Cinobufagin Promotes Cell Cycle Arrest and Apoptosis to Block Human Esophageal Squamous Cell Carcinoma Cells Growth via the p73 Signalling Pathway.

Cinobufagin isolated from traditional Chinese herbs has antitumour, anaesthetic, analgesic and anti-inflammatory effects. Recently, the antitumour activity of cinobufagin has attracted increasing attention from researchers. However, the anticancer activity of this drug on esophageal cancer cells and the precise mechanism are unclear. In this study, we determined the inhibitory effect of cinobufagin on the growth of three esophageal squamous cell carcinoma cell lines and explored its underlying mechanism. EC-109, Kyse-150, and Kyse-520 cells were treated with different concentrations of cinobufagin. The results of the Cell Counting Kit-8 (CCK-8) and clone formation assays showed that cinobufagin significantly reduced cell proliferation in a dose- and time-dependent manner. Also, flow cytometry and Hoechst 33342 staining indicated that the inhibition of growth induced by cinobufagin was mediated by G2/M cell cycle arrest and apoptosis. In addition, the expression of proteins related to cell cycle arrest and apoptosis was assessed by real-time quantitative (q)RT-PCR and Western blot. The results showed that cinobufagin caused G2/M arrest via upregulation of p21 and Wee1 and downregulation of cyclin B1 and Cdc2 at the mRNA and protein levels and induced apoptosis via upregulation of cleaved caspase-3, Puma and Noxa expression and an increased Bax/Bcl-2 ratio. Other data further showed that cinobufagin increased p73 expression and decreased Mdm2 expression, whereas p53 expression was not significantly changed. Taken together, these results suggest that growth inhibition of cinobufagin in esophageal cancer cells might act through the p73 pathway and its downstream molecules.