积雪草酸通过Wnt/β-catenin通路逆转白血病K562/ADR细胞的多药耐药性研究  

Reversal Rolesand Its Mechanism of Asiatic Acidon Multidrug Resistancein K562/ADR Cells Through the Wnt/β-catenin Pathway

作　　者：张婷[1] 刘永蛟[1] 张蕾[1] 周欣宇 贾秀红[1] ZHANG Ting;LIU Yong-Jiao;ZHANG Lei;ZHOU Xin-Yu;JIA Xiu-Hong(Department of Pediatrics,Binzhou Medical University Hospital,Binzhou 256603,Shandong Province,China)

机构地区：[1]滨州医学院附属医院儿科,山东滨州256603

出　　处：《中国实验血液学杂志》2024年第6期1696-1703,共8页Journal of Experimental Hematology

基　　金：山东省自然科学基金(ZR2014HL032);山东省医药卫生科技发展计划项目(2017WS038)。

摘　　要：目的:探讨积雪草酸(asiatic acid,AA)对人耐阿霉素(adriamycin,ADR)慢性髓系白血病K562/ADR细胞多药耐药的逆转作用及其机制。方法:采用CCK-8法检测K562细胞和K562/ADR细胞对ADR的耐药性;CCK-8法检测AA对K562/ADR细胞活力的影响及对ADR敏感性的增强作用;无毒剂量的AA(10、20μmol/L)处理K562/ADR细胞后,流式细胞术检测细胞内ADR的平均荧光强度;实时荧光定量聚合酶链反应(RT-qPCR)检测多药耐药相关蛋白1(multidrug resistance protein-1,MRP1)、P-糖蛋白(P-glycoprotein,P-gp)、β-连环蛋白(β-catenin)、原癌基因(C-myc)、细胞周期蛋白D1(cyclinD1)的mRNA表达水平;蛋白免疫印迹法(Western blot)法检测MRP1、P-gp、β-catenin、C-myc、cyclinD1蛋白的表达水平。20μmol/L AA联合Wnt/β-catenin通路激动剂WAY-262611(5μmol/L)处理K562/ADR细胞后,Western blot法检测细胞内MRP1、P-gp、β-catenin、C-myc、cyclinD1蛋白表达水平的变化。结果:CCK-8检测结果显示,K562/ADR细胞的耐药性是K562细胞的56.57倍,具有稳定耐药性,差异具有统计学意义(P<0.05)。AA可呈浓度依赖性抑制K562/ADR细胞的增殖活力(r=0.9666)。与0μmol/L AA组相比,10、20μmol/L AA组可明显增强细胞内ADR的平均荧光强度(P<0.05),逆转细胞对ADR的耐药性(P<0.05),明显下调细胞中MRP1、P-gp、β-catenin、C-myc和cyclinD1 mRNA和蛋白的表达(P均<0.05)。与单用20μmol/L AA组相比,20μmol/L AA+WAY组细胞中MRP1、P-gp、β-catenin、C-myc、cyclinD1蛋白表达水平均明显升高(P均<0.05)。结论:AA呈浓度依赖性抑制K562/ADR细胞增殖,逆转该细胞对ADR的耐药性,其逆转机制可能与抑制Wnt/β-catenin信号通路后下调耐药相关蛋白MRP1和P-gp的表达有关。Objective:To investigate the reversal effect and mechanism of asiatic acid(AA)on multidrug resistance in human adriamycin(ADR)chronic myeloid leukemia K562/ADR cells.Methods:CCK-8 assay was used to detect the resistance of K562 cells and K562/ADR cells to ADR.CCK-8 assay was used to detect the effect of AA on K562/ADR cell viability and adriamycin sensitization.After K562/ADR cells were treated with non-toxic doses of AA(10,20μmol/L),the average fluorescence intensity of ADR was detected by flow cytometry.Real-time quantitative PCR was used to detect the expression levels of MRP1,P-gp,β-catenin,C-myc and cyclinD1 mRNA.Western blot was used to detect the expression levels of MRP1,P-gp,β-catenin,C-myc and cyclinD1 proteins.Western blot assay was used to detect the expression levels of MRP1,P-gp,β-catenin,C-myc and cyclinD1 proteins in K562/ADR cells treated with 20μmol/L AA and Wnt/β-catenin pathway agonist WAY-262611(5μmol/L).Results:The CCK-8 assay showed that the drug resistance of K562/ADR cells was 56.57 times that of K562 cells,showing stable drug resistance,and the difference was statistically significant(P<0.05).AA inhibited the proliferative activity of K562/ADR cells in a concentration-dependent manner(r=0.9666).Compared with 0μmol/L AA group,the 10 and 20μmol/L AA groups could significantly enhance the average fluorescence intensity of intracellular ADR(P<0.05),and reverse the cell resistance to ADR(P<0.05).The mRNA and protein expressions of MRP1,P-gp,β-catenin,C-myc and cyclinD1 in cells were down-regulated(P<0.05).Compared with 20μmol/L AA group,the expression levels of MRP1,P-gp,β-catenin,C-myc and cyclinD1 protein in 20μmol/L AA+WAY group were significantly increased(P<0.05).Conclusion:AA inhibits K562/ADR cell proliferation in a concentration-dependent manner and reverse their resistance to ADR,the reversal mechanism may be related to the down-regulation of MRP1 and P-gp expression after inhibiting Wnt/β-catenin signaling pathway.

关 键 词：积雪草酸 慢性髓系白血病 多药耐药 Wnt/β-catenin信号通路 

分 类 号：R733.72[医药卫生—肿瘤]