OBJECTIVEto investigate the effects of antidiabetic drug metformin on proliferation and apoptosis in human hepatocellular carcinoma cell line Huh-7 cells.

METHODSHuh-7 cells were treated with metformin at different concentrations. Cell viability was determined by MTT assay. Cell apoptosis and CD133(+) expression rate were detected by flow cytometery (FCM). Expressions of PTEN, Akt, p-Akt, Bcl-2, Bax proteins in the cells were measured by Western blot. The effect of metformin on the hepatosphere formation was observed in the serum-free suspension culture. Reverse transcription-polymerase chain reaction (RT-PCR) was used to validate the expression levels of stemness marker genes CD133, β-catenin, and ABCG2 mRNA in the hepatospheres.

RESULTSThe proliferation of Huh-7 cells was inhibited by metformin in a dose- and time-dependent manner. The early and late cell apoptosis rates induced by metformin at dose of 10 mmol/L for 48 hrs were (22.29 ± 0.8)% and (13.87 ± 1.2)%, respectively, and 25 mmol/L for 48 hrs (15.28 ± 2.1)% and (25.89 ± 2.3)%, respectively. Western blotting results revealed that the expression of CD133, phosphorylated Akt and the Bcl-2/Bax ratio were downregulated, and PTEN was upregulated in the Huh-7 cells after treated with 25 mmol/L metformin for 48 hrs. Metformin inhibited the formation of hepatospheres. Metformin also downregulated the expression of several cancer stem cells (CSCs)-related genes which are involved in the signaling pathways governing the self-renewal, proliferation and differentiation of CSCs in the hepatospheres.

CONCLUSIONSMetformin inhibits the proliferation of human hepatocellular carcinoma Huh-7 cells and enhances their apoptosis in vitro. It may be related to the downregulation of PI3K/Akt signal pathway and selectively targeting CD133(+) cells.