Objective: To investigate the effects of bromodomain-containing protein 4 (BRD4) inhibitor GSK525762A on the proliferation and apoptosis of primary common B-cell acute lymphoblastic leukemia (common B-ALL) cells from adult patients, then to further explore the possible mechanisms.
Methods: Purified leukemia cells from 14 common B-ALL adult patients (4 Ph⁺ and 10 Ph⁻ cases) were obtained by flow cytometry sorting, and maintained in a mimic bone marrow microenvironment culture system for short-term culture. Leukemia cells were treated with various concentrations of GSK525762A. The inhibitory effects of BRD4 inhibitor on common B-ALL leukemia cells were measured by CCK-8 assay and the apoptosis of those cells was determined by AnnexinⅤ/7-AAD staining using flow cytometry. The transcripts of c-MYC, CDK6 and Bcl-2 were detected by quantitative RT-PCR, and the expression of c-MYC, CDK6 and Bcl-2 proteins were detected via Western blot.
Results: GSK525762A could inhibit the proliferation of leukemia cells from all 14 common B-ALL patients in a dose-dependent manner, the median value of IC50 was 256.25 (90.64-1 378.39)nmol/L. GSK525762A could promote cells apoptosis of B-ALL leukemia cells in a dose-dependent manner, the median apoptosis rates respectively were 45.17%(9.38%-70.91%), 66.02% (24.36%-96.34%) and 89.29% (39.29%-99.37%) after treated by 500, 1 000 and 2 500 nmol/L GSK525762A. GSK525762A has a similar effect on Ph⁺ ALL and Ph⁻ B-ALL, but the effect of proliferation inhibition and apoptosis enhancement on Ph+ B-ALL is weaker than that on Ph⁻ B-ALL. Compared with vehicle control group, the levels of c-MYC, Bcl-2 and CDK6 transcripts in leukemic cells were reduced after treatment for 24 h and 48 h by 1 000 nmol/L GSK525762A, and there are no significant differences in the downregulation of c-MYC and CDK6 mRNA between Ph⁺ and Ph⁻ B-ALL; however, the inhibitory effect on Bcl-2 transcription was weaker in Ph⁺ B-ALL cells than that in Ph⁻ B-ALL cells. Moreover, c-MYC, Bcl-2 and CDK6 protein levels decreased in GSK525762A treated group.
Conclusion: GSK525762A could strongly inhibit the proliferation of common B-ALL and trigger apoptosis; meanwhile it has certain effects against Ph⁺ ALL in vitro. The effect may be achieved by down-regulation of c-MYC, CDK6 and Bcl-2 expression.
目的: 探讨4型含Bromo结构域蛋白（BRD4）拮抗剂对普通型急性B淋巴细胞白血病（B-ALL）患者原代白血病细胞的作用及可能机制。
方法: 采用流式细胞术分选14例普通型B-ALL患者（Ph+ B-ALL 4例，Ph−B-ALL 10例）白血病细胞，在模拟骨髓微环境条件下进行短期培养；给予BRD4拮抗剂GSK525762A处理后，采用CCK-8法检测细胞增殖抑制率，用AnnexinⅤ/7-AAD法检测细胞凋亡率，荧光定量PCR法检测c-MYC、CDK6、Bcl-2 mRNA表达水平，Western blot法检测c-MYC、CDK6、Bcl-2蛋白的表达。
结果: GSK525762A对14例普通型B-ALL患者原代白血病细胞均有抑制增殖作用，且呈剂量依赖性，其中位IC50值为256.25（90.64~1 378.39）nmol/L。500、1 000、2 500 nmol/L GSK525762A作用后中位细胞凋亡率分别为45.17%（9.38%~70.91%）、66.02%（24.36%~96.34%）、89.29%（39.29%~99.37%）。GSK525762A对Ph+与Ph−B-ALL细胞具有类似的作用，但对Ph+ B-ALL细胞抑制增殖和诱导凋亡作用均弱于Ph−B-ALL细胞。1 000 nmol/L GSK525762A处理白血病细胞24、48 h后，c-MYC、CDK6和Bcl-2 mRNA表达水平下降，其中对Ph+和Ph−B-ALL细胞c-MYC、CDK6 mRNA的下调作用差异无统计学意义，而对Ph+ B-ALL细胞Bcl-2 mRNA表达水平的下调弱于对Ph−B-ALL细胞；GSK525762A处理后白血病细胞c-MYC、CDK6、Bcl-2蛋白表达水平均下调。
结论: GSK525762A可抑制普通型B-ALL原代细胞的增殖，并促进其凋亡，且对Ph+ ALL细胞在体外具有一定作用。该作用可能通过下调c-MYC、CDK6和Bcl-2而实现。