Effects of β-hydroxy-β-methylbutyrate on skeletal muscle loss in a mouse model of esophageal squamous cell carcinoma

Introduction

Loss of skeletal muscle mass adversely affects the prognosis of patients with cancer (1). Decreased skeletal muscle mass can lead to decreased activity due to frailty, and respiratory muscle weakness can increase pneumonia risk. Additionally, as increased inflammatory responses due to tumor-host interactions are associated with decreased skeletal muscle mass, muscle loss may increase the risk of tumor recurrence, metastasis, and cancer-related mortality (2,3).

Esophageal cancer is a highly malignant tumor with invasive growth and rapid expansion. Patients are often malnourished before treatment due to dysphagia from advanced tumors. Furthermore, patients often experience progressive loss of skeletal muscle mass due to stress from treatments such as surgery and chemotherapy, as well as systemic and local inflammation associated with malignancy. Esophageal cancer surgery is one of the most invasive gastrointestinal surgeries, and patients often experience a decrease in skeletal muscle mass and decline in quality of life (QOL) postoperatively (4). Notably, a significant decrease in skeletal muscle mass occurs during preoperative chemotherapy for esophageal cancer, and patients with decreased skeletal muscle mass following neoadjuvant chemotherapy before surgery exhibit lower overall survival rates and recurrence-free survival rates (5). Therefore, maintaining or increasing skeletal muscle mass in esophageal cancer survivors is a crucial management goal for improving the prognosis of patients while maintaining their QOL.

The skeletal muscle is a site of continuous protein catabolism and anabolism (6,7). Normal muscle protein metabolism requires an adequate supply of amino acids, particularly essential amino acids. Essential amino acids and exercise directly stimulate protein synthesis in muscle cells. Among amino acids, leucine, a branched-chain amino acid, is the most potent protein synthesis stimulator, promoting muscle protein synthesis through the mechanistic target of rapamycin (mTOR) pathway (8,9). During cachexia-induced invasion, inflammatory cytokines inactivate the mammalian target of the rapamycin pathway, resulting in decreased skeletal muscle synthesis. Furthermore, muscle protein degradation pathways, including the ubiquitin-proteasome, autophagy, and apoptosis pathways, are activated, resulting in accelerated skeletal muscle protein degradation. β-Hydroxy-β-methylbutyrate (HMB) is a metabolite of leucine, and approximately 5% of ingested leucine is metabolized to HMB. HMB has known effects on muscle protein synthesis, inhibition of protein degradation, muscle strength, and the suppression of excessive inflammatory responses (10). HMB exerts a potent muscle protein synthesis effect, stronger than leucine (11). In animal study, HMB preserves lean body mass and attenuates protein breakdown by downregulating the increased expression of key regulators of the ubiquitin-proteasome proteolytic pathway and stimulating protein synthesis (12). However, the effect of HMB on skeletal muscle loss in patients with esophageal cancer remains unclear. In addition, although reports exist on the effects of HMB on skeletal muscle mass, its effects on muscle function, such as grip strength, have not been examined. This study aimed to elucidate the effects of HMB on skeletal muscle loss in patients with esophageal squamous cell carcinoma (ESCC) using mouse models of ESCC. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://atm.amegroups.com/article/view/10.21037/atm-25-126/rc).

Methods

The human ESCC cell line TE-8 was obtained from the Riken Cell Bank (Tsukuba, Japan). The cells were cultured in RPMI-1640 (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum at 37 °C in a CO₂ incubator. Nine-week-old male BALB/cAJcl-nu/nu mice were purchased from CLEA Japan Inc. (Tokyo, Japan). Cultured TE-8 cells were injected subcutaneously into the backs of the mice using a 27-gauge needle. The total number of tumor cells injected per mouse was 5×10⁶, and 10 mice with ESCC were generated. Ten ESCC-bearing mice were used in this study, along with three additional mice that did not undergo malignant cell transplantation (n=13). To reduce variability, only male mice were used in this study. The 13 mice were randomly divided into three groups, and investigators were blinded to basic pre-transplant data. The groups included (I) the non-tumor control group (n=3) comprising non-ESCC mice fed a normal diet, (II) the ESCC + HMB group (n=5) comprising ESCC-bearing mice fed HMB, and (III) the ESCC control group (n=5) comprising ESCC mice fed a normal diet. All the mice were provided free access to a standard mouse diet and water. In addition to the intake of a standard diet, the mice in the ESCC + HMB group were given 50 mg/kg of HMB dissolved in 200 µL of distilled water orally at 1 h before the cell transplantation and then once daily for 5 days per week until day 44. A powdered Ca-HMB product was used as the HMB source (DNS, Daiichi Sankyo Healthcare Co., LTD, Tokyo, Japan). The dosage for mice was determined based on the recommended daily intake of 3000 mg of Ca-HMB for humans. HMB was force-fed to the mice by a fixed researcher. The mice in the ESCC control group were provided with distilled water by the same schedule for the same period. The observation period was 44 days, and the body weight, grip strength, and tumor diameter of the mice were measured every 7 days. A protocol was prepared before the study without registration. All animal experiments were performed under a project license (No. 24043) granted by the Ethics Review Committee for Animal Experimentation of Tokai University School of Medicine, in compliance with the national or institutional guidelines for the care and use of animals.

Grip strength (limb grip strength) was measured using a grip strength meter (MK-380Si; Muromachi Kikai, Tokyo, Japan). Each mouse was allowed to grip a horizontal bar connected to a force gauge, and the tail was gently pulled back until the grip was released (Figure 1). The peak tension was automatically recorded when the mouse released the bar. The measurements were repeated five times, and the average of the top three values was recorded as the maximum grip strength. Lower grip strength values indicate weaker muscles. The tumor diameters in the mice were measured using a slide caliper, and the tumor volumes were calculated using the following formula (13): a × b²/2, where a is the largest and b is the smallest of the two dimensions. After the final administration on day 44, the body weight, grip strength, and tumor diameter of the mice were measured. The mice were sacrificed using sodium pentobarbital (120 mg/kg, intraperitoneal injection), and the skeletal muscle tissue (bilateral gastrocnemius muscles) was removed and weighed.

Figure 1 Grip strength test of the mice.

Statistical analysis

A t-test or Mann-Whitney U test was used for comparisons between any two groups. For multiple comparisons, Tukey’s test was applied. Significant differences between the two independent groups were determined using Student’s t-test. A paired t-test was used to assess differences between two variables in the same mice. The Mann-Whitney U test, a nonparametric test, was performed when the data were non-normally distributed. One-way repeated measures analysis of variance was used to assess temporal changes in the same mice. An overall group-by-time interaction test was also conducted. Statistical significance was set at P≤0.05. A post-hoc power analysis was performed to confirm that the sample size was adequate for detecting differences in the primary outcome, body-weight-adjusted gastrocnemius muscle weight. Based on the observed means and standard deviations in the ESCC control and ESCC + HMB groups, the effect size (Cohen’s d) was 2.85. With a sample size of 5 in each group, the achieved power (1−β) was 0.95 at a significance level (α) of 0.05.

Results

Twelve of the 13 mice were sacrificed on day 44 at the end of the scheduled observation period, and the gastrocnemius muscles were resected. The remaining one mouse in the ESCC + HMB group died due to tumor collapse on day 43. Therefore, its body weight and grip strength could not be measured on day 43, and the gastrocnemius muscles could not be resected on day 44.

Figure 2 indicates the conditions of the mice before transplantation. No differences in body weight or grip strength before transplantation were observed among the three groups (Figure 2A, body weight, P=0.22; Figure 2B, grip strength, P=0.76). Figure 3 indicates the condition of the mice after ESCC transplantation. No significant difference in the mean tumor volume was observed between the ESCC control and ESCC + HMB groups (Figure 4A, P=0.33). Furthermore, no significant difference in tumor weight was observed between the ESCC control and ESCC + HMB groups at the time of sampling on day 44 (Figure 4B, P=0.42). Figure 5 presents the percentage changes in the body weights of the mice during the observation period. ESCC-bearing mice exhibited a decreasing trend in body weight over the latter half of the observation period, whereas non-ESCC (healthy) mice maintained their weight. However, the difference in weight change between the ESCC and non-ESCC groups was not statistically significant (non-tumor control group vs. ESCC control group, P>0.99; non-tumor control group vs. ESCC + HMB group, P=0.61; ESCC control group vs. ESCC + HMB group, P=0.52). Grip strength was analyzed after normalization to body weight (Figure 6). Grip strength was significantly higher in the ESCC + HMB group than in the ESCC control group (P=0.03). No significant difference in grip strength was observed between the ESCC + HMB group and non-tumor control group or between the non-tumor control group and ESCC control group (ESCC + HMB group vs. non-tumor control group, P=0.94; non-tumor control group vs. ESCC control group, P=0.08). Gastrocnemius muscle weight was significantly higher in the ESCC + HMB group than it was in the ESCC control group (Figure 7A, P=0.05). The ratio of gastrocnemius muscle weight to body weight was significantly higher in the ESCC + HMB group than in the ESCC control group (Figure 7B, P<0.01). No difference in grip strength or gastrocnemius muscle weight was observed between non-tumor mice and ESCC mice (grip strength: P=0.35; gastrocnemius muscle weight: P=0.37).

Figure 2 Condition of the mice before the tumor transplantation on day 1. (A) Body weight; (B) grip strength. ESCC, esophageal squamous cell carcinoma; HMB, β-hydroxy-β-methylbutyrate.

Figure 3 Condition of the mice after the tumor transplantation. ESCC, esophageal squamous cell carcinoma; HMB, β-hydroxy-β-methylbutyrate.

Figure 4 Tumor volumes and weight. (A) Changes of the tumor volume. (B) Tumor weight at the time of sampling on day 44. ESCC, esophageal squamous cell carcinoma; HMB, β-hydroxy-β-methylbutyrate.

Figure 5 Changes in body weight. ESCC, esophageal squamous cell carcinoma; HMB, β-hydroxy-β-methylbutyrate.

Figure 6 Changes in grip strength. ESCC, esophageal squamous cell carcinoma; HMB, β-hydroxy-β-methylbutyrate.

Figure 7 Weight of the gastrocnemius muscle at the time of sampling on day 44. (A) Weight of the gastrocnemius muscle; (B) ratio of the gastrocnemius muscle weight to the body weight. ESCC, esophageal squamous cell carcinoma; HMB, β-hydroxy-β-methylbutyrate.

Discussion

In this study, grip strength was significantly higher in the ESCC + HMB group than in the ESCC control group. Additionally, the gastrocnemius muscle weight at the time of sampling and ratio of gastrocnemius muscle weight to body weight were significantly higher in the ESCC + HMB group than those in the ESCC control group. No significant differences in grip strength or gastrocnemius muscle weight were observed between the ESCC + HMB and non-tumor groups. HMB administration to mice transplanted with ESCC cells appeared to preserve grip strength and gastrocnemius muscle weight at the same levels as those in the non-tumor control group. This result indicates that HMB helps maintain both skeletal muscle mass and muscle function, including grip strength. No difference in grip strength or gastrocnemius muscle weight was observed between non-tumor mice and ESCC mice. This is because the ESCC + HMB group included the ESCC mice, and HMB may have been useful in maintaining skeletal muscle.

Recent advances in thoracoscopic surgery and robot-assisted surgery for esophageal cancer have enabled thorough lymph node dissection while maintaining the minimally invasive nature of the procedure. However, the 5-year survival rate after surgery for esophageal cancer remains 59.9%, and further improvements are required (14). In addition to improving surgical techniques, developing adequate perioperative supportive care methods is essential to improve prognosis after surgery for esophageal cancer. Nutritional therapy during the perioperative period in patients with esophageal cancer is becoming increasingly important as a powerful supportive therapy aimed at improving both short- and long-term surgical outcomes. Many patients with esophageal cancer are older adults, and a decline in QOL after surgery is a concern. As long-term survival improves, preventing a decline in QOL and supporting reintegration into social life are becoming critical priorities for these patients.

Patients with esophageal cancer experience skeletal muscle loss due to a combination of poor oral intake, decreased activity, and invasive treatment. Multimodal therapies, including surgery, chemotherapy, and chemoradiotherapy, are highly invasive and cause skeletal muscle loss. In the present study, we focused on cancer-associated cachexia caused by systemic and local inflammation as an important cause of skeletal muscle loss. ESCC-bearing mice tended to lose more body weight than the non-tumor control mice. ESCC control group mice that were administered only distilled water exhibited a marked decrease in gastrocnemius muscle weight, suggesting progressive skeletal muscle loss due to cachexia. However, grip strength and gastrocnemius muscle weight in the ESCC + HMB group were maintained at the same levels as those in the non-tumor control group, suggesting that HMB is useful in treating cachexia associated with skeletal muscle loss in a mouse model of ESCC.

HMB activates the mTOR pathway (promotes muscle anabolism), increases the expression of growth hormones and insulin-like growth factor-1 (promotes muscle anabolism and suppresses degradation), promotes cholesterol synthesis (enhances tissue repair), promotes muscle satellite cell proliferation (enhances muscle regeneration), enhances Ca release from the sarcoplasmic reticulum, suppresses autophagy, and suppresses excessive inflammatory reactions. Therefore, HMB may effectively suppress skeletal muscle loss (15). Experimental study performed using in vitro methods and animal models of muscle wasting indicated that HMB may be effective in a number of disorders, notably under conditions of enhanced proteolysis in cancer (11). Specifically, in most studies, the action of HMB is mediated by the attenuation of proteasome activity and protein breakdown rather than by stimulating protein synthesis. In an in vivo study using a cancer cachexia model, oral HMB administration suppressed weight loss and skeletal muscle loss while increasing the phosphorylation of key anabolic molecules, such as p70S6K and mTOR, suggesting that these actions are mediated by improved protein anabolism in muscle (16).

To investigate whether HMB can reduce tumor burden, Nunes et al. administered the anti-catabolic agent HMB to adult Walker 256 tumor-bearing rats, a cancer cachexia model, and reported that it exerted anti-tumor and anti-cachexia effects, mediated by the tumor cell nuclear factor κB pathway (17). This suggests that HMB could serve as a potential nutritional strategy in cancer treatment. In our study, no significant difference was observed in the change in tumor volume between the ESCC control group and ESCC + HMB group. However, we observed a trend toward smaller tumors in the ESCC + HMB group, warranting further investigation. In human study, positive results have been observed in cancer-related cachexia (15). A few studies have reported that oral HMB administration is useful for increasing body weight and fat-free mass in patients with cancer-related cachexia (18,19). However, the effect of HMB on skeletal muscle loss associated with cachexia in patients with cancer remains unclear. The present results demonstrate the efficacy of HMB in mouse models of ESCC with cachexia. Future studies should confirm the mechanisms by which HMB counteracts cachexia and corroborate these physiological findings with molecular biological evidence.

The present study also has some limitations. The sample size was small and lacked blinding during grip strength measurements. Therefore, the results should be interpreted with caution. In future studies, larger groups will strengthen confidence in the conclusions. Only male mice were used to reduce variability in this study. However, sex differences may also influence metabolism, inflammation, and muscle atrophy. Our model utilizes subcutaneous tumor implantation in otherwise healthy, young mice with continuous access to food. This differs from human esophageal cancer in several ways, where patients often experience reduced food intake due to tumor obstruction and are typically older with comorbidities. The mouse model primarily captures the inflammatory aspects of cachexia but not the mechanical nutritional impediments. Moreover, HMB was administered preventively (starting before tumor establishment), whereas in clinical practice, interventions are usually initiated after cancer diagnosis. These differences indicate that the results, although promising, may not directly translate into clinical outcomes. In this study, only TE-8 was used. Notably, different cancer models may respond differently to HMB. Therefore, the generalizability of the protective effects of HMB across other cancer types and cachexia models remains to be determined.

In this animal study, one mouse in the ESCC + HMB group died on day 43 after tumor transplantation due to tumor collapse. Therefore, final weight and grip strength measurements and sampling could not be performed in this mouse. In an animal model of debilitating cachexia, the timing of assessment is crucial, and this may be considered a limitation of this study. The results of analysis of the data on the body weight and grip strength of all the 13 mice up to day 36, when it was still possible to conduct the measurements in all the mice, revealed that grip strength was significantly higher in the ESCC + HMB group than that in the ESCC control group, suggesting that HMB administration may be effective even during progression of tumor-induced cachexia. In this study, gastrocnemius weight measurements were evaluated at one endpoint (day 44). In mice with progressive cachexia, setting multiple endpoints would lead to an increase in the number of mice sacrificed. As this was our first attempt, we did not set multiple endpoints, and instead aimed to minimize the number of animals sacrificed. This one-time evaluation provides a snapshot but does not provide a trajectory of muscle loss. Muscle weakness is a dynamic process that may appear earlier or later than the selected endpoint, and whether the effects of HMB appear or persist should be investigated in future studies. Additionally, this study did not include molecular analyses to clarify how HMB administration contributes to skeletal muscle maintenance. HMB contributes to an increase in skeletal muscle mass by activating the mTOR pathway; however, the detailed molecular mechanism is unclear. Future studies should investigate the expression of skeletal muscle-related signaling proteins (such as mTOR, adenosine monophosphate-kinase, 4E-binding protein 1, muscle RING finger protein-1, atrogin-1, and p70S6K) using western blotting and reverse transcriptase-polymerase chain reaction. Such analyses could clarify how HMB administration contributes to muscle maintenance at the molecular level. As HMB suppresses excessive inflammatory responses, quantifying plasma interleukin-6 (IL-6) concentrations in mice and examining the effect of the anti-inflammatory action of HMB on skeletal muscle are crucial.

HMB is a commercially available dietary supplement. It is a metabolite of the essential amino acid leucine, naturally present in protein-rich foods, but only in very small amounts. HMB supplementation can be a versatile supportive therapy, as it can be easily and safely administered to many patients with malignant tumors. Based on our results from a mouse model, HMB administration may preserve skeletal muscle mass and contribute to improving the quality of daily and social lives of many esophageal cancer survivors. In the next phase, a large clinical study is necessary to confirm the effects of HMB on cancer-related cachexia.

Conclusions

HMB administration to ESCC-bearing mice maintained grip strength and gastrocnemius muscle weight at levels comparable to those of non-transplanted (non-ESCC) mice. To clarify that HMB administration is effective in treating skeletal muscle loss that occurs during multidisciplinary treatment of patients with aggressive esophageal cancer, future studies should investigate the mechanisms by which HMB counteracts cachexia and confirm these physiological findings with molecular biological evidence.

Acknowledgments

The authors would like to thank Dr. Kota Fukai of the Department of Preventive Medicine, Tokai University School of Medicine for helping with the statistical analysis. We would also like to thank the Medical Science College Office of Tokai University for their technical assistance and Dr. Tetsuro Nishihira, the depositor of the TE-series cell lines.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://atm.amegroups.com/article/view/10.21037/atm-25-126/rc

Data Sharing Statement: Available at https://atm.amegroups.com/article/view/10.21037/atm-25-126/dss

Peer Review File: Available at https://atm.amegroups.com/article/view/10.21037/atm-25-126/prf

Funding: This study was supported by the Japanese Foundation for Multidisciplinary Treatment of Cancer.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-25-126/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All animal experiments were performed under a project license (No. 24043) granted by the Ethics Review Committee for Animal Experimentation of Tokai University School of Medicine, in compliance with the national or institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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