Montelukast Performs Synergistic Effect with Carfilzomib in Multiple Myeloma by Inhibiting the Deubiquitinase UCHL1

Research Article

Austin J Cancer Clin Res. 2022; 9(1): 1101.

Montelukast Performs Synergistic Effect with Carfilzomib in Multiple Myeloma by Inhibiting the Deubiquitinase UCHL1

Yu W, Wei W, Peng R, Chen H, Zhou N, Wu L, Shi H, Chen X, Wang D, Wei J, Zhao W and Zhou F*

Zhabei Central Hospital, Jing’an District, Shanghai, China

*Corresponding author: Fan Zhou, Zhabei Central Hospital, Jing’an District, Shanghai, No.619, Zhonghua Xin Road, China

Received: March 02, 2022; Accepted: March 25, 2022; Published: April 01, 2022


Immunomodulators play an important role in combination chemotherapy of multiple myeloma. Besides, it has been reported that leukotriene-D4 receptors play an important role in carcinogenesis. The current study was carried out to assess the possible antitumor effects of montelukast (both an immunomodulator and a CysLT1R antagonist) both in single and in combination with carfilzomib in multiple myeloma cells and the potential mechanisms. We performed our experiment using cell apoptosis assay, cell cycle assay, western blot analysis, mitochondrial transmembrane potential, and chemical proteomics based on protein activity. The results showed that montelukast inhibits cell proliferation and leads to cell apoptosis in multiple myeloma cell lines. Then we performed combination of montelukast and carfilzomib on multiple myeloma cells to explore whether they are synergistic. We demonstrated that the combination treatment leads to stronger effect of cell apoptosis. Furthermore, we demonstrated the combination effect is performed independent of the leukotriene-D4 receptors inhibition. Given that protesome inhibitor leads to accumulation of ubiquitin by inhibiting the degradation of these proteins, we tested ubiquitin levels of different groups. We found that montelukast induces ubiquitin accumulation by inhibiting Ubiquitin-Specific Protease UCHL1. All of these lead to the activity of indoplasmic reticulum stress and therefore result in cell apoptosis. This brings us a new idea in the treatment of multiple myeloma. Since montelukast is cheap and widely used in clinic, if it is proved to be effective in the treatment of multiple myeloma, it will greatly reduce the economic pressure of patients and bring new hope to patients.

Keywords: Montelukast; Carfilzomib; Multiple myeloma; Cell apoptosis; UCHL1


UPS: Ubiquitin-Proteasome System; MM: Multiple Myeloma; MNK: Montelukast; CFZ: Carfilzomib; CI: Combination Index; PNK: Pranlukast; ZFK: Zafirlukast


Multiple myeloma (MM) is a clonal plasma cell neoplasm involving the bone marrow and extramedullary sites, manifested by anemia, hypercalcemia, impairment of renal function and bone destruction [1]. Over the past decades, the introduction of novel agents has improved clinical outcomes of MM patients. However, nearly all patients will relapse and require subsequent therapy finally [2,3]. Proteasome inhibitors are commonly used in the treatment of multiple myeloma. Of it, bortezomib is recommended as the frontline and effective in newly diagnosed MM. It significantly prolongs patient survival [4,5]. However, several deficiencies which limit its clinical application. Firstly, treatment is often limited by dose-limiting side effects, most due to peripheral neuropathy. Besides, prolonged continuous treatment often induces drug resistance. Moreover, bortezomib is usually not effective in refractory or relapsed setting of MM patients. Carfilzomib is the second generation of proteasome inhibitor. It is commonly used in relapsed cases with prior resistant to bortezomib [6-8]. But the outcome is also not so satisfactory. Consequently, a novel agent that could increase proteasome inhibitors sensitivity and overcome resistance would be clinically meaningful.

It is well known that proteasome inhibitors perform their effect by inhibiting proteasomes which play critical roles in the ubiquitinproteasome system (UPS). The UPS plays an important role in different cellular processes by targeted destruction of proteins, such as cell cycle progression, receptor down-regulation, gene transcription and apoptosis [9-11]. It has been an important target in the treatment of malignancies [12-14]. This process includes two specific and sequential steps: ubiquitination and proteasomal degradation. Ubiquitin is a small modifier molecule that labels proteins in a highly specific manner. Ubiquitination is a stepwise cascade of enzymatic reactions and requires the ubiquitin activating enzyme, ubiquitin conjugating enzyme and the ubiquitin ligase [15,16]. This ubiquitination process can be reversed by deubiquitinating enzymes. The balance between ubiquitination and deubiquitination activities regulates the level and activity of the protein substrates and thus cell homeostasis [17]. Proteins with a poly-ubiquitination will be degraded by the 26S proteasome. The 26S proteasome also known as the “proteasome” is a large (more than 2000kDA) multi-protein complex present in the nucleus and cytoplasm of all eukaryotic cells. It is composed of one 20S core particle and two 19S regulatory particles. The 19S regulatory particle binds a polyubiquitin chain and cleaves it from the substrate and recycles the ubiquitin. The substrate is then denaturated/unfolded and subsequently degraded into small peptides [18,19].

Immunomodulator is another important component of combination chemotherapy for multiple myeloma. As the earliest and most widely used immunomodulator in the clinic, montelukast (mnk), a selective reversible cys-leukotriene-1 receptor (LTD4 receptor) antagonist, is commonly used in the treatment of asthma. Leukotrienes (LTs) are biologically active fatty acids derived from the oxidative metabolism of arachidonic acid via a 5-lipooxygenase (5-LOX) pathway (Wenzel, 1998). 5-LOX activity leads to the formation of unstable LTA4, which can be converted into either LTB4 or several cysteinyl LT (CysLT; e.g. LTC4, LTD4, and LTE4), the latter are components of a slow-reacting substance of anaphylaxis. It is reported that 5-LOX is expressed by a wide variety of tumor cells and tissues. Therefore, LTs maybe potential targets in the treatment of malignancies [20-22]. Montelukast selectively block the cysteinyl leukotriene 1 receptor (CysLT1R). Based on these reports, we investigated whether montelukast perform antitumor effect in multiple myeloma. Furthermore, we explored the combination effect of montelukast and proteasome inhibitor carfilzomib. Finally, we searched for the potential mechanisms of these effects.


Cell culture and viability assay

The MM cell lines RPMI-8226, NCI-H929, and MM1S were routinely cultured in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (Gibco-BRL, Gaithersburg, MD, USA) in a humidified atmosphere with 5% CO2 at 37°C.


Montelukast and carfilzomib was purchased from Selleckchem (Houston, TX, USA). The antibodies used were as follows: anti-poly (ADP-ribose) polymerase-1 (PARP-1) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-caspase-9, anti-caspase-3, anti-β-actin, ubiquitin, OTUB1, UCHL1, UCHL3 and RPS27A (Cell Signaling Technology, Beverly, MA, USA).

Cell apoptosis assay

Cell apoptosis was measured using a fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA) following the manufacturer’s instructions. After treatment with montelukast, carfilzomib respectively or in combination for 48 hours, cells were harvested, washed twice with ice-cold binding buffer, and resuspended with 100μl buffer. Annexin V-FITC (5μl) was then added to the suspended cells and mixed gently. This preparation was incubated for 5 minutes in the dark. Following incubation, 5μl propidium iodide was added and mixed gently. Incubation was continued for another 3 minutes. Finally, cells were analyzed by flow cytometry.

Cell cycle assay

After treatment, cells were harvested, washed twice with phosphate-buffered saline (PBS), and fixed with 75% cold ethanol at 4°C overnight. The next day, cells were incubated with RNase (100mg/ ml) for 30 minutes at 37°C. Cells were stained with propidium iodide (PI; Sigma) (250mg/ml) and incubated for another 15 minutes in the dark. Cells were then analyzed by flow cytometry.

Western blot analysis

Cells were harvested and lysed with lysis buffer. After quantification, protein extracts were equally loaded to 6%-15% sodium dodecyl sulfate–polyacrylamide gels, electrophoresed, and transferred to nitrocellulose membrane (Amersham Bioscience, Little Chalfont, UK). When completed, the membrane was blocked with 5% milk in PBS for 30 minutes at a temperature of 25ºC. The membranes were washed three times with Tris-buffered saline containing 0.05% Tween-20 and incubated with antibodies overnight at 4°C. The next day, the membranes were washed three times and horseradish peroxidase (HRP)-linked secondary antibody was added. The mixture was allowed to stand for 1-hour at a temperature of 25ºC. Signals were detected by chemiluminescence phototope-HRP kit (Merck Millipore, USA) according to manufacturer’s instructions. HRP-linked anti-β-actin antibody was used as the loading control.

Mitochondrial transmembrane potential

After treatment, cells were harvested, washed twice with phosphate-buffered saline (PBS). Then remove the supernatant and resuspend cells with 200μl PBS. Rhodamine123 was added to the cells to be tested so that the final concentration was 10μg/ml. Incubate for 30 minutes at a temperature of 37°C. Centrifugal at 2000 rpm for 10 minutes and remove unbound dyes. Centrifugal and wash cells with PBS (2000rpm for 10 minutes). Remove the supernatant and resuspend cells with 100μl PBS. Adding 5μl PI dyestuff into the cell suspension and mix them well. Stand for 15 minutes in the dark at room temperature. Adding 300μl PBS buffer and collecting the fluorescence signal of FL1-H/FL2-H excited at 488nm by flow cytometer.

Chemical proteomics based on protein activity

Cells treated were harvested and washed by PBS. Then entrifugal at 2000 rpm for 5 minutes and remove the supernatant. Make up non denatured cracking solution and resuspend cells well with appropriate solution. Incubate for 60 minutes at a temperature of 4°C to crack cell proteins. Centrifugal at 12000 rpm for 10 minutes and remove the supernatant into a new EP tube. Quantitative the proteins. Take 50μg proteins and incubate with different chemicals for 2 hours at room temperature. And then add 1μM HA-Ub-CI/Br/VME probe into the mixture. Incubate for 15 minutes at room temperature and add in 5x SDS the next step. Cooking the protein at a temperature of 98°C for 5 minutes. Finally, detecting proteins using HA antibody by western blot.


To explore the effect of mnk in MM cells, we treated H929 and RPMI-8226 cell lines with mnk in different concentration and tested cell growth inhibition 48 hours later (Figure 1A). We found that mnk inhibits cell growth in a concentration-dependent manner. MM cell growth was completely inhibited treated with mkn in 80-100μmol/L. And then, we detected cell apoptosis-related proteins in MM cells that have been treated in the same way. As showed in Figure 1B, PARP-1, caspase-3 and caspase-9 were obviously cleaved in highconcentration group. It was suggested that mnk induces MM cell apoptosis in high concentration.