孟鲁司特钠靶向USP2诱导骨髓瘤细胞凋亡的实验研究

doi:10.19401/j.cnki.1007-3639.2025.09.005

摘要/Abstract

摘要：

背景与目的： 多发性骨髓瘤（multiple myeloma，MM）细胞内泛素特异性肽酶2（ubiquitin-specific peptidase 2，USP2）通过去泛素化作用调控细胞内蛋白降解及稳态平衡，参与MM细胞增殖和存活，靶向抑制MM细胞内USP2活性可以调控其生物学行为。本研究旨在探究白三烯受体拮抗剂孟鲁司特钠对MM细胞内USP2的调控作用及其机制。方法： 应用纯化USP2蛋白及其底物蛋白谷胱甘肽S-转移酶（glutathione S-transferase，GST）标签的泛素核糖体融合蛋白52（ubiquitin A-52 residue ribosomal protein fusion product，UbA52）即GST-UbA52蛋白构建体外去泛素化反应体系，观察孟鲁司特钠对USP2去泛素化酶活性的影响。以MM细胞系MM1.S和H929细胞为模型，采用细胞热迁移分析检测孟鲁司特钠与细胞内USP2的相互结合效力。以不同浓度的孟鲁司特钠处理MM1.S和H929细胞，应用蛋白质印迹法（Western blot）检测细胞内USP2蛋白及其靶向调控分子[包括细胞周期调控分子细胞周期蛋白D1（cyclin D1，CCND1）和细胞周期蛋白A1（cyclin A1，CCNA1），经典信号转导通路分子KRAS和葡萄糖调节蛋白78（glucose regulated protein 78kD，GRP78），以及凋亡通路相关分子C/EBP同源蛋白（C/EBP-homologous protein，CHOP）]水平的改变。利用慢病毒载体构建过表达USP2的H929-OE和MM1.S-OE细胞以及低表达USP2的H929-LE和MM1.S-LE细胞，采用细胞计数试剂盒-8（cell counting kit-8，CCK-8）和流式细胞术检测孟鲁司特钠处理后H929、H929-OE、H929-LE、MM1.S、MM1.S-OE和MM1.S-LE细胞增殖抑制率和凋亡率。结果： 孟鲁司特钠可以抑制USP2介导的GST-UbA52蛋白降解，并呈浓度依赖性[半数抑制浓度（half inhibitory concentration，IC₅₀）为3.814 μmol/L]。同时孟鲁司特钠能够增加USP2蛋白加热环境（49.1、53.2和56.4 ℃）的稳定性。经孟鲁司特钠处理后，MM1.S和H929细胞内CCND1、CCNA1和KRAS蛋白表达明显降低，而GRP78和CHOP蛋白表达显著升高。进一步研究发现40 μmol/L孟鲁司特钠处理24 h后，H929-OE细胞增殖抑制率和凋亡率分别为（37.68±1.10）%和（18.99±0.26）%，MM1.S-OE细胞增殖抑制率和凋亡率分别为（24.48±0.49）%和（33.29±0.75）%，显著低于H929和MM1.S细胞[H929：（57.19±1.93）%和（45.65±0.24）%；MM1.S：（50.04±0.53）%和（40.25±0.91）%；P均<0.05，n=3]；而低表达USP2的H929-LE和MM1.S-LE细胞增殖抑制率和凋亡率明显升高[H929-LE-1#：（80.70±1.60）%和（89.08±0.49）%；H929-LE-2#：（75.30±3.80）%和（82.41±1.07）%；MM1.S-LE-1#：（70.64±0.84）%和（67.63±0.21）%；MM1.S-LE-2#：（68.47±1.32）%和（85.90±0.18）%；P均<0.05，n=3]。结论： 孟鲁司特钠能够与MM细胞内泛素-蛋白酶体调控分子USP2结合并抑制其去泛素化酶活性，进而调控USP2靶蛋白表达并激活内质网应激，诱导MM细胞增殖阻滞和凋亡。

关键词: 多发性骨髓瘤, 孟鲁司特钠, 泛素特异性肽酶2, 去泛素化, 细胞凋亡

Abstract:

Background and purpose: Intracellular deubiquitylating enzymes, such as ubiquitin-specific peptidase 2 (USP2), play a pivotal role in regulating protein degradation and cellular homeostasis by modulating protein ubiquitin deconjugation, which have been implicated in the proliferation and survival of multiple myeloma (MM) cells. Targeting the inhibition of USP2 activity in MM cells might modulate their biological behavior. This study aimed to investigate regulatory effects of the leukotriene receptor antagonist montelukast sodium on USP2 in MM cells and its subsequent biological effects. Methods: An in vitro deubiquitination reaction system was established using purified USP2 protein and its substrate, the glutathione S-transferase (GST) tagged ubiquitin A-52 residue ribosomal protein fusion product (UbA52), known as GST-UbA52 protein. This system was used to characterize inhibitory effects of montelukast sodium on USP2 deubiquitinase activity. The MM cell lines MM1.S and H929 were used as in vitro models. Cellular thermal shift assay (CETSA) was subsequently employed to test interaction mode between montelukast sodium and USP2 in MM cells. Western blot assay was applied to detect expression levels of USP2 and its targeting regulators, including cell cycle supervisors cyclin D1 (CCND1) and cyclin A1 (CCNA1), classical signaling transducer KRAS and glucose regulated protein 78kD (GRP78), as well as apoptotic molecule C/EBP-homologous protein (CHOP) in MM1.S and H929 cells before and after the treatment with different concentrations of montelukast sodium. MM cells with either overexpression (H929-OE, MM1.S-OE) or knockdown (H929-LE, MM1.S-LE) of USP2 were generated using a lentiviral vector. Cell counting kit-8 (CCK-8) and flow cytometry were utilized to detect the proliferation and apoptotic rates of H929-OE, MM1.S-OE, H929-LE and MM1.S-LE cells treated with montelukast sodium. Results: Montelukast sodium was found to inhibit USP2 mediated degradation of GST-UbA52 protein in a concentration-dependent manner, with a half inhibitory concentration (IC₅₀) of 3.814 μmol/ L. Additionally, montelukast sodium significantly enhanced the thermal stability of USP2 at temperatures of 49.1, 53.2 and 56.4 ℃. It was also shown that montelukast sodium could down-regulate expressions of CCND1, CCNA1 and KRAS, while increase levels of GRP78 and CHOP in MM1.S and H929 cells. Furthermore, after treating with 40 μmol/L montelukast sodium for 24 h, the proliferation inhibition and apoptotic rate of H929-OE cells reached to (37.68±1.10)% and (18.99±0.26)%, while the proliferation inhibition and apoptotic rate of MM1.S-OE cells reached to (24.48±0.49)% and (33.29±0.75)%, which were significantly lower than those in H929 and MM1.S cells [H929: (57.19±1.93)% and (45.65±0.24)%; MM1.S: (50.04±0.53)% and (40.25±0.91)%; P<0.05, n=3]. Conversely, the proliferation inhibition and apoptotic rates of H929-LE and MM1.S-LE cells were significantly higher [H929-LE-1#: (80.70±1.60)% and (89.08±0.49)%; H929-LE-2#: (75.30±3.80)% and (82.41±1.07)%; MM1.S-LE-1#: (70.64±0.84)% and (67.63±0.21)%; MM1.S-LE-2#: (68.47±1.32)% and (85.90±0.18)%; P<0.05, n=3]. Conclusion: Montelukast sodium can target ubiquitin proteasome regulator USP2 and inhibit its deubiquitylating activity, which may modulate USP2 directing protein and trigger endoplasmic reticulum stress to induce cell cycle arrest and apoptosis in MM cells.

Key words: Multiple myeloma, Montelukast sodium, Ubiquitin-specific peptidase 2, Deubiquitylation, Cell apoptosis

中图分类号:

R738.1

杜承蓉, 王莹莹, 唐勇, 姚一芸, 吴英理, 朱琦. 孟鲁司特钠靶向USP2诱导骨髓瘤细胞凋亡的实验研究[J]. 中国癌症杂志, 2025, 35(9): 850-858.

DU Chengrong, WANG Yingying, TANG Yong, YAO Yiyun, WU Yingli, ZHU Qi. Experimental study on montelukast sodium inducing apoptosis in multiple myeloma cells via targeting intracellular USP2 protein[J]. China Oncology, 2025, 35(9): 850-858.

图/表 4

参考文献 20

[1]	COWAN A J, GREEN D J, KWOK M, et al. Diagnosis and management of multiple myeloma: a review[J]. JAMA, 2022, 327(5): 464-477. doi: 10.1001/jama.2022.0003 pmid: 35103762
[2]	VINCENT RAJKUMAR S. Multiple myeloma: 2024 update on diagnosis, risk-stratification, and management[J]. Am J Hematol, 2024, 99(9): 1802-1824. doi: 10.1002/ajh.27422 pmid: 38943315
[3]	BONACCI T, EMANUELE M J. Dissenting degradation: deubiquitinases in cell cycle and cancer[J]. Semin Cancer Biol, 2020, 67(Pt 2): 145-158. doi: 10.1016/j.semcancer.2020.03.008 pmid: 32201366
[4]	XU F H, XU X D, DENG H H, et al. The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression[J]. Transl Oncol, 2024, 49: 102104.
[5]	KUANG Z A, LIU X J, ZHANG N, et al. USP2 promotes tumor immune evasion via deubiquitination and stabilization of PD-L1[J]. Cell Death Differ, 2023, 30(10): 2249-2264.
[6]	WANG Y Y, ZHANG Y P, LUO H, et al. Identification of USP2 as a novel target to induce degradation of KRAS in myeloma cells[J]. Acta Pharm Sin B, 2024, 14(12): 5235-5248. doi: 10.1016/j.apsb.2024.08.019 pmid: 39807309
[7]	TONG J, YU Q, XU W B, et al. Montelukast enhances cytocidal effects of carfilzomib in multiple myeloma by inhibiting mTOR pathway[J]. Cancer Biol Ther, 2019, 20(3): 381-390. doi: 10.1080/15384047.2018.1529112 pmid: 30359543
[8]	Integrated DNA Technologies[EB/OL]. [2023-02-11]. https://sg.idtdna.com/site/order/designtool/index/CRISPR_CUSTOM.
[9]	LI Y N, LI S J, WU H J. Ubiquitination-proteasome system (UPS) and autophagy two main protein degradation machineries in response to cell stress[J]. Cells, 2022, 11(5): 851.
[10]	SOGBEIN O, PAUL P, UMAR M, et al. Bortezomib in cancer therapy: mechanisms, side effects, and future proteasome inhibitors[J]. Life Sci, 2024, 358: 123125.
[11]	PARK J, CHO J, SONG E J. Ubiquitin-proteasome system (UPS) as a target for anticancer treatment[J]. Arch Pharm Res, 2020, 43(11): 1144-1161. doi: 10.1007/s12272-020-01281-8 pmid: 33165832
[12]	ZANGOOIE A, TAVOOSI S, ARABHOSSEINI M, et al. Ubiquitin-specific proteases (USPs) in leukemia: a systematic review[J]. BMC Cancer, 2024, 24(1): 894.
[13]	KITAMURA H, HASHIMOTO M. USP2-related cellular signaling and consequent pathophysiological outcomes[J]. Int J Mol Sci, 2021, 22(3): 1209.
[14]	ZHANG S L, GUO Y, ZHANG S J, et al. Targeting the deubiquitinase USP2 for malignant tumor therapy (review)[J]. Oncol Rep, 2023, 50(4): 176.
[15]	CHEN S Y, LIU Y Q, ZHOU H C. Advances in the development ubiquitin-specific peptidase (USP) inhibitors[J]. Int J Mol Sci, 2021, 22(9): 4546.
[16]	ZHANG J R, LIU S Y, LI Q, et al. The deubiquitylase USP2 maintains ErbB2 abundance via counteracting endocytic degradation and represents a therapeutic target in ErbB2-positive breast cancer[J]. Cell Death Differ, 2020, 27(9): 2710-2725.
[17]	LIU D, FAN Y F, LI J, et al. Inhibition of cFLIP overcomes acquired resistance to sorafenib via reducing ER stress-related autophagy in hepatocellular carcinoma[J]. Oncol Rep, 2018, 40(4): 2206-2214.
[18]	WERMUTH H R, BADRI T, TAKOV V. Montelukast. In StatPearls. StatPearls Publishing[EB/OL]. (2023-03-22)[2025-03-07]. https://www.ncbi.nlm.nih.gov/books/NBK459301.
[19]	TSAI M J, CHANG W A, CHUANG C H, et al. Cysteinyl leukotriene pathway and cancer[J]. Int J Mol Sci, 2022, 23(1): 120.
[20]	TIAN Y C, LIU K, WU D D, et al. The discovery of potent USP2/USP8 dual-target inhibitors for the treatment of breast cancer via structure guided optimization of ML364[J]. Eur J Med Chem, 2024, 268: 116275.

Group	Samples n	H929		MM.1S
Group	Samples n	Proliferation inhibition rate/%	Apoptosis rate/%	Proliferation inhibition rate/%	Apoptosis rate/%
Control	3	57.19±1.93	45.65±0.24	50.04±0.53	40.25±0.91
USP2-OE	3	37.68±1.10^*	18.99±0.26^**	24.48±0.49^**	33.29±0.75^**
USP2-LE-1#	3	80.70±1.60^**	89.08±0.49^**	70.64±0.84^**	67.63±0.21^**
USP2-LE-2#	3	75.30±3.80^*	82.41±1.07^**	68.47±1.32^**	85.90±0.18^**
F value		143.7	5 664	1 233	3 290