As a conclusion, both A549 and H1299 cells comprise stem-like cells with self-renewal ability.
Keywords: Lung cancer, Cancer stem-like properties, Self-renewal.
Introduction:
Lung cancer is the main cause of cancer deaths worldwide with both high mortality and
morbidity rates due to the limited therapeutic options (Kalemkerian et al., 2013; Siegel et al.,
2016). Approximately 80% of lung cancers are non-small cell lung cancers, which are non-
neuroendocrine tumors including adenocarcinomas, large cell carcinoma and squamous cell
carcinoma (Parsons et al., 2010; Siegel et al., 2016). Even with the most advanced imaging,
staging, surgical strategies, chemotherapy, radiotherapy, as well as personalized treatments
with targeted therapies such as EGFR tyrosine kinase inhibitors and ALK inhibitors in specific
patients group, the five-year survival rate for patients is only 5–15% (Kato et al., 2004; Winton
et al., 2005; Leon et al.,2016).
The stem cell model of carcinogenesis suggests that cancers arise from cells with stem-like
characteristics as a result of the impaired regulation of self-renewal pathways. Such
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dysregulation stimulates the expansion of this cell population that then may undergo additional
genetic or epigenetic modifications to turn into a fully transformed clone (Wicha et al., 2006).
Tumor heterogeneity and the presence of small subsets of cells with stem-like properties called cancer stem cells (CSCs) have been reported in almost all malignancies during the past decade
(Leon et al.,2016). CSCs typically exhibit many features of embryonic or tissue stem cells including slow growth rates and insensitivity to chemotherapy and/or radiation therapy,
therefore new treatment modalities that selectively target these cells are needed to control stem cell survival, proliferation, and differentiation (Jordan et al., 2006).
Increasing evidence proposes that lung cancer incorporates a cancer stem cell subpopulation
responsible for the onset, spread, and metastasis of the disease (Ho et al.,2007; Eramo et
al.,2008; Levina et al.,2008; Bertolini et al.,2009; Jiang et al.,2009, O’Flaherty et al.,2012;
Wang et al.,2013). Due to the fact that CSCs cells have been implicated in tumorigenicity,
cancer progression, and therapeutic resistance, the study of cancer stem cells in lung cancer
would provide important insights into the understanding of CSCs biology, expansion, and
maintenance, as well as for the development of novel diagnostic and prognostic tests and
exploring novel therapeutic targets for combating this disease (Pine et al.,2008; Peacock and
Watkins ,2008; Miyata et al.,2015; Zakaria et al.,2017). However, each type of tumor
comprises different progenitor or stem cell types that are controlled by various molecular
pathways, which results in variation in the expression of markers in lung cancer subtypes
making these cells more difficult to be identified and targeted (Zakaria et al.,2017).
The ability to self-renew and produce differentiated progeny constitutes the basic features of
cancer stem cells (Ponti et al., 2005; Eramo et al., 2008). Tumorspheroids are a typical
descriptor of cancer stem cells that can maintain stemness profiles in proliferation,
differentiation and under serum-free culturing conditions (Weiswald et al., 2015). Sphere
formation assay has been widely used as a surrogate assay for the enrichment of cancer stem
cells or cancer cells with stem-like characteristics from solid tumors. Tumorspheroids culture
systems are based on the ability of stem/progenitor cells to survive and form floating 3-
dimensional spheroid bodies in semisolid media, such as collagen or Matrigel or in low
attachment plates, in serum-free growth medium containing epidermal growth factor and basic
fibroblast growth factor (Ishiguro et al.,2017). Spheroid cultures as a method of enrichment
for cancer stem cells have been used in many human cancers and cancer cell lines including
glioma, melanoma, sarcoma, lung cancer, renal cancer, and rhabdomyosarcoma (Singh et al.,
2003;Fang et al., 2005;Gibbs et al., 2005; Eramo et al., 2008; Zhong et al., 2010 ; Walter et
al., 2011).
The in vitro extreme limiting dilution assay is used to determine the frequency of cancer stem
cells (CSCs) enriched suspension cultures obtained from a primary tumor sample or a cell line
and grown in a serum-free medium containing growth factors. In addition, in vitro and in vivo
limiting dilution assays (LDAs) can be used to determine the effect of a specific treatment or
genetic knockdown strategy on the cancer stem cell frequency (CSC) in a cancer cell
population of a given cell line or a primer tumor sample (Agro and O'Brien, 2015).
In view of all that has been mentioned so far, this study aims to assess cancer stem-like properties in A549 and H1299 non-small cell lung cancer cells.
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Materials and Methods:
Cell culture:
A549 and H1299 non-small cell lung cancer cell lines were maintained in RPMI-1640 medium
supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin. Cells were
grown in a humidified incubator at 37°C and 5% CO2 (Zakaria et al., 2015).
Spheroid formation assay:
To enrich for A549 and H1299 CSCs, they were plated at ~100 cells per well in ultra- low
attachment 96-well plates (Corning, New York, USA) in spheroid culture medium containing
serum-free medium DMEM-F12K (1:1) supplemented with 20 ng/ml EGF, 10 ng/ml bFGF,
1x B27 supplement, 1x insulin, 0.4% Bovine Serum Albumin and 1 % PenStrep. To prevent
cell aggregation, methylcellulose was added at a concentration of 1.5% to the culture medium.
The cells were incubated at 37°C for 14 days according to the previously published protocol
(Leung et al., 2010) with slight modifications. At the end of the incubation period; spheroids
were counted using an inverted microscope. The area, diameter, and size of the spheres were
analyzed using Image J-NIH software.
Extreme Limiting dilution assay:
Extreme Limiting Dilution Analysis (ELDA) was performed as described by Hu and Smyth
(2009). Briefly, cells derived from A549 and H1299 NSCLC cell lines were plated with 100
µl of serum-free medium per well at 100, 100, 10 and 1 cell seeding densities (24 wells per
dilution) on ultra- low attachment 96-well plates and incubated for 14 days. At the end of the
14 days, the number of wells showing the lung tumor spheroids formation was calculated. The
frequency of the spheroid-forming cells in the A549 and H1299 cell lines was calculated using
the ELDA webtool at http://bioinf.wehi.edu.au/software/elda (Bioinf.wehi.edu.au, 2018)
(Kanwar et al., 2010).
Statistical Analysis:
All data are expressed as mean ± SD were and calculated using Office Excel 2016 (Microsoft
Corporation, USA) The statistical difference between groups were evaluated for significance
using the Student's t test. Those with a P value equal 0.05 or less were considered statistically significant (Han et al., 2012; Jiang et al., 2016).
Results:
Spheroid formation assay results:
To determine whether a population of self-renewing cancer stem cells is present in A549 and
H1299 non-small cell lung cancer cell lines, the cancer stem-like cell population was enriched
in these cell lines. For this purpose, cells were cultured in a serum-free sphere formation
inducing conditions using a medium consisting of DEMEM /F12 (1:1) supplemented with
0.4% BSA, 1x B27, 20ng/ml EGF, 10ng/ml bFGF and 1x insulin and were seeded in culture
plates with ultra-low adhesion properties. The growth potential of floating spheroid colonies
of A549 and H1299 stem-like cells was examined. A few of the cells remaining after the first
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164
cell death rapidly proliferated and formed lung tumor spheroids. The medium was changed
four times a week during the 14 days incubation period. After 10-14 days of culture, ball-like
spheres were observed. Images of tumor spheroids were taken with an inverted microscope
using 4x, 10x, 20x and 40x objective lenses to observe and analyze spheroids (Figure 1 and
Figure 2). The spheroid analysis was performed using image processing algorithms of the
ImageJ 1.51r software program.
To optimize the seeding density of each cell line, the number of cells required to form spheroids in preliminary tests was determined. Spheroid formation was observed by an inverted microscope at the following cell densities: 1x104, 5x10 3, 3x103, 1x103, 500 and 200 cells/ml. The seeding density to form H1299 and A549 spheroids with a mean diameter of 95.25 ± 41.03 and 80.70 ± 31.72μm, respectively, is about 1000 cells/ml.
A549 and H1299 cell lines were seeded at a density of 100 cells/well in a total of 24 wells
from an ultra-low adherent 96 well plates to obtain new spheroids. The percentage of the
spheroid formation efficiency (SFE%) was calculated by dividing the total number of formed
spheroids by the total number of seeded living cells and multiplying by 100. The sphere
forming efficiencies of A549 and H1299 parental cells were approximately 4.14 ± 1.06% and
5.81 ± 1.28%, respectively. Cells derived from both cell lines had the ability to form tumor
spheroids. However, the number of tumorspheroids and their size were different. The H1299
cell line produced more and larger tumorspheres when compared to the A549 cell line. This
suggests that each cell line contains a different number of stem-like cells. The spheroids were
round, with regular borders and a larger size than the cancer cells. As a result, both A549 and
H1299 cell lines have self-renewal abilities; however, we observe that this ability is more
pronounced and higher in the H1299 cell line.
(A) (B)
(C) (D)
1
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Figure 1. (A) H1299 adherent monolayer parental cells. Scale bar is 100μm. Original magnification 20x. (B), (C) and (D) Tumor spheroids derived from H1299 cells after 14 days
of culture under serum-free conditions. The seeding density was 1000 cells/ml per well. Scale bar is 100μm. Original magnification is 10x. (B) The diameters of the first, second and third spheres
are 76.82,115.45 and 71.47μm, respectively. (C) The diameters of the first and second spheres are 78.66, and 86.42μm, respectively. (D) The diameters of the first and second spheres are 74.57, and
92.80μm, respectively.
(A) (B)
1
(C) (D)
1 2
1
2 3
Figure 2. (A) A549 adherent monolayer parental cells. Scale bar is 100μm. Original
magnification 20x. (B), (C) and (D) Tumor spheroids derived from A549 cells after 14 days of
culture under serum-free conditions. The seeding density was 1000 cells/ml per well. Scale bar
is 100μm. Original magnification is 10x. (B) The diameter of the sphere is 87.14μm. (C) The
diameters of the first and second spheres are 87.1 and 98.56μm, respectively. (D) The diameters of
the first, second and third spheres are 76.53,78.04 and 108.79μm, respectively.
Extreme Limiting dilution assay results:
A549 and H1299 cells were examined for their ability to regenerate themselves using
limiting dilution analysis (Table 1). Typical spheroids were formed after 14 days when
A549 and H1299 cells were grown in a 96-well culture plate at extreme limiting dilutions.
A549 and H1299 single cells (24 single cells per experiment) formed new spheroids at 23.5
± 43.7% and 62.5 ± 49.4%, respectively. The frequency of sphere-forming cells (stem-like
cells) was 6.06-fold higher for H1299 than that of A549 cells (Table 1, and Figure 3).
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As a result, these observations show that both A549 and H1299 spheroids are composed of a heterogeneous cell population with self-renewal capacity (stem-like cells) or without (non-stem-like cells or differentiated cells).
Discussion:
Increasing evidence has shown that NSCLC enhanced tumorigenicity, as in other solid cancer
types, is driven by a tumor cell population that exhibits stem cell-like characteristics such as
self-renewal, differentiation, cell mobility and therapy resistance, and referred to as cancer
stem cells (CSC) or tumor-initiating cells (TIC) (Eramo et al.,2008; Bertolini et al.,2009;
Magee et al.,2012;Visvader et al.,2012). Further investigation of the CSCs population would
provide a deeper understanding of the regulatory mechanisms involved in their maintenance
and facilitate the development of therapeutic strategies to control lung cancer in the long term
(Liu et al.,2013).
Table1. The frequency of tumor spheroids formed by A549 and H1299 cells. 95% CI: 95% confidence interval.
Cell number/well Number of wells
Number of wells showing
spheroids
A549 H1299 A549 H1299
1000 24 24 24 24
100 24 24 21 24
10 24 24 19 21
1 17 24 4 15
Spheroid-forming frequency 1/19.65 1/ 3.24
(% 95 CI)
P value 1.79e-11=0,0000000000179
Table 2. The diameters of spheroids formed by A549 and H1299 cells.
A549 H1299
Diameter Mean (μm) 80.70 ± 31.72μm 95.25 ± 41.03μm
P value 0.00003
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H1299
A549
Figure 3. A log-fraction graph of the limiting dilution model fitting the data in Table 4.1. The slope of the line is the log-active cell fraction. The dotted lines give the 95% confidence interval. The data value with zero negative response at dose 1000 for A549 cells and the data value with zero negative response at dose 100 for H1299 cells are represented by triangular downward values.
To establish a suitable system for the study of CSCs in NSCLC, we used the sphere formation
assay, which is a marker-independent approach. For this purpose, we compared the sphere-
forming capacities of A549 and H1299 cells. In serum-free and non-adherent culture
conditions, the non-CSCs undergo anoikis but CSCs survive and give rise to tumorspheres
(Charpentier and Martin,2013) thus, theoretically, the number of spheroids in the culture is
reflective of the number of CSCs in the sample.
Sphere numbers at different cell seeding densities showed a good correlation for both A549
and H1299 cell lines. However, H1299 cells were more potent than A549 cells in sphere
formation. In terms of the frequency of sphere-forming cells (stem-like cells), it was found to
be 6.06-fold higher for H1299 cells compared to A549 cells (Table 1). H1299 cells formed
spheres of an average of about 95.25 ± 41.03μm in diameter, whereas A549 cells formed
spheres of an average of about 80.70 ± 31.72μm in diameter (Table 2). Overall, the frequency
of sphere-forming cells in H1299 cells was significantly higher than that of A549 cells (Table
1), indicating a higher cancer stem cell content in this cell line. These results are in accordance
with recent studies, reporting decreased sphere formation potential (Chung et al.,2015;
Holmboe et al.,2017) and in vivo tumorigenicity of A549 compared to H1299 cells (Chung et
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168
al.,2015). Taken together, these results suggest that H1299 cells could be a suitable model
system for studying CSCs in NSCLC.
Conclusion:
Recently the cancer stem cells are implicated in tumor initiation, progression, invasion,
metastasis, relapse and resistance to chemotherapy and radiotherapy. Since the resistance to
conventional chemotherapeutic agents is becoming a growing phenomenon, there is an urgent
need to develop novel drugs that specifically target the cancer stem cell populations in tumors.
Thus, it is of great value to conduct future studies to broaden our current knowledge of the
biology of cancer stem cells and the underlying molecular regulatory mechanisms of their
behavior.
Acknowledgments:
Financial support from Gaziantep university BAP (Project no.FEF. DT.17.12) is gratefully acknowledged.
Conflicts of interest:
The authors declare no conflicts of interest.
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Investigation of Apoptotic Effect of Betulinic Acid in Renal Cancer Cells
Merve Nur Ataş1, Barış Ertuğrul1, Elif Sinem İplik2, Bedia Çakmakoğlu1, Arzu Ergen1
1Istanbul University, Aziz Sancar Institute of Experimental Medicine, Department of Molecular
Medicine, Istanbul, Turkey 2Istanbul Yeni Yuzyil University, Faculty of Pharmacy, Istanbul, Turkey
E-mail: [email protected]
ABSTRACT
Kidney cancer or renal cancer is the fourteenth most frequent cancer in the world. With its
increasing incidence in the last few years, the renal cell carcinoma is the most common kidney
cancer type. Apart from the environmental factors, increasing activities of expressions of genes
and/or their pathways make cells become cancerous. AKT-1 and mTOR promote cell growth,
proliferation and survival, participate in cellular homeostasis and have ability to suppress
apoptosis in various ways.
Betulinic acid which is a lupane-type pentacyclic triterpenoid is derived from various plants.
Betulinic acid exhibits wide range biological activities such as anti-inflammatory, anti-viral,
anti-bacterial and particularly anti-cancer activities. It is known that betulinic acid promotes
apoptosis in cancer cells along with no toxic effect in normal cells. This effect makes betulinic
acid a potential anti-cancer drug.
In our study, clear cell renal carcinoma cell line CAKI-2 and healthy cell line MRC-5 are used
to research the effect of betulinic acid on apoptosis and gene expression levels. In order to
identify the toxic effect of betulinic acid, WST-1 and to detect apoptosis Annexin-V were
conducted. Expression analysis of AKT-1 and mTOR genes were conducted with Real-Time
PCR to measure the effect of betulinic acid in gene expressions levels.
As a result of this study, the apoptotic activity of betulinic acid on kidney cancer cell line was
detected with Annexin-V. In gene expression analysis, there was statistically significant
decrease in AKT-1 expression level while mTOR expression level was increased. Betulinic
acid with its apoptotic effect on renal cancer cell line and non-toxic effect on normal cell line
is a potential anti-cancer drug promising for future studies.
Keywords: Betulinic acid, renal cancer, apoptosis
Acknowledgment: The present work was supported by the Research Fund of Istanbul University.
Project No: 31565
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INTRODUCTION
Renal cancer accounts for about 2-3% of all adult malignant tumors [1]. The most common type
of kidney cancer is renal cell carcinoma (RCC), which is %90 of all renal cancer cases and
occurs in renal tubular epithelial cells [2]. Considering the continuous increase in the incidence
of renal cell carcinoma due to risk factors such as smoking, alcohol use, hypertension and
obesity [3], it has become extremely important to establish new treatment options to improve
the quality of life and, more importantly, survival ratio of patients after diagnosis.
The treatment options of RCC are, surgery and nephrectomy in the treatment of localized RCC
and systemic treatment for metastatic RCC or in cases of recrudescence after local treatment
[4]. Systemic treatment is designated according to the molecular biology of the RCC subtypes
that patients have. Immune checkpoint inhibitors, mTOR inhibitors, and approved drugs are
among the systemic treatment options [5].
AKT/mTOR pathway which plays a key role in regulation of cell growth, proliferation,
survival, metabolism and cellular homeostasis, is one of the pathways with overactivation in
various types of cancer including renal cell carcinoma [6]. AKT-1 and mTOR are members of
this pathway [7]. Any mutation occurring in the components of AKT/mTOR pathway may lead
to tumor development [8].
Betulinic acid is a lupane-type pentacyclic triterpenoid, which consists of six isoprene units,
and is isolated from various plants [9]. It has various biological activities such as anti-viral,
anti-bacterial, anti-inflammatory, particularly anti-cancer activities [10]. Although betulinic
acid is known to exhibit apoptotic effect through the mitochondria-dependent pathway,
studying different molecules and pathways will help to improve its known effects.
In this context, the purpose of this study is the investigation of apoptotic activity of betulinic
acid in renal cell carcinoma cells and examination of its effects on apoptosis-associated genes,
AKT-1 and mTOR.
MATERIALS AND METHODS
Cell culture
The CAKI-2 clear cell renal cell carcinoma and MRC-5 lung fibroblast cell lines were acquired
from American Type Culture Collection (ATCC, USA). The CAKI-2 (ATCC® HTB-47™) and
MRC-5 (ATCC® CCL-171™) cell lines were cultured in McCoy’s 5A and EMEM medium
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respectively, and supplemented with %1 Penicilin-Streptomycin and %10 Fetal Bovine Serum
(FBS) at 37 oC in %5 CO2
Reagents and assay kits
All cell culture reagents were purchased from Thermo Fisher Scientific (USA). Betulinic acid
and WST-1 cell proliferation assay kit were purchased form Sigma-Aldrich (USA) and Roche
Life Sciences (Germany), respectively. Annexin-V kit was purchased from Merck Millipore
(USA) and The RNA isolation, cDNA synthesis and qPCR assay kits were acquired from Jena
Bioscience (Germany).
Cell proliferation analysis
The cell lines were seeded at a density of 104 cells per well in 96-well plates. After 24 h
incubation, were treated with increasing concentration of betulinic acid (1, 2.5, 5, 7.5, 10, 25
and 50µM) for 24, 48 and 72 h. WST-1 solution (10µl) was applied to each well and the well
plates were incubated for 4 hours at 37oC. The reaction was measured at 460 nm using
microplate ELISA reader (Thermo Fisher Scientific, Germany).
The analysis of apoptotic activity
The cells were seeded at density of 105 per well in 6-well plates and were treated with different
concentrations of betulinic acid (25 and 50µM for 24h). At the end of 24h, the cells were
suspended with 1xbinding buffer at a concentration of 1x106 cells/mL. Following this, 5 µl
Annexin V-FITC and 5 µl PI were added to 100 µl of suspension cells. The cells were incubated
for 20 min at room temperature in dark. The results were analyzed using flow cytometry.
qPCR gene expression analysis
After each cell line was treated with determined concentrations of betulinic acid (25 and 50µM
for 24h), total RNA was extracted from cells and cDNA synthesis was performed from RNA
samples. It was determined fold changes in mRNA levels of AKT-1, mTOR and housekeeping
gene (GAPDH). The fold change was calculated as 2-∆∆CT.
Statistical analysis
Statistical analysis was performed using SPSS 21.0 program. The significance limit was
accepted as p <0.05. Mann-Whitney-U test was used for inter-group evaluation.
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RESULTS
Betulinic acid reduced cell viability in renal cancer cells
We investigated the cytotoxic effects of betulinic acid on CAKI-2 and MRC-5 cell lines using
the WST-1 colorimetric cell proliferation assay. The cells were seeded and incubated with
increasing concentration (1-50µM) of betulinic acid for 24, 48 and 72h. WST-1 assay was
performed at the end of each incubation period. The analysis showed that cell viability of CAKI-
2 cells treated with 25 and 50 µM betulinic acid decreased significantly. Nevertheless, it was
observed that betulinic acid treatment had no significant effect on cell viability of MRC-5 cells.
Betulinic acid enhanced apoptosis in renal cancer cells
In order to detect apoptosis and necrosis in betulinic acid treated cells, we performed Annexin
V assay. In CAKI-2 cell line, apoptotic cell death was observed %7.5 at 25 µM and %14.25 at
50 µM doses of betulinic acid for 24h incubation period (p<0.05). In the healthy cell line MRC-
5, it was observed that betulinic acid treatment had no effect on apoptosis of cells.
Betulinic acid changed AKT-1 and mTOR gene expression levels
In CAKI-2 cells treated with 25 µM betulinic acid, 0.41-fold change in AKT-1 expression
(p=0,034) and 1.61-fold change in mTOR gene expression (p=0,037) was observed. On the
other hand, in 50 µM betulinic acid treatment, 0.38-fold change in AKT-1gene expression
(p=0,037) and 1.94-fold change in mTOR gene expression was detected. These results showed
that betulinic acid caused a statistically significant decrease in AKT-1 gene expression while
increasing mTOR gene expression.
DISCUSSION
Renal cancer is the most lethal type of cancer among urological cancers. In recent years there
has been an increasing interest in natural compounds derived from plants in search of new
treatment options. Betulinic acid, a natural compound obtained from the bark of various plants,
is a member of the triterpenoids group and has many biological activities. Betulinic acid
performs the anti-cancer activity, mainly through the mitochondrial pathway [11].
Recently, many studies have been conducted to identify the anti-cancer activity of betulinic
acid. Lee et al. extracted potential anti-cancer compounds including betulinic acid, from Cornus
walteri. In ovarian cancer cell line A2780, among other compounds, betulinic acid showed the
highest toxic effect on cells. Annexin-V analysis showed that betulinic acid induced cell
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apoptosis in dose-dependent manner and enhanced apoptosis by modulating Bcl-2 protein levels
[12].
Yang et al. RCC cell lines 786-O and ACHN in their study on the anticancer effects of betulinic
acid; It has been found to have an anti-proliferative effect on cells, induce apoptosis via
mitochondrial pathway by decreasing BCL-2 expression and increasing BAX and caspase 3
expression [13].
Apart from its effects to mitochondrial pathway, betulinic acid affects different molecules and
pathways in apoptosis. In cervical cancer cells HeLa, Xu et al. investigated the apoptotic effects
of betulinic acid and found that betulinic acid treatment caused significant decrease in PI3K
(p110a), PI3K (p85), p-AKT(Ser473) and p-AKT (Thr308) in time and dose dependent manner
[14].
In this study, we determined the apoptotic effect of betulinic acid on CAKI-2 renal cell
carcinoma cell line. We performed WST-1, Annexin-V, and Real-Time PCR to determine the
effects of betulinic acid on cell viability, apoptosis and AKT-1 and mTOR gene expression
levels in time and dose dependent manner. According to the results, betulinic acid reduced cell
viability, induced cell apoptosis and significantly reduced AKT-1 gene expression.
In conclusion, apoptotic activity of betulinic acid and its potential effect on AKT/mTOR
pathway suggest that betulinic acid could be a potential anti-cancer drug. With further studies,
using different analysis methods, the effect of betulinic acid on the AKT / mTOR pathway in
RCC or in different cancer types can be better elucidated.
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Identification of lncRNA molecules involved in the regulation of EGF-induced of cellular
proliferation
EGF ile indüklenen hücresel proliferasyonda rol oynayan lncRNA moleküllerinin
belirlenmesi
Esra Bozgeyik
Tekirdag Namik Kemal University, Faculty of Medicine, Department of Medical Biology, Tekirdag,
Turkey.
Özet
Akciğer kanseri, dünya çapında kansere bağlı ölümlerin önde gelen nedenlerinden biridir. 2019
kanser istatistiklerine göre, 230000'den fazla yeni akciğer kanseri vakasının ortaya çıkacağı ve
143000 kişinin akciğer kanseri nedeniyle öleceği tahmin edilmektedir . Ayrıca, lncRNA'lar
(long non-coding RNA), protein kodlama kapasitesine sahip olmayan ve çoğalma, büyüme,
bölünme, farklılaşma, hayatta kalma ve hücrelerin göçü gibi hayati hücresel süreçlerde önemli
rol oynayan 200 nükleotitten daha uzun fonksiyonel RNA molekülleridir. Çok çeşitli biyolojik
fonksiyonlarından dolayı, bu transkriptlerin deregülasyonu, akciğer kanseri de dahil olmak
üzere bütün kanserlerin moleküler patogenezine büyük ölçüde katkıda bulunur. EGF, hücre
proliferasyonunun düzenlenmesinde rol oynayan anahtar faktördür. EGF, normal fizyolojik
işlemlerin sürdürülmesi için çok temel bir protein olduğundan, EGF tarafından indüklenen
sinyal yolaklarının düzenlenmesinde meydana gelen bozukluklar, insan kanserlerinin bazı
belirgin özelliklerine katkıda bulunur. Bu nedenle, EGF ile indüklenen hücresel proliferasyonda
rol oynayan lncRNA moleküllerinin belirlenmesi, insan kanserlerinin moleküler patolojisinin
aydınlatılması adına önem arz etmektedir. Bu nedenle bu çalışmada biz EGF ile indüklenen
hücresel proliferasyonda farklı eksprese edilen lncRNA moleküllerini belirlemeyi amaçladık.
Sonuç olarak, MALAT1, ANRIL ve MCM3AP-AS1 lncRNA'ların ekspresyon seviyelerinin
arttığı, LOC105372161 ve MEG3'ün ekspresyon seviyelerinin azaldığı bulundu. Ancak,
yalnızca LOC105372161'in ifadesindeki değişikliklerin istatistiksel olarak anlamlı olduğu
bulundu. Bu sonuçlar, LOC105372161'in EGF tarafından indüklenen hücre proliferasyonun
düzenlenmesinde rol oynayabileceğini ve BCL2'nin LOC105372161'in varsayılan hedefi
olabileceğini göstermektedir.
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Abstract
Lung cancer is one of the leading causes of cancer-related deaths worldwide. According to 2019
cancer statistics, it is estimated that over 230000 new lung cancer cases will occur and 143000
people will die due to lung cancer. Moreover, lncRNAs (long non-coding RNA) are functional
RNA molecules longer than 200 nucleotides that do not have protein coding capacity and play
important roles in the vital cellular processes such as proliferation, growth, division,
differentiation, survival, and migration of cells. Due to their wide range of biological functions,
deregulation of these transcripts highly contributes to molecular pathogenesis of cancers
including lung cancer. EGF is key factor that is involved in the regulation of cell proliferation.
Since EGF is a very chief protein for the maintenance of normal physiological processes,
deregulation of EGF-induced signaling significantly contributes to the several hallmark
capabilities of human cancers. Thus, determination of lncRNA molecules involved in the
regulation of EGF-induced promotion of cellular proliferation is of great interest to illuminate
the molecular pathology of human cancers. Accordingly, in this study we aimed to identify
differentially expressed lncRNAs involved in the regulation of EGF-induced stimulation of
cellular proliferation. As a result, expression levels of MALAT1, ANRIL and MCM3AP-AS1
lncRNAs was found to be increased whereas expression levels of LOC105372161 and MEG3
were found to be decreased. However, only changes in the expression of LOC105372161 were
found to be statistically significant, suggesting that LOC105372161 is involved in the
regulation of EGF-induced cell proliferation and BCL2 might be the putative target of
LOC105372161.
Keywords: EGF, lncRNA, lunc cancer, VIM-AS1
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Introduction
Lung cancer is one of the leading causes of cancer-related deaths worldwide. According to 2019
cancer statistics, it is estimated that approximately 230000 people will be diagnosed with lung
cancer and 143000 people will die due to lung cancer [1]. Moreover, lncRNAs (long non-coding
RNA) are functional RNA molecules longer than 200 nucleotides that do not have protein
coding capacity. LncRNAs can interact with certain proteins with their specific secondary and
tertiary structures and bind to certain regions of the DNA to influence the expression of genes
in the relevant location. Studies show that lncRNAs play important roles in normal
physiological processes as well as in the pathogenesis of complex diseases such as cancer [2].
Epidermal growth factor (EGF) is a growth factor that plays important roles in proliferation,
growth, division, differentiation, survival, and migration of cells. EGF is a ligand for the
epidermal growth factor receptor (EGFR) and binding of EGF to EGFR leads to growth factor
induced autophosphorylation of the receptor and activation of downstream signaling cascades
promoting cell proliferation. Although EGF is a very important protein for the maintenance of
normal physiological processes, deregulation of EGF-induced signaling significantly
contributes to the several hallmark capabilities of cancers [3, 4]. Cancer cells may acquire
increased expression of growth factors to stimulate cognate receptors for autocrine signaling.
In an alternative way, they may also stimulate neighboring cells to supply these growth factor
ligands themselves for sustaining proliferation. Accordingly, an increased level of EGF
provides a good model for stimulation of cell proliferation and studying mediators of cellular
proliferation. It is also evident that several lncRNAs are involved in the cellular proliferation
and deregulation of these molecules leads to increased and sustained proliferation of cancer
cells.
Thus, identification of lncRNA molecules involved in the regulation of EGF-induced
stimulation of cellular proliferation is of great interest to understand better about the molecular
pathology of human cancers. Accordingly, here we aimed to determine lncRNAs involved in
the regulation of EGF-induced stimulation of cellular proliferation.
Material and Methods
Cell culture
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The lung cancer cell line, A549, was cultured in a carbon dioxide incubator at 37 °C, containing
5 % CO2 and in 95% humidified medium. For the propagation of cells, DMEM (Dulbecco's
Modified Eagle's Medium, Sigma-Aldrich, Germany) containing 10 % FBS (fetal bovine
serum, Sigma-Aldrich, Germany) was used.
EGF treatment
For the EGF treatments, cells were seeded to 6-well plates at a concentration of 106 per milliliter
and wells were treated with the increasing concentrations of EGF as 50 ng/ml, 100 ng/ml, 200
ng/ml, 400 ng/ml. after 24 hours of incubation cells were collected using Trypsin-EDTA
solution.
RNA isolation and qPCR
RNA isolation was performed with the Qiagen RNeasy RNA isolation kit (Qiagen, Germany).
RNA concentrations were determined with NanoDrop® ND-1000 spectrophotometer. RT2
First Strand Kit (Qiagen, Germany) was then used to synthesize single chain cDNA and
recommended protocols of manufacturer were followed. miScript SYBR® Green PCR Kit
(Qiagen, Germany) was used in combination with gene-specific primers for each lncRNA
molecule to determine lncRNA expression levels between the groups. GAPDH
(Glyceraldehyde-3-Phosphate Dehydrogenase) and ACTB (beta (β)-actin) were used as
reference genes.
Statistical analysis
Ct (cycle threshold) values obtained from quantitative PCR were calculated and analyzed by 2-
ΔCt method where ΔCt = Hedef gen – Referans Gen [5]. The t test was used for comparisons,
and Mann Whitney U test was used for samples that did not show normal distribution. Also,
fold-change analysis was performed. For the fold-change analysis, 2-ΔΔCt (ΔΔCt=[ Treated
(Target gene - Reference gene) - Control (Target gene – Reference gene)]) [5]. For all results,
p <0.05 was considered statistically significant.
Results
Differential expressions of lncRNAs after EGF treatments
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After treatment of A549 cells with different concentrations of EGF, lncRNA expression levels
were analyzed. As a result, while expression levels of LOC105372161 and MEG3 decreased
with increasing EGF concentrations, MALAT1, ANRIL and MCM3AP-AS1 lncRNAs
expression levels was found to be not significantly altered (Figure 1A, 2B).
Figure 1. Expression of lncRNAs in EGF-treated A549 cells. Heatmap (A), fold change (B).
Particularly, the expression level of MEG3 was found to be decreased in cells treated with 400
ng/ml and 200 ng/ml of EGF compared to untreated samples. However, this change was not
statistically significant (p400 ng/ml EGF =0.0728, p200 ng/ml EGF = 0.0676). In addition,
ANRIL expression levels was also found to be decreased with increasing EGF concentrations,
but this change was not statistically significant (p400 ng/ml EGF = 0.8958) (Figure 2). The
expression level LOC105372161 was found to be lower in cells treated with 400 ng/ml and 200
ng/ml EGF compared to untreated cells and this change was statistically significant (p400 ng/ml
EGF = 0.0042, p200 ng/ml EGF = 0.0095) (Figure 2).
Discussion
Lung cancer is the most common cause of cancer-related deaths in the world. Therefore, there
is an urgent need to fully elucidate the molecular mechanisms of this disease. Accumulating
evidence suggest lncRNAs play a chief role in the development and progression of human
malignancies including lung cancer. Recent studies have shown that lncRNAs are important
regulatory molecules that play a role in controlling various cellular activities [6]. Therefore,
determination and/or identification of non-coding counterparts of coding genes in molecular
pathogenesis of lung cancer are very important for developing more efficient targeted
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therapeutics. Here, lncRNA expression levels in EGF-stimulated A549 lung cancer cells were evaluated
and LOC105372161 and MEG3 lncRNAs were found to be differentially expressed with the increasing
EGF concentrations.
Figure 2. Relative gene expression changes of lncRNAs according to different EGF concentrations.
However, only changes in LOC105372161 expression was found to be statistically significant.
Low expression of MEG3 was consistent with the previous findings but this change was not
enough to be statistically significant. However, changes in LOC105372161 expression was
found to be statistically significant. Reduction of LOC105372161, which is transcribed from
opposite strand of BCL2, expression levels in EGF-induced cell proliferation indicates that this
lncRNA might be involved in the negative regulation of BCL2 anti-apoptotic gene. Yet, more
comprehensive studies are needed to delineate the functions of this lncRNA in tumor formation.
References
• Siegel, R.L., K.D. Miller, and A. Jemal, Cancer statistics, 2019. CA: a cancer journal
for clinicians, 2019. 69(1): p. 7-34.
• Bhan, A., M. Soleimani, and S.S. Mandal, Long Noncoding RNA and Cancer: A New
Paradigm. Cancer Res, 2017. 77(15): p. 3965-3981.
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• Johnson, L., et al., Epidermal growth factor and expression of specific genes: effects on cultured rat pituitary cells are dissociable from the mitogenic response. Proceedings of the National Academy of Sciences, 1980. 77(1): p. 394-398.
• Jorissen, R.N., et al., Epidermal growth factor receptor: mechanisms of activation and signalling, in The EGF receptor family. 2003, Elsevier. p. 33-55.
• Schmittgen, T.D. and K.J. Livak, Analyzing real-time PCR data by the comparative C T method. Nature protocols, 2008. 3(6): p. 1101.
• Cech, T.R. and J.A. Steitz, The noncoding RNA revolution—trashing old rules to forge new ones. Cell, 2014. 157(1): p. 77-94.
• Tano, K., et al., MALAT‐1 enhances cell motility of lung adenocarcinoma cells by influencing the expression of motility‐related genes. FEBS letters, 2010. 584(22): p.
4575-4580.
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Yeni antikanser ilaç keşfinde genomik ve metabolomik: Fırsatlar ve zorluklar
Ekrem Kum, Süleyman Özakın, Ebru İnce*
Dicle Üniversitesi Fen Fakültesi Biyoloji Bölümü 21280 Diyarbakır
Özet
Elli yıldan uzun bir süredir, mikroorganizmalardan ve bitkilerden elde edilen küçük
organik moleküller çok sayıda kullanışlı kanser kemoterapötik ilacına kaynak teşkil etmiştir.
Doğal ürünlerin bu tip öncü bileşiklerinin tarama çalışmaları son yıllarda ivme kazanmış,
karasal bitki ve mikroorganizmalar kadar, deniz fauna ve florasını oluşturan bileşenlerinin de
anti-kanser aktiviteleri araştırılmaya başlanmıştır. Doğal ürünlerin biyosentezlerinin daha iyi
anlaşılması ile beraber, geleneksel ilaç veya ilaç öncülü tarama çalışmaları yerini genomik
bilginin kimyasal verilerle kombine edilmesi prensibe dayalı farklı stratejilere bırakmıştır. Anti-
kanser dahil olmak üzere farklı biyolojik aktivite ve emsalsiz kimyasal çeşitliliğe sahip
mikrobiyal doğal ürünlerin üreticisi olan Aktinomisetler kompleks bir yaşam döngüsüne ve
diğer bakteriler ile kıyaslandığında büyük genomlara sahiptirler. Yeni nesil dizileme
teknolojileri sayesinde genomlarında biyoaktif doğal ürün sentezinden sorumlu yüksek
miktarda biyosentetik gen kümesine sahip oldukları bulunmuştur. Bu makalede; mikrobiyal
kaynaklı yeni biyoaktif bileşik keşfinde genomik ve metabolomik tarama stratejileri, kullanılan
biyoinformatik programlar, avantaj veya zorluklarına değinilerek, son yıllarda bu teknoloji ile
elde edilmiş yeni anti-kanser ilaçların altı çizilmiştir.
Anahtar kelimeler: Doğal ürünler, anti-kanser , genomik, metabolomik, LC-MS/MS.
• Giriş
Doğal ürünler; mikroorganizmalar, bitkiler ve omurgasızlar tarafından üretilen küçük
organik moleküllerdir. Aynı zamanda sekonder metabolit olarak adlandırılan doğal ürünler
çeşitli ve olağandışı kimyasal yapılara ve küçük moleküler kütlelere sahiptirler. Hem kimyasal
yapı çeşitliliği hem de farklı biyolojik aktivitelere sahip olmalarından ötürü şu an klinikte
kullanılan birçok ilaç doğal ürün veya onların kimyasal modifikasyonlarından elde
edilmektedir.
Doğal ürünler alkaloidler, terpenoidler, flavonoidler, nükleositler, ribozomal olmayan
peptidler (NRP), poliketidler (PK) olmak üzere çeşitli temel gruplara ayrılmaktadır. Bilinen
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doğal ürünlerin yaklaşık % 60’ını terpenoidler oluşturmaktadır. Terpenoidler bitkiler, mantarlar
ve mikroorganizmalarca sentezlenen biyoaktif metabolitlerdir. Terpenoidlerin biyosentezi
izopentil ünitelerinin substrat olarak kullanılmasıyla gerçekleştirilmektedir. Klinikte güncel
olarak kullanılan antikanser ilaç taxol (Paclitaxol®) en iyi bilinen terpenoidlerdendir.
Mikrobiyal doğal ürünler biyoaktif bileşiklerin en önemli sınıfını oluşturmaktadır.
Bunlar arasında antibiyotikler, antifungallar, antitümör ajanlar, toksinler, antihelmitikler,
immunbaskılayıcılar ve sideroforları da içeren, işlevsel olarak birbirinden oldukça farklı
biyokimyasal sınıflara dahil bileşikler mevcuttur. Mikrobiyal kaynaklı doğal ürünlerin en
önemli üreticilerinden biri aktinomisetlerdir. Örneğin, anti-kanser kemoterapötik ilaçlar olan
bleomycin (Blenoxane®) (1) Streptomyces verticillus, doxorubicin (Adriamycin) (2)
Streptomyces peucetius tarafından üretilen doğal ürünlerdir.
(1) (2)
Farmakolojik ve biyolojik açıdan önemli onbinlerce PK ve NRP keşfedilmiş olmasına
rağmen, yalnızca 350 antimikrobiyal molekül bu pazarda yerini bulmuştur (Demain 2009). Son
kırk yıl içerisinde, mikroorganizmalardan markete ulaşabilen ürünlerin sayısı hatırı sayılır bir
biçimde azalmıştır. Bundan dolayı yeni doğal ürünlerin keşfi ve alternatif stratejilerle bu doğal
ürünlerin kimyasal potansiyelini arttırmak önem arz etmektedir.
Geleneksel yöntemler biyolojik aktivite temelli olup, organik ekstraktlardan analitik
yöntemlerle biyoaktif bileşiklerin izolasyonu ve karakterizasyonu prensibine dayanır.
Ekstraktlarda bilinen metabolitlerin yeniden izolasyonu dereplikasyon olarak
adlandırılmaktadır. Dereplikasyon biyolojik aktivite temelli doğal ürün araştırmalarında
karşılaşılan önemli sorunlardan biridir. Özellikle 1990’lı yıllardan günümüze kadar yeni
metabolit keşif oranlarının düşmesinin temel nedeni dereplikasyon sorunudur. Doğal ürün
araştırmalarında yeni yöntemlerin geliştirilmesinin temel amaçlarından biri dereplikasyon
sorunun üstesinden gelmektir. Özellikle bilinen metabolitlerin saflaştırma aşamasına geçmeden
daha ekstrakt aşamasında tespiti için çeşitli veritabanları ve informatik programlar
geliştirilmektedir.
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(4) Genomik Yaklaşımlarla İlaç Taramaları
Yeni genomların keşfedilmesi neticesinde çoğu mikroorganizmanın şimdiye kadar
üretmiş olduğu doğal ürünlerden daha fazlasını üretme potansiyeline sahip olduğu
anlaşılmaktadır. Zira bu mikroorganizmalar standart laboratuvar koşullarında kültüre
edildiklerinde doğal ürünleri sentezleyen biyosentetik gen kümeleri ya aktifleşememekte
veyahut çok düşük seviyelerde ifade olabilmektedirler. Uygun fiziksel veya kimyasal uyarıcılar
olmadığı takdirde uyku halinde olup klasik kültür koşullarında ifade edilemeyen bu genler
kriptik olarak adlandırılırlar. Özellikle aktinomisetlerin genomları incelendiğinde, kompleks
yapılı çok sayıda doğal ürün biyosentezinden sorumlu kriptik gen kümesinin varlığı ortaya
çıkarılmıştır (Bentley ve ark. 2002, Hosaka ve ark. 2013).
Genomik araştırmaları ile değişik varyetede birçok organizmadan elde edilen büyük
miktarda DNA sekans verisi ulusal veri tabanlarında depolanmaktadır. Araştırıcıların erişimine
açık olan DNA sekans verileri yeni ilaç veya ilaç öncülü bileşiklerin keşfi için yeni
biyoinformatik araçlardan faydalanılmasının yolu açmaktadır. Genomik temelli yaklaşımlar,
daha önce standart fermentasyon koşulları altında keşfedilemeyen yeni doğal ürünlerin açığa
çıkarılması için geliştirilmektedir. Dizilenen bu genomlar sonucunda yeni doğal ürünlerin
keşfedilmesi, genom madenciliği vasıtasıyla ise bunların biyosentetik yolaklarına ulaşılması
hedeflenmektedir. Genomda izi sürülen kriptik genlerin, aynı zamanda uygun ortam
koşullarında ifadesi sağlanmaktadır (Şekil 1).
Şekil 1. Genom madenciliği (Choi ve ark. 2015)
Sıra dışı habitatlara adaptasyon sağlamış mikroorganizmaların standart laboratuvar
koşullarında kültüre edilmeleri oldukça zordur. Bu amaçla yeni kültür metotları geliştirilmesine
rağmen mikroorganizmaların ancak %1’i standart koşullarda kültüre edilebilmektedir.
Genomik teknolojiler, sayesinde kültüre edilebilen veya edilemeyen mikroorganizmaların
böyle bileşiklerin sentezi için yüksek biyosentetik kapasiteye sahip olduğunu ortaya koymuştur.
Genom tarama yönteminin en temel bileşenleri biyoinformatik programlardır. Son dönemde
geliştirilen ‘antiSMASH ve ‘NapDOS’ biyoinformatik programları genomdaki doğal ürün
genlerinden yola çıkarak bileşiğin iskelet yapısı hakkında önemli bilgiler vermektedir. Buna en
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güzel örnek miksobakteri Sorangium cellulosum’da tanımlanan anti-mitotik ve anti-neoplastik
bir ajan olan epothilone’un yapısının biyoinformatik programlarca ana iskelet yapıya yüksek
benzerlikte tahmin edilmiş olmasıdır (Li ve ark. 2009) (3)-Tahmini Yapı, (4)-Gerçek Yapı.
(3) (4)
Çeşitli veri tabanlarında analiz edilmeyi bekleyen binlerce genom dizilimi bulunmaktadır.
Bu dizilimlerin analizlerinin kimyasal verilerle desteklenmesi doğal ürün araştırmalarındaki en
yeni stratejidir.
28. Metabolomik Yaklaşımlarla İlaç Taramaları
Biyolojik sistemlerde bulunan küçük moleküler ağırlığa sahip metabolitleri global
seviyede analiz edebilen metabolomik çalışmalarında kütle spektroskopisi (MS) en yaygın
kullanılan yaklaşımlardan biridir. Bir ekstrakt içinde çok düşük konsantrasyonlardaki
metabolitlerin tespitinde, hem oldukça hassas olması hem de bileşiğin kimyasal yapısı hakkında
bilgi vermesinden dolayı, yeni bileşik araştırmalarında geniş bir kullanım alanına sahiptir.
Genel olarak metabolomik; hedefe odaklı analizler (targeted) ve herhangi bir hedefe
odaklanmayan analizler (non-targeted) olmak üzere ikiye ayrılır. Hedefe odaklı metabolomik,
ilgili metabolik yolaklarda kesin miktar tayini yapılmış metabolitleri içerirken, herhangi bir
hedefe odaklanmayan metabolomik ön yargısız bir şekilde birçok metabolitin ölçümünde
kullanılan bir yaklaşımdır. Kütle spektroskopisi; sıvı kromatografi (LC-MS), nükleer manyetik
rezonans (LC-MS-NMR), gaz kromatografisi (GC-MS) gibi yöntemler ile kombine olma
özelliğine sahiptir. İkili kütle spektrometresi (MS/MS) ise en az iki aşamada kütle analizini
gerçekleştiren genel bir yaklaşımdır.
Doğal ürünlerin toplu moleküler ağı (Global Natural Products Social Molekuler
Network-GNPS) en yaygın ve kullanışlı veri tabanlarından biridir.
(5) Moleküler ağ analizleri
Moleküler ağ analizi geçtiğimiz on yıl içinde geliştirilmiş yeni bir dereplikasyon ve
metabolomiks yöntemidir. Bu yöntem herhangi bir ekstrakt ve biyolojik örnek içinde bulunan
metabolitlerin MS/MS verilerinin analizine dayanmaktadır. MS/MS, analiz edilen iyonların
fragmentasyonuna neden olduğundan bileşiklerin kimyasal yapısı hakkında oldukça önemli
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bilgi vermektedir. Bu sebepten dolayı doğal ürün araştırmalarında MS/MS analizleri önemli
bir kullanım alanına sahiptir. Hibrit MS sistemindeki ilk analizör tarafından tespit edilen iyona
ait fragment iyonlar, ikinci analizör tarafından tespit edilerek sıralı MS (tandem MS) veya
MS/MS verisi olarak çeşitli veri tabanlarında ve programlarda analiz edilmek üzere
kullanılmaktadır. GNPS (Global Natural Products Social Molekuler Network), dereplikasyon,
bilinen bileşiklerin yeni analoglarının bulunması ve aynı zamanda yeni bileşik keşfi için en
yaygın kullanılan veri tabanlarından biridir (Tablo 1).
Tablo 1. Doğal ürün tarama veri tabanları.
Veri Tabanı Web Adresi
GNPS www.gnps.ucsd.edu
Norine www.bioinfo.lifl.fr
Scifinder www.cas.org
Metlin www.scripps.edu
Massbank www.massbank.jp
AntiBase www.user.gwdg.de
Marinlit www.chem.canterbury.ac.zn
Drugbank www.drugbank.ca
HMDB www.hmdb.ca
MS/MS analizi sonucunda benzer fragmentasyona sahip iyonlar kimyasal yapı olarak
birbirine benzerlik gösterir. Şekil 2’de görüldügü gibi moleküler network analiziyle benzer
fragmentasyona sahip iyonlar network içinde birlikte gruplandırılmaktadır. Bu gruplandırma
benzer fragment iyon sayısı ve MS/MS pik yoğunluğuna göre program tarafından vektörel
büyüklük (kozin değeri) olarak hesaplanır.
Şekil 2. GNPS ile moleküler ağ analizi (Wang ve ark. 2016).
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1 MS rehberli genom madenciliği
Son yıllarda doğal ürün araştırmalarında genomik bilginin kimyasal verilerle kombine
edilmesi prensibine dayalı farklı stratejiler geliştirilmiştir. Özellikle MS/MS analizi sonucunda
elde edilen metabolit fragmentasyon verisinin, biyoinformatik temelli genom taramaları
sonuçlarıyla birleştirilmesiyle ‘peptidogenomik’ ve ‘glikogenomik’ tarama stratejileri
geliştirilmiştir. Her iki stratejinin temelinde, MS verisiyle genomik bilginin değerlendirilmesi
amaçlanmaktadır.
MS/MS fragmentasyonu sonucunda peptidler (ribozomal ve ribozomal olmayan) daha
küçük peptidlere parçalanmaktadır. Bu parçalanma sonucunda peptid yapısından kopan her
aminoasit spesifik kütle kayma değerine sahip olmaktadır. Bu değere bağlı olarak peptidin
yapısındaki aminoasitler belirlenebilmektedir. Fragmentasyon temelli aminoasit bilgisi,
biyoinformatik programlarca peptid sentezinde olası subsrat olarak kullanılan aminoasit
sonuçlarıyla karşılaştırılıp doğal ürün biyosentezinden sorumlu gen kümesine ulaşılmaktadır.
Peptidogenomik olarak adlandırılan bu yöntem ile stenothricins örneğinde olduğu gibi yeni
doğal ürünler keşfedilmektedir (Kersten ve ark. 2011).
Glikozilasyon, doğal ürün biyosentezinde en yaygın görülen modifikasyon
reaksiyonlarındandır. Glikozilasyon reaksiyonlarının metabolitlerin hücresel haberleşme,
moleküler tanıma ve biyolojik aktivite açısından oldukça önemli olduğu bilinmektedir.
Lomaiviticin ve landomycine gibi glikolize doğal ürünlerin biyolojik aktivite potensinin şeker
üniteleriyle ilgili olduğu düşünülmektedir. Ribozomal (nocathiacin), ribozomal olmayan
(vancomycin), alkaloid (staurosporine) (5) poliketid (erythromycin), ve terpen
(phenalinolactone A) gibi birbirinden oldukça farklı yapılara sahip biyoaktif glikolize doğal
ürünler mikroorganizmalarca sentezlenmektedir.
(5) (6)
Glikolize doğal ürünler yapılarında bulunan şeker ünitesine göre MS/MS analizinde
spesifik fragmentasyon sinyali verirler. Bu sinyaller doğrultusunda doğal ürün yapısındaki
şeker ünitesi tespit edilebilmektedir. Glikozil transferaz, 2,3 dehidrataz, metil transferaz,
aminotransferaz ve nükleotit transferaz glikolize doğal ürün biyosentez gen kümelerinde
bulunan spesifik genlerdir. Doğal ürün biyosentezinden sorumlu mikoorganizmanın
genomunda biyoinformatik programlarla bu spesifik genlerin taranması sonucunda olası
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biyosentez yolakları tespit edilmektedir. MS/MS verisinden glikolize doğal ürün biyosentez
gen kümesine ulaşılması ‘glikogenomik’ olarak adlandırılmaktadır (Kersten ve ark. 2013). Bu
yöntemle son yıllardaki en büyük keşif bir deniz aktinomiseti olan Salinispora tropica
tarafından üretilen salinosporamide A (Marizomib) (6), potansiyel bir antikanser ajanı olarak
saptanan güçlü bir proteazom inhibitörüdür. Multipl miyelomun tedavisi için faz II insan klinik
denemeleri devam etmektedir.
Kaynaklar
Bentley, S.D., Chater, K.F., Cerdeno-Tarraga, A.M., Challis, G.L., Thomson, N.R., James, K.D.,
Harris, D.E., Quail, M.A., Kieser, H., Harper, D., Bateman, A., Brown, S., Chandra, G., Chen, C.W.,
Collins, M., Cronin, A., Fraser, A., Goble, A., Hidalgo, J., Hornsby, T., Howarth, S., Huang, C.H.,
Kieser, T., Larke, L., Murphy, L., Oliver, K., O’Niel, S., Rabbinowitsch, E., Rajandream, M.A.,
Rutherford, K., Rutter, S., Seeger, K., Saunders, D., Sharp, S., Squares, R., Squares, S., Taylor, K.,
Warren, T., Wietzorrek, A., Woodward, J., Barrell, B.G., Parkhill, J., Hopwood, D.A. 2002. Complete
Genome Sequence of the Model Actinomycete Streptomyces coelicolor A3(2). Nature, 417: 141-147.
Choi, S.S., Kim, H.J., Lee, H.S., Kim, P., Kim, E.S. 2015. Genome mining of rare actinomycetes
and cryptic pathway awakening. Process Biochemistry, 50 :1184–1193.
Demain, A.L. 2009. Antibiotics: Natural Products Essential to Human Health. Med Res Rev, 29: 821-
842.
Hosaka, T., Ochi K. 2013. New Strategies for Drug Discovery: Activation of Silent or Weakly
Expressed Microbial Gene Cluster. Apll Microbiol Biotechnol, 97: 87-98.
Kersten RD, Yang YL, Xu Y, Cimermancic P, Nam SJ, Fenical W, Fischbach MA, Moore BS,
Dorrestein PC (2011). A mass spectrometry-guided genome mining approach for natural product
peptidogenomics. Nat Chem Biol 7: 794-802.
Kersten RD, Ziemert N, Gonzalez DJ, Duggan BM, Nizet V, Dorrestein PC, Moore BS (2013).
Glycogenomics as a mass spectrometry-guided genome-mining method for microbial glycosylated
molecules. P Natl Acad Sci USA 110: 4407-4416.
Li, M.H.T., Ung, P.M.U., Zajkowski, J., Tsodikova, S.G., Sherman, D.H. 2009. Automated
Genome Mining for Natural Product. BMC. Bioinfo. 10: 185-195.
Wang M, Carver JJ, Phelan VV, Sanchez LM, Garg N, Peng Y, Nguyen DD, Watrous J, Kapono
CA, Luzzatto-Knaan T et al. (2016). Sharing and community curation of mass spectrometry data
with Global Natural Products Social Molecular Networking. Nat Biotech 34: 828-837.
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A Useful Tool for Distinguishing Gene/Pseudogene Variants Detected via NGS:
Haplotype Analysis
Esra Arslan Ateş1,2, Ahmet İlter Güney2, Tuba GÜNEL1 1Istanbul University, Faculty of Science, Department of Molecular Biology and Genetics, Istanbul.
2Marmara University Pendik Training and Research Hospital, Department of Medical Genetics,
Istanbul.
Correspondence: [email protected]
Abstract
Introduction: Lynch Syndrome is an inherited cancer prone disease mostly associated with
colon, endometrium, ovarian and gastrointestinal tract cancers. Mutations affecting missmatch
repair genes (MLH1, PMS2, MSH2, MSH6) are responsible from Lynch Syndrome. PMS2 has
a psedudogene called PMS2CL showing a high sequence similarity to PMS2 gene.
Here we report a novel mutation in PMS2 gene associated with Lynch Syndrome which was
proven to be on the active gene using haplotype analysis.
Case Report: A 69 year-old woman referred to us because of endometrium cancer. She had a
history of colon cancer diagnosed at the age of 66. Endometrial tumor showed high
microsatellite instability and MLH1 and PMS2 expression loss were detected in
immunohistochemical analysis. There were no other cancer history in her family. BRAF V600E
mutation and MLH1 promoter hypermethyation were not detected in tumor tissue. On the basis
of immunohystochemical findings MLH1 and PMS2 genes were screened for germline
mutations from peripheral blood lymphocytes via next generation sequencing. We detected a
novel heterozygous 47bp insertion (c.1362_1407dup; p.Pro470Valfs*3) in exon 11 of PMS2
gene. The false positive result due to pseudogene was excluded by haplotype analysis using the
SNPs that allow us to distinguish PMS2 and PMS2CL.
Conclusion: PMS2 gene is a MMR system component which has an important role in DNA
replication and homologous recombination, located on chromosome 7p and consist of 15 exons.
There is a pseudogene, PMS2CL, which shows 98% sequence similarity to exons 9 and 11–15
of PMS2 and it is important to distinguish the variations between gene and pseudogene
sequences. NGS data enable us to assess the haplotypes in an amplicon using the distinct SNPs
on gene and psedudogene.
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GİRİŞ
Lynch Sendromu, diğer adıyla Herediter Nonpolipozis Kolon Kanseri (HNPCC) kolorektal
kanserler başta olmak üzere gastrointestinal traktus kanserleri, endometrium kanseri, meme
kanseri gibi kanserlerle karşımıza çıkan kalıtımsal bir kanser yatkınlık sendromudur. Tüm
kolorektal kanserlerin %3’ü ve ednfometrial kanserlerin %3 kadarı Lynch Sendromu ile ilişkili
olarak değerlendirilmiştir [1,2]. Hatalı eşleşme tamir genlerindeki (MMR genleri: MLH1,
MSH2, MSH6, PMS2 ve EPCAM) mutasyonlar Lynch Sendromuna neden olmaktadır [3].
Otozomal dominant kalıtım özelliği dolayısıyla pedigri analizinde vertikal kalıtım özelliği
gösteren aile öyküsü varlığı, tümör dokusunda mikrosatellit instabilitesi ve MMR genlerinde
ekspresyon kaybı saptanması tanıya yönlendiren özelliklerdendir [4].
PMS2 geni Lynch Sendromlu olguların yaklaşık %15’inden sorumlu olup, MMR sisteminin
önemli bir komponentidir. MLH1 ile birlikte bir komleks oluşturarak MMR sürecinde anahtar
bir rol oynar [5]. PMS2 geninin yüksek homoloji gösteren çok sayıda psödogeni bulunmaktadır.
Bu durum moleküler genetik tanıyı zorlaştıran önemli bir problemdir. PMS2CL PMS2 geni
ekzon 9, 10-15 ile yüksek homoloji gösteren bir psödogendir. Bu problemin üstesinden gelmek
için PMS2’ye özgü olan ekzon 10’dan dizayn edilen bir primer kullanmak üzere elde edilen
cDNA’dan Long Range PCR dizaynı kullanılan bir yöntemdir[6].
OLGU
Tarafımıza endometrium kanseri tanısı ile yönlendirilen 69 yaşında kadın olgu polikliniğimizde
değerlendirldi. Olgunun 3 yıl önce kolon kanseri tanısı nedeniyle yapılan rutin takiplerinde
endometrial kitle saptanması üzerine yapılan total abdominal histerektomi öyküsü mevcuttu.
Pedigri analizinde kanser öyküsü bulunmayan hasta izole vaka olarak değerlendirildi. Patolojik
değerlendirmesinde endometrial kanser tanısı alan hastanın tümör dokusunda yüksek
mikrosatellit instabilitesi ve PMS2 ekspresyon kaybı saptandı, MLH1 hipermetilasyonu ve
BRAF V600E mutasyonu saptanmadı. Hastadan bu bulgularla ACMG kriterlerine göre Lynch
Sendromu ön tanısıyla moleküler genetik analiz planlandı [4]. Periferik kandan DNA
izolasyonu (QIAamp DNA Mini Kit, QIAGEN Germantown, MD USA) sonrası MLH1 ve
PMS2 genleri Illumina MiSeq platformunda, HNPCC MASTR Plus (Multiplicom,
Agilent,CA,USA) kiti kullanılarak yeni nesil dizi analizi yöntemiyle değerlendirildi. MLH1
geninde mutasyon saptanmayan olguda PMS2 geni ekzon 11’de heterozigot 47bç bir
duplikasyon saptandı (c.1362_1407dup; p.Pro470Valfs*3) (Şekil 1). Mutasyon veri
tabanlarında tanımlı değildi, çerçeve kaymasına yol açarak erken stop kodonuna sebep olması
beklenmesi dolayısıyla patojenik bir varyant olarak değerlendirildi. Ancak pseödogen
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dolayısıyla tahmin ettiğimiz üzere kopya sayı analizinde ekzon 11’in 4 kopya olduğu
görünmekteydi (Şekil 2). Amplikon değerlendirildiğinde bu bölgede PMS2CL ile PMS2
arasında kısmen düşük bir homoloji olduğu dikkat çekiciydi (%96). Farklı olan nükleotidlerin
değerlendirilmesi ile yapılan haplotip analizinde insersiyonun olduğu okumaların tamamıyla
PMS2 genine ait olduğu ortaya koyuldu (Şekil 3).
Şekil 1: PMS2 geni ekzon 11 47bç insersiyon IGV görüntüsü
Şekil 2: PMS2 CNV analizi
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Şekil 3: PMS2 ve PMS2CL dizileri
Sarı: Farklı nükleotidler Yeşil: Hastada saptanan varyasyonlar
Kırmızı : insersiyon bölgesi
TARTIŞMA
Psödogenler moleküler tanıyı zorlaştıran bir problem olarak karşımıza çıkmaktadır. Gen ile
psödogenler arasındaki minimal farklılıkları kullanarak saptanan mutasyonun gen veya
psödogen üzerinde olduğunu belirlemek ek yöntemlerle gerektirmektedir. Bu durum tanıda
gecikmelere yol açması ve ek olanaklar gerektirmesi bakımından klinik pratikte sıkıntılara yol
açmaktadır. Bu çalışmada yeni nesil dizileme yönteminin tek bir DNA ipliğine ait diziyi ortaya
koyma avantajı kullanılarak amplikondaki tek nükleotid varyasyonları (SNVs) analiz edilmiş
ve amplikonun kopyalandığı DNA molekülünün tamamıyla PMS2 genine ait olduğu
gösterilmiştir. Varyasyonun psödogene ait olma şüphesi durumunda amplikonun bilgi verici
SNV’ler açısısndan değerlendirilmesi ek tetkik gereksinimini ortadan kaldırarak zaman
kazandırmakla birlikte gereksiz harcamaları da azaltacaktır.
KAYNAKLAR
1- Boland, C. R. (2005). Evolution of the nomenclature for the hereditary colorectal cancer
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3- Kuiper, R. P., Vissers, L. E. L. M., Venkatachalam, R., Bodmer, D., Hoenselaar, E., Goossens, M.,
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4- American College of Medical Genetics technical standards and guidelines for genetic testing for
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la Chapelle A. 2004. Mismatch repair gene PMS2: diseasecausing germline mutations are frequent in
patients whose tumors stain negative for PMS2 protein, but paralogous genes obscure mutation
detection and interpretation.
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