华蟾素通过诱导细胞凋亡抑制人宫颈癌Caski细胞生长

Cinobufacini Inhibits Proliferation of Human Caski Cervical Cancer Cells by Inducing Apoptosis

目的:探讨中药华蟾素对人宫颈癌Caski细胞生长及凋亡的影响。方法:MTT法检测细胞增殖抑制率,流式细胞术分析细胞周期和鉴定凋亡细胞;琼脂糖凝胶电泳分析DNA片段化;Western Blot分析不同浓度药物处理后细胞胞浆中细胞色素C的含量,荧光免疫组化法观察细胞中活性氧的含量变化,Dioc6染色法检测线粒体膜电位。结果:华蟾素能显著抑制人宫颈癌Caski细胞增殖,抑制效应呈时间和剂量依赖性,其48h的半数抑制浓度值(IC50)为1.68μg/mL;华蟾素可诱导DNA片段化,升高细胞中活性氧(ROS)含量、促进细胞色素C由线粒体向胞浆中释放,降低线粒体膜电位,同时抑制细胞分裂,将细胞阻滞在G2/M期。结论:华蟾素能显著性抑制Caski细胞增殖,其机制可能与增加细胞内活性氧的生成而诱导内源性细胞凋亡和抑制细胞分裂有关,具有用于宫颈癌临床治疗的潜在应用价值。

Objective： To investigate the effects of cinobufacini on proliferation and apoptosis in human Caski cervical cancer ceils. Method： MTT assay was used to assay cell proliferation; the flow cytometry assay was used to determine cell cycle; DNA fragrnenta-tion assays was used to evaluate apoptosis ; western blot was used to analyze cytochrome c content in the cytosol; the level of reactive oxy-gen species （ROS） was determined by fluorescence histochemistry; the mitochondrial membrane potential was determined by Dioc6 - stai-ning method. Result： Cinobufacini inhibited proliferation of Caski cells significantly in the time - and dose - dependent manners. The me-dian inhibition concentration （IC50） at 48h is 1. 68μg/mL. Cinobufacini treatment resulted in DNA fragmentation, elevated ROS levels, increased release of cytochrome C from mitochondria to the cytosol and decreased mitochondrial membrane potential. In the same time, cinobufacini inhibited cell division and arrested Caski cells at the G2/M phase. Conclusion： Cinobufotalin significant inhibit proliferation of Caski cells. The underlying mechanism is associated with increased ROS generation, which induced endogenous apoptosis and inhibited cell division. The results of this study suggest that cinobufacini has a potential value for the clinical treatment of cervical cancer.