Carnosol ameliorated cancer cachexia-associated myotube atrophy by targeting P5CS and its downstream pathways.

Introduction: Carnosol exhibited ameliorating effects on muscle atrophy of mice developed cancer cachexia in our previous research. Method: Here, the ameliorating effects of carnosol on the C2C12 myotube atrophy result from simulated cancer cachexia injury, the conditioned medium of the C26 tumor cells or the LLC tumor cells, were observed. To clarify the mechanisms of carnosol, the possible direct target proteins of carnosol were searched using DARTS (drug affinity responsive target stability) assay and then confirmed using CETSA (cellular thermal shift assay). Furthermore, proteomic analysis was used to search its possible indirect target proteins by comparing the protein expression profiles of C2C12 myotubes under treatment of C26 medium, with or without the presence of carnosol. The signal network between the direct and indirect target proteins of carnosol was then constructed. Results: Our results showed that, Delta-1-pyrroline-5-carboxylate synthase (P5CS) might be the direct target protein of carnosol in myotubes. The influence of carnosol on amino acid metabolism downstream of P5CS was confirmed. Carnosol could upregulate the expression of proteins related to glutathione metabolism, anti-oxidant system, and heat shock response. Knockdown of P5CS could also ameliorate myotube atrophy and further enhance the ameliorating effects of carnosol. Discussion: These results suggested that carnosol might ameliorate cancer cachexia-associated myotube atrophy by targeting P5CS and its downstream pathways.

Carnosol attenuated atrophy of C2C12 myotubes induced by tumour-derived exosomal miR-183-5p through inhibiting Smad3 pathway activation and keeping mitochondrial respiration.

Cancer-derived exosomes are involved in the development of cancer cachexia. Carnosol, which exhibited ameliorating effects on cancer cachexia of C26 tumour-bearing mice in our previous study, alleviated atrophy of C2C12 myotubes induced by exosomes of C26 tumour cells in the present study. MiR-183-5p was found to be rich in C26 cells and C26 exosomes, and miR-183-5p mimic could directly induce atrophy of C2C12 myotubes. Carnosol at 5 to 20 µM could dose-dependently ameliorate the myotube atrophy induced by miR-183-5p. Four and a half LIM domain protein 1 (FHL1) was shown to be the direct target of miR-183-5p. Increase in myostatin, p-Smad3, MuRF-1, Atrogin-1, HIF-1α and p-STAT3 and decrease in mitochondrial respiration were also induced by miR-183-5p mimic in C2C12 myotubes. Carnosol could not affect the decrease in FHL-1 and the activation of STAT3 pathway but could significantly alleviate the increase in myostatin, p-Smad3, MuRF-1, Atrogin-1 and the decrease in mitochondrial respiration induced by miR-183-5p. The protective effects of carnosol on myotubes against atrophy of C2C12 myotubes induced by miR-183-5p, based on both its inhibiting effects on MuRF-1 and Atrogin-1-mediated protein degradation and its ability of keeping the mitochondrial respiration, might contribute to its ameliorating effects on cancer cachexia.