Hsp90抑制剂HDN-1诱导急性早幼粒细胞白血病NB4细胞凋亡的研究

A research of apoptosis-inducing effects of APL cells NB4 by an Hsp90 inhibitor HDN-1

目的探讨南极土壤来源真菌的次级代谢产物HDN-1对人急性粒细胞白血病NB4细胞的增殖抑制,诱导凋亡作用及其机制。方法采用MTT法检测HDN-1对NB4细胞的增殖抑制作用;DNA结合染料Hoechst 33324染色,Western Blot实验,流式细胞仪检测细胞凋亡以及凋亡相关蛋白的变化。结果 HDN-1能显著抑制NB4细胞的增殖,并呈剂量-时间依赖性;HDN-1作用后,细胞核形态发生明显变化且流式细胞仪分析表明NB4细胞凋亡数量明显增加;HDN-1可引起Caspase-3,8,9激活,bid减少,线粒体中凋亡蛋白Bcl-2,Mcl-1的表达降低,以及γ-H2AX、Parp剪切带增加。但是NB4细胞中融合蛋白PML-RARα的表达不能被HDN-1所抑制。结论 HDN-1诱导凋亡是通过Caspase依赖的线粒体途径和死亡受体途径共同介导的,PML-RARα不是Hsp90的客户蛋白,其诱导凋亡机制可能是通过抑制Hsp90的其他蛋白表达进行的。

Objective To investigate the mechanisms of proliferation inhibition and inducing apoptosis of human acute promyelocytic leukimic cells NB by HDN-1, a secondary metabolite of Antarctic soil fun- gi. Methods MTT assay was applied to examine the effect of HDN-1 on the proliferation inhibition of NIM cells. Hoechst 33324 staining, Western Blot and FCM were used to detect the apoptosis of NB4 cells. Results HDN-1 could inhibit the proliferation and induce apoptosis in NB4 cells in time- and dose- dependent manners. Typical morphological changes were observed under the treatment of HDN-1. Western Blot showed cleavage of the caspase-3,8, 9 zymogen protein and an 89kDa subunit cleavage product of ADP-ribose Parp after treatment of HDN-1. Moreover, Western Blot also revealed that the expression of anti-apoptotic protein bcl-2 and Mcl-1 was up-regulated, and PML-RARα could not de- grade after treatment of HDN-1. FCM figures also showed the effect of HDN-1 on the proliferation in- hibition of NPA cells. Conclusion All these results suggested that HDN-1 induced apoptosis of NB4 cells by caspase-dependent pathway and death receptor pathway. PML-RARa wasn＇t client protein of Hsp90, so the mechanism of HDN-1-induced apoptotic could be inhibition other client protein of Hsp90.