5-Fluorouracil-based chemotherapy disrupts autophagy flux and protein synthesis in cultured myotubes: a role for mTORC1 signaling.

5-Fluorouracil-based chemotherapies, such as FOLFOX (5-fluorouracil, leucovorin, oxaliplatin), are used to treat colon cancer but also induce skeletal muscle toxicities. While FOLFOX can disrupt muscle autophagy signaling in vivo, the upstream mechanisms underlying this effect, as well as the roles of mTORC1 and AMPK signaling, require further investigation. We investigated whether 5-FU, 5-FU + leucovorin, oxaliplatin, or the combined FOLFOX regimen disrupts protein synthesis or autophagy flux through altered mTORC1/AMPK signaling in C2C12 myotubes with or without tumor cell-conditioned media, and whether these effects were readily reversible. We also examined the effects of rapamycin or metformin administration on FOLFOX-induced disruptions to muscle protein synthesis and autophagy. FOLFOX was added to myotube cultures for 24 hours; recovery was assessed by removing FOLFOX from the culture media for an additional 24 hours. Cultured Colon-26 tumor cells (CT26) were used for conditioned media. Autophagy flux was assessed using Bafilomycin A1 and LC3BII/I immunoblotting. FOLFOX and CT26 each decreased myotube diameter and protein synthesis compared to the vehicle. CT26, but not FOLFOX, increased STAT3 phosphorylation. FOLFOX, but not CT26, increased Atrogin-1 protein, while MURF-1 protein was increased by both CT26 and FOLFOX. FOLFOX reduced autophagy flux, AMPK, and ULK1(S555) phosphorylation, which did not recover upon FOLFOX removal. Rapamycin, but not metformin, altered autophagy flux in FOLFOX-treated myotubes and restored suppressed AMPK phosphorylation without increasing protein synthesis. We report that FOLFOX and oxaliplatin disrupt myotube mTORC1/AMPK regulation of autophagy flux, which was not easily reversible. However, rapamycin treatment increased autophagy flux in FOLFOX-treated myotubes.