Dual sources of melatonin and evidence for different primary functions.

This article discusses data showing that mammals, including humans, have two sources of melatonin that exhibit different functions. The best-known source of melatonin, herein referred to as Source #1, is the pineal gland. In this organ, melatonin production is circadian with maximal synthesis and release into the blood and cerebrospinal fluid occurring during the night. Of the total amount of melatonin produced in mammals, we speculate that less than 5% is synthesized by the pineal gland. The melatonin rhythm has the primary function of influencing the circadian clock at the level of the suprachiasmatic nucleus (the CSF melatonin) and the clockwork in all peripheral organs (the blood melatonin) via receptor-mediated actions. A second source of melatonin (Source # 2) is from multiple tissues throughout the body, probably being synthesized in the mitochondria of these cells. This constitutes the bulk of the melatonin produced in mammals and is concerned with metabolic regulation. This review emphasizes the action of melatonin from peripheral sources in determining re-dox homeostasis, but it has other critical metabolic effects as well. Extrapineal melatonin synthesis does not exhibit a circadian rhythm and it is not released into the blood but acts locally in its cell of origin and possibly in a paracrine matter on adjacent cells. The factors that control/influence melatonin synthesis at extrapineal sites are unknown. We propose that the concentration of melatonin in these cells is determined by the subcellular redox state and that melatonin synthesis may be inducible under stressful conditions as in plant cells.

Melatonin/Sericin Wound Healing Patches: Implications for Melanoma Therapy.

Melatonin and sericin exhibit antioxidant properties and may be useful in topical wound healing patches by maintaining redox balance, cell integrity, and regulating the inflammatory response. In human skin, melatonin suppresses damage caused by ultraviolet radiation (UVR) which involves numerous mechanisms associated with reactive oxygen species/reactive nitrogen species (ROS/RNS) generation and enhancing apoptosis. Sericin is a protein mainly composed of glycine, serine, aspartic acid, and threonine amino acids removed from the silkworm cocoon (particularly Bombyx mori and other species). It is of interest because of its biodegradability, anti-oxidative, and anti-bacterial properties. Sericin inhibits tyrosinase activity and promotes cell proliferation that can be supportive and useful in melanoma treatment. In recent years, wound healing patches containing sericin and melatonin individually have attracted significant attention by the scientific community. In this review, we summarize the state of innovation of such patches during 2021-2023. To date, melatonin/sericin-polymer patches for application in post-operational wound healing treatment has been only sparingly investigated and it is an imperative to consider these materials as a promising approach targeting for skin tissue engineering or regenerative dermatology.

Evolutionary formation of melatonin and vitamin D in early life forms: insects take centre stage.

Melatonin, a product of tryptophan metabolism via serotonin, is a molecule with an indole backbone that is widely produced by bacteria, unicellular eukaryotic organisms, plants, fungi and all animal taxa. Aside from its role in the regulation of circadian rhythms, it has diverse biological actions including regulation of cytoprotective responses and other functions crucial for survival across different species. The latter properties are also shared by its metabolites including kynuric products generated by reactive oxygen species or phototransfomation induced by ultraviolet radiation. Vitamins D and related photoproducts originate from phototransformation of ∆5,7 sterols, of which 7-dehydrocholesterol and ergosterol are examples. Their ∆5,7 bonds in the B ring absorb solar ultraviolet radiation [290-315 nm, ultraviolet B (UVB) radiation] resulting in B ring opening to produce previtamin D, also referred to as a secosteroid. Once formed, previtamin D can either undergo thermal-induced isomerization to vitamin D or absorb UVB radiation to be transformed into photoproducts including lumisterol and tachysterol. Vitamin D, as well as the previtamin D photoproducts lumisterol and tachysterol, are hydroxylated by cyochrome P450 (CYP) enzymes to produce biologically active hydroxyderivatives. The best known of these is 1,25-dihydroxyvitamin D (1,25(OH)2D) for which the major function in vertebrates is regulation of calcium and phosphorus metabolism. Herein we review data on melatonin production and metabolism and discuss their functions in insects. We discuss production of previtamin D and vitamin D, and their photoproducts in fungi, plants and insects, as well as mechanisms for their enzymatic activation and suggest possible biological functions for them in these groups of organisms. For the detection of these secosteroids and their precursors and photoderivatives, as well as melatonin metabolites, we focus on honey produced by bees and on body extracts of Drosophila melanogaster. Common biological functions for melatonin derivatives and secosteroids such as cytoprotective and photoprotective actions in insects are discussed. We provide hypotheses for the photoproduction of other secosteroids and of kynuric metabolites of melatonin, based on the known photobiology of ∆5,7 sterols and of the indole ring, respectively. We also offer possible mechanisms of actions for these unique molecules and summarise differences and similarities of melatoninergic and secosteroidogenic pathways in diverse organisms including insects.

Unraveling Endothelial Cell Migration: Insights into Fundamental Forces, Inflammation, Biomaterial Applications, and Tissue Regeneration Strategies.

Cell migration is vital for many fundamental biological processes and human pathologies throughout our life. Dynamic molecular changes in the tissue microenvironment determine modifications of cell movement, which can be reflected either individually or collectively. Endothelial cell (EC) migratory adaptation occurs during several events and phenomena, such as endothelial injury, vasculogenesis, and angiogenesis, under both normal and highly inflammatory conditions. Several advantageous processes can be supported by biomaterials. Endothelial cells are used in combination with various types of biomaterials to design scaffolds promoting the formation of mature blood vessels within tissue engineered structures. Appropriate selection, in terms of scaffolding properties, can promote desirable cell behavior to varying degrees. An increasing amount of research could lead to the creation of the perfect biomaterial for regenerative medicine applications. In this review, we summarize the state of knowledge regarding the possible systems by which inflammation may influence endothelial cell migration. We also describe the fundamental forces governing cell motility with a specific focus on ECs. Additionally, we discuss the biomaterials used for EC culture, which serve to enhance the proliferative, proangiogenic, and promigratory potential of cells. Moreover, we introduce the mechanisms of cell movement and highlight the significance of understanding these mechanisms in the context of designing scaffolds that promote tissue regeneration.

Role of Cyclins and Cytoskeletal Proteins in Endometriosis: Insights into Pathophysiology.

Endometriosis is a gynecological condition where endometrium-like tissue grows outside the uterus, posing challenges in understanding and treatment. This article delves into the deep cellular and molecular processes underlying endometriosis, with a focus on the crucial roles played by cyclins and cytoskeletal proteins in its pathogenesis, particularly in the context of Epithelial-Mesenchymal Transition (EMT). The investigation begins by examining the activities of cyclins, elucidating their diverse biological roles such as cell cycle control, proliferation, evasion of apoptosis, and angiogenesis among ectopic endometrial cells. A comprehensive analysis of cytoskeletal proteins follows, emphasizing their fundamental biological roles and their specific significance to endometriotic cell features. This review sheds light on the interconnected pathways through which cyclins and cytoskeletal proteins converge, contributing to the genesis and progression of endometriosis. Understanding these molecular complexities not only provides insight into the underlying causes of the disease but also holds promise for the development of specific therapeutic approaches, ushering in a new era in the management of this devastating disorder.

Melanogenesis Is Directly Affected by Metabolites of Melatonin in Human Melanoma Cells.

Melatonin (N-acetyl-5-methoxytryptamine, MEL), its kynurenic (N1-acetyl-N2-formyl-5-methoxykynurenine, AFMK) and indolic derivatives (6-hydroxymelatonin, 6(OH)MEL and 5-methoxytryptamine, 5-MT) are endogenously produced in human epidermis. Melatonin, produced by the pineal gland, brain and peripheral organs, displays a diversity of physiological functions including anti-inflammatory, immunomodulatory, and anti-tumor capacities. Herein, we assessed their regulatory effect on melanogenesis using amelanotic (A375, Sk-Mel-28) and highly pigmented (MNT-1, melanotic) human melanoma cell lines. We discovered that subjected compounds decrease the downstream pathway of melanin synthesis by causing a significant drop of cyclic adenosine monophosphate (cAMP) level, the microphthalmia-associated transcription factor (MITF) and resultant collapse of tyrosinase (TYR) activity, and melanin content comparatively to N-phenylthiourea (PTU, a positive control). We observed a reduction in pigment in melanosomes visualized by the transmission electron microscopy. Finally, we assessed the role of G-protein-coupled seven-transmembrane-domain receptors. Obtained results revealed that nonselective MT1 and MT2 receptor antagonist (luzindole) or selective MT2 receptor antagonist (4-P-PDOT) did not affect dysregulation of the melanin pathway indicating a receptor-independent mechanism. Our findings, together with the current state of the art, provide a convenient experimental model to study the complex relationship between metabolites of melatonin and the control of pigmentation serving as a future and rationale strategy for targeted therapies of melanoma-affected patients.

Melatonin and Its Metabolites Can Serve as Agonists on the Aryl Hydrocarbon Receptor and Peroxisome Proliferator-Activated Receptor Gamma.

Melatonin is widely present in Nature. It has pleiotropic activities, in part mediated by interactions with high-affinity G-protein-coupled melatonin type 1 and 2 (MT1 and MT2) receptors or under extreme conditions, e.g., ischemia/reperfusion. In pharmacological concentrations, it is given to counteract the massive damage caused by MT1- and MT2-independent mechanisms. The aryl hydrocarbon receptor (AhR) is a perfect candidate for mediating the latter effects because melatonin has structural similarity to its natural ligands, including tryptophan metabolites and indolic compounds. Using a cell-based Human AhR Reporter Assay System, we demonstrated that melatonin and its indolic and kynuric metabolites act as agonists on the AhR with EC50's between 10-4 and 10-6 M. This was further validated via the stimulation of the transcriptional activation of the CYP1A1 promoter. Furthermore, melatonin and its metabolites stimulated AhR translocation from the cytoplasm to the nucleus in human keratinocytes, as demonstrated by ImageStream II cytometry and Western blot (WB) analyses of cytoplasmic and nuclear fractions of human keratinocytes. These functional analyses are supported by in silico analyses. We also investigated the peroxisome proliferator-activated receptor (PPAR)γ as a potential target for melatonin and metabolites bioregulation. The binding studies using a TR-TFRET kit to assay the interaction of the ligand with the ligand-binding domain (LBD) of the PPARγ showed agonistic activities of melatonin, 6-hydroxymelatonin and N-acetyl-N-formyl-5-methoxykynuramine with EC50's in the 10-4 M range showing significantly lower affinities that those of rosiglitazone, e.g., a 10-8 M range. These interactions were substantiated by stimulation of the luciferase activity of the construct containing PPARE by melatonin and its metabolites at 10-4 M. As confirmed by the functional assays, binding mode predictions using a homology model of the AhR and a crystal structure of the PPARγ suggest that melatonin and its metabolites, including 6-hydroxymelatonin, 5-methoxytryptamine and N-acetyl-N-formyl-5-methoxykynuramine, are excellent candidates to act on the AhR and PPARγ with docking scores comparable to their corresponding natural ligands. Melatonin and its metabolites were modeled into the same ligand-binding pockets (LBDs) as their natural ligands. Thus, functional assays supported by molecular modeling have shown that melatonin and its indolic and kynuric metabolites can act as agonists on the AhR and they can interact with the PPARγ at high concentrations. This provides a mechanistic explanation for previously reported cytoprotective actions of melatonin and its metabolites that require high local concentrations of the ligands to reduce cellular damage under elevated oxidative stress conditions. It also identifies these compounds as therapeutic agents to be used at pharmacological doses in the prevention or therapy of skin diseases.

Ultraviolet Radiation-Induced Mitochondrial Disturbances Are Attenuated by Metabolites of Melatonin in Human Epidermal Keratinocytes.

Melatonin (N-acetyl-5-methoxytryptamine) is recognized as an effective antioxidant produced by the pineal gland, brain and peripheral organs, which also has anti-inflammatory, immunomodulatory, and anti-tumour capacities. Melatonin has been reported as a substance that counteracts ultraviolet radiation B (UVB)-induced intracellular disturbances. Nevertheless, the mechanistic actions of related molecules including its kynurenic derivatives (N1-acetyl-N2-formyl-5-methoxykynurenine (AFMK)), its indolic derivatives (6-hydroxymelatonin (6(OH)MEL) and 5-methoxytryptamine (5-MT)) and its precursor N-acetylserotonin (NAS) are only poorly understood. Herein, we treated human epidermal keratinocytes with UVB and assessed the protective effect of the studied substances in terms of the maintenance of mitochondrial function or their radical scavenging capacity. Our results show that UVB caused the significant elevation of catalase (CAT) and superoxide dismutase (Mn-SOD), the dissipation of mitochondrial transmembrane potential (mtΔΨ), a reduction in ATP synthesis, and the enhanced release of cytochrome c into cytosol, leading subsequently to UVB-mediated activation of the caspases and apoptosis (appearance of sub-G1 population). Our findings, combined with data reported so far, indicate the counteracting and beneficial actions of melatonin and its molecular derivatives against these deleterious changes within mitochondria. Therefore, they define a path to the development of novel strategies delaying mitochondrial aging and promoting the well-being of human skin.

Melatonin: A Potential Regulator of DNA Methylation.

The pineal gland-derived indoleamine hormone, melatonin, regulates multiple cellular processes, ranging from chronobiology, proliferation, apoptosis, and oxidative damage to pigmentation, immune regulation, and mitochondrial metabolism. While melatonin is best known as a master regulator of the circadian rhythm, previous studies also have revealed connections between circadian cycle disruption and genomic instability, including epigenetic changes in the pattern of DNA methylation. For example, melatonin secretion is associated with differential circadian gene methylation in night shift workers and the regulation of genomic methylation during embryonic development, and there is accumulating evidence that melatonin can modify DNA methylation. Since the latter one impacts cancer initiation, and also, non-malignant diseases development, and that targeting DNA methylation has become a novel intervention target in clinical therapy, this review discusses the potential role of melatonin as an under-investigated candidate epigenetic regulator, namely by modulating DNA methylation via changes in mRNA and the protein expression of DNA methyltransferases (DNMTs) and ten-eleven translocation (TET) proteins. Furthermore, since melatonin may impact changes in the DNA methylation pattern, the authors of the review suggest its possible use in combination therapy with epigenetic drugs as a new anticancer strategy.

Melatonin and TGF-ß-Mediated Release of Extracellular Vesicles.

The immune system, unlike other systems, must be flexible and able to "adapt" to fully cope with lurking dangers. The transition from intracorporeal balance to homeostasis disruption is associated with activation of inflammatory signaling pathways, which causes modulation of the immunology response. Chemotactic cytokines, signaling molecules, and extracellular vesicles act as critical mediators of inflammation and participate in intercellular communication, conditioning the immune system's proper response. Among the well-known cytokines allowing for the development and proper functioning of the immune system by mediating cell survival and cell-death-inducing signaling, the tumor necrosis factor α (TNF-α) and transforming growth factor ß (TGF-ß) are noteworthy. The high bloodstream concentration of those pleiotropic cytokines can be characterized by anti- and pro-inflammatory activity, considering the powerful anti-inflammatory and anti-oxidative stress capabilities of TGF-ß known from the literature. Together with the chemokines, the immune system response is also influenced by biologically active chemicals, such as melatonin. The enhanced cellular communication shows the relationship between the TGF-ß signaling pathway and the extracellular vesicles (EVs) secreted under the influence of melatonin. This review outlines the findings on melatonin activity on TGF-ß-dependent inflammatory response regulation in cell-to-cell communication leading to secretion of the different EV populations.

Mechanism of Extracellular Vesicle Secretion Associated with TGF-ß-Dependent Inflammatory Response in the Tumor Microenvironment.

Extracellular vesicles (EVs) serve as central mediators in communication between tumor and non-tumor cells. These interactions are largely dependent on the function of the endothelial barrier and the set of receptors present on its surface, as endothelial cells (ECs) are a plenteous source of EVs. The molecular basis for EV secretion and action in the tumor microenvironment (TME) has not been fully elucidated to date. Emerging evidence suggests a prominent role of inflammatory pathways in promoting tumor progression and metastasis. Although transforming growth factor ß (TGF-ß) is a cytokine with strong immunomodulatory and protective activity in benign and early-stage cancer cells, it plays a pro-tumorigenic role in advanced cancer cells, which is known as the "TGF-ß paradox". Thus, the aim of this review is to describe the correlation between EV release, TGF-ß-dependent inflammation, and dysregulation of downstream TGF-ß signaling in the context of cancer development.

Scaffolds Loaded with Dialdehyde Chitosan and Collagen-Their Physico-Chemical Properties and Biological Assessment.

In this work, dialdehyde chitosan (DAC) and collagen (Coll) scaffolds have been prepared and their physico-chemical properties have been evaluated. Their structural properties were studied by Fourier Transform Infrared Spectroscopy with Attenuated Internal Reflection (FTIR-ATR) accompanied by evaluation of thermal stability, porosity, density, moisture content and microstructure by Scanning Electron Microscopy-SEM. Additionally, cutaneous assessment using human epidermal keratinocytes (NHEK), dermal fibroblasts (NHDF) and melanoma cells (A375 and G-361) was performed. Based on thermal studies, two regions in DTG curves could be distinguished in each type of scaffold, what can be assigned to the elimination of water and the polymeric structure degradation of the materials components. The type of scaffold had no major effect on the porosity of the materials, but the water content of the materials decreased with increasing dialdehyde chitosan content in subjected matrices. Briefly, a drop in proliferation was noticed for scaffolds containing 20DAC/80Coll compared to matrices with collagen alone. Furthermore, increased content of DAC (50DAC/50Coll) either significantly induced the proliferation rate or maintains its ratio compared to the control matrix. This delivery is a promising technique for additional explorations targeting therapies in regenerative dermatology. The using of dialdehyde chitosan as one of the main scaffolds components is the novelty in terms of bioengineering.

Assessment of Melatonin-Cultured Collagen/Chitosan Scaffolds Cross-Linked by a Glyoxal Solution as Biomaterials for Wound Healing.

Chitosan (CTS) and collagen (Coll) are natural biomaterials that have been extensively used in tissue engineering or wound healing applications, either separately or as composite materials. Most methods to fabricate CTS/Coll matrices employ chemical crosslinking to obtain solid and stable scaffolds with the necessary porosity and mechanical properties to facilitate regeneration. In this study, we comparatively assessed the physicochemical properties of 3D scaffolds loaded with a cross-linker, glyoxal. Using a scanning electron microscope, we evaluated the microstructure of resultant matrices and their mechanistic testing by the determination of the compressive modulus (Emod), the maximum force (Fmax), thermogravimetric analysis (TG), Fourier Transform Infrared Spectroscopy-Attenuated Total Reflectance (FTIR-ATR), and proliferation rate in vitro using human epidermal keratinocytes and dermal fibroblasts cultured in presence of melatonin solution (10-5 M). We observed that enhanced content of collagen (50CTS/50Coll or 20CTS/80Coll compared to 80CTS/20Coll) significantly elevated the physicochemical capacities of resultant materials. Besides, presence of 5% glyoxal increased porosity, Emod and Fmax, compared to scaffolds without glyoxal. Finally, keratinocytes and dermal fibroblasts cultured on subjected matrices in presence of melatonin revealed a prominently enhanced growth rate. This indicates that the combination of glyoxal and melatonin make it imperative to consider these materials as a promising approach for targeting skin tissue engineering or regenerative dermatology.

Protective Role of Melatonin and Its Metabolites in Skin Aging.

The skin, being the largest organ in the human body, is exposed to the environment and suffers from both intrinsic and extrinsic aging factors. The skin aging process is characterized by several clinical features such as wrinkling, loss of elasticity, and rough-textured appearance. This complex process is accompanied with phenotypic and functional changes in cutaneous and immune cells, as well as structural and functional disturbances in extracellular matrix components such as collagens and elastin. Because skin health is considered one of the principal factors representing overall "well-being" and the perception of "health" in humans, several anti-aging strategies have recently been developed. Thus, while the fundamental mechanisms regarding skin aging are known, new substances should be considered for introduction into dermatological treatments. Herein, we describe melatonin and its metabolites as potential "aging neutralizers". Melatonin, an evolutionarily ancient derivative of serotonin with hormonal properties, is the main neuroendocrine secretory product of the pineal gland. It regulates circadian rhythmicity and also exerts anti-oxidative, anti-inflammatory, immunomodulatory, and anti-tumor capacities. The intention of this review is to summarize changes within skin aging, research advances on the molecular mechanisms leading to these changes, and the impact of the melatoninergic anti-oxidative system controlled by melatonin and its metabolites, targeting the prevention or reversal of skin aging.

Melatonin: highlighting its use as a potential treatment for SARS-CoV-2 infection.

Numerous pharmaceutical drugs have been repurposed for use as treatments for COVID-19 disease. These drugs have not consistently demonstrated high efficacy in preventing or treating this serious condition and all have side effects to differing degrees. We encourage the continued consideration of the use of the antioxidant and anti-inflammatory agent, melatonin, as a countermeasure to a SARS-CoV-2 infection. More than 140 scientific publications have identified melatonin as a likely useful agent to treat this disease. Moreover, the publications cited provide the rationale for the use of melatonin as a prophylactic agent against this condition. Melatonin has pan-antiviral effects and it diminishes the severity of viral infections and reduces the death of animals infected with numerous different viruses, including three different coronaviruses. Network analyses, which compared drugs used to treat SARS-CoV-2 in humans, also predicted that melatonin would be the most effective agent for preventing/treating COVID-19. Finally, when seriously infected COVID-19 patients were treated with melatonin, either alone or in combination with other medications, these treatments reduced the severity of infection, lowered the death rate, and shortened the duration of hospitalization. Melatonin's ability to arrest SARS-CoV-2 infections may reduce health care exhaustion by limiting the need for hospitalization. Importantly, melatonin has a high safety profile over a wide range of doses and lacks significant toxicity. Some molecular processes by which melatonin resists a SARS-CoV-2 infection are summarized. The authors believe that all available, potentially beneficial drugs, including melatonin, that lack toxicity should be used in pandemics such as that caused by SARS-CoV-2.

The Characterization of Scaffolds Based on Dialdehyde Chitosan/Hyaluronic Acid.

In this work, two-component dialdehyde chitosan/hyaluronic acid scaffolds were developed and characterized. Dialdehyde chitosan was obtained by one-step synthesis with chitosan and sodium periodate. Three-dimensional scaffolds were prepared by the lyophilization method. Fourier transform infrared spectroscopy (FTIR) was used to observe the chemical structure of scaffolds and scanning electron microscopy (SEM) imaging was done to assess the microstructure of resultant materials. Thermal analysis, mechanical properties measurements, density, porosity and water content measurements were used to characterize physicochemical properties of dialdehyde chitosan/hyaluronic acid 3D materials. Additionally, human epidermal keratinocytes (NHEK), dermal fibroblasts (NHDF) and human melanoma cells (A375 and G-361) were used to evaluate cell viability in the presence of subjected scaffolds. It was found that scaffolds were characterized by a porous structure with interconnected pores. The scaffold composition has an influence on physicochemical properties, such as mechanical strength, thermal resistance, porosity and water content. There were no significant differences between cell viability proliferation of all scaffolds, and this observation was visible for all subjected cell lines.

Evaluation of Polymeric Matrix Loaded with Melatonin for Wound Dressing.

The development of scaffolds mimicking the extracellular matrix containing bioactive substances has great potential in tissue engineering and wound healing applications. This study investigates melatonin-a methoxyindole present in almost all biological systems. Melatonin is a bioregulator in terms of its potential clinical importance for future therapies of cutaneous diseases. Mammalian skin is not only a prominent melatonin target, but also produces and rapidly metabolizes the multifunctional methoxyindole to biologically active metabolites. In our methodology, chitosan/collagen (CTS/Coll)-contained biomaterials are blended with melatonin at different doses to fabricate biomimetic hybrid scaffolds. We use rat tail tendon- and Salmo salar fish skin-derived collagens to assess biophysical and cellular properties by (i) Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR), (ii) thermogravimetric analysis (TG), (iii) scanning electron microscope (SEM), and (iv) proliferation ratio of cutaneous cells in vitro. Our results indicate that melatonin itself does not negatively affect biophysical properties of melatonin-immobilized hybrid scaffolds, but it induces a pronounced elevation of cell viability within human epidermal keratinocytes (NHEK), dermal fibroblasts (NHDF), and reference melanoma cells. These results demonstrate that this indoleamine accelerates re-epithelialization. This delivery is a promising technique for additional explorations in future dermatotherapy and protective skin medicine.

Differential and Overlapping Effects of Melatonin and Its Metabolites on Keratinocyte Function: Bioinformatics and Metabolic Analyses.

We investigated the effects of melatonin and its selected metabolites, i.e., N1-Acetyl-N2-formyl-5-methoxykynurenamine (AFMK) and 6-hydroxymelatonin (6(OH)Mel), on cultured human epidermal keratinocytes (HEKs) to assess their homeostatic activities with potential therapeutic implications. RNAseq analysis revealed a significant number of genes with distinct and overlapping patterns, resulting in common regulation of top diseases and disorders. Gene Set Enrichment Analysis (GSEA), Reactome FIViZ, and Ingenuity Pathway Analysis (IPA) showed overrepresentation of the p53-dependent G1 DNA damage response gene set, activation of p53 signaling, and NRF2-mediated antioxidative pathways. Additionally, GSEA exhibited an overrepresentation of circadian clock and antiaging signaling gene sets by melatonin derivatives and upregulation of extension of telomere signaling in HEKs, which was subsequently confirmed by increased telomerase activity in keratinocytes, indicating possible antiaging properties of metabolites of melatonin. Furthermore, Gene Ontology (GO) showed the activation of a keratinocyte differentiation program by melatonin, and GSEA indicated antitumor and antilipidemic potential of melatonin and its metabolites. IPA also indicated the role of Protein Kinase R (PKR) in interferon induction and antiviral response. In addition, the test compounds decreased lactate dehydrogenase A (LDHA) and lactate dehydrogenase C (LDHC) gene expression. These results were validated by qPCR and by Seahorse metabolic assay with significantly decreased glycolysis and lactate production under influence of AFMK or 6(OH)Mel in cells with a low oxygen consumption rate. In summary, melatonin and its metabolites affect keratinocytes' functions via signaling pathways that overlap for each tested molecule with some distinctions.

Mitochondrial function is controlled by melatonin and its metabolites in vitro in human melanoma cells.

Melanoma is a leading cause of cancer deaths worldwide. Although immunotherapy has revolutionized the treatment for some patients, resistance towards therapy and unwanted side effects remain a problem for numerous individuals. Broad anti-cancer activities of melatonin are recognized; however, additional investigations still need to be elucidated. Herein, using various human melanoma cell models, we explore in vitro the new insights into the regulation of melanoma by melatonin and its metabolites which possess, on the other side, high safety profiles and biological meaningful. In this study, using melanotic (MNT-1) and amelanotic (A375, G361, Sk-Mel-28) melanoma cell lines, the comparative oncostatic responses, the impact on melanin content (for melanotic MNT-1 melanoma cells) as well as the mitochondrial function controlled by melatonin, its precursor (serotonin), a kynuric (N1 -acetyl-N2 -formyl-5-methoxykynuramine, AFMK) and indolic pathway (6-hydroxymelatonin, 6(OH)MEL and 5-methoxytryptamine, 5-MT) metabolites were assessed. Namely, significant disturbances were observed in bioenergetics as follows: (i) uncoupling of oxidative phosphorylation (OXPHOS), (ii) attenuation of glycolysis, (iii) dissipation of mitochondrial transmembrane potential (mtΔΨ) accompanied by (iv) massive generation of reactive oxygen species (ROS), and (v) decrease of glucose uptake. Collectively, these results together with previously published reports provide a new biological potential and make an imperative to consider using melatonin or its metabolites for complementary future treatments of melanoma-affected patients; however, these associations should be additionally investigated in clinical setting.

Expression of antimicrobial peptide genes oscillates along day/night rhythm protecting mice skin from bacteria.

Antimicrobial peptides (AMPs) are important components of the innate immune system and are involved in skin protection against environmental insults and in wound healing. Herein, we assessed the gene expression of chemerin (Rarres2), cathelicidin CRAMP (Camp), and three ß-defensins (Defb1, Defb3, and Defb14) in mouse skin during light/dark cycle (LD 12:12) and constant darkness (DD). Next, we examined the survival of bacteria applied on the skin at specific times during the day. We found that the expression of Rarres2, Camp, and Defb1 was the highest at 4 h after the beginning of darkness, during high activity of mice. These rhythms, however, were not maintained under DD in the skin but were present in the liver. This indicated that in the case of skin, a circadian input was masked by daily changes of light in the environment. In contrast, Defb3 and Defb14 showed the highest mRNA levels when the mice slept, and these rhythmic mRNA oscillations were maintained under DD. This shows that Rarres2, Camp, and Defb1 levels in the skin are correlated with high locomotor activity in mice and they are controlled by daily changes of light and dark. Alternatively, oscillations in the mRNA levels of Defb3 and Defb14 seem to protect skin and heal wounds during sleep. These rhythms are maintained under DD, indicating that they are regulated by a circadian clock. Our study suggests that daily AMP expression affects the survival of bacteria on the surface of skin, which depends on the phase of AMP cycling.