Cinobufagin induced cell apoptosis and protective autophagy through the ROS/MAPK signaling pathway

Abstract Purpose Cinobufagin(CB), an cardiotonic steroid isolated from the skin and parotid venom glands of the toad Bufo bufo gargarizans Cantor, has reported to have a significant anti-cancer effect on various cancers. However, the effect of CB on ovarian cancers was none reported. Herein, the present study aimed to investigate the therapeutic effect of cinobufagin on the ovarian cancer cells and elucidate the underlying mechanism. Methods Cell viability in our work was assessed via MTT. Cell apoptosis was detected by flow cytometry analysis and Hoechst 33258. Autophagy was defined by confocal microscopy after infected with mRFP-GFP-LC3 dual fluorescence adenovirus. Reactive oxygen species (ROS) was investigated by flow cytometry. The level of marker proteins involved in autophagy, apoptosis and ROS/MAPK signaling pathway were determined by western blot. Results Cinobufagin significantly reduced the viability and induced apoptotic cell death of human ovarian cancer cell lines SKOV-3 and A2780. MRFP-GFP-LC3 infection elaborated that cinobufagin could promote cell autophagy. Moreover, autophagy inhibitor 3-methyladenine (3-MA) markedly enhanced the cinobufagin-induced apoptosis. In addition, treatment with cinobufagin could dramatically increase the expression of ROS and then activate the phosphorylation of MAPK family proteins, including ERK 1/2, JNK and p38. What's more, the reaction of apoptosis and autophagy induced by cinobufagin treatment could be reversed by p38 inhibitor SB203580 and JNK inhibitor SP600125 as well as ROS exclusive inhibitor antioxidant N -acetyl cysteine (NAC). Conclusions Our findings provide clues concluding that cinobufagin could induce cell apoptosis and protective autophagy through the ROS/MAPK signaling pathway in human ovarian cancer cells.