Sonexai Kidoikhammouan M.Sc. Student, Department of Biochemistry Faculty of Medicine, KKU

1. TNP- 470 as a potential adjuvant Therapy for Cholangiocarcinoma SonexaiKidoikhammouan M.Sc. Student, Department of BiochemistryFaculty of Medicine, KKU Advisory committees: Assoc. Prof. Dr. Chaisiri Wongkham Dr. WunchanaSeubwai Dr. AtitSilsilivanit External examiners: Assoc.Prof.Dr. SopitWongkham Assis.Prof.Dr. ChariyaHahnvajanawong 17th August, 2012

2. Contents Introduction Hypothesis and Research questions Objectives Conceptual framework Experimental design Anticipated outcomes Research Plan • Cholangiocarcinoma(CCA) epimeology and treatment • Methionine aminopeptidase2(MetAP2) Expression in cancers including in CCA • Definition, function , and inhibitors of MetAP2

3. Introduction Cholangiocarcinoma (CCA) • Intrahepatic CCA (ICC) • Extrahepatic CCA (ECC)

4. Epidemiology of CCA Bragazzi et al., 2011

5. CCA alternative treatments • Surgery • Adjuvant therapy • Chemotherapy • Radiation therapy

6. Surgery correlated with survival and recurrence rate in CCA patients However, this regimen is still giving low survival, but high recurrence rate Further more, some patients can not undergo such regimen

7. Association between response rate and median time survival using Chemotherapy Nevertheless, chemotherapy is still giving low response rate and median survival time RR, response rate; MST, median survival time; 5-FU, 5-fl uorouracil; FA, folinic acid;

8. Radiotherapy • 2-year survival was 80% and 4-year survival 30% • Polistinaet al. 2011 The need of targeted molecules with novel chemotherapy and adjuvant therapeutic strategies for diagnosis and treatment of CCA patients are increasing nowadays • External beam radiotherapy 19.1 mo survival time • Jiang et al., 2010 • Survival time were 12.9 mo in patients who received EBRT • Váleket al., 2007 EBRT; external beam radiation therapy

9. Serial analysis of gene expression(SAGE) K4 ; Low invasive cell s K3; High invasive cells K2D; Poorly differentiated adenocarcinoma K1; Metastatic tumor (intrahepatic metastasis from cholangiocarcinoma primary tumor)

10. Comparison of MetAP2 expressions Normal biliary cells Hyperplastic and dysplastic bile duct epithelia Lymph node with metastatic CCA Well differentiated tubular CCA Sawanyawisuth et al., 2007

11. Comparison of MetAP2 expressions in bile duct epithelia Sawanyawisuth et al., 2007

12. Expression and association of MetAP2 in cancers

13. MetAP2 structure Addlagattaet al., 2005

14. How does MetAP2 work? MetAP2 Met

15. MetAP2 inhibitors Cell growth inhibition Anti-angiogenesis Dattaet al.,2009

16. Why we choose TNP-470? 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) Wang et al. 2008

17. Anti-tumor activity of TNP- 470; in vitro

18. Anti - tumor activity of TNP- 470; in vitro; animal model

19. Effect of TNP- 470 in clinical trial RR; response rate ND, no determine

20. 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.

21. Research questions 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?

22. Objectives • 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.

23. Conceptual framework Background Cancers with high expression of MetAP2 Enhance Angiogenesis High metastasis High proliferation MetAP2 inhibitors

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

25. 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, Doxorubicinand Gemcitabine) Determine the molecular mechanism Genes related to apoptosis (Casepase3, Bax, Bcl-2, p38 ) Genes related to metastasis (c-Myc, MMP2 , MMP9)

26. 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

27. Research plans

28. Pilot study

29. Expression of CCA cell line

30. Effect of TNP-470 on CCA cells proliferation

31. Effect of TNP-470 on CCA cell migration KKU - M213 KKU - M214 Vehicle TNP - 470 (1.25 µg/ml)

32. Effect of TNP-470 on CCA cell invasion KKU - M213 KKU - M214 Vehicle TNP - 470 (1.25 µg/ml)

33. Determination molecular mechanism

34. Acknowledgements External examiners

35. Thank you very much

36. Structure of fumagillin and TNP-470 Ingber et al., 1990

37. SAGE process • Isolate the mRNAof an input sample. • Extract a small chunk of sequence from a defined position of each mRNAmolecule. • Link these small pieces of sequence together to form a long chain • Clone these chains into a vector which can be taken up by bacteria. • Sequence these chains using modern high-throughput DNA sequencers • Process this data with a computer to count the small sequence tags

38. 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.

39. Pathological feature of CCA patients and expression of MetAP2 in primary tissue

40. Effect of fumagillin in CCA cells Sawanyawisuth et al., 2007

41. Effect of fumagillin on cell growth Hou et al. (2009).

42. Immune suppression in murine

43. Mass forming Blechacz et al., 2011

44. periductal-infiltrating type Blechacz et al., 2011

45. The intraductal-growth type Blechacz et al., 2011

46. Criteria for diagnosis • Tumor stage, • Tumor location • Growth pattern

47. Diagnosis of cholangiocarcinoma • Diagnosis of intrahepaticcholangiocarcinoma Require histopathology and is a diagnosis of exclusion; a pathologic staging system • The diagnosis of perihilarcholangiocarcinoma 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 extrahepaticcholangiocarcinoma can usually be confirmed by cytology; stage is highly dependent upon depth of invasion of surrounding structures Blechacz et al., 2011

48. Risk factors for CCA Blechacz et al., 2011

49. CCA classification Blechacz et al., 2011

50. CCA subtype