In Vitro And In Vivo Antitumoral Action Of Metformin On Hepatocellular Carcinoma And The Possible Mechanism

Aims:Metformin is a biguanide that has been widely used to treat type2diabetes. Several studies have shown that metformin is also effective in treating cancer, including hepatocellular carcinoma (HCC). The objective of this study was to evaluate the antitumor effects of metformin in HCC, as well as to investigate the potential molecular target(s) of metformin-mediated antitumor activity.Methods:HCC cell lines HepG2, Huh7, SMMC7721, Be17402and human normal liver cell line L02were cultured. Cells were treated with different concentrations of metformin and then the cell proliferation, cell cycle and cell apoptosis were measured.Target protein levels were measured by Western blot analysis. Target gene mRNA levels were measured by Real-time PCR.To investigate whether Livin protein and the AMPK/mTOR pathway were involved in the antitumor effects of metformin in HCC, Livin siRNA and AMPKal/2siRNA carried by Lipofectamine2000were transfected into cells.HCC xenograft tumors were established in athymic nude mice, and tumor growth was monitored during metformin treatment.The expression of cell proliferation-related and cell apoptosis- related proteins in tumor tissues and normal liver tissues were detected by immunohistochemical analysis. The serum insulin and IGF-1levels in tumor-bearing mice were detected by ELISA.Results:Cultured cells were treated with metformin (1mM,5mM,10mM,15mM) for72hours. The proliferation of HCC cells was significantly inhibited by metformin treatment in a dose-dependent manner(P<0.05). Meanwhile, the viabilities of the HCC cells were significantly lower than that of the normal human liver cells after metformin treatment (P <0.05).Metformin treatment induced G0/G1phase cell cycle arrest and cell apoptosis in HCC cells (P<0.05), however, failed to affect the cell cycle distribution and cell apoptosis in normal liver cells.Livin siRNA transfection caused growth inhibition of HCC cells, induced G0/G1phase cell cycle arrest and cell apoptosis (P<0.05). However, it failed to affect the cell cycle distribution and cell apoptosis in normal liver cells. Metformin treatment decreased the expression level of Livin protein in HCC cells in a dose-dependent manner. However, it failed to affect the expression level of Livin protein in normal liver cells. Livin mRNA expression levels of HCC cells and normal liver cells were not affected by metformin treatment.After AMPKa siRNA transfection for72hours, the growth of HepG2cells was not significantly affected, but the inhibitory effect of metformin on the growth of HepG2cells was significantly inhibited. In HepG2cells, Metformin treatment increased AMPK phosphorylation levels and reduced p70S6K protein phosphorylation levels in a dose-dependent manner. Meformin treatment failed to decrease Livin protein levels and p70S6K phosphorylation levels after cells were transfected with AMPKa siRNA.In nude mice ectopic xenograft model, metformin treatment significantly reduced tumor growth in a dosage-dependent manner (P <0.05).In nude mice orthotopic xenograft model, metformin treatment significantly reduced tumor growth in a dosage-dependent manner (P-0.05). In the xenograft tumor tissues, metformin treatment significantly decreased Ki67index and increased Cleaved caspase3index (P<0.05). However, metformin treatment failed to affect the Ki67index and Cleaved caspase3index in normal liver tissues. Metformin treatment increased AMPK phosphorylation levels and decreased Livin protein expression levels in the xenograft tumor tissues in a dosage-dependent manner. Metformin treatment did not cause any pathological changes in organs of nude mice according to HE staining analysis. Metformin treatment didn't affect body weight, blood glucose and serum insulin, IGF-1levels in tumor-bearing mice. Conclusion:In vitro and in vivo, Metformin inhibits the growth of HCC cells through inducing G0/G1phase cell cycle arrest and cell apoptosis, however, the growth of normal liver cells was not affected.Metformin decreases Livin protein level in HCC cells which causes tumor specific growth inhibition.Metformin activates AMPK/mTOR pathway and subsequently inhibits Livin protein expression at the mRNA translation level.