Abstract
Cancer cachexia, characterized by progressive skeletal muscle loss, is common in advanced malignancies and correlates with poor prognosis. Cisplatin, a widely used chemotherapeutic, is linked to muscle atrophy, but its mechanisms remain unclear. Recent studies implicate endoplasmic reticulum (ER) stress in muscle disorders; however, its role in chemotherapy-induced muscle atrophy is unknown. This study examined the effects of five anticancer agents—cisplatin, 5-fluorouracil, vincristine, irinotecan, and cyclophosphamide—on mouse skeletal muscle. Quadriceps muscle mass, gene expression related to protein synthesis (IGF-1), degradation (MuRF1, atrogin-1), ER stress (Ddit3/CHOP, Atf4, sXbp-1), and inflammation (TNF-α, IL-1β, COX2) were analyzed. Despite similar body weight loss, cisplatin-treated mice showed a significant reduction in muscle mass compared to dietary-restricted controls. Only cisplatin upregulated MuRF1 and atrogin-1 and downregulated IGF-1. Inflammatory markers were unaffected. Notably, cisplatin induced ER stress genes Ddit3, Atf4, and sXbp1. These findings suggest cisplatin promotes muscle atrophy via ER stress activation and protein degradation, independently of reduced food intake or inflammation. Targeting ER stress may help prevent chemotherapy-induced muscle wasting. Further studies are needed to clarify mechanisms and develop protective strategies.
INTRODUCTION
We previously administered cisplatin, a platinum-based drug that is commonly used to treat various cancers, to mice and measured their skeletal muscle weight and muscle fiber diameter. The results showed significant reductions in skeletal muscle mass and muscle fiber diameter (Sakai et al., 2020). Skeletal muscle mass is regulated by a balance between anabolic and catabolic pathways. The insulin-like growth factor-1 (IGF-1)/mTOR pathway promotes protein synthesis and muscle growth, while catabolic processes are mediated by the ubiquitin–proteasome system through FoxO transcription factors and muscle-specific E3 ubiquitin ligases such as MuRF1 and MAFbx (atrogin-1) (Bodine et al., 2001). To explore the mechanisms underlying cisplatin-induced muscle atrophy, we also analyzed the gene expression in the skeletal muscle of treated mice. Compared with dietary-restricted controls with weight loss similar to that of cisplatin-treated mice, cisplatin markedly upregulated MuRF1 and atrogin-1 at the mRNA and protein levels. This upregulation of MuRF1 and atrogin-1 was associated with the FoxO pathway activation. Although hepatic IGF-1 expression remained unchanged, local IGF-1 levels in the muscle reduced, and the IGF-1/mTOR pathway, which is critical for muscle growth, was suppressed (Sakai et al., 2021). Taken together, these results demonstrate that cisplatin induces muscle atrophy by concurrently enhancing protein degradation pathways and suppressing protein synthesis pathways in skeletal muscle. Cancer cachexia, characterized by substantial skeletal muscle loss, is common in advanced cancer (Setiawan et al., 2023). Studies have reported poor survival outcomes associated with muscle loss during chemotherapy in patients with various types of cancers (Armstrong et al., 2020). These findings suggest that cancer and cisplatin treatment contribute to muscle mass reduction.
The endoplasmic reticulum (ER) stress response is a cellular defense mechanism triggered by misfolded protein accumulation in the ER. Normally, the ER ensures proper folding and processing of secretory and membrane proteins. However, stressors such as hypoxia, nutrient deprivation, or drug exposure impair ER function, causing ER stress. In response, cells activate the unfolded protein response (UPR) to restore homeostasis. The UPR is mediated by three sensors: inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Activated IRE1 splices X-box binding protein 1 (Xbp-1) mRNA to produce sXBP-1, a transcription factor that induces genes for protein folding and degradation. IRE1 signaling also promotes ATF4, which regulates amino acid metabolism, redox balance, and apoptosis. PERK phosphorylates eukaryotic translation initiation factor 2α (eIF2α), transiently reducing global protein synthesis. ATF6 is cleaved in the Golgi and moves to the nucleus to activate stress-response genes. These pathways help restore ER integrity, but prolonged stress can lead to apoptosis and dysfunction (Chen et al., 2023).
ER stress is increasingly recognized as a potential pathogenic factor in various muscle disorders, suggesting that targeting ER stress response pathways represents a promising therapeutic approach for disuse-induced muscle atrophy (Wang et al., 2023). However, it remains unclear whether chemotherapeutic agents other than cisplatin affect muscle mass or whether ER stress contributes to cisplatin-induced muscle atrophy. In this study, we examined the effects of five widely used chemotherapeutic agents—cisplatin, 5-fluorouracil, vincristine, irinotecan, and cyclophosphamide—on the expression of genes regulating skeletal muscle mass, focusing on those involved in protein synthesis and degradation. We also analyzed changes in ER stress-related gene expression, given their potential role in muscle disorders.
MATERIALS AND METHODS
Animals and schedule for the anticancer drug injections
All animal experiments were approved by the Animal Welfare Committee of Hoshi University of Pharmacy and Life Sciences (Approval No. P21-059 and P24-096). Male C57BL/6J mice (8–9 weeks old, 3-4 mice/group) were sourced from Tokyo Laboratory Animals Science Co. (Tokyo, Japan). They received intraperitoneal injections of cisplatin (3 mg/kg, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 5-fluorouracil (50 mg/kg, FUJIFILM Wako Pure Chemical Corporation), vincristine (0.5 mg/kg, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), irinotecan (60 mg/kg, Alfresa Pharma Corporation, Osaka, Japan) and cyclophosphamide (100 mg/kg, FUJIFILM Wako Pure Chemical Corporation) once daily for 4 days (days 0–3), with saline administered as a control. The doses of anticancer drugs were selected based on previously reported repeated-dose mouse models that are widely used and have been shown to be pharmacologically effective without inducing severe systemic toxicity in mice (Zhang et al., 2018; Starobova et al., 2019; Chen et al., 2013; Tsarovsky et al., 2023). In addition, as a control for weight loss induced by cisplatin-containing anticancer drugs, a dietary restriction (DR) group was used, designed based on the method described in a previous report (Sakai et al., 2020).
Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Isolation of total RNA from skeletal muscle samples and reverse transcription were conducted as previously described (Sakai et al., 2020). Primers used for qRT-PCR of Xbp-1 and sXbp-1 were as previously reported (Yoon et al., 2019). The primer sets used are listed in the Supplemental materials.
Immunoblots
Tissue and cell sample preparation, along with immunoblotting, was carried out following the previously established protocol (Sakai et al., 2014; Sakai et al., 2020). In brief, tissue homogenates and cell lysates were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with Tris-buffered saline containing 2.5% non-fat dry milk, 2.5% bovine serum albumin, and 0.1% Tween 20. Primary antibodies used in the analysis included rabbit anti-MuRF1 (1:1,000 dilution, ECM Biosciences, KY, USA), mouse anti-Atf4 (1:1,000 dilution, Santa Cruz Biotechnology), and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (Gapdh) (1:10,000 dilution, Proteintech). Secondary antibodies conjugated with horseradish peroxidase (HRP) included horse anti-mouse immunoglobulin G (IgG) (1:3,000 dilution, Cell Signaling Technology, MA, USA) and goat anti-rabbit IgG (1:3,000 dilution, Cell Signaling Technology). Protein expression levels were quantified by normalizing MuRF1 and Atf4 signals to the Gapdh housekeeping protein. Total protein loading was assessed using Ponceau S staining.
Statistical analysis
All data are presented as mean ± standard error of the mean (SEM). Statistical comparisons were performed using one-way ANOVA followed by Bonferroni/Dunn or Dunnett's multiple comparison test, as appropriate. Error bar calculations and analysis of significance tests were performed using GraphPad Prism version 10 for macOS (GraphPad Software, Inc.). Statistical significance was defined as a p-value less than 0.05.
RESULTS
Effects of dietary restriction (DR) and intraperitoneal chemotherapy on body weight, quadriceps muscle mass, and skeletal muscle gene expression related to protein synthesis and degradation in mice
Because the administration of the anticancer drug induces weight loss, two control groups were included in this study: a vehicle-treated group and a DR group that experienced weight loss comparable to that caused by the drug (Fig. 1A). Despite having equivalent body weights, mice treated with cisplatin exhibited considerably reduced quadriceps muscle mass compared with the DR controls (Fig. 1B). These findings suggest that muscle atrophy occurs due to reduced food intake or nutritional deficiency and that cisplatin directly contributes to muscle atrophy.
MuRF1 and atrogin-1 are established markers of muscle atrophy that cause increased gene expression. Moreover, we previously reported that cisplatin treatment in mice induces muscle atrophy accompanied by the upregulation of these genes (Sakai et al., 2020). Based on this, we investigated the expression of these genes in the skeletal muscle of mice treated with various anticancer drugs. Among the anticancer drugs tested in this study, only cisplatin induced a significant increase in the gene expression of MuRF1 and atrogin-1 (Fig. 1C and D), along with a significant decrease in IGF-1 gene expression (Fig. 1E). Because inflammation plays a key role in skeletal muscle atrophy, partly through the activation of inflammatory cytokines (Ji et al., 2022), we evaluated the gene expression of the proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β). Given that both cytokines are known to induce cyclooxygenase-2 (COX2) expression (Kuldo et al., 2005), changes in COX2 mRNA levels were also assessed. However, there were no significant differences in the gene expression levels among all the anticancer drug-treated and DR groups (Fig. 1F–H).
Changes in ER stress response-related gene expression in the quadriceps muscle of mice following DR and intraperitoneal administration of various cytotoxic anticancer drugs
It is well established that the gene expression of DNA damage-inducible transcript 3 (Ddit3)/ C/EBP homologous protein (CHOP), Activating transcription factor 4 (Atf4), and Xbp-1 is elevated during ER stress response (Chen et al., 2023). Therefore, we investigated changes in the expression of these genes in the skeletal muscle during the administration of anticancer drugs. The expression of all these genes was significantly increased only in the cisplatin-treated group compared with the DR group (Fig. 2A-C). Furthermore, the gene expression of sXbp-1, an active spliced form and transcriptional activator of Xbp-1, was also significantly elevated (Fig. 2D).
Changes in protein levels of MuRF1 and Atf4 in the quadriceps muscle of mice following DR and intraperitoneal administration of cisplatin
Increased expression of genes causing muscle atrophy and increased expression of ER stress-related genes were observed. Therefore, we focused on the cisplatin-treated group and investigated the protein expression levels of these genes. In skeletal muscle from cisplatin-treated mice, protein expression levels of MuRF1 and Atf4 were significantly elevated compared to the control and DR groups. However, no obvious changes were observed in Ponceau-S staining or Gapdh band density across all groups (Fig. 3).
DISCUSSION
In this study, the gene expression of the muscle atrophy markers MuRF1 and atrogin-1 were markedly upregulated only in the cisplatin-treated group. These findings align with those of previous studies (Sakai et al., 2020; Sakai et al., 2021), which demonstrated that cisplatin treatment induces muscle atrophy and increases the expression of these atrogenes. Notably, among the various anticancer drugs tested, only cisplatin resulted in this specific upregulation, indicating the involvement of a unique mechanism of muscle toxicity. Furthermore, the expression of IGF-1, which plays a crucial role in promoting muscle protein synthesis and maintaining muscle mass, was markedly reduced in the cisplatin group. These changes suggest that cisplatin disrupts the balance between protein synthesis and degradation in the muscle tissue, leading to progressive muscle loss.
In connection with these findings, the expression of the genes related to ER stress Ddit3/CHOP, Atf4, and Xbp-1 was selectively upregulated in the cisplatin-treated group. Notably, sXbp-1, an active transcription factor produced during ER stress, was markedly upregulated only in this group. sXbp-1 plays a central role in regulating the expression of genes involved in protein folding, ER-associated degradation (ERAD), and the maintenance of ER homeostasis. These findings suggest that cisplatin induces ER stress in skeletal muscles, which may contribute to muscle atrophy. ER stress can disrupt cellular homeostasis and trigger apoptosis, particularly in metabolically active tissues such as the skeletal muscles. The upregulation of sXbp-1 may represent an adaptive response; however, prolonged activation could result in maladaptive effects and muscle degradation. In the present study, ATF4 and CHOP were examined as markers that mainly reflect activation of the PERK pathway, and to some extent the ATF6 pathway, while XBP1 reflects activation of the IRE1 pathway. Accordingly, cisplatin-induced skeletal muscle toxicity may involve coordinated activation of multiple UPR pathways rather than a single pathway.
In this study, increased expression of MuRF1 and ATF4 in the quadriceps femoris muscle of cisplatin-treated mice was confirmed at the protein level by immunoblotting. This further supports that altered protein metabolism and ER stress-related signaling are associated with cisplatin-induced skeletal muscle atrophy. Previous report has demonstrated suppression of the IGF-1/PI3K/Akt signaling pathway following cisplatin administration (Sakai et al., 2021). However, the causal relationship between this pathway and ER stress signaling in skeletal muscle atrophy remains unclear. At present, it is difficult to determine whether ER stress occurs upstream or downstream of IGF-1/PI3K/Akt signaling, and these pathways may interact with each other depending on the situation. Furthermore, pharmacological inhibition of ER stress would likely yield new insights into the mechanisms of cisplatin-induced skeletal muscle atrophy. However, as this study was designed as a letter article for comparative analysis and hypothesis generation, such functional experiments are beyond its scope and represent an important topic for future research.
One limitation of the present study is that each anticancer drug was examined at a single dose. Although the selected doses were based on commonly used dosing regimens in experimental mouse models, further dose–response analyses would help clarify whether skeletal muscle toxicity differs among anticancer agents in a dose-dependent manner. Furthermore, in addition to alterations in protein metabolism, differences in tissue distribution or retention of cisplatin in skeletal muscle may also contribute to its myotoxicity. Because the present study did not assess intramuscular drug concentrations, this possibility cannot be excluded and warrants further investigation. Together, these results indicate that cisplatin-induced muscle atrophy occurs independently of reduced food intake, inflammatory pathways or signals and involves three key mechanisms: activation of the ubiquitin–proteasome system via MuRF1 and atrogin-1, suppression of anabolic IGF-1 signaling, and induction of ER stress pathways. This study identifies ER stress as a novel contributor to chemotherapy-induced muscle wasting and a potential therapeutic target. Further research is needed to clarify whether modulating ER stress can prevent muscle atrophy during cisplatin treatment and improve outcomes and quality of life in cancer patients.
ACKNOWLEDGMENTS
The authors would like to thank Enago (www.enago.jp) for the English language review.
Funding
This study was funded by JSPS (Japan Society for the Promotion of Science) KAKENHI Grant-in-Aid for Scientific Research (C), Grant Number 22K06869 (to H.S.) and Hoshi University Otani Research Grants 2022. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Conflict of interest
The authors declare that there is no conflict of interest.
Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Author contributions
Conceptualization: Hiroyasu Sakai
Funding acquisition: Hiroyasu Sakai
Investigation: Shinki Soga, Hayato Nanri, Ryunosuke Ichikawa, Yuya Chigusa, Yui Urase, Shiori Yonamine, Takayuki Ogiwara
Supervision: Kumiko Ogawa
Writing – original draft: Shinki Soga, Hayato Nanri
Writing – review & editing: Hiroyasu Sakai, Risako Kon, Nobutomo Ikarashi, Yoshihiko Chiba, Tomoo Hosoe, Kumiko Ogawa
Ethical approval and consent to participate
All animal experiments were approved by the Animal Welfare Committee of Hoshi University of Pharmacy and Life Sciences (Approval No. P21-059 and P24-096).
Patient consent for publication
Not applicable.
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