土槿皮乙酸诱导人胃癌SGC7901细胞凋亡及机制

目的研究土槿皮乙酸(pseudolaric acid B,PAB)诱导人胃癌SGC7901细胞株凋亡及其分子机制。方法MTT法检测细胞增殖的抑制作用,电镜观察细胞的形态学变化,An-nexinV/PI双染法检测细胞早期凋亡率,罗丹明染色流式细胞仪检测线粒体膜电位,Western blot检测PAB对caspase-3、caspase-9剪切片断,bcl-2、bax蛋白表达,及对MAPKs通路相关蛋白,包括p-ERK、ERK、p-JNK、JNK、p-p38、p38表达的影响。结果PAB对人胃癌SGC7901细胞株生长具有明显的抑制作用,并呈时间和浓度依赖性。电镜结果显示0.5μmol·L-1PAB作用24 h后,核染色体聚集、边集,72 h凋亡小体形成。用0.5μmol·L-1PAB处理SGC7901细胞12、24、48、72 h,AnnexinV/PI双染检测细胞的早期凋亡,其早期凋亡率分别为10.06%、15.98%,23.12%,29.06%,而对照组的早期凋亡率仅为0.9%(P<0.05)。罗丹明染色检测线粒体膜电位,0.5μmol·L-1浓度的PAB作用于SGC7901细胞12 h后,线粒体膜电位开始下降并随时间延长而逐渐增加,12、24、48、72h线粒体膜电位下降分别为26.36%、33.26%、41.28%、52.13%(P<0.05或0.01)。0.5μmol·L-1浓度的PAB作用于SGC7901细胞24h后,Western blot法检测发现caspase-3、caspase-9 2种蛋白均出现断裂片断,并随着时间的增加断裂更明显。进一步研究发现,PAB处理SGC7901细胞后,bax蛋白表达升高,bcl-2蛋白表达下降,同时JNK、p38的磷酸化水平明显升高,ERK表达水平下降。结论PAB具有明显的细胞毒作用,能诱导SGC7901细胞凋亡,JNK和p38途径激活后通过对bax和bcl-2表达的调控,导致线粒体膜电位的下降,进而激活caspase最终导致细胞死亡,可能是其诱导胃癌细胞凋亡的分子机制之一。

Molecular mechanisms of pseudolaric acid B induced apoptosis in SGC7901 cells

Aim To study molecular mechanisms of pseudolaric acid B(PAB) induced apoptosis in SGC7901 cells.Methods Cytotoxicity was analyzed by MTT.Electron microscopy was used to observe morphological changes.Early apoptotic cells stained with AnnexinV /PI and the loss of mitochondrial membrane potential(ΔΨm) stained with Rhodamine123 were examined using flow cytometry.The protein expression of ERK、JNK、p38 、bax 、bcl-2、 caspase-3 and-9 were detected by Western blot analysis.Results PAB could induce apoptosis in human SGC7901 cells,as characterized by morphological changes,the loss of ΔΨm,activation of caspase-3 and-9.Molecular data showed that PAB decreased bcl-2 protein and increased bax protein.Moreover,PAB could activate c-Jun NH2-terminal kinase(JNK) and p38 kinase.Conclusions These findings indicated that JNK-and p38 kinase mediated mitochondrial pathways might be involved in PAB-induced apoptosis and enhanced the understanding of the anticancer function of PAB in herbal medicine.