BNIP3-Related Mitophagy-Associated Signaling in Cancer-Induced Myotube Atrophy: Modulation by 5-Fluorouracil and In Vitro Mechanistic Validation

Background: Chemotherapy, an indispensable treatment for patients with colorectal cancer (CRC), often results in some adverse effects, such as sarcopenia. This study investigates the role of Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3)-related mitophagy signaling in tumor cell-mediated myotube atrophy and its modulation by 5-fluorouracil (5-FU)-induced alterations in tumor secretory phenotype in vitro. It aims to understand the causal relationship between chemotherapy and myotube atrophy in CRC.Methods: Gene expression datasets (GSE142340, GSE153412, GSE167186) and The Cancer Genome Atlas (TCGA) data were evaluated using differential expression analysis and weighted gene co-expression network analysis (WGCNA) to identify key candidate genes linking CRC chemotherapy and sarcopenia. In vitro, C2C12 myotubes were exposed to conditioned medium from C26 colon cancer cells to establish a cancer-induced atrophy model. Myotube morphology was assessed by hematoxylin and eosin (H&E) staining. The expression of BNIP3, myotube atrophy-related proteins (MHC, MyoD, MuRF-1, Atrogin-1), and mitophagy regulators (PINK1, PRKN) was examined by western blotting. BNIP3 overexpression was performed to validate its functional role.Results: BNIP3 was identified as a candidate gene shared by CRC chemotherapy-related transcriptome changes and the age-related sarcopenia dataset. In vitro, 5-FU significantly reduced C26 cell viability. In the cancer-induced myotube atrophy model, 5-FU treatment increased myotube diameter (p < 0.05), downregulated BNIP3 expression (p < 0.01), restored MHC and MyoD levels (p < 0.001), suppressed MuRF-1 and Atrogin-1 (p < 0.001), and was associated with reduced expression of the mitophagy-related proteins PINK1 and PRKN (p < 0.01). Rescue experiments using BNIP3 overexpression reversed these effects (p < 0.01), highlighting its functional role.Conclusion: BNIP3 is a molecular node in tumor cell-mediated myotube atrophy through mitophagy-associated signaling in vitro. 5-FU attenuates cancer cell-conditioned medium-induced myotube atrophy via downregulation of BNIP3-related signaling, consistent with a tumor cell-conditioned medium-mediated effect, although residual 5-FU and direct effects cannot be fully excluded.