莪术二酮对MDA-MB-231细胞周期和凋亡的影响

Effect of Curdione on MDA-MB-231 Cell Cycle and Apoptosis

目的:探讨莪术二酮对三阴性乳腺癌细胞MDA-MB-231增殖、凋亡和细胞周期的作用。方法:体外培养MDA-MB-231细胞,以卡培他滨作为阳性对照,采用细胞增殖与活性检测(CCK-8)法检测不同浓度的莪术二酮(125,250,500,1000,2000μmol·L-1)作用细胞24,48 h后对细胞活力的影响;选取3个有效抑制增殖的莪术二酮浓度(250,500,1000μmol·L^(-1))进行后续实验。流式细胞术结合碘化丙啶(PI)染色法检测莪术二酮对细胞周期的影响;增设高浓度组(2000μmol·L^(-1)),用JC-1法检测莪术二酮对细胞线粒体膜电位的影响,并采用流式细胞术结合Annexin V-FITC/PI双染色法检测细胞凋亡的情况;蛋白免疫印迹法(Western blot)检测莪术二酮作用后细胞中周期调控和凋亡相关蛋白表达的变化。结果:与空白组比较,250,500,1000,2000μmol·L^(-1)莪术二酮对细胞增殖有显著抑制作用(P<0.01),效果呈浓度和时间依赖性,24 h和48 h的半抑制浓度(IC50)分别为1607,1401μmol·L-1;250,500,1000μmol·L^(-1)莪术二酮可将细胞阻滞在G1期;250μmol·L^(-1)莪术二酮对细胞线粒体膜电位无影响,500,1000,2000μmol·L^(-1)莪术二酮可使细胞线粒体膜电位明显下降(P<0.05,P<0.01);250,500,1000μmol·L^(-1)莪术二酮可使凋亡细胞比例明显增加(P<0.05,P<0.01);各浓度莪术二酮可使B淋巴细胞瘤-2相关X蛋白(Bax)/B淋巴细胞瘤-2(Bcl-2)明显增加(P<0.05,P<0.01),半胱氨酸天冬氨酸蛋白水解酶-3(Caspase-3)蛋白表达量无明显变化,而Caspase-9,cleaved Caspase-9,cleaved Caspase-3,p53和p21蛋白表达量均有所增加(P<0.05)。结论:一定浓度的莪术二酮能抑制MDA-MB-231细胞的增殖,可能与其阻滞细胞周期、诱导细胞凋亡有关。

Objective:To investigate the effects of curdione on the proliferation,apoptosis and cell cycle of triple negative breast cancer cell line MDA-MB-231.Method:MDA-MB-231 cells were cultured in vitro with capecitabine(positive control)and curdione at different concentrations(125,250,500,1 000,and2 000μmol·L^(-1)),respectively,for detecting their viability using the cell counting kit-8(CCK-8)at 24 and 48 h.Three effective inhibitory concentrations(250,500,and 1 000μmol·L-1)against cell proliferation were selected for subsequent experiments.The effect of curdione on cell cycle was determined by flow cytometry combined with propidium iodide(PI)staining.After the set-up of high-concentration(2 000μmol·L^(-1))group,the effect of curdione on cell mitochondrial membrane potential was measured by JC-1(5,5,6,6-tetrachloro-1,1,3,3-tetraethy lbenzimidazolylcarbocyanine iodide)staining,followed by the detection of cell apoptosis by flowcytometry combined with Annexin V-FITC/PI double staining.The changes in cell cycle status and apoptosisrelated protein expression following curdione intervention were assayed by Western blot.Result:Compared with the blank control,curdione at 250,500,1 000,and 2 000μmol·L^(-1) significantly inhibited the proliferation of MDA-MB-231 cells(P<0.01),exhibiting a concentration-and time-response relationship.The half maximal inhibitory concentration(IC50)values at 24 and 48 h were 1 607 and 1 401μmol·L^(-1),respectively.Curdione at250,500,and 1 000μmol·L^(-1) arrested cells in G1phase.Curdione at 250μmol·L^(-1) had no effect on cell mitochondrial membrane potential,which,however,declined significantly in the 500,1 000,and 2 000μmol·L-1groups(P<0.05,P<0.01).Curdione at 250,500,and 1 000μmol·L-1obviously increased the proportion of apoptotic cells(P<0.05,P<0.01).Curdione at each concentration elevated the Bcl-2-associated Xprotein(Bax)/B-cell lymphoma 2(Bcl-2)ratio(P<0.05,P<0.01),but did not change the cysteinyl aspartatespecific protease-3(Caspase-3)expression.The protein expression levels of Caspase-9,cleaved Caspase-9,cleaved Caspase-3,p53,and p21 were up-regulated(P<0.05).Conclusion:A certain concentration of curdione inhibits the proliferation of MDA-MB-231 cells,which may be related to its efficacy in arresting cell cycle and inducing apoptosis.