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 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.     

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 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 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.     

Melatonin receptors in PC3 human prostate tumor cells

Melatonin, secreted nocturnally by the pineal gland, can bind to human benign prostate epithelial cells and attenuate their growth and viability. In the present study, melatonin binding and responses were explored in the human steroid-independent PC3 prostatic tumor cells. PC3 cells bound 125I-melatonin with low affinity (Kd ca. 0.9 nM) at high as well as low cell density. Melatonin enhanced cGMP and 3H-thymidine incorporation at low, but attenuated them at high cell density. In addition, melatonin inhibited cAMP at low, but augmented it at high cell density. These effects were associated with an increase in cell count at low- but not high-density cultures. Pertussis toxin treatment suppressed 125I-melatonin binding and ablated all the effects of melatonin on 3H-thymidine incorporation, cAMP, and cGMP at both cell densities. Cholera toxin treatment failed to block the effects of melatonin on 3H-thymidine incorporation, but prevented the modulation by melatonin of cAMP at low and cGMP at high cell density. The cGMP analog 8-Br-cGMP, inhibited melatonin's effects on 3H-thymidine incorporation at both cell densities. H89, a protein kinase A inhibitor, prevented melatonin's effects on 3H-thymidine incorporation at low but not high cell density. These results provide the first demonstration of direct interaction of melatonin with hormone-insensitive prostate tumor cells. The melatonin receptors in the PC3 cells are coupled to pertussis toxin-sensitive G proteins to induce cell density-dependent changes in cGMP, cAMP, and cell growth.     

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.     

Melatonin receptors in the rat brain and pituitary

Abstract: 2-(¹²⁵I)iodomelatonin binding has been mapped and characterized in the brain and pituitary of the male laboratory rat using quantitative in vitro autoradiography. Specific binding was defined as that completely displaced in the presence of 1 μM melatonin. In the brain high levels of binding were localized over the suprachiasmatic nucleus (SCN), the area postrema (AP), and the spinal tract of the trigeminal nerve (Sp5). Lower densities of binding were found over the medial preoptic area (MPA), the septohypothalamic nuclei (SHy), the anterior hypothalamic area (AHA), the nuclei of the lateral olfactory tract (LOT), the paraventricular (PV), anteroventral (AV) and intermediodorsal (IMD) nuclei of the thalamus, the medial region of the lateral habenular (Lhb), the nuclei of the stria medullaris (SM), the basolateral (BL) and medial (ME) amygdaloid nuclei, the ventromedial nuclei (VMH), the arcuate nuclei (Arc), the subiculum of the hippocampus (S) and the lateral mammillary nuclei (LM). High levels of binding were also present over the pars tuberalis of the pituitary (PT) and the anterior and posterior cerebral arteries (CA). In both neuronal and non-neuronal areas, specific binding was time dependent and partially reversible in the presence of 1 μM melatonin. Binding was also saturable and of high affinity with dissociation constants (Kd) in the low picomolar range and was significantly inhibited in the presence of 104M guanosine 5'-0-(3-thiotriphosphate) (GTPγS) and 150 mM NaCl in all regions examined, indicating the presence of high affinity G-protein coupled melatonin receptors.     

Melatonin enhances alkaline phosphatase activity in differentiating human adult mesenchymal stem cells grown in osteogenic medium via MT2 melatonin receptors and the MEK/ERK (1/2) signaling cascade

The goals of this study were to determine (a) if melatonin enhances human adult mesenchymal stem cell (hAMSC) differentiation into osteoblasts as assessed by measuring alkaline phosphatase (ALP) enzyme activity, and (b) identify potential signal transduction pathways that mediate this process. ALP activity significantly increased in hAMSCs following a 10-day incubation in osteogenic medium, relative to hAMSCs incubated in basal growth medium alone. Melatonin (50 nm), added in combination with the osteogenic medium, significantly increased ALP activity relative to osteogenic medium alone. Co-exposure of hAMSCs to osteogenic medium supplemented with melatonin and either pertussis toxin or the melatonin receptor antagonists, luzindole or 4P-PDOT (MT2 receptor selective), inhibited the melatonin-induced increase in ALP activity, indicating the involvement of melatonin receptors, in particular, MT2 receptors. Assessment of melatonin receptor function following exposure to osteogenic medium containing either vehicle or melatonin produced dichotomous results. That is, if the differentiation of hAMSCs into an osteoblast was induced by osteogenic medium alone, then 2-[125I]-iodomelatonin binding and melatonin receptor function increased. However, examination of melatonin receptor function following chronic melatonin exposure, an exposure that resulted in a 50% enhancement in ALP activity, revealed that these receptors were desensitized. This was reflected by a complete loss in specific 2-[125I]-iodomelatonin binding as well as melatonin efficacy to inhibit forskolin-induced cAMP accumulation. Further characterization of the mechanisms underlying melatonin's effects on these differentiation processes revealed that MEK (1/2) and ERK (1/2), epidermal growth factor receptors, metalloproteinase and clathrin-mediated endocytosis were essential while PKA was not. Our results are consistent with a role for melatonin in osteoblast differentiation. If so, then, the decrease in plasma melatonin levels observed in humans during late adulthood may further enhance susceptibility to osteoporosis.     

Melatonin promotes regeneration of injured motor axons via MT1 receptors

Melatonin is an ancient multi‐tasking molecule produced by the pineal gland and by several extrapineal tissues. A variety of activities has been ascribed to this hormone in different physiological and pathological contexts, but little is known about its role in peripheral neuroregeneration. Here, we have exploited two different types of injury to test the capability of melatonin to stimulate regeneration of motor axons: (a) the acute and reversible presynaptic degeneration induced by the spider neurotoxin α‐Latrotoxin and (b) the compression/transection of the sciatic nerve. We found that in both cases melatonin administration accelerates the process of nerve repair. This pro‐regenerative action is MT₁‐mediated, and at least in part due to a sustained activation of the ERK1/2 pathway. These findings reveal a receptor‐mediated, pro‐regenerative action of melatonin in vivo that holds important clinical implications, as it posits melatonin as a safe candidate molecule for the treatment of a number of peripheral neurodegenerative conditions.     

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.     

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 regulates the transcription of βAPP‐cleaving secretases mediated through melatonin receptors in human neuroblastoma SH‐SY5Y cells

Melatonin is involved in the control of various physiological functions, such as sleep, cell growth and free radical scavenging. The ability of melatonin to behave as an antioxidant, together with the fact that the Alzheimer‐related amyloid β‐peptide (Aβ) triggers oxidative stress through hydroxyl radical‐induced cell death, suggests that melatonin could reduce Alzheimer's pathology. Although the exact etiology of Alzheimer's disease (AD) remains to be established, excess Aβ is believed to be the primary contributor to the dysfunction and degeneration of neurons that occurs in AD. Aβ peptides are produced via the sequential cleavage of β‐secretase β‐site APP‐cleaving enzyme 1 (BACE1) and γ‐secretase (PS1/PS2), while α‐secretase (ADAM10) prevents the production of Aβ peptides. We hypothesized that melatonin could inhibit BACE1 and PS1/PS2 and enhance ADAM10 expression. Using the human neuronal SH‐SY5Y cell line, we found that melatonin inhibited BACE1 and PS1 and activated ADAM10 mRNA level and protein expression in a concentration‐dependent manner and mediated via melatonin G protein‐coupled receptors. Melatonin inhibits BACE1 and PS1 protein expressions through the attenuation of nuclear factor‐κB phosphorylation (pNF‐κB). Moreover, melatonin reduced BACE1 promoter transactivation and consequently downregulated β‐secretase catalytic activity. The present data show that melatonin is not only a potential regulator of β/γ‐secretase but also an activator of α‐secretase expression through the activation of protein kinase C, thereby favoring the nonamyloidogenic pathway over the amyloidogenic pathway. Altogether, our findings suggest that melatonin may be a potential therapeutic agent for reducing the risk of AD in humans.     

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.     

Melatonin protects against oxidative stress in granular corneal dystrophy type 2 corneal fibroblasts by mechanisms that involve membrane melatonin receptors

Considering that oxidative stress plays a role in corneal fibroblast degeneration during granular corneal dystrophy type 2 (GCD2) and melatonin is an effective antioxidant, we examined the ability of melatonin to protect against oxidative stress-induced cell death of primary cultured normal and GCD2-homozygous corneal fibroblasts. Melatonin treatment protected primary cultured normal and GCD2 corneal fibroblasts from paraquat (PQ)-induced oxidative stress and caused increased expression levels of Cu/Zn-superoxide dismutase (SOD1) and glutathione reductase (GR) in both types of cells. Interestingly, catalase expression increased in normal corneal fibroblasts, but decreased in GCD2 corneal fibroblasts after melatonin treatment. Melatonin also reduced the levels of intracellular reactive oxygen species and H(2)O(2) in both cell types. In addition, the selective melatonin receptor antagonist luzindole blocked melatonin-induced expression of SOD1 and GR. The expression levels of melatonin receptors 1A (MT1) and 1B (MT2) were significantly higher in GCD2 corneal fibroblasts than in normal cells. These results suggest that increased expression of melatonin receptors may be involved in the defense mechanisms against oxidative stress in GCD2 corneal fibroblasts, and melatonin may have potential therapeutic implications for GCD2 treatment.     

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 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 mitochondrial-mediated apoptosis in human myeloid HL-60 cells

Abstract:  The role of melatonin in the mediation of apoptotic events has recently gained attention, especially after recent studies have reported that melatonin exerts antiapoptotic actions in normal cells but may activate proapoptotic pathways in some tumor cells. Here, we have evaluated the effect of melatonin on apoptosis in the human leukemia cell line HL-60. Melatonin treatment (1 m m ) induced a significant increase in caspase-3 and -9 activities. The effect of melatonin on the activation of caspases was time dependent, reaching a maximum after 12 hr of stimulation, and then decreasing to a minimum after 72 hr. Treatment with melatonin also evoked mitochondrial membrane depolarization and permeability transition pore induction, which caused loss of mitochondrial staining by calcein, and increased cell death by apoptosis/necrosis as demonstrated by propidium iodide positive-staining of cells after 72 hr of stimulation. In addition, the exposure of cells to melatonin resulted in an activation and association of the proapoptotic proteins Bax and Bid, as well as promoting detectable increases in the expression of both proteins. We conclude that melatonin has proapoptotic and/or oncostatic effects in the human myeloid cell line HL-60.