5-Fluorouracil-based chemotherapies disrupt autophagy and protein synthesis in cultured myotubes: a role for mTORC1 signaling

Over the past 25 years, colorectal cancer rates in the United States have been rising in adults under the age of fifty. 5-Fluorouracil based chemotherapies, such as the FOLFOX (5-fluorouracil, leucovorin, oxaliplatin) regimen, are used to treat colorectal cancer but also induce skeletal muscle toxicities. FOLFOX can disrupt muscle autophagy signaling in vivo, however, the role of upstream autophagy regulators, mTORC1 and AMPK, in this disrupted regulation requires further investigation. We investigated whether 5-FU, 5-FU + leucovorin, oxaliplatin, or the combined FOLFOX regimen disrupts protein synthesis and autophagy flux through altered mTORC1/AMPK signaling in C2C12 myotubes with or without tumor cell-conditioned media. We also examined the effects of rapamycin administration on FOLFOX-induced disruptions to muscle protein synthesis and autophagy. Conditioned media were collected from Colon-26 (CT26) cell culture and added to day 5 differentiated myotube cultures for 48 h. FOLFOX was added to day 6 myotube cultures for 24 h. Recovery was assessed by removing FOLFOX from culture media for an additional 24 hours. Protein synthesis and autophagy flux were assessed using Puromycin and Bafilomycin A1, respectively. Rapamycin was dosed for 48 h to inhibit mTORC1. FOLFOX, CT26, and FOLFOX+CT26 all decreased myotube diameter (p=0.028, p=0.015) and protein synthesis (p< 0.001) compared to vehicle, but rps6 phosphorylation was not affected. Protein synthesis was not altered by 5-FU, 5-FU + leucovorin, or oxaliplatin treatment. FOLFOX and FOLFOX+CT26, but not CT26 alone, reduced autophagy flux, AMPK (T172) (p< 0.001), ULK1 (S757) (p=0.002), and ULK1(S555) (p< 0.001) phosphorylation compared to the vehicle. While 5-FU and 5-FU + leucovorin decreased AMPK phosphorylation, only oxaliplatin treatment reduced autophagy flux (p=0.024, p< 0.001). The FOLFOX effect was not readily reversable and did not recover after FOLFOX removal. Rapamycin increased autophagy flux in FOLFOX-treated myotubes and restored suppressed AMPK phosphorylation (p< 0.001) but did not rescue suppressed protein synthesis. We report that FOLFOX and oxaliplatin disrupt myotube mTORC1/AMPK regulation of autophagy flux, which was not readily reversible. Rapamycin treatment partially restored the FOLFOX suppression of myotube autophagy flux. Funding This work was supported by the National Institutes of Health (National Cancer Institute) grant R21 CA-231131 to JC. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.