组蛋白H3K27甲基化抑制剂EPZ005687对U937细胞和正常骨髓CD34^+细胞凋亡、增殖及细胞周期的影响

Effects of H3K27 Methylation Inhibitor EPZ005687 on Apoptosis,Proliferation and Cell Cycle of U937 Cells and Normal CD34 Positive Cells

本研究目的在于探讨组蛋白H3K27甲基化抑制剂新药EPZ005687对白血病细胞系U937细胞和正常骨髓CD34+细胞的凋亡、增殖抑制和细胞周期的影响。以不同浓度的EPZ005687作用于U937细胞,在不同时间点采用Annexin V/PI法检测细胞凋亡,WST-1法检测细胞增殖,7-AAD流式细胞术检测法检测细胞周期,免疫化学法检测H3K27组蛋白甲基化活性。结果表明:EPZ005687显著诱导U937细胞的凋亡,在0.5、1、5和10μmol/L浓度下作用于U937细胞48 h后,其凋亡率分别为3.96%±0.79%、5.74%±0.73%、13.34%±1.77%和25.24%±2.55%,而EPZ005687对正常骨髓CD34+细胞的凋亡影响较小;在0.5、1、5和10μmol/L浓度下CD34+细胞凋亡率分别为3.64%±0.62%、4.28%±0.99%、6.18%±1.19%和7.56%±1.34%;0.5、1、5和10μmol/L浓度的EPZ005687分别作用于U937细胞12 h至96 h,作用CD34+细胞1至5 d,明显观察到EPZ005687显著抑制U937细胞的增殖且呈剂量依赖性,而对正常CD34+细胞的增殖抑制并不明显。细胞周期分析显示,1μmol/L EPZ005687作用72 h可使U937细胞明显阻滞于G1期(64.18%±13.27%vs 49.43%±12.54%),S期细胞比例明显下降低(9.67%±2.61%vs 15.26%±5.58%),而正常CD34+细胞因多数细胞位于G1期,S期细胞较少而不受其影响。进一步的H3K27组蛋白甲基化检测分析显示,EZP005687可明显地降低U937细胞的H3K27组蛋白甲基化,而不降低正常CD34+细胞的H3K27组蛋白甲基化。结论:组蛋白H3K27甲基化抑制剂EPZ005687明显抑制U937细胞的增殖,诱导细胞凋亡和细胞周期阻滞,但对正常造血细胞CD34+影响较小,可作为一种潜在的血液肿瘤治疗药物应用于临床。

The aim of this study was to investigate the effects of H3K27 methylation inhibitor EPZ005687 on the apoptosisproliferation and cell cycle of U937 cells and normal CD34 ＋ cells.The U937 cells and normal CD34 ＋ cells were treated with different concentration of EPZ005687 at different time points.The apoptosis rate was determined by Annexin V /PI staining.The cell proliferation and cell cycle was determined using WST-1 assay and 7-AAD assayrespectively.The activity of H3K27 methylation was detected by chemiluminescent immunoassay.The results showed that the EPZ005687 induced an obvious apoptosis of U937 cells.The apoptotic rate was 3.96% ± 0.79%5.74% ±0.73%13.34% ± 1.77% and 25.24% ± 2.55% in U937 cells treated with 0.5and 10 μ/L EPZ005687 for48 hoursrespectively.HoweverEPZ005687 had rare effect on normal bone marrow（ NBM） CD34 ＋ cells.The apoptotic rate was 3.64% ± 0.62%4.28% ± 0.99%6.18% ± 1.19% and 7.56% ± 1.34% after U937 cells were treated with 0.5and 10 μ/L EPZ005687 for 48 hoursrespectively.EPZ005687 inhibited obviously the proliferation of U937 cells but had weak effect on the proliferation of NBMCD34 ＋ cells.The inhibitory effect of EPZ005687 on U937 cells was time-dependent after treated with 0.5and 10 μ/L EPZ005687 from 12 to 96 hours.EPZ005687 induced Gphase blocking（ G1%,64.18% ± 13.27% vs 49.43% ± 12.54%）and decreased the percentage of cells in S phase（ 9.67% ± 2.61% vs26% ± 5.58%）in U937 cells.HoweverEPZ005687 had no effect on the cell cycle of NBMCD34 ＋ cells.In additionEPZ005687 produced obviously depletion of H3K27 methylation in U937 cells（ P〈0.05）,but hardly had effect on the H3K27 methylation of NBMCD34 ＋ cells.It is concluded that the EPZ005687 inhibites proliferationinduces apoptosis and cell cycle blocking in Gphase in leukemia cells.This agent may have potential value in clinical application.