Escames, Germaine

Metabolic rewiring in glioblastoma (GBM) is linked to intra- and extracellular pH regulation. In this study, we sought to characterize the role of melatonin on intracellular pH modulation and metabolic consequences to identify the mechanisms of action underlying melatonin oncostatic effects on GBM tumor initiating cells. GBM tumor initiating cells were treated at different times with melatonin (1.5 and 3.0 mM). We analyzed melatonin’s functional effects on GBM proliferation, cell cycle, viability, stemness, and chemo-radiosensitivity. We then assessed the effects of melatonin on GBM metabolism by analyzing the mitochondrial and glycolytic parameters. We also measured the intracellular and extracellular pH. Finally, we tested the effects of melatonin on a mouse subcutaneous xenograft model. We found that melatonin downregulated LDHA and MCT4, decreasing lactate production and inducing a decrease in intracellular pH that was associated with an increase in ROS and ATP depletion. These changes blocked cell cycle progression and induced cellular death and we observed similar results in vivo. Melatonin’s cytotoxic effects on GBM were due, at least in part, to intracellular pH modulation, which has emerged as a newly identified mechanism, providing new insights into the oncostatic effect of melatonin on GBM.

Metabolic reprogramming, which is characteristic of cancer cells that rapidly adapt to the hypoxic microenvironment and is crucial for tumor growth and metastasis, is recognized as one of the major mechanisms underlying therapeutic resistance. Mitochondria, which are directly involved in metabolic reprogramming, are used to design novel mitochondria-targeted anticancer agents. Despite being targeted by melatonin, the functional role of mitochondria in melatonin’s oncostatic activity remains unclear. In this study, we aim to investigate the role of melatonin in mitochondrial metabolism and its functional consequences in head and neck cancer. We analyzed the effects of melatonin on head and neck squamous cell carcinoma (HNSCC) cell lines (Cal-27 and SCC-9), which were treated with 100, 500, and 1500 µM of melatonin for 1, 3, and 5 days, and found a connection between a change of metabolism following melatonin treatment and its effects on mitochondria. Our results demonstrate that melatonin induces a shift to an aerobic mitochondrial metabolism that is associated with changes in mitochondrial morphology, function, fusion, and fission in HNSCC. We found that melatonin increases oxidative phosphorylation (OXPHOS) and inhibits glycolysis in HNSCC, resulting in increased ROS production, apoptosis, and mitophagy, and decreased cell proliferation. Our findings highlight new molecular pathways involved in melatonin’s oncostatic activity, suggesting that it could act as an adjuvant agent in a potential therapy for cancer patients. We also found that high doses of melatonin, such as those used in this study for its cytotoxic impact on HNSCC cells, might lead to additional effects through melatonin receptors.

Abstract Glioblastoma (GBM) is the most common form of malignant primary brain cancer in adults with a median survival of only 15 months. Therefore, new therapies to suppress malignant brain cancer are needed. Brain Tumor Initiating Cells (BTICs) are a GBM subpopulation of cells with a highly glycolytic profile that are thought to be responsible of the resistance of GBM to treatments. Metabolic reprogramming allows tumor cells to survive in unsupportive microenvironments. Manipulating tumor metabolism to counteract GBM resistance arises as a powerful approach with minimum effects in normal counterparts. At pharmacological concentrations, melatonin displays oncostatic properties. This is thought to be due to an increase in mitochondrial oxidative phosphorylation through the effects of melatonin in mitochondria, key organelle in metabolic homeostasis. We hypothesize that melatonin could alter BTIC metabolism, by inducing an anti-Warburg effect and as consequence, melatonin will decrease the viability of GBM cells and tumor growth. We found that treatment of GBM cell lines with 3mM melatonin significantly altered tumor cell metabolism. We observed that melatonin downregulated the lactate symporter MCT4 (p<0.002), inducing a significant intracellular accumulation of lactate (p<0.002) while decreasing it in the extracellular media (p<0.001). This was followed by a decrease in the internal pH (p<0.002). These effects were compensated by an increase in the oxygen consumption rate (OCR) followed by decay that leaded to an increase in ROS production (p<0.001). All these changes result in a depletion of cellular ATP (p<0.001) and eventually drove to a decrease in the proliferation (p<0.001) and cell death (p<0.001). When applied in vivo we observed a significant reduction in the tumor growth (p<0.001), volume (p<0.002) and weight (p<0.002), as well as a drop in the proliferation marker ki67 (p<0.001) and a fibrosis increase in treated tumors. These results position melatonin as a strong therapeutic candidate for GBM therapy.

Abstract Around fifteen to thirty percent of stage IV breast cancer metastasizes to the brain, severely decreasing the quality of life of these patients by causing neurological decline and eventually death. In metastatic cancers there is a small subset of cells in the primary tumor bulk called Metastatic Tumor Initiating Cells (MTICs) which are able to escape and produce a niche establishment at distal sites where they can quickly become resistant to surgery and radiation. Melatonin has shown an inhibitory role in the viability and invasiveness of breast cancer and in modulating the expression of proteins related to Breast Cancer Stem Cells (BCSCs). These findings suggest its potential anti-metastatic role in different breast cancer cell lines. In this study we aimed to evaluate the effects of melatonin treatment in vitro for breast cancer brain metastasis. The cell line MDA-BT was originally obtained from MDA-MB-231, passed through the rat’s heart and then isolated once engrafted as a tumor in the brain. After a dose response assay, cells were treated with melatonin at doses of 1500 and 3000 µM for 48hrs. Clonogenic assay, MTT, as well as a stem cell signature through RT-qPCR, including CD44, CD24 and ALDH1 markers, were performed to evaluate the malignancy of the MTICs. The results showed that melatonin at high doses impacts morphology, declines viability, reduces colony formation ability, and decreases stemness in MDA-BT cells. Therefore, our findings highlight melatonin as a relevant therapeutic candidate to target breast cancer brain metastases.

Head and neck cancer is the sixth leading cancer by incidence worldwide. Unfortunately, drug resistance and relapse are the principal limitations of clinical oncology for many patients, and the failure of conventional treatments is an extremely demoralizing experience. It is therefore crucial to find new therapeutic targets and drugs to enhance the cytotoxic effects of conventional treatments without potentiating or offsetting the adverse effects. Melatonin has oncostatic effects, although the mechanisms involved and doses required remain unclear. The purpose of this study is to determine the precise underlying mitochondrial mechanisms of melatonin, which increase the cytotoxicity of oncological treatments, and also to propose new melatonin treatments in order to alleviate and reverse radio- and chemoresistant processes. We analyzed the effects of melatonin on head and neck squamous cell carcinoma (HNSCC) cell lines (Cal-27 and SCC-9), which were treated with 0.1, 0.5, 1, and 1.5 mM melatonin combined with 8 Gy irradiation or 10 μM cisplatin. Clonogenic and MTT assays, as well as autophagy and apoptosis, involving flow cytometry and western blot, were performed in order to determine the cytotoxic effects of the treatments. Mitochondrial function was evaluated by measuring mitochondrial respiration, mtDNA content (RT-PCR), and mitochondrial mass (NAO). ROS production, antioxidant enzyme activity, and GSH/GSSG levels were analyzed using a fluorometric method. We show that high concentrations of melatonin potentiate the cytotoxic effects of radiotherapy and CDDP in HNSCC, which are associated with increased mitochondrial function in these cells. In HNSCC, melatonin induces intracellular ROS, whose accumulation plays an upstream role in mitochondria-mediated apoptosis and autophagy. Our findings indicate that melatonin, at high concentrations, combined with cisplatin and radiotherapy to improve its effectiveness, is a potential adjuvant agent.