Melatonin biological activity and binding sites in human melanoma cells

The effects of melatonin, N-acetylserotonin and serotonin on the growth and tyrosinase activity of SK-Mel 23 and SK-Mel 28 human melanoma cell lines were investigated. Binding assays were also performed to establish the nature of the binding site. SK-Mel 28 cells were responsive to melatonin and its precursors, exhibiting a decrease in growth and an increase in tyrosinase activity after a 72 hr treatment. N-acetylserotonin was as potent as melatonin, the minimal effective concentration (MEC, which is defined as the smallest concentration that elicits a measurable biological response, significantly different from control) being 10-8 m. Serotonin was the least potent (MEC = 10-6 m). Both melatonin antagonists, prazosin and luzindole, exhibited no effect per se and reversed both responses to melatonin. SK-Mel 23 cells, however, showed no significant responses to the indoleamines. Competition binding assays in SK-Mel 28 cells demonstrated the presence of binding sites to 2-[125 I]-iodomelatonin, which was displaced by the unlabelled hormone, by both antagonists, and by N-acetylserotonin. The curve adjustment of the displacement values with melatonin suggests the existence of two binding sites, with the following Ki values: 1.0 x 10-10 m and 6.5 x 10-6 m. Ki values for acetylserotonin, prazosin and luzindole were, respectively, 3.8 x 10-8 m, 1.2 x 10-8 m, and 8.3 x 10-6 m. Surprisingly, in SK-Mel 23 cells, melatonin and luzindole were able to compete with the radioligand, with Ki values of 3.1 x 10-8 and 2.4 x 10-8 m, respectively. Our data suggest that SK-Mel 28 cells probably possess high affinity binding sites to melatonin and, in addition, MT3 low affinity binding sites, because N-acetylserotonin was as effective as the native hormone, and prazosin effectively blocked the actions of melatonin. Both sites are functional as demonstrated by the blockade promoted by both luzindole and prazosin on the proliferative and melanogenic responses. Although growth and tyrosinase activity of SK-Mel 23 cells were not affected by melatonin or its precursors, this cell line possesses high affinity binding sites, which may be non-functional, or trigger responses other than the ones herein investigated.     

Molecular deficiency (ies) in MT1 melatonin signaling pathway underlies the melatonin‐unresponsive phenotype in MDA‐MB‐231 human breast cancer cells

Melatonin has been shown repeatedly to inhibit the growth of human breast tumor cells in vitro and in vivo. Its antiproliferative effects have been well studied in MCF‐7 human breast cancer cells and several other estrogen receptor α (ERα)‐positive human breast cancer cell lines. However, the MDA‐MB‐231 breast cancer cell line, an ERα‐negative cell line widely used in breast cancer research, has been shown to be unresponsive to melatonin's growth‐suppressive effect in vitro. Here, we examined the effect of melatonin on the cell proliferation of several ERα‐negative breast cancer cell lines including MDA‐MB‐231, BT‐20, and SK‐BR‐3 cells. Although the MT1 G‐protein‐coupled receptor is expressed in all three cell lines, melatonin significantly suppressed the proliferation of SK‐BR‐3 cells without having any significant effect on the growth of MDA‐MB‐231 and BT‐20 cells. We confirmed that the MT1‐associated Gα proteins are expressed in MDA‐MB‐231 cells. Further studies demonstrated that the melatonin unresponsiveness in MDA‐MB‐231 cells may be caused by aberrant signaling downstream of the Gαi proteins, resulting in differential regulation of ERK1/2 activity.     

Melatonin increases proliferation of cultured neural stem cells obtained from adult mouse subventricular zone

Abstract: Melatonin, a circadian rhythm–promoting molecule secreted mainly by the pineal gland, has a variety of biological functions and neuroprotective effects including control of sleep–wake cycle, seasonal reproduction, and body temperature as well as preventing neuronal cell death induced by neurotoxic substances. Melatonin also modulates neural stem cell (NSC) function including proliferation and differentiation in embryonic brain tissue. However, the involvement of melatonin in adult neurogenesis is still not clear. Here, we report that precursor cells from adult mouse subventricular zone (SVZ) of the lateral ventricle, the main neurogenic area of the adult brain, express melatonin receptors. In addition, precursor cells derived from this area treated with melatonin exhibited increased proliferative activity. However, when cells were treated with luzindole, a competitive inhibitor of melatonin receptors, or pertussis toxin, an uncoupler of Gi from adenylate cyclase, melatonin‐induced proliferation was reduced. Under these conditions, melatonin induced the differentiation of precursor cells to neuronal cells without an upregulation of the number of glia cells. Because stem cell replacement is thought to play an important therapeutic role in neurodegenerative diseases, melatonin might be beneficial for stimulating endogenous neural stem cells.     

Differential growth inhibitory effect of melatonin on two endometrial cancer cell lines

The effect of melatonin on endometrial cancer cell growth was investigated using two cell lines, SNG-II and Ishikawa, which are different in their estrogen receptor status. A physiological concentration of melatonin (10(-9) M) showed no growth inhibitory effect on SNG-II cells, which are estrogen receptor-negative at all cell densities and incubation times. In contrast, melatonin significantly inhibited Ishikawa cells, which are estrogen receptor-positive at all cell densities tested after 96 hr incubation. The greatest inhibition of Ishikawa cell growth was observed at 10(-9) M melatonin, compared with other supra (10(-6), 10(-8) M) or subphysiological concentrations (10(-10), 10(-12) M). This growth inhibitory effect of melatonin on Ishikawa cells was completely blocked by 10(-10) to 10(-8) M concentrations of 17-beta estradiol administration. Pretreatment with luzindole, which is a selective melatonin receptor antagonist, prior to the addition of melatonin also blocked the inhibitory effect of melatonin on Ishikawa cells. This is the first study to demonstrate an anti-proliferative effect of physiological melatonin on endometrial cancer cells in vitro. The present study revealed that melatonin also inhibits the growth of estrogen receptor positive endometrial cancer cells and that this effect of the pineal indole may be mediated by both steroid and melatonin receptors.     

Melatonin influences somatostatin secretion from human pancreatic δ‐cells via MT1 and MT2 receptors

Melatonin is an effector of the diurnal clock on pancreatic islets. The membrane receptor‐transmitted inhibitory influence of melatonin on insulin secretion is well established and contrasts with the reported stimulation of glucagon release from α‐cells. Virtually, nothing is known concerning the melatonin‐mediated effects on islet δ‐cells. Analysis of a human pancreatic δ‐cell model, the cell line QGP‐1, and the use of a somatostatin‐specific radioimmunoassay showed that melatonin primarily has an inhibitory effect on somatostatin secretion in the physiological concentration range. In the pharmacological range, melatonin elicited slightly increased somatostatin release from δ‐cells. Cyclic adenosine monophosphate (cAMP) is the major second messenger dose‐dependently stimulating somatostatin secretion, in experiments employing the membrane‐permeable 8‐Br‐cAMP. 8‐Br‐cyclic guanosine monophosphate proved to be of only minor relevance to somatostatin release. As the inhibitory effect of 1 nm melatonin was reversed after incubation of QGP‐1 cells with the nonselective melatonin receptor antagonist luzindole, but not with the MT2‐selective antagonist 4‐P‐PDOT (4‐phenyl‐2‐propionamidotetraline), an involvement of the MT1 receptor can be assumed. Somatostatin release from the δ‐cells at low glucose concentrations was significantly inhibited during co‐incubation with 1 nm melatonin, an effect which was less pronounced at higher glucose levels. Transient expression experiments, overexpressing MT1, MT2, or a deletion variant as a control, indicated that the MT1 and not the MT2 receptor was the major transmitter of the inhibitory melatonin effect. These data point to a significant influence of melatonin on pancreatic δ‐cells and on somatostatin release.     

Melatonin influences insulin secretion primarily via MT(1) receptors in rat insulinoma cells (INS-1) and mouse pancreatic islets

Several studies have revealed that melatonin affects the insulin secretion via MT(1) and MT(2) receptor isoforms. Owing to the lack of selective MT(1) receptor antagonists, we used RNA interference technology to generate an MT(1) knockdown in a clonal β-cell line to evaluate whether melatonin modulates insulin secretion specifically via the MT(1) receptor. Incubation experiments were carried out, and the insulin concentration in supernatants was measured using a radioimmunoassay. Furthermore, the intracellular cAMP was determined using an enzyme-linked immunosorbent assay. Real-time RT-PCR indicated that MT(1) knockdown resulted in a significant increase in the rIns1 mRNA and a significantly elevated basal insulin secretion of INS-1 cells. Incubation with melatonin decreased the amount of glucagon-like peptide 1 or inhibited the glucagon-stimulated insulin release of INS-1 cells, while, in MT(1) -knockdown cells, no melatonin-induced reduction in insulin secretion could be found. No decrease in 3-isobutyl-1-methylxanthine-stimulated intracellular cAMP in rMT(1) -knockdown cells was detectable after treatment with melatonin either, and immunocytochemistry proved that MT(1) knockdown abolished phosphorylation of cAMP-response-element-binding protein. In contrast to the INS-1 cells, preincubation with melatonin did not sensitize the insulin secretion of rMT(1) -knockdown cells. We also monitored insulin secretion from isolated islets of wild-type and melatonin-receptor knockout mice ex vivo. In islets of wild-type mice, melatonin treatment resulted in a decrease in insulin release, whereas melatonin treatment of islets from MT(1) knockout and MT(1/2) double-knockout mice did not show a significant effect. The data indicate that melatonin inhibits insulin secretion, primarily via the MT(1) receptor in rat INS-1 cells and isolated mouse islets.     

Melatonin induces apoptosis in human neuroblastoma cancer cells

Low concentrations (nanomolar) of melatonin had been previously shown to inhibit cell proliferation in several cancer cell lines as well as in experimental animal models. Additionally, cell growth inhibition and differentiation of prostate cancer cell lines by high concentrations (micromolar to millimolar) of melatonin have been recently reported. In the present paper, we show the induction of apoptosis by high doses of melatonin in the human neuroblastoma cell line SK-N-MC. We found accumulation of cells in the G2/M cell cycle phase and induction of cellular death, measured as lactate dehydrogenase (LDH) released into the culture medium, under millimolar concentration of melatonin. Apoptosis was evaluated using 4,6-diamidino-2-phenylindole staining, DNA gel electrophoresis, electron microscopy, and annexin V binding. Apoptosis progressed through the classical pathway, which involves caspase-3 activation. Cell death was dose and time-dependent; the lowest effective concentration of melatonin was 100 microm. Treatment with 1 mm melatonin for 6 days induced cell death in 75% of the cells. This novel finding shows that a nontoxic natural indoleamine may be potential therapy for some types of human neuroblastomas.     

Expression of melatonin receptor MT1 in cells of human invasive ductal breast carcinoma

In humans, two main types of membrane melatonin receptors have been identified, MT1 and MT2. Expression of MT1 in neoplastic cells seems to increase the efficacy of melatonin's oncostatic activity. The purpose of this study was to determine the distribution and the intensity of MT1 expression in breast cancer cells and to correlate it with clinicopathological factors. Immunohistochemical studies (IHC) were conducted on 190 cases of invasive ductal breast carcinomas (IDC) and molecular studies were performed on 29 cases of frozen tumor fragments and selected breast cancer cell lines. Most of the studied tumors manifested a membranous/cytoplasmic IHC expression of MT1. In IDC, the MT1 expression was higher than in fibrocystic breast disease. MT1 expression was higher in estrogen receptor positive (ER+) and HER2 positive (HER2+) tumors. Triple negative tumors (TN) manifested the lowest MT1 expression level. The lowest MT1 protein expression level was noted in the TN breast cancer cell line MDA‐MB‐231 compared with ER+ cell lines MCF‐7 and SK‐BR‐3. MT1 mRNA expression was negatively correlated with the malignancy grade of the studied IDC cases. Moreover, higher MT1 expression was associated with patients' longer overall survival (OS) in the group of ER+ breast cancers and treated with tamoxifen. Multivariate analysis indicated that MT1 was an independent prognostic factor in the ER+ tumors for OS and event‐free survival in the ER+ tumors. The results of this study may point to a potential prognostic and therapeutic significance of MT1 in IDC.     

Melatonin resynchronizes dysregulated circadian rhythm circuitry in human prostate cancer cells

Abstract: Prostate cancer (PCa) is a major age‐related malignancy as increasing age correlates with increased risk for developing this neoplasm. Similarly, alterations in circadian rhythms have also been associated with the aging population and cancer risk. The pineal hormone melatonin is known to regulate circadian rhythms, which is under the control of a core set of genes: Period 1, 2, 3 (Per 1–3); Cryptochrome 1, 2 (Cry 1, 2); Clock, and Bmal 1, 2. Melatonin levels have been shown to decrease in patients with cancer and exogenous melatonin exhibits antiproliferative effects against certain cancers. In this study, we challenged the hypothesis that melatonin imparts antiproliferative effects in prostate cancer via resynchronization of deregulated core clock circuitry. We found that Clock and Per2 protein levels were downregulated whereas Bmal1 protein levels were upregulated in PCa cells, compared to normal prostate cells. Additionally, employing automated quantitative analysis of a microarray containing human tissues, we found that compared to benign tissues, Clock and Per2 levels were downregulated, whereas Bmal1 levels were upregulated in PCa and other proliferative prostatic conditions. Overexpression of Per2 was found to result in a significant loss of PCa cell growth and viability. Interestingly, melatonin treatment resulted in an increase in Per2 and Clock and a reduction in Bmal1 in PCa cells. Further, melatonin treatment resulted in a resynchronization of oscillatory circadian rhythm genes (Dbp and Per2). Our data support our hypothesis and suggest that melatonin should be thoroughly investigated as an agent for the management of PCa and other age‐related malignancies.     

Melatonin antagonizes apoptosis via receptor interaction in U937 monocytic cells

Among the non-neurological functions of melatonin, much attention is being directed to the ability of melatonin to modulate the immune system, whose cells possess melatonin-specific receptors and biosynthetic enzymes. Melatonin controls cell behaviour by eliciting specific signal transduction actions after its interaction with plasma membrane receptors (MT(1), MT(2)); additionally, melatonin potently neutralizes free radicals. Melatonin regulates immune cell loss by antagonizing apoptosis. A major unsolved question is whether this is due to receptor involvement, or to radical scavenging considering that apoptosis is often dependent on oxidative alterations. Here, we provide evidence that on U937 monocytic cells, apoptosis is antagonized by melatonin by receptor interaction rather than by radical scavenging. First, melatonin and a set of synthetic analogues prevented apoptosis in a manner that is proportional to their affinity for plasma membrane receptors but not to their antioxidant ability. Secondly, melatonin's antiapoptotic effect required key signal transduction events including G protein, phospholipase C and Ca(2+) influx and, more important, it is sensitive to the specific melatonin receptor antagonist luzindole.     

Genetic deletion of MT1 and MT2 melatonin receptors differentially abrogates the development and expression of methamphetamine‐induced locomotor sensitization during the day and the night in C3H/HeN mice

Abstract: This study explored the role of the melatonin receptors in methamphetamine (METH)‐induced locomotor sensitization during the light and dark phases in C3H/HeN mice with genetic deletion of the MT₁ and/or MT₂ melatonin receptors. Six daily treatments with METH (1.2 mg/kg, i.p.) in a novel environment during the light phase led to the development of locomotor sensitization in wild‐type (WT), MT₁KO and MT₂KO mice. Following four full days of abstinence, METH challenge (1.2 mg/kg, i.p.) triggered the expression of locomotor sensitization in METH‐pretreated but not in vehicle (VEH)‐pretreated mice. In MT₁/MT₂KO mice, the development of sensitization during the light phase was significantly reduced and the expression of sensitization was completely abrogated upon METH challenge. During the dark phase the development of locomotor sensitization in METH‐pretreated WT, MT₁KO and MT₂KO mice was statistically different from VEH‐treated controls. However, WT and MT₂KO, but not MT₁KO mice receiving repeated VEH pretreatments during the dark phase expressed a sensitized response to METH challenge that is of an identical magnitude to that observed upon 6 days of METH pretreatment. We conclude that exposure to a novel environment during the dark phase, but not during the light phase, facilitated the expression of sensitization to a METH challenge in a manner dependent on MT₁ melatonin receptor activation by endogenous melatonin. We suggest that MT₁ and MT₂ melatonin receptors are potential targets for pharmacotherapeutic intervention in METH abusers.     

Distribution and density of melatonin receptors in human main pancreatic islet cell types

Recent investigations of our group established that melatonin modulates hormone secretion of pancreatic islets via melatonin receptor types MT1 and MT2. Expression of MT1 and MT2 has been shown in mouse, rat, and human pancreatic islets as well as in the β‐, α‐, and δ‐cell lines INS‐1, αTC1.9, and QGP‐1. In view of these earlier investigations, this study was performed to analyze in detail the distribution and density of melatonin receptors on the main islet cell types in human pancreatic tissue obtained from nondiabetic and type 2 diabetic patients. Immunohistochemical analysis established the presence of MT1 and MT2 in β‐, α‐, and δ‐cells, but notably, with differences in receptor density. In general, the lowest MT1 and MT2 receptor density was measured in α‐cells compared to the 2 other cell types. In type 2 diabetic islets, MT1 and MT2 receptor density was increased in δ‐cells compared to normoglycemic controls. In human islets in batch culture of a nondiabetic donor, an increase of somatostatin secretion was observed under melatonin treatment while in islets of a type 2 diabetic donor, an inhibitory influence could be observed, especially in the presence of 5.5 mmol/L glucose. These data suggest the following: i) cell‐type‐specific density of MT1 and MT2 receptors in human pancreatic islets, which should be considered in context of the hormone secretion of islets, ii) the influence of diabetes on density of MT1 and MT2 as well as iii) the differential impact of melatonin on somatostatin secretion of nondiabetic and type 2 diabetic islets.     

Sleep well. Untangling the role of melatonin MT1 and MT2 receptors in sleep

The pharmacological potential of targeting selectively melatonin MT1 or MT2 receptors has not yet been exploited in medicine. Research using selective MT1/MT2 receptor ligands and MT1/MT2 receptor knockout mice has indicated that the activation of MT2 receptors selectively increases non‐rapid eye movement (NREM) sleep whereas MT1 receptors seem mostly implicated in the regulation of REM sleep. Moreover, MT1 knockout mice show an increase in NREM sleep, while MT2 knockout a decrease, suggesting an opposite role of these two receptors. A recent paper in mice by Sharma et al (J Pineal Res, 2018, e12498) found that MT1 but not MT2 receptors are expressed on orexin neurons in the perifornical lateral hypothalamus (PFH). Moreover, after injecting melatonin or luzindole into the mouse PFH, the authors suggest that melatonin promotes NREM sleep because activates PFH MT1 receptors, which in turn inhibit orexin neurons that are important in promoting arousal and maintaining wakefulness. In this commentary, we have critically commented on some of these findings on the bases of previous literature. In addition, we highlighted the fact that no conclusions could be drawn on the melatonin receptor subtype mediating the effects of melatonin on sleep because the authors used the non‐selective MT1/MT2 receptors antagonist luzindole. More solid research should further characterize the pharmacological function of these two melatonin receptors in sleep.     

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.