Cinobufacini from the Skin of Bufo bufo gargarizans Induces Apoptosis,Possibly via Activation of the Wnt/β-Catenin Pathway,in Human Osteosarcoma Cells

Abstract

Cinobufacini (huachansu) is a traditional Chinese medicine extracted from the skin of Bufo bufo gargarizans, which is used in clinical cancer therapy. The purpose of this study was to investigate the signaling pathways regulating cinobufacini-induced apoptosis in the osteosarcoma cell line, U₂OS. We used 3-[4,5-dimethylthiazol- 2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay to evaluate the effects of cinobufacini on cell proliferation in U₂OS cells. Changes in cell morphology and apoptosis were detected by TUNEL staining. The expression of apoptosis-related and Wnt/β-catenin pathway proteins was detected by immunofluorescence, RT-PCR, and western blot analysis. Our data indicated that cinobufacini significantly inhibited cell proliferation in a dose- and time-dependent manner. Marked changes in cell morphology and apoptosis rate were clearly observed after cinobufacini treatment. The Wnt/β-catenin pathway was activated, and β-catenin expression was positive in cells after treatment. Further, protein expression of bax was increased, whereas bcl-2 was decreased, resulting in an increased bax/bcl-2 ratio. Moreover, after cinobufacini treatment, the expression of Wnt/β-catenin pathway-related proteins was similar to controls. Taken together, our study indicates that cinobufacini can induce apoptosis in U₂OS cells, likely through activating the Wnt/β-catenin pathway.