From Chronodisruption to Sarcopenia: The Therapeutic Potential of Melatonin.

Sarcopenia is an age-related condition that involves a progressive decline in muscle mass and function, leading to increased risk of falls, frailty, and mortality. Although the exact mechanisms are not fully understood, aging-related processes like inflammation, oxidative stress, reduced mitochondrial capacity, and cell apoptosis contribute to this decline. Disruption of the circadian system with age may initiate these pathways in skeletal muscle, preceding the onset of sarcopenia. At present, there is no pharmacological treatment for sarcopenia, only resistance exercise and proper nutrition may delay its onset. Melatonin, derived from tryptophan, emerges as an exceptional candidate for treating sarcopenia due to its chronobiotic, antioxidant, and anti-inflammatory properties. Its impact on mitochondria and organelle, where it is synthesized and crucial in aging skeletal muscle, further highlights its potential. In this review, we discuss the influence of clock genes in muscular aging, with special reference to peripheral clock genes in the skeletal muscle, as well as their relationship with melatonin, which is proposed as a potential therapy against sarcopenia.

The Relationship between Clock Genes, Sirtuin 1, and Mitochondrial Activity in Head and Neck Squamous Cell Cancer: Effects of Melatonin Treatment.

The circadian clock is a regulatory system, with a periodicity of approximately 24 h, which generates rhythmic changes in many physiological processes, including mitochondrial activity. Increasing evidence links chronodisruption with aberrant functionality in clock gene expression, resulting in multiple diseases such as cancer. Melatonin, whose production and secretion oscillates according to the light-dark cycle, is the principal regulator of clock gene expression. In addition, the oncostatic effects of melatonin correlate with an increase in mitochondrial activity. However, the direct links between circadian clock gene expression, mitochondrial activity, and the antiproliferative effects of melatonin in cancers, including head and neck squamous cell carcinoma (HNSCC), remain largely unknown. In this study, we analyzed the effects of melatonin on HNSCC cell lines (Cal-27 and SCC9), which were treated with 500 and 1000 µM melatonin. We found that the antiproliferative effect of melatonin is not mediated by the Bmal1 clock gene. Additionally, high doses of melatonin were observed to result in resynchronization of oscillatory circadian rhythm genes (Per2 and Sirt1). Surprisingly, the resynchronizing effect of melatonin on Per2 and Sirt1 did not produce alterations in the oscillation of mitochondrial respiratory activity. These results increase our understanding of the possible antiproliferative mechanisms in melatonin in the treatment of head and neck squamous cell carcinoma and suggest that its antiproliferative effects are independent of clock genes but are directly related to mitochondrial activity.

Patient-derived tumor models in cancer research: Evaluation of the oncostatic effects of melatonin.

The development of new anticancer therapies tends to be very slow. Although their impact on potential candidates is confirmed in preclinical studies, ∼95 % of these new therapies are not approved when tested in clinical trials. One of the main reasons for this is the lack of accurate preclinical models. In this context, there are different patient-derived models, which have emerged as a powerful oncological tool: patient-derived xenografts (PDXs), patient-derived organoids (PDOs), and patient-derived cells (PDCs). Although all these models are widely applied, PDXs, which are created by engraftment of patient tumor tissues into mice, is considered more reliable. In fundamental research, the PDX model is used to evaluate drug-sensitive markers and, in clinical practice, to select a personalized therapeutic strategy. Melatonin is of particular importance in the development of innovative cancer treatments due to its oncostatic impact and lack of adverse effects. However, the literature regarding the oncostatic effect of melatonin in patient-derived tumor models is scant. This review aims to describe the important role of patient-derived models in the development of anticancer treatments, focusing, in particular, on PDX models, as well as their use in cancer research. This review also summarizes the existing literature on the anti-tumoral effect of melatonin in patient-derived models in order to propose future anti-neoplastic clinical applications.

Intratumoral injection of melatonin enhances tumor regression in cell line-derived and patient-derived xenografts of head and neck cancer by increasing mitochondrial oxidative stress.

Head and neck squamous cell carcinoma present a high mortality rate. Melatonin has been shown to have oncostatic effects in different types of cancers. However, inconsistent results have been reported for in vivo applications. Consequently, an alternative administration route is needed to improve bioavailability and establish the optimal dosage of melatonin for cancer treatment. On the other hand, the use of patient-derived tumor models has transformed the field of drug research because they reflect the heterogeneity of patient tumor tissues. In the present study, we explore mechanisms for increasing melatonin bioavailability in tumors and investigate its potential as an adjuvant to improve the therapeutic efficacy of cisplatin in the setting of both xenotransplanted cell lines and primary human HNSCC. We analyzed the effect of two different formulations of melatonin administered subcutaneously or intratumorally in Cal-27 and SCC-9 xenografts and in patient-derived xenografts. Melatonin effects on tumor mitochondrial metabolism was also evaluated as well as melatonin actions on tumor cell migration. In contrast to the results obtained with the subcutaneous melatonin, intratumoral injection of melatonin drastically inhibited tumor progression in HNSCC-derived xenografts, as well as in patient-derived xenografts. Interestingly, intratumoral injection of melatonin potentiated CDDP effects, decreasing Cal-27 tumor growth. We demonstrated that melatonin increases ROS production and apoptosis in tumors, targeting mitochondria. Melatonin also reduces migration capacities and metastasis markers. These results illustrate the great clinical potential of intratumoral melatonin treatment and encourage a future clinical trial in cancer patients to establish a proper clinical melatonin treatment.

Role of Melatonin in Cancer: Effect on Clock Genes.

The circadian clock is a regulatory system, with a periodicity of approximately 24 h, that generates rhythmic changes in many physiological processes. Increasing evidence links chronodisruption with aberrant functionality in clock gene expression, resulting in multiple diseases, including cancer. In this context, tumor cells have an altered circadian machinery compared to normal cells, which deregulates the cell cycle, repair mechanisms, energy metabolism and other processes. Melatonin is the main hormone produced by the pineal gland, whose production and secretion oscillates in accordance with the light:dark cycle. In addition, melatonin regulates the expression of clock genes, including those in cancer cells, which could play a key role in the numerous oncostatic effects of this hormone. This review aims to describe and clarify the role of clock genes in cancer, as well as the possible mechanisms of the action of melatonin through which it regulates the expression of the tumor's circadian machinery, in order to propose future anti-neoplastic clinical treatments.

Primary head and neck cancer cell cultures are susceptible to proliferation of Epstein-Barr virus infected lymphocytes.

BACKGROUND: New concepts for a more effective anti-cancer therapy are urgently needed. Experimental flaws represent a major counter player of this development and lead to inaccurate and unreproducible data as well as unsuccessful translation of research approaches into clinics. In a previous study we have created epithelial cell cultures from head and neck squamous cell carcinoma (HNSCC) tissue. METHODS: We characterize primary cell populations isolated from human papillomavirus positive HNSCC tissue for their marker expression by RT-qPCR, flow cytometry, and immunofluorescence staining. Their sensitivity to MDM2-inhibition was measured using cell viability assays. RESULTS: Primary HNSCC cell cultures showed the delayed formation of spheroids at higher passages. These spheroids mimicked the morphology and growth characteristics of other established HNSCC spheroid models. However, expression of epithelial and mesenchymal markers could not be detected in these cells despite the presence of the HNSCC stem cell marker aldehyde dehydrogenase 1 family member A1. Instead, strong expression of B- and T-lymphocytes markers was observed. Flow cytometry analysis revealed a heterogeneous mixture of CD3 + /CD25 + T-lymphocytes and CD19 + B-lymphocytes at a ratio of 4:1 at passage 5 and transformed lymphocytes at late passages (≥ passage 12) with CD45 + CD19 + CD20 + , of which around 10 to 20% were CD3 + CD25 + CD56 + . Interestingly, the whole population was FOXP3-positive indicative of regulatory B-cells (Bregs). Expression of transcripts specific for the Epstein-Barr-virus (EBV) was detected to increase in these spheroid cells along late passages, and this population was vulnerable to MDM2 inhibition. HPV + HNSCC cells but not EBV + lymphocytes were detected to engraft into immunodeficient mice. CONCLUSIONS: In this study we present a primary cell culture of EBV-infected tumor-infiltrating B-lymphocytes, which could be used to study the role of these cells in tumor biology in future research projects. Moreover, by describing the detailed characteristics of these cells, we aim to caution other researchers in the HNSCC field to test for EBV-infected lymphocyte contaminations in primary cell cultures ahead of further experiments. Especially researchers who are interested in TIL-based adopted immunotherapy should exclude these cells in their primary tumor models, e.g. by MDM2-inhibitor treatment. BI-12-derived xenograft tumors represent a suitable model for in vivo targeting studies.

Understanding the Mechanism of Action of Melatonin, Which Induces ROS Production in Cancer Cells.

Reactive oxygen species (ROS) constitute a group of highly reactive molecules that have evolved as regulators of important signaling pathways. In this context, tumor cells have an altered redox balance compared to normal cells, which can be targeted as an antitumoral therapy by ROS levels and by decreasing the capacity of the antioxidant system, leading to programmed cell death. Melatonin is of particular importance in the development of innovative cancer treatments due to its oncostatic impact and lack of adverse effects. Despite being widely recognized as a pro-oxidant molecule in tumor cells, the mechanism of action of melatonin remains unclear, which has hindered its use in clinical treatments. The current review aims to describe and clarify the proposed mechanism of action of melatonin inducing ROS production in cancer cells in order to propose future anti-neoplastic clinical applications.

Melatonin drives apoptosis in head and neck cancer by increasing mitochondrial ROS generated via reverse electron transport.

The oncostatic effects of melatonin correlate with increased reactive oxygen species (ROS) levels, but how melatonin induces this ROS generation is unknown. In the present study, we aimed to elucidate the two seemingly opposing actions of melatonin regarding its relationship with free radicals. We analyzed the effects of melatonin on head and neck squamous cell carcinoma cell lines (Cal-27 and SCC-9), which were treated with 0.5 or 1 mM melatonin. We further examined the potential effects of melatonin to induce ROS and apoptosis in Cal-27 xenograft mice. Here we report 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 led to 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 membrane potential and CoQ10 H2 /CoQ10 ratio to elevate mitochondrial ROS production, which are essential conditions for RET. We found that genetic manipulation of cancer cells with alternative oxidase, which transfers electrons from QH2 to oxygen, inhibited melatonin-induced ROS generation, and apoptosis. RET restored the melatonin-induced oncostatic effect, highlighting the importance of RET as the site of ROS production. These results illustrate that RET and ROS production are crucial factors in melatonin's effects in cancer cells and establish the dual effect of melatonin in protecting normal cells and inducing apoptosis in cancer cells.

Effect of 5-Azacitidine Treatment on Redox Status and Inflammatory Condition in MDS Patients.

This study focused on the impact of the treatment with the hypomethylating agent 5-azacitidine on the redox status and inflammation in 24 MDS patients. Clinical and genetic features of MDS patients were recorded, and peripheral blood samples were used to determine the activity of the endogenous antioxidant defense system (superoxide dismutase, SOD; catalase, CAT; glutathion peroxidase, GPx; and reductase, GRd, activities), markers of oxidative damage (lipid peroxidation, LPO, and advanced oxidation protein products, AOPP). Moreover, pro-inflammatory cytokines and plasma nitrite plus nitrate levels as markers of inflammation, as well as CoQ10 plasma levels, were also measured. Globally, MDS patients showed less redox status in terms of a reduction in the GSSG/GSH ratio and in the LPO levels, as well as increased CAT activity compared with healthy subjects, with no changes in SOD, GPx, and GRd activities, or AOPP levels. When analyzing the evolution from early to advanced stages of the disease, we found that the GPx activity, GSSG/GSH ratio, LPO, and AOPP increased, with a reduction in CAT. GPx changes were related to the presence of risk factors such as high-risk IPSS-R or mutational score. Moreover, there was an increase in IL-2, IL-6, IL-8, and TNF-α plasma levels, with a further increase of IL-2 and IL-10 from early to advanced stages of the disease. However, we did not observe any association between inflammation and oxidative stress. Finally, 5-azacitidine treatment generated oxidative stress in MDS patients, without affecting inflammation levels, suggesting that oxidative status and inflammation are two independent processes.

The Impact of Melatonin Supplementation and NLRP3 Inflammasome Deletion on Age-Accompanied Cardiac Damage.

To investigate the role of NLRP3 inflammasome in cardiac aging, we evaluate here morphological and ultrastructural age-related changes of cardiac muscles fibers in wild-type and NLRP3-knockout mice, as well as studying the beneficial effect of melatonin therapy. The results clarified the beginning of the cardiac sarcopenia at the age of 12 months, with hypertrophy of cardiac myocytes, increased expression of ß-MHC, appearance of small necrotic fibers, decline of cadiomyocyte number, destruction of mitochondrial cristae, appearance of small-sized residual bodies, and increased apoptotic nuclei ratio. These changes were progressed in the cardiac myocytes of 24 old mice, accompanied by excessive collagen deposition, higher expressions of IL-1α, IL-6, and TNFα, complete mitochondrial vacuolation and damage, myofibrils disorganization, multivesicular bodies formation, and nuclear fragmentation. Interestingly, cardiac myocytes of NLRP3-/- mice showed less detectable age-related changes compared with WT mice. Oral melatonin therapy preserved the normal cardiomyocytes structure, restored cardiomyocytes number, and reduced ß-MHC expression of cardiac hypertrophy. In addition, melatonin recovered mitochondrial architecture, reduced apoptosis and multivesicular bodies' formation, and decreased expressions of ß-MHC, IL-1α, and IL-6. Fewer cardiac sarcopenic changes and highly remarkable protective effects of melatonin treatment detected in aged cardiomyocytes of NLRP3-/- mice compared with aged WT animals, confirming implication of the NLRP3 inflammasome in cardiac aging. Thus, NLRP3 suppression and melatonin therapy may be therapeutic approaches for age-related cardiac sarcopenia.

Melatonin Targets Metabolism in Head and Neck Cancer Cells by Regulating Mitochondrial Structure and Function.

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.

Protective Effects of Melatonin on the Skin: Future Perspectives.

When exposed to hostile environments such as radiation, physical injuries, chemicals, pollution, and microorganisms, the skin requires protective chemical molecules and pathways. Melatonin, a highly conserved ancient molecule, plays a crucial role in the maintenance of skin. As human skin has functional melatonin receptors and also acts as a complete system that is capable of producing and regulating melatonin synthesis, melatonin is a promising candidate for its maintenance and protection. Below, we review the studies of new metabolic pathways involved in the protective functions of melatonin in dermal cells. We also discuss the advantages of the topical use of melatonin for therapeutic purposes and skin protection. In our view, endogenous intracutaneous melatonin production, together with topically-applied exogenous melatonin and its metabolites, represent two of the most potent defense systems against external damage to the skin.

Melatonin Enhances Cisplatin and Radiation Cytotoxicity in Head and Neck Squamous Cell Carcinoma by Stimulating Mitochondrial ROS Generation, Apoptosis, and Autophagy.

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.

Combination of melatonin and rapamycin for head and neck cancer therapy: Suppression of AKT/mTOR pathway activation, and activation of mitophagy and apoptosis via mitochondrial function regulation.

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.