藤黄酸对肾癌786-O细胞活力和对HUVEC细胞血管形成能力的抑制作用及其分子机制

Inhibitory effect and molecular mechanism of gambogic acid on viability of renal carcinoma cell 786-O and angiogenic ability of HUVECs

目的观察藤黄酸(GA)对肾癌786-O细胞活力和对HUVEC细胞血管形成能力的影响及其分子机制。方法不同浓度的GA处理786-O细胞一定时间后,分别采用Alamar blue法、流式细胞术和免疫印迹,检测GA对786-O细胞活力、周期分布、凋亡率及凋亡标志物聚ADP核糖聚合酶(poly ADP-ribose polymerase,PARP)降解片段的影响;GA对HUVEC细胞的作用除进行上述检测外,还利用划痕实验法和Transwell小室法检测细胞横向及纵向迁移能力变化,反映GA对HUVEC细胞血管形成能力的影响;免疫印迹检测P21Waf1/Cip1、Bax、缺氧诱导因子(HIF-1α)、血管内皮生长因子(VEGF)和泛素化蛋白;特异性荧光蛋白酶体底物肽降解法检测细胞内糜蛋白酶样蛋白酶体活性。结果 GA能够剂量依赖性抑制786-O和HUVEC细胞的活力、诱导细胞G2-M期阻滞和凋亡,同时增加P21Waf1/Cip1、Bax和泛素化蛋白水平;能够减弱HUVEC细胞横向和纵向迁移能力,伴随HIF-1α的稳定性增加和VEGF表达下调;能够抑制细胞内糜蛋白酶样蛋白酶体活性。结论 GA通过诱导细胞G2-M周期阻滞和凋亡而显著抑制786-O和HUVEC细胞活力,并能有效抑制HUVEC细胞血管形成能力,其机制可能与蛋白酶体活性抑制所导致的底物蛋白P21Waf1/Cip1、Bax和HIF-1α的稳定性改变,及HIF-1α调控的下游蛋白VEGF表达水平的变化有关。

Objective To explore the inhibitory effect and molecular mechanism of gambogic acid （GA）on the viability of renal carcinoma cell 786-0 and angiogenic ability of human umbilical vein endothelial cells （HUVECs）. Methods Alamar blue assay was used to examine the viability of 786-0 cells and HUVECs after treatment with GA. Cell cycle distribution and cell apoptosis induced by GA were detected by flow cytometry, and cleaved-PARP protein which was identified as an apoptotic marker was examined by immunoblotting method. Scratch migration assay and Transwell migration assay were utilized to determine the ability of migration of HUVECs after GA treatment, to reflect influence of GA on the angiogenic ability of HUVECs. Im- munoblotting method was used to detect the expression of P21wafl/cipl , Bax, HIF-la, VEGF and ubiquitinated-proteins. Pepti- dase activity assay was employed to examine chymotrypsin-like proteasome activity. Results GA remarkably inhibited 786-0 cells and HUVECs viability in a dose-dependent manner. G2-M phase cell cycle arrest and significant apoptosis were induced by GA in 786-0 cells and HUVECs, accompanied with upregulation of P21wafl/cipx , Bax and ubiquitinated-proteins. GA suppressed the ability of migration of HUVECs, decreased VEGF expression but increased HIF-la expression. Furthermore, chymotrypsinlike proteasome activity was remarkably inhibited by GA treatment both in 786-0 ceils and HUVECs. Conclusion Treatment with GA can inhibit 786-0 ceils and HUVECs viability via induction of cell cycle arrest and cell apoptosis, and can suppress angiogenic ability of HUVECs. This inhibitory effect of GA may be associated with proteasome substrates stability alteration, which is mediated by proteasome inhibition.