藤黄酸调节急性白血病细胞HL-60核孔蛋白Nup88的意义

Effects of gambogic acid on the regulation of nucleoporin Nup88 in HL-60 cells

目的观察藤黄酸对白血病细胞株HL-60增殖和凋亡的影响及其对核孔蛋白Nup88的调控作用，探讨二者间的相互关系。方法采用二苯基溴化四氮唑蓝（MTT）比色法检测细胞增殖活性；Armexin-VFITC／PI双标法和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；逆转录聚合酶链反应（RT-PCR）检测藤黄酸对白血病细胞内Nup88基因表达的影响；共聚焦显微镜下观察Nup88蛋白的分布情况。结果藤黄酸能明显抑制HL-60细胞的增殖，其抑制作用呈时间、剂量依赖性，其12h的IC50为1．797μmol/L。藤黄酸具有较强的诱导白血病细胞凋亡的效应，0．4μmol/L藤黄酸即能诱导15．1％的HL-60细胞发生凋亡。当藤黄酸浓度达到1．6μmol/L时，总凋亡率为79．0％，且该效应并不依赖于细胞周期阻滞作用。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蛋白和mRNA的表达水平明显下降，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HL-60白血病细胞的增殖，并诱导其凋亡；藤黄酸诱导的核孔蛋白Nup88的重新分布和表达量的下调可能参与了其诱导凋亡作用。

Objective To investigate the effect of gambogic acid （GA） on cell proliferation and induction of apoptosis in HL-60 cells in vitro, as well as the regulation of nucleoporin Nup88 to explore the relationship between them. Methods The effect of GA on the growth of HL-60 cells was determined by MTT assay. Apoptosis was detected with Hoechst 33258 staining and annexin-V FITC/PI double-labeled flow cytometry. The influence on cell cycle was studied by a propidium iodide method. Both flow cytometry （FCM） and RT-PCR techniques were applied to assess the expression of Nup88, whereas the localization of Nup88 was determined by confocal laser scanning microscopy. Results GA presented striking inhibitory effect on proliferation of HL-60 cells in vitro and induction of apoptosis in a time- and dose-dependent manner. However, no obvious influence was found on the cell cycle in HL-60 cells. The ICso value for 12 h was 1.797 μmol/L. 15.1% of HL-60 cells went apoptosis when treated with 0.4 μmmol/L GA for 12 h. When the dose of GA was increased to 1.6 μmmol/L, more than half of cells were apoptotic. On the other hand, the expression level of Nup88 was down-regulated in HL-60 cells induced by GA in a dose-dependent manner. The distribution of Nup88 was also changed from widely dispersed in both nucleus and cytoplasm to that only localized at the cytoplasmic side of nuclear membrane, occasionally in the cytoplasm sporadically. Conclusion GA exhibites remarkable inhibitory effect on cell proliferation in leukemic cells and inducing apoptosis in HL-60 cells in a cell cycle-independent manner, which might correspond to the regulation of the expression as well as the distribution of nucleoporin Nup88. It may become a new remedy for treatment for acute leukemia.