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3.84 μmol/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。
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 Abstract: 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 CDDO-Me 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 including β-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 pLVX-USP2a (pLVX-USP2a group) plasmids. After CDDO-Me treatment, the protein levels of β-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 3.84 μmol/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 of β-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 with the pLVX group, there were more viable cells in the pLVX-USP2a group and the cells also did not undergo cycle arrest.
Conclusion·CDDO-Me can inhibit the activity of USP2a in TNBC cells, inhibit the proliferation of TNBC cells and induce apoptosis.
Key words: bardoxolone methyl (CDDO-Me), ubiquitin-specific protease 2a (USP2a), triple negative breast cancer (TNBC), ubiquitin-specific protease inhibitor screening system, β-catenin, tumor necrosis factor receptor-associated factor 6 (TRAF6) PDF全文下载地址:
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