神经鞘磷脂合成酶2与阿霉素致乳腺癌细胞线粒体损伤的相关性

Correlation between SMS2 and adriamycin induced mitochondrial damage in breast cancer cells

目的:探讨神经鞘磷脂合成酶2(SMS2)的表达与阿霉素(ADR)致乳腺癌细胞线粒体损伤的相关性.方法:采用过表达慢病毒感染法构建并筛选SMS2过表达的乳腺癌MCF-7细胞.CCK-8实验检测细胞对ADR的IC50值,选择适当浓度的ADR药物作用条件处理细胞后,使用MitoSOX试剂染色检测线粒体ROS水平,检测细胞内ATP水平以评估线粒体功能;电镜观察线粒体超微结构的异同;Western blot检测线粒体细胞色素C的释放水平和细胞凋亡相关蛋白Cleaved-PARP、Cleaved-Caspase 3、Bcl-2、Bax表达水平.结果:SMS2过表达的MCF-7细胞对ADR的IC50值为(2.42±0.073)μmol/L,而对照组细胞IC50值为(0.62±0.036)μmol/L(P<0.01).2μmol/L ADR处理细胞24 h后,SMS2过表达的MCF-7细胞内线粒体ROS水平降低(P<0.05);细胞内ATP水平增加(P<0.05);电镜观察示线粒体结构性损伤减轻;线粒体细胞色素C的释放水平降低(P<0.05);抗凋亡蛋白Bcl-2表达增加,促凋亡蛋白Cleaved-PARP、Cleaved-Caspase 3、Bax的表达减少(P<0.05).结论:SMS2过表达可能通过减轻ADR对乳腺癌MCF-7细胞线粒体的损伤作用,进而抑制细胞凋亡.

Objective:To explore the association between the expression of sphingomyelin synthase 2(SMS2)and the adriamycin-induced injury to the mitochondria in breast cancer cells.Methods:Breast cancer cells MCF-7 with stable SMS2 overexpressed were constructed by lentivirus infection.CCK-8 assay was used to detect the IC50 value of adriamycin in MCF-7 cells and appropriate concentration of adriamycin was then selected for MCF-7 cells treatment.After treatment,ROS level of mitochondria was measured by MitoSOX reagent staining in MCF-7 cells,intracellular ATP level was detected to evaluate mitochondrial function,and ultrastructure of mitochondria was observe by transmission electron microscope as well.The release level of mitochondria cytochrome C and the expression of apoptosis-associated protein,including Cleaved-PARP,Cleaved-Caspase 3,Bcl-2 and Bax,were analyzed using Western blotting.Results:The IC50 value of adriamycin was(2.42±0.073)μmol/L in SMS2 overexpressed MCF-7 cells,while the IC50 value was(0.62±0.036)μmol/L in control cells(P<0.01).After exposing to adriamycin(2μmol/L)for 24 h,the ROS level was significantly down-regulated in mitochondria of SMS2 overexpressed MCF-7 cells(P<0.05),intracellular ATP level was obviously up-regulated(P<0.05),and alleviated mitochondrial damage was observe in ultrastructure.The release level of mitochondria cytochrome C was decreased(P<0.05),the expression levels of pro apoptotic proteins Cleaved-PARP,Cleaved-Caspase 3 and Bax were decreased while the expression level of anti-apoptotic protein Bcl-2 was increased(P<0.05).Conclusion:Overexpression of SMS2 inhibited cell apoptosis by reducing the mitochondrial damage caused by adriamycin in MCF-7 cells.