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