钙磷酸蛋白C对乳癌细胞MDA-MB-435S凋亡的影响

Effect of Calphostin C on apoptosis of breast carcinoma cell MDA-MB-435S

目的:研究钙磷酸蛋白C对乳癌细胞MDA-MB-435S凋亡的影响及相关机制。方法:采用倒置显微镜、琼脂糖凝胶电泳和流式细胞术等方法观察钙磷酸蛋白C处理前后MDA-MB-435S细胞形态、基因组DNA断裂和凋亡细胞数的变化;采用RT-PCR检测钙磷酸蛋白C处理前后MDA-MB-435S细胞凋亡相关基因bcl-XL、bcl-2、IAP-1、bax及Smac mRNA表达的变化;采用Western blotting法测定Bax和Bcl-2蛋白的表达。结果:与MDA-MB-435S亲本细胞(对照组)相比,钙磷酸蛋白C处理后的MDA-MB-435S细胞(实验组)出现凋亡形态学特征(如胞膜发泡、染色质凝集、凋亡小体形成等)改变,显示特征性的DNA"梯状"断裂变化,凋亡细胞数明显增多。RT-PCR和Western blotting结果显示:与对照组相比,实验组细胞中促凋亡基因bax和Smac mRNA表达上调,抗凋亡基因bcl-2mRNA表达下调,但抗凋亡基因bcl-XL和IAP-1mRNA表达没有明显变化;实验组Bax蛋白表达上调,Bcl-2蛋白表达下调。结论:钙磷酸蛋白C可诱导MDA-MB-435S凋亡,该作用可能与上调bax和Smac基因表达及下调bcl-2基因表达有关。

Aim：To investigate the effect of Calphostin C on the apoptosis of breast carcinoma cell MDA-MB-435S.Methods：The changes of morphology,DNA cleavage,and number of apoptosis of MDA-MB-435S cells before and after treated with Calphostin C were observed by inverted microscope,agarose gel electrophoresis,and flow cytometry.The expressions of apoptosis-related genes bcl-2,bcl-XL,bax,IAP-1 and Smac mRNA were detected by RT-PCR.Bax and Bcl-2 proteins were detected by Western blotting assay.Results：Compared with the parental MDA-MB-435S cells （ control group） , morphological changes of apoptosis （ blowing up in membrance, chromatic agglutination, and apoptotic bodies, etc） , the appearance of ＂DNA ladder＂ by agarose gel electrophoresis, and the significant increase of the apoptosis by flow cytometry in the MDA-MB-435S cells treated with Calphostin C （experimental group） were found. The expressions of bax and Smac mRNA were higher, and the expression of bcl-2 mRNA was lower, while the expressions of bcl-XL and IAP-I mRNA had no significant changes in experinental group. The expression of Bax protein was higher, and that of Bcl-2 protein was lower in experimetal group. Conclusion： Calphostin C could induce the apoptosis of MDA-MB-435S cells, which maybe due to the up-regulating expressions of bax and Smac, and the down-regulating expression of bcl-2.