Zeste基因增强子同源物2抑制剂GSK126体外对急性白血病和淋巴瘤细胞增殖及凋亡的影响

摘要

目的 探讨Zeste基因增强子同源物2(EZH2)抑制剂GSK126体外对急性白血病和淋巴瘤细胞增殖和细胞凋亡的作用.方法 采用CCKˉ8法测定不同浓度GSK126作用不同时间对人T淋巴细胞白血病CEM细胞、人急性单核细胞白血病U937细胞和人伯基特淋巴瘤Raji细胞增殖的影响,采用Annexin V／PI双染法测定GSK126对细胞凋亡的影响,采用实时荧光定量聚合酶链反应(qPCR)检测GSK126对EZH2、bclˉ2、bclˉxL基因表达的影响.结果 作用24、48、72 h后,与相应阴性对照组(0 μmol／L)比较,5、10、15、20、25 μmol／L GSK126 对 CEM 细胞,5、10、15、20 μmol／L GSK126 对U937细胞和5、10、15、20、25、30 μmol／L GSK126对Raji细胞的增殖均具有抑制作用,且呈浓度和时间依赖性(均P＜0.05). GSK126作用于CEM、U937、Raji细胞48 h的半数抑制浓度(IC50)分别为(13.46±0.83)、(11.65±1.02)、(15.00±0.19)μmol／L.与相应阴性对照组(0 μmol／L)比较,8、12、16 μmol／L GSK126均可不同程度促进CEM、U937细胞凋亡,凋亡率差异均有统计学意义(F＝167.995, P＜0.01;F＝158.400,P＜0.01);而Raji细胞,16 μmol／L GSK126较阴性对照组有更高的细胞凋亡率,差异有统计学意义(t＝47.998,P＜0.05). 8、12、16 μmol／L GSK126均较阴性对照组降低CEM、U937、Raji细胞EZH2 mRNA的表达水平(F＝82.035,P＜0.05;F＝252.712,P＜0.05;F＝690.536,P＜0.01)和凋亡相关基因 bclˉ2、bclˉxL mRNA 的表达水平(bclˉ2:F＝1 900.525,P＜0.01;F＝431.324,P＜0.01;F＝216.184,P＜0.01;bclˉxL:F＝256.751,P＜0.01;F＝147.019,P＜0.01;F＝209.325,P＜0.01).结论 EZH2对急性白血病和淋巴瘤的发生发展有重要作用,GSK126作为EZH2高选择性小分子抑制剂之一,可为血液系统恶性肿瘤临床用药提供新的可能.%Objective To investigate the effects of a novel enhancer of Zeste homolog 2 (EZH2) inhibitor GSK126 in vitro on the cell proliferation and apoptosis in acute leukemia and lymphoma cells. Methods CCKˉ8 assay was used to measure the effects of GSK126 with different concentrations and time on the cell proliferation in human Tˉcell acute lymphoblastic leukemia (TˉALL) CEM cell line, acute monocytic leukemia U937 cell line and Burkitt lymphoma Raji cell line. Annexin V/PI double staining method was used to determine the effects of GSK126 on the cell apoptosis. The effect of GSK126 on the mRNA levels of EZH2, bclˉ2 and bclˉxL were measured by using quantitative polymerase chain reaction (qPCR). Results After the function for 24 h, 48 h and 72 h, compared to the negative control group (0 μmol/L), 5, 10, 15, 20, 25 μmol/L GSK126 treatment showed a significant doseˉand timeˉdependent cell proliferation suppression in CEM cells; 5, 10, 15, 20 μmol/L GSK126 treatment showed a significant doseˉand timeˉdependent cell proliferation suppression in U937 cells; 5, 10, 15, 20, 25, 30 μmol/L GSK126 treatment showed a significant timeˉdependent suppression in Raji cells (all P< 0.05). The 48 h 50% inhibitory concentration ( IC 50) of GSK126 on CEM, U937, Raji cells was (13.46 ±0.83), (11.65 ±1.02), (15.00 ±0.19) μmol/L, respectively. GSK126 treatment with the doses of 8, 12 and 16 μmol/L promoted the apoptosis of CEM and U937 cells compared with the negative control group (0 μmol/L), and there were statistical differences in the apoptosis rate (F＝ 167.995, P< 0.01; F＝ 158.400, P< 0.01). In Raji cells, only 16 μmol/L GSK126 treatment had a higher cell apoptosis rate compared with the control group (t＝ 47.998, P< 0.05). Furthermore, 8, 12 and 16 μmol/L GSK126 treatment suppressed the expressions of EZH2 mRNA in CEM, U937 and Raji cells compared with the control group (F＝82.035, P<0.01; F＝ 252.712, P< 0.01; F＝ 690.536, P< 0.01), and the expressions of bclˉ2 and bclˉxL mRNA (bclˉ2: F＝ 1 900.525, P< 0.01; F＝ 431.324, P< 0.01; F＝216.184, P<0.01; bclˉxL: F＝256.751, P<0.01; F＝147.019, P<0.01; F＝209.325, P<0.01). Conclusion EZH2 plays an important role in the occurrence and development of acute leukemia and lymphoma. GSK126 as a high selective inhibitor of EZH2 might be a potential new drug in treatment of hematological malignancies.