土槿皮乙酸B不通过Fas途径诱导人乳腺癌MCF-7细胞凋亡和M期周期阻滞

Pseudolaric acid B induced apoptosis and mitotic arrest circumventing Fas receptor pathway in MCF-7 cells

目的初探土槿皮乙酸B(PAB)诱导人乳腺癌MCF-7细胞凋亡和周期阻滞。方法MTT法测定PAB对MCF-7细胞的生长抑制情况;相差显微镜观察细胞形态变化;荧光显微镜观察经Hoechst33258染色的DNA变化;流式细胞仪检测用PI染色后MCF-7细胞的周期分布;免疫印迹实验检测相关蛋白的表达。结果PAB时间剂量依赖性的抑制MCF-7细胞生长。4μmol.L-1PAB在24h使DNA皱缩,在36和48h使DNA皱缩的细胞死亡。4μmol.L-1PAB时间依赖性的促进PARP〔poly-(ADP-ribose)polymerase〕的剪切。在36hPAB剂量依赖性的增加cdc2和核内cyclinB1的表达。Fas拮抗性抗体UB2不影响PAB诱导的凋亡,但Fas激动性抗体CH11促进PAB诱导的凋亡。并且UB2不影响PAB诱导的细胞周期阻滞,Fas激动性抗体CH11不影响G1和S期,但促进凋亡特征性的亚二倍体峰生成。结论4μmol.L-1的PAB通过凋亡方式可明显地抑制MCF-7细胞生长,并促进M期阻滞。Fas途径不参与PAB诱导的凋亡和周期阻滞。

Aim To study the mechanisms of Pseudolatic acid B （PAB）-induced MCF-7 cell apoptosis and mitotic arrest. Methods MTT assay was performed to assess the cell growth inhibition, contrast phase microscope was used to observe cellular morphologie alteration, and the change of DNA was detected by fluorescent microscopy. The distribution of cell cycle was determined by flow cytometric analysis of propidium iodide staining, and the protein expression was examined by Western blot analysis. Results PAB inhibited MCF-7 cell growth in a dose- and time-dependent man- ner. 4 pumol · L^-1 PAB induced DNA condensation at 24 h. PAB cleaved PARP in a time-dependent man-ner. At 36 h, PAB up-regulated the expression of cdc 2 and nuclear cyclin B1. Fas antagonistic antibody UB2 had no effect on apoptosis, but agonistie antibody CHll enhanced the apoptosis induced by PAB. UB2 exerted no effect on cell cycle arrest, and CH11 had the same action as UB2 except for reducing the mitotic arrest through enhancing apoptotic subdiploid peak. Conclusion PAB inhibited MCF-7 cell growth through mitotic arrest and apoptosis. Apoptosis and mitotic arrest were independent of Fas pathway.