The Research on the Anti-glioma Effect and Mechanism of Cinobufagin

Objective To study the effect of cinobufagin (CB) on the proliferation inhibition and induction of apoptosis in glioblastoma cell lines U87 and its molecular mechanism. Methods A gradient concentration (0-20 μmol/L) of CB was used to treat the U87 glioma cells for 6 h, 12 h, 24 h and 48 h, respectively. Cell viabilities were determined by CCK-8 assay to discover the effects of different concentrations of CB on the proliferation of glioma cells. Different concentrations (1-20 μmol/L) of CB were used to treat the U87 glioma cells for 12 h and 24 h, hochest33342 staining assay was used to assess the apoptosis levels. Immunofluorescence staining was used to determine the expression of growth related proteins phospho-protein kinase B(T308)〔 p-AKT(T308)〕 in U87 glioma cells after being treated with CB for 24 h. Western blot was used to determine the apoptotic related proteins (BAX, cleaved-caspase 3, cleaved-caspase 9) and growth related proteins 〔phospho-inositide 3-kinase (p-PI3K), p-AKT(T308), p-AKT(S473), phospho-ribosomal protein S6 kinase (PS6), phospho-4E-binding protein 1 (p-4EBP1)〕. Results A significant effect of CB on the proliferation inhibition and induction of apoptosis in U87 glioma cells in a time- and dose-dependent manner was observed. Treatment with CB induced the expression levels of apoptosis-related protein, cleaved-caspase 3 and BAX, and the PI3K-AKT-4EBP1 signaling pathway related proteins p-AKT(T308) and p-4EBP1 were decreased. Conclusion CB can inhibit U87 glioma cells growth and induce apoptosis, which may involve the PI3K-AKT-4EBP1 and BAX-caspase signaling pathways.