Oncostatic action of melatonin: facts and question marks

The paper presents the data concerning the in vivo effects of melatonin on experimentally-induced tumors in animals and the in vitro effects on animal and human tumor cells. The majority of experimental tumors responded to the melatonin treatment with growth inhibition. However, some negative or opposite results (i.e. stimulation of tumor instead of inhibition) were also reported. Some of the negative results can be attributed to the improper timing of melatonin administration. Melatonin was also shown to inhibit the growth of several animal and human tumor cell lines in vitro. On the basis of these experiments, a hypothesis of the oncostatic action of melatonin was put forward. The mechanism of the postulated action is complex and probably includes: 1) modulation of the endocrine system; 2) modulation of the immune system; 3) the direct oncostatic action of melatonin on tumor cells. The latter includes the recently discovered anti-oxidative action which probably plays an important role in the countering the DNA damage during the radiation challenge or the exposure to chemical carcinogens. It also includes the antiproliferative and pro-apoptotic effects exerted via melatonin receptors expressed by tumor cells. The involvement of the membrane melatonin receptors is mainly assumed. However, the recent data from our and other laboratories suggest also the involvement of RZR/ROR receptors (the putative melatonin nuclear receptors) in both melatonin-induced proliferation inhibition and apoptosis.     

Nuclear Melatonin Receptors

Current opinions on the potential role of orphan nuclear retinoid receptors of the ROR/RZR subfamily in regulatory activities of the pineal gland hormone melatonin are reviewed. The mechanisms of receptor–DNA interactions and known coactivators, tissue peculiarities of the expression of different receptor isoforms, and its regulation are described. The spectrum of probable targets for regulation by the receptors, the most promoted of which in this aspect being the immune and central nervous systems, is traced. It is clear that for final adoption of the orphan ROR/RZR receptors, there is need for a full collaboration of endocrinologists for solution of the still debatable questions whether and under which situations melatonin does serve as a physiological modulator of the activities of these receptors.     

Melatonin-induced stimulation of rat corpus epididymal epithelial cell proliferation.

Stimulation of rat epididymal epithelial cell proliferation by melatonin was demonstrated by thymidine incorporation and flow cytometric analyses. The stimulatory effect of melatonin was dependent on the hormone concentration and the duration of cell exposure to the hormone. Maximal stimulation of [3H]thymidine incorporation into epididymal epithelial cells by melatonin was observed at 1 x 10(-9) M 5alpha-dihydrotestosterone in medium, while lower or higher concentrations of androgen attenuated the stimulatory effect of melatonin. Interestingly, a nuclear melatonin receptor agonist (1-[3-allyl-4-oxothiazolidine-2-ylidene]-4-methyl-thiosemi-carb azone, CGP 52608) induced opposite effect on epithelial cell proliferation to that produced by melatonin. Our data suggest that melatonin-induced stimulation of rat epididymal epithelial cell proliferation is not likely to be mediated by nuclear receptor. Furthermore, sequential changes of cell cycle distribution with melatonin treatment also supports a stimulatory action of melatonin on epididymal epithelial cell proliferation.     

Biological basis and possible physiological implications of melatonin receptor-mediated signaling in the rat epididymis

The mammalian epididymis plays an important role in sperm maturation, an important process of male reproduction. Specific high-affinity 2-[(125)I]iodomelatonin binding sites, satisfying the pharmacokinetic properties of specific receptors, have been found in the rat corpus epididymis, suggesting a direct melatonin action on epididymal physiology. Subsequent molecular and cell biology studies have identified these 2-[(125)I]iodomelatonin binding sites to be mt(1) (MEL(1A)) and MT(2) (MEL(1B)) melatonin receptor subtypes. Changes in the binding characteristics of these receptors in the rat corpus epididymis in response to castration and steroid hormones like testosterone and hydrocortisone indicated that these membrane melatonin receptors are biologically functional receptors, whose activities are differentially regulated by testosterone and hydrocortisone. These melatonin receptors are coupled to pertussis toxin (PTX)-sensitive G(i) protein and probably participate in androgenic and adrenergic regulation of rat corpus epididymal epithelial cell functions. Furthermore, rat corpus epididymal epithelial cell proliferation was stimulated by melatonin, whose action was dependent on the concentration and duration of exposure to the hormone. Interestingly, an MT(2) receptor ligand (4-phenyl-2-propionamidotetraline, 4-P-PDOT) induced a stimulatory effect on epididymal epithelial cell proliferation similar to that produced by melatonin. In contrast, a nuclear melatonin receptor agonist (1-[3-allyl-4-oxo-thiazolidine-2-ylidene]-4-methyl-thiosemi-car bazone , CGP52608) and 8-bromo-cAMP inhibited epididymal epithelial cell proliferation. Taken together, our data lead us to postulate that one of the possible physiological functions of melatonin on the rat epididymis is the stimulation of mt(1) and MT(2) melatonin receptors resulting in the inhibition of cAMP signaling and an increase in epithelial cell proliferation.     

Melatonin induces gamma-glutamylcysteine synthetase mediated by activator protein-1 in human vascular endothelial cells.

In the present study, we show that melatonin induces the expression of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme of glutathione (GSH) synthesis, in ECV304 human vascular endothelial cells. One micromolar melatonin induced the expression of gamma-GCS mRNA followed by an increase in the concentration of GSH with a peak at 24 h. An electrophoretic mobility shift assay showed that melatonin stimulates the DNA-binding activity of activator protein-1 (AP-1) as well as retinoid Z receptor/retinoid receptor-related orphan receptor alpha (RZR/RORalpha). ECV304 cells transiently transfected with a plasmid containing the gamma-GCS promoter-luciferase construct showed increased luciferase activity when treated with melatonin. The melatonin-dependent luciferase activity was found in the gamma-GCS promoter containing AP-1 site. The luciferase activity mediated by AP-1 was repressed in the promoter containing RZR/RORalpha site. In addition, cell cycle analysis showed that melatonin increases the number of cells in the G0/G1 phase; however, treatment of the cells with buthionine sulfoximine, a specific inhibitor of gamma-GCS, abolished the effect of melatonin on the cell cycle, suggesting induction of cell arrest by melatonin requires GSH. As conclusion, induction of GSH synthesis by melatonin protects cells against oxidative stress and regulates cell proliferation.     

Nuclear receptors are involved in the enhanced IL-6 production by melatonin in U937 cells

This report shows that melatonin enhances IL-6 production by U937 cells via a nuclear receptor-mediated mechanism. Resting U937 cells only express membrane (mt1) melatonin receptors. In these cells, melatonin did not modify basal production of IL-6 or when activated by PMA plus lipopolysaccharide, a treatment that downregulates the expression of mt1 receptor. However, in U937 cells activated with IFN-gamma, which induces the expression of the ROR alpha 1 and ROR alpha 2 nuclear receptors and represses the expression of the mt1 receptor, melatonin can activate IL-6 production. These results show that the expression of nuclear melatonin receptor but not membrane receptors is sufficient for melatonin to activate cytokine production in human lymphocytic and monocytic cell lines.     

Melatonin inhibits the proliferation of retinal pigment epithelial (RPE) cells in vitro

Summary The possible antiproliferative effect of melatonin on retinal pigment epithelial (RPE) cells in vitro was investigated. Bovine RPE cells cultured in Ham’s F12 medium supplemented with 10% fetal bovine serum had a nuclear density of 73.6 ± 6.1 nuclei/mm² at 72 h after seeding. The nuclear density at this time-point was doubled if either 50 or 100 ng/ml human epidermal growth factors (hEGF) was added to the culture medium. When these hEGF-stimulated cells were treated with melatonin from 10 to 500 pg/ml, the proliferation was suppressed with a dose-response relationship. At 250 and 500 pg/ml melatonin, the nuclear densities of the melatonin-treated cells were similar to those of the control cells. Using mitotically active SV-40 transformed human fetal RPE cells cultured in a serum-free medium, melatonin was also shown to be antiproliferative. In the presence of 500 pg/ml melatonin, the proliferation of these cells was inhibited to 77% as compared to the control. These results were further supported by the reduced [H³]thymidine uptake in the melatonin-treated cells. We propose that melatonin, at physiologic concentrations, has an antiproliferative effect, and that cultured RPE cells stimulated to proliferate by either hEGF treatment or SV-40 transfection are responsive to melatonin. Melatonin may either inhibit mitosis in actively dividing cells or modulate hEGF action.     

Melatonin synthesized by Jurkat human leukemic T cell line is implicated in IL-2 production

Human lymphocytes have recently been described as an important physiological source of melatonin (N-acetyl-5-methoxytryptamine), which could be involved in the regulation of the human immune system. On the other hand, stimulation of IL-2 production by exogenous melatonin has been shown in the Jurkat human lymphocytic cell line. Furthermore, both melatonin membrane and nuclear receptors are present in these cells. In this study, we show that the necessary machinery to synthesize melatonin is present and active in resting and stimulated Jurkat cells. Accordingly, we have found that cells synthesize and release melatonin in both conditions. Therefore, we investigated whether endogenous melatonin produced by Jurkat cells was involved in the regulation of IL-2 production. When melatonin membrane and nuclear receptors were blocked using specific antagonists, luzindole and CGP 55644, respectively, we found that IL-2 production decreased, and this drop was reverted by exogenous melatonin. Additionally, PHA activation of Jurkat cells changed the profile of melatonin nuclear receptor mRNA expression. A previous study showed that exogenous melatonin is able to counteract the decrease in IL-2 production caused by prostaglandin E2 (PGE2) in human lymphocytes via its membrane receptor. In our model, when we blocked the melatonin membrane receptor with luzindole, the inhibitory effect of PGE2 on IL-2 production was higher. Therefore, we have demonstrated the physiological role of endogenous melatonin in this cell line. These findings indicate that endogenous melatonin synthesized by human T cells would contribute to regulation of its own IL-2 production, acting as an intracrine, autocrine, and/or paracrine substance.     

Exogenously applied melatonin (N-acetyl-5-methoxytryptamine) affects flowering of the short-day plant Chenopodium rubrum

Melatonin ( N -acetyl-5-methoxytryptamine) is an animal hormone synthesized predominantly at night. It often serves as a signal of darkness that regulates circadian rhythmicity and photoperiodism. Melatonin has also been found in algae and higher plants, including the short-day flowering plant Chenopodium rubrum . To test its involvement in plant photoperiodism, melatonin solutions were applied to the cotyledons and plumules of 5-day-old-seedlings of Chenopodium rubrum L., ecotype 374. ³H-labelled melatonin was readily taken up by the plants and was very stable for a period of 37 h from application. Treatment with 100 and 500 µ M melatonin significantly reduced flowering of plants exposed to a single inductive 12-h darkness. Melatonin was efficient only when applied before lights off or during the first half of the dark period. This indicates that melatonin affects some early steps of the transition to flowering. However, it had no effect on the period or phase of a circadian rhythm in photoperiodic time measurement. Melatonin agonists (2-I-melatonin, 6-Cl-melatonin, CGP 52608) and 5-hydroxytryptamine also reduced flowering, whereas 5-methoxytryptamine did not. The results demonstrate that exogenous melatonin is able to influence the early stages of photoperiodic flower induction and/or flower development in a higher plant. Possible mechanisms for this effect are discussed.     

Antioxidant activity of melatonin in Chinese hamster ovarian cells: changes in cellular proliferation and differentiation

Melatonin is an endogenously generated molecule with free radical scavenging and antioxidant properties. Here, we studied the antiproliferative role of melatonin and other antioxidants on transformed Chinese hamster ovarian cells. Melatonin reduces cell proliferation in a dose- and time-dependent manner. Natural antioxidants which appear in edible plants including resveratrol and vitamin E mimicked the effect of melatonin. Flow cytometer analysis revealed that melatonin treatment reduces the number of cells in S-phase and increases cells in both G0/G1 and G2/M gaps. In addition, melatonin, as well as trolox, caused a clear morphological change by inducing the cells to become spindle shaped and fibroblast-like. Its effect is a reversible phenomenon that disappeared when melatonin was withdrawn from the culture medium. GSH levels are increased after melatonin treatment but pharmacologically blockade of GSH synthesis did not abolish melatonin's antiproliferative effect. Reduction of cell proliferation and the apparent induction of cell differentiation overlapped with melatonin's ability to change the intracellular redox state of CHO cells. We conclude that the cellular redox state may be involved in cellular transformation caused by antioxidants such as melatonin and trolox.     

Melatonin increases p53 and p21WAF1 expression in MCF-7 human breast cancer cells in vitro.

The aim of the present work was to study whether melatonin, at physiological concentrations, exerts its antiproliferative effects on MCF-7 human breast cancer cells by inducing the expression of some of the proteins involved in the control of the cell cycle. MCF-7 cells were cultured for 48 h in DMEM media containing either melatonin (1 nM) or the diluent (0.001% ethanol). At this concentration, after 48 hours of incubation, melatonin reduced the number of viable cells in relation to controls. The decreased cell proliferation was coincident with a significant increase in the expression of p53 as well as p21WAF1 proteins. These results demonstrate that melatonin inhibits MCF-7 cell proliferation by inducing an arrest of cell cycle dependent on an increased expression of p21WAF1 protein, which is mediated by the p53 pathway.     

The circadian nuclear receptor RORα negatively regulates cerebral ischemia–reperfusion injury and mediates the neuroprotective effects of melatonin

Disruptions of the circadian rhythm and reduced circulating levels of the circadian hormone melatonin predispose to ischemic stroke. Although the nuclear receptor RORα is considered as a circadian rhythm regulator and a mediator of certain melatonin effects, its potential role in cerebral ischemia-reperfusion (CI/R) injury and in the neuroprotective effects of melatonin remain undefined. Here, we observed that CI/R injury in RORα-deficient mice was associated with greater cerebral infarct size, brain edema, and cerebral apoptosis compared with wild-type model. In contrast, transgenic mice with brain-specific overexpression of RORα versus non-transgenic controls exerted significantly reduced infarct volume, brain edema and apoptotic response induced by CI/R. Mechanistically, RORα deficiency was found to exacerbate apoptosis pathways mediated by endoplasmic-reticulum stress and mitochondria and aggravate oxidative/nitrative stress after CI/R. Further studies revealed that RORα deficiency intensified the activation of nuclear factor-κB signaling induced by CI/R. Given the emerging evidence of RORα as an essential melatonin activity mediator, we further investigated the RORα roles in melatonin-exerted neuroprotection against acute ischemic stroke. Melatonin treatment significantly decreased infarct volume and cerebral apoptosis; mitigated endoplasmic reticulum stress and mitochondrial dysfunction; and inhibited CI/R injury-induced oxidative/nitrative stress and nuclear factor-κB activation, which was eradicated in RORα-deficient mice. Collectively, current findings suggest that RORα is a novel endogenous neuroprotective receptor, and a pivotal mediator of melatonin's suppressive effects against CI/R injury.     

Melatonin blocks the activation of estrogen receptor for DNA binding.

The present study shows that melatonin prevents, within the first cell cycle, the estradiol-induced growth of synchronized MCF7 breast cancer cells. By using nuclear extracts of these cells, we first examined the binding of estradiol-estrogen receptor complexes to estrogen-responsive elements and found that the addition of estradiol to whole cells activates the binding of the estrogen receptor to DNA whereas melatonin blocks this interaction. By contrast, melatonin neither affects the binding of estradiol to its receptor nor the receptor nuclear localization. Moreover, we also show that addition of estradiol to nuclear extracts stimulates the binding of estrogen receptor to DNA, but this activation is also prevented by melatonin. The inhibitory effect caused by melatonin is saturable at nanomolar concentrations and does not appear to be mediated by RZR nuclear receptors. The effect is also specific, since indol derivatives do not cause significant inhibition. Furthermore, we provide evidence that melatonin does not interact with the estrogen receptor in the absence of estradiol. Together, these results demonstrate that melatonin interferes with the activation of estrogen receptor by estradiol. The effect of melatonin suggests the presence of a receptor that, upon melatonin addition, destabilizes the binding of the estradiol-estrogen receptor complex to the estrogen responsive element.     

Expression of mRNA for growth hormone-releasing hormone and splice variants of GHRH receptors in human malignant bone tumors

Splice variants (SV) of receptors for growth hormone-releasing hormone (GHRH) have been found in several human cancer cell lines. GHRH antagonists inhibit growth of various human cancers, including osteosarcomas and Ewing's sarcoma, xenografted into nude mice or cultured in vitro and their antiproliferative action could be mediated, in part, through these SV of GHRH receptors. In this study, we found mRNA for the SV(1) isoform of GHRH receptors in human osteosarcoma line MNNG/HOS and SK-ES-1 Ewing's sarcoma line. We also detected mRNA for GHRH, which is apparently translated into the GHRH peptide and secreted by the cells, as shown by the presence of GHRH-like immunoreactivity in the conditioned media of cell cultures. In proliferation studies in vitro, the growth of SK-ES-1 and MNNG/HOS cells was dose-dependently inhibited by GHRH antagonist JV-1-38 and an antiserum against human GHRH. Our study indicates the presence of an autocrine stimulatory loop based on GHRH and SV(1) of GHRH receptors in human sarcomas. The direct antiproliferative effects of GHRH antagonists on malignant bone tumors appear to be exerted through the SV(1) of GHRH receptors on tumoral cells.     

Intracellular redox state as determinant for melatonin antiproliferative vs cytotoxic effects in cancer cells

Melatonin is an endogenous indolamine, classically known as a light/dark regulator. Besides classical functions, melatonin has also showed to have a wide range of antitumoral effects in numerous cancer experimental models. However, no definite mechanism has been described to explain the whole range of antineoplasic effects. Here we describe a dual effect of melatonin on intracellular redox state in relation to its antiproliferative vs cytotoxic actions in cancer cells. Thus, inhibition of proliferation correlates with a decrease on intracellular reactive oxygen species (ROS) and increase of antioxidant defences (antioxidant enzymes and intracellular gluthation,GSH levels), while induction of cell death correlates with an increase on intracellular ROS and decrease of antioxidant defences. Moreover, cell death can be prevented by other well-known antioxidants or can be increased by hydrogen peroxide. Thus, tumour cell fate will depend on the ability of melatonin to induce either an antioxidant environment--related to the antiproliferative effect or a prooxidant environment related to the cytotoxic effect.     

Melatonin enhancement of splenocyte proliferation is attenuated by luzindole, a melatonin receptor antagonist

In addition to marked seasonal changes in reproductive, metabolic, and other physiological functions, many vertebrate species undergo seasonal changes in immune function. Despite growing evidence that photoperiod mediates seasonal changes in immune function, little is known regarding the neuroendocrine mechanisms underlying these changes. Increased immunity in short days is hypothesized to be due to the increase in the duration of nightly melatonin secretion, and recent studies indicate that melatonin acts directly on immune cells to enhance immune parameters. The present study examined the contribution of melatonin receptors in mediating the enhancement of splenocyte proliferation in response to the T cell mitogen Concanavalin A in mice. The administration of luzindole, a high-affinity melatonin receptor antagonist, either in vitro or in vivo significantly attenuated the ability of in vitro melatonin to enhance splenic lymphocyte proliferation during the day or night. In the absence of melatonin or luzindole, splenocyte proliferation was intrinsically higher during the night than during the day. In the absence of melatonin administration, luzindole reduced the ability of spleen cells to proliferate during the night, when endogenous melatonin concentrations are naturally high. This effect was not observed during the day, when melatonin concentrations are low. Taken together, these results suggest that melatonin enhancement of splenocyte proliferation is mediated directly by melatonin receptors on splenocytes and that there is diurnal variation in splenocyte proliferation in mice that is also mediated by splenic melatonin receptors.     

Melatonin and alcohol-related disorders

ABSTRACT

This review concerns the current knowledge of melatonin and alcohol-related disorders. Chronobiological effects of ethanol are related to melatonin suppression and in relation to inflammation, stress, free radical scavenging, autophagy and cancer risk. It is postulated that both alcohol- and inflammation-induced production of reactive oxygen species (ROS) alters cell membrane properties leading to tissue dysfunction and, subsequent further ROS production. Lysosomal enzymes are often used to assess the relationships between intensified inflammation states caused by alcohol abuse and oxidative stress as well as level of tissue damage estimated by the increased release of cellular enzymes into the extracellular space. Studies have established a link between alcoholism and desynchronosis (circadian disruption). Desynchronosis results from the disorganization of the body’s circadian time structure and is an aspect of the pathology of chronic alcohol intoxication. The inflammatory conditions and the activity of lysosomal enzymes in acute alcohol poisoning or chronic alcohol-dependent diseases are in most cases interrelated. Inflammation can increase the activity of lysosomal enzymes, which can be regarded as a marker of lysosomal dysfunction and abnormal cellular integrity. Studies show alcohol toxicity is modulated by the melatonin (Mel) circadian rhythm. This hormone, produced by the pineal gland, is the main regulator of 24 h (sleep-wake cycle) and seasonal biorhythms. Mel exhibits antioxidant properties and may be useful in the prevention of oxidative stress reactions known to be responsible for alcohol-related diseases. Naturally produced Mel and exogenous sources in food can act in free radical reactions and activate the endogenous defense system. Mel plays an important role in the normalization of the post-stress state by its influence on neurotransmitter systems and the synchronization of circadian rhythms. Acting simultaneously on the neuroendocrine and immune systems, Mel optimizes homeostasis and provides protection against stress.

Abbreviations: ROS, reactive oxygen species; Mel, melatonin; SRV, resveratrol; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; ANT, arylalkylamine-N-acetyltransferase; EC cells, gastrointestinal enterochromaffin cells; MT1, melatonin high-affinity nanomolecular receptor site; MT2, melatonin low-affinity nanomolecular receptor site; ROR/RZR, orphan nuclear retinoid receptors; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, reduced form of glutathione; GSSG, oxidized form of glutathione; TAC, total antioxidant capacity; ONOO?^–, peroxynitrite radical; NCAM, neural cell adhesion molecules; LPO, lipid peroxidation; α-KG, α-ketoglutarate, HIF-1α, Hypoxia-inducible factor 1-α, IL-2, interleukin-2; HPA axis, hypothalamic-pituitary-adrenal axis; Tph1, tryptophan hydroxylase 1; AA-NAT, arylalkylamine-N-acetyltransferase; AS-MT, acetylserotonin O-methyltransferase; NAG, N-acetyl-beta-D-glucosaminidase; HBA1c glycated hemoglobin; LPS, lipopolysaccharide; AAP, alanyl-aminopeptidase; β-GR, β-glucuronidase; β-GD, β-galactosidase; LAP, leucine aminopeptidase.