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

作　　者：余静静[1] 胡萌[2] 谢仁古丽·阿力木 张曼 曲建华[1] YU Jingjing;HU Meng;Xierenguli ALIMU;ZHANG Man;QU Jianhua(Hematology Center,the First Affiliated Hospital of Xinjiang Medical University,Urumqi,Xinjiang,830000;Department of Hematology,the First Affiliated Hospital of Henan University of Science and Technology,Luoyang,Henan,471003)

机构地区：[1]新疆医科大学第一附属医院血液病中心,新疆乌鲁木齐830000 [2]河南科技大学第一附属医院血液科,河南洛阳471003

出　　处：《实用临床医药杂志》2025年第5期26-30,35,共6页Journal of Clinical Medicine in Practice

基　　金：国家自然科学基金项目(81860034);“天山创新团队计划”项目(2022D14008);“天山英才”医药卫生高层次人才培养计划领军人才项目(TSYC202301A006)。

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G_(0)/G_(1)期,其中两药联合组G_(0)/G_(1)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。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 GSK3βinhibitors induced U937 cell arrest in the G_(0)/G_(1) phase,with a significant increase in the proportion of cells in the G_(0)/G_(1) phase in the combination group(P<0.05).The combination of DNMT1 inhibitor with ERK1,HIPK2,and GSK3βinhibitors 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 i

关 键 词：抑制剂 DNA甲基转移酶1 急性髓系白血病 外胚层信号调节激酶1 同源结构域相互作用蛋白激酶2 糖原合成酶激酶3Β 靶向治疗 细胞凋亡 蛋白阻滞 

分 类 号：R733.71[医药卫生—肿瘤] R730.57[医药卫生—临床医学] R329.2