鱼藤素对乳腺癌细胞MDA-MB-231线粒体通透性转换孔的作用  

Effect of deguelin on mitochondrial permeability transition pore of human breast cancer cell line MDA-MB-231

作　　者：杜佳[1] 邓华瑜[1] 姜蓉[1] 陈黎[1] 

机构地区：[1]重庆医科大学病理生理学教研室干细胞与组织工程研究室,重庆400016

出　　处：《第二军医大学学报》2010年第4期374-379,共6页Academic Journal of Second Military Medical University

摘　　要：目的研究鱼藤素对乳腺癌细胞MDA-MB-231线粒体通透性转换孔的作用。方法MTT法检测鱼藤素对MDA-MB-231细胞的增殖抑制作用;AnnexinⅤ-FITC/PI双染流式细胞术检测细胞凋亡率;罗丹明123单染法观察线粒体膜电位变化;蛋白免疫印迹法检测细胞质中Cytc的表达;分光光度法检测caspase-3蛋白活性;Fluo-3/AM荧光指示剂检测细胞内钙离子浓度变化;RT-PCR和蛋白免疫印迹法检测Bcl-2和Bax mRNA及其蛋白表达。结果鱼藤素对MDA-MB-231细胞增殖具有明显的抑制作用,呈时效和量效依赖关系(P<0.05);鱼藤素处理细胞后,细胞线粒体膜电位降低,细胞质内Cytc蛋白表达水平升高,caspase-3活性明显提高,与未处理组相比差异具有统计学意义(P<0.01)。流式细胞术检测显示细胞凋亡率和细胞内钙离子浓度随鱼藤素浓度的增加而增多;RT-PCR和蛋白印迹检测结果发现鱼藤素能使Bcl-2 mRNA和蛋白表达减少,而Bax表达增加。结论鱼藤素对乳腺癌MDA-MB-231细胞的增殖抑制、诱导凋亡作用可能与细胞内钙超载,Bcl-2和Bax表达改变影响线粒体通透性转换孔开放,诱导线粒体膜电位降低及Cytc释放有关。Objective To investigate the effect of deguelin on mitochondrial permeability transition pore of human breast cancer cell line MDA-MB-231.Methods The inhibitory effect of deguelin on cell proliferation was determined by MTT assay;cell apoptosis rate was analyzed by flow cytometry with Annexin Ⅴ FITC/PI double staining;MDA-MB-231 cells were stained by Rhodamine123 to detect the changes of mitochondrial transmembrane potential by FCM;alteration of protein of Cyt c outside of mitochondria was detected by Western blotting analysis;caspase-3 activity was assessed by colorimetric assay;and MDA-MB-231 cells were stained by Fluo-3 /AM to detect changes of intracellular Ca2 ＋ concentration by FCM.The expression of Bcl-2 and Bax was examined by RT-PCR and Western blotting analysis.Results Deguelin significantly inhibited the growth of MDA-MB-231 cells in a time-and dose-dependent manner（P 0.05）.After treatment with deguelin,mitochondrial transmembrane potential was decreased,the expression of Cyt c outside of mitochondria was increased,and caspase-3 activity was significantly increased compared with negative control group（P 0.01）.FCM analysis showed that the apoptotic rate of MDA-MB-231 cells and intracellular Ca2 ＋ concentration increased gradually with the increase of deguelin concentration.RT-PCR and Western blotting analysis showed that the expression of Bcl-2 mRNA and protein was down-regulated and that of Bax was up-regulated after deguelin treatment.Conclusion Deguelin can inhibit proliferation and induce apoptosis of MDA-MB-231 cells,and the induction of apoptosis might be related to increased intracellular Ca2 ＋ concentration and changes of mitochondrial permeability transition pore induced by altered Bcl-2,Bax expression.

关 键 词：鱼藤素 乳腺肿瘤 BCL-2 BAX 钙离子 

分 类 号：R737.9[医药卫生—肿瘤]