异土木香内酯通过介导ROS 产生及线粒体损伤诱导人宫颈癌Hela细胞凋亡

目的:探讨异土木香内酯通过介导ROS 产生及线粒体损伤诱导人宫颈癌Hela 细胞凋亡机制。方法:不同终浓度异土木香内酯体外诱导Hela 细胞24 h 及加入抑制剂NAC 作为实验组,以正常细胞作为对照组,MTT 测定细胞活性;Hoechst 33258 染色观察Hela 细胞凋亡细胞核变化;流式细胞仪检测细胞凋亡及周期和ROS 产生变化和MMP 水平;Western blot 方法测定细胞色素C 蛋白、Bcl-2、Bax 和半胱氨酸天冬氨酸蛋白酶(Caspase-3)蛋白表达水平。结果:异土木香内酯以浓度依赖性抑制Hela 细胞生长;20 mol/ L 和40 mol/ L 终浓度异土木香内酯处理Hela 细胞24 h 后,细胞核出现典型的核固缩及核碎裂凋亡形态,细胞凋亡率增加,可抑制细胞生长于S 期,细胞内均可诱导ROS 的产生。ROS 抑制剂NAC 可以明显阻断异土木香内酯对Hela 细胞生长的抑制作用,降低细胞凋亡率;20 mol/ L 和40 mol/ L 终浓度异土木香内酯处理Hela 细胞24 h后,Bax 蛋白表达水平明显增加而Bcl鄄2 蛋白表达水平明显降低,同时Caspase-3 蛋白也被活化,出现cleaved Caspase 蛋白,线粒体内细胞色素C 蛋白释放增加。结论:异土木香内酯在体外可通过介导ROS 产生及线粒体损伤来抑制人宫颈癌Hela 细胞生长且诱导其凋亡,且伴随Bax 蛋白表达上调、Bcl-2 蛋白表达下调及Caspase-3 活化相关变化。

Isoalantolactone induces apoptosis in human cervical cancer Hela cells through ROS generation and Mitochondrial dysfunction

Objective:To investigate the induction of apoptosis by isoalantolactone in human cervical cancer Hela cells is mediated through ROS generation and Mitochondrial dysfunction.Methods: Cells were treated with isoalantolactone in a dose-dependent manner in the presence or absence of NAC for 24 h as the experimental group,and the normal cells were used as control Group.Cell viabilities were determined by the MTT assay;the nuclear morphology of Hela cells were observed under fluorescence microscope using the Hoechst 33258 staining;apoptosiscell cycle and reactive oxygen species(ROS) and mitochondrial membrane potential(MMP) were measured by flow cytometry;the protein expression levels of cytochrome C,Bcl-2,Bax and Caspase-3 were detected by Western blot.Results: In the present study,we found that isoalantolactone inhibits growth in a dose-dependent manner in Hela cells.Further studies revealed that Hela cells were treated with 20 and 40 mol/ L isoalantolactone for 24 h,after which we could observe the fragmented nuclei and the increased apoptosis rate.And we also found that isoalantolactone arrested the cell cycle at S phase and increased generation of reactive oxygen species and dissipation of mitochondrial membrane potential in Hela cells.While pretreatment with NAC obviously blocked the apoptotic and inhibition effect of isoalantolactone indicating that induction of apoptosis is ROS-dependent,Western blot study showed that isoalantolactone increased the expression of Bax and cleaved Caspase-3 and decreased the expression of Bcl-2 with concomitant release of cytochrome C from mitochondria into cytosol.Conclusion: Isoalantolactone could inhibit the proliferation and induce the apoptosis of human cervical cancer Hela cells in vitro through mediating ROS generation and Mi-tochondrial dysfunction,the mechanism of which is also accompanied by up-regulation of Bax expression,down-regulation of Bcl-2 expression,activation of Caspase-3 and release of cytochrome C.