氯化两面针碱通过抑制p53泛素化降解诱导宫颈癌细胞凋亡的机制

Mechanism of nitidine chloride inducing apoptosis of cervical cancer cells by inhibiting p53 ubiquitination degradation

目的探究氯化两面针碱(nitidine chloride,NC)对宫颈癌细胞凋亡的影响及其作用机制。方法以宫颈癌细胞系HeLa及SiHa为对象,通过CCK-8法和克隆形成实验检测NC的细胞毒性作用及对细胞生长的抑制作用。通过TUNEL法检测NC对宫颈癌细胞凋亡的影响,并采用Western blot法检测凋亡相关蛋白表达情况。运用免疫共沉淀实验、泛素化实验及Western blot实验分析NC对宫颈癌细胞中p53与E6AP蛋白相互作用、p53泛素化修饰水平、p53蛋白稳定性的影响。结果NC可明显抑制宫颈癌细胞增殖、诱导宫颈癌细胞凋亡,可抑制宫颈癌细胞中抑癌蛋白p53与泛素蛋白连接酶E6AP相互作用,降低p53泛素化修饰水平,延缓p53降解,增加p53蛋白表达水平。结论NC可通过抑制p53泛素化降解,提升p53蛋白表达水平,恢复其抑癌功能,发挥抗宫颈癌作用。

Aim To investigate the effect of nitidine chloride(NC)on the apoptosis of cervical cancer cells and its mechanism.Methods Cervical cancer cell lines HeLa and SiHa were selected as subjects.The cytotoxicity of NC and its inhibitory effect on cell growth were detected by CCK-8 assay and clone formation assay.The effect of NC on the apoptosis of cervical cancer cells was detected by TUNEL assay,and the expression of apoptosis-related proteins was detected by Western blot.The effects of NC on the interaction between p53 and E6AP protein,the level of p53 ubiquitination modification and the stability of p53 protein in cervical cancer cells were analyzed by immunoprecipitation assay,ubiquitination assay and Western blot assay.Results NC could significantly inhibit the proliferation and induce apoptosis of cervical cancer cells.NC could inhibit the interaction between tumor suppressor protein p53 and E6AP in cervical cancer cells,reduce the level of p53 ubiquitination modification,delay the degradation of p53 and increase the expression level of p53 protein.Conclusions NC can inhibit the ubiquitination and degradation of p53,improve the expression level of p53 protein,restore its tumor suppressor function,and thus play an anti-cervical cancer role.