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.

Age and Chronodisruption in Mouse Heart: Effect of the NLRP3 Inflammasome and Melatonin Therapy.

Age and age-dependent inflammation are two main risk factors for cardiovascular diseases. Aging can also affect clock gene-related impairments such as chronodisruption and has been linked to a decline in melatonin synthesis and aggravation of the NF-κB/NLRP3 innate immune response known as inflammaging. The molecular drivers of these mechanisms remain unknown. This study investigated the impact of aging and NLRP3 expression on the cardiac circadian system, and the actions of melatonin as a potential therapy to restore daily rhythms by mitigating inflammaging. We analyzed the circadian expression and rhythmicity of clock genes in heart tissue of wild-type and NLRP3-knockout mice at 3, 12, and 24 months of age, with and without melatonin treatment. Our results support that aging, NLRP3 inflammasome, and melatonin affected the cardiac clock genes expression, except for Rev-erbα, which was not influenced by genotype. Aging caused small phase changes in Clock, loss of rhythmicity in Per2 and Rorα, and mesor dampening of Clock, Bmal1, and Per2. NLRP3 inflammasome influenced the acrophase of Clock, Per2, and Rorα. Melatonin restored the acrophase and the rhythm of clock genes affected by age or NLRP3 activation. The administration of melatonin re-established murine cardiac homeostasis by reversing age-associated chronodisruption. Altogether, these results highlight new findings about the effects aging and NLRP3 inflammasome have on clock genes in cardiac tissue, pointing to continuous melatonin as a promising therapy to placate inflammaging and restore circadian rhythm in heart muscle. Additionally, light microscopy analysis showed age-related morphological impairments in cardiomyocytes, which were less severe in mice lacking NLRP3. Melatonin supplementation preserved the structure of cardiac muscle fibers in all experimental groups.

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.

The Impact of Melatonin and NLRP3 Inflammasome on the Expression of microRNAs in Aged Muscle.

Muscular aging is a complex process and underlying physiological mechanisms are not fully clear. In recent years, the participation of the NF-kB pathway and the NLRP3 inflammasome in the chronic inflammation process that accompanies the skeletal muscle's aging has been confirmed. microRNAs (miRs) form part of a gene regulatory machinery, and they control numerous biological processes including inflammatory pathways. In this work, we studied the expression of four miRs; three of them are considered as inflammatory-related miRs (miR-21, miR-146a, and miR-223), and miR-483, which is related to the regulation of melatonin synthesis, among other targets. To investigate the changes of miRs expression in muscle along aging, the impact of inflammation, and the role of melatonin in aged skeletal muscle, we used the gastrocnemius muscle of wild type (WT) and NLRP3-knockout (NLRP3-) mice of 3, 12, and 24 months-old, with and without melatonin supplementation. The expression of miRs and pro-caspase-1, caspase-3, pro-IL-1ß, bax, bcl-2, and p53, was investigated by qRT-PCR analysis. Histological examination of the gastrocnemius muscle was also done. The results showed that age increased the expression of miR-21 (p < 0.01), miR-146a, and miR-223 (p < 0.05, for both miRs) in WT mice, whereas the 24-months-old mutant mice revealed decline of miR-21 and miR-223 (p < 0.05), compared to WT age. The lack of NLRP3 inflammasome also improved the skeletal muscle fibers arrangement and reduced the collagen deposits compared with WT muscle during aging. For the first time, we showed that melatonin significantly reduced the expression of miR-21, miR-146a, and miR-223 (p < 0.05 for all ones, and p < 0.01 for miR-21 at 24 months old) in aged WT mice, increased miR-223 in NLRP3- mice (p < 0.05), and induced miR-483 expression in both mice strains, this increase being significant at 24 months of age.

Melatonin/Nrf2/NLRP3 Connection in Mouse Heart Mitochondria during Aging.

Aging is a major risk for cardiovascular diseases (CVD). Age-related disorders include oxidative stress, mitochondria dysfunction, and exacerbation of the NF-κB/NLRP3 innate immune response pathways. Some of the molecular mechanisms underlying these processes, however, remain unclear. This study tested the hypothesis that NLRP3 inflammasome plays a role in cardiac aging and melatonin is able to counteract its effects. With the aim of investigating the impact of NLRP3 inflammasome and the actions and target of melatonin in aged myocardium, we analyzed the expression of proteins implied in mitochondria dynamics, autophagy, apoptosis, Nrf2-dependent antioxidant response and mitochondria ultrastructure in heart of wild-type and NLRP3-knockout mice of 3, 12, and 24 months-old, with and without melatonin treatment. Our results showed that the absence of NLRP3 prevented age-related mitochondrial dynamic alterations in cardiac muscle with minimal effects in cardiac autophagy during aging. The deficiency of the inflammasome affected Bax/Bcl2 ratio, but not p53 or caspase 9. The Nrf2-antioxidant pathway was also unaffected by the absence of NLRP3. Furthermore, NLRP3-deficiency prevented the drop in autophagy and mice showed less mitochondrial damage than wild-type animals. Interestingly, melatonin treatment recovered mitochondrial dynamics altered by aging and had few effects on cardiac autophagy. Melatonin supplementation also had an anti-apoptotic action in addition to restoring Nrf2-antioxidant capacity and improving mitochondria ultrastructure altered by aging.

Lack of retinoid acid receptor-related orphan receptor alpha accelerates and melatonin supplementation prevents testicular aging.

The role of retinoid acid receptor-related orphan receptor alpha (RORα) on male reproductive functions during aging is unclear. Here, we analyze the morphological changes in the testis of both young and aged RORα-deficient mice, with and without melatonin supplementation. Young mutants showed vacuolation, degeneration and pyknosis of spermatogenic epithelium and Sertoli cells. Aged mutants showed atrophy of the seminiferous tubules and absence of mitotic spermatogenic cells. Absence of sperms in many tubules, loss of acrosomal cap, vacuolation and hypertrophy of Sertoli cells were detected in aged mice, with a significant reduction in the number of seminiferous tubules and a significant increase in the number of Leydig cells and telocytes. Repair in seminiferous tubules and interstitial tissues with enhancement of spermatogenesis was observed in melatonin-treated aged mice. Young mutants overexpressed VEGF that was weaker in aged animals and observed only in the spermatocytes, while melatonin increased VEGF expression in spermatocytes and spermatids. Caspase 3 increased in both young and aged mutant mice in all seminiferous tubules and interstitium; caspase 3 immunostaining in seminiferous tubules, however, showed a normal pattern of apoptosis with melatonin supplementation. The present study reports that age-dependent testicular changes in RORα mutant mice were recovered by melatonin treatment.

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.

Lack of NLRP3 Inflammasome Activation Reduces Age-Dependent Sarcopenia and Mitochondrial Dysfunction, Favoring the Prophylactic Effect of Melatonin.

To investigate the role of NLRP3 inflammasome in muscular aging, we evaluated here the morphological and functional markers of sarcopenia in the NLRP3-knockout mice, as well as the beneficial effect of melatonin supplementation. The gastrocnemius muscles of young (3 months), early-aged (12 months), and old-aged (24 months) NLRP3-knockout female mice were examined. Moreover, locomotor activity and apoptosis were assessed. The results revealed early markers of sarcopenia at the age of 12 months, including reduction of lactate, ratio of muscle weight to body weight, muscle fibers number, and mitochondrial number. Increased interstitial tissues, apoptosis, and muscle fibers area, as well as mitochondrial damage were detected, with little muscular activity effects. In the old-aged, these alterations progressed with a reduction in locomotor activity, mitochondrial cristae destruction, nuclear fragmentation, tubular aggregates (TAs) formation, and increased frailty index. Oral melatonin supplementation preserved the normal muscular structure, muscle fibers number, and muscular activity in old age. Melatonin enhanced lactate production, recovered mitochondria, inhibited TAs formation, reduced apoptosis, and normalized frailty index. The fewer sarcopenic changes as well as the highly detectable prophylactic effects of melatonin treatment reported here in the muscle of NLRP3-knockout mice comparing with that previously detected in wild-type mice, confirming NLRP3 inflammasome implication in muscular aging and sarcopenia onset and progression.

Expression of modified tocopherol content and profile in sunflower tissues.

BACKGROUND: Alpha-tocopherol is the predominant tocopherol form in sunflower seeds. Sunflower lines that accumulate increased levels of beta-, gamma- and delta-tocopherol in seeds as well as lines with reduced and increased total seed tocopherol content have been developed. The objective of this research was to evaluate whether the modified tocopherol levels are expressed in plant tissues other than seeds. RESULTS: Lines with increased levels of beta-, gamma- and delta-tocopherol in seeds also possessed increased levels of these tocopherols in leaves, roots and pollen. Correlation coefficients for the proportion of individual tocopherols in different plant tissues were significantly positive in all cases, ranging from 0.68 to 0.97. A line with reduced tocopherol content in seeds also showed reduced content in roots and pollen. CONCLUSION: Genetic modifications producing altered seed tocopherol profiles in sunflower are also expressed in leaves, roots and pollen. Reduced total seed tocopherol content is mainly expressed at the root and pollen level. The expression of tocopherol mutations in other plant tissues will enable further studies on the physiological role of tocopherols and could be of interest for early selection for these traits in breeding programmes.