补骨脂乙素通过多途径诱导人乳腺癌MCF-7细胞死亡

Isobavachalcone induces cell death through multiple pathways in human breast cancer MCF-7 cells

目的探究补骨脂乙素(IBC)对人乳腺癌MCF-7细胞死亡的作用及其可能机制。方法使用不同浓度IBC处理MCF-7细胞24、48、72 h后,用MTT法检测IBC对MCF-7细胞增殖的影响;将MCF-7细胞设置10、20、40μmol/L IBC处理组,用Annexin V-FITC/PI双染结合流式细胞仪、荧光显微镜检测IBC对MCF-7细胞凋亡的影响;免疫印迹法检测凋亡、自噬相关蛋白(Bax、Bcl-2、Akt、p-Akt、p62、LC3)表达的变化;透射电镜观察40μmol/L IBC处理MCF-7后的细胞亚显微结构;利用JC-1试剂盒、ATP试剂盒和活性氧试剂盒检测IBC对细胞线粒体功能的影响。结果MTT实验结果显示,IBC具有抑制MCF-7细胞增殖的作用,并呈现浓度和时间依赖性,其作用24、48、72 h的IC50值分别为38.46、31.31、28.26μmol/L。IBC诱导MCF-7细胞发生凋亡,呈现浓度依赖性。预处理凋亡抑制剂z-VAD-fmk后,其细胞存活率显著高于单独处理IBC组(P<0.05),而程序性坏死抑制剂Nec-1没有保护作用。免疫印迹结果显示IBC增加促凋亡蛋白Bax(P<0.05)表达,下调Bcl-2(P<0.05)、Akt(P<0.05)及其Ser-473位的磷酸化水平(P<0.05)而诱导凋亡。同时,随着IBC浓度的增加,自噬相关蛋白p62的表达逐渐增加,LC3-II/I比值升高,透射电镜下也观察到IBC处理的MCF-7细胞胞浆内有自噬泡以及自噬小体。试剂盒检测到IBC导致线粒体膜电位降低、细胞内ATP水平下降、产生和积聚活性氧(ROS)(P<0.05)。结论IBC诱导人乳腺癌MCF-7细胞发生凋亡和自噬等多种死亡形式,可成为今后抗乳腺癌创新药物研发的先导化合物。

Objective To explore the effects of isobavachalcone(IBC)on cell death of human breast cancer MCF-7 cells and explore the possible mechanism.Methods MCF-7 cells were treated with different concentrations of IBC,and the changes in cell proliferation were assessed using MTT assay.Apoptosis of MCF-7 cells following treatment with 10,20,and 40μmol/L IBC was analyzed using flow cytometry with annexin V-FITC/PI double staining and fluorescence microscopy,and the expressions of apoptosis-and autophagy-related proteins(Bax,Bcl-2,Akt,p-Akt,p62,and LC3)were detected with Western blotting.Electron microscopy was used to observe the changes in submicrostructure of the cells following treatment with 40μmol/L IBC.JC-1 assay kit,ATP assay kit,and reactive oxygen species(ROS)kit were used to determine the effect of IBC on mitochondrial function of the cells.Results MTT assay showed that IBC significantly inhibited the proliferation of MCF-7 cells in a concentration-and time-dependent manner,with IC50 values of 38.46,31.31,and 28.26μmol/L at 24,48,and 72 h,respectively.IBC also concentration-dependently induced apoptosis of MCF-7 cells.IBC-induced cell death was inhibited by z-VAD-fmk,a caspase inhibitor(P<0.05),but not by the necroptosis inhibitor necrostatin-1(Nec-1).Western blotting showed that IBC-induced MCF-7 cell apoptosis by increasing Bax expression and down-regulating the expressions of Bcl-2,Akt and p-Akt-473(all P<0.05).With the increase of IBC concentration,the expression of autophagy-related protein p62 and the LC3-II/I ratio increased progressively.Electron microscopy revealed the presence of autophagic bodies in IBC-treated MCF-7 cells.IBC treatment also resulted in decreased mitochondrial membrane potential and intracellular ATP level and increased ROS accumulation in MCF-7 cells(P<0.05).Conclusion IBC is capable of inducing both apoptosis and autophagy in MCF-7 cells,suggesting the potential value of IBC as a lead compound in the development of anti-breast cancer agents.