白皮杉醇对三阴性乳腺癌细胞系MDA-MB-468抗肿瘤作用及机制

Anti-tumor Effect of Piceatannol on Triple Negative Breast Cancer Cell Line MDA-MB-468 and Its Mechanism

目的:考察白皮杉醇(PIC)对三阴性乳腺癌细胞MDA-MB-468增殖、凋亡及细胞周期的作用,并对其作用机制进行探讨。方法:采用噻唑蓝(MTT)比色法考察PIC(0,2.5,5.0,10.0,20.0,40.0,80.0,160.0μmol·L-1)对三阴性乳腺癌MDA-MB-468细胞成活率的影响,并计算半抑制率(IC50);采用碘化丙啶(PI)染色观察PIC(5.0,10.0,20.0μmol·L^(-1))对MDA-MB-468细胞周期的影响;采用细胞凋亡检测(Annexin V-FITC/PI)双染法观察PIC(5.0,10.0,20.0μmol·L^(-1))对三阴性乳腺癌MDA-MB-468细胞的诱导凋亡作用;采用蛋白免疫印迹法(Western blot)观察不同浓度PIC(5.0,10.0,20.0μmol·L^(-1))对MDA-MB-468细胞增殖、凋亡蛋白的影响,并用Western blot对分泌型糖蛋白Wnt/β-连环蛋白(Wnt/β-catenin)信号通路相关蛋白进行检测。结果:MTT比色法结果显示,与空白组比较,PIC(5.0,10.0,20.0,40.0,80.0,160.0μmol·L^(-1))可呈浓度依赖性地抑制MDA-MB-468细胞的增殖(P<0.05,P<0.01),IC50为(39.4±4.6)μmol·L^(-1);作用48 h,PIC(5.0,10.0,20.0μmol·L^(-1))呈浓度依赖性地升高MDA-MB-468细胞处于细胞周期G0/G1期细胞(P<0.01);呈浓度依赖性地诱导MDA-MB-468细胞凋亡(P<0.01),其中,PIC(20.0μmol·L^(-1))诱导细胞凋亡率为49.87%;PIC(10.0,20.0μmol·L^(-1))可明显降低MDA-MB-468细胞中β-catenin及原癌基因(C-myc),黏附因子(CD44)蛋白的表达水平(P<0.05,P<0.01);PIC(5.0,10.0,20.0μmol·L^(-1))可显著抑制蛋白激酶B(Akt)磷酸化,p38丝裂原活化蛋白激酶(p38 MAPK)蛋白的磷酸化,B淋巴细胞瘤-2(Bcl-2)的蛋白表达水平(P<0.01);增强半胱氨酸天冬氨酸蛋白酶-3(Caspase-3),Bcl-2相关X蛋白(Bax)和磷酸化β-catenin的蛋白表达(P<0.01)。结论:PIC可能通过抑制Wnt/β-catenin信号通路抑制MDA-MB-468细胞增殖,并将细胞周期阻滞在G0/G1期,从而诱导其凋亡。

Objective:To investigate the effect of piceatannol(PIC)on the proliferation,apoptosis and cell cycle of MDA-MB-468 triple negative breast cancer cells and its mechanism.Method:The methylthiazolyldiphenyl-tetrazoliu bromide(MTT)colcorimetry method was used to investigate the effect of different concentrations of PIC(0,2.5,5.0,10.0,20.0,40.0,80.0,160.0μmol·L-1)on the cell viabilities of triple negative breast cancer MDA-MB-468 cells and calculate the half maximal inhibitory concentration(IC50)value,the effect of different concentrations of PIC(5.0,10.0,20.0μmol·L^(-1))on the cell cycle of MDA-MB-468 were investigated by flow cytometry with propidium iodide(PI)staining.The apoptotic effect of PIC(5.0,10.0,20.0μmol·L^(-1))on MDA-MB-468 cells in triple negative breast cancer was investigated by flow cytometry with cell apoptosis detection Annexin V-FITC and PI double staining.Western blot was used to investigate the effect of different concentrations of PIC(5.0,10.0,20.0μmol·L^(-1))on the proliferation and apoptosis of MDA-MB-468 cells and detect the expressions of secreted glycoprotein Wnt/β-catenin pathway related proteins.Result:MTT results showed that compared with the blank group,PIC could inhibit the proliferation of MDA-MB-468 cells in a concentration-dependent manner(P<0.05,P<0.01),with IC50at(39.4±4.6)μmol·L^(-1).Compared with the blank group,PIC could increase the percentage of MDA-MB-468 cells in G0/G1phase about cell cycle in a concentration-dependent manner (P<0.01).Compared with the blank group,5.0,10.0,20.0μmol·L^(-1)PIC could induce apoptosis of MDA-MB-468 cells for 48 h(P<0.01),and the apoptosis rate of MDA-MB-468 cells reached 49.87%when treated with 20.0μmol·L^(-1)for 48 h.Compared with the blank group,PIC could significantly reduce the expressions ofβ-catenin,proto-oncogene(C-myc)and adhesion factor(CD44)proteins in MDA-MB-468 cells,significantly inhibit the phosphorylation of protein kinase B(Akt)and p38 mitogen activated protein kinase(p38 MAPK)proteins and the protein expression of B lymphocyte tumor-2(Bcl-2),and enhance cysteine aspartic acid protease-3(Caspase-3),Bcl-2 related X protein(Bax)and phosphorylatedβ-catenin protein expression(P<0.01).Conclusion:PIC may inhibit the proliferation of MDA-MB-468 cells by inhibiting the Wnt/β-catenin signaling pathway,block the cell cycle in G0/G1 phase,and induce its apoptosis.