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UNIVERSITI PUTRA MALAYSIA
INDUCTION OF APOPTOSIS IN A549 CELLS IN VITRO BY BOESENBERGIN A ISOLATED FROM BOESENBERGIA ROTUNDA
NORBAITI BINTI MOHD ISA
IB 2013 20
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INDUCTION OF APOPTOSIS IN A549 CELLS IN VITRO BY
BOESENBERGIN A ISOLATED FROM BOESENBERGIA ROTUNDA
By
NORBAITI BINTI MOHD ISA
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfillment of the Requirements for the Degree of Master of
Science
July 2013
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All material contained within the thesis, including without limitation text, logos, icons, photographs and all other artwork, is copyright material of Universiti Putra Malaysia unless otherwise stated. Use may be vary of any material contained within the thesis for non-commercial purposes from the copyright holder. Commercial use of material may only be made with the express, prior, written permission of Universiti Putra Malaysia. Copyright © Universiti Putra Malaysia
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Abstract of thesis presented to the senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science
INDUCTION OF APOPTOSIS IN A549 CELLS IN VITRO BY
BOESENBERGIN A ISOLATED FROM BOESENBERGIA ROTUNDA
By
NORBAITI BINTI MOHD ISA
July 2013
Chairman: Ahmad Bustamam Abdul, PhD
Faculty: Institute of Bioscience
Boesenbergin A (BA) is a chalcone isolated from Boesenbergia rotunda,
containing methoxyl and hydroxyl groups in its structure. These groups have
been reported to exert anti-proliferative, antioxidant and anti-inflammation
activities. However, there has been no literature reported on the biological
activities of BA and its effects on lung cancer. The current in vitro study was
designed to investigate the apoptotic induction of BA in A549 cells, as well
as its antioxidant and anti-inflammatory activities. Human hepatocellular
carcinoma cell (HepG2), human prostate cancer cell (PC3), colon
adenocarcinoma cell (HT-29), non-small cell lung cancer cell (A549) and
normal hepatic cells (WRL-68) were used to evaluate the cytotoxicity of BA
using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
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assay. The antioxidant activity of BA was assessed by the Oxygen Radical
Absorbance Capacity (ORAC) assay and compared to quercetin as a
standard reference antioxidant. The anti-inflammatory assay was performed
by inducing murine monocytic macrophage cell line (RAW 264.7) using 100
U/mL of interferon-γ (IFN-γ) and 5 µg/mL of lipopolysaccharide (LPS) with or
without the presence of BA, and compared concurrently to L-Nitro-Arginine
Methyl Ester (L-NAME) as positive control. The production of nitric oxide
(NO) after inducing the inflammation was then assessed using Griess
reaction. Multiparametric High Content Screening (HCS) assay was
conducted to detect changes in total nuclear intensity (chromatin
condensation and fragmentation), cell permeability, mitochondrial membrane
potential and release of cytochrome c. Caspase assay for caspase-8, -9 and
-3/7 were analyzed using luminescence reader, whereas DNA fragmentation
was detected through gel electrophoresis. To measure the production of
intracellular ROS, 2′,7′-dichlorofluorescin diacetate (DCFH-DA) was used.
Cell cycle analysis was performed using Flow Cytometer. Expression of
Bax, Bcl2 and Hsp70 protein were detected by using the western blot
analysis.
Toxic effect of BA on different cell types, reported as IC50, yielded 20.22 ±
3.15, 10.69 ± 2.64, 20.31 ± 1.34, 94.10 ± 1.19, and 9.324 ± 0.24 µg/mL for
A549, PC3, HepG2, HT-29 and WRL-68, respectively. ORAC results are
reported as the equivalent concentration of trolox that produces the same
level of antioxidant activity as the samples tested at 20µg/mL. BA displayed
low antioxidant activity, when the results of ORAC assay were reported as
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trolox equivalents, where BA (20 µg/mL) and quercetin (5 µg/mL) were
equivalent to a trolox concentration of 11.91 ± 0.23 and 160.97 ± 0.02 µM,
respectively. For the anti-inflammatory assay, the results clearly showed a
concentration-dependent decrease in NO production in the induced RAW
264.7 cells, with a significantly low level being evident even at 25 and 50 µM
of BA concentrations, with a level of 29.69 ± 2.5 and 25.69 ± 2.0 µM of NO
production respectively. The anti-inflammatory activity of BA was significant
between concentration of 25 µM to 50 µM but with no significant cytotoxicity
towards RAW 264.7 cells at 50 µM. The anti cancer activities of BA was
investigated using A549 cell line, as BA was found to be cytotoxic to A549
cells (IC50< 30 µg/mL). The HCS results showed the BA-induced A549 cells
exhibited a concentration-dependent increase in total nuclear intensity, cell
permeability and cytochrome c release, whereas the cells mitochondrial
membrane potential was decreasing (p<0.05), (p<0.01). Luminescence
assay for caspase-8, -9 and -3/7 revealed an elevation of all three caspases
in concentration-dependent manner (p<0.05). A549 cells treated with BA
showed DNA fragmentation at the concentration of 20 and 50 µg/mL of BA.
Production of ROS was also elevated in a concentration-dependent manner,
indicated by the increase of DCF fluorescence intensity in treated cells
(p<0.05). Protein analysis by western blot showed an increase of Bax, whilst
Bcl2 and Hsp70 protein were subsequently decreased due to time-
dependent treatment (p<0.05). Cell cycle analysis by flow cytometer showed
a significant concentration-dependent increase in the sub G1 phase
(p<0.05). A slight increase of cell arrest was observed in S phase but it was
not statistically significant (p>0.05), which confirmed cell cycle arrest was not
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induced in treated A549 cells. Collectively, results presented in this study
demonstrate that BA inhibited the proliferation of non-small lung cancer in
vitro, leading to a possible induction of apoptosis, which was later confirmed
to be induced through the extrinsic and intrinsic pathways. Moreover the role
of free radicals was significantly found to be elevated with concomitant
decrease in Hsp70. This compound also showed low antioxidant capacity
and significant anti-inflammatory activity. BA can be a potential drug to treat
lung cancer and inflammation.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains
PENGARUHAN APOPTOSIS DI DALAM SEL A549 IN VITRO OLEH
BOESENBERGIN A YANG DIASINGKAN DARIPADA BOESENBERGIA
ROTUNDA
Oleh
NORBAITI BINTI MOHD ISA
Julai 2013
Pengerusi: Ahmad Bustamam Abdul, PhD
Fakulti: Institut Biosains
Boesenbergin A (BA) ialah sejenis calkon daripada Boesenbergia rotunda
yang mengandungi kumpulan methoxyl dan hydroxyl di dalam strukturnya.
Kumpulan ini pernah dilaporkan mengandungi aktiviti anti-proliferatif,
antioksida dan anti-radang. Walaubagaimanapun, belum terdapat sorotan
literatur yang melaporkan tentang aktiviti biologi BA dan kesannya terhadap
kanser paru-paru. Kajian in vitro kini direka bertujuan untuk menyiasat
pencetusan apoptosis oleh BA pada sel A549, juga aktiviti antioksida dan
anti-radangnya. Sel kanser hati manusia (HepG2), sel kanser prostat
manusia (PC3), sel kanser kolon (HT-29), sel kanser paru-paru bukan kecil
(A549) dan sel hati normal (WRL-68) telah digunakan untuk menilai
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sitotoksisiti BA dengan menggunakan asai MTT. Aktiviti antioksida BA dinilai
melalui asai ORAC lalu dibandingkan dengan quercetin yang bertindak
sebagai anti oksida rujukan standard. Asai anti-radang telah dilaksanakan
dengan meransang sel murine monocytic macrophage (RAW 264.7)
menggunakan 100 U/mL interferon-y (IFN-y) dan 5 µg/mL
lipopolysaccharide (LPS) dengan atau tanpa kehadiran BA, dan
dibandingkan dengan L-Nitro-Arginine Methyl Ester (L-NAME) yang
bertindak sebagai kawalan positif. Penghasilan nitric oxide (NO) selepas
meransang keradangan kemudiannya dinilai menggunakan reaksi Griess.
Multiparametrik High Content Screening (HCS) dijalankan bagi mengesan
perubahan dalam intensiti nuklear seluruh (kondensasi nuclear dan
fragmentasi), ketembusan sel, potensi membran mitokondria dan pelepasan
sitokrom c. Asai caspase untuk caspase-8, -9 dan -3/7 telah dianalisis
menggunakan pembaca luminesen, sementara fragmentasi DNA dikesan
melalui kaedah gel elektroforesis. Bagi mengukur penghasilan ROS
intraselular, 2′,7′-dichlorofluorescin diacetate (DCFH-DA) digunakan. Kitaran
sel analisis pula dilaksanakan dengan menggunakan flow cytometer.
Ekspresi protein Bax, Bcl2 dan Hsp70 dikesan menggunakan analisis
western blot.
Kesan toksik BA terhadap jenis sel yang berlainan, dilaporkan sebagai IC50,
masing-masing bernilai 20.22 ± 3.15, 10.69 ± 2.64, 20.31 ± 1.34, 94.10 ±
1.19, dan 9.324 ± 0.24 µg/mL untuk A549, PC3, HepG2, HT-29 and WRL-
68. Keputusan ORAC dilaporkan sebagai kepekatan setaraf trolox yang
menghasilkan aktiviti anti oksida yang sama paras dengan sampel yang diuji
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pada 20 µg/mL. BA menunjukkan aktiviti anti oksida yang rendah apabila
keputusan ORAC dilaporkan sebagai setaraf trolox, di mana BA (20 µg/mL)
dan quercetin (5 µg/mL) adalah setaraf kepada kepekatan trolox iaitu 11.91
± 0.23 dan 160.97 ± 0.02 µM. Bagi asai anti-radang, keputusannya jelas
menunjukkan peningkatan secara kebergantungan-kepekatan dalam
penghasilan NO dalam sel RAW 264.7 yang diransang, dengan paras
rendah BA secara siknifikan menjadi bukti walau pada kepekatan 25 dan 50
µM, dengan penghasilan NO masing-masing pada paras 29.69 ± 2.5 dan
25.69 ± 2.0 µM. Aktiviti anti-radang BA adalah siknifikan antara kepekatan
25 µM hingga 50 µM tetapi tanpa sitotoksisiti yang siknifikan terhadap sel
RAW 264.7 pada 50 µM. Aktiviti anti-kanser BA yang selanjutnya disiasat
dengan menggunakan sel A549 memandangkan BA didapati bersifat
sitotoksik terhadap sel A549 (IC50<30 µg/mL). Keputusan HCS menunjukkan
bahawa sel A549 yang diransang oleh BA mempamerkan peningkatan
secara kebergantungan-kepekatan dalam intensiti nuklear seluruh,
ketembusan sel dan pelepasan sitokrom c, manakala potensi membran
mitokondria pula menurun (p<0.05), (p<0.01). Asai luminesen untuk
caspase-8,-9 dan -3/7 mendedahkan peningkatan bagi ketiga-tiga caspase
secara kebergantungan-kepekatan (p<0.05). Sel A549 yang dirawat dengan
BA menunjukkan fragmentasi DNA pada kepekatan 20 dan 50 µg/mL
(p<0.05). Penghasilan ROS ditingkatkan juga secara kebergantungan-
kepekatan, diindikasi oleh peningkatan intensiti fluoresen dalam sel yang
dirawat (p<0.05). Analisis protein oleh western blot menunjukkan
peningkatan Bax, sementara protein Bcl2 dan Hsp70 menurun berikutan
rawatan secara kebergantungan-masa (p<0.05). Analisis kitaran sel oleh
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flow cytometer menunjukkan peningkatan secara kebergantungan-
kepekatan pada fasa sub G1 (p<0.05). Sedikit peningkatan pada
penangkapan sel dapat dilihat pada fasa S, akan tetapi ia tidak siknifikan
secara statistik (p>0.05), yang membuktikan bahawa penangkapan sel tidak
dicetuskan oleh BA pada sel A549 yang dirawat. Secara keseluruhan,
keputusan yang dipersembahkan dalam kajian ini menunjukkan bahawa BA
menghalang proliferasi ke atas sel kanser paru-paru bukan-besar secara in
vitro, membawa kepada pendorongon apoptosis, yang selanjutnya disahkan
didorong melalui laluan ekstrinsik dan intrinsik. Tambahan pula, peranan
radikal bebas didapati secara meningkat secara siknifikan dengan dituruti
penurunan Hsp70. Kompaun ini juga menunjukkan kapasiti antioksida yang
rendah dan aktiviti anti-radang yang siknifikan. BA berpotensi menjadi ubat
untuk merawat kanser paru-paru dan keradangan.
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ACKNOWLEDGEMENTS
First and foremost, I would like to express tremendous gratitude, respect and
admiration for my supervisor, Ahmad Bustamam Hj Abdul, PhD. Throughout
my studies, I have learned from his wisdom and experience, and benefited
from his continuous guidance and support. His enthusiasm and commitment
to this research project is deeply appreciated and undoubtly invaluable. He
has been the most wonderful mentor and teacher. My utmost appreciation
also goes to Prof. Dr. Rasedee Abdullah and Assoc. Prof. Dr. Siddig Ibrahim
Abdelwahab, that without their continuous support, encouragement, help,
and advice, I was not be able to continue and complete this thesis.
I also want to thank Assoc. Prof. Dr. Syam Mohan for his help and support.
He’s not only a senior, but also a brother to me. Special thanks to all my
fellow colleagues and staffs at UPM Makna Cancer Research Laboratory.
I also wish to thank my mother, Puan Norkiah Adnan for her unconditional
love and support.
Not to forget my lovely housemates: Suzita, Norliana and Nabila for lending
your ears and sharing your ideas.
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APPROVAL
I certify that a Thesis Examination Committee has met on 4th July 2013 to conduct the final examination of Norbaiti binti Mohd Isa on her Master of Science thesis entitled “INDUCTION OF APOPTOSIS IN A549 CELLS IN VITRO BY BOESENBERGIN A ISOLATED FROM BOESENBERGIA ROTUNDA” in accordance with the Universities and University Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U. (A) 106] 15 March 1998. The Committee recommends that the student be awarded the Master of Science. Members of Thesis Examination Committee were as follows: Md. Zuki Abu Bakar, PhD Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Chairman) Hazilawati Hamzah, PhD Senior Lecturer Faculty of Veterinary Medicine Universiti Putra Malaysia (Internal Examiner) Sharida Fakurazi, PhD Associate Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Internal Examiner) Vijayapandi Pandy, PhD Associate Professor Faculty of Medicine Universiti Malaya (External Examiner) ________________________________ NORITAH OMAR, PhD Assoc. Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia
Date:
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This thesis submitted to the Senate of University Putra Malaysia and has been accepted as fulfillment of the requirement for the degree of Master of Science. The members of the Supervisory Committee are as follows: Ahmad Bustamam Hj Abdul, PhD
Senior Lecturer Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Chairman) Rasedee Bin Abdullah, PhD
Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member) Siddig Ibrahim Abdelwahab, PhD
Associate Professor Medical Research Centre Jazan University Kingdom of Saudi Arabia (Member)
_________________________ BUJANG BIN KIM HUAT, PhD
Professor and Dean School of Graduate Studies Universiti Putra Malaysia
Date:
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DECLARATION
I declare that the thesis is my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously, and is not concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other institution.
NORBAITI BINTI MOHD ISA
Date: 4th July 2013
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TABLE OF CONTENTS Page
ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS
ii vi x xi xiii xviii xix xxi
CHAPTER
1
INTRODUCTION
1
2 LITERATURE REVIEW
7
2.1 Natural Products
7
2.2 Drug Discovery from Plant-Derived Substances
8
2.3 Plant Derived Antioxidant Compounds
10
2.4 Plant Derived Anti Inflammatory Compounds
11
2.5 Plant Derived Anti Cancer Compounds
12
2.6 Natural Compounds from Local Medicinal Plants
13
2.7 Zingiberaceae
14
2.8 Boesenbergia Rotunda
15
2.8.1 Biological Activities of B. rotunda
17
2.9 Disorders and diseases
18
2.9.1 Oxidative stress
18
2.9.2 Inflammation
20
2.9.3 Cancer
21
2.10 Biology of Tumour
24
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2.11 Lung Cancer
24
2.12 Non Small Cell Lung Cancer
26
2.13 A549 Cell Line
27
2.14 Cell Death
27
2.14.1 Necrosis
28
2.14.2 Apoptosis
29
2.14.3 Morphological and biochemical characteristics of apoptosis
30
2.14.4 Apoptosis Pathways
31
2.15 Caspases
34
2.16 Bcl2 Family
37
2.17 Hsp70 Protein 2.18 Reactive Oxygen Species (ROS)
38
39
2.19 Cell Cycle
39
3 MATERIALS AND METHODS
43
3.1 Isolation of boesenbergin A
43
3.2 In vitro cytotoxicity effects of boesenbergin A 3.2.1 Cell Culture Condition 3.2.2 Cell Viability Assay
43
43
43
3.3 ORAC Antioxidant Activity Assay
44
3.4 Anti Inflammatory Assay 45 3.4.1 Cell Culture and Stimulation
45
3.4.2 Nitrite Determination Using the Griess Assay
46
3.4.3 RAW 264.7 Cell Viability
47
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3.5 Multiparametric High Content Screening (HCS) Assay
47
3.6 Caspase -8, -9 and -3/7 assay 3.7 DNA Laddering
49
49
3.8 Measurement of reactive oxygen species generation
50
3.9 Protein Detection by Western Blotting 3.9.1 Extraction of Whole Protein from the Cell
51
51 3.9.2 Western Blotting Analysis
3.10 Cell Cycle Analysis 3.11 Statistical Analysis
51
53
54
4 RESULTS
55
4.1 Cytotoxic Assay
55
4.2 Orac Antioxidant Activity Assay
58
4.3 Effect of Boesenbergin A on NO production and RAW 264.7 cell viability
59
4.4 Multiparametric High Content Screening (HCS) Assay
61
4.5 Caspase-8, -9 and -3/7 Assay
4.6 DNA Laddering
64
65
4.7 Boesenbergin A induces ROS formation
66
4.8 Western Blot Analysis 67
4.9 Flow Cytometric Analysis of Cell Cycle 71 5
DISCUSSION
74