低氧诱导因子抑制剂对白血病细胞株HIF-1α和VEGF表达及诱导细胞凋亡作用的研究

Effects of Hypoxia-Inducible Factor Inhibitor on Expression of HIF-1α and VEGF and Induction of Apoptosis in Leukemic Cell Lines

本研究探讨低氧诱导因子抑制剂3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole(YC-1)对人急性白血病病细胞低氧诱导因子1α(hypoxia inducible factor1α,HIF-1α)和血管内皮因子(vascular endothelial growth factor,VEGF)的调节及诱导细胞凋亡作用。应用DAPI染色检查凋亡细胞的形态;流式细胞术检测细胞凋亡率;RT-PCR检测K562、U937、Jurkat白血病细胞株hif-1α和vegf mRNA表达以及YC-1对U937细胞hif-1α、vegf mRNA表达影响;Western blot检测YC-1对U937细胞HIF-1α和VEGF蛋白表达,以及BAX、BCL-2、caspase-3蛋白变化,分析YC-1诱导U937细胞凋亡的可能机制。结果表明:在3种白血病细胞株均可见hif-1α和vegf mRNA表达。4μmol/L YC-1处理U937细胞(0、8、16和24小时)后,可见凋亡细胞出现的典型凋亡形态特征;流式细胞术检测的细胞0、8、16和24小时凋亡率分别为(4.87±0.70)%、(27.27±2.00)%、(51.53±2.81)%及(60.5±3.20)%;vegf mRNA表达下调,而hif-1αmRNA表达无明显变化;HIF-1α蛋白、VEGF蛋白和BCL-2蛋白表达下调,而BAX和caspase-3蛋白表达上调,BAX/BCL-2比率升高并呈时间依赖性(r=0.973,p<0.01)。结论:3种白血病细胞株均显示hif-1α和vegf mRNA表达,YC-1可以抑制白血病细胞HIF-1α蛋白表达,进而下调VEGF,并通过上调BAX/BCL-2比率、caspase-3蛋白表达诱导细胞凋亡。

This study was purposed to investigate the effect of a hypoxia-inducible factorinhibitor （YC-1） on expression of hypoxia-inducible factor 1α （HIF-1α） and vascular endothelial growth factor （VEGF） as well as induction of aproptosis in leukemic cell lines. RT-PCR was used to determine the levels of HIF-1α mRNA and VEGF mRNA in K562 ,U937 and Jurkat cells. After treatment of U937 cell with 4 μmol/L YC-l, cell apoptosis was assayed by DAPI staining under fluorescent microscope and flow cytometry with Annexin V-FITC/PI staining; the expression levels of HIF-1α mRNA and VEGF mRNA were measured with RT-PCR; the expression levels of HIF-1α, VEGF, BAX, BCL-2 and caspase-3 proteins were measured by Western blot. The results showed that HIF-1α mRNA and VEGF mRNA were expressed in all three leukemia cell lines. After treatment of U937 cell with 4 μmol/L YC-1 for 0,8,16 and 24 hours, the changes of morphologic features of U937 cells could be observed under fluorescent microscope and the apoptotic rates significantly increased in time-dependent manner, they were （4.87 ± 0.70）%, （27.27 ± 2.00）%, （51.53 ± 2.81 ） and （60.5 ± 3.20） % respectively, the expression levels of VEGF mRNA reduced, while the expression levels of HIF-1α mRNA had no obviously changes. Furthermore, the expression of HIF-1α, VEGF and BCL-2 decreased, while the expression of BAX and caspase-3 increased, the ratio of BAX/BCL-2 increased in time-dependent manner （ r = 0. 973, p 〈0.01 ）. It is concluded that HIF-1α mRNA and VEGF mRNA are all expressed in in K562, U937 and Jurkat cells, YC-1 has significant effect on down-regulating the protein expression of HIF-1α and VEGF, and induces the apoptosis in U937. The mechanism of apoptosis in leukemic cells may involve in up-regulating BAX/BCL-2 ratio and expression of protein caspase-3.