甘草查尔酮A对人乳腺癌MDA-MB-231细胞凋亡的影响

Effect of Licochalcone A on Apoptosis in Human Breast Cancer MDA-MB-231 Cells

目的:观察甘草查尔酮A对人乳腺癌MDA-MB-231细胞凋亡的影响,并探讨其可能作用机制。方法:细胞增殖与活性检测(CCK-8)法检测不同浓度甘草查尔酮A对MDA-MB-231细胞存活率的影响;甘草查尔酮A(10,20,40μmol·L^(-1))作用MDA-MB-231细胞24 h,分别用细胞凋亡试剂盒(Annexin V-FITC/PI)检测细胞凋亡情况;荧光探针法(DCFA-DA)检测细胞内活性氧(ROS)水平,荧光探针(JC-1)法检测细胞线粒体膜电位(MMP);蛋白免疫印迹法(Western blot)检测细胞凋亡相关蛋白B细胞淋巴瘤-2(Bcl-2),B细胞淋巴瘤-2相关X蛋白(Bax)的表达及内质网应激相关蛋白(CHOP),转录激活因子4(ATF4),蛋白激酶R样内质网激酶(PERK),磷酸化蛋白激酶R样内质网激酶(p-PERK),真核翻译起始因子2α(eIF2α),磷酸化真核翻译起始因子2α(p-eIF2α)表达。结果:与空白组比较,随着甘草查尔酮A浓度增大,从甘草查尔酮A 5μmol·L^(-1)开始,细胞存活率明显降低(P<0.05),其半数抑制浓度(IC50)为19.05μmol·L^(-1);甘草查尔酮A(10,20,40μmol·L^(-1))组细胞凋亡明显升高(P<0.05),甘草查尔酮A 40μmol·L^(-1)时细胞凋亡率达30.2%(P<0.05);甘草查尔酮A(10,20,40μmol·L^(-1))使抗凋亡蛋白Bcl-2的表达明显降低(P<0.05),促凋亡蛋白Bax表达明显升高(P<0.05),且呈浓度依赖性;甘草查尔酮A(10,20,40μmol·L^(-1))明显升高细胞内ROS水平(P<0.05),降低线粒体MMP水平(P<0.05),导致线粒体功能障碍,呈浓度依赖性;甘草查尔酮A(10,20,40μmol·L^(-1))诱导内质网应激,使内质网应激相关蛋白CHOP,ATF4表达明显增多,磷酸化(p)-PERK,p-eIF2α表达明显升高(P<0.05),呈浓度依赖性。结论:甘草查尔酮A可能通过增加细胞内ROS水平,降低MMP引起线粒体功能障碍和内质网应激诱导MDA-MB-231细胞凋亡。

Objective:To investigate the effect of licochalcone A(LCA)on apoptosis in human breast cancer MDA-MB-231 cells,and to explore its possible mechanism. Method: MDA-MB-231 cells were treated with LCA of different concentrations,and cell counting kit-8(CCK-8)assay was used to detect the cell viability.The cells were treated with LCA(10,20,and 40 μmol·L^(-1))for 24 h,and apoptosis was detected by Annexin V staining with fluorescein isothiocyanate(FITC)and propidium iodide(PI)(Annexin V-FITC/PI). The level of intracellular reactive oxygen species(ROS)was detected by 2 ′,7 ′-dichlorodihydrofluorescein diacetate(DCFADA)fluorescent probe. Mitochondrial membrane potential(MMP)was detected by 5,5 ′,6,6 ′-tetrachloro-1,1 ′, 3, 3 ′-tetraethyl-imidacarbocyanine(JC-1) fluorescence probe. Western blot was used to detect the expression of cell apoptosis-related proteins,such as B-cell lymphoma-2(Bcl-2)and Bcl-2-associated X protein(Bax),and endoplasmic reticulum(ER)stress-related proteins,such as C/EBP homologous protein(CHOP),activating transcription factor 4(ATF4),protein kinase R-like ER kinase(PERK),p-PERK,eukaryotic translation initiation factor 2 alpha(eIF2α),and p-eIF2α. Result: With the increase in the drug concentration(starting from 5 μmol·L^(-1)),the cell viability decreased(P<0.05)with IC50 of 19.05 μmol·L^(-1) as compared with the normal group. Additionally,the apoptosis rates of the LCA groups(10,20,40 μmol·L^(-1))significantly increased(P<0.05),which reached 30.2%(P<0.05)at LCA concentration of 40 μmol·L^(-1). LCA(10,20,and40 μmol·L^(-1))decreased the expression of Bcl-2(P<0.05)and increased Bax expression(P<0.05)in a dosedependent manner. Besides,the intracellular ROS level was elevated(P<0.05)and mitochondrial MMP was reduced(P<0.05)after LCA(10,20,and 40 μmol·L^(-1))treatment in a dose-dependent manner,leading to mitochondrial dysfunction. LCA(10,20,and 40 μmol·L^(-1))induced ER stress to up-regulate the expression of CHOP,ATF4,p-PERK,and p-eIF2α(P<0.05)in a dose-dependent manner. Conclusion: LCA can induce MDA-MB-231 cell apoptosis by increasing intracellular ROS level and reducing MMP to trigger mitochondrial dysfunction and ER stress.