Synergistic Anti-tumer Effect Of Metformin Combined With Sorafenib On Hepatocellular Carcinoma HepG2 Cells

Objective:The early clinical manifestations of hepatocellular carcinoma are atypical,and the progression of the disease is fast.When the patient is diagnosed,it may have reached the middle and late stage.It has lost the best period of early surgical treatment,and the prognosis is poor.The treatment of advanced hepatocellular carcinoma has become the focus of attention.Sorafenib is the first-line treatment for advanced hepatocellular carcinoma,but long term use will cause resistance to sorafenib.Therefore,finding new ways to increase sorafenib anti-tumor efficacy is particularly important.Recently metformin has been reported to have an anti-tumor effect.To investigate the anti-proliferation and apoptosis induction effects metformin in combination with sorafenib on hepatocellular carcinoma cells HepG2 and to explore the potential mechanism.Methods:The anti-proliferation effects of either metformin or sorafenib alone or their combination on HepG2 cells were assessed by MTT.The IC₅₀ values of two single drug and the CI values were calculated.The morphological changes were observed by fluorescent microscope.The apoptosis ratios were analyzed by FCM.The expression of phospho-Erk?p-Erk??phospho-Akt?p-Akt?proteins and apoptosis-related proteins Bcl-2 and Bax were detected by Western blot.Results:MTT assay indicated that the IC₅₀ values of metformin and sorafenib on HepG2cells were?9.87±1.52mmol/L?and?5.65±0.71??mol/L,the anti-proliferation effect of combination with metformin and sorafenib was more remarkably than the two single drugs?P<0.01?,the combination with metformin and sorafenib exhibited a synergistic effect?CI<1?.Fluorescent microscope observed the apoptosis morphological changes were more obvious in the combination group.Flow cytometry indicated that the apoptosis rate of metformin and sorafenib was?13.94±0.40?%and?16.54±0.09?%,the apoptosis rate of combination group was significantly higher than single drug group?P<0.01?,which was?42.09±1.12?%.Western blot indicated that metformin combinated with sorafenib can significantly decrease the expression levels of p-Erk?p-Akt?Bcl-2 and increase the expression level of Bax of HepG2 cells,as compared with the control and the single drugs.Conclusion:The combination of metformin and sorafenib performs a synergistic anti-tumor effect on HepG2 cells.Mechanism may be related to apoptosis and down-regulation the expression of p-Erk and p-Akt.