摘要
目的:研究新合成的靛玉红衍生物吲哚-3-取代物A的体外抗肿瘤作用及可能机制。方法:以不同浓度的吲哚-3-取代物A作用于HepG2细胞,MTT法检测细胞活力;观察HepG2细胞的集落形成;DAPI染色观察细胞核的变化;Annexin-V/PI双染检测细胞凋亡率;分别采用JC-1和Fluo-3/AM染色检测HepG2细胞线粒体膜电位和胞内Ca2+含量;蛋白质印迹检测Bcl-2、Bax的表达和Caspase-3、9的活化。结果:吲哚-3-取代物A对HepG2细胞的抑制作用呈时间浓度依赖性,且可抑制HepG2细胞克隆集落的形成。吲哚-3-取代物A作用于HepG2细胞后,细胞表现出明显的核固缩、碎裂等凋亡特征,Annexin-V/PI双染结果显示HepG2细胞出现明显的凋亡,细胞凋亡相关的Caspase-3、9活化也明显增加。同时,吲哚-3-取代物A处理可降低Bcl-2的蛋白表达水平,提高Bax的蛋白表达水平,呈浓度依赖性。此外,吲哚-3-取代物A能降低线粒体膜电位并诱发细胞内钙离子超载。结论:吲哚-3-取代物A能够诱导HepG2细胞出现明显的细胞凋亡,其机制可能与损伤线粒体,活化线粒体介导的细胞凋亡通路密切相关。
Objective: To investigate the anti-tumor effects and possible mechanisms of a novel indole-3-derivate indirubin in vitro. Methods: Human HepG2 cells were exposed to different concentrations of indole-3-derivate A,cell proliferation was detected by MTT assay and cell colony formation of HepG2 cells treated with indole-3-derivate A was also observed. Then the nuclei changes were observed by staining with DAPI and cell apoptosis was detected by Annexin-V / PI staining. Meanwhile,mitochondrial membrane potential and intracellular free Ca2+ content were detected with JC-1 and Fluo-3 / AM staining,respectively.Moreover,the protein levels of Bcl-2,Bax and activation of Caspase-3,9 were examined by western blotting. Results: Indole-3-derivate A inhibited the proliferation and colony formation of HepG2 cells in a doseand time-dependent way. Besides that,it was found that nucleus morphology was showing pyknosis and fragmentation and early apoptotic cells were significantly increased by Annexin-V / PI staining after indole-3-derivate A treatment. Moreover,western blotting showed indole-3-derivate A activated Caspase-3,9,decreased the expression of Bcl-2 and increased the expression of Bax in a dose-dependent manner. In addition,indole-3-derivate A caused a loss of mitochondrial membrane potential and intracellular Ca2+ overloading. Conclusions: Indole-3-derivate A could significantly inhibit proliferation of HepG2 cells and the mechanisms might be related to mitochondria dysfunction and activation of mitochondria-dependent apoptotic pathway.
出处
《江苏大学学报(医学版)》
CAS
2015年第1期38-42,48,共6页
Journal of Jiangsu University:Medicine Edition