ABSTRACT
To investigate the apoptosis-inducing effect of cinobufacini (Huachansu) on human hepatoma cell line HepG2 and its possible mechanism of action, HepG2 cells were treated with different concentrations of cinobufacini. Cell proliferation was measured by methylthiazolyl tetrazolium (MTT) assay. The morphological changes of apoptosis were observed by Hoechst 33258 staining. Cell cycle distribution and apoptotic rate were evaluated by flow cytometry (FCM). Quantitative real-time RT-PCR and Western blotting analysis were used to detect the mRNA and protein expressions of apoptosis related factors Bcl-2, Bax and p53. The results indicated that cinobufacini could inhibit the proliferation of HepG2 cells in a dose and time dependent manner. Remarkable morphological changes of apoptosis including cytoplasmic and nuclear condensation and partition of cytoplasm were observed by Hoechst 33258 staining. According to FCM analysis, HepG2 cells were arrested in G2/M phase and the apoptotic rate increased with the increase of the concentration of cinobufacini. Both the mRNA and protein expressions of Bax and p53 were up-regulated while Bcl-2 expression down-regulated. Thus, cinobufacini could inhibit the proliferation and induce apoptosis of human hepatoma cell line HepG2. Furthermore, up regulation of Bax and p53 as well as down regulation of Bcl-2 expressions may be one of the important apoptotic inducing mechanisms.