Head and neck squamous cell carcinoma (HNSCC) clearly involves activation of the Akt mammalian target of rapamycin (mTOR) signalling pathway. However, the effectiveness of treatment with the mTOR inhibitor rapamycin is often limited by chemoresistance. Melatonin suppresses neoplastic growth via different mechanisms in a variety of tumours. In this study, we aimed to elucidate the effects of melatonin on rapamycin-induced HNSCC cell death and to identify potential cross-talk pathways. We analysed the dose-dependent effects of melatonin in rapamycin-treated HNSCC cell lines (Cal-27 and SCC-9). These cells were treated with 0.1, 0.5 or 1 mmol/L melatonin combined with 20 nM rapamycin. We further examined the potential synergistic effects of melatonin with rapamycin in Cal-27 xenograft mice. Relationships between inhibition of the mTOR pathway, reactive oxygen species (ROS), and apoptosis and mitophagy reportedly increased the cytotoxic effects of rapamycin in HNSCC. Our results demonstrated that combined treatment with rapamycin and melatonin blocked the negative feedback loop from the specific downstream effector of mTOR activation S6K1 to Akt signalling, which decreased cell viability, proliferation and clonogenic capacity. Interestingly, combined treatment with rapamycin and melatonin-induced changes in mitochondrial function, which were associated with increased ROS production, increasing apoptosis and mitophagy. This led to increase cell death and cellular differentiation. Our data further indicated that melatonin administration reduced rapamycin-associated toxicity to healthy cells. Overall, our findings suggested that melatonin could be used as an adjuvant agent with rapamycin, improving effectiveness while minimizing its side effects.

Multiple studies reporting mitochondrial impairment in Parkinson's disease (PD) involve knockout or knockdown models to inhibit the expression of mitochondrial-related genes, including parkin, PINK1, and DJ-1 ones. Melatonin has significant neuroprotective properties, which have been related to its ability to boost mitochondrial bioenergetics. The meaning and molecular targets of melatonin in PD are yet unclear. Zebrafish are an outstanding model of PD because they are vertebrates, their dopaminergic system is comparable to the nigrostriatal system of humans, and their brains express the same genes as mammals. The exposure of 24 hpf zebrafish embryos to MPTP leads to a significant inhibition of the mitochondrial complex I and the induction of sncga gene, responsible for enhancing γ-synuclein accumulation, which is related to mitochondrial dysfunction. Moreover, MPTP inhibited the parkin/PINK1/DJ-1 expression, impeding the normal function of the parkin/PINK1/DJ-1/MUL1 network to remove the damaged mitochondria. This situation remains over time, and removing MPTP from the treatment did not stop the neurodegenerative process. On the contrary, mitochondria become worse during the next 2 days without MPTP, and the embryos developed a severe motor impairment that cannot be rescued because the mitochondrial-related gene expression remained inhibited. Melatonin, added together with MPTP or added once MPTP was removed, prevented and recovered, respectively, the parkinsonian phenotype once it was established, restoring gene expression and normal function of the parkin/PINK1/DJ-1/MUL1 loop and also the normal motor activity of the embryos. The results show, for the first time, that melatonin restores brain function in zebrafish suffering with Parkinson-like disease.

To gain insight into the mechanism of sarcopenia and the protective effect of melatonin, the gastrocnemius muscles of young (3-4 months), early-aged (12 months), and old-aged (24 months) wild-type C57BL/6J female mice were examined by magnetic resonance and microscopy. Locomotor activity, lactate production, and nuclear apoptosis were also assessed. The results support the early onset of sarcopenia at 12 months of age, with reduction of muscle fiber number, muscle weight/body weight ratio, lactate, and locomotor activity. Lipid droplet infiltration and autophagosomes were also detected. These changes driven little effects on the early-aged muscle, but they got worse in old-aged animals by the progressive damage of the muscle. Old-aged muscle showed a reduction of the mitochondrial number, a destruction of the mitochondrial cristae, and swelling. Tubular aggregates and nucleic acid fragmentation were the most striking findings in old-aged muscle, reflecting a broad damage with loss of autophagy efficacy. Oral melatonin administration conserved the normal muscular architecture, weight, muscle fiber number, and activity in the old age; it stimulated lactate production, prevented mitochondrial damage and tubular aggregates, and reduced the percentage of apoptotic nuclei in aged muscles. Altogether, gastrocnemius muscle showed age-mediated signs of sarcopenia that were reduced by melatonin treatment.