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Introduction

Uterine leiomyosarcoma (Ut-LMS) is a rare and aggressive malignant tumor that originates from the smooth muscle cells of the uterus (1). Ut-LMS constitutes approximately 1–2% of all uterine malignancies and is characterized by poor prognosis, high recurrence rates, and a tendency to metastasize (2,3). Pathologically, Ut-LMS is characterized by atypical spindle cells with considerable nuclear pleomorphisms, high mitotic activity, and areas of necrosis. These characteristics distinguish it from benign leiomyomas (fibroids) that lack malignant potential (4,5). Ut-LMS diagnosis typically requires histopathological examinations, which are often supplemented by immunohistochemical staining, to confirm the smooth muscle origin of the tumor cells (6). Clinically, patients with Ut-LMS may present with nonspecific symptoms, such as abnormal uterine bleeding, pelvic pain, and a palpable mass (1). Owing to its aggressive nature, Ut-LMS often presents at an advanced stage, and early diagnosis is challenging. Imaging techniques, including ultrasound, magnetic resonance imaging, and computed tomography, aid in the evaluation of tumor size, extent, and metastatic spread; however, definitive diagnosis relies on tissue biopsy (7).

Depending on the extent of the disease, Ut-LMS treatment primarily involves surgical resection, often in the form of total hysterectomy with or without bilateral salpingo-oophorectomy (1). Conservative treatment for patients who wish to preserve fertility may be an option; however, it requires close and intensive follow-up (8). Although laparoscopic approaches provide minimally invasive options, the abdominal incision approach remains commonly used owing to its lower cost and ease of application (9). Despite surgical intervention, the high recurrence rate of Ut-LMS necessitates additional therapeutic strategies. Owing to the limited data on this rare tumor, preoperative diagnosis is challenging, and uncertainty regarding optimal postoperative management render treatment decisions complex (8). Adjuvant therapies, including radiation and chemotherapy, have been explored; however, their efficacy remains limited, and the optimal treatment regimen remains a subject of ongoing research (10). Therefore, factors, such as safety, fertility preservation, long-term prognosis, and cost should be carefully considered when determining the best surgical approach (8).

Proteasome inhibitors are a class of compounds that target the ubiquitin-proteasome system, which is a critical pathway for degrading misfolded, damaged, or unnecessary proteins within cells (11). By inhibiting proteasome activity, these inhibitors disrupt cellular protein homeostasis and trigger stress responses, which results in programmed cell death (apoptosis) in some cases (12). MG132 is a potent peptide aldehyde-based inhibitor that primarily blocks the chymotrypsin-like activity of proteasomes, thereby resulting in accumulation of ubiquitinated proteins (13,14). Hence, MG132 is widely used in experimental settings to study proteasome function, apoptosis induction, and stress response pathways (15,16). Furthermore, MG132 is utilized to investigate signaling mechanisms, such as nuclear factor kappa B (NF-κB) pathway inhibition, which is crucial in inflammation and immune responses (17,18).

Clinically, proteasome inhibitors, including MG132, have shown notable therapeutic potential, particularly in cancer treatment (19,20). Moreover, MG132 serves as a foundational compound for developing Food and Drug Administration (FDA)-approved drugs, such as bortezomib and carfilzomib, which are used to treat multiple myeloma and mantle cell lymphoma (21). However, in patients with metastatic sarcomas, including leiomyosarcoma, single-agent bortezomib showed minimal efficacy and led to early study termination after the first stage of patient accrual (22). In addition to oncology, MG132 has been used in preclinical studies to explore its role in neurodegenerative diseases, such as Alzheimer's and Parkinson's disease, where protein aggregation is a hallmark (23,24). Furthermore, its anti-inflammatory properties and ability to modulate oxidative stress suggest its potential application in the treatment of inflammatory, cardiovascular, and metabolic disorders (25–27). Accordingly, continued research on MG132 and its related inhibitors may uncover new therapeutic avenues and deepen our understanding of proteasome biology.

However, the effect of MG132 on the growth of Ut-LMS remains poorly understood. Therefore, this study aimed to investigate the effects of MG132 on Ut-LMS cells by focusing on the relevant molecular mechanisms and cellular responses. By elucidating the underlying processes, this study will provide valuable insights that will contribute to the development of more effective therapeutic strategies for Ut-LMS. This will ultimately enhance patient outcomes and offer renewed hope to individuals affected by this challenging malignancy.

Materials and methods

Cell lines and reagents

Human Ut-LMS cancer cell lines (SK-LMS-1, SK-UT-1, and SK-UT-1B) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). These cells were cultured in Minimal Essential Medium (LM007-07; Gyeongsan, South Korea, Welgene) supplemented with 10% fetal bovine serum (SH30919.03; Hyclone, Logan, UT, USA) and 1% streptomycin-penicillin (15140122; Gibco, Waltham, MA, USA) and were maintained in a humidified incubator at 37°C and 5% CO2. MG132 was obtained from Selleckchem (S2619; Houston, TX, USA), and dimethyl sulfoxide (DMSO; LPS Solution) was used as the control. N-acetyl-L-cysteine (NAC; A7250) and catalase (CAT; C1345) were purchased from Sigma-Aldrich (St. Louis, MI, USA).

The 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay

Cells were seeded in 96-well plates at a density of 5,000 cells/well and allowed to adhere overnight. After treatment with various concentrations of MG132 for 24 h, 20 µl of MTT solution (5 mg/ml, M2128; Sigma-Aldrich) was added to each well and incubated for 2 h at 37°C. The resulting formazan crystals were dissolved by adding 150 µl of DMSO to each well, and the absorbance was measured at 570 nm using an INNO microplate spectrophotometer (LTek, Seongnam, South Korea). Cell viability was calculated as a percentage of the untreated control group.

Lactate dehydrogenase (LDH) release assay

The cells were cultured in 96-well plates and treated with MG132 for 24 h. After treatment, LDH release was measured using a Dyne LDH Plus Cytotoxicity Assay Kit (GBL-P500; Dyne Bio, Seongnam, South Korea) following the manufacturer's instructions. LDH PLUS Reaction Mixture (100 µl) was added to each well, and the plate was gently mixed. Thereafter, the reaction was allowed to proceed in the dark for 30 min at room temperature. The absorbance of the samples was measured at 490 nm using a microplate reader. Absorbance values were normalized to those of the control group, thus allowing for comparison between treated and untreated samples.

Protein preparation and western blotting analysis

Ut-LMS cells were treated with varying concentrations of MG132 (0–2 µM) for 24 h. Following treatment, the cells were lysed using radioimmunoprecipitation assay buffer (R2002; Biosesang, Yongin, South Korea) supplemented with a protease inhibitor cocktail (04693132001; Roche, Basel, Switzerland). Subsequently, equal amounts of protein were denatured by heating and were resolved using 12–15% sodium dodecyl-sulfate polyacrylamide gel electrophoresis. The separated proteins were transferred onto polyvinylidene fluoride membranes (IPVH00010; Merck Millipore, Burlington, MA, USA) and blocked with 5% skim milk (262100; BD Difco, Franklin Lakes, NJ, USA) for 1 h at room temperature. The membranes were then incubated overnight at 4°C with gentle shaking using primary antibodies. The following primary antibodies were used: caspase-3 (9665S), cleaved caspase-3 (9664S), caspase-9 (9502S), poly-adenosine diphosphate ribose polymerase (PARP; 9542S), p21 (2947S), p27 (3686T), p53 (2527S), light chain 3 (LC3) beta (2775S), β-actin (4967S; Cell Signaling Technology, Danvers, MA, USA), and ubiquitin (BML-PW0930; Enzo Biochem Inc., Farmingdale, NY, USA). After washing with Tris-buffered saline with 0.1% Tween® 20 detergent, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (7074S or 7076S; Cell Signaling Technology) for 1 h at room temperature. Protein signals were detected using the Western Pico ECL Kit (PICO-250; LPS Solution) and visualized using an Azure c280 chemiluminescent imaging system. The intensity of the protein bands was quantified using ImageJ version 1.53a software to analyze relative protein expression levels.

Apoptotic profile analysis

The apoptotic profile of Ut-LMS cells was assessed using the Muse Annexin V & Dead Cell Kit (MCH100105; Luminex, Austin, TX, USA) according to the manufacturer's instructions. Ut-LMS cells were seeded in six-well plates at a density of 5×10 cells/well and treated with various concentrations of MG132 for 24 h. After treatment, the cells were detached using trypsin, harvested by centrifugation, and resuspended in 100 µl of fresh medium. Subsequently, 100 µl of Muse Annexin V & Dead Cell reagent was added, and the mixture was incubated for 20 min at room temperature in the dark. The stained cells were then analyzed using a Guava Muse Cell Analyzer to evaluate apoptotic and dead cell populations.

Flow cytometric evaluation of cell cycle distribution

Cell cycle distribution was analyzed using the Muse Cell Cycle Kit (MCH100106; Luminex) following the manufacturer's protocol. Cells were harvested, centrifuged to form a pellet, and fixed in 70% ethanol at −20°C for 3 h. After fixation, the cells were stained with the Muse Cell Cycle Reagent and incubated in the dark for 30 min. Thereafter, the stained samples were analyzed using a Guava Muse Cell Analyzer to evaluate the distribution of cells across different cell cycle phases.

Assessment of autophagy using flow cytometry

Autophagy induction by MG132 was assessed using the Muse Autophagy LC3-Antibody Based Kit (MCH200109; Luminex) and Muse Cell Analyzer following the manufacturer's instructions. Both treated and untreated cells were cultured, detached, and incubated with anti-LC3 Alexa Fluor 555 and 1X Autophagy Reagent for 30 min in the dark. After incubation, the cells were resuspended in 1X assay buffer and analyzed using a Muse Cell Analyzer. The level of autophagy was then quantified by calculating the ratio of fluorescence intensity in the treated samples relative to the controls.

ROS quantification using flow cytometry

ROS production was quantified using flow cytometry with the Muse Oxidative Stress Kit (cat. no. MCH100111; Luminex) according to manufacturer's instructions. Cells were seeded at 5×104 cells/well in six-well plates and cultured at 37°C for 24 h. The cells were then treated with 0, 0.25, 0.5, or 1 µM MG132, diluted in fresh medium, and incubated for 24 h. After treatment, the cells were detached, resuspended in 1X Assay Buffer, and incubated with the Muse Oxidative Stress Reagent working solution for 30 min at 37°C. Samples were then analyzed using a Guava Muse Cell Analyzer (cat. no. 0500-3115; Luminex), and the data were processed using Muse analysis software (version 1.5; Luminex).

Statistical analyses

Statistical analyses were performed using GraphPad Prism software (version 8.0). Data are expressed as the mean ± standard deviation. One-way analysis of variance was used to compare multiple groups, followed by Tukey's post hoc test or Dunnett's multiple comparison test, as appropriate. Statistical significance was set at *P<0.05 and **P<0.01.

Results

Assessment of MG132 cytotoxicity in Ut-LMS cell lines

To evaluate the cytotoxic effects of MG132 (Fig. 1A), Ut-LMS cell lines (SK-LMS-1, SK-UT-1B, and SK-UT-1) were treated with varying concentrations (0–1 or 2 µM) of MG132 for 24 h. Cytotoxicity was then evaluated using the MTT and LDH release assays. As shown in Fig. 1B, all three Ut-LMS cell lines exhibited a dose-dependent decrease in viability as MG132 concentrations increased. This indicated that MG132 effectively reduced cell viability in a concentration-dependent manner. Additionally, LDH release assays were conducted on Ut-LMS cells treated with MG132 for 24 h, which confirmed enhanced membrane damage. As the concentration of MG132 increased, all three cell lines showed a considerable increase in LDH release (Fig. 1C). Moreover, a relative increase in ubiquitinated proteins was observed in Ut-LMS cells treated with 0.5 and 1 µM MG132 for 24 h compared with those in control cells (Fig. S1). This indicated that Ut-LMS cell lines experienced reduced cell viability and membrane damage. These findings demonstrate that MG132 induces cytotoxicity in Ut-LMS cells, rendering it a promising therapeutic agent for uterine smooth muscle tumors.

Figure 1. MG132 decreases cell viability in Ut-LMS cells. (A) Chemical structure of MG132. (B) Cell viability of Ut-LMS cell lines (SK-LMS-1, SK-UT-1B and SK-UT-1) treated with the indicated MG132 concentrations for 24 h, assessed using the MTT assay. (C) LDH release assay results for MG132-treated Ut-LMS cells. Cells were treated with the indicated MG132 concentrations for 24 h, and the amount of LDH released into the culture medium was measured. Data are presented as means ± SD. All experiments were performed in triplicate. *P<0.05 and **P<0.01 compared to the control group, or where indicated. Ut-LMS, uterine leiomyosarcoma; LDH, lactate dehydrogenase; SD, standard deviation; MTT, 2,5-diphenyl-2H-tetrazolium bromide.

Assessment of apoptosis induction by MG132 in Ut-LMS cell lines

To investigate the pro-apoptotic effects of MG132 in Ut-LMS cells, apoptotic markers were examined using Annexin V and 7-AAD staining via flow cytometry after treating SK-LMS-1, SK-UT-1B, and SK-UT-1 cell lines with MG132 (0, 0.25, 0.5, or 1 µM) for 24 h. Apoptosis markedly increased in a dose-dependent manner upon MG132 treatment in all three cell lines (Fig. 2A). To further confirm apoptosis induction, western blot analysis was performed to evaluate the expression of apoptosis-related proteins including PARP, caspase-3, and caspase-9. The concentrations of MG132 (0.5 and 1 µM) were carefully selected based on the cell viability data. Following MG132 treatment, increased cleaved PARP and cleaved caspase-3 levels were observed in all three cell lines, whereas no changes in cleaved caspase-9 levels were detected in any of the cell lines (Fig. 2B). These results confirmed that MG132 induced apoptosis via activation of apoptosis-related markers in all three Ut-LMS cell lines.

Figure 2. MG132 induces apoptosis and cell death in Ut-LMS cells. (A) Annexin V and 7-AAD analysis of apoptosis and cell death in MG132-treated SK-LMS-1 (upper panels), SK-UT-1B (middle panels) and SK-UT-1 (bottom panels) cells. Cells were treated with MG132 (0.25, 0.5 and 1 µM) for 24 h, followed by flow cytometric analysis to determine the percentage of cells in the live, early apoptosis, late apoptosis and dead cell phases. Quantitative data are presented in the right panel. (B) Western blot analysis of apoptosis-related proteins, including PARP, cleaved caspase-3 and caspase-9, in MG132-treated Ut-LMS cell lines. Cells were treated with MG132 (0.5 and 1 µM) for 24 h, and protein expression levels were assessed using specific antibodies. Band intensities are shown in the right panel, with β-actin serving as a loading control. Representative results from three independent experiments are shown. Data are expressed as means ± SD. *P<0.05 and **P<0.01. Ut-LMS, uterine leiomyosarcoma; SD, standard deviation; PARP, poly-adenosine diphosphate ribose polymerase.

Effects of MG132 on cell cycle progression and regulatory proteins in Ut-LMS cells

To investigate the impact of MG132 on cell cycle progression in Ut-LMS cells, cell cycle distribution was analyzed using propidium iodide staining, followed by flow cytometry after treating the cells with varying concentrations of MG132 for 24 h. As shown in Fig. 3A, MG132 treatment induced a G2/M phase arrest at concentrations of 0.5 and 1 µM in SK-LMS-1 and SK-UT-1 cells, whereas no significant changes in cell cycle distribution were observed in SK-UT-1B cells. Western blot analysis was performed to evaluate the expression of cell cycle regulatory proteins, including p21, p27, and p53, in Ut-LMS cells (Fig. 3B). The p21 expression was markedly upregulated by MG132 in all three cell lines. In contrast, p27 levels pronouncedly decreased in all three cell lines following MG132 treatment. However, a differential response was observed in p53 expression; its expression decreased in SK-LMS-1 cells, increased in SK-UT-1B cells, and showed no significant changes in SK-UT-1 cells. These findings suggest that MG132 differentially regulates cell cycle progression and expression of cell cycle-related proteins in Ut-LMS cell lines, thereby highlighting the distinct cellular responses to treatment.

Figure 3. MG132 regulates cell cycle arrest and modulates the expression of cell cycle regulatory proteins in Ut-LMS cells. (A) Cell cycle distribution in SK-LMS-1, SK-UT-1B and SK-UT-1 cells treated with MG132 (0.5 and 1 µM) for 24 h, analyzed using PI staining and flow cytometry. The subG1 peak was removed through gating to focus solely on the pure cell cycle changes. Quantitative data representing the percentage of cells in each phase of the cell cycle (G0/G1, S and G2/M) are shown on the right panel. (B) Western blot analysis of cell cycle regulatory proteins, including p21, p27 and p53, in MG132-treated Ut-LMS cell lines. Cells were treated with MG132 (0.5 and 1 µM) for 24 h, and protein expression levels were analyzed using specific antibodies. Band intensities are shown in the right panel, with β-actin serving as a loading control. Data are presented as means ± SD. *P<0.05 and **P<0.01. Ut-LMS, uterine leiomyosarcoma; SD, standard deviation; PI, propidium iodide.

Effects of MG132 on autophagy in Ut-LMS cell lines

Autophagy is an essential cellular mechanism that maintains homeostasis under normal and stressful conditions (28). To assess the induction of autophagy, LC3 protein levels, a marker of autophagosome formation, were evaluated using flow cytometry and western blotting. Following MG132 treatment at the indicated concentrations (0.5 and 1 µM) for 24 h, flow cytometry analysis revealed an increase in autophagy induction in SK-LMS-1, SK-UT-1B, and SK-UT-1 cells (Fig. 4A). Consistent with the flow cytometry results, western blot analysis showed elevated LC3 II levels in all three cell lines, thereby indicating the conversion of LC3 I to LC3 II, which is a hallmark of autophagy induction (Fig. 4B). These findings suggest that MG132 promotes autophagy in Ut-LMS cells, thus potentially contributing to its cellular effects.

Figure 4. MG132 modulates autophagy in Ut-LMS cells. (A) Flow cytometry analysis of LC3 protein levels in MG132-treated SK-LMS-1, SK-UT-1B and SK-UT-1 cells. Cells were treated with MG132 (0.5 and 1 µM) for 24 h, followed by staining with an anti-LC3 antibody. The flow cytometry analysis quantified the number of LC3-positive cells, which reflected autophagy induction. (B) Western blot analysis of LC3 expression in MG132-treated Ut-LMS cell lines. Elevated LC3 II levels in cells indicate the conversion from LC3 I to LC3 II, a hallmark of autophagy induction. Cells were treated with MG132 (0.5 and 1 µM) for 24 h, and LC3 I and LC3 II protein expression levels were measured. Band intensities are shown in the right panel, with β-actin serving as a loading control. Data are represented as means ± SD. *P<0.05 and **P<0.01. Ut-LMS, uterine leiomyosarcoma; SD, standard deviation; LC3, light chain 3.

Effects of MG132 on intracellular ROS levels in Ut-LMS cells

Excessive ROS accumulation in cells leads to oxidative stress, which damages nucleic acids, lipids, proteins, membranes, and mitochondria (29). To assess whether MG132 increases ROS levels in Ut-LMS cells, cells were treated with varying concentrations of MG132 (0.5 and 1 µM) for 24 h, followed by flow cytometry analysis. In SK-LMS-1 cells, no significant changes in ROS levels were observed after MG132 treatment (Fig. 5A, upper panels). Based on the results shown in Fig. 2A and these findings, apoptosis induced by MG132 in SK-LMS-1 cells appeared to occur independently of ROS production. However, ROS levels were increased in SK-UT-1B and SK-UT-1 cells treated with MG132 at the indicated concentrations for 24 h (Fig. 5A, middle and bottom panels). Therefore, to investigate whether ROS production contributes to MG132-induced apoptosis in SK-UT-1B and SK-UT-1 cells, cells were treated with MG132 (1 µM) with or without the ROS scavengers NAC and CAT (30,31). In SK-UT-1B cells, NAC and CAT had no significant effect on MG132-induced apoptosis (Fig. S2A). However, NAC effectively reduced MG132-induced apoptosis (Fig. 5B), whereas CAT had no significant effect (Fig. S2B) in SK-UT-1 cells. These results suggest that MG132 induced ROS-mediated apoptosis in SK-UT-1 cells, whereas MG132-induced apoptosis in SK-LMS-1 and SK-UT-1B cells occurs in an ROS-independent manner.

Figure 5. MG132 induces ROS-mediated apoptosis in SK-UT-1 cells. (A) Flow cytometry analysis of intracellular ROS levels in MG132-treated SK-LMS-1, SK-UT-1 and SK-UT-1B cells. Cells were treated with MG132 (0.5 and 1 µM) for 24 h, followed by staining with the ROS-sensitive dye dihydroethidium. The percentage of ROS-positive cells is shown in the histogram (right panel). (B) Apoptosis analysis in SK-UT-1 cells treated with 1 µM MG132 for 24 h, in the presence or absence of the ROS scavenger NAC (5 mM). The data are expressed as means ± SD; n=3. **P<0.01. ROS, reactive oxygen species; NAC, N-acetyl-L-cysteine; SD, standard deviation.

Discussion

The findings of this study (Fig. 6) provide valuable insights into the molecular mechanisms underlying the effects of MG132 on Ut-LMS cell lines. The study emphasizes the therapeutic potential of MG132 and the distinct responses observed in different cell lines. Ut-LMS is a rare and aggressive malignancy of the uterine smooth muscle cells, characterized by poor prognosis and considerable challenges in treatment and management. Thus, the study findings contribute to the growing understanding of this disease and offer new perspectives on potential therapeutic strategies for difficult-to-treat malignancies.

Figure 6. MG132 exerts its antitumor effects in Ut-LMS cells by modulating multiple cellular pathways, including apoptosis, cell cycle regulation, apoptosis and ROS-mediated effects, in a cell-line-specific manner. LDH, lactate dehydrogenase; ROS, reactive oxygen species; NAC, N-acetyl-L-cysteine; Ut-LMS, uterine leiomyosarcoma; PARP, poly-adenosine diphosphate ribose polymerase; LC3, light chain 3; NAC, N-acetyl-L-cysteine.

This study demonstrated that MG132 induces various forms of cell death in Ut-LMS cell lines. MG132 induced cytotoxicity in SK-LMS-1, SK-UT-1B, and SK-UT-1 cells, with a dose-dependent decrease in cell viability and increased LDH release observed in all three cell lines. Furthermore, MG132 treatment affected cell cycle progression, resulting in G2/M arrest in SK-LMS-1 and SK-UT-1 cells. However, no significant changes were observed in SK-UT-1B cells. In addition, MG132 induced apoptosis, with ROS-independent apoptosis observed in SK-LMS-1 and SK-UT-1B cells, and ROS-dependent apoptosis in SK-UT-1 cells. Additionally, MG132 differentially regulated the expression of cell cycle proteins (p21, p27, and p53) in each cell line and induced autophagy, as evidenced by increased LC3 II levels. This suggests that MG132 simultaneously activates multiple pathways, thereby disrupting key survival mechanisms.

This study also revealed crucial differences in protein expression and cellular behavior across these cell lines. MG132 treatment decreased p53 expression in SK-LMS-1 cells, whereas it increased p53 expression in SK-UT-1B cells. Furthermore, ROS analysis demonstrated variability; SK-LMS-1 cells exhibited higher basal ROS levels, whereas MG132 treatment elevated ROS levels in SK-UT-1B and SK-UT-1 cells solely. These findings underscore the molecular heterogeneity of Ut-LMS cell lines and align with previous reports of variability in cell doubling times and tumor formation rates among these models (32).

The differences between MG132-treated SK-LMS-1, SK-UT-1, and SK-UT-1 cell lines in this study could be attributed to several key factors. A detailed comparison of the cell line characteristics based on ATCC product sheets supports these observations. SK-UT-1B and SK-UT-1, which are derived from the uterus of a 75-year-old female, differ in pathology. SK-UT-1B is associated with Grade III endometrial leiomyosarcoma (LMS), whereas SK-UT-1 originates from a mesodermal mixed tumor. In contrast, SK-LMS-1 cells, which are derived from the vulva of a 43-year-old female patient with LMS, exhibited fibroblast morphology indicative of its stromal tissue origin. However, SK-UT-1B and SK-UT-1 display epithelial morphology consistent with uterine and endometrial tissues. Therefore, these differences in tissue origin are likely to contribute to variations in molecular pathways that subsequently influence drug responses. For example, the expression levels of key proteins, such as p21 and p27, and ROS levels, play key roles in the response of each cell line to MG132-induced proteasome inhibition and stress. Additionally, according to the ATCC product sheet, SK-UT-1B is a subclone derived from SK-UT-1. Although SK-UT-1, similar to SK-LMS-1, harbors two independent p53 point mutations, some reports suggest that SK-UT-1B retains wild-type p53 (33,34). Owing to these conflicting findings, the p53 status of SK-UT-1B remains unclear, and further sequencing or validation may be necessary for confirmation. These factors collectively contributed to the variability in MG132-induced signaling observed in this study. Therefore, future studies should investigate these differences in detail to gain a comprehensive understanding of the diverse mechanisms underlying Ut-LMS progression and therapeutic responses.

In addition to MG132, research on proteasome inhibitors has markedly expanded, thereby contributing to advancements in cancer treatment by targeting the ubiquitin-proteasome system (19,20). Proteasome inhibitors, such as bortezomib, carfilzomib, ixazomib, and marizomib, have demonstrated efficacy in inducing apoptosis and disrupting critical cellular processes in various malignancies (20,35). Bortezomib is the first FDA-approved proteasome inhibitor and is notably effective in treating multiple myeloma (MM) by inhibiting the NF-κB pathway and stabilizing pro-apoptotic factors, thereby improving survival rates (36). Despite the associated cardiovascular risks, carfilzomib, which is a second-generation inhibitor, provides sustained proteasome inhibition and has shown enhanced efficacy in combination therapies for relapsed/refractory MM (37,38). Ixazomib is the first oral proteasome inhibitor that is convenient for outpatient treatment and is effective in extending progression-free survival in patients with MM (39). Furthermore, marizomib has emerged as a promising candidate because of its ability to cross the blood-brain barrier, thus showing potential for treating glioblastoma by inducing oxidative stress and apoptosis in cancer cells (40). Research on the mechanisms of action of these proteasome inhibitors is ongoing to optimize treatment protocols and expand their use to other cancer types, including Ut-LMS. These efforts highlight the critical role of proteasome inhibition in contemporary cancer therapy, with ongoing studies focusing on improving efficacy, reducing toxicity, and enhancing patient outcomes.

This study had some limitations. Although the cell death mechanisms and therapeutic potential of MG132 were investigated, the full extent of its effects across different Ut-LMS cell lines is not yet clear. In addition, the long-term toxicity of MG132 and its potential synergistic effects in combination with other treatments have not yet been explored. Therefore, further research is needed to address these aspects and enhance current understanding of the mechanism by which MG132 can be effectively and safely incorporated into treatment strategies for Ut-LMS.

In conclusion, this study demonstrates the anticancer potential of MG132 in Ut-LMS cell lines, thereby revealing diverse and cell-specific responses. The variability in the effects of MG132 on these cell lines underscores the molecular heterogeneity of this aggressive malignancy and emphasizes the importance of personalized therapeutic approaches. Therefore, the study findings provide a strong foundation for further research to elucidate the precise mechanisms underlying MG132-induced cell death and to explore its synergistic potential in combination therapies.

Supplementary Material

Supporting Data

Acknowledgements

Not applicable.

Funding

This study was supported by the Basic Science Research Program through the National Research Foundation of Korea, funded by the Ministry of Education (grant no. NRF-2022R1l1A1A01053069) and the Korean Government (Ministry of Science and Information and Communication Technology) (grant no. RS-2022-00166501).

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

HL and SS designed the experiments and revised the manuscript accordingly. HL and HJ conducted the experiments and wrote the manuscript. HJ and SS performed the data analysis. All authors have read and approved the final manuscript. HL and HJ confirm the authenticity of all the raw data.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

References