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

doi:10.3969/j.issn.1674-8115.2021.08.005

Abstract

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)

CLC Number:

Q591.2

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.

Figures/Tables 6

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

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

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

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

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

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

TrendMD

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