华蟾素诱导AMPK磷酸化促进结直肠癌细胞免疫原性凋亡的抗肿瘤机制研究

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

关键词:  华蟾素  免疫原性死亡  腺苷酸蛋白激酶  结直肠癌

Key words:  cinobufagin  immunogenic cell death  adenylate protein kinase  colorectal cancer

摘要:

目的 探讨AMPK在华蟾素（cinobufagin，CBG）诱导结直肠癌HCT116细胞发生免疫原性死亡的作用。方法 体外培养结直肠癌HCT116细胞，并将细胞分为对照组、CBG组、CBG+AMPK抑制剂组（抑制剂组）和CBG+AMPK siRNA干扰组（干扰组）；用MTT法检测细胞增殖，LIVE/DEAD^TM Viability/Cytotoxicity Kit试剂盒观察细胞死亡情况，流式细胞仪技术检测细胞凋亡比例、钙网蛋白（calreticulin，CRT）含量，ELISA法检测培养基中HMGB1含量，Western blotting方法检测AMPK、磷酸化的AMPK（p-AMPK）的表达水平。结果 2.5，5，10，25，50，100，250，500 nmol·L^-1的CBG处理48 h后，细胞增殖抑制率显著增加，IC₅₀为（53.03±5.36）nmol·L^-1。25，50 nmol·L^-1的CBG显著增加p-AMPK表达。与对照组比较，CBG组细胞凋亡比例、cleaved-caspase 3表达、CRT和HMGB1含量增加，Bcl-2表达下降；与CBG组相比，抑制剂组和干扰组细胞凋亡比例、cleaved-caspase 3表达、CRT和HMGB1含量明显下降，Bcl-2表达增加。结论 CBG通过激活AMPK诱发免疫原性死亡，抑制结直肠癌细胞的增殖。

Abstract:

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.