Study of the mitochondria as a new therapeutic target against head and neck cancerevaluation of melatonin effects

Abstract

Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancer by incidence worldwide. Despite therapeutic and diagnostic advances, survival rates are still only about 50%. Drug resistance and relapse are the principal limitations of clinical oncology for many patients. It is therefore crucial to find new therapeutic targets and drugs to enhance the cytotoxic effects of conventional treatments without potentiating the adverse effects. Reactive oxygen species (ROS) constitute a group of highly reactive molecules that have involved as regulators of important signaling pathways, but they are also involved in some pathologies such as cancer. In this context, tumor cells have an altered redox balance compared to normal cells, which can be targeted as an antitumoral therapy by increasing ROS levels and by decreasing the capacity of the antioxidant system, leading to programmed cell death. Regarding that, melatonin is of particular importance in the development of innovative cancer treatments due to the regulation of ROS production in both normal and cancer cells. It has been demonstrated that melatonin has a dual effect, reducing ROS production in normal cells but increasing them in tumoral cells. However, despite the antioxidant effect of melatonin in normal cells has been described, the pro-oxidant mechanism of action of melatonin in tumoral cells remains unclear, which has hindered its use in clinical treatments. In the present study, we aimed to elucidate the mechanism of action of melatonin to induce ROS production in in head and neck squamous cancer cells. We analyzed the effects of melatonin on HNSCC cell lines (Cal‐27 and SCC‐9), which were treated with 0.5 or 1mM melatonin. We further examined the potential effects of melatonin to induce ROS and apoptosis in Cal‐27 xenograft mice. We measured ROS production using fluorometric methods and apoptosis levels by flow cytometry in HNSCC cells alone or in presence of N-acetyl cysteine (NAC) and mitochondrial inhibitors. In addition, we analyzed some mitochondrial parameters, such as: OXPHOS expression by Western Blot, mitochondria complex (CI–CV) activity by spectrophotometric analysis, mitochondrial respiration by SeaHorse and with a Clark oxygen electrode, and ATP levels by spectrophotometric technics. Likewise, CoQ levels by liquid chromatography–mass spectrometer (UPLC-MS/MS), mitochondrial membrane potential by fluorimetric methods and UCPs expressions by western blot were studied. We also performed a metabolomics study by UPLC-MS/MS, as well as an analysis of mitochondrial supercomplexes conformation by blue native gel electrophoresis (BNGE). Finally, we tested melatonin pro-oxidant and apoptotic effects in vivo in Cal-27 expressing alternative oxidase (AOX) protein, which avoid reverse electron transport (RET) ROS production, and in vivo inducing Cal‐27 wild type and Cal- 27 expressing AOX xenograft mice. We demonstrated that melatonin mediates apoptosis in head and neck cancer by driving mitochondrial reverse electron transport (RET) to induce ROS production. Melatonin‐induced changes in tumoral metabolism and increased mitochondrial activity which, in turn, induced ROS‐dependent mitochondrial uncoupling. Interestingly, mitochondrial complex inhibitors, including rotenone, abolished the ROS elevation indicating that melatonin increased ROS generation via RET. Melatonin also increased the membrane potential and CoQ10H2/CoQ10 ratio to elevate mitochondrial ROS production, which are essential conditions for RET. We found that genetic manipulation of Cal-27 cells with alternative oxidase, which transfers electrons from QH2 to oxygen, inhibited melatonin‐induced ROS generation, and apoptosis, highlighting the importance of RET as the site of ROS production. These results illustrate that RET and ROS production are crucial factors for melatonin's effects in cancer cells and establish the dual effect of melatonin, to protect normal cells and to induce apoptosis in cancer cells. Our data show, for the first time, that melatonin induces ROS complex I RET and leads to apoptosis from excessive ROS production, only at high doses, in Cal-27 and SCC9 cells. This pathway clarifies the proposed mechanism of action of melatonin inducing ROS production in cancer cells in order to propose future anti-neoplastic clinical applications.