The linkage between cell cycle S phase arrest and apoptosis on human hepatocellular carcinoma HepG2 induced by Na~+,K~+-ATPase inhibitors via regulating proteins associated with cell cycle / 中国药理学通报

ABSTRACT

Aim To investigate the effect of ouabain and cinobufogenin on cell proliferation,apoptosis and cell cycle on HepG2,and explore their molecular mechanism.Methods The anti-proliferative effect on HepG2 cells was determined by MTT assay.The HepG2 cells were stained with Hoechst 33342,and its morphological changes were observed under fluorescence microscope;The cell cycle was measured by flow cytometry.The Cyclin A1,CDK 2,PCNA and p21~(CIP1) expression levels of HepG2 cells treated with ouabain and cinobufogenin were dectected in mRNA and protein by Real time PCR and Western blot.Results Ouabain and cinobufogenin could inhibit cell proliferation on HepG2 cells,and the inhibitory effects were in time and dose dependent manners.The HepG2 cells treated with ouabain and cinobufogenin showed the typical morphological features of apoptosis.Cell cycle analysis showed that the S phase of HepG2 cells treated with ouabain and cinobufogenin increased significantly compared with the control group.Real-time quantitative PCR and Western blot results showed that ouabain and cinobufogenin could down-regulate Cyclin A1,CDK 2,and PCNA expressions(P<0.05)and up-regulate p21~(CIP1) expression(P<0.05).Conclusion Nα~+,K~+-ATPase inhibitor has the anti-proliferative effect on HepG2 cells and induce apoptosis and S phase arrest.These effects might be related with proteins associated with cell cycle closely.