Abstract
Metformin (Met) is a widely used antidiabetic drug and has demonstrated interesting anticancer effects in various cancer models, alone or in combination with chemotherapeutic drugs. The aim of the present study is to investigate the synergistic effect of Met with cisplatin (Cis) on the tumor growth inhibition of gallbladder cancer cells (GBC-SD and SGC-996) and explore the underlying mechanism. Cells were treated with Met and/or Cis and subjected to cell viability, colony formation, apoptosis, cell cycle, western blotting, xenograft tumorigenicity assay and immunohistochemistry. The results demonstrated that Met and Cis inhibited the proliferation of gallbladder cancer cells, and combination treatment with Met and Cis resulted in a combination index < 1, indicating a synergistic effect. Co-treatment with Met and Cis caused G0/G1 phase arrest by upregulating P21, P27 and downregulating CyclinD1, and induced apoptosis through decreasing the expression of p-PI3K, p-AKT, and p-ERK. In addition, pretreatment with a specific AKT activator (IGF-1) significantly neutralized the pro-apoptotic activity of Met + Cis, suggesting the key role of AKT in this process. More importantly, in nude mice model, Met and Cis in combination displayed more efficient inhibition of tumor weight and volume in the SGC-996 xenograft mouse model than Met or Cis alone. Immunohistochemistry analysis suggests the combinations greatly suppressed tumor proliferation, which is consistent with our in vitro results. In conclusion, our findings indicate that the combination therapy with Met and Cis exerted synergistic antitumor effects in gallbladder cancer cells through PI3K/AKT/ERK pathway, and combination treatment with Met and Cis would be a promising therapeutic strategy for gallbladder cancer patients.
Similar content being viewed by others
References
Alao JP (2007) The regulation of cyclin D1 degradation: roles in cancer development and the potential for therapeutic invention. Mol Cancer 6:24
Cai X, Hu X, Cai B, Wang Q, Li Y, Tan X et al (2013) Metformin suppresses hepatocellular carcinoma cell growth through induction of cell cycle G1/G0 phase arrest and p21CIP and p27KIP expression and downregulation of cyclin D1 in vitro and in vivo. Oncol Rep 30:2449–2457
Chou TC (2010) Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res 70:440–446
Citro S, Miccolo C, Meloni L, Chiocca S (2015) PI3K/mTor mediate mitogen-dependent HDAC1 phosphorylation in breast cancer: a novel regulation of estrogen receptor expression. J Mol Cell Biol 7:132–142
Goetze TO, Paolucci V (2010) Adequate extent in radical re-resection of incidental gallbladder carcinoma: analysis of the German registry. Surg Endosc 24:2156–2164
Jurado R, López-Flores A, Álvarez-Figueroa A, García-López P (2007) Synergistic effects of mifepristone on the cytotoxicity of cisplatin in cervical carcinoma cell lines and tumors grown in athymic mice. BMC Cancer 7:A18
Kelly PN, Strasser A (2011) The role of Bcl-2 and its pro-survival relatives in tumourigenesis and cancer therapy. Cell Death Differ 18:1414–1424
Kim KM, Heo DR, Lee J, Park JS, Baer MG, Yi JM et al (2015) 5,3′-dihydroxy-6,7,4′-trimethoxyflavanone exerts its anticancer and antiangiogenesis effects through regulation of the Akt/mTor signaling pathway in human lung cancer cells. Chem Biol Interact 225:32–39
Li L, Li X, Wang L, Du W, Guo R, Liang H et al (2012) Matrine inhibits breast cancer growth via miR-21/PTEN/Akt pathway in MCF-7 cells. Cell Physiol Biochem 30:631–641
Li L, Han R, Xiao H, Lin C, Wang Y, Liu H et al (2014a) Metformin sensitizes EGFR-TKI-resistant human lung cancer cells in vitro and in vivo through inhibition of IL-6 signaling and EMT reversal. Clin Cancer Res 20:2714–2726
Li T, Song T, Ni L, Yang G, Song X, Wu L et al (2014b) The p-ERK–p-c-Jun–cyclinD1 pathway is involved in proliferation of smooth muscle cells after exposure to cigarette smoke extract. Biochem Biophys Res Commun 453:316–320
Liang CZ, Zhang JK, Shi Z, Liu B, Shen CQ, Tao HM (2012) Matrine induces caspase-dependent apoptosis in human osteosarcoma cells in vitro and in vivo through the upregulation of Bax and Fas/FasL and downregulation of Bcl-2. Cancer Chemoth Pharm 69:317–331
Liu J, Li M, Song B, Jia C, Zhang L, Bai X et al (2013a) Metformin inhibits renal cell carcinoma in vitro and in vivo xenograft. Urol Oncol 31:264–270
Liu TY, Tan ZJ, Jiang L, Gu JF, Wu XS, Cao Y et al (2013b) Curcumin induces apoptosis in gallbladder carcinoma cell line GBC-SD cells. Cancer Cell Int 13:64
Liu J, Yao Y, Ding H, Chen R (2014) Oxymatrine triggers apoptosis by regulating Bcl-2 family proteins and activating caspase-3/caspase-9 pathway in human leukemia HL-60 cells. Tumor Biol 35:5409–5415
Liu Y, Bi T, Wang G, Dai W, Wu G, Qian L et al (2015) Lupeol inhibits proliferation and induces apoptosis of human pancreatic cancer PCNA-1 cells through AKT/ERK pathways. Naunyn Schmiedeberg’s Arch Pharmacol 388:295–304
Liu Y, Bi T, Dai W, Wang G, Qian L, Shen G et al (2016a) Lupeol enhances inhibitory effect of 5-fluorouracil on human gastric carcinoma cells. Naunyn Schmiedeberg’s Arch Pharmacol 389:477–484
Liu Y, Bi T, Wang Z, Wu G, Qian L, Gao Q et al (2016b) Oxymatrine synergistically enhances antitumor activity of oxaliplatin in colon carcinoma through PI3K/AKT/mTOR pathway. Apoptosis 21:1398–2407
Liu Y, Bi T, Shen G, Li Z, Wu G, Wang Z et al (2016c) Lupeol induces apoptosis and inhibits invasion in gallbladder carcinoma GBC-SD cells by suppression of EGFR/MMP-9 signaling pathway. Cytotechnology 68:123–133
Lu Z, Hunter T (2010) Ubiquitylation and proteasomal degradation of the p21 (Cip1), p27 (Kip1) and p57 (Kip2) CDK inhibitors. Cell Cycle 9:2342–2352
Miyoshi H, Kato K, Iwama H, Maeda E, Sakamoto T, Fujita K et al (2014) Effect of the anti-diabetic drug metformin in hepatocellular carcinoma in vitro and in vivo. Int J Oncol 2419:322–332
Nair V, Pathi S, Jutooru I, Sreevalsan S, Basha R, Abdelrahim M et al (2013) Metformin inhibits pancreatic cancer cell and tumor growth and downregulates Sp transcription factors. Carcinogenesis 34:2870–2879
Peng M, Huang Y, Tao T, Peng C, Su Q, Xu W et al (2016) Metformin and gefitinib cooperate to inhibit bladder cancer growth via both AMPK and EGFR pathways joining at Akt and Erk. Sci Rep 6:28166
Piulats JM, Jiménez L, García del Muro X, Villanueva A, Viñals F, Germà- Lluch JR (2009) Molecular mechanisms behind the resistance of cisplatin in germ cell tumours. Clin Transl Oncol 11:780–786
Qu Z, Zhang Y, Liao M, Chen Y, Zhao J, Pan Y (2012) In vitro and in vivo antitumoral action of metformin on hepatocellular carcinoma. Hepatol Res 42:922–933
Reid KM, Ramos-De la Medina A, Donohue JH (2007) Diagnosis and surgical management of gallbladder cancer: a review. J Gastrointest Surg 11:671–681
Shank JJ, Yang K, Ghannam J, Cabrera L, Johnston CJ, Reynolds RK et al (2012) Metformin targets ovarian cancer stem cells in vitro and in vivo. Gynecol Oncol 127:390–397
Teicher BA, Linehan WM, Helman LJ (2012) Targeting cancer metabolism. Clin Cancer Res 18:5537–5545
Tennant DA, Duran RV, Gottlieb E (2010) Targeting metabolic transformation for cancer therapy. Nat Rev Cancer 10:267–277
Wang S, Yu S, Shi W, Ge L, Yu X, Fan J et al (2011) Curcumin inhibits the migration and invasion of mouse hepatoma Hca-F cells through down-regulating caveolin-1 expression and epidermal growth factor receptor signaling. IUBMB Life 63:775–782
Wang L, Li X, Zhou Y, Shi H, Xu C, He H et al (2014) Downregulation of miR-133 via MAPK/ERK signaling pathway involved in nicotine-induced cardiomyocyte apoptosis. Naunyn Schmiedeberg’s Arch Pharmacol 387:197–206
Wu J, Chen C, Zhao KN (2013) Phosphatidylinositol 3-kinase signaling as a therapeutic target for cervical cancer. Curr Cancer Drug Tar 13:143–156
Zhang S, ZhangY ZY, Wang J, Ye J, Zhang S et al (2012a) Matrine induces apoptosis in human acute myeloid leukemia cells via the mitochondrial pathway and Akt inactivation. PLoS ONE 7:e46853
Zhang Y, Liu X, Zhang J, Li L, Liu C (2012b) The expression and clinical significance of PI3K, p-Akt and VEGF in colon cancer. Oncol Lett 4:763–766
Zhu A, Hong T, Hezel A, Kooby D (2010) Current management of gallbladder carcinoma. Oncologist 15:168–181
Acknowledgements
This study was supported by the Program for Young Scientist in Science and Education of Suzhou City (No. KJXW2014053), the Program for Young Scientist in Science and Education of Wujiang No. 1 People’s Hospital (No. 201724).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no conflict of interest.
Rights and permissions
About this article
Cite this article
Bi, T., Zhu, A., Yang, X. et al. Metformin synergistically enhances antitumor activity of cisplatin in gallbladder cancer via the PI3K/AKT/ERK pathway. Cytotechnology 70, 439–448 (2018). https://doi.org/10.1007/s10616-017-0160-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10616-017-0160-x