过氧麦角甾醇线粒体靶向衍生物体内外抗乳腺癌作用研究

Anti-breast cancer effect of a mitochondrion-targeted derivative of ergosterol peroxide in vitro and in vivo

研究过氧麦角甾醇线粒体靶向衍生物(Mito-EP)对乳腺癌的影响及作用机制。采用MTT法检测不同浓度Mito-EP(0、0.075、0.15、0.3、0.6、1.2、2.4μmol·L^(-1))作用于MDA-MB-231细胞后增殖抑制情况。将实验分为空白对照组,低、中、高浓度(0.15、0.3、0.6μmol·L^(-1))Mito-EP组及过氧麦角甾醇(EP)0.6μmol·L^(-1)组,培养48 h后,采用流式细胞术检测细胞凋亡率、活性氧水平、线粒体膜电位变化以及细胞周期分布,并在激光共聚焦显微镜下观察细胞凋亡、细胞活性氧及细胞线粒体膜电位变化;通过构建4T1皮下移植瘤模型体内验证Mito-EP对乳腺癌的抑制作用;采用Western blot法检测细胞及肿瘤组织内B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)、细胞色素C(Cyt C)、cleaved caspase-7、cleaved caspase-9蛋白表达水平。结果表明,Mito-EP可呈浓度依赖性地降低MDA-MB-231细胞增殖率;与空白对照组相比,EP 0.6μmol·L^(-1)组细胞凋亡率、活性氧水平、线粒体膜电位变化不明显。与空白对照组相比,Mito-EP处理组细胞凋亡率明显增加(P<0.05);活性氧水平明显升高(P<0.05);线粒体膜电位明显降低(P<0.05);Bax、Cyt C、cleaved caspase-7、cleaved caspase-9蛋白表达显著上调,Bcl-2蛋白表达显著下调(P<0.05);肿瘤体积和质量明显减小。综上所述,Mito-EP可能通过激活线粒体凋亡途径促进乳腺癌细胞凋亡。

This study aims to explore the effect and mechanism of a mitochondrion-targeted derivative of ergosterol peroxide(Mito-EP)on breast cancer.The methyl thiazolyl tetrazolium(MTT)assay was employed to examine the proliferation of MDA-MB-231 cells treated with different concentrations(0,0.075,0.15,0.3,0.6,1.2,and 2.4μmol·L^(-1))of Mito-EP.Cells were grouped for treatment with water(blank control),low,medium,and high concentrations(0.15,0.3,and 0.6μmol·L^(-1))of Mito-EP,and ergosterol peroxide(EP)(0.6μmol·L^(-1)).After the cells were treated for 48 h,flow cytometry was employed to examine the apoptosis rate,reactive oxygen species(ROS)level,mitochondrial membrane potential,and cell cycle distribution,and the apoptosis,ROS,and mitochondrial membrane potential were observed by laser confocal microscopy.A mouse model bearing subcutaneous xenograft tumor was established by injecting 4T1 cell suspension and used to study the inhibitory effect of Mito-EP on breast cancer.Western blot was employed to determine the protein levels of B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(Bax),cytochrome C(Cyt C),cleaved caspase-7,and cleaved caspase-9 in cells and the tumor tissue.The results showed that Mito-EP reduced the proliferation rate of MDA-MB-231 cells in a concentration-dependent manner.Compared with the blank control group,EP(0.6μmol·L^(-1))caused slight changes in the apoptosis rate,ROS level,and mitochondrial membrane potential.However,Mito-EP increased the apoptosis rate,elevated the ROS level,decreased mitochondrial membrane potential,up-regulated the protein levels of Bax,Cyt C,cleaved caspase-7,and cleaved caspase-9,and down-regulated the protein level of Bcl-2(all P<0.05).Moreover,Mito-EP reduced the tumor volume and weight.In summary,Mito-EP may promote apoptosis in breast cancer cells by activating the mitochondrial apoptosis pathway.