Evaluation of the interaction between melatonin and rapamycin for head and neck cancer therapy

Resumen

Head and neck squamous cell carcinoma (HNSCC) is the most common malignant neoplasm arising in the upper aerodigestive tract (UADT), and has the sixth highest incidence among cancers worldwide. Despite therapeutic and diagnostic advances, survival rates are still only about 50%. This is because HNSCC often involves local recurrences, distant metastases, and second primary tumours. Chemoresistance development also constitutes a major obstacle in HNSCC management. There is clearly a need for alternative treatment strategies. Cancer arises because of accumulation of genetic and/or epigenetic changes in cancer-associated genes. Among them, alterations in the PI3K /AKT/mTOR pathway are consistently observed in various cancers. Several studies suggest that mTOR signalling is important in HNSCC progression, metastasis, and therapy resistance. Unexpected Akt activation leads to activation of mTORC1, which contributes to cell growth, proliferation, and metabolism, and promotes tumorigenesis. Rapamycin, first isolated as a secondary metabolite production from Streptomyces hygroscopicus, is an allosteric inhibitor of mTORC1. Innumerable scientific and clinical studies have proved its anticancer activity, but only in a limited subset of cancer types. Rapamycin analogues, such as temsirolimus and everolimus, are under investigation for HNSCC treatment, but these drugs show poor tolerability and treatment leads to therapy resistance. Rapamycin-induced loss of the feedback inhibition of Akt may actually increase cancer cell survival, potentially resulting in more severe tumours. One means of overcoming this problem may be a combination therapeutic strategy in which the feedback inhibition of Akt is neutralized using other anticancer agents. Melatonin (N-acetyl-5-methoxytryptamine) is a potent free radical scavenger with anti-oxidant and anti-inflammatory properties, which has well-known functions in immune system regulation, modulation of mitochondrial activity, and regulation of cell death and autophagy. Moreover, melatonin has attracted attention as a natural oncostatic agent that can suppress neoplastic growth in a variety of tumours. Possible mechanisms of melatonin oncostasis induce direct and indirect antioxidant effects, regulation of metabolism, anti-angiogenic and antimetastatic effects, the capacity to decrease telomerase activity and the ability to regulate the immune system. Notably, melatonin also induces downregulation of the phosphorylation of mTOR and Akt in various cancers. Melatonin shows synergistic anticancer activity and amelioration of the side effects of several chemotherapies and thus is a potential adjuvant in chemotherapy, with the ability to protect normal cells from a variety of insults. In light of these properties, melatonin seems potentially useful as a combined anti-cancer agent to enhance the therapeutic effect of rapamycin and to minimize the toxic side effects of this drug in HNSCC. mTOR inhibitor treatment is associated with a number of adverse events in cancer patients, with mucositis being the most frequent adverse effect leading to dose reduction or interruption of therapy. We recently demonstrated that a melatonin gel prevents mucosal disruption and ulcer formation associated with oral mucositis. In the present study, we aimed to elucidate the effects of melatonin on rapamycin-induced HNSCC cell death, and to identify potential cross-talk pathways. We analyzed 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 mM 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 rapamycin inhibited the downstream target of mTOR signaling effectively but also increased Akt expression. The combined treatment with rapamycin and melatonin blocked the negative feedback loop from S6K1 to Akt signaling. The combined treatment decreased cell viability, proliferation, and clonogenic capacity. Interestingly, combined treatment with rapamycin and melatonin induced changes in mitochondrial function. Melatonin enhanced the cytotoxic effects of rapamycin by augmenting the number of dysfunctional mitochondria. Combined treatment with rapamycin and melatonin elevated ROS generation compared with rapamycin alone in a dose dependent manner. Nevertheless, ROS accumulation resulted in severe oxidative damage of mitochondria and, therefore, in mitophagy. This led to increased 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.