藤黄酸对急性白血病细胞U937增殖和凋亡的影响及对核孔蛋白Nup88的调控作用

Effect on proliferation and apoptosis and regulation on nucleoporin Nup88 in acute leukemic cell U937 by gambogic acid

目的观察藤黄酸对白血病细胞株U937增殖和凋亡的影响及其对核孔蛋白Nup88的调控作用,研究藤黄酸诱导凋亡作用与其调节Nup88的相互关系。方法采用MTT比色法检测细胞增殖活性,Annexin-V FITC/PI双标法及Hoechst33258染色法分析细胞凋亡的改变,流式细胞术分析细胞周期和细胞内核孔蛋白Nup88的改变,RT-PCR检测藤黄酸对白血病细胞内Nupp88基因表达的调控作用;共聚焦显微镜观察Nup88蛋白的细胞分布情况。结果藤黄酸能明显抑制U937细胞的增殖,其抑制作用呈时间、剂量依赖性,其24h的IC50为(1.019±0.134)mg/L;此外,藤黄酸还具有诱导U937细胞凋亡和G0/G1期阻滞的作用。核孔蛋白Nup88弥漫分布于白血病细胞的核浆之间,以细胞浆和核膜为主,经藤黄酸干预后,Nup88的蛋白和mRNA表达水平明显下降,主要集中于核膜的胞浆面,偶见胞浆中表达。结论藤黄酸能明显抑制U937白血病细胞的增殖,并诱导其凋亡。而藤黄酸诱导的核孔蛋白Nup88的重新分布以及表达量的下调可能参与了其诱导凋亡作用。

Objective To investigate the effect on proliferation and apoptosis and the regulation on nucleoporin Nup88 in acute leukemic cell U937 by gambogic acid （GA） in vitro. To study the relationship between the effect and the regulation. Methods The effect of GA on the growth of U937 cells was studied by MTT assay. Apoptosis was detected through Annexin-V FITC/PI double-labeled cytometry and Hoechst 33258 staining. The influence on cell cycle was studied by a propidium iodide method. Both flow cytometry （FCM） and RT-PCR technologies were applied to assessing the expression of Nup88, whereas, the cell localization of Nup88 was determined by using confocal microscopy method. Results GA present ed striking proliferation inhibition, as well as apoptosis induction potency on U937 cells in vitro in a timeand dose-dependent manner. The IC50 value for 24 h was （1. 019±0. 134） mg/L. Moreover, cells treated with GA showed accumulation in G0/G1 phase and reduction in the percentage of cells in S phase. The expression of Nup88 was down-regulated in U937 cells by GA in a dose-dependent manner, and the disposition of Nup88 was also switched from widely dispersed in both nucleus and cytoplasm to situate only at the cytoplasmic side of nuclear rim, occasionally in cytoplasm sporadically. Conclusion GA exhibits the potent proliferation inhibition, apoptosis induction, and cell-cycle arrest in leukemia cells, which might correspond to the down-regulation of the expression, as well as the disposition, of nucleoporin Nup88.