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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

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Contents Introduction Hypothesis and Research questions Objectives Conceptual framework Experimental design Anticipated outcomes Research Plan 2

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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

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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

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Criteria for diagnosis 1. Tumor stage, 2.Tumor location 3.Growth pattern 46

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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

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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

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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

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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

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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

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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

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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, )

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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

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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

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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

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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

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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, )

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Conceptual framework (cont) 100 CCA cell lines high expression of MetAP2 High proliferation High metastasis Enhance chemotherapeutic drug MetAP2 inhibitor: TNP-470 ?? Hypothesis ?

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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

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High MetAP2 expression in cancer Metastasis Cell cycle G1 arrest Migration Apoptosis TNP-470 Invasion ERK1/2 Cell growth Angiogenesis Adhesions VEGF

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