西达本胺联合(+)-JQ-1通过破坏DNA损伤反应途径杀伤MLL重排急性髓系白血病细胞  

Chidamide Combined with(+)-JQ-1 to Kill MLL-R earrangement Acute Myeloid Leukemia Cells by Disrupting the DNA Damage Response Pathway

作　　者：张青 李凤梅 王玮[1] 张志华[1] 张荣娟[1] 马铭帅 王丽红[1] ZHANG Qing;LI Feng-Mei;WANG Wei;ZHANG Zhi-Hua;ZHANG Rong-Juan;MA Ming-Shuai;WANG Li-Hong(Department of Hematology,The Affiliated Hospital of Chengde Medical University,Chengde 067000,Hebei Province,China)

机构地区：[1]承德医学院附属医院血液科,河北承德067000

出　　处：《中国实验血液学杂志》2024年第5期1323-1333,共11页Journal of Experimental Hematology

基　　金：承德市应用技术研究与开发专项科技计划(202305B081)。

摘　　要：目的:探讨西达本胺与BRD4抑制剂(+)-JQ-1联合对混合谱系白血病基因重排急性髓系白血病(MLL-r AML)细胞的DNA损伤及修复机制的影响。方法:分别将MLL-r AML细胞系Molm-13、MV4-11细胞和非MLL-r AML细胞系Kasumi细胞分成对照组(contr)、西达本胺组(chida)、(+)-JQ-1组、联合组(combi) 4组。采用CCK-8检测Molm-13细胞活力,以确定西达本胺和(+)-JQ-1的给药浓度。采用流式细胞术检测细胞周期变化,Western blot检测凋亡相关因子Bcl-2、Bax、caspase-3的表达。利用免疫荧光检测DNA损伤标志物γH2AX;Western blot检测DNA损伤因子γH2AX,DNA损伤检查点激酶p-ATR、p-CHK1、p-ATM、p-CHK2和DNA损伤修复因子Rad51、53BP1蛋白的表达情况;qRT-PCR检测DNA损伤修复因子Rad51、53BP1 mRNA的表达情况。结果:在药物西达本胺(300 nmol/L)和(+)-JQ-1(400 nmol/L)的联合作用下,MLL-r AML细胞系Molm-13、MV4-11中,与contr组相比,combi组G1期细胞比例升高;非MLL-r AML细胞系Kasumi中,与contr组相比,combi组G1期细胞比例升高(P <0.05)。在Molm-13、MV4-11细胞系中,与contr组相比,combi组DNA损伤标志物γH2AX表达水平升高(P <0.05),DNA损伤检查点和损伤修复因子p-ATR、p-CHK1、p-ATM、p-CHK2、Rad51、53BP1表达水平降低(P <0.05);而在Kasumi细胞系中,与contr组相比,combi组上述因子部分表达无明显变化(P>0.05),部分因子的表达趋势与MLL-r AML细胞系相反。在MLL-r AML细胞系Molm-13、MV4-11中,与contr组相比,combi组Bax和caspase-3蛋白表达水平升高,而Bcl-2蛋白表达水平下降(P <0.05);非MLL-r AML细胞系Kasumi细胞中,与contr组相比,combi组凋亡因子的表达无明显变化(P>0.05)。结论:西达本胺联合(+)-JQ-1可抑制MLL-r AML细胞的增殖,并通过抑制DNA损伤反应途径抑制此类白血病细胞保护性自我修复的启动,最终增加细胞的凋亡,而非MLL-r AML细胞却无类似的结果。Objective:To investigate the mechanism of DNA damage and repair in MLL rearranged acute myeloid leukemia(MLL-r AML)cells by the combination of Chidamide and the BRD4 inhibitor(+)-JQ-1.Methods:MLL-r AML cell lines Molm-13,MV4-11 and non-MLL-r AML cell line Kasumi were divided into control group(contr),Chidamide group(chida),(+)-JQ-1 group and Combination group(combi),respectively.Cell viability of Molm-13.was measured by CCK-8 to determine optimal the concentrations of Chidamide and(+)-JQ-1.The cell cycle was detected by flow cytometry,and apoptosis-related factors Bcl-2,Bax and caspase-3 were detected by Western blot.DNA damage markerγH2AX was detected by immunofluorescence.The protein expressions of DNA damage factorγH2 AX,DNA damage checkpoint kinases p-ATR,p-CHK1,p-ATM,p-CHK2 and DNA damage repair factors Rad51 and 53 BP1 were detected by Westem blot.The ex pression of DNA damage repair factors Rad51 and 53BP1 mRNA was detected by qRT-PCR.Results:Under the treatment of Chidamide(300 nmol/L)and(+)-JQ-1(400 nmol/L),the proportion of G,phase cells in MLL-AML cell lines Molm-13 and MV4-11 was increased in combination group compared with control group.In non-MLL-r AML cell line Kasumi,compared with control group,the proportion of G phase cells in combination group was increased(P<0.05).In Molm-13 and MV4-11 cell lines,compared with control group,the expression level of DNA damage markerγH2AX in combination group was increased(P<0.05).The expression levels of DNA damage checkpoint and damage repair factors p-ATR,p-CHK1,p-ATM,p-CHK2,Rad51,53BP1 were decreased(P<0.05).In Kasumi cell line,compared with control group,there was no significant change in the expression of some of the above factors in combination group(P>0.05),but the expression trend of some factors was opposite.In MLL-r AML cell lines Molm-13 and MV4-11,compared with control group,the expression levels of Bax and caspase-3 protein were increased in combination group,while the expression levels of Bcl-2 protein were decreased(P<0.05).In non-MLL-r AML ce

关 键 词：混合谱系白血病基因 BRD4 西达本胺 DNA损伤反应 急性髓系白血病 

分 类 号：R733.71[医药卫生—肿瘤]