木香烃内酯对人乳腺癌SK-BR-3细胞增殖、迁移和凋亡的影响及机制研究

Study on the Effects of Costunolide on the Proliferation,Migration and Apoptosis of Human Breast Cancer SK-BR-3 Cells and Its Mechanism

目的:研究木香烃内酯对乳腺癌SK-BR-3细胞增殖、迁移和凋亡的影响及机制。方法:取对数生长期的SK-BR-3细胞,分别加入不同浓度(10、20、30、40、50μmol/L)的木香烃内酯作用24、48、72 h,采用CCK-8法检测细胞的增殖抑制率。将细胞分为空白对照组和木香烃内酯10、20、30μmol/L浓度组,采用Hoechst 33258荧光染色法观察细胞形态及凋亡情况,并计算细胞凋亡率;采用细胞划痕试验检测细胞的迁移能力,并计算迁移率;采用Western blotting法检测细胞中B淋巴细胞瘤因子2(Bcl-2)、Bcl-2相关X蛋白(Bax)、半胱氨酸蛋白酶3(Caspase-3)和分裂型Caspase-3(Cleaved Caspase-3)蛋白的相对表达水平。结果:木香烃内酯对SK-BR-3细胞具有显著的增殖抑制作用(P<0.05或P<0.01),且呈现浓度和时间依赖趋势。空白对照组细胞轮廓清晰、形态规则、贴壁较好;与空白对照组比较,木香烃内酯10、20、30μmol/L浓度组细胞数量明显减少,细胞结构松散、体积缩小、间隙变大,且多数细胞轮廓消失、变圆,贴壁不良;细胞迁移率和Bcl-2蛋白的相对表达水平均显著降低,细胞凋亡率和Bax、Caspase-3及Cleaved Caspase-3蛋白的相对表达水平均显著升高(P<0.05或P<0.01)。结论:木香烃内酯能抑制人乳腺癌SK-BR-3细胞增殖和迁移,诱导其凋亡,其作用机制可能与上调Bax、Caspase-3和Cleaved Caspase-3表达,下调Bcl-2表达有关。

OBJECTIVE:To study the effects of costunolide on the proliferation,migration and apoptosis of breast cancer SK-BR-3 cells and its mechanism.METHODS:SK-BR-3 cells in logarithmic growth period were collected and cultured with different concentrations(10,20,30,40,50μmol/L)of costunolide for 24,48,72 h.Inhibitory rate of costunolide on cell proliferation was detected with CCK-8.The cells were divided into blank control group and costunolide group(10,20,30μmol/L).Hoechst 33258 fluorescence was used to observe the morphology and apoptosis of cells,and apoptotic rate of cells were calculated.Cell scratch test was used to detect the migration ability of cells and calculate the migration rate.Western blotting was used to detect the relative expression level of Bcl-2,Bax,Caspase-3 and Cleaved Caspase-3 in cells.RESULTS:The proliferation of SK-BR-3 cells were significantly inhibited by costunolide(P<0.05 or P<0.01),and it shows a trend of concentration and time dependence.In the blank control group,cells possessed clear contour,regular shape and good adherence.Compared with blank control group,the number of cells were decreased significantly in 10,20,30μmol/L costunolide groups,the cell structure was loose,the volume was reduced,and the gap became larger,and most of the cell contour disappeared and became round,the cell adherence was poor;cell migration rate and Bcl-2 protein relative expression level were decreased significantly,while apoptosis rate and the relative expression level of Bax,Caspase-3 and Cleaved Caspase-3 protein were significantly increased(P<0.05 or P<0.01).CONCLUSIONS:Costunolide can inhibit the proliferation and migration,and induce apoptosis of human breast cancer SK-BR-3 cells,mechanism of which may be through up-regulating the expression of Bax,Caspase-3 and Cleaved Caspase-3 while down-regulating the expression of Bcl-2.