ZM447439对乳腺癌细胞运动能力及凋亡的影响  

Effects of Aurora kinase inhibitor ZM447439 on mobility and apoptosis of basal-like breast cancer cells

作　　者：张月[1] 王耀一[1] 武雪亮[1] 张志生[1] 杨修明 姜洋 乔志飞[1] 梁晚平[1] 薛军[1] ZHANG Yue;WANG Yaoyi;WU Xueliang;ZHANG Zhisheng;YANG Xiuming;JIANG Yang;QIAO Zhifei;LIANG Wanping;XUE Jun(Department of Mammography Surgery,First Affiliated Hospital of Hebei North University,Zhangjiakou 075000,China)

机构地区：[1]河北北方学院附属第一医院乳腺外科,张家口075000

出　　处：《山西医科大学学报》2021年第5期552-558,共7页Journal of Shanxi Medical University

基　　金：张家口市科学研究计划自筹经费项目(1821065D)。

摘　　要：目的研究ZM447439对三阴性乳腺癌(TNBC)MDA-MB-231细胞运动能力和凋亡的影响及其作用机制。方法用不同浓度ZM447439处理MDA-MB-231细胞,MTT法检测细胞24,48 h增殖抑制率。PI染色流式细胞术检测多倍体细胞形成,免疫荧光检测细胞多核形成。AnnexinⅤ/PI双染流式细胞术观察不同浓度下ZM447439诱导MDA-MB-231细胞凋亡情况。Western blotting检测CyclinB1和Aurora激酶,Histone H3,Cofilin-1,cdc25c,cdc2及其磷酸化水平的改变,凋亡相关蛋白Bcl-2,Bax,PARP的表达变化。划痕实验及趋化实验观察细胞迁移和趋化能力。结果不同浓度Aurora激酶抑制剂ZM447439(0,0.01,0.1,1,10μmol/L)处理MDA-MB-231细胞24 h后,细胞增殖抑制率分别为(1.21±0.01)%,(3.11±0.11)%,(19.27±0.17)%,(22.72±0.24)%和(62.23±0.03)%;不同浓度ZM447439(0,0.01,0.1,1,10μmol/L)处理MDA-MB-231细胞48 h后,细胞增殖抑制率分别为(3.45±0.02)%,(15.61±0.14)%,(39.35±0.25)%,(43.46±0.13)%和(84.17±0.01)%。流式细胞术结果显示,1μmol/L ZM447439作用于乳腺癌细胞48 h可观察到多倍体细胞,并且免疫荧光显示多核形成。0.1,1,10μmol/L ZM447439作用处理MDA-MB-231细胞后,与对照组(0μmol/L)相比,细胞凋亡率均明显升高[(17.4±2.2)%,(28.5±3.1)%,(51.8±5.4)%vs(0.6±0.2)%,P<0.05]。0,0.01,0.1,1,10μmol/L ZM447439处理细胞,随着药物浓度增加,磷酸化cdc25c,cdc2的表达逐渐增加,CyclinB1、Aurora激酶及Cofilin-1的磷酸化表达逐渐减少,抗凋亡蛋白Bcl-2表达逐渐减少,促凋亡蛋白Bax、PARP蛋白剪切逐渐增加。与0μmol/L相比,0.01,0.1,1,10μmol/L ZM447439可显著延长划痕愈合时间,减少趋化穿膜细胞个数,差异均有统计学意义(P<0.05)。结论ZM447439可通过抑制Aurora激酶及Cofilin-1磷酸化水平而抑制TNBC细胞增殖、运动,从而抑制侵袭及转移,诱导凋亡。Objective To study the effects of Aurora kinase inhibitor ZM447439 on the apoptosis,motility of triple negative breast cancer cells MDA-MB-231,and explore its possible mechanism.Methods MDA-MB-231 cells were treated with different concentrations of ZM447439.MTT assay was used to detect the cell proliferation inhibition rate at 24,48 h.The formation of polyploid was tested by propidium iodide(PI)staining flow cytometry,and the formation of multinucleation was detected by immunofluorescence.AnnexinⅤ/PI double staining flow cytometry was used to observe the apoptosis of MDA-MB-231 cells induced by ZM447439 at different concentrations.The expression of apoptosis-related proteins(Bcl-2,Bax and PARP),CyclinB1 and phosphorylation levels of Aurora kinase,Histone H3,cdc25c,cdc2 and Cofilin-1 were analyzed by Western blotting.Scratch test and chemotaxis test were used to observe the effects on cell migration and chemotaxis.Results The inhibition rates of cell proliferation were(1.21±0.01)%,(3.11±0.11)%,(19.27±0.17)%,(22.72±0.24)%,(62.23±0.03)%after treated with 0,0.01,0.1,1,10μmol/L ZM447439 for 24 h,respectively.The inhibition rates of cell proliferation were(3.45±0.02)%,(15.61±0.14)%,(39.35±0.25)%,(43.46±0.13)%,(84.17±0.01)%after treated with 0,0.01,0.1,1,10μmol/L ZM447439 for 48 h,respectively.After treated with 1μmol/L ZM447439 for 48 h,the polyploid cells were detected by flow cytometry and the multinucleation was observed by immunofluorescence.The apoptosis rate of MDA-MB-231 cells was(17.4±2.2)%,(28.5±3.1)%,(51.8±5.4)%after treated with 0.1,1,10μmol/L ZM447439,which was significantly higher than that in control group[(0.6±0.2)%,P<0.05].The expression levels of p-cdc25c,p-cdc2,PRAP and Bax proteins were up-regulated,while the expression levels of p-Aurora A,p-histone H3,p-Cofilin-1,CyclinB1,Bcl-2 proteins were down-regulated.Compared with 0μmol/L ZM447439,0.01,0.1,1,10μmol/L ZM447439 significantly extended the healing time of scratches,and decreased the cell migration in a dose-dependent manner(

关 键 词：乳腺癌 AURORA激酶 Cofilin-1 细胞运动 凋亡 

分 类 号：R737.9[医药卫生—肿瘤]