去氢木香内酯诱导乳腺癌SK-BR-3细胞凋亡的机制研究

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。

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

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 were down-regulated significantly(P<0.05,P<0.01).Conclusion Dehydrocostuslactone can induce apoptosis and cell cycle arrest of human breast cancer SK-BR-3 cells,and its mechanism may be through regulating Bax/Bcl-2/Caspase-3 apoptosis signal pathway to inhibit the anti apoptosis ability of breast cancer cells.