CDDO-Me对三阴性乳腺癌细胞泛素特异性蛋白酶2a活性及细胞增殖的抑制作用  

Inhibition of CDDO-ME on ubiquitin-specific protease 2a activity and cell proliferation in triple negative breast cancer cells

作　　者：季艳杰 罗浩 蔡海燕 刘欣宇 金诗佳 粟深月 徐含章[1] 雷虎[1] 吴英理[1] JI Yan-jie;LUO Hao;CAI Hai-yan;LIU Xin-yu;JIN Shi-jia;SU Shen-yue;XU Han-zhang;LEI Hu;WU Ying-li(Department of Pathophysiology,Key Laboratory of Cell Differentiation and Apoptosis of Ministry of Education,Shanghai Jiao Tong University School of Medicine,Shanghai 200025,China;Department of Pathology,School of Basic Medicine,Weifang Medical University,Weifang 261053,China)

机构地区：[1]上海交通大学医学院病理生理学教研室,细胞分化与凋亡教育部重点实验室,上海200025 [2]潍坊医学院基础医学院临床病理系,潍坊261053

出　　处：《上海交通大学学报（医学版）》2021年第8期1025-1032,共8页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家蛋白质机器与生命过程调控重点专项(2017YFA0505200);山东省自然科学基金(ZR2020QH095)。

摘　　要：目的·在三阴性乳腺癌(triple negative breast cancer,TNBC)细胞中研究筛选得到的泛素特异性蛋白酶2a(ubiquitin-specific protease 2a,USP2a)抑制剂甲基巴多索隆(bardoxolone methyl,CDDO-Me)对USP2a活性及细胞增殖的影响。方法·利用泛素特异性蛋白酶抑制剂筛选系统筛选得到USP2a抑制剂CDDO-Me,采用分子对接分析技术预测CDDO-Me与USP2a的结合情况,采用细胞热迁移实验(cellular thermal shift assay,CETSA)检测CDDO-Me与3种TNBC细胞株内USP2a蛋白的结合情况。通过Western blotting检测USP2a的底物β连环素(β-catenin)和肿瘤坏死因子受体相关因子6(tumor necrosis factor receptor-associated factor 6,TRAF6)蛋白水平以及凋亡相关蛋白胱天蛋白酶3(caspase3)和聚腺苷二磷酸核糖聚合酶1[poly(ADP-ribose)polymerase1,PARP1]的变化。利用细胞活性检测试剂盒8(cell counting kit-8,CCK8)检测CDDO-Me对TNBC细胞增殖的影响。MDA-MB-468细胞瞬时转染pLVX(pLVX组)或pLVX-USP2a(pLVX-USP2a组)质粒,经CDDO-Me处理后,采用Western blotting检测β-catenin和TRAF6的蛋白水平,流式细胞术检测细胞周期以及台盼蓝拒染法检测活细胞数。结果·CDDO-Me在体外可以抑制USP2a活性,50%抑制浓度为3.84μmol/L。分子对接分析结果显示,CDDO-Me可以和USP2a的His456残基之间形成氢键,和Phe409、Tyr514残基之间具有疏水相互作用。CETSA实验结果显示,CDDO-Me能够和3种TNBC细胞中的USP2a蛋白结合。Western blotting结果显示,CDDO-Me可以下调USP2a底物β-catenin和TRAF6的蛋白水平,而相同浓度的CDDO-Me处理USP2a过表达的MDA-MB-468细胞,发现β-catenin和TRAF6蛋白水平未出现明显降低。CDDO-Me呈剂量依赖性抑制TNBC细胞的增殖,使细胞发生caspase3活化和PARP1的剪切并且导致细胞出现S期和G2/M期阻滞。与pLVX组相比,pLVX-USP2a组活细胞数目更多,细胞也未出现周期阻滞。结论·CDDO-Me可以抑制TNBC细胞中USP2a的活性,并且抑制TNBC细胞的增殖,诱导其凋亡。Objective·To explore the effect of bardoxolone methyl(CDDO-Me),an inhibitor of ubiquitin-specific protease 2a(USP2a)screened in vitro,on USP2a activity and cell proliferation in the triple negative breast cancer(TNBC)cells.Methods·Ubiquitin-specific protease inhibitor screening system was used to screen USP2a inhibitors and CDDO-Me was obtained.Molecular docking technology was used to analyze the interaction of CDDOMe and USP2a.Cellular thermal shift assay(CETSA)was used to detect the interaction between CDDO-Me and USP2a protein in three TNBC cell lines.Western blotting was used to detect the changes of USP2a substrates includingβ-catenin and tumor necrosis factor receptor-associated factor 6(TRAF6)protein levels and apoptosis-related proteins including caspase3 and poly(ADP-ribose)polymerase 1(PARP1).Cell counting kit-8(CCK8)was used to detect the effect of CDDO-Me on the proliferation of TNBC cells.MDA-MB-468 cells were transiently transfected with pLVX(pLVX group)or pLVXUSP2a(pLVX-USP2a group)plasmids.After CDDO-Me treatment,the protein levels ofβ-catenin and TRAF6 were detected by Western blotting,the cell cycle was detected by flow cytometry,and the number of viable cells was detected by trypan blue exclusion method.Results·CDDO-Me inhibited the activity of USP2a in vitro,and half-maximal inhibitory concentration was 3.84μmol/L.The results of molecular docking analysis showed that CDDO-Me formed a hydrogen bond with His456 residue of USP2a,and had hydrophobic interactions with Phe409 and Tyr514 residues.CETSA results showed that CDDO-Me binded to the USP2a protein in the three TNBC cells.The results of Western blotting showed that CDDO-Me down-regulated the protein levels ofβ-catenin and TRAF6,while the two USP2a substrates did not decrease in the USP2a-overexpressed MDA-MB-468 cells treated by the same concentration of CDDO-Me.CDDO-Me inhibited the proliferation of TNBC cells in a dose-dependent manner,caused caspase3 activation and PARP1 cleavage,and led to S phase and G2/M phase arrest.Compared wi

关 键 词：甲基巴多索隆 泛素特异性蛋白酶2a 三阴性乳腺癌 泛素特异性蛋白酶抑制剂筛选系统 Β连环素 肿瘤坏死因子受体相关蛋白6 

分 类 号：Q591.2[生物学—生物化学] R979.1[医药卫生—药品]