盐霉素对乳腺癌干细胞增殖和凋亡的影响及其作用机制  

Effects of salinomycin on proliferation and apoptosis of breast cancer stem cells

作　　者：曹露 吴开祥 张亚 孙雯雯 杨会 张树鹏 CAO Lu;WU Kaixiang;ZHANG Ya;SUN Wenwen;YANG Hui;ZHANG Shupeng(Pathology Department,the Second Affiliated Hospital of Shandong First Medical University,Taian 271000,China)

机构地区：[1]山东第一医科大学第二附属医院病理科,山东泰安271000

出　　处：《山东医药》2021年第25期41-45,共5页Shandong Medical Journal

基　　金：山东省医药卫生科技发展计划项目(202002060874);山东第一医科大学学术提升计划(2019QL017)。

摘　　要：目的探讨盐霉素对乳腺癌干细胞增殖和凋亡的影响及其作用机制。方法体外培养MCF-7细胞形成微球体,取第3代微球体细胞,分别加入CD44-FITC、CD24-PE,采用流式细胞术检测乳腺癌干细胞百分比。取第3代微球体细胞,分别给予0、0.5、2、5、7、9μmol/L盐霉素干预12、24、48 h,采用MTT法检测乳腺癌干细胞盐霉素干预后细胞活性;取第3代微球体细胞,分别给予0、2.5、5、9μmol/L盐霉素干预24 h,倒置相差显微镜下观察乳腺癌干细胞形态学变化;取第3代微球体细胞,分别给予0、2、5、9μmol/L盐霉素干预24 h,分别采用FITC-Annexin V/PI双染法、PI单染法检测乳腺癌干细胞凋亡率和细胞周期;取第3代微球体细胞,分别给予0、0.5、2、5、9μmol/L盐霉素干预24 h,采用Western blotting法检测凋亡相关蛋白Caspase-3、Caspase-9、PARP、β-catenin、Survivin、Bcl-2、Bax表达。结果经流式细胞仪鉴定,在悬浮微球细胞中CD44^(+)/CD24^(-/low)表型的乳腺癌干细胞比例为(87.0±0.4)%,在MCF-7细胞中为(40.1±1.0)%,二者比较P<0.05。不同浓度盐霉素均能抑制乳腺癌干细胞存活,并且其抑制作用呈时间和浓度依赖性(P均<0.05)。倒置相差显微镜下观察,盐霉素干预后乳腺癌微球体逐渐解聚,密度降低,细胞大小不一、极度不规则,肿瘤细胞内部出现颗粒,细胞出现水肿、固缩,细胞碎片产生,并且随着盐霉素浓度升高,凋亡现象越来越明显。随着盐霉素浓度升高,乳腺癌干细胞凋亡率逐渐升高,并且在凋亡过程中可能伴G2/M期阻滞(P均<0.05)。随着盐霉素浓度升高,乳腺癌干细胞Caspase-3、Caspase-9、PARP活性片段以及Bax表达逐渐升高,而β-catenin、Survivin、Bcl-2表达逐渐降低(P均<0.05)。结论盐霉素能够抑制乳腺癌干细胞增殖并诱导其凋亡,其机制可能与抑制β-catenin-Survivin-Caspase-3通路和干扰Wnt信号通路活性有关。Objective To investigate the effects and mechanism of salinomycin on the proliferation and apoptosis of breast cancer stem cells.Methods The MCF-7 cells were cultured in vitro to form microspheres,and the third-genera⁃tion microspheres were added to CD44-FITC and CD24-PE,respectively.The percentage of breast stem cells was detected by flow cytometry.The third-generation microspheric cells were treated with salinomycin(0,0.5,2,5,7,and 9μmol/L)for 12,24 and 48 h,respectively.MTT assay was used to detect the effect of salinomycin on the proliferation of breast can⁃cer stem cells.The third-generation microspheric cells were treated with salinomycin(0,2.5,5,and 9μmol/L)for 24 h,and the inverted phase-contrast microscope was used to observe the morphological change of breast cancer stem cells.The third-generation microspheric cells were treated with salinomycin(0,2,5,and 9μmol/L)for 24 h,and FITC-Annexin V/PI apoptosis detection kit and PI staining assay were used to detect the apoptosis rate and cell cycle of the breast cancer stem cells.The third-generation microspheric cells were treated with salinomycin(0,0.5,2,5,and 9μmol/L)for 24 h,and Western blotting was used to detect the expression levels of apoptosis-related proteins Caspase-3,Caspase-9,PARP,β-catenin,Survivin,Bcl-2,and Bax.Results Flow cytometry showed that the ratio of CD44^(+)/CD24^(-/low) breast cancer stem cells in the microspheres was 87.0%±0.4%,which was 40.1%±1.0%in the MCF-7 cells,with statistically signifi⁃cant difference(P<0.05).MTT assay showed that different concentrations of salinomycin could inhibit the activity of breast cancer stem cells,with a concentration-and time-dependent manner(P<0.05).The inverted phase-contrast micro⁃scope showed that breast cancer microspheres gradually depolymerized after the intervention of salinomycin,the density de⁃creased,the cell size was different and extremely irregular,and granules appeared in the tumor cells,the cells had ede⁃ma,pyknosis,and cell debris.As the concentration of salinomyci

关 键 词：乳腺癌 盐霉素 肿瘤干细胞 细胞增殖 细胞凋亡 

分 类 号：R737.9[医药卫生—肿瘤]