激活AKT和ERK可拮抗N-去甲基克拉霉素对HeLa细胞的凋亡诱导作用

N-demethyl-clarithromycin induced HeLa apoptosis through regulating Akt and ERK activity

目的研究Akt和ERK的激活在N-去甲基克拉霉素诱导HeLa细胞凋亡中作用。方法MTT法测定N-去甲基克拉霉素对HeLa细胞的细胞毒作用;荧光染色观察细胞形态学变化;用琼脂糖凝胶电泳检测DNA片段化;用Western blot法分析药物对蛋白质表达的影响。结果N-去甲基克拉霉素明显诱导HeLa细胞凋亡,其作用呈明显的时间依赖性。Akt抑制剂和ERK抑制剂(PD98059)能促进N-去甲基克拉霉素诱导HeLa细胞凋亡,形态学观察可见凋亡小体的形成,琼脂糖凝胶电泳可见凋亡典型的DNA条带。Western blot结果显示,N-去甲基克拉霉素作用HeLa细胞24h后,Akt和磷酸化Akt蛋白表达减少,同时ERK和磷酸化ERK蛋白表达也减少。结论N-去甲基克拉霉素通过激活Akt和ERK对其诱导HeLa细胞凋亡起拮抗作用。

Aim To study the mechanisms of N-demethyl-clarithromycin-induced apoptosis in human cervical cancer cell line HeLa. Methods MTT, photomicroscopical observation, DNA agarose gel electrophoresis, LDH release and Western blot were used for apoptosis assay. Results N-demethyl-clarithremycin inhibited growth of HeLa in a time-dependent manner. Apoptotic bodies were found with Hoechst 33258 staining after treatment with 60 μmol · L^-1 N-demethylclarithremycin. DNA fragmentation was observed in N-demethyl-clarithromycin treated HeLa cells. The Akt inhibitor and the ERK inhibitor （PD98059） increased cell death. The expression of anti-apoptotic protein Akt, phosphorylated-Akt, ERK and phosphorylated-ERK decreased. Conclusion N-demethyl-clarithromycin induces HeLa apoptosis through Akt and ERK expression and phosphorylation.