上海交通大学学报（医学版）

上海交通大学学报（医学版） >

2021 , Vol. 41 >Issue 8: 1025 - 1032

DOI: https://doi.org/10.3969/j.issn.1674-8115.2021.08.005

论著 · 基础研究

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

  • 季艳杰 ,
  • 罗浩 ,
  • 蔡海燕 ,
  • 刘欣宇 ,
  • 金诗佳 ,
  • 粟深月 ,
  • 徐含章 ,
  • 雷虎 ,
  • 吴英理
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  • 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
季艳杰（1994—），女，硕士生；电子信箱：jiyanjie@sjtu.edu.cn
季艳杰（1994—），女，硕士生；电子信箱：jiyanjie@sjtu.edu.cn
吴英理，电子信箱：wuyingli@shsmu.edu.cn。

网络出版日期: 2021-07-28

基金资助

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)

收起

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

  • Yan-jie JI ,
  • Hao LUO ,
  • Hai-yan CAI ,
  • Xin-yu LIU ,
  • Shi-jia JIN ,
  • Shen-yue SU ,
  • Han-zhang XU ,
  • Hu LEI ,
  • Ying-li WU
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  • 1.Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
    2.Department of Pathology, School of Basic Medicine, Weifang Medical University, Weifang 261053, China
WU Ying-li, E-mail: wuyingli@shsmu.edu.cn.

Online published: 2021-07-28

Supported by

National Key Project of Protein Machine and Life Process Regulation(2017YFA0505202);Shandong Provincial Natural Science Foundation(ZR2020QH095)

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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

本文引用格式

季艳杰 , 罗浩 , 蔡海燕 , 刘欣宇 , 金诗佳 , 粟深月 , 徐含章 , 雷虎 , 吴英理 . 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[J]. 上海交通大学学报（医学版）, 2021 , 41(8) : 1025 -1032 . DOI: 10.3969/j.issn.1674-8115.2021.08.005

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)

参考文献

1 Priolo C, Tang D, Brahamandan M, et al. The isopeptidase USP2a protects human prostate cancer from apoptosis[J]. Cancer Res, 2006, 66(17): 8625-8632.
2 Tao BB, He H, Shi XH, et al. Up-regulation of USP2a and FASN in gliomas correlates strongly with glioma grade[J]. J Clin Neurosci, 2013, 20(5): 717-720.
3 Kim J, Alavi Naini F, Sun Y, et al. Ubiquitin-specific peptidase 2a (USP2a) deubiquitinates and stabilizes β-catenin[J]. Am J Cancer Res, 2018, 8(9): 1823-1836.
4 Shi Y, Solomon LR, Pereda-Lopez A, et al. Ubiquitin-specific cysteine protease 2a (USP2a) regulates the stability of aurora-A[J]. J Biol Chem, 2011, 286(45): 38960-38968.
5 Wei T, Biskup E, Gjerdrum LM, et al. Ubiquitin-specific protease 2 decreases p53-dependent apoptosis in cutaneous T-cell lymphoma[J]. Oncotarget, 2016, 7(30): 48391-48400.
6 He X, Li Y, Li C, et al. USP2a negatively regulates IL-1β- and virus-induced NF-κB activation by deubiquitinating TRAF6[J]. J Mol Cell Biol, 2013, 5(1): 39-47.
7 Wang YY, Yang YX, Zhe H, et al. Bardoxolone methyl (CDDO-Me) as a therapeutic agent: an update on its pharmacokinetic and pharmacodynamic properties[J]. Drug Des Devel Ther, 2014, 8: 2075-2088.
8 Jing B, Liu M, Yang L, et al. Characterization of naturally occurring pentacyclic triterpenes as novel inhibitors of deubiquitinating protease USP7 with anticancer activity in vitro[J]. Acta Pharmacol Sin, 2018, 39(3): 492-498.
9 Wang Z, Xie W, Zhu M, et al. Development of a highly reliable assay for ubiquitin-specific protease 2 inhibitors[J]. Bioorg Med Chem Lett, 2017, 27(17): 4015-4018.
10 Morris GM, Goodsell DS, Halliday RS, et al. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function[J]. J Comput Chem, 1998, 19(14): 1639-1662.
11 Renatus M, Parrado SG, D'Arcy A, et al. Structural basis of ubiquitin recognition by the deubiquitinating protease USP2[J]. Structure, 2006, 14(8): 1293-1302.
12 Qin D, Wang W, Lei H, et al. CDDO-Me reveals USP7 as a novel target in ovarian cancer cells[J]. Oncotarget, 2016, 7(47): 77096-77109.
13 He J, Lee HJ, Saha S, et al. Inhibition of USP2 eliminates cancer stem cells and enhances TNBC responsiveness to chemotherapy[J]. Cell Death Dis, 2019, 10(4): 285.
14 Borella R, Forti L, Gibellini L, et al. Synthesis and anticancer activity of CDDO and CDDO-Me, two derivatives of natural triterpenoids[J]. Molecules, 2019, 24(22): 4097.
15 Medina MA, Oza G, Sharma A, et al. Triple-negative breast cancer: a review of conventional and advanced therapeutic strategies[J]. Int J Environ Res Public Health, 2020, 17(6): 2078.
16 Zhou L, Wang ZY, Yu SB, et al. CDDO-Me elicits anti-breast cancer activity by targeting LRP6 and FZD7 receptor complex[J]. J Pharmacol Exp Ther, 2020, 373(1): 149-159.
17 Shen H, Li L, Yang S, et al. Regulatory role of tumor necrosis factor receptor-associated factor 6 in breast cancer by activating the protein kinase B/glycogen synthase kinase 3β signaling pathway[J]. Mol Med Rep, 2017, 16(2): 2269-2273.
18 Kim EH, Deng C, Sporn MB, et al. CDDO-methyl ester delays breast cancer development in BRCA1-mutated mice[J]. Cancer Prev Res (Phila), 2012, 5(1): 89-97.
19 Yu J, Qin B, Moyer AM, et al. DNA methyltransferase expression in triple-negative breast cancer predicts sensitivity to decitabine[J]. J Clin Invest, 2018, 128(6): 2376-2388.
20 Bishop RT, Marino S, Carrasco G, et al. Combined administration of a small-molecule inhibitor of TRAF6 and docetaxel reduces breast cancer skeletal metastasis and osteolysis[J]. Cancer Lett, 2020, 488: 27-39.
21 Wang Q, Ma S, Song N, et al. Stabilization of histone demethylase PHF8 by USP7 promotes breast carcinogenesis[J]. J Clin Invest, 2016, 126(6): 2205-2220.
22 Gibellini L, Pinti M, Bartolomeo R, et al. Inhibition of Lon protease by triterpenoids alters mitochondria and is associated to cell death in human cancer cells[J]. Oncotarget, 2015, 6(28): 25466-25483.
23 Beinke C, Scherthan H, Port M, et al. Triterpenoid CDDO-Me induces ROS generation and up-regulates cellular levels of antioxidative enzymes without induction of DSBs in human peripheral blood mononuclear cells[J]. Radiat Environ Biophys, 2020, 59(3): 461-472.
24 El-Ashmawy M, Delgado O, Cardentey A, et al. CDDO-Me protects normal lung and breast epithelial cells but not cancer cells from radiation[J]. PLoS One, 2014, 9(12): e115600.
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