Q39在缺氧条件下诱导人白血病K562细胞凋亡  

Q39 induces apoptosis in human leukemia cell line K562 in hypoxia

作　　者：张博[1] 翁勤洁[1] 陈中婷[1] 姜发琴[1] 盛荣[1] 胡永洲[1] 何俏军[1] 杨波[1] 

机构地区：[1]浙江大学药学院,浙江杭州310058

出　　处：《浙江大学学报（医学版）》2007年第3期261-266,共6页Journal of Zhejiang University(Medical Sciences)

基　　金：国家自然科学基金项目(20602030;C03020705);浙江省科技厅项目(2006C23002).

摘　　要：目的:探索3-(4-溴苯基)-2-(乙砜基)-6-甲基喹喔啉-1,4-二氧化物(Q39)在缺氧条件下诱导人白血病K562细胞凋亡的作用机制。方法:1MTT法测定Q39对K562细胞的体外抑制作用,计算其半数抑制浓度(IC50)。24,6-Diamidino-2-Phenylindole(DAPI)荧光染料染色观察细胞的凋亡情况。3流式细胞术测定K562细胞凋亡率。4JC-1染色法观察Q39对K562细胞线粒体膜电位(△Ψm)的影响。5Western-blotting法检测低氧条件下细胞内procaspase-3、cleaved caspase-3、PARP、Bax、Bcl-2和HIF-1α蛋白表达的变化。结果:在缺氧(3%O2)条件下,Q39对K562细胞表现出较强的体外抑制增殖作用,IC50为(0.21±0.05)μmol/L。经DAPI染色证实,Q39作用6h后开始诱导K562细胞凋亡,后期细胞体积缩小,并出现凋亡小体。流式细胞术结果显示:K562细胞与Q39共孵育0、6、12和24h的凋亡率分别为2.8%、3.2%、5.9%和19.2%。JC-1染色实验结果显示:孵育0、6、12、24和48h后Q39使K562细胞的线粒体△Ψm呈明显下降趋势,并且具有时间依赖关系。Western-blotting结果显示:Q39降低K562细胞的HIF-1α、procaspase-3和Bcl-2蛋白表达,明显增加Bax和cleaved caspase-3蛋白表达,并且促使PARP裂解。结论:Q39在缺氧条件下对K562细胞有较好的抑制作用,并能通过降低HIF-1α蛋白表达和调节其他凋亡相关蛋白表达,促使K562细胞线粒体△Ψm下降,诱导细胞凋亡。Q39诱导细胞凋亡可能是通过线粒体和HIF-1α信号转导通路实现的。Objective ： To determine whether a novel compound, 3- （4-bromophenyl）-2- （ethyl sulfonyl）-6-methylquinoxaline 1,4-dioxide （Q39）,induces apoptosis in human leukemia cell line k562 in hypoxic environment. Methods： MTT assay was used to determine the 50% inhibitory concentrations （IC50）. Flow cytometry and DAPI staining were employed to determine the apoptosis; JC-1 staining was used to determine mitochondria membrane potential （△ψm）; Western-blotting was used to determine protein expression of procaspase-3,cleaved caspase-3,PARP, Bax, Bcl-2 and HIF-1α. Results： In hypoxic environment, Q39 exerted higher antiproliferative activity in K562 cells,and the IC50 value was （0. 21±0. 05）μmol/L. The apoptotic phenomenon was observed at 6 h after cells exposed to Q39,and apoptotic body emerged as exposure time increased. After K562 cells were incubated with Q39 for 0,6,12 and 24 h,the ratio of apoptotic cells was 2. 8%, 3. 2%, 5. 9% and 19. 2%,respectively. By fluorescence stain assay,an significant △ψm loss in K562 cells induced by Q39 was shown in a time-dependent manner. Western blot assay demonstrated that Q39 decreased the protein expression of Bcl-2, procaspase -3 ,and HIF-1α,meanwhile increased protein expression of Bax and cleaved caspase-3, and induced the cleavage of PARP. Conclusions： The novel compound Q39 exhibits great anticancer activity against K562 cells in hypoxic environment. Q39 can downregulate the protein expression of HIF-1α,and regulate the apoptosis-related protein expression to cause a drop of △ψm, suggesting that mitochondria and HIF-1α pathway might be involved in the antiproliferative effect of Q39.

关 键 词：K562细胞 缺氧 细胞凋亡/药物作用 白血病/病理学 线粒体 流式细胞术 

分 类 号：R733.7[医药卫生—肿瘤]