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

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。

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

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.