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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 purpose of this study was to investigate the effect of EPZ005687, a histone H3K27 methylation inhibitor, on the apoptosis, proliferation inhibition and cell cycle of leukemia cell line U937 and normal bone marrow CD34 + cells. The apoptosis of U937 cells was induced by different concentrations of EPZ005687, apoptosis was detected by Annexin V / PI at different time points, cell proliferation was detected by WST-1 assay, cell cycle was detected by flow cytometry with 7-AAD, H3K27 histone methylation activity. The results showed that EPZ005687 significantly induced the apoptosis of U937 cells, and the apoptosis rates of U937 cells were 3.96% ± 0.79% and 5.74% ± 0.73% at the concentrations of 0.5, 1, 5 and 10 μmol / L for 48 h, respectively. 13.34% ± 1.77% and 25.24% ± 2.55%, respectively, while EPZ005687 had little effect on the apoptosis of CD34 + cells. The apoptotic rates of CD34 + cells at the concentrations of 0.5, 1, 5 and 10μmol / L were 3.64% ± 0.62% EPZ005687 at concentrations of 0.5, 1, 5 and 10 μmol / L, respectively, for 12 h to 96 h, respectively, for 1 to 5 days on CD34 + cells, It was clearly observed that EPZ005687 significantly inhibited the proliferation of U937 cells in a dose-dependent manner, while inhibition of proliferation of normal CD34 + cells was not significant. Cell cycle analysis showed that 1μmol / L EPZ005687 could significantly arrest U937 cells in G1 phase (64.18% ± 13.27% vs 49.43% ± 12.54%) for 72 h, and significantly decreased in S phase cells (9.67% ± 2.61% vs 15.26% ± 5.58%). However, most of the normal CD34 + cells were located in G1 phase and S phase cells were less affected by them. Further H3K27 histone methylation assay analysis showed that EZP005687 significantly reduced H3K27 histone methylation in U937 cells without reducing H3K27 histone methylation in normal CD34 + cells. Conclusion: EPZ005687, a histone H3K27 methylation inhibitor, significantly inhibits the proliferation of U937 cells and induces apoptosis and cell cycle arrest. However, it has little effect on CD34 + of normal hematopoietic cells and can be used as a potential hematological tumor drug in clinical.