Effects of GHSC-73 on proliferation and cell cycle of human hepatocellular carcinoma HepG2 cells

Objective:To investigate the effects of β-D-glucosyl-(1-4)-α-L-thevetosides of 17β-digitoxigenin (GHSC-73),an isolate from the seeds of Cerbera manghas L.,on cell growth and cell cycle regulation of human hepatocellular carcinoma cell line HepG2,and to discuss the related mechanism.Methods:HepG2 cells were treated with different concentrations (0-80 μmol/L) of GHSC-73.Cell viability was determined using MTT assay at 24,48,and 72 h after treatment.Cell cycle distribution was assessed by flow cytometry after propidium iodide (PI) staining.Expression of the S phase associated genes GADD153,cyclin D1,cyclin A2,DHFR,TYMS,and p21 was determined by real-time RT-PCR before and after GHSC-73 treatment.Results:GHSC-73 inhibited the cell proliferation of HepG2 cells in a dose-and time-dependent manner.The values of IC50 were (5.18±0.21),(0.37±0.08),and (1.66±0.16) μmol/L at 24,48,and 72 h,respectively.Compared with control group,the proportion of cells in S phase increased in the treatment group with the prolongation of treatment,the cells in G0/G1 phase gradually decreased (P0.05),and cells in G2/M phase remained unchanged,indicating that GHSC-73 blocked HepG2 cell in S phase.Real-time RT-PCR showed that cyclin A2,DHFR,and TYMS were down-regulated,and p21,GADD153,and cyclin D1 were up-regulated,which might be associated with the GHSC-73-induced S phase arrest in HepG2 cells.Conclusion:GHSC-73 can inhibit growth of HepG2 cells by inducing S phase arrest; down-regulation of cyclin A2,DHFR,and TYMS genes and up-regulation of p21,GADD153,and cyclin D1 genes might participate in the induction of arrest.