AMPK Activators Induce Cell Cycle Arrest By Regulating P21^CIP, P27^KIP, Cyclin D1 In Hepatocellular Carcinoma Cell Line, HepG2

Background and ObjectivesAMP-activated protein kinase (AMPK) is an energy-sensing/signaling. Recent researches have shown that activators of AMPK, metformin and AICAR have cytotoxicity effects on tumor cells, and the inhibition of cell growth is possibly associated with the effect of cell cycle arrest. It has been rarely reported about whether metformin and AICAR can inhibit the growth and affect the cell cycle process of hepatocellular carcinoma cells (HCC). The mechanisms of growth inhibition and cell cycle arrest of AMPK have not been well understood. In this study, we investigate the effect on proliferation and cell cycle inhibition of metformin and AICAR on HCC cell line, HepG2 and clarify its possible molecule mechanisms. This will provide evidances for AMPK as a new target of anti-HCC therapy.Materials and MethodsA human HCC cell line, HepG2 was cultured in RPMI-1640 with 10% fetal bovine serum supplemented with 100 U/ml penicillin and 100 U/ml streptomycin.HepG2 cells were treated with different concentrations of metformin (0, 2.5, 5.0, 10.0, 20.0 mM) or AICAR (0, 50, 100, 250, 500μM ) for 24, 48, 72h. MTT assay was used to evaluate the influence of metformin or AICAR on the proliferation of HepG2 cells.HepG2 cells were cultured in the medium containing 10mM of metformin or 500μM of AICAR for 72h, then stained by propidium iodide (PI) in single staining method and cell cycle was detected by flow cytometry (FCM).HepG2 cells were cultured in medium containing metformin of 10mM or AICAR of 500μM for 24h, and the untreated cells were taken as control. The phosphorylation of AMPK and expression of p21^CIP, p27^KIP, cyclin D1, CDC2, CDK2 were detected by Western Blot.HepG2 cells were cultured in the medium containing 10mM of metformin or 500μM of AICAR for 24h, the apoptosis of HepG2 cells was observed by DAPI staining. Results1. Phosphorylation of AMPK in HepG2 cells increased comparing with the control groups after treatment by metformin or AICAR.2. Metformin or AICAR inhibits the growth of HepG2 cells in a dose and time-dependent manner comparing with the control groups.3. Flow cytometry indicated that cell cycle arrest was induced in HepG2 cells at G0-G1 phase by metformin or AICAR treatment, comparing with the control group.4. Metformin or AICAR up-regulated the expression of p21^CIP and p27^KIP, down-regulated the expression of cyclin D1 in HepG2 cells through activation of AMPK. The expression of CDC2, CDK2 did not change significantly.5. Nuclear fragmentation (apoptosis) was observed by DAPI stained in HepG2 cells by metformin or AICAR treatment.Conclusions1. AMPK activators induce the proliferation inhibition in HepG2 cells.2. AMPK activators induce cell cycle arrest by inhibiting G1/S check point.3. AMPK activators up-regulate the expression of p21^CIP, p27^KIP and down-regulating cyclin D1 through activation of AMPK.The changes in CDC2 and CDK2 expressions are not significant.4. Apoptosis was induced by AMPK activators in HepG2 cells.In conclusion, AMPK activators induce cell cycle arrest at G0-G1 phase by up-regulating p21^CIP, p27^KIP and down-regulating cyclin D1 through activation of AMPK in HCC cells.