Bufarenogin induces intrinsic apoptosis via Bax and ANT cooperation

Toads have high medicinal value and have been used for medicinal purposes since the Tang Dynasty period (7th-10th Century AD). Bufarenogin, an active anti-tumor constituent of toad venom, shows anti-tumor activity. In this study, we investigated the inhibitory effects of bufarenogin on the growth and metastasis of colorectal cancer (CRC), particularly its effects on mediating intrinsic signaling pathways that initiate apoptosis. An orthotopic CRC model was established in nude mice via surgical orthotopic implantation to investigate tumor growth. Immunohistochemistry, immunofluorescence, and Western blotting assays were performed to evaluate protein expression. The in vitro results revealed the anti-proliferative effect of bufarenogin against CRC cells. Bufarenogin caused cell death via apoptosis, as revealed by Annexin V/7-amino-actinomycin D double staining, which was verified using a pan-caspase inhibitor. Bufarenogin induced B-cell lymphoma 2-associated X protein (Bax)-dependent intrinsic apoptosis, as demonstrated by mitochondrial translocation of Bax and cytoplasm release of HCT116 wild-type cells and cytochrome C (soluble pro-apoptotic factors). Additionally, we showed that adenine-nucleotide translocator interacted with Bax. Bufarenogin induced intrinsic apoptosis through the cooperation of Bax and adenine-nucleotide translocator and inhibited the metastasis and growth of orthotopical CRC cells.