Antitumor effects by Wilfoside C3N treatment in ECA109 cells.

C21 steroidal glycoside (C21) is one of the most bioactive compounds of Cynanchum auriculatum Royle, known as Baishouwu, and possesses potent antitumor activity. Wilfoside C3N is one of the two most abundant and active C21 in it. The aim of this study was to further investigate the antitumor activity of C3N and to clarify its signaling pathway. The growth inhibition ofECA109 cells induced by C3N was assessed. Western blot analysis, reverse-transcription PCR, caspase-2 and Fas activity assay, and knockdown of caspase-2 with siRNA were used to study the apoptotic mechanisms. We showed that C3N inhibited the proliferation of ECA109 cells moderately in a dose and time-dependent manner and induced apoptosis in the ECA109 cell line through a mitochondrial pathway by triggered cytochrome c release from the mitochondria, with caspase-2 functioning upstream of caspase-9 rather than association with Fas and caspase-8. Furthermore, C3N-driven apoptotic events were associated with downregulation of Bcl-2. These results suggest that C3N-induced apoptosis of ECA109 cells in vitro was dependent on caspase-2 or mitochondria or caspase-9 and independent of the Fas-FasL or caspase-8 pathway.