Study on the Anti-tumor Mechanism of Cinobufacin-induced AMPK Phosphorylation to Promote Immunogenic Apoptosis of Colorectal Cancer Cells

OBJECTIVE To investigate the role of AMPK in cinobufagin(CBG) induced immunogenic death of colorectal cancer HCT116 cells. METHODS The colorectal cancer HCT116 cells were cultured in vitro and divided into four group including control group, CBG group, CBG+AMPK inhibitor group(named inhibitor group) and CBG+AMPK siRNA group(named interference group). Cell survival was detected by MTT. The dying cells were detected by LIVE/DEAD^TM Viability/Cytotoxicity Kit. The apoptosis and calreticulin(CRT) content were detected by flow cytometry. The expression of AMPK, phosphorylated AMPK(p-AMPK) were detected by Western blotting. RESULTS After treatment with 2.5, 5, 10, 25, 50, 100, 250, 500 nmol·L^-1 CBG for 48 h, the inhibition rate of HCT116 increased significantly and the half-inhibitory concentration(IC₅₀) was (53.03±5.36)nmol·L^-1. Dosage of 25, 50 nmol·L^-1 CBG treatment significantly increased the expression of p-AMPK. Compared with control group, the apoptotic cells, the expression of cleaved-capapse3, the content of CRT and HMGB1increased, while the expression of Bcl-2 decreased in CBG group. Compared with CBG group, the apoptotic cells, the expression of cleaved-capapse3, the content of CRT and HMGB1 decreased, wherea the expression of Bcl-2 increased in inhibitor group and interference group. CONCLUSION CBG induces immunogenic death by activating AMPK and inhibits the proliferation of colorectal cancer cells.