Thesis title: Oxytocin, as a hormonal treatment for cachexia in preclinical models
Introduction. Physiological Oxytocin (OT) affects the CNS, the uterus, and the mammary gland; lately, OT has been shown to promote myogenic differentiation and muscle regeneration. OT levels decrease with aging, and its exogenous administration counteracts sarcopenia in aged mice. Cachexia is a syndrome associated with chronic diseases, such as cancer, characterized by weight loss and severe muscle wasting. Cachexia interferes with therapy and lowers patients’ quality of life. Several pharmacological and hormonal treatments are currently proposed against cancer cachexia, with, for instance, the Ghrelin-R agonist Anamorelin in phase III clinical trial. However, to date, cachexia syndrome remains incurable while still accounting for about 20% of the deaths of cancer patients.
Methods. We exploited an in vitro model based on L6C5 myoblast cell cultures exposed to C26 tumor-conditioned medium (C26-CM). In vivo, a regeneration assay was performed by freeze injury on the Tibialis anterioris in mice, followed by intramuscular injections with OT, TNF, or both. Lastly, a fragment of the C26 tumor was grafted to 8-week-old male BALB/C mice. Gastrocnemius muscles and myogenic cells were analyzed by morphometric/morphologic and molecular analyses. To assess the impact of C26 and OT treatment on protein anabolism, we used BONCAT (bio-orthogonal non-canonical amino acid tagging) technology to identify the newly synthesized ANL (azidonorleucine) labeled proteins, using tRNA methionine synthase mutant (MetRSL274G) mouse and human myoblasts.
Results. We recorded in cancer patients a lower level of circulating OT that specifically associated with cachexia, suggesting that the treatment with OT may rescue muscle mass. As a preliminary, in vitro experiments showed inhibition of differentiation by the C26-CM, reversed by the addition of OT, which established a proof of principle of the efficacy of OT against the effects of tumor-derived factors. We then observed in vivo that OT accelerated muscle regeneration following focal injury, a process that is inhibited by the pro-inflammatory cytokine TNF, used to mimic the effects of inflammation on muscle homeostasis. To further confirm the efficacy of OT in counteracting the C26-dependent skeletal muscle wasting in a preclinical model, C26-tumor-bearing mice were daily injected with OT. We observed a rescue of skeletal mass, muscle fiber cross-sectional area, and of the classical markers of protein catabolism, resulting in a recovery of the bodyweight of the animals. In particular, we focused our interest on the effects of the tumor and OT on protein anabolism, an aspect of muscle metabolism that has been poorly investigated to date. In myoblasts co-cultured with tumor cells, we observed a decrease of the newly synthesized proteins, a phenomenon that is globally rescued by OT treatment. The proteins affected mainly by the tumor cells or the OT treatment are involved in multiple biological functions related to muscle homeostasis and crosstalk with other organs. These include muscle atrophy, regeneration, development, function; stem cell behavior; inflammation and immunity; cancer, bone, and nerve tissue survival and metabolism.
Conclusions. We show for the first time that OT is efficacious to counteract the effects of tumor-derived factors, which inhibit myogenic differentiation, muscle regeneration and lead to muscle wasting in tumor-bearing mice. Significantly, we did not limit our analysis to protein catabolism, which accounts for muscle fiber atrophy. Still, we extended our investigation to the synthesis of proteins and peptides by muscle cells to see how this is influenced by the presence of tumor cells, contributing to the cachectic phenotype, and if OT treatment rescues muscle cell metabolism. That is the case for most factors included in our analysis, indicating that the tumor dramatically alters muscle biology and that OT significantly contributes to the rescue of healthy muscle metabolism and function. Since OT is already authorized for clinical use, these results could readily be translated into effective clinical practice to prevent and/or treat cachexia in cancer patients.