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

doi:10.3969/j.issn.1674-8115.2021.08.005

上海交通大学学报(医学版) ›› 2021, Vol. 41 ›› Issue (8): 1025-1032.doi: 10.3969/j.issn.1674-8115.2021.08.005

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¹(), 罗浩²(), 蔡海燕¹, 刘欣宇¹, 金诗佳¹, 粟深月¹, 徐含章¹, 雷虎¹, 吴英理¹()

^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

出版日期:2021-08-28 发布日期:2021-07-28
通讯作者: 吴英理 E-mail:jiyanjie@sjtu.edu.cn;wuyingli@shsmu.edu.cn
作者简介: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
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)

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

Yan-jie JI¹(), Hao LUO²(), Hai-yan CAI¹, Xin-yu LIU¹, Shi-jia JIN¹, Shen-yue SU¹, Han-zhang XU¹, Hu LEI¹, Ying-li WU¹()

^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

Online:2021-08-28 Published:2021-07-28
Contact: Ying-li WU E-mail:jiyanjie@sjtu.edu.cn;wuyingli@shsmu.edu.cn
Supported by:
National Key Project of Protein Machine and Life Process Regulation(2017YFA0505202);Shandong Provincial Natural Science Foundation(ZR2020QH095)

摘要/Abstract

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)

中图分类号:

Q591.2

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.

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. Inhibition of CDDO-ME on ubiquitin-specific protease 2a activity and cell proliferation in triple negative breast cancer cells[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(8): 1025-1032.

图/表 6

图1 CDDO-Me在体外对USP2a活性的抑制作用Note：A. The inhibitory effects of a series of natural compounds on USP2a-detected by the USP inhibitor screening system. B. The inhibitory effects of different concentrations of CDDO-Me on USP2a. C. The dose-effect curve of CDDO-Me. The unit of CDDO-Me concentration is μmol·L-1.

Fig 1 Inhibition of CDDO-Me on the activity of USP2a in vitro

图2 USP2a与CDDO-Me结合的分子对接分析Note：CDDO-Me is shown in orange sticks. USP2a is shown in grey cartoon, and residues provided for interactions with CDDO-Me in USP2a are shown in grey sticks. The dashed lines in yellow represent hydrogen bonds.

Fig 2 Predicted interaction between CDDO-Me and USP2a by molecular docking

图3 CETSA实验检测CDDO-Me与USP2a在TNBC细胞内的结合情况Note：A. MDA-MB-231 cells. B. MDA-MB-468 cells. C. MDA-MB-231-LN2 cells.

Fig 3 Interaction between CDDO-Me and USP2a in the TNBC cells detected by CETSA assay

图4 CDDO-Me对MDA-MB-468细胞中USP2a底物蛋白水平的影响Note：A. The effect of CDDO-Me on the levels of USP2a substrates in the MDA-MB-468 cells detected by Western blotting.B. The effect of CDDO-Me on the levels of USP2a substrates in the MDA-MB-468 cells transfected with pLVX or pLVX-USP2a detected by Western blotting.

Fig 4 Effects of CDDO-Me on the levels of USP2a substrate proteins in the MDA-MB-468 cells

图5 CDDO-Me对TNBC细胞增殖和凋亡的影响Note：A. The inhibitive effect of CDDO-Me on the proliferation of TNBC cells by CCK8. B?D. The apoptosis-related proteins detected by Western blotting in the MDA-MB-231 (B), MDA-MB-468 (C) and MDA-MB-231-LN2 (D) cells treated with different concentrations of CDDO-Me for 24 h.

Fig 5 Effects of CDDO-Me on the proliferation and apoptosis of TNBC cells

图6 MDA-MB-468细胞中过表达USP2a对CDDO-Me增殖抑制作用的影响Note：A. The USP2a protein levels in the MDA-MB-468 cells transfected with pLVX or pLVX-USP2a plasmid detected by Western blotting. B. The number of viable cells in the MDA-MB-468 cells transfected with pLVX or pLVX-USP2a plasmid after the treatment with different concentrations of CDDO-Me for 24 h detected by trypan blue exclusion method. ①P=0.001, ②P=0.017, ③P=0.002, compared with the pLVX group. C/D. Flow cytometric detection of cell cycles in the MDA-MB-468 cells transfected with pLVX (C) or pLVX-USP2a (D) plasmid.

Fig 6 Effects of overexpression of USP2a in the MDA-MB-468 cells on CDDO-Me proliferation inhibition

参考文献 24

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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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

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