Q39在缺氧条件下诱导人白血病K562细胞凋亡

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。

Q39 induces apoptosis in human leukemia cell line K562 in hypoxia

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 (DeltaPsim); Western-blotting was used to determine protein expression of procaspase-3, cleaved caspase-3, PARP, Bax, Bcl-2 and HIF-1alpha. RESULTS: In hypoxic environment, Q39 exerted higher antiproliferative activity in K562 cells, and the IC50 value was (0.21+/- 0.05) micromol/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 Delta Psim 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-1alpha, 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-1alpha, and regulate the apoptosis-related protein expression to cause a drop of DeltaPsim, suggesting that mitochondria and HIF-1alpha pathway might be involved in the antiproliferative effect of Q39.