PHⅡ-7作用于慢性粒细胞白血病细胞k562的机制研究

摘要

目的： PHⅡ-7是以靛玉红为模板合成的一种抗肿瘤药物，并有显著逆转耐药作用。实验室前期研究发现在人慢性粒细胞白血病k562细胞中，PHⅡ-7可与烯醇化酶ENO1相结合。本研究旨在探究PHⅡ-7通过ENO1对k562细胞体外抑癌作用机制。方法构建ENO1表达稳定干扰细胞株k562-shENO1和对照细胞株k562-shCON。通过MTT法检测PHⅡ-7对k562-shCON和k562-shENO1细胞株的生长抑制作用；通过细胞计数法检测k562-shCON和k562-shENO1细胞株生长差异；采用流式细胞仪测定细胞凋亡水平。采用逆转录PCR，实时荧光定量PCR和免疫印迹技术检测相关基因表达水平。结果通过real-time-PCR和免疫印迹实验验证k562-shCON和k562-shENO1干扰细胞株成功建立。k562-shENO1和k562-shCON细胞株体外生长无显著差异，PHⅡ-7对k562-shENO1和k562-shCON细胞株IC50无显著性差异。PHⅡ-7可诱导k562-shENO1和k562-shCON细胞株凋亡，并可激活caspase3、caspase9和PARP等凋亡相关蛋白。与k562-shCON相比较，k562-shENO1细胞对高浓度PHⅡ-7诱导凋亡作用更敏感。结论烯醇化酶ENO1与PHⅡ-7在人慢性粒白血病k562细胞中作用密切相关，干扰ENO1表达能在一定程度上增强PHⅡ-7的抑癌作用。%Objective PHⅡ-7 was a novel anti-cancer drug and was designed according to indirubin which was the active agent of traditional Chinese herb Danggui Longhui Wan. PHⅡ-7 was revealed to be a novel multi-functional drug which could not only in-hibit cancer progression but also overcome multi-drug resistance. Our previous results reveal that PHⅡ-7 could interact with ENO1 in chronic myeloid leukemia k562 cell and our aim is to investigate the anti-tumor mechanism of PHⅡ-7 through affecting ENO1 in chronic myeloid leukemia k562 cell. Methods In our studies, stable ENO1 interfering chronic myeloid leukemia cell k562-shENO1 cell and control cell k562-shCON cell were screened by hygromycin. We used MTT assay to research the cytotoxicity and proliferation of k562-shCON and k562-shENO1 cells. Cell growth was assayed by counting cell numbers after phenol blue dyeing. Cell cycle and apoptosis was assayed by flow cytometer. And the expressions of relative genes on RNA level were assayed by real-time PCR and ex-pressions of proteins were assayed by western blot. All experiments were repeated three times. Results Chronic myeloid leukemia k562-shENO1 cell and control cell k562-shCON cell were identified by real-time PCR and western blot, and results indicated that both interfering cells were valid. The growth of k562-shENO1 cell and k562-shCON cell had no difference, and IC50 of PHⅡ-7 in k562-shENO1 cell and k562-shCON cell also had no significant difference in vitro. PHⅡ-7 could induce apoptosis by increasing the expres-sions of cl-PARP, cl-caspase3, and cl-caspase9 in k562-shENO1cell and k562-shCON cell. k562-shENO1 cell was more sensitive to higher concentration of PHⅡ-7, compared with k562-shCON cell. Conclusion ENO1 is closely associated with the effects of PHⅡ-7 in chronic myeloid leukemia k562 cell. Interfering the expression of ENO1 can enhance the anti-tumor of PHⅡ-7 in some extent.