Photophysical studies on melatonin and its receptor agonists

Previous work has demonstrated that melatonin inhibits the growth of both dermal and uveal melanoma cells. Recent clinical trials have found that melatonin is an efficacious treatment for metastatic dermal melanoma. The goal of this study was to provide further insight into the oncostatic mechanism(s) of melatonin. The inhibition of the growth of uveal melanoma cells is dose-dependent (0.1-10 nM) within the range of endogenous melatonin concentrations (2 nM) found in the human aqueous humor. We know that this inhibition of growth is receptor-mediated, at least in part, because uveal melanoma cell growth was also blocked by the agonists of melatonin receptors. There are two known membrane receptors for melatonin (Mel(1a) and Mel(1b)) and one known nuclear receptor (Mel2). To determine if singlet oxygen production and/or quenching contributed to the growth inhibition of melatonin, we examined the photophysical properties of melatonin and its agonists. Using flash photolysis, we determined that melatonin and its membrane receptor agonist 6-chloromelatonin (Mel(1a-b), Lilly, Indianapolis, IN) produced very little singlet oxygen (psidelta = 0.073 and psidelta = 0.01, respectively). There was no detectable singlet oxygen phosphorescence at 1,270 nm for the nuclear receptor agonist CG-52608 (Mel2, Novartis, Basel, Switzerland). In contrast, the agonist of the Mel(1b) receptor, S-20098 (Servier, Paris, France), produced singlet oxygen with a quantum efficiency of psidelta = 0.34. Singlet oxygen was quenched by melatonin and 6-chloromelatonin at approximately the same rate (6.1 x 10(7) M(-1)s(-1) and 6.0 x 10(7) M(-1)s(-1) in CD3OD), while the rate of quenching for the nuclear receptor agonist CG-52608 and membrane receptor agonist S-20098 was less (2.2 x 10(7) M(-1)s(-1) and 1.5 x 10(7) M(-1) s(-1), respectively). It appears that the production of singlet oxygen by melatonin would not be sufficient to directly block the proliferation of melanoma cells, but may activate gene products that could contribute to the oncostatic effect.     

Melatonin binding sites in estrogen receptor-positive cells derived from human endometrial cancer

Our previous work showed that melatonin (N-acetyl-5-methoxytryptamine) inhibits proliferation of the human endometrial cancer cell line, Ishikawa, which is estrogen receptor-positive. The aim of the present study was to determine whether Ishikawa cells possess membrane melatonin receptors. Binding of the radioligand 2-[125I]-iodomelatonin to membrane preparations obtained from Ishikawa cells was detectable, saturable and stable. Scatchard analysis revealed that the dissociation constant (Kd) of the binding sites was 179.0 pm (similar to that of the MT2 [Mel1b] melatonin receptor subtype), and that the concentration (Bmax) of the binding sites was 12.9 fmol/mg protein. Luzindole, a selective MT2 melatonin receptor antagonist, significantly suppressed binding of 2-[125I]-iodomelatonin at all concentrations tested (10(-8) to 10(-4) m). These results suggest that the MT2 melatonin receptor subtype is present in the membranes of Ishikawa cells, and that the antiproliferative effect of melatonin on Ishikawa cells is mediated via the MT2 receptor. This may have implications for the use of melatonin in endometrial cancer therapy.     

Melatonin receptor subtype MT2 (Mel 1b) and not mt1 (Mel 1a) is associated with melatonin-induced enhancement of cell-mediated and humoral immunity

Individuals of many vertebrate species undergo seasonal changes in immune function in addition to marked seasonal changes in reproductive, metabolic, and other physiological processes. Despite growing evidence that photoperiod mediates seasonal changes in immunity, little is known regarding the neuroendocrine mechanisms underlying these changes. Enhanced immune function in short days is correlated with increased duration of nightly melatonin secretion, and recent studies indicate that melatonin can act directly on immune cells to enhance immune function. It remains unknown, however, which melatonin receptor subtype mediates immune enhancement by melatonin. The present study examined the contribution of specific melatonin receptor subtypes, mt1 (Mel 1a) and MT2 (Mel 1b), in mediating melatonin-induced enhancement of cell-mediated and humoral immune function in mice. Melatonin enhanced both splenocyte proliferation and anti-keyhole limpet hemocyanin (KLH) IgG concentrations in both wild-type (WT) and mice lacking a functional gene for melatonin receptor mt1 (mt1 -/-), suggesting that the mt1 receptor does not mediate these responses. In addition, luzindole, an MT2 receptor antagonist, attenuated melatonin-induced enhancement of splenocyte proliferation in both WT and mt1 -/- mice. Taken together, these results suggest that receptor subtype mt1 is not necessary for mediating melatonin-induced enhancement of immune function and provide the first evidence for a specific melatonin receptor subtype, MT2, that may be involved in melatonin-induced immune enhancement.     

2[125I]Iodomelatonin binding and interaction with beta-adrenergic signaling in chick heart/coronary artery physiology

2[125I]Iodomelatonin ([125I]Mel) binding sites were characterized on membrane preparations of young chick hearts. [125I]Mel binding was rapid, saturable, stable, reversible, specific and of picomolar affinity and femtomolar density. Guanosine 5'-O-(3-thiotriphosphate) significantly lowered the binding affinity by one- to twofold, supporting G-protein linkage of melatonin receptors. Binding was detected as early as embryonic day-9 (E9), and increased steadily peaking at E13 before it slowly declined to about 15% of the peak level a week posthatch. Specific [125I]Mel binding was significantly increased by in ovo administration of inotropic agents dopamine and isoproterenol. Melatonin or 2-iodo-N-butanoyl-tryptamine inhibited isoproterenol-stimulated cAMP accumulation in primary heart cell cultures and the effect was attenuated after pretreatment with pertussis toxin (PTX). Localization of melatonin receptors using autoradiography showed intense labeling in the coronary arteries in all age groups whereas those in the myoblasts decreased as the heart matured. While the myoblasts and undifferentiated developing coronary arteries expressed melatonin MT1 receptor subtype in E11 hearts as detected by immunostaining with anti-MT1 receptor serum, immunoreactivities were observed mostly on the endothelium/subendothelium and smooth muscle cells of the well developed coronary vessels in posthatch hearts. Collectively, our data suggest the presence of PTX-sensitive, G protein-coupled melatonin receptors, whose expression is up-regulated by dopamine and isoproterenol, in the chick heart. Activation of these receptors, which include MT1 subtype, may modulate beta-adrenergic receptor-mediated cAMP signaling in the control of chick heart and coronary artery physiology.     

Identification and functional characterization of melatonin Mel 1a receptors in pancreatic beta cells: potential role in incretin-mediated cell function by sensitization of cAMP signaling

Melatonin receptors are expressed within the pancreatic islets of Langerhans, and melatonin induces a direct effect on insulin secretion ex-vivo. Here, we report the endogenous expression of the melatonin Mel 1a receptor in the INS-1 pancreatic beta cell line. Pharmacological characterization of the receptor using a CRE-luciferase reporter gene demonstrated its functional activity in INS-1 cells, displaying the characteristic signaling properties of the G(i/o) coupled receptor. Acute melatonin treatment of INS-1 cells in the presence of either forskolin or the incretin hormone glucagon-like peptide 1 (GLP-1) caused an attenuation of the responses in insulin secretion, insulin promoter activity, and CRE mediated gene expression, consistent with its effects in inhibiting cAMP mediated signal transduction. However, prolonged exposure (12 h) of INS-1 cells to melatonin treatment resulted in a sensitization of cAMP mediated responses to forskolin and GLP-1. Insulin secretion, insulin promoter activity and CRE mediated gene expression levels were augmented compared with responses without melatonin pre-treatment in INS-1 cells. In isolated rat islets, insulin secretion was enhanced following melatonin pre-treatment both in the absence and presence of GLP-1 or forskolin. This phenomenon reflects observations reported in other cell types expressing the melatonin Mel 1a receptor, and may represent the first evidence of a specific physiological role for melatonin-induced sensitization.     

MT1 and MT2 melatonin receptors are expressed in nonoverlapping neuronal populations

Melatonin (MLT) exerts its physiological effects principally through two high‐affinity membrane receptors MT1 and MT2. Understanding the exact mechanism of MLT action necessitates the use of highly selective agonists/antagonists to stimulate/inhibit a given MLT receptor. The respective distribution of MT1 and MT2 within the CNS and elsewhere is controversial, and here we used a “knock‐in” strategy replacing MT1 or MT2 coding sequences with a LacZ reporter. The data show striking differences in the distribution of MT1 and MT2 receptors in the mouse brain: whereas the MT1 subtype was expressed in very few structures (notably including the suprachiasmatic nucleus and pars tuberalis), MT2 subtype receptors were identified within numerous brain regions including the olfactory bulb, forebrain, hippocampus, amygdala and superior colliculus. Co‐expression of the two subtypes was observed in very few structures, and even within these areas they were rarely present in the same individual cell. In conclusion, the expression and distribution of MT2 receptors are much more widespread than previously thought, and there is virtually no correspondence between MT1 and MT2 cellular expression. The precise phenotyping of cells/neurons containing MT1 or MT2 receptor subtypes opens new perspectives for the characterization of links between MLT brain targets, MLT actions and specific MLT receptor subtypes.     

Intracellular second messengers involved in melatonin signal transduction in chicken splenocytes in vitro

The pineal hormone melatonin exhibits immunomodulatory activity well documented in mammals and birds. The mechanism of melatonin action within the immune system is, however, poorly understood. In mammalian immune cells in vitro, melatonin acts mainly as an antiapoptotic, oncostatic and antiproliferative agent, and these effects are exerted via specific receptors or are related to its free radical scavenging activity. In previous studies we have found that in short-term chicken splenocyte cultures in vitro melatonin stimulated basil proliferation and inhibited that stimulated with phytohemagglutinin, a T-cell mitogen. This paper is devoted to the involvement of membrane receptors, previously characterised by us as MT2 (Mel(1b)) and Mel(1c) subtypes, in the above mentioned melatonin effects in chicken splenocyte cultures. For this purpose, in present study a nonselective melatonin receptor antagonist, luzindole, and the selective MT2 blocker, 4P-PDOT, were used. The effect of melatonin on second messengers, cyclic adenosine-3',5'-monophosphate (cAMP) and inositol-1,4,5-trisphosphate (IP(3)), involved in the regulation of proliferation, was examined. We have found that the stimulation of proliferation occurs via Mel(1c) receptor and is associated with the changes in intracellular second messengers concentration: a decrease in cAMP and an increase in IP(3). In contrast, in mitogen-activated splenocytes, melatonin-induced inhibition of proliferation is mediated by MT2 receptors and is related to cAMP accumulation, as well as a decrease in IP(3). In conclusion, we have demonstrated that the stimulatory and inhibitory effect of melatonin on chicken splenocytes in vitro, dependent on the magnitude of cell stimulation, resulted from two different subtypes of membrane receptors.     

Melatonin induces the expression of gonadotropin-inhibitory hormone in the avian brain

We recently identified a novel hypothalamic neuropeptide inhibiting gonadotropin release in quail and termed it gonadotropin-inhibitory hormone (GnIH). Cell bodies and terminals containing the dodecapeptide GnIH are localized in the paraventricular nucleus (PVN) and median eminence, respectively. To understand the physiological role of GnIH, we investigated the mechanisms that regulate GnIH expression. In this study, we show that melatonin originating from the pineal gland and eyes induces GnIH expression in the quail brain. Pinealectomy (Px) combined with orbital enucleation (Ex) (Px plus Ex) decreased the expression of GnIH precursor mRNA and content of mature GnIH peptide in the diencephalon, which includes the PVN and median eminence. Melatonin administration to Px plus Ex birds caused a dose-dependent increase in expression of GnIH precursor mRNA and production of mature peptide. The expression of GnIH was photoperiodically controlled and increased under short-day photoperiods, when the duration of melatonin secretion increases. To identify the mode of melatonin action on GnIH induction, we investigated the expression of Mel(1c), a melatonin receptor subtype, in GnIH neurons. In situ hybridization of Mel(1c) mRNA combined with immunocytochemistry for GnIH revealed that Mel(1c) mRNA was expressed in GnIH-immunoreactive neurons in the PVN. Melatonin receptor autoradiography further revealed specific binding of melatonin in the PVN. These results indicate that melatonin is a key factor for GnIH induction. Melatonin appears to act directly on GnIH neurons through its receptor to induce GnIH expression. This is the first demonstration, to our knowledge, of a direct action of melatonin on neuropeptide induction in any vertebrate class.     

Melatonin exerts oncostatic capacity and decreases melanogenesis in human MNT‐1 melanoma cells

Melanogenesis is a key parameter of differentiation in melanocytes and melanoma cells; therefore, search for factors regulating this pathway are strongly desired. Herein, we investigated the effects of melatonin, a ubiquitous physiological mediator that is found throughout animals and plants. In mammals, the pineal gland secretes this indoleamine into the blood circulation to exert an extensive repertoire of biological activities. Our in vitro assessment indicates an oncostatic capacity of melatonin in time‐dependent manner (24, 48, 72 hours) in highly pigmented MNT‐1 melanoma cells. The similar pattern of regulation regarding cell viability was observed in amelanotic Sk‐Mel‐28 cells. Subsequently, MNT‐1 cells were tested for the first time for evaluation of melanin/melatonin interaction. Thus primary, electron paramagnetic resonance (EPR) spectroscopy demonstrated that melatonin reduced melanin content. Artificially induced disturbances of melanogenesis by selected inhibitors (N‐phenylthiourea or kojic acid) were slightly antagonized by melatonin. Additionally, analysis using transmission electron microscopy has shown that melatonin, particularly at higher dose of 10^?3 mol/L, triggered the appearance of premelanosomes (stage I‐II of melanosome) and MNT‐1 cells synthesize de novo endogenous melatonin shown by LC‐MS. In conclusion, these studies show a melanogenic‐like function of melatonin suggesting it as an advantageous agent for treatment of pigmentary disorders.     

Rapid modulation of the silent information regulator 1 by melatonin after hypoxia‐ischemia in the neonatal rat brain

Increasing evidence indicates that melatonin possesses protective effects toward different kinds of damage in various organs, including the brain. In a neonatal model of hypoxia‐ischemia (HI), melatonin was neuroprotective and preserved the expression of the silent information regulator 1 (SIRT1) 24 hours after the insult. This study aimed to gain more insight into the role of SIRT1 in the protective effect of melatonin after HI by studying the early (1 hour) modulation of SIRT1 and its downstream targets, and the consequences on necrosis, apoptosis, autophagy, and glial cell activation. We found that melatonin administered 5 minutes after the ischemic insult significantly reduced necrotic cell death assessed 1 hour after its administration. In parallel, we found a reduced activation of the early phases of intrinsic apoptosis, detected by reduced BAX translocation to the mitochondria and preservation of the mitochondrial expression of cytochrome C, indicating a reduced outer mitochondrial membrane permeabilization in the melatonin‐treated ischemic animals. These effects were concomitant to increased expression and activity of SIRT1, reduced expression and acetylation of p53, and increased autophagy activation. Melatonin also reduced HI‐induced glial cells activation. SIRT1 was expressed in neurons after HI and melatonin but not in reactive glial cells expressing GFAP. Colocalization between SIRT1 and GFAP was found in some cells in control conditions. In summary, our results provide more insight into the connection between SIRT1 and melatonin in neuroprotection. The possibility that melatonin‐induced SIRT1 activity might contribute to differentiate neuronal progenitor cells during the neurodegenerative process needs to be further investigated.     

MT-1 melatonin receptor expression increases the antiproliferative effect of melatonin on S-91 murine melanoma cells

Melatonin, a derivative of tryptophan that is present in all vertebrates, was first described in bovine pineal gland. It is known that melatonin is a highly conserved molecule, present also in unicellular organisms and plants. Several effects of melatonin have been described, including receptor- and non-receptor-mediated actions. Herein, we studied the effects of melatonin on in vitro and in vivo cell proliferation of Cloudman S-91 murine melanoma cells. We demonstrated that melatonin treatment significantly inhibits S-91 melanoma cell proliferation in vitro (EC50 = 10-7 m) as well as reduces tumor growth in vivo. We also demonstrated that melatonin directly increases the activity of the antioxidant enzymes catalase and glutathione peroxidase. These effects are most likely triggered through the direct intracellular action of melatonin, since the presence of receptors could not be demonstrated in this cell line. Expression of MT-1 melatonin receptor by stable transfection, mediated a dramatic antiproliferative melatonin effect (EC50 = 10-10 m) in S-91 cells. The expressed receptor is negatively coupled to the adenylyl cyclase/cyclic AMP signaling pathway via Gi protein. These results suggest that expression of the MT-1 melatonin receptor in melanoma cells is a potential alternative approach to specifically target cells in cancer therapeutic treatment.     

Endothelin-1 Induces CXCL1 and CXCL8 Secretion in Human Melanoma Cells

The endothelin pathway plays a critical role in melanoma tumor progression by a variety of mechanisms that enhance tumor cell growth, invasion, and metastasis. Here, we investigate the effect of this pathway on CXC chemokine expression in human melanoma cells and melanocytes. As determined by ELISA, endothelin-1 (ET-1) induces CXCL1 and CXCL8 secretion in three human melanoma cell lines in a concentration-dependent fashion. These responses are mediated by the endothelin-B receptor and are sustained over a 40 h time course. ET-1 does not induce CXCL1 secretion in primary human melanocytes but ET-3, an endothelin isoform, induces a low level of CXCL1 secretion in certain cultures. Neither ET-1 nor ET-3 induces secretion of CXCL8 in primary human melanocytes; thus, this response may be specific for melanocytic cells that have undergone malignant transformation. We have previously demonstrated that ET-1 induces changes in the expression of adhesion molecules in melanoma cells such that invasion and metastasis are favored. This study demonstrates that ET-1 additionally induces secretion of CXC chemokines critical for melanoma metastasis and tumor progression.     

Melatonin decreases cell proliferation and induces melanogenesis in human melanoma SK‐MEL‐1 cells

Abstract: Melatonin is an indoleamine synthesized in the pineal gland, and after its release into the blood, it has an extensive repertoire of biological activities, including antitumoral properties. In this study, we found that melatonin reduced the growth of the human melanoma cells SK‐MEL‐1. The antiproliferative effect was associated with an alteration in the progression of the phases of the cell cycle and also with an increase in tyrosinase activity, the key regulatory enzyme of melanogenesis. Antagonists for melatonin membrane receptors (luzindole and 4‐P‐PDOT) and the general G‐coupled receptor inhibitor, pertussis toxin, did not prevent the melatonin‐induced cell growth arrest; this suggests a mechanism independent of G‐coupled membrane receptors. In contrast, p38 mitogen‐activated protein kinase (p38 MAPK) signaling pathway seems to play a significant role in cell growth inhibition by melatonin. The indoleamine‐induced phosphorylation of p38 MAPK and the effect on cell proliferation were abrogated by the specific inhibitor SB203580 . Furthermore, comparative studies with known antioxidants such as N‐acetyl‐l ‐cysteine and trolox indicate that the growth of SK‐MEL‐1 cells is highly sensitive to antioxidants.