蓝萼甲素通过PI3K/Akt信号通路诱导三阴性乳腺癌MDA-MB-231细胞凋亡

Glaucocalyxin A induces triple-negative breast cancer MDA-MB-231 cell apoptosis through PI3K/Akt signaling pathway

目的研究蓝萼甲素(GLA)诱导三阴性乳腺癌MDA-MB-231细胞凋亡及其对PI3K/Akt信号通路的影响。方法 MTT实验检测GLA对乳腺癌细胞MDA-MB-231、MDA-MB-435s,正常乳腺上皮细胞MCF-10a的增殖抑制活性;倒置显微镜观察GLA对MDA-MB-231细胞的形态的影响;Annexin V/PI双标记流式细胞术检测GLA诱导MDA-MB-231细胞凋亡;qRT-PCR检测GLA干预后,细胞中Bax、Bcl-2、Bcl-xl mRNA表达水平变化;Western blot检测PI3K、Akt、p-PI3K、p-Akt、Bax、Bcl-2、Bcl-xl蛋白表达水平变化。结果 GLA呈剂量依赖性抑制MDA-MB-231和MDA-MB-435s细胞生长,IC50分别为5.11、6.32μmol·L^-1;GLA作用MDA-MB-231细胞48 h后,贴壁细胞间隙增大,形态变圆;GLA可诱导MDA-MB-231细胞凋亡,8μmol·L^-1 GLA作用48 h细胞凋亡率达到61.65%;qRT-PCR和Western blot结果显示,GLA下调MDA-MB-231细胞中Bcl-2、Bcl-xl mRNA和蛋白质水平,上调Bax mRNA和蛋白质水平,下调p-PI3K,p-Akt蛋白水平,PI3K,Akt蛋白水平基本不变。结论 GLA在一定浓度范围内可有效抑制人乳腺癌细胞的增殖,并通过抑制PI3K/Akt信号通路诱导MDA-MB-231细胞凋亡。

Aim To investigate the effects of glaucocalyxin A(GLA)on triple-negative breast cancer MDA-MB-231 cell apoptosis and PI3K/Akt signaling pathway.Methods MTT assay was performed to determine the proliferation inhibition effects of GLA on MDA-MB-231,MDA-MB-435s and MCF-10a cells.The morphology of MDA-MB-231cells was observed by inverted microscopy.Apoptosis on MDA-MB-231 cells was observed by Annexin V/propidium iodide staining.The mRNA expression of Bax,Bcl-2 and Bcl-xl genes was assessed by qRT-PCR.Bax,Bcl-2,Bcl-xl,PI3K,Akt,p-PI3K and p-Akt protein expression levels were examined by Western blot.Results GLA inhibited the viability of MDA-MB-231 and MDA-MB-435s cells in a dose-dependent manner,and the value of IC50 was 5.11,6.32μmol·L^-1 respectively.The inverted microscope observed that the number of adherent cells treated with GLA decreased,the arrangement was loose,and the morphology became round.GLA could induce MDA-MB-231 cell apoptosis,and the cell apoptotic rate at 48 h and 8μmol·L^-1 GLA was 61.65%.According to qRT-PCR and Western blot,the mRNA expressions and protein expressions of Bcl-2 and Bcl-xl down-regulated,Bax up-regulated,and the phosphorylation levels of PI3K and Akt down-regulated after treated with GLA.Conclusions GLA could inhibit the proliferation of human breast cancer cells and induce apoptosis of triple-negative breast cancer MDA-MB-231 cells within a certain concentration range,and the mechanism may be related to the inhibition of PI3K/Akt signaling pathway.