高级检索

DNA甲基转移酶1抑制剂联合外胚层信号调节激酶1、同源结构域相互作用蛋白激酶2及糖原合成酶激酶3β治疗复发难治性急性髓系白血病的细胞实验研究

Experimental study on treatment of relapsed and refractory acute myeloid leukemia with DNA methyltransferase 1 inhibitor combined with extracellular signal-regulated kinase 1, homeodomain-interacting protein kinase 2, and glycogen synthase kinase 3β inhibitors

    摘要
  • 摘要:
    目的 探讨DNA甲基转移酶1(DNMT1)抑制剂联合外胚层信号调节激酶1(ERK1)、同源结构域相互作用蛋白激酶2(HIPK2)及糖原合成酶激酶3β(GSK3β)抑制剂协同诱导复发难治性急性髓系白血病(AML)细胞凋亡和蛋白阻滞的机制。
    方法 基于表达综合数据库(GEO)、癌症基因组图谱(TCGA)数据库以及Expression2Kinases等数据库筛选出ERK1、HIPK2和GSK3β等3个治疗靶点。DNMT1抑制剂单独或联合ERK1、HIPK2或GSK3β抑制剂处理人AML细胞系U937细胞; 采用CCK-8法检测细胞活力; 采用流式细胞术分析细胞凋亡率，采用碘化丙啶单染色法染色法(PI)测定细胞周期分布; 采用实时荧光定量逆转录聚合酶链反应(RT-qPCR)检测DNMT1、ERK1、HIPK2和GSK3β的mRNA表达水平; 采用免疫印迹法检测DNMT1、ERK1、HIPK2和GSK3β的蛋白表达水平。
    结果 DNMT1抑制剂能显著抑制U937细胞的细胞活力(P < 0.05)，显著诱导U937细胞凋亡和周期阻滞(P < 0.05); DNMT1抑制剂与ERK1、HIPK2或GSK3β抑制剂联合使用时，细胞活力和细胞凋亡率显著降低(P < 0.05)。DNMT1抑制剂及其与ERK1、HIPK2、GSK3β抑制剂联合诱导U937细胞停留在G0/G1期，其中两药联合组G0/G1期比例显著增高(P < 0.05)。DNMT1抑制剂与ERK1、HIPK2、GSK3β抑制剂联合能显著降低U937细胞DNMT1、ERK1、HIPK2和GSK3β的mRNA表达水平(P < 0.05); DNMT1抑制剂与ERK1、HIPK2、GSK3β抑制剂联合能显著降低U937细胞DNMT1、ERK1、HIPK2和GSK3β的蛋白表达水平(P < 0.05)。
    结论 DNMT1抑制剂联合ERK1、HIPK2和GSK3β抑制剂可协同诱导复发难治性AML细胞凋亡和蛋白阻滞，为联合靶向治疗AML提供一种新的策略。

     

    Abstract:
    Objective To investigate the mechanism of DNA methyltransferase 1 (DNMT1) inhibitor combined with extracellular signal-regulated kinase 1 (ERK1), homeodomain-interacting protein kinase 2 (HIPK2), and glycogen synthase kinase 3β (GSK3β) inhibitors in synergistically inducing apoptosis and protein arrest in relapsed and refractory acute myeloid leukemia (AML) cells.
    Methods Three therapeutic targets, including ERK1, HIPK2, and GSK3β, were screened based on the Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA) database, and Expression 2 Kinases database. Human AML cell line U937 cells were treated with DNMT1 inhibitor alone or combined with ERK1, HIPK2, or GSK3β inhibitors. Cell viability was detected using the CCK-8 method. Apoptosis rate was analyzed by flow cytometry, and cell cycle distribution was determined by propidium iodide (PI)staining. The mRNA expression levels of DNMT1, ERK1, HIPK2, and GSK3β were detected by real-time fluorescent quantitative reverse transcriptionpolymerase chain reaction (RT-qPCR). Protein expressionlevels of DNMT1, ERK1, HIPK2, and GSK3β were detected by immunoblotting.
    Results DNMT1 inhibitor significantly inhibited the cell viability of U937 cells, and significantly induced apoptosis and cell cycle arrest in U937 cells (P < 0.05). When DNMT1 inhibitor was combined with ERK1, HIPK2, or GSK3β inhibitors, cell viability and apoptosis rate were significantly reduced (P < 0.05). DNMT1 inhibitor alone or its combination with ERK1, HIPK2, and GSK3β inhibitors induced U937 cell arrest in the G0/G1 phase, with a significant increase in the proportion of cells in the G0/G1 phase in the combination group (P < 0.05). The combination of DNMT1 inhibitor with ERK1, HIPK2, and GSK3β inhibitors significantly reduced the mRNA expression levels in DNMT1, ERK1, HIPK2, and GSK3β in U937 cells (P < 0.05). Similarly, the combination therapy significantly reduced the protein expression levels of DNMT1, ERK1, HIPK2, and GSK3β in U937 cells (P < 0.05).
    Conclusion DNMT1 inhibitor combined with ERK1, HIPK2, and GSK3β inhibitors can synergistically induce apoptosis and protein arrest in relapsed and refractory AML cells, providing a novel strategy for combined targeted therapy of AML.

     

    • HTML全文
    • 参考文献(21)
    • 相关文章
    • 施引文献
  • 资源附件(0)

/

返回文章
返回