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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 the mitochondrial permeability transition of breast cancer cell line MDA-MB-231. Methods MTT assay was used to detect the inhibitory effect of deguelin on the proliferation of MDA-MB-231 cells. AnnexinⅤ-FITC / PI double staining was used to detect the apoptotic rate. Mitochondrial membrane potential was measured by rhodamine 123 single staining. Western blotting was used to detect the expression of Cytc in cytoplasm; the activity of caspase-3 protein was detected by spectrophotometry; the change of intracellular calcium concentration was detected by Fluo-3 / AM fluorescent indicator; Bcl-2 and Bax mRNA were detected by RT- And its protein expression. Results deguelin significantly inhibited the proliferation of MDA-MB-231 cells in a time and dose-dependent manner (P <0.05). After deguelin treatment, degranulation of mitochondrial membrane potential and expression of Cytc in the cytoplasm The activity of caspase-3 was significantly increased compared with the untreated group (P <0.01). Flow cytometry showed that apoptosis rate and intracellular calcium concentration increased with the increase of deguelin concentration. The results of RT-PCR and Western blot showed that deguelin decreased the expression of Bcl-2 mRNA and protein, Bax expression increased. Conclusion The inhibition of deguelin on the proliferation of breast cancer MDA-MB-231 cells may be related to the increase of intracellular calcium overload, the alteration of Bcl-2 and Bax expression, the opening of mitochondrial permeability transition pore and the decrease of mitochondrial membrane potential Cytc release related.