1 Bal MM, Ramadwar M, Deodhar K, Shrikhande S. Pathology of Gallbladder
Carcinoma: Current Understanding and New Perspectives. Pathol Oncol Res.
2015;21:509-525.
2 Ha TY, Yoon YI, Hwang S, Park YJ, Kang SH, Jung BH, et al. Effect of Reoperation
on Long-Term Outcome of pT1b/T2 Gallbladder Carcinoma After Initial Laparoscopic
Cholecystectomy. J Gastrointest Surg. 2015;19:298–305.
3 Doval DC, Azam S, Sinha R, Batra U, Mehta A. Expression of epidermal growth
factor receptor, p53, Bcl2, vascular endothelial growth factor, cyclooxygenase-2, cyclin
D1, human epidermal receptor-2 and Ki-67: Association with clinicopathological
profiles and outcomes in gallbladder carcinoma. J Carcinog. 2014;13:10:3903-9.
Levy
AD,
Murakata
LA,
Rohrmann
CA
Jr.
Gallbladder
carcinoma:
radiologic-pathologic correlation. Radiographics. 2001;21(2):295–314. questionnaire,
549–555.
5 Goetze TO. Gallbladder carcinoma: Prognostic factors and therapeutic options. World
J Gastroenterol. 2015;21(43):12211–7.
26
6 Soraya J Kaewpitoon, Ryan A Loyd, Ratana Rujirakul, Sukij Panpimanmas, Likit
Matrakool,
Taweesak
Tongtawee,
et
al.
Benefits
of
Metformin
Use
for
Cholangiocarcinoma. Asian Pac J Cancer Prev. 2015;16(18):8079–83.
7 Ren HB, Yu T, Liu C, Li YQ. Diabetes mellitus and increased risk of biliary tract
cancer: systematic review and meta-analysis. Cancer Causes Control. 2011;22:837–47.
8 Palmer WC, Patel T. Are common factors involved in the pathogenesis of primary
liver cancers? A meta-analysis of risk factors for intrahepatic cholangiocarcinoma. J
Hepatol. 2012;57:69–76.
9 Ben Sahra I, Laurent K, Loubat A, Giorgetti-Peraldi S, Colosetti P, Auberger P, et al.
The antidiabetic drug metformin exerts an antitumoral effect in vitro and in vivo
through a decrease of cyclin D1 level. Oncogene. 2008;27:3576–86.
10 Anisimov VN, Egormin PA, Piskunova TS, Popovich IG, Tyndyk ML, Yurova MN,
et al. Metformin extends life span of HER-2/neu transgenic mice and in combination
with melatonin inhibits growth of transplantable tumors in vivo. Cell Cycle.
2010;9:188–97.
11 Z Bao B, Wang Z, Ali S, Ahmad A, Azmi AS, Sarkar SH, et al. Metformin Inhibits
27
Cell Proliferation, Migration and Invasion by Attenuating CSC Function Mediated by
Deregulating miRNAs in Pancreatic Cancer Cells. Cancer Prev Res (Phila).
2012;5(3):355–64.
12 Kato K, Gong J, Iwama H, Kitanaka A, Tani J, Miyoshi H, et al. The antidiabetic
drug metformin inhibits gastric cancer cell proliferation in vitro and in vivo. Mol
Cancer Ther. 2012;11:549–60.
13 Fujihara S, Kato K, Morishita A, Iwama H, Nishioka T, Chiyo T, et al. Antidiabetic
drug metformin inhibits esophageal adenocarcinoma cell proliferation in vitro and in
vivo. Int J Oncol. 2015;46(5):2172–80.
14 Miyoshi H, Kato K, Iwama H, Maeda E, Sakamoto T, Fujita K, et al. Effect of the
anti-diabetic drug metformin in hepatocellular carcinoma in vitro and in vivo. Int J
Oncol. 2014;45:322–32.
15 Morishita A, Masaki T. miRNA in hepatocellular carcinoma. Hepatol Res.
2015;45:128–41.
16 Fujimori T, Kato K, Fujihara S, Iwama H, Yamashita T, Kobayashi K, et al.
Antitumor effect of metformin on cholangiocarcinoma: In vitro and in vivo studies.
28
Oncol Rep. 2015;34(6):2987–96.
17 Pernicova I, Korbonits M. Metformin-mode of action and clinical implications for
diabetes and cancer. Nat Rev Endocrinol. 2014;10:143–56.
18 Kourelis TV, Siegel RD. Metformin and cancer: new applications for an old drug.
Med Oncol. 2012;29:1314–27.
19 Hui AM, Cui X, Makuuchi M, Li X, Shi YZ, Takayama T. Decreased p27(Kip1)
expression and cyclin D1 overexpression, alone and in combination, influence
recurrence and survival of patients with resectable extrahepatic bile duct carcinoma.
Hepatology. 1999;30(5):1167–73.
20 Sugimachi K, Aishima S, Taguchi K, Tanaka S, Shimada M, Kajiyama K, et al. The
role of overexpression and gene amplification of cyclin D1 in intrahepatic
cholangiocarcinoma. J Hepatol. 2001;35(1):74–9.
21 Ling S, Feng T, Ke Q, Fan N, Li L, Li Z, et al Metformin inhibits proliferation and
enhances chemosensitivity of intrahepatic cholangiocarcinoma cell lines. Oncol Rep.
2014;31(6):2611–8.
22 Laemmli UK. Cleavage of structural proteins during the assembly of the head of
29
bacteriophage T4. Nature. 1970;227:680–5.
23 Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from
polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc
Natl Acad Sci USA. 1979;76:4350–4.
24 D'Incalci M, Colombo T, Ubezio P, Nicoletti I, Giavazzi R, Erba E, et al. The
combination of yondelis and cisplatin is synergistic against human tumor xenografts.
Eur J Cancer. 2003;39:1920–6.
25 Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J, et al. Role of
AMP-activated protein kinase in mechanism of metformin action. J Clin Invest.
2001;108(8):1167–74.
26 Chaiteerakij R, Yang JD, Harmsen WS, Slettedahl SW, Mettler TA, Fredericksen ZS,
et al. Risk factors for intrahepatic cholangiocarcinoma: association between metformin
use and reduced cancer risk. Hepatology. 2013;57(2):648–55.
27 Han S, Kim HY, Park K, Lee MS, Kim HJ, Kim YD. Expression of p27Kip1 and
cyclin D1 proteins is inversely correlated and is associated with poor clinical outcome
in human gastric cancer. J Surg Oncol. 1999;71:147–54.
30
28 Aoyagi K, Koufuji K, Yano S, Murakami N, Terasaki Y, Yamasaki Y, et al.
Immunohisto- chemical study on the expression of cyclin D1 and E in gastric cancer.
Kurume Med J. 2000;47:199–203.
29 Masaki T, Shiratori Y, Rengifo W, Igarashi K, Yamagata M, Kurokohchi K, et al.
Cyclins and cyclin- dependent kinases: Comparative study of hepatocellular carcinoma
versus cirrhosis. Hepatology. 2003;37:534–43.
30 Masaki T, Shiratori Y, Rengifo W, Igarashi K, Matsumoto K, Nishioka M, et al.
Hepatocellular carcinoma cell cycle: study of Long-Evans cinnamon rats. Hepatology.
2000;32:711–20.
31 Sherr CJ. Mammalian G1 cyclins. Cell. 1993;73:1059–65.
32 Sherr CJ. G1 phase progression: cyclin g on cue. Cell. 1994;79:551–5.
33 Pollak M. Insulin and insulin-like growth factor signaling in neoplasia. Nat Rev
Cancer. 2008;8:915–28.
34 Narayanan R, Kim HN, Narayanan NK, Nargi D, Narayanan B. Epidermal growth
factor-stimulated human cervical cancer cell growth is associated with EGFR and cyclin
D1 activation, independent of COX-2 expression levels. Int J Oncol. 2011;40:13–20.
31
35 Oki M, Yamamoto H, Taniguchi H, Adachi Y, Imai K, Shinomura Y. Overexpression
of the receptor tyrosine kinase EphA4 in human gastric cancers. World J Gastroenterol.
2008;14:5650–6.
36 Li Y, Jin L, Ye F, Ma Q, Yang Z, Liu D, et al. Isoform expression of EPHA 10 protein
mediate breast cancer progression by regulating the E-cadherin and
β-catenin complex.
Oncotarget. 2017;8:30344–56.
37 Zhang JH, Wang LH, Li XJ, Wang AP, Reng LQ, Xia FG, et al. Expression of
Ang-2/Tie-2 and PI3K/AKT in Colorectal Cancer. ASIAN PACIFIC JOURNAL OF
CANCER PREVENTION. 2014:15:8651-6.
38 Lipimski CA, Tran NL, Menashi E, Rohl C, Kloss J, Bay RC, et al. The tyrosine
kinase pyk2 promotes migration and invasion of grioma cells. Neoplasia.
2005;7:435–45.
39 Yoshida R, Sasaki T, Minami Y, Hibino Y, Okumura S, Sado M, et al. Activation of
Src signaling mediates acquired to ALK inhibition in lung cancer. Int J Oncol.
2017;51:1533–40.
40 Simone V, Brunetti O, Lupo L, Testini M, Maiorano E, Simone M, et al. Targeting
32
Angiogenesis in Biliary Tract Cancers: An Open Option. Int J Mol Sci. 2017;18(2):418.
41 Lv D, Zhang Y, Kim HJ, Zhang L, Ma X. CCL5 as a potential immunotherapeutic
target in triple-negative breast cancer. Cell Mol Immunol. 2013;10:30310.
42 Principe DR, Doll JA, Bauer J, Jung B, Munshi HG, Bartholin L, et al. TGF-β:
duality of function between tumor prevention and carcinogenesis. J Natl Cancer Inst.
2014;106, doi:10.1093/jnci/djt369.
43 Gong Y, Scott E, Lu R, Xu Y, Oh WK, Yu Q. TIMP-1 promote accumulation of
cancer associated fibroblast and cancer progression. PLoS One. 2013;8: e77366. 2013.
44 He D, Wang J, Zhang C, Shan B, Deng X, Li B, et al. Down-regulation of
miR-675-5p contributes to tumor progression and development by targeting
pro-tumorigenic GPR55 in non-small cell lung cancer. Mol Cancer. 2015;14:73.
45 Zhu M, Chen Q, Liu X, Sun Q, Zhao X, Deng R, et al. lncRNA H19/miR-675 axis
represses prostate cancer metastasis by targeting TGFBI. FEBS J. 2014;281:3766–75.
46 Schmitz KJ, Helwig J, Bertram S, Sheu SY, Suttorp AC, Seggewiss J, et al.
Differential expression of microRNA-675, microRNA-139-3p and microRNA-335 in
benign and malignant adrenocortical tumours. J Clin Pathol. 2011;64:529–35.
33
Figure legends
Figure 1. Metformin inhibits the proliferation of cultured GBC cells. NOZ,
TGBC14TKB, and TGBC24TKB cells were seeded in 96-well plates. Metformin (1, 5,
and 10 mmol/L) or vehicle was added to the culture medium; cells were subjected to the
CCK assay at 24–72 h after metformin treatment. Cell viability was assayed daily from
0 to 72 h; viability of metformin-treated cells at each time point differed significantly
from the control cells (P* < 0.05).
34
Figure 2. (A) Flow cytometric analysis of proliferating NOZ cells at 24 h after addition
of 10 mmol/L metformin (Met). Metformin blocked the cell cycle at G0/G1. Results are
representative of three independent experiments. (B) Expressions of cyclin D1, Cdk4,
Cdk6, cyclin E, phosphorylated Rb (pRb), and Rb in NOZ cells at 24, 48, and 72 h after
the addition of 10 mmol/L metformin. Protein expression was assayed by western
blotting.
Figure 3. (A) Template showing the location of tyrosine kinase antibodies spotted onto a
human phospho-RTK array. (B) Representative expression of various phosphorylated
tyrosine kinase receptors in NOZ cells treated with or without metformin. (C) The
densitometric of ratios p-Tie, p-PYK, p-ALK, p-EGFR, p-EphA4, and p-EphA10 spots
of metformin-treated to untreated cells were 31.8 %, 32.1 %, 8.4 %, 72.1 %,1.3 %, and
14.6%, respectively.
Figure 4. (A) Template showing the location of angiogenesis related proteins spotted
onto a human angiogenesis array. (B) Representative expression of various
35
angiogenesis-related proteins in NOZ cells treated with or without metformin. (C) The
densitometric ratios of RANTES, TGF-β, and TIMP spots of the metformin-treated cells
to untreated cells were 0.21 times,, 20 times and 0.37 times, respectively (Fig. 4C).
, respectively.
Figure 5. Hierarchical clustering of NOZ cells with and without metformin treatment.
NOZ cells were clustered according to the expression profiles of 35 miRNAs
differentially expressed in NOZ cells with and without metformin treatment. The
analyzed samples are shown in columns and the miRNAs are presented in rows. The
miRNA clustering color scale shown at the top depicts relative expression levels of the
miRNAs, with red and blue representing high and low expression levels, respectively.
Figure 6. In vivo anti-tumor effects of metformin on established gall bladder cancer
xenografts in nude mice. NOZ cells were implanted subcutaneously into the flanks of
nude mice. When tumors became palpable, 0 or 2 mg metformin was injected
intraperitoneally for 23 days, 5 times per week. (A) Representative images of gross
36
NOZ tumors from nude mice treated with vehicle (i) or 2 mg metformin (ii). (B) Tumor
growth curves in control and metformin groups. Tumor volume (mm3) was calculated as
(tumor length (mm) × tumor width (mm) 2)/2. Tumors were significantly smaller in
metformin-treated than in vehicle-treated mice. Each point represents the mean ±
standard deviation of six animals. *P = 0.01 by two-way ANOVA
Table 1. Statistical results and chromosomal locations of miRNAs in NOZ cells treated
with or without metformin.
Figure 1
Met (-)
G0/G1：36.3％
S：47.4％
G2/M：16.3％
72h
Met (+)
Figure 2
G0/G1：50.6％
S：35.2％
G2/M：14.1％
Met
(10mM)
(-)
24h
(+)
(-)
48 h
(+)
(-)
72h
(+)
β-actin
Rb
pRb
Cdk6
Cdk4
CyclinE
CyclinD1
Tie-2
Tie-2
Met (-)
ALK
150
Axl
PYK
PYK
Reference
Spots
Met (+)
ALK
EphB3
EphB1
TrkB
MSP R
PYK
EphB2
TrkC
PDGF Rα
EphA10
EphA10
EphB4
VEGF R1
PDGF Rβ
Flt-3
met+
50
100
met-
EphA10
met+
150
50
50
met+
100
100
met-
150
150
ALK
met-
50
EphA5
EphA2
ROR1
50
Tie-2
DDR2
EphA1
C-Ret
100
DDR1
MuSK
M-CSF R
100
150
ALK
VEGF R3
Figure 3
EphA4
EphA4
EphB6
VEGF R2
SCF R
EphA10
EphA3
ROR2
met-
EphA4
met-
PYK
PBS
EphA4
Tie-1
EphA7
IGF-1 R
EphA6
Insulin R
TrkA
FGF R4
Tie-2
FGF R3
HGF R
FGF R2α
Mer
FGF R1
Dtk
ErbB4
ErbB2
EGF R
ErbB3
Reference
Spots
Reference
Spots
met+
met+
RANTES
IFN gamma
RANTES
RANTES
BLANK
BLANK
GRO
PLGF
PLGF
BLANK
BLANK
Met (-)
IFN gamma
GRO
TIMP-1
POS
POS
TGF beta1
POS
POS
RANTES
BLANK
BLANK
Figure 4
TIMP-1
BLANK
BLANK
TIMP-1
TIMP-1
IL-6
IL-6
NEG
NEG
Met (+)
TGF beta1
TGF beta 1
TGF beta 1
IGF-1
IGF-1
NEG
NEG
BLANK
BLANK
TIMP-2
TIMP-2
IL-8
IL-8
ANG
ANG
BLANK
BLANK
THPO
THPO
Leptin
Leptin
EGF
EGF
50
100
150
1000
2000
3000
50
100
150
NEG
NEG
VEGF
VEGF
POS
POS
VEGF-D
VEGF-D
met-
met-
met+
met+
TIMP-1
TGF beta1
met+
PDGF-BB
PDGF-BB
b FGF
b FGF
RANTES
met-
MCP-1
MCP-1
ENA-78
ENA-78
Metformin
Figure 5
Control
Figure 6
i) Control
ii) Met 2 mg/d
...
論文の公開元へ
