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

1

Sonexai KidoikhammouanM.Sc. Student, Department of Biochemistry

Faculty of Medicine, KKU

17th 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 WongkhamAssis.Prof.Dr. Chariya Hahnvajanawong

Contents

Introduction

Hypothesis and Research questions

Objectives

Conceptual framework

Experimental design

Anticipated outcomes

Research Plan

2

Cholangiocarcinoma(CCA) epimeology and treatment Methionine aminopeptidase2(MetAP2) Expression in cancers

including in CCA Definition, function , and inhibitors of MetAP2

Introduction

Intrahepatic CCA (ICC) Extrahepatic CCA (ECC)

3

Cholangiocarcinoma (CCA)

Epidemiology of CCA

4Bragazzi et al., 2011

CCA alternative treatments

Surgery

Adjuvant therapy

Chemotherapy

Radiation therapy

5

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

Hilar 298 42 ND Igami et al., 2010

Liver resection ECC 34 20 ND Guglielmi et al.,2009

Liver resection ICC 45 35 30 Yedibela et al., 2009

Liver resection ICC 97 31.1 ND Palik et al. 2008

Liver resection ICC 44 63 ND DeOliveira et al. 2007

Liver resection with lymphadenectomy Hilar 26 21 27 Rea et al. 2005

Liver resection ICC 34 32 62 Casavilla et al. 1997

However, this regimen is still giving low survival, but high recurrence rate

Further more, some patients can not undergo such regimen

Association between response rate and median time survival using Chemotherapy

7

Drug n RR(%) MST (Months) Reference

Capecitabine 40 32.5 9.4 Furuse et al.,2008

Gemcitabine 40 17.5 7.6 Okusaka et al., 2006

Gemcitabine/capecitabine 45 32 14 Cho et al.,2005

Gem/cisplatin 40 28 9 Thongprasert et al., 2005

Gemcitabine/5-FU 27 33 5.3 Knox et al., 2004

5-FU/FA 30 7 14.8 Malik et al., 2003

5-FU/oxaliplatin 16 56 10 Nehls et al. 2002

Irinotecan 36 8 6.1 Sanz-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

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 Válek 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

Serial analysis of gene expression(SAGE)

9

K4 ; Low invasive cell sK3; High invasive cellsK2D; Poorly differentiated adenocarcinomaK1; Metastatic tumor (intrahepatic metastasis from cholangiocarcinoma primary tumor)

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

Comparison of MetAP2 expressions in bile duct epithelia

11

Sawanyawisuth et al., 2007

Expression and association of MetAP2 in cancers

12

Cancer Expression in cancer

Clinical finding Reference

Colon High Proliferation

Apoptosis

Selvakumar et al., 2009

Cholangiocarcinoma High Proliferation

Metastasis

Sawanyawisuth et al.,

2007

Neuroblastoma High Angiogenesis Morowitz et al., 2005

Hepatocellular carcinoma High Tumor growth,

Metastasis

Sheen et al., 2005

Mesothelioma High Proliferation Catalano et al. ,2001

MetAP2 structure

13

Addlagatta et al., 2005

How does MetAP2 work?

14

Met

MetAP2

STABILITY

TRANSLOCATION

TARGETING

MetAP2 inhibitors

15

Cell growth inhibition

Anti-angiogenesis

Datta et al.,2009

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)

Anti-tumor activity of TNP- 470; in vitro

17

Cell type Result Reference

FU-MMT-1 cells Anti angiogenesis Naganuma et al.,2011

Endothelial cell

B16F10 melanomaG1 arrest Hines et al., 2010

Wanget al.,2008

Fetal mouse bone cell Vasculature disruption

Anti angiogenesis

Wijngaarden et al., 2010

B16F10 (murine melanoma) Induction apoptosis Okrój et al.,2006

Human pancreatic Growth inhibition Hotz et al., 2001

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

18

Cell type Result Reference

Human uterine carcinosarcroma Tumor growth Naganuma et al.,2011

Human gioblastoma Tumor growth Yao et al., 2010

Murine neuroblastomaProliferation

ApoptosisChesler, et al.,2007

Sarcoma Tumor growth Kanamori et al.,2007

Human Wilms tumor cells Antiangiogenesis Huang et al.,2004

Effect of TNP- 470 in clinical trial

19

Cancer type Dose (mg/m2)

n RR(%) Reference

Solid tumor (lung, sarcoma, thymoma)

60 17 24 Tran et al., 2004

Lung cancer 60 32 33 Herbst et al., 2002

Kaposi’s sarcoma 10-70 38 ND Dezube et al.,1998

Cervical cancer 9.3 -71.2 18 ND Kudelka et al.,1998

Renal carcinoma 60 33 3 Stadler et al.,1999

Prostate 71 33 ND Stadler et al.,1994

RR; response rateND, no determine

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

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?

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.

Conceptual framework

23

Background

Cancers with high expression of MetAP2

High proliferation

High metastasis

Enhance Angiogenesis

MetAP2 inhibitors

Conceptual framework (cont)

24

CCA cell lines high expression of MetAP2

High proliferation

High metastasis

Enhance chemotherapeutic drug

MetAP2 inhibitor: TNP-470

? ?

Hypothesis

?

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 )

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

Research plansActivities 2011 2013

Apr - Jun Jul-Sep Oct-Dec Jan-Mar

1Literature 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 *

Pilot study

28

Expression of CCA cell line

29

M055 M139 M156 M213 M214 KKU100 MMNK10

0.5

1

1.5

2

2.5

3

3.5

4 3.55

1.0420.892000000000002

2.928

1.3281.205

1

Met

AP2

exp

ress

ion

leve

l (2-

ΔC

t)

Effect of TNP-470 on CCA cells proliferation

30

Effect of TNP-470 on CCA cell migration

31

Vehicle

TNP - 470 (1.25 µg/ml)

KKU - M213 KKU - M214

Effect of TNP-470 on CCA cell invasion

32

Vehicle

TNP - 470 (1.25 µg/ml)

KKU - M214KKU - M213

Determination molecular mechanism

33MMP2 MMP9 VCAM1 C-MYC

0

0.2

0.4

0.6

0.8

1

0.1780.044 0.00160000

000000001

1

0.00032000000000000

20.00780000000000002 0.0024

0.27

KKU-M214

VehicleTNP-470

Expr

essi

on le

vel (

2-ΔC

t)

MMP2 MMP9 VCAM1 C-MYC0

0.2

0.4

0.6

0.8

1

0.1

0.91

0.011

1

0.0540.19

0 0.054

KKU-M213

VehicleTNP-470

Expr

essi

on le

vel (

2-ΔC

t)

Acknowledgements

34

External examiners

35

Thank you very much

Structure of fumagillin and TNP-470

36

Ingber et al., 1990

SAGE process1. 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 chain4. Clone these chains into a vector which can be taken

up by bacteria.5. Sequence these chains using modern high-

throughput DNA sequencers6. Process this data with a computer to count the small

sequence tags

37

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

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

39

Effect of fumagillin in CCA cells

40Sawanyawisuth et al., 2007

Effect of fumagillin on cell growth

41

Hou et al. (2009).

Immune suppression in murine

42

Mass forming

43

Blechacz et al., 2011

periductal-infiltrating type

44


The intraductal-growth type

45


Criteria for diagnosis

1. Tumor stage, 2. Tumor location 3. Growth pattern

46

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


Risk factors for CCA

48Blechacz et al., 2011

CCA classification

49Blechacz et al., 2011

CCA subtype

50

Adjuvant chemoradiation therapy

51

Adjuvant chemoradiation therapy• 5-year survival; 5-FU plus radiotherapy 35% and

surgical resection alone 27%

Hughes et al. 2007

ERK

52

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 effectsVomitingPoor appetite sores in mouth, lips, or throat hair loss or thinning (may include face and body hair) diarrhea dry, flaky, cracking skin

GemcitabineGemcitabine 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

Irinotecan

prevents DNA from unwinding by inhibition of topoisomerase 1

55

Side-effectsThe 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.

ImmunosuppressionThe 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.

http://en.wikipedia.org/wiki/Loperamide

http://en.wikipedia.org/wiki/Lomotil

http://en.wikipedia.org/wiki/White_blood_cell

http://en.wikipedia.org/wiki/Neutrophil

http://en.wikipedia.org/wiki/Neutropenia

cisplatinthese 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

Folinic acidFolinic 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

Cell cycle control

58

Regulates cell growth and Protein synthesis

59

P67/MetAP2

αβ

ϒ

eIF2αSpecific kinase

α β

ϒ

αβ

ϒeIF2αSpecific kinase α β

ϒ

P

P67/MetAP2

ERK1/2

P67/MetAP2

ERK1/2

P

P

Inhibition of Protein synthesis

Inhibition of Cell Growth

Protein synthesis

Deglycocetylation ?

Effect of TNP-470 on angiogenesis

60Naganuma et al., 2011

Effect of TNP-470 on mouse xenograft

61Yao, Zhao et al. 2010

Effect ofTNP-470 on cell cycle

62

TNP-470 arrest cell cycle at G1 phase

Wang et al. 2008

63

Wang et al. 2008

Reversible inhibition of MetAP2 catalytic activity by A-800141

64Wang et al. 2008

Western blot analysis of cell cycle proteins in HUVEC treated with MetAP2 inhibitors.

6565Wang et al. 2008

MetAP2 inhibition results in formation of cellular GAPDH variants with an unprocessed N-terminal methionine.

66Wang et al. 2008

Effects on growth of the FU-MMT-1 xenografts

67

Emoto et al., 2007

Anti-tumor effect of radiation response by combined treatment with angiogenesis inhibitor, TNP-470, in oral squamous cell

carcinoma

68Shintani et al., 2006

TNP-470 promotes initial vascular sprouting in xenograft

Huang et al., 2004

Malignant Progression and Blockade of Angiogenesis

Hitting the mother lode of tumor angiogenesis

Post-translational myristoylation: Fat matters in cellular life and death

CCA

73Khan, S., et al., 2005

74

MetAP2 inhibitors Target gene in cell cycle arrest

Target gene in apoptosis

Cell type reference

fumagillin G1 arrestcyclinE2 Bcl-2

Bcl-2

Colorectal ,hepatocellular carcinoma

HumanMesothelioma

Hou.L.et.al(2009)Sheen,I.et.al(2005)Catalano,A.et.al(2001)

TNP-470 or AGM-1470

G1 arrest P53, p21, p27 p-RB cyclinE

p-RB CDK/cyclinG0 arrest

HUVEC

HUVEC

BAEC

Zhang,Y.et.al(2000)

Abe,J.et.al(1994)Antoine, N. et.al(1994)

A-800141 G1 arrest p53 and p21 p-RB

human neuroblastoma

Wang,J.et.al(2007)

75

MetAP2 inhibitors Target gene in cell cycle arrest

Target gene in apoptosis

Cell type reference

A-353700 G1 arrest p-RB cyclinA

Carcinoma,Sarcoma , neuroblastoma

Wang,J.et.al(2003)

hybrid of 1-deoxynojirimycin(DNJ) and an aryl-1,2,3-triazole

G1 arrest cyclin D1 ERK1/2

BAEC Zhao,Y. et.al.(2008)

IDR-803, IDR-804,CKD-732

G1 arrest p21

HUVEC Chun,E.et.al(2005)

PPI-2458 G1 arrest HUVEC Bainbridge, J.et.al.(2007)

bovine aortic endothelial cell (BAEC)

MetAP2

76Addlagatta et al. 2005

Enzymes similarity Dissimilarity

MetAP1MetAP1 Could compensate when MetAP2 is inactive (in yeast)

Mn2+ in active site

Play role in the G2/M phase of cell cycle

MetAP2

MetAP2 inhibition leads to G1 arrest

number of keyRespects ( 60-aa insert)

retinoblastoma protein

77

Cell cycle control

78

Criteria for choosing chemotherapeutic drugs

79

1. Different mechanism from cytotoxic agent

2. Side effect

3. Cost

Signal transduction

80

Expression of MMP-2 in bladder cancer

81Seiler et al., 2011

Expression of MMP-9 in bladder cancer

82Seiler et al., 2011

MetAP2 specific substrates

83

1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)2. cyclophilinA

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

PI staining

8585




- Proliferation- MTT assay- Cell cycle and apoptosis - flow cytometry


Chemotherapeutic sensitizing(5-FU and Gemcitabine )


Genes related to proliferation(ERK1/2, cyclinE, p21, Rb)

Genes related to metastasis(ICAM1, ALCAM, MMP2 , MMP9)

Genes related to apoptosis(bcl-2, p53, )

SYBR

868686











The sulphorhodamine (SRB) assay

87878787











Combination index

8888888888











Effect of TNP-470 in the treatment on uterine carcinosarcoma in vivo

89

Etiology

90Yongvanit et al. 2011

Wound healing

91

Information of CCA cell lines

92

Cell lines Gender Age (years old) Histological typeKKU-M055 Male 56 Poorly differentiated

KKU-M139 Female 53 Squamous cell carcinoma

KKU-M156 Male 68 Moderately differentiated

KKU-M213 Male 58 Adenosquamous carcinoma

KKU-M214 Male 52 Moderately differentiated

KKU-100

KKU-OCA17

Female

Male

65

38

Poorly differentiated

Well differentiated

TNP-470 binding

93Wang et al., 2003

9494949494

MetAP2

MetastasisCell cycle G1 arrest

ICAM1, ALCAM, MMP2 , MMP9

Apoptosis induction

TNP-470

Casepase3 p21

UpregulationDownregulatio

n

Bcl-2CyclinD

95

MetAP2

MetastasisCell cycle G1 arrest

MMP2/9

Apoptosis

TNP-470

Casepase3 p21


n

Bcl-2CyclinD c-Myc

ERK1/2

Hypothesis

Tentative model for role of MetAP2 in CCA development

96

MetAP2

MetastasisCell cycle

MMP2/9

Apoptosis

TNP-470

Casepase3 p21


n

Bcl-2CyclinD c-Myc

ERK1/2

Hypothesis

Tentative model for role of MetAP2 in CCA development

CCA cell lines with high MetAP2 expression

MetastasisGrowth

Cell apoptosis

TNP-470

Cell cycle


Chemotherapeutic sensitizing(5-FU and Gemcitabine)


Cell cycle and apoptosis(flow cytometry)

Proliferation(MTT assay)

G1 phase arrest

Determine the molecular mechanism(real time PCR, western blot)

Genes related to metastasis


98

Cancers with high MetAP2 expression

MetastasisGrowth

C-MycCell apoptosis

TNP-470

Casepase3 Upregulation

Downregulation

Bcl-2

CyclinD

HypothesisG1 phase arrest


? ??


- Invasion assay - Migration assay - Adhesion assayCell cycle and apoptosis

(flow cytometry)Proliferation(MTT assay)

MMP2 MMP9VCAM1

Experimental design

99








Genes related to cell cycle(p21, cyclinD)

Genes related to metastasis(ICAM1, c-Myc, MMP2 , MMP9)


Conceptual framework (cont)

100

CCA cell lines high expression of MetAP2

High proliferation

High metastasis


MetAP2 inhibitor: TNP-470

? ?

Hypothesis

?

MetastasisGrowth

Cell apoptosis

ERK1/2

Cell cycle


Chemotherapeutic sensitizing(5-FU and Gemcitabine)


Cell cycle and apoptosis(flow cytometry)


G1 phase arrest

Determine the molecular mechanism(real time PCR, western blot)

Genes related to metastasis




High MetAP2 expression in cancer

Metastasis

Cell cycle G1 arrest MigrationApoptosis

TNP-470

Invasion

ERK1/2

Cell growth Angiogenesis

Adhesions

VEGF

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

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