去氢木香内酯诱导乳腺癌SK-BR-3细胞凋亡的机制研究  

Effects of Dehydrocostuslactone on the Apoptosis of Human Breast Cancer SK-BR-3 Cells and Its Mechanism

作　　者：马强[1] 陈洁 熊书[1] 李国利[1] 罗娇 杨贞妮 孙厚良[1] 邓雪松[1] 苗加伟[1] MA Qiang;CHEN Jie;XIONG Shu;LI Guoli;LUO Jiao;YANG Zhenni;SUN Houliang;DENG Xuesong;MIAO Jiawei(Chongqing Engineering Research Center of Antitumor Natural Drugs,Department of Basic Medicine,Chongqing Three Gorges Medical College,Chongqing 404120,China)

机构地区：[1]重庆三峡医药高等专科学校基础医学部重庆市抗肿瘤天然药物工程技术研究中心,重庆404120

出　　处：《中药新药与临床药理》2021年第2期200-207,共8页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：重庆市教育委员会科学技术研究项目(KJ1725385,KJQN201802712,KJQN201902701,KJQN201802711);重庆市自然科学基金项目(cstc2018jcyjAX0469);重庆三峡医药高等专科学校自然科学基金重点项目(2016xzz03)。

摘　　要：目的观察去氢木香内酯(Dehydrocostuslactone,Dehy)对乳腺癌SK-BR-3细胞增殖和凋亡的影响,并探讨其分子机制。方法体外培养乳腺癌SK-BR-3细胞,分别加入不同浓度Dehy(5、10、20、30、40、50、60、80、100μmol·L-1)作用24、48、72 h,采用CCK-8法检测细胞增殖抑制率。将SK-BR-3细胞分为空白对照组(0μmol·L-1)和Dehy 10、20、30μmol·L-1浓度组,干预48 h后,利用倒置显微镜观察乳腺癌SK-BR-3细胞的形态;采用流式细胞术检测细胞周期;Annexin V-FITC/PI双染法检测细胞凋亡率;Hoechst 33258荧光染色法检测细胞凋亡的形态学变化;Western Blot法检测Bcl-2、Bax、Caspase-3和Cleaved Caspase-3蛋白的表达。结果Dehy干预SK-BR-3细胞24、48、72 h后的IC50值分别为24.84、19.39、11.45μmol·L-1,且呈现浓度和时间依赖性。与空白对照组比较,Dehy 10、20、30μmol·L-1浓度组的SK-BR-3细胞数量明显减少,细胞结构松散、轮廓消失、变圆,贴壁不良;Dehy 10、20、30μmol·L-1浓度组的G2/M期细胞比例及细胞凋亡率均显著升高(P<0.05,P<0.01),呈明显的浓度依赖性;Dehy 10、20、30μmol·L-1浓度组的SK-BR-3细胞出现不同程度的细胞核浓染、固缩及碎裂等凋亡现象,且细胞核致密浓染的比例显著升高(P<0.05,P<0.01);Dehy 10、20、30μmol·L-1浓度组的Bax、Caspase-3和Cleaved Caspase-3蛋白表达显著上调(P<0.05,P<0.01),Bcl-2蛋白表达明显下调(P<0.05,P<0.01),Bax/Bcl-2比值明显升高(P<0.05,P<0.01)。结论Dehy能够抑制乳腺癌SK-BR-3细胞的增殖及诱导其凋亡,可能与其调控Bax/Bcl-2/Caspase-3凋亡信号通路来抑制乳腺癌细胞的抗凋亡能力有关。Objective To observe the effect of dehydrocostuslactone on the proliferation and apoptosis of human breast cancer SK-BR-3 cells and explore the molecular mechanism.Methods Human breast cancer cells were cultured in vitro and treated with different concentrations(5,10,20,30,40,50,60,80,100μmol·L-1)of dehydrocostuslactone for 24,48 and 72 h.Inhibitory rate of dehydrocostuslactone on cell proliferation was detected with CCK-8.The cells were divided into blank control group(0μmol·L-1)and dehydrocostuslactone groups(10,20,30μmol·L-1).The cell morphology was observed under inverted microscope.Flow cytomwas adopted to detect proliferation cycles of cells and apoptosis was analyzed by Annexin V-FITC/propidium iodide(PI)double staining.The morphological changes of cells were observed by Hochest 33258 staining.Western Blot was used to detect the expression levels of Bax,Bcl-2,Caspase-3 and cleaved Caspase-3proteins in SK-BR-3 cells.Results IC50 values were 24.84μmol·L-1,19.39μmol·L-1 and 11.45μmol·L-1 after cells were treated with Dehy for 24 h,48 h and 72 h,which showed a concentration and time dependent manner.Compared with blank control group,the number of cells were decreased significantly in 10,20,30μmol·L-1 dehydrocostuslactone groups,the cell structure was loose,the volume was reduced,and the gap became larger;most of the cell contour disappeared and became round,the cell adherence was poor.The percentages of cells at G2/M phase and apoptosis in Dehy 10,20,30μmo)l·L-1 groups cells were significantly increased(P<0.05,P<0.01),also with a concentration-dependent manner.Hochest 33258 staining showed apoptotic morphology such as nuclear staining,pyknosis and fragmentation appeared in Dehy 10,20,30μmol·L-1 groups SK-BR-3 cells,and the proportion of nuclear dense staining was significantly increased(P<0.05,P<0.01).The protein expression levels of Bax,Caspase-3 and cleaved Caspase-3 were up-regulated significantly in Dehy 10,20,30μmol·L-1 groups(P<0.05,P<0.01),while the expression levels of Bcl-2 protein

关 键 词：去氢木香内酯 人乳腺癌SK-BR-3细胞 增殖 细胞周期 凋亡 机制 Bax/Bcl-2/Caspase-3信号通路 

分 类 号：R285.5[医药卫生—中药学]