TNP- 470 as a potential adjuvant Therapy
for Cholangiocarcinoma
Sonexai Kidoikhammouan
M.Sc. Student, Department of Biochemistry
Faculty of Medicine, KKU
Advisory committees:
Assoc. Prof. Dr. Chaisiri Wongkham
Dr. Wunchana Seubwai
Dr. Atit Silsilivanit
External examiners:
Assoc.Prof.Dr. Sopit Wongkham
Assis.Prof.Dr. Chariya Hahnvajanawong
17th August, 2012
1
Contents
Introduction
Hypothesis and Research questions
Objectives
Conceptual framework
Experimental design
Anticipated outcomes
Research Plan
2
Introduction
Cholangiocarcinoma (CCA)
 Intrahepatic CCA (ICC)
 Extrahepatic CCA (ECC)
3
Epidemiology of CCA
Bragazzi et al., 2011
4
CCA alternative treatments
 Surgery
 Adjuvant therapy
 Chemotherapy
 Radiation therapy
5
Surgery correlated with survival and recurrence
rate in CCA patients
Methods
Portal vein and
hepatic artery
resection
CCA
subtype
Hilar
n
Five-year
survival
rate (%)
Recurrence
(%)
298
42
ND
Reference
Igami et al., 2010
et al.,
However, this regimen is still giving low survival, but Guglielmi
high recurrence
ECC
34
20
ND
2009
rate
Liver resection
Liver resection
ICC
45
35
30
Yedibela et al., 2009
Further more, some patients can not undergo such regimen
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
6
Association between response rate and median time
survival using Chemotherapy
n
RR(%)
MST
(Months)
Capecitabine
40
32.5
9.4
Furuse et al.,2008
Gemcitabine
40
17.5
7.6
Okusaka et al., 2006
Drug
Reference
Nevertheless, chemotherapy is still giving low
Gemcitabine/capecitabine
45
32
Cho et al.,2005
14
response rate and median survival time
Gem/cisplatin
40
28
9
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
Thongprasert et al., 2005
RR, response rate; MST, median survival time; 5-FU, 5-fl uorouracil; FA, folinic acid; 7
Radiotherapy
 2-year survival was 80% and 4-year survival 30%
Polistina et al. 2011
The need of targeted molecules with novel chemotherapy and
adjuvant
therapeutic
strategies
External
beam radiotherapy
19.1for
mo diagnosis
survival timeand treatment of
CCA patients are increasing nowadays
Jiang et al., 2010
 Survival time were 12.9 mo in patients who received EBRT
Válek et al., 2007
EBRT; external beam radiation therapy
8
Serial analysis of gene expression(SAGE)
K4 ;
K3;
K2D;
K1;
Low invasive cell s
High invasive cells
Poorly differentiated adenocarcinoma
Metastatic tumor (intrahepatic metastasis from cholangiocarcinoma primary tumor)
9
Comparison of MetAP2 expressions
Normal biliary cells
Hyperplastic and dysplastic bile duct epithelia
Well differentiated tubular CCA
Lymph node with metastatic CCA
Sawanyawisuth et al., 2007
10
Comparison of MetAP2 expressions in bile duct epithelia
Sawanyawisuth et al., 2007
11
Expression and association of MetAP2 in cancers
Cancer
Expression
Clinical finding
Reference
in cancer
Colon
High
Proliferation
Selvakumar et al., 2009
Apoptosis
Cholangiocarcinoma
High
Proliferation
Sawanyawisuth
Metastasis
2007
et
Neuroblastoma
High
Angiogenesis
Morowitz et al., 2005
Hepatocellular carcinoma
High
Tumor growth,
Sheen et al., 2005
Metastasis
Mesothelioma
High
Proliferation
Catalano et al. ,2001
12
al.,
MetAP2 structure
Addlagatta et al., 2005
13
How does MetAP2 work?
Met
14
MetAP2 inhibitors
Cell growth inhibition
Anti-angiogenesis
Datta et al.,2009
15
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
16
Anti-tumor activity of TNP- 470; in vitro
Cell type
Result
Reference
FU-MMT-1 cells
Anti angiogenesis
Naganuma et al.,2011
Endothelial cell
G1 arrest
Hines et al., 2010
B16F10 melanoma
Fetal mouse bone cell
Wanget al.,2008
Vasculature disruption
Wijngaarden et al., 2010
Anti angiogenesis
B16F10 (murine melanoma)
Induction apoptosis
Okrój et al.,2006
Human pancreatic
Growth inhibition
Hotz et al., 2001
17
Anti - tumor activity of TNP- 470; in vitro;
animal model
Cell type
Result
Reference
Human uterine carcinosarcroma
Tumor growth
Naganuma et al.,2011
Human gioblastoma
Tumor growth
Yao et al., 2010
Proliferation
Murine neuroblastoma
Chesler, et al.,2007
Apoptosis
Sarcoma
Human Wilms tumor cells
Tumor growth
Antiangiogenesis
Kanamori et al.,2007
Huang et al.,2004
18
Effect of TNP- 470 in clinical trial
Cancer type
Dose
(mg/m2)
n
RR(%)
Solid tumor (lung, sarcoma,
thymoma)
60
17
24
Tran et al., 2004
Lung cancer
60
32
33
Herbst et al., 2002
10-70
38
ND
Dezube et al.,1998
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
Kaposi’s sarcoma
Cervical cancer
Reference
RR; response rate
ND, no determine
19
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
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 antitumor activity of chemotherapeutic drugs in CCA cell lines?
21
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.
22
Conceptual framework
Background
Cancers with high expression of MetAP2
High
proliferation
High
metastasis
Enhance
Angiogenesis
MetAP2 inhibitors
23
Conceptual framework (cont)
Hypothesis
CCA cell lines high expression of MetAP2
MetAP2 inhibitor: TNP-470
?
High
proliferation
?
High
metastasis
?
Enhance
chemotherapeutic drug
24
Experimental design
Selected CCA cell lines with high expression of MetAP2
Study the effect of TNP - 470,
MetAP2 inhibitor
Growth
Metastasis
- Proliferation
- MTT assay
- Cell cycle and apoptosis
- Flow cytometry
- Invasion assay
- Migration assay
- Adhesion assay
Chemotherapeutic drug
response
Chemotherapeutic sensitizin
(5-FU, Cisplatin, Doxorubicin and Ge
Determine the molecular mechanism
Genes related to apoptosis
(Casepase3, Bax, Bcl-2, p38 )
Genes related to metastasis
(c-Myc, MMP2 , MMP9)
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 plans
2011
Activities
Apr - Jun
1
2
Jul-Sep
2013
Oct-Dec
Jan-Mar
Literature review
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
4.5
MetAP2 expression level (2-ΔCt)
4
3.55
3.5
2.928
3
2.5
2
1.328
1.042
1.5
1.205
1.00
0.892
1
0.5
0
M055
M139
M156
M213
M214
KKU100
MMNK1
29
Effect of TNP-470 on CCA cells proliferation
30
Effect of TNP-470 on CCA cell migration
KKU - M213
KKU - M214
Vehicle
TNP - 470
(1.25 µg/ml)
31
Effect of TNP-470 on CCA cell invasion
KKU - M213
KKU - M214
Vehicle
TNP - 470
(1.25 µg/ml)
32
Determination molecular mechanism
Expression level (2-ΔCt)
KKU-M213
1
1
0.91
0.8
0.6
Vehicle
0.4
0.2
0.1
TNP-470
0.19
0.054
0.011
0
MMP2
MMP9
VCAM1
KKU-M214
Expression level (2-ΔCt)
0.054
0
C-MYC
1
1
0.8
0.6
Vehicle
0.4
0.2
0
0.27
0.178
TNP-470
0.00032 0.044 0.0078 0.0016 0.0024
MMP2
MMP9
VCAM1
C-MYC
33
Acknowledgements
34
Thank you very much
35
Structure of fumagillin and TNP-470
Ingber et al., 1990
36
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 highthroughput DNA sequencers
6. 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
Sawanyawisuth et al., 2007
40
Effect of fumagillin on cell growth
Hou et al. (2009).
41
Immune suppression in murine
42
Mass forming
Blechacz et al., 2011
43
periductal-infiltrating type
Blechacz et al., 2011
44
The intraductal-growth type
Blechacz et al., 2011
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
Blechacz et al., 2011
47
Risk factors for CCA
Blechacz et al., 2011
48
CCA classification
Blechacz et al., 2011
49
CCA subtype
50
Adjuvant chemoradiation therapy
 Adjuvant chemoradiation therapy
• 5-year survival; 5-FU plus radiotherapy 35% and
surgical resection alone 27%
Hughes et al. 2007
51
ERK
52
Capecitabine convert to 5-FU
 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
53
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.
Side effects
weakness, loss of appetite, headache, cough, chills, and
muscle aches); hair loss; infection (fever, chills, sore throat);
54
Irinotecan
prevents DNA from unwinding by inhibition
of topoisomerase 1
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.
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.
55
cisplatin
these platinum complexes react in vivo, binding to
and causing crosslinking of DNA which ultimately
triggers apoptosis (programmed cell death)
Side effects
Nephrotoxicity (kidney damage)
Neurotoxicity (nerve damage)
Ototoxicity (hearing loss)
56
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
Cell cycle control
58
Regulates cell growth and Protein synthesis
ϒ
P67/MetAP2
α
Protein synthesis
β
Deglycocetylation ?
ϒ
P67/MetAP2
P
ERK1/2
β
α
P
eIF2α
Specific
kinase
ERK1/2
P67/MetAP2
eIF2α
Specific
kinase
Inhibition of Cell Growth
ϒ
α
ϒ
β
P
α
β
Inhibition of Protein
synthesis
59
Effect of TNP-470 on angiogenesis
Naganuma et al., 2011
60
Effect of TNP-470 on mouse xenograft
Yao, Zhao et al. 2010
61
Effect ofTNP-470 on cell cycle
TNP-470 arrest cell cycle at G1 phase
62
Wang et al. 2008
Wang et al. 2008
63
Reversible inhibition of MetAP2 catalytic
activity by A-800141
Wang et al. 2008
64
Western blot analysis of cell cycle proteins in HUVEC treated
with MetAP2 inhibitors.
Wang et al. 2008
65
MetAP2 inhibition results in formation of cellular GAPDH variants with an
unprocessed N-terminal methionine.
Wang et al. 2008
66
Effects on growth of the FU-MMT-1 xenografts
Emoto et al., 2007
67
Anti-tumor effect of radiation response by combined treatment
with angiogenesis inhibitor, TNP-470, in oral squamous cell
carcinoma
Shintani et al., 2006
68
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
Khan, S., et al., 2005
73
MetAP2 inhibitors
fumagillin
Target gene in
cell cycle arrest
Target gene in
apoptosis
G1 arrest
cyclinE2
Bcl-2
Bcl-2
TNP-470 or
AGM-1470
A-800141
Cell type
reference
Colorectal ,
Hou.L.et.al
hepatocellular (2009)
carcinoma
Sheen,I.et.al
(2005)
Human
Catalano,A.et.al
Mesothelioma (2001)
G1 arrest
P53, p21, p27
p-RB
cyclinE
HUVEC
Zhang,Y.et.al
(2000)
p-RB
CDK/cyclin
G0 arrest
HUVEC
Abe,J.et.al
(1994)
Antoine, N. et.al
(1994)
G1 arrest
p53 and p21
p-RB
human
neuroblastom
a
BAEC
Wang,J.et.al
(2007)
74
MetAP2 inhibitors
Target gene in
cell cycle arrest
Target gene in
apoptosis
Cell type
reference
A-353700
G1 arrest
p-RB
cyclinA
Carcinoma,
Wang,J.et.al
Sarcoma ,
(2003)
neuroblastoma
hybrid of
1-deoxynojirimycin
(DNJ) and an aryl1,2,3-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)
75
MetAP2
Enzymes
similarity
Dissimilarity
MetAP1
MetAP1 Could compensate when
MetAP2 is inactive (in yeast)
Play role in the G2/M phase of cell
cycle
Mn2+
 MetAP2 inhibition leads to G1
arrest
in active site
MetAP2
 number of key
Respects ( 60-aa insert)
Addlagatta et al. 2005
76
retinoblastoma protein
77
Cell cycle control
78
Criteria for choosing chemotherapeutic drugs
1. Different mechanism from cytotoxic agent
2. Side effect
3. Cost
79
Signal transduction
80
Expression of MMP-2 in bladder cancer
Seiler et al., 2011
81
Expression of MMP-9 in bladder cancer
Seiler et al., 2011
82
MetAP2 specific substrates
1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
2. cyclophilinA
83
BCL2 (B-cell leukemia/lymphoma 2)
antiapoptosis, through a possibly complex process;
dimerization, especially with BAX; role of the BCL2 antiapoptosis 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
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 and Gemcitabine )
Determine the molecular mechanism
Genes related to apoptosis
(bcl-2, p53, )
Genes related to proliferation
(ERK1/2, cyclinE, p21, Rb)
Genes related to metastasis
(ICAM1, ALCAM, MMP2 , MMP9)
85
SYBR
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 and Gemcitabine )
Determine the molecular mechanism
Genes related to apoptosis
(bcl-2, p53, )
Genes related to proliferation
(ERK1/2, cyclinE, p21, Rb)
Genes related to metastasis
(ICAM1, ALCAM, MMP2 , MMP9)
86
The sulphorhodamine (SRB) assay
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 and Gemcitabine )
Determine the molecular mechanism
Genes related to apoptosis
(bcl-2, p53, )
Genes related to proliferation
(ERK1/2, cyclinE, p21, Rb)
Genes related to metastasis
(ICAM1, ALCAM, MMP2 , MMP9)
87
Combination index
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 and Gemcitabine )
Determine the molecular mechanism
Genes related to apoptosis
(bcl-2, p53, )
Genes related to proliferation
(ERK1/2, cyclinE, p21, Rb)
Genes related to metastasis
(ICAM1, ALCAM, MMP2 , MMP9)
88
Effect of TNP-470 in the treatment on
uterine carcinosarcoma in vivo
89
Etiology
Yongvanit et al. 2011
90
Wound healing
91
Information of CCA cell lines
Cell lines
Gender Age (years old) Histological type
KKU-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
Female
65
Poorly differentiated
KKU-OCA17
Male
38
Well differentiated
92
TNP-470 binding
Wang et al., 2003
93
MetAP2
TNP-470
Cell cycle G1 arrest
p21
CyclinD
Apoptosis induction
Bcl-2
Casepase3
Metastasis
ICAM1, ALCAM, MMP2 , MMP9
Upregulation
Downregulation
94
Tentative model for role of MetAP2 in CCA development
MetAP2
ERK1/2
TNP-470
Cell cycle G1 arrest
p21
CyclinD
Apoptosis
Bcl-2
Metastasis
Casepase3
MMP2/9
c-Myc
Hypothesis
Upregulation
Downregulation
95
Tentative model for role of MetAP2 in CCA development
MetAP2
ERK1/2
TNP-470
Cell cycle
p21
Apoptosis
CyclinD
Bcl-2
Metastasis
Casepase3
MMP2/9
c-Myc
Hypothesis
Upregulation
Downregulation
96
CCA cell lines with high MetAP2
expression
TNP-470
Growth
n
)
cycle
Metastasis
Cell cycle and apoptosis
(flow cytometry)
- Invasion assay
- Migration assay
- Adhesion assay
Determine the molecular mechanism
(real time PCR, western blot)
Cell apoptosis
Enhan
chemotherape
Chemotherapeuti
(5-FU and Gem
Prolifera
(MTT as
Genes related to metastasis
Cancers with high MetAP2
expression
TNP-470
?
?
Growth
?
Enhance chemo
drug
Metastasis
- Invasion assay
- Migration assay
- Adhesion assay
Cell cycle and apoptosis
(flow cytometry)
C-Myc
VCAM1
MMP2
Chemotherapeuti
(5-FU and Gem
MMP9
Cell apoptosis
Hypothe
Bcl-2
Casepase3
Upregula
Downreg
98
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 and Gemcitabine )
Determine the molecular mechanism
Genes related to apoptosis
(bcl-2, p53, )
Genes related to cell cycle
(p21, cyclinD)
Genes related to metastasis
(ICAM1, c-Myc, MMP2 , MMP9)
99
Conceptual framework (cont)
Hypothesis
CCA cell lines high expression of MetAP2
MetAP2 inhibitor: TNP-470
?
High
proliferation
?
High
metastasis
?
Enhance
chemotherapeutic drug
100
Selected CCA cell lines with high expression of MetAP2
Study the effect of TNP - 470, MetAP2 inhibitor
Growth
n
)
cycle
Metastasis
Cell cycle and apoptosis
(flow cytometry)
- Invasion assay
- Migration assay
- Adhesion assay
Determine the molecular mechanism
(real time PCR, western blot)
Cell apoptosis
Enhan
chemotherape
Chemotherapeuti
(5-FU and Gem
Prolifera
(MTT as
Genes related to metastasis
High MetAP2 expression in cancer
ERK1/2
TNP-470
Cell growth
e G1 arrest
Angiogenesis
Metastasis
VEGF
Apoptosis
Migration
Invasion
Adhesions