Transcript of 1 Sonexai Kidoikhammouan M.Sc. Student, Department of Biochemistry Faculty of Medicine, KKU 17 th...
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- 1 Sonexai Kidoikhammouan M.Sc. Student, Department of
Biochemistry Faculty of Medicine, KKU 17 th August, 2012 Advisory
committees: Assoc. Prof. Dr. Chaisiri Wongkham Dr. Wunchana Seubwai
Dr. Atit Silsilivanit TNP- 470 as a potential adjuvant Therapy for
Cholangiocarcinoma External examiners: Assoc.Prof.Dr. Sopit
Wongkham Assis.Prof.Dr. Chariya Hahnvajanawong
- Slide 2
- Contents Introduction Hypothesis and Research questions
Objectives Conceptual framework Experimental design Anticipated
outcomes Research Plan 2
- Slide 3
- Introduction Intrahepatic CCA (ICC) Extrahepatic CCA (ECC) 3
Cholangiocarcinoma (CCA)
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- Epidemiology of CCA 4 Bragazzi et al., 2011
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- CCA alternative treatments Surgery Adjuvant therapy
Chemotherapy Radiation therapy 5
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- Surgery correlated with survival and recurrence rate in CCA
patients 6 Methods CCA subtype n Five-year survival rate (%)
Recurrence (%) Reference Portal vein and hepatic artery resection
Hilar29842NDIgami et al., 2010 Liver resection ECC 3420ND Guglielmi
et al., 2009 Liver resectionICC453530 Yedibela et al., 2009 Liver
resectionICC9731.1ND Palik et al. 2008 Liver
resectionICC4463NDDeOliveira et al. 2007 Liver resection with
lymphadenectomy Hilar262127Rea et al. 2005 Liver
resectionICC343262Casavilla et al. 1997 However, this regimen is
still giving low survival, but high recurrence rate Further more,
some patients can not undergo such regimen
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- Association between response rate and median time survival
using Chemotherapy 7 DrugnRR(%) MST (Months) Reference
Capecitabine4032.59.4Furuse et al.,2008 Gemcitabine
4017.57.6Okusaka et al., 2006 Gemcitabine/capecitabine45 3214Cho et
al.,2005 Gem/cisplatin 40289 Thongprasert et al., 2005
Gemcitabine/5-FU27335.3Knox et al., 2004 5-FU/FA 30714.8Malik et
al., 2003 5-FU/oxaliplatin 165610Nehls et al. 2002 Irinotecan
3686.1Sanz-Altamira et al., 2002 RR, response rate; MST, median
survival time; 5-FU, 5-fl uorouracil; FA, folinic acid;
Nevertheless, chemotherapy is still giving low response rate and
median survival time
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- Radiotherapy 8 2-year survival was 80% and 4-year survival 30%
Polistina et al. 2011 Survival time were 12.9 mo in patients who
received EBRT Vlek et al., 2007 EBRT; external beam radiation
therapy External beam radiotherapy 19.1 mo survival time Jiang et
al., 2010 The need of targeted molecules with novel chemotherapy
and adjuvant therapeutic strategies for diagnosis and treatment of
CCA patients are increasing nowadays
- Slide 9
- Serial analysis of gene expression(SAGE) 9 K4 ; Low invasive
cell s K3; High invasive cells K2D; Poorly differentiated
adenocarcinoma K1; Metastatic tumor (intrahepatic metastasis from
cholangiocarcinoma primary tumor)
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- Comparison of MetAP2 expressions 10 Normal biliary cells
Hyperplastic and dysplastic bile duct epithelia Well differentiated
tubular CCA Lymph node with metastatic CCA Sawanyawisuth et al.,
2007
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- Comparison of MetAP2 expressions in bile duct epithelia 11
Sawanyawisuth et al., 2007
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- Expression and association of MetAP2 in cancers 12 Cancer
Expression in cancer Clinical findingReference
ColonHighProliferation Apoptosis Selvakumar et al., 2009
CholangiocarcinomaHigh Proliferation Metastasis Sawanyawisuth et
al., 2007 NeuroblastomaHighAngiogenesisMorowitz et al., 2005
Hepatocellular carcinomaHigh Tumor growth, Metastasis Sheen et al.,
2005 MesotheliomaHighProliferationCatalano et al.,2001
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- MetAP2 structure 13 Addlagatta et al., 2005
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- How does MetAP2 work? 14 Met
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- MetAP2 inhibitors 15 Cell growth inhibition Anti-angiogenesis
Datta et al.,2009
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- Why we choose TNP-470? 16 Wang et al. 2008 TNP-470 gives high
potential on antitumor activity and endothelial cell growth more
than other MetAP2 inhibitors such as fumagillin, bestatine and
anthranilic acid sulfonamide (Wang et al. 2008; Ingber et al.,
1990)
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- Anti-tumor activity of TNP- 470; in vitro 17 Cell
typeResultReference FU-MMT-1 cellsAnti angiogenesisNaganuma et
al.,2011 Endothelial cell B16F10 melanoma G 1 arrestHines et al.,
2010 Wanget al.,2008 Fetal mouse bone cell Vasculature disruption
Anti angiogenesis Wijngaarden et al., 2010 B16F10 (murine
melanoma)Induction apoptosisOkrj et al.,2006 Human pancreaticGrowth
inhibitionHotz et al., 2001
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- Anti - tumor activity of TNP- 470; in vitro; animal model 18
Cell typeResultReference Human uterine carcinosarcromaTumor
growthNaganuma et al.,2011 Human gioblastomaTumor growthYao et al.,
2010 Murine neuroblastoma Proliferation Apoptosis Chesler, et
al.,2007 SarcomaTumor growthKanamori et al.,2007 Human Wilms tumor
cellsAntiangiogenesisHuang et al.,2004
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- Effect of TNP- 470 in clinical trial 19 Cancer typeDose (mg/m 2
) nRR(%)Reference Solid tumor (lung, sarcoma, thymoma) 601724Tran
et al., 2004 Lung cancer603233Herbst et al., 2002 Kaposis
sarcoma10-7038NDDezube et al.,1998 Cervical cancer9.3
-71.218NDKudelka et al.,1998 Renal carcinoma60333Stadler et
al.,1999 Prostate7133NDStadler et al.,1994 RR; response rate ND, no
determine
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- Hypothesis Suppression of MetAP2 activity by TNP-470 can
inhibit proliferation, migration/invasion and enhance anti-tumor
activity of chemotherapeutic drugs in CCA cell lines. 20
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- Research questions 21 1. Does supplementation of TNP-470
inhibit the proliferation, migration and invasion of CCA cell
lines? 2. What is molecular mechanism by which TNP-470 affects the
proliferation, migration and invasion of CCA cell lines? 3. Can
supplementation of TNP-470 enhances the anti- tumor activity of
chemotherapeutic drugs in CCA cell lines?
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- Objectives 22 1. To determine the effect of TNP470 on
proliferation, migration and invasion of CCA cell lines. 2. To
identify the molecular mechanism by which TNP470 affects
proliferation, migration and invasion of CCA cell lines. 3. To
explore the possibility of using TNP470 as an adjuvant therapy of
CCA.
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- Conceptual framework 23 Background Cancers with high expression
of MetAP2 High proliferation High metastasis Enhance Angiogenesis
MetAP2 inhibitors
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- Conceptual framework (cont) 24 CCA cell lines high expression
of MetAP2 High proliferation High metastasis Enhance
chemotherapeutic drug MetAP2 inhibitor: TNP-470 ?? Hypothesis
?
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- Experimental design Selected CCA cell lines with high
expression of MetAP2 Study the effect of TNP - 470, MetAP2
inhibitor Growth Metastasis Chemotherapeutic drug response -
Proliferation - MTT assay - Cell cycle and apoptosis - Flow
cytometry - Invasion assay - Migration assay - Adhesion assay
Chemotherapeutic sensitizing (5-FU, Cisplatin, Doxorubicin and
Gemcitabine) Determine the molecular mechanism Genes related to
metastasis (c-Myc, MMP2, MMP9) Genes related to apoptosis
(Casepase3, Bax, Bcl-2, p38 )
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- Anticipated outcomes TNP-470 and its combination with
chemotherapeutic drugs will be the basic knowledge for treatment of
CCA patient in the future Part of this thesis outcome will be
presented in a national/international scientific conference At
least one publications in an international journal are expected
26
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- Research plans Activities 20112013 Apr - Jun Jul-SepOct-Dec
Jan-Mar 1 Literature review 2 MetAP2 expression in CCA cell lines
3. Proposal examination * 4. Investigation of MetAP2 functions on
metastasis of CCA Cell proliferation, adhesion, migration and
invasion assays 5. Investigation of underlying mechanism by which
MetAP2 play roles in the particular function cell cycle and
apoptosis analysis Determine molecular gene 6.Data analysis and
thesis writing 7.Manuscript preparation 8.Thesis defense *
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- Pilot study 28
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- Expression of CCA cell line 29
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- Effect of TNP-470 on CCA cells proliferation 30
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- Effect of TNP-470 on CCA cell migration 31 Vehicle TNP - 470
(1.25 g/ml) KKU - M213KKU - M214
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- Effect of TNP-470 on CCA cell invasion 32 Vehicle TNP - 470
(1.25 g/ml) KKU - M214KKU - M213
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- Determination molecular mechanism 33
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- Acknowledgements 34
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- 35 Thank you very much
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- Structure of fumagillin and TNP-470 36 Ingber et al., 1990
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- SAGE process 1.Isolate the mRNA of an input sample. 2.Extract a
small chunk of sequence from a defined position of each mRNA
molecule. 3.Link these small pieces of sequence together to form a
long chain 4.Clone these chains into a vector which can be taken up
by bacteria. 5.Sequence these chains using modern high- throughput
DNA sequencers 6.Process this data with a computer to count the
small sequence tags 37
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- Conclusion CCA is a malignant cancer, its early state for
diagnosis and very poor prognosis because its low response to
treatments. The need of targeted molecules with novel
chemopreventive and adjuvant therapeutic strategies for diagnosis,
prognosis, and treatment of CCA patients have been increasing in
nowadays. Overexpression of MetAP2 play a crucial role in several
cancers especially in CCA development. Inhibition of MetAP2
activity by its inhibitors is lethal for cancers No studies
regarding effects of TNP-470 adjuvant with chemotherapeutic drugs
in CCA has been reported, therefore, this effects are of our
interest. 38
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- Pathological feature of CCA patients and expression of MetAP2
in primary tissue 39
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- Effect of fumagillin in CCA cells 40 Sawanyawisuth et al.,
2007
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- Effect of fumagillin on cell growth 41 Hou et al. (2009).
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- Immune suppression in murine 42
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- Mass forming 43 Blechacz et al., 2011
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- periductal-infiltrating type 44 Blechacz et al., 2011
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- The intraductal-growth type 45 Blechacz et al., 2011
- Slide 46
- Criteria for diagnosis 1. Tumor stage, 2.Tumor location
3.Growth pattern 46
- Slide 47
- Diagnosis of cholangiocarcinoma Diagnosis of intrahepatic
cholangiocarcinoma Require histopathology and is a diagnosis of
exclusion; a pathologic staging system The diagnosis of perihilar
cholangiocarcinoma is often made clinically, and is aided by
cytologic fluorescent in situ hybridization studies; staging
systems for this subtype of cholangiocarcinoma are still evolving
Diagnosis of distal extrahepatic cholangiocarcinoma can usually be
confirmed by cytology; stage is highly dependent upon depth of
invasion of surrounding structures 47 Blechacz et al., 2011
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- Risk factors for CCA 48 Blechacz et al., 2011
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- CCA classification 49 Blechacz et al., 2011
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- CCA subtype 50
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- Adjuvant chemoradiation therapy 51 Adjuvant chemoradiation
therapy 5-year survival; 5-FU plus radiotherapy 35% and surgical
resection alone 27% Hughes et al. 2007 Adjuvant chemoradiation
therapy 5-year survival; 5-FU plus radiotherapy 35% and surgical
resection alone 27% Hughes et al. 2007
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- ERK 52
- Slide 53
- Capecitabine convert to 5-FU 53 phosphorylated 5-FU is
converted to its deoxynucleoside, which inhibits DNA synthesis by
blocking the functions of a key enzyme in DNA replication-
thymidylate synthetase. phosphorylated and incorporated into RNA
where it causes miscoding and halts protein synthesis. Side effects
Vomiting Poor appetite sores in mouth, lips, or throat hair loss or
thinning (may include face and body hair) diarrhea dry, flaky,
cracking skin
- Slide 54
- Gemcitabine Gemcitabine diphosphate effectively inhibits
ribonucleotide reductase inducing a depletion of cellular
deoxynucleotides (dNTP). On the one hand this will inhibit DNA
synthesis by lack of sufficient DNA precursors. 54 weakness, loss
of appetite, headache, cough, chills, and muscle aches); hair loss;
infection (fever, chills, sore throat); Side effects
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- Irinotecan prevents DNA from unwinding by inhibition of
topoisomerase 1 55 Side-effects The most significant adverse
effects of irinotecan are severe diarrhea and extreme suppression
of the immune system. Diarrhea Irinotecan-associated diarrhea is
severe and clinically significant, sometimes leading to severe
dehydration requiring hospitalization or intensive care unit
admission. This side-effect is managed with the aggressive use of
antidiarrheals such as loperamide or Lomotil with the first loose
bowel movement.loperamideLomotil Immunosuppression The immune
system is adversely impacted by irinotecan. This is reflected in
dramatically lowered white blood cell counts in the blood, in
particular the neutrophils. The patient may experience a period of
neutropenia (a clinically significant decrease of neutrophils in
the blood) while the bone marrow increases white cell production to
compensate.white blood cell neutrophilsneutropenia
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- cisplatin these platinum complexes react in vivo, binding to
and causing crosslinking of DNA which ultimately triggers apoptosis
(programmed cell death) 56 Nephrotoxicity (kidney damage)
Neurotoxicity (nerve damage) Ototoxicity (hearing loss) Side
effects
- Slide 57
- Folinic acid Folinic acid, therefore, allows for some
purine/pyrimidine synthesis to occur in the presence of
dihydrofolate reductase inhibition, so that some normal DNA
replication and RNA transcription processes can proceed. 57
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- Cell cycle control 58
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- Regulates cell growth and Protein synthesis 59 P67/MetAP2 eIF2
Specific kinase eIF2 Specific kinase P P67/MetAP2 ERK1/2 P67/MetAP2
ERK1/2 P P Inhibition of Protein synthesis Inhibition of Cell
Growth Protein synthesis Deglycocetylation ?
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- Effect of TNP-470 on angiogenesis 60 Naganuma et al., 2011
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- Effect of TNP-470 on mouse xenograft 61 Yao, Zhao et al.
2010
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- Effect ofTNP-470 on cell cycle 62 TNP-470 arrest cell cycle at
G 1 phase Wang et al. 2008
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- 63 Wang et al. 2008
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- Reversible inhibition of MetAP2 catalytic activity by A-800141
64 Wang et al. 2008
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- Western blot analysis of cell cycle proteins in HUVEC treated
with MetAP2 inhibitors. 65 Wang et al. 2008
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- MetAP2 inhibition results in formation of cellular GAPDH
variants with an unprocessed N-terminal methionine. 66 Wang et al.
2008
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- Effects on growth of the FU-MMT-1 xenografts 67 Emoto et al.,
2007
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- Anti-tumor effect of radiation response by combined treatment
with angiogenesis inhibitor, TNP-470, in oral squamous cell
carcinoma 68 Shintani et al., 2006
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- TNP-470 promotes initial vascular sprouting in xenograft Huang
et al., 2004
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- Malignant Progression and Blockade of Angiogenesis
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- Hitting the mother lode of tumor angiogenesis
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- Post-translational myristoylation: Fat matters in cellular life
and death
- Slide 73
- CCA 73 Khan, S., et al., 2005
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- MetAP2 inhibitorsTarget gene in cell cycle arrest Target gene
in apoptosis Cell typereference fumagillinG 1 arrest cyclinE 2
Bcl-2 Colorectal, hepatocellular carcinoma Human Mesothelioma
Hou.L.et.al (2009) Sheen,I.et.al (2005) Catalano,A.et.al (2001)
TNP-470 or AGM-1470 G 1 arrest P53, p21, p27 p-RB cyclinE p-RB
CDK/cyclin G 0 arrest HUVEC BAEC Zhang,Y.et.al (2000) Abe,J.et.al
(1994) Antoine, N. et.al (1994) A-800141G 1 arrest p53 and p21 p-RB
human neuroblastom a Wang,J.et.al (2007) 74
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- MetAP2 inhibitorsTarget gene in cell cycle arrest Target gene
in apoptosis Cell typereference A-353700G 1 arrest p-RB cyclinA
Carcinoma, Sarcoma, neuroblastoma Wang,J.et.al (2003) hybrid of
1-deoxynojirimycin (DNJ) and an aryl- 1,2,3-triazole G 1 arrest
cyclin D1 ERK1/2 BAEC Zhao,Y. et.al. (2008) IDR-803, IDR-804,
CKD-732 G 1 arrest p21 HUVECChun,E.et.al (2005) PPI-2458G 1
arrestHUVECBainbridge, J.et.al.(2007) 75 bovine aortic endothelial
cell (BAEC)
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- MetAP2 76 Addlagatta et al. 2005 EnzymessimilarityDissimilarity
MetAP1 MetAP1 Could compensate when MetAP2 is inactive (in yeast)
Mn 2+ in active site Play role in the G2/M phase of cell cycle
MetAP2 MetAP2 inhibition leads to G1 arrest number of key Respects
( 60-aa insert)
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- retinoblastoma protein 77
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- Cell cycle control 78
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- Criteria for choosing chemotherapeutic drugs 79 1.Different
mechanism from cytotoxic agent 2.Side effect 3.Cost
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- Signal transduction 80
- Slide 81
- Expression of MMP-2 in bladder cancer 81 Seiler et al.,
2011
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- Expression of MMP-9 in bladder cancer 82 Seiler et al.,
2011
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- MetAP2 specific substrates 83 1.Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) 2.cyclophilinA
- Slide 84
- BCL2 (B-cell leukemia/lymphoma 2) antiapoptosis, through a
possibly complex process; dimerization, especially with BAX; role
of the BCL2 anti- apoptosis members in forming complexes with
caspase-9 and APAF1 (homolog of the nematode CED-4), which prevent
them to initiate the protease cascade (through caspase-3 cytochrome
C dependent activation and) leading to apoptosis 84
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- PI staining 85 Selected CCA cell lines with high expression of
MetAP2 Study the effect of TNP - 470, MetAP2 inhibitor Growth
Metastasis Chemotherapeutic drug response - Proliferation - MTT
assay - Cell cycle and apoptosis - flow cytometry - Proliferation -
MTT assay - Cell cycle and apoptosis - flow cytometry - Invasion
assay - Migration assay - Adhesion assay - Invasion assay -
Migration assay - Adhesion assay Chemotherapeutic sensitizing (5-FU
and Gemcitabine ) Chemotherapeutic sensitizing (5-FU and
Gemcitabine ) Determine the molecular mechanism Genes related to
proliferation (ERK1/2, cyclinE, p21, Rb) Genes related to
proliferation (ERK1/2, cyclinE, p21, Rb) Genes related to
metastasis (ICAM1, ALCAM, MMP2, MMP9) Genes related to metastasis
(ICAM1, ALCAM, MMP2, MMP9) Genes related to apoptosis (bcl-2, p53,
) Genes related to apoptosis (bcl-2, p53, )
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- SYBR 86 Selected CCA cell lines with high expression of MetAP2
Study the effect of TNP - 470, MetAP2 inhibitor Growth Metastasis
Chemotherapeutic drug response - Proliferation - MTT assay - Cell
cycle and apoptosis - flow cytometry - Proliferation - MTT assay -
Cell cycle and apoptosis - flow cytometry - Invasion assay -
Migration assay - Adhesion assay - Invasion assay - Migration assay
- Adhesion assay Chemotherapeutic sensitizing (5-FU and Gemcitabine
) Chemotherapeutic sensitizing (5-FU and Gemcitabine ) Determine
the molecular mechanism Genes related to proliferation (ERK1/2,
cyclinE, p21, Rb) Genes related to proliferation (ERK1/2, cyclinE,
p21, Rb) Genes related to metastasis (ICAM1, ALCAM, MMP2, MMP9)
Genes related to metastasis (ICAM1, ALCAM, MMP2, MMP9) Genes
related to apoptosis (bcl-2, p53, ) Genes related to apoptosis
(bcl-2, p53, )
- Slide 87
- The sulphorhodamine (SRB) assay 87 Selected CCA cell lines with
high expression of MetAP2 Study the effect of TNP - 470, MetAP2
inhibitor Growth Metastasis Chemotherapeutic drug response -
Proliferation - MTT assay - Cell cycle and apoptosis - flow
cytometry - Proliferation - MTT assay - Cell cycle and apoptosis -
flow cytometry - Invasion assay - Migration assay - Adhesion assay
- Invasion assay - Migration assay - Adhesion assay
Chemotherapeutic sensitizing (5-FU and Gemcitabine )
Chemotherapeutic sensitizing (5-FU and Gemcitabine ) Determine the
molecular mechanism Genes related to proliferation (ERK1/2,
cyclinE, p21, Rb) Genes related to proliferation (ERK1/2, cyclinE,
p21, Rb) Genes related to metastasis (ICAM1, ALCAM, MMP2, MMP9)
Genes related to metastasis (ICAM1, ALCAM, MMP2, MMP9) Genes
related to apoptosis (bcl-2, p53, ) Genes related to apoptosis
(bcl-2, p53, )
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- Combination index 88 Selected CCA cell lines with high
expression of MetAP2 Study the effect of TNP - 470, MetAP2
inhibitor Growth Metastasis Chemotherapeutic drug response -
Proliferation - MTT assay - Cell cycle and apoptosis - flow
cytometry - Proliferation - MTT assay - Cell cycle and apoptosis -
flow cytometry - Invasion assay - Migration assay - Adhesion assay
- Invasion assay - Migration assay - Adhesion assay
Chemotherapeutic sensitizing (5-FU and Gemcitabine )
Chemotherapeutic sensitizing (5-FU and Gemcitabine ) Determine the
molecular mechanism Genes related to proliferation (ERK1/2,
cyclinE, p21, Rb) Genes related to proliferation (ERK1/2, cyclinE,
p21, Rb) Genes related to metastasis (ICAM1, ALCAM, MMP2, MMP9)
Genes related to metastasis (ICAM1, ALCAM, MMP2, MMP9) Genes
related to apoptosis (bcl-2, p53, ) Genes related to apoptosis
(bcl-2, p53, )
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- Effect of TNP-470 in the treatment on uterine carcinosarcoma in
vivo 89
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- Etiology 90 Yongvanit et al. 2011
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- Wound healing 91
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- Information of CCA cell lines 92 Cell linesGenderAge (years
old)Histological type KKU-M055Male56Poorly differentiated
KKU-M139Female53Squamous cell carcinoma KKU-M156Male68Moderately
differentiated KKU-M213Male58Adenosquamous carcinoma
KKU-M214Male52Moderately differentiated KKU-100 KKU-OCA17 Female
Male 65 38 Poorly differentiated Well differentiated
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- TNP-470 binding 93 Wang et al., 2003
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- 94 MetAP2 Metastasis Cell cycle G1 arrest ICAM1, ALCAM, MMP2,
MMP9 Apoptosis induction TNP-470 Casepase3 p21 Upregulation
Downregulation Bcl-2 CyclinD
- Slide 95
- 95 MetAP2 Metastasis Cell cycle G1 arrest MMP2/9 Apoptosis
TNP-470 Casepase3 p21 Upregulation Downregulation Bcl-2 c-Myc
ERK1/2 Hypothesis Tentative model for role of MetAP2 in CCA
development
- Slide 96
- 96 MetAP2 Metastasis Cell cycle MMP2/9 Apoptosis TNP-470
Casepase3 p21 Upregulation Downregulation Bcl-2 c-Myc ERK1/2
Hypothesis Tentative model for role of MetAP2 in CCA
development
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- CCA cell lines with high MetAP2 expression MetastasisGrowth
C-Myc Cell apoptosis TNP-470 Casepase3 p21 Bcl-2 ERK1/2 Cell cycle
Enhance chemotherapeutic drug Chemotherapeutic sensitizing (5-FU
and Gemcitabine) - Invasion assay - Migration assay - Adhesion
assay Cell cycle and apoptosis (flow cytometry) Proliferation (MTT
assay) MMP2MMP9VCAM1 G1 phase arrest Determine the molecular
mechanism (real time PCR, western blot) Genes related to metastasis
Proliferation (MTT assay) Cyclin D
- Slide 98
- 98 Cancers with high MetAP2 expression Metastasis Growth C-Myc
Cell apoptosis TNP-470 Casepase3 p21 Upregulation Downregulation
Bcl-2 ERK1/2 Hypothesis G1 phase arrest Cell cycle Enhance
chemotherapeutic drug ??? Chemotherapeutic sensitizing (5-FU and
Gemcitabine ) Chemotherapeutic sensitizing (5-FU and Gemcitabine )
- Invasion assay - Migration assay - Adhesion assay - Invasion
assay - Migration assay - Adhesion assay Cell cycle and apoptosis
(flow cytometry) Cell cycle and apoptosis (flow cytometry)
Proliferation (MTT assay) Proliferation (MTT assay) MMP2 MMP9
VCAM1
- Slide 99
- Experimental design 99 Selected CCA cell lines with high
expression of MetAP2 Study the effect of TNP - 470, MetAP2
inhibitor Growth Metastasis Chemotherapeutic drug response -
Proliferation - MTT assay - Cell cycle and apoptosis - flow
cytometry - Proliferation - MTT assay - Cell cycle and apoptosis -
flow cytometry - Invasion assay - Migration assay - Adhesion assay
- Invasion assay - Migration assay - Adhesion assay
Chemotherapeutic sensitizing (5-FU and Gemcitabine )
Chemotherapeutic sensitizing (5-FU and Gemcitabine ) Determine the
molecular mechanism Genes related to cell cycle (p21, cyclinD)
Genes related to cell cycle (p21, cyclinD) Genes related to
metastasis (ICAM1, c-Myc, MMP2, MMP9) Genes related to metastasis
(ICAM1, c-Myc, MMP2, MMP9) Genes related to apoptosis (bcl-2, p53,
) Genes related to apoptosis (bcl-2, p53, )
- Slide 100
- Conceptual framework (cont) 100 CCA cell lines high expression
of MetAP2 High proliferation High metastasis Enhance
chemotherapeutic drug MetAP2 inhibitor: TNP-470 ?? Hypothesis
?
- Slide 101
- MetastasisGrowth C-Myc Cell apoptosis Casepase3 p21 Bcl-2
ERK1/2 Cell cycle Enhance chemotherapeutic drug Chemotherapeutic
sensitizing (5-FU and Gemcitabine) - Invasion assay - Migration
assay - Adhesion assay Cell cycle and apoptosis (flow cytometry)
Proliferation (MTT assay) MMP2MMP9VCAM1 G1 phase arrest Determine
the molecular mechanism (real time PCR, western blot) Genes related
to metastasis Proliferation (MTT assay) Cyclin D Selected CCA cell
lines with high expression of MetAP2 Study the effect of TNP - 470,
MetAP2 inhibitor
- Slide 102
- High MetAP2 expression in cancer Metastasis Cell cycle G1
arrest Migration Apoptosis TNP-470 Invasion ERK1/2 Cell growth
Angiogenesis Adhesions VEGF
- Slide 103