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.
ABSTRACT
OBJECTIVE:To investigate the effect of ouabain and cinobufogenin on the apoptosis of HepG2 cell in order to study its apoptosis mechanism through ERK signaling pathway. METHODS:The activity of Na+/K+-ATPase was detected with HepG2 cell as target. The morphological changes of HepG2 cell and DNA injury were determined by Hoechst 33342 fluorescent staining and single cell gel electrophoresis (SCGE) respectively. The changes of the concentration of calcium ion were investigated by Fura-3 AM, and the expressions of Caspase-3 and ERK were detected by Western blotting assay. RESULTS:Ouabain and cinobufogenin inhibited the activity of Na+/K+-ATPase in HepG2 cell. Hoechst 33342 fluorescent staining showed that the clear apoptotic characteristics of HepG2 cell. The results of SCGE demonstrated that DNA double-strand of HepG2 cell was injured by ouabain and cinobufogenin. The concentration of calcium ion also increased after the treatment of ouabain and cinobufogenin according to the result of Fura-3 AM assay. The results of Western blot proved that ouabain and cinobufogenin promoted the production of p-ERK and the activition of caspase-3. CONCLUSION:Ouabain and cinobufogenin can induce the apoptosis of HepG2 cell and DNA damage, the increase of free calcium concentration and the activity of Caspase-3. ERK signaling pathway plays an important role in the apoptosis of HepG2 induced by ouabain and cinobufogenin.