Inhibitory Effect Of Melatonin Research Articles

Abstract 5423: Melatonin inhibits aerobic glycolysis (Warburg effect) and fatty acid metabolic signaling in human leiomyosarcoma.

Abstract In the United States alone this year approximately 3,500 people will be diagnosed with leiomyosarcoma (LMS), a rare, malignant soft tissue cancer derived from smooth muscle cells, and about 36% will perish from this disease, despite the current medical interventions of surgery, radio- and chemotherapy. Epidemiological studies have indicated that the risk of many types of epithelial cancer including breast, prostate, colorectal, and endometrial cancer is increased in night-shift workers. These individuals experience circadian disruption in response to ocular exposure to light at night, which suppresses the nocturnal circadian production of melatonin by the pineal gland. In previous studies, we demonstrated that human LMS perfused with rodent donor blood containing physiological nocturnal levels of melatonin (1nM) showed a complete inhibition of LA uptake, 13-HODE release, and marked reduction in tumor cAMP levels and [3H]thymidine incorporation into tumor DNA. Here we showed the in vivo effects of a physiological, nocturnal concentration of melatonin on tumor proliferative activity mediated via aerobic glycolysis (Warburg effect) and LA metabolic signaling in tissue-isolated LMS xenografts perfused in situ in nude rats with human donor blood. Following tumor implantation, LMS xenografts exhibited latency-to-onset and growth rates that were 22 days and 0.21 ± 0.04 g/day, respectively; mean tumor weights were 5.1 ± 0.2 g (n = 72). The tissue-isolated human LMS xenografts in nude rats perfused in situ for 60 min with human donor blood containing melatonin resulted in a significant reduction in tumor aerobic glycolysis that included a 30-50% reduction in glucose uptake, lactate release, O2 uptake and CO2 production. Additionally, melatonin induced a complete inhibition of LA uptake, 13-HODE release, ERK 1/2, Akt, GSK3β (Ser9), and NF-kB (p65) phosphorylation, as well as significant reductions in tumor cAMP levels, DNA content and [3H]thymidine incorporation into tumor DNA. Addition of the non-selective MT1/MT2 melatonin antagonist S20928 (1 μM) completely reversed the inhibitory effects of melatonin on aerobic glycolysis, LA metabolic signaling and proliferative activity in LMS xenografts. Moreover, melatonin at physiological concentrations (1 nM) induced a 30-50% inhibition in cell proliferation in culture, and suppressed cell invasion by 30% in the transwell assay. These results demonstrate that in human LMS nocturnal melatonin levels directly and rapidly inhibit tumor growth activity and invasion via suppression of the Warburg effect and LA metabolic and other related signaling mechanisms. An understanding of this novel signaling pathway for the control of aerobic glycolysis and LA metabolism in human leiomyosarcoma could lead to new circadian-based approaches for cancer therapy and/or prevention of rare but highly aggressive mesenchymally-derived solid tumors. Citation Format: Lulu Mao, Robert T. Dauchy, Erin M. Dauchy, Melissa A. Wren, Samantha D. Zeringue, Victoria P. Belancio, David E. Blask, Steven M. Hill. Melatonin inhibits aerobic glycolysis (Warburg effect) and fatty acid metabolic signaling in human leiomyosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5423. doi:10.1158/1538-7445.AM2013-5423 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Melatonin inhibits the proliferation of human osteosarcoma cell line MG-63

It seems established that the onset of osteosarcoma and the reduction in melatonin production run in parallel; this suggests that the decline in the cancer-inhibiting agent, melatonin, may contribute to the occurrence of osteosarcoma and that melatonin supplementation may have promise for preventing the development and progression of this condition. There is, however, no direct evidence regarding an antiproliferative effect of melatonin in osteosarcoma cells. In the current study, we examined whether melatonin inhibits the proliferation of human osteosarcoma cell line MG-63. MTT staining showed that at 4mM–10mM concentrations, melatonin significantly reduced the MG-63 cell proliferation in a dose-dependent and time-dependent manner. Flow cytometry documented that 4mM melatonin significantly increased the fraction of cells in the G0/G1 phase of the cell cycle, while simultaneously reducing the proportion in the S and G2/M phases. Western blot and real-time PCR analyses further confirmed that melatonin's inhibitory effect was possibly because of downregulation of cyclin D1 and CDK4, related to the G1 phase, and of cyclin B1 and CDK1, related to the G2/M phase. There was no downregulation of cyclin E, CDK2, and cyclin A, which are related to G1/S transition and S phase. These findings provide evidence that melatonin may significantly inhibit human osteosarcoma cell proliferation in a dose-dependent and time-dependent manner and this inhibition involves the downregulation of cyclin D1, CDK4, cyclin B1 and CDK1.

Effect of Melatonin on the Proliferation and Differentiation of Chondrocytes from Rat Vertebral Body Growth Plate In Vitro

Purpose: Abnormal growth of vertebral body growth plate (VBGP) is considered as one of the etiologic factors in the adolescent idiopathic scoliosis (AIS). It was well-known that melatonin was correlated with the emergence and development of AIS. This study aimed to investigate the effect of melatonin on rat VBGP chondrocytes in vitro.Methods:Chondrocytes were isolated from rat VBGP, and treated with or without melatonin. Cell proliferation was measured by the Alamar Blue assay. Gene expression of collagen type II and aggrecan were evaluated by real-time PCR. Expression of the melatonin receptors (MT1, MT2), proliferating cell nuclear antigen (PCNA, a cell proliferation marker), Sox9 (a chondrocytic differentiation marker) and Smad4 (a common mediator in regulating the proliferation and differentiation of chondrocytes) were detected by Western blotting.Results: Expression of melatonin receptors (MT1, MT2) were detected in the rat VBGP chondrocytes. Melatonin, at 10 and 100 µg/mL concentration, significantly inhibited the proliferation of VBGP-chondrocytes and the gene expression of collagen type II and aggrecan, and down-regulated the protein expression of PCNA, Sox9 and Smad4. In addition, the inhibitory effect of melatonin was reversed by luzindole, a melatonin receptor antagonist.Conclusions: These results suggest that melatonin at high concentrations can inhibit the proliferation and differentiation of VBGP chondrocytes, which might give some new insight into the pathogenic mechanism of AIS.

Melatonin inhibits nitric oxide signaling by increasing PDE5 phosphorylation in coronary arteries

Melatonin inhibits nitric oxide (NO)-induced relaxation of coronary arteries. We tested the hypothesis that melatonin increases the phosphorylation of phosphodiesterase 5 (PDE5), which increases the activity of the enzyme and thereby decreases intracellular cGMP accumulation in response to NO and inhibits NO-induced relaxation. Sodium nitroprusside (SNP) and 8-Br-cGMP caused concentration-dependent relaxation of isolated coronary arteries suspended in organ chambers for isometric tension recording. In the presence of melatonin, the concentration-response curve to SNP, but not 8-Br-cGMP, was shifted to the right. The effect of melatonin on SNP-induced relaxation was abolished in the presence of the PDE5 inhibitors zaprinast and sildenafil. Melatonin markedly inhibited the SNP-induced increase in intracellular cGMP in coronary arteries, an effect that was also abolished by zaprinast. Treatment of coronary arteries with melatonin caused a nearly fourfold increase in the phosphorylation of PDE5, which increased the catalytic activity of the enzyme and thereby increased the degradation of cGMP to inactive 5'-GMP. Melatonin-induced PDE5 phosphorylation was markedly attenuated in the presence of the PKG1 inhibitors DT-2 or Rp-8-Br-PET-cGMPS and in those arteries in which PKG1 expression was first downregulated by 24-h incubation with SNP before exposure to melatonin. The selective MT(2) receptor antagonist 4-phenyl-2-propionamidotetralin completely blocked the stimulatory effect of melatonin on PDE5 phosphorylation as well as the inhibitory effect of melatonin on SNP-induced relaxation and intracellular cGMP. Thus, in coronary arteries, melatonin acts via MT(2) receptors and PKG1 to increase PDE5 phosphorylation, resulting in decreased cGMP accumulation in response to NO and impaired NO-induced vasorelaxation.

Melatonin Supplementation Decreases Prolactin Synthesis and Release in Rat Adenohypophysis: Correlation With Anterior Pituitary Redox State and Circadian Clock Mechanisms

In the laboratory rat, a number of physiological parameters display seasonal changes even under constant conditions of temperature, lighting, and food availability. Since there is evidence that prolactin (PRL) is, among the endocrine signals, a major mediator of seasonal adaptations, the authors aimed to examine whether melatonin administration in drinking water resembling in length the exposure to a winter photoperiod could affect accordingly the 24-h pattern of PRL synthesis and release and some of their anterior pituitary redox state and circadian clock modulatory mechanisms. Melatonin (3 µg/mL drinking water) or vehicle was given for 1 mo, and rats were euthanized at six time intervals during a 24-h cycle. High concentrations of melatonin (>2000 pg/mL) were detected in melatonin-treated rats from beginning of scotophase (at 21:00 h) to early photophase (at 09:00 h) as compared with a considerably narrower high-melatonin phase observed in controls. By cosinor analysis, melatonin-treated rats had significantly decreased MESOR (24-h time-series average) values of anterior pituitary PRL gene expression and circulating PRL, with acrophases (peak time) located in the middle of the scotophase, as in the control group. Melatonin treatment disrupted the 24-h pattern of anterior pituitary gene expression of nitric oxide synthase (NOS)-1 and -2, heme oxygenase-1 and -2, glutathione peroxidase, glutathione reductase, Cu/Zn- and Mn-superoxide dismutase, and catalase by shifting their acrophases to early/middle scotophase or amplifying the maxima. Only the inhibitory effect of melatonin on pituitary NOS-2 gene expression correlated temporally with inhibition of PRL production. Gene expression of metallothionein-1 and -3 showed maxima at early/middle photophase after melatonin treatment. The 24-h pattern of anterior pituitary lipid peroxidation did not vary after treatment. In vehicle-treated rats, Clock and Bmal1 expression peaked in the anterior pituitary at middle scotophase, whereas that of Per1 and Per2 and of Cry1 and Cry2 peaked at the middle and late photophase, respectively. Treatment with melatonin raised mean expression of anterior pituitary Per2, Cry1, and Cry2. In the case of Per1, decreased MESOR was observed, although the single significant difference found between the experimental groups when analyzed at individual time intervals was increase at early scotophase in the anterior pituitary of melatonin-treated rats. Melatonin significantly phase-delayed expression of Per1, Per2, and Cry1, also phase-delayed the plasma corticosterone circadian rhythm, and increased the amplitude of plasma corticosterone and thyrotropin rhythms. The results indicate that under prolonged duration of a daily melatonin signal, rat anterior pituitary PRL synthesis and release are depressed, together with significant changes in the redox and circadian mechanisms controlling them. (Author correspondence: danielcardinali@uca.edu.ar; danielcardinali@fibertel.com.ar)

Melatonin suppresses doxorubicin‐induced premature senescence of A549 lung cancer cells by ameliorating mitochondrial dysfunction

Melatonin is an indolamine that is synthesized in the pineal gland and shows a wide range of physiological functions. Although the anti-aging properties of melatonin have been reported in a senescence-accelerated mouse model, whether melatonin modulates cellular senescence has not been determined. In this study, we examined the effect of melatonin on anticancer drug-induced cellular premature senescence. We found that the doxorubicin (DOX)-induced senescence of A549 human lung cancer cells and IMR90 normal lung cells was substantially inhibited by cotreatment with melatonin in a dose-dependent manner. Mechanistically, the DOX-induced G2/M phase cell cycle arrest and the decrease in cyclinB and cdc2 expression were not affected by melatonin. However, the DOX-induced increase in intracellular levels of ROS, which is necessary for premature senescence, was completely abolished upon melatonin cotreatment. In addition, the reduction in mitochondrial membrane potential that occurs upon DOX treatment was inhibited by melatonin. An aberrant increase in mitochondrial respiration was also significantly suppressed by melatonin, indicating that melatonin ameliorates the mitochondrial dysfunction induced by DOX treatment. The treatment of A549 cells with luzindole, a potent inhibitor of melatonin receptors, failed to prevent the effects of melatonin treatment on mitochondrial functions and premature senescence in cells also treated with DOX; this suggests that melatonin suppresses DOX-induced senescence in a melatonin receptor-independent manner. Together, these results reveal that melatonin has an inhibitory effect of melatonin on premature senescence at the cellular level and that melatonin protects A549 cells from DOX-induced senescence. Thus, melatonin might have the therapeutic potential to prevent the side effects of anticancer drug therapy.

Melatonin inhibits NO‐dependent activation of large conductance, calcium‐activated K channels (BKCa) in porcine coronary artery smooth muscle cells

We recently demonstrated that melatonin inhibits NO‐induced relaxation of porcine coronary arteries by activating MT2 melatonin‐receptors (JPET 336: 127, 2011). We tested the hypothesis that melatonin inhibits NO/cGMP‐dependent activation of BKCa channels in coronary artery smooth muscle cells. Melatonin (10−7M) inhibited sodium nitroprusside (SNP)‐induced relaxation of isolated coronary arteries but had no effect on relaxation induced by 8‐Br‐cGMP, a stable, cell permeable cGMP analog. ODQ (10−5 M) completely inhibited SNP‐induced relaxation, as well as the increase in intracellular cGMP levels in response to SNP. In patch clamp studies, SNP (10−5 M)‐induced increases in BKCa currents were abolished in the presence of ODQ (10−5 M). Melatonin (10−7M) inhibited both the voltage‐dependent and SNP‐induced increase in BKCa currents, but had no effect on BKCa currents elicited by 8‐Br‐cGMP (10−3M). The inhibitory effect of melatonin on the voltage‐dependent and SNP‐induced increase in BKCa currents was abolished by either the PDE inhibitor, zaprinast (10−5M), or the MT2‐receptor antagonist, 4P‐PDOT (10−7 M). Coronary artery relaxation in response to NO involves the cGMP‐dependent activation of BKCa channels. Melatonin impairs NO‐induced relaxation of porcine coronary arteries by inhibiting the cGMP‐dependent activation of BKCa channels in coronary smooth muscle cells. (Supported by NIH HL77204)

Inhibitory effects of melatonin on the lipopolysaccharide‐induced CC chemokine expression in BV2 murine microglial cells are mediated by suppression of Akt‐induced NF‐κB and STAT/GAS activity

Melatonin influences sleep and circadian rhythm, and it has anti-inflammatory functions. However, the mechanism of its anti-inflammatory roles is not well understood. In our studies, we show that melatonin blocked lipopolysaccharide (LPS)-induced CCL2 (monocyte chemotactic protein-1; MCP-1), CCL5 (Regulated upon Activation, Normal T-cell Expressed, and Secreted), and CCL9 (macrophage inflammatory protein-1γ) chemokine mRNA expression in BV2 murine microglial cells. Melatonin markedly inhibited LPS-induced Akt phosphorylation and NF-κB activation. Furthermore, melatonin inhibited LPS-induced STAT1/3 phosphorylation and interferon-gamma activated sequence (GAS)-driven transcriptional activity. Interestingly, these effects were not associated with reactive oxygen species scavenging effects of melatonin or melatonin receptor signal pathways. Taken together, our results suggested that melatonin has anti-inflammatory functions through down-regulation of chemokine expression by inhibition of NF-κB and STAT/GAS activation in LPS-stimulated BV2 murine microglial cell line.

Short-term effects of melatonin and pinealectomy on serotonergic neuronal activity across the light–dark cycle

Melatonin (MLT) and serotonin (5-HT) are two biosynthetically related compounds implicated in several common physiological functions and the etiology of mood disorders. How they interact, though, is not yet fully understood. In this study, single-unit extracellular recordings were used to monitor dorsal raphe nucleus (DR) 5-HT neuronal activity in anesthetized rats, under basal conditions (CTRL), in response to MLT administration, and after pinealectomy (PX) across the light-dark cycle. Under basal conditions, the number of spontaneously active 5-HT neurons and their firing rate were both significantly lower in the dark phase. In the light phase, administration of MLT at low doses (0.5-1 mg/kg, i.v.) decreased 5-HT firing activity. This inhibitory effect of MLT was completely blocked by the MT₁/MT₂ receptor antagonist luzindole, but not by the selective MT(2) receptor antagonist 4P-PDOT, the selective 5-HT(1A) receptor antagonist WAY100635, or by the α₂ adrenoceptor antagonist idazoxan. In the opposite experiment, PX increased 5-HT firing activity in the dark phase, and this was reversed by MLT administration (1 mg/kg, i.v.). Finally, in a forced swim test, MLT (1 mg/kg, i.p.) increased immobility time and decreased swimming behavior. Together, these results suggest that nocturnal MLT secretion imposes tonic inhibitory control over a sub-population of DR 5-HT neurons. This MLT-induced decrease in 5-HT neurotransmission may represent a biological mechanism underlying mood disorders characterized by increased MLT secretion, such as seasonal affective disorder.

Melatonin inhibits insulin secretion in rat insulinoma β-cells (INS-1) heterologously expressing the human melatonin receptor isoform MT2

Melatonin exerts some of its effects via G-protein-coupled membrane receptors. Two membrane receptor isoforms, MT1 and MT2, have been described. The MT1 receptor is known to inhibit second messenger cyclic adenosine monophosphate (cAMP) signaling through receptor-coupling to inhibitory G-proteins (G(i) ). Much less is known about the MT2 receptor, but it has also been implicated in signaling via G(i) -proteins. In rat pancreatic β-cells, it has recently been reported that the MT2 receptor plays an inhibitory role in the cyclic guanosine monophosphate (cGMP) pathway. This study addresses the signaling features of the constitutively expressed human recombinant MT2 receptor (hMT2) and its impact on insulin secretion, using a rat insulinoma β-cell line (INS-1). On the basis of a specific radioimmunoassay, insulin secretion was found to be more strongly reduced in the clones expressing hMT2 than in INS-1 controls, when incubated with 1 or 100 nm melatonin. Similarly, cAMP and cGMP levels, measured by specific enzyme-linked immunosorbent assays (ELISAs), were reduced to a greater extent in hMT2 clones after melatonin treatment. In hMT2-expressing cells, the inhibitory effect of melatonin on insulin secretion was blocked by pretreatment with pertussis toxin, demonstrating the coupling of the hMT2 to G(i) -proteins. These results indicate that functional hMT2 expression leads to the inhibition of cyclic nucleotide signaling and a reduction in insulin release. Because genetic variants of the hMT2 receptor are considered to be risk factors in the development of type 2 diabetes, our results are potentially significant in explaining and preventing the pathogenesis of this disease.

Expression of functional melatonin MT1 receptors in equine luteal cells: in vitro effects of melatonin on progesterone secretion

In the present study, we analysed the molecular mechanism(s) by which melatonin directly affects ovarian function in the mare. In Experiment 1, follicles and corpora lutea (CL) were collected from slaughterhouse ovaries and analysed for melatonin (MT(1)) receptor mRNA and protein. In Experiment 2, CL were collected from slaughterhouse ovaries and cultured in Dulbecco's modified Eagle's medium-F12 medium (control medium) supplemented with 50 ng mL(-1) equine chorionic gonadotrophin (eCG), 1 nM-1 μM melatonin, 1 μM forskolin or 1 μM luzindole. Explants were cultured for 3 h in the presence of these drugs. Conditioned media were analysed for progesterone production; luteal cells were analysed for cholesterol side-chain cleavage enzyme (P450scc), a steroidogenic enzyme that converts cholesterol into pregnenolone. Both MT(1) receptor mRNA and protein were expressed in follicles and CL. Melatonin inhibited both the eCG- and forskolin-stimulated production of progesterone, as well as the forskolin-stimulated expression of P450scc, in equine luteal cells and the effect was dose-dependent. The inhibitory effect of melatonin was blocked by luzindole, a non-selective melatonin MT(1) and MT(2) receptor antagonist. The data support the presence of functional melatonin receptors in luteal cells and a regulatory role for melatonin in the endocrine function of the equine CL.

Inhibitory effects of melatonin on the expression of phosphorylation p38 mitogen-activated protein kinase during acute lung injury in rats

To investigate the protective effect of melatonin (MT) on lung tissues during acute lung injury (ALI) in rats and its possible mechanism. Seventy-two Sprague-Dawley (SD) rats were randomly divided into three groups: control group, lipopolysaccharide (LPS) group and MT treatment group. In LPS group and MT treatment group, 1 ml/kg of LPS (200 mug/200 mul) was administered through the airway. In MT group, 10 mg/kg of MT was injected intraperitoneally before and after administration of LPS, and 1 ml/kg of ethanol-normal saline was given in control rats. The rats were sacrificed at 3, 6, 10 hours after administration of LPS, and the lung tissue was obtained for determination of the contents of nitrogen monoxide (NO) and malondialdehyde (MDA), and activity of superoxide dismutase (SOD). In addition, the expression of phosphorylation p38 mitogen-activated protein kinase (p-p38 MAPK) in lung tissue was assayed with immunohistochemistry staining. Compared with control group, SOD activity (U/mg) decreased significantly in LPS group (3 hours: 73.78+/-3.62 vs. 112.69+/-3.26, 6 hours : 66.07+/-2.31 vs. 117.85+/-1.96, 10 hours: 55.13+/-5.26 vs. 118.27+/-2.16, all P<0.01), but NO (micromol/mg), MDA (nmol/mg) content and the expression of p-p38 MAPK [absorbance (A) value] increased obviously (NO: 8.19+/-0.48 vs. 2.32+/-0.20 at 3 hours, 11.71+/-0.27 vs. 2.08+/-0.15 at 6 hours, 16.53+/-0.60 vs. 2.76+/-0.21 at 10 hours; MDA: 11.43+/-0.68 vs. 2.86+/-0.21 at 3 hours, 19.63+/-1.29 vs. 2.85+/-0.19 at 6 hours, 26.63+/-2.00 vs. 2.84+/-0.28 at 10 hours; p-p38 MAPK: 0.340+/-0.020 vs. 0.238+/-0.019 at 3 hours, 0.410+/-0.016 vs. 0.218+/-0.024 at 6 hours, 0.578+/-0.066 vs. 0.238+/-0.036 at 10 hours, all P<0.01). The administration of MT mitigated above contents significantly [SOD (U/mg) was 86.02+/-2.81, 80.87+/-3.40, 94.46+/-5.03, NO (micromol/mg) was 3.80+/-0.28, 5.32+/-0.22, 7.24+/-0.52, MDA (nmol/mg) was 8.18+/-0.84, 7.84+/-0.78, 6.43+/-1.06, and p-p38 MAPK (A value) was 0.311+/-0.018, 0.312+/-0.019, 0.314+/-0.021 at 3, 6, 10 hours, respectively, P<0.05 or P<0.01]. MT possessed protective effect on lung tissues during ALI by its antioxidation effect and inhibition of over- expression of p-p38 MAPK.

Melatonin inhibits NO‐induced cGMP accumulation by increasing PDE5 phosphorylation in coronary arteries

Melatonin impairs NO‐induced relaxation by inhibiting the NO‐induced increase in cGMP in coronary arteries. We tested the hypothesis that the inhibitory effect of melatonin on NO‐signaling is due to increased phosphodiesterase type‐5 (PDE5) activation. Melatonin (10−7M) inhibited sodium nitroprusside (SNP) induced relaxation of isolated coronary arteries but had no effect on relaxation induced by 8‐Br‐cGMP. The inhibitory effect of melatonin on SNP‐induced relaxation was abolished in the presence of either the PDE inhibitor, zaprinast (10−5M) or the MT2‐receptor antagonist, 4P‐PDOT (10−7 M). Melatonin (10−7M) markedly reduced the SNP‐induced increase in cGMP levels in coronary arteries, and this effect of melatonin was significantly attenuated in the presence of either zaprinast (10−5M) or 4P‐PDOT (10−7 M). Immunohistochemical analysis confirmed localization of MT2‐receptors in the vascular smooth muscle cell layer. Immunoblot analysis demonstrated PDE5 expression in coronary arteries. Incubation of the tissues with melatonin (10−7M) caused a significant increase in PDE5 phosphorylation, which was abolished in the presence of 4P‐PDOT (10−7 M). The data indicate that melatonin increases the catalytic activity of PDE5 by activating MT2‐receptors in coronary arteries, resulting in decreased cGMP accumulation in response to NO and impaired NO‐induced vasorelaxation. (Supported by NIH HL77204)

Effect of naloxone on the inhibitory effect of melatonin on the release of arginine-vasopressin induced by physical exercise in man

This study was performed in order to establish whether endogenous opioids play a role in the inhibitory effect of melatonin on arginine-vasopressin (AVP) response to physical exercise. Seven healthy men underwent four bicycle ergometer tests until exhaustion [exercise control test, exercise plus naloxone (2 mg injected plus 5 mg infused intravenously), exercise plus melatonin (6 mg), exercise plus melatonin plus naloxone]. Plasma AVP concentrations, non endocrine physiological parameters (NEPP) and biochemical parameters were evaluated during all tests. NEPP and biochemical values had a similar pattern during all tests. Physical exercise significantly increased the AVP levels. The pre-treatment with melatonin inhibited the AVP response to physical exercise. In contrast, naloxone had no effect on AVP rise during exercise, when given alone, whereas it abolished the negative effect of melatonin on AVP response to physical exercise. Our data indicate that naloxone-sensitive endogenous opiates mediate the inhibitory modulation exerted by melatonin on the AVP response to physical exercise.

Inhibitory effects of melatonin on sulfatase and 17β-hydroxysteroid dehydrogenase activity and expression in glioma cells

Melatonin interacts with estradiol at the estrogen receptor level in different kinds of neoplasias and also regulates the expression and the activity of some enzymes involved in the biosynthesis of estrogens in peripheral tissues. Glioma cells express estrogen receptors and have the ability to synthesize estrogens locally. Since melatonin inhibits the growth of C6 cells, and this indoleamine has been demonstrated to be capable of decreasing aromatase expression and activity in these cells, the aim of the present study was to analyze whether the regulation of the sulfatase, the enzyme that catalyzes the rate-limiting step in the conversion of estrogen sulfates to estrogens, and 17beta-hydroxysteroid dehydrogenase, the enzyme which converts the relatively inactive estrone to the most potent 17beta-estradiol, could be involved in the inhibition of glioma cell growth by melatonin. We found that melatonin decreases the growth of C6 glioma cells and reduces the sulfatase and 17beta-hydroxysteroid dehydrogenase activity. Finally, we demonstrated that melatonin downregulates sulfatase and 17beta-hydroxysteroid dehydrogenase mRNA steady state levels in these glioma cells. By analogy to the implications of these enzymes in other forms of estrogen-sensitive tumors, it is conceivable that their modulation by melatonin may play a role in the growth of glioblastomas.

Inhibitory effect of melatonin on lung oxidative stress induced by respiratory syncytial virus infection in mice

Previous research has shown that antioxidant (butylated hydroxyanisole) treatment ameliorates respiratory syncytial virus (RSV)-induced disease and lung inflammation. Melatonin has been reported to exhibit a wide varieties of biological effects, including antioxidant and anti-inflammation, and has no evident toxicity and side effect. But it is not known whether melatonin would modify RSV-induced lung disease and oxidative stress. The present study was to establish the involvement of oxidative stress in the pathogenesis of RSV-induced lung inflammation, and to investigate the protective effect of administration of melatonin in mice with RSV-induced oxidative pulmonary injury for 4 days. Malondialdehyde (MDA), an end product of lipid peroxidation, and glutathione (GSH) and superoxide dismutase (SOD) and nitric oxide (NO) levels were evaluated in lung tissue homogenates by spectrophotometry. Hydroxyl radical (.-OH), one of the indicators of free radical formation, was also detected in lung homogenates by Fenton reaction. Tumor necrosis factor-a (TNF-a) concentrations in mouse serum were measured with ELISA assay. The results demonstrated that the mice intranasally inoculated with RSV resulted in oxidative stress changes by increasing NO, MDA and .-OH levels, and decreasing GSH and SOD activities, whereas administration of melatonin significantly reversed all these effects. Furthermore, melatonin inhibited production of proinflammatory cytokines such as TNF-a in serum of RSV-infected mice. These results suggest that melatonin ameliorates RSV-induced lung inflammatory injury in mice via inhibition of oxidative stress and proinflammatory cytokine production and may be as a novel therapeutic agent in virus-induced pulmonary infection.

Adipocyte differentiation is inhibited by melatonin through the regulation of C/EBPβ transcriptional activity

Considering that melatonin has been implicated in body weight control, this work investigated whether this effect involves the regulation of adipogenesis. 3T3-L1 preadipocytes were induced to differentiate in the absence or presence of melatonin (10(-3) m). Swiss-3T3 cells ectopically and conditionally (Tet-off system) over-expressing the 34 kDa C/EBPbeta isoform (Swiss-LAP cells) were employed as a tool to assess the mechanisms of action at the molecular level. Protein markers of the adipogenic phenotype were analyzed by Western blot. At 36 hr of differentiation of 3T3-L1 preadipocytes, a reduction of PPARgamma expression was detected followed by a further reduction, at day 4, of perilipin, aP2 and adiponectin protein expression in melatonin-treated cells. Real-time PCR analysis also showed a decrease of PPARgamma (60%), C/EBPalpha (75%), adiponectin (30%) and aP2 (40%) mRNA expression. Finally, we transfected Swiss LAP cells with a C/EBPalpha gene promoter/reporter construct in which luciferase expression is enhanced in response to C/EBPbeta activity. Culture of such transfected cells in the absence of tetracycline led to a 2.5-fold activation of the C/EBPalpha promoter. However, when treated with melatonin, the level of C/EBPalpha promoter activation by C/EBPbeta was reduced by 50% (P = 0.05, n = 6). In addition, this inhibitory effect of melatonin was also reflected in the phenotype of the cells, since their capacity to accumulate lipids droplets was reduced as confirmed by the poor staining with Oil Red O. In conclusion, melatonin at a concentration of 10(-3 ) m works as a negative regulator of adipogenesis acting in part by inhibiting the activity of a critical adipogenic transcription factor, C/EBPbeta.

An unexpected effect of 5-MCA-NAT in chick retinal development

Luzindole is an unselective antagonist of the melatonin receptors and melatonin's other binding sites, although some exceptions have been observed in chick retinal neurodevelopment, where this unselective antagonist does not block melatonin's inhibitory effect on the adenylate cyclase enzyme, probably due to the presence of some other melatonin receptor(s) or binding site(s). The present study investigated the modulation of cyclic adenosine 3′-5′-monophosphate (cAMP) levels via MT3 melatonin-binding sites, located within the QR2 (dihydronicotinamide riboside: quinone oxidoreductase 2) enzyme, by observing the response to luzindole. Embryonic and post-hatch retinas, incubated with a selective agonist for the MT3 melatonin-binding site 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT, 10 or 100nM), had an increase in cAMP accumulation relative to control retinas. Luzindole (5μM) inhibited the 5-MCA-NAT stimulatory effect at all ages tested. The agonist 5-MCA-NAT enhanced the melatonin inhibitory effect on cAMP levels stimulated by forskolin (5μM), but not the stimulatory forskolin effect. The results suggest that MT3 melatonin-binding sites are present in embryonic and post-hatch chick retinas and that luzindole more selectively blocks the 5-MCA-NAT effect on cAMP accumulation than it blocks the melatonin inhibitory effect via G protein-coupled receptors in chick retinal neurodevelopment.

Melatonin inhibits human fibroblast‐like synoviocyte proliferation via extracellular signal‐regulated protein kinase/P21CIP1/P27KIP1 pathways

The excessive proliferation and migration of synoviocytes are well-characterized phenomena that play key roles in the pathophysiology of rheumatoid arthritis (RA). Melatonin has been shown to have potent anti-proliferative effect in various cancer cells such as breast and prostate cancer cells. In this study, we examined the role of melatonin on synoviocyte proliferation in primary cultured human fibroblast-like synoviocytes (FLSs) by analyzing protein expression of P21(CIP1) (P21) and P27(KIP1) (P27), the cyclin-dependent kinase inhibitors that are important in cell cycle control, and the phosphorylation of mitogen-activated protein kinases (MAPKs). RA-FLS proliferation was determined by a [(3)H]-thymidine incorporation assay. Western blot analysis was applied to examine the underlying mechanisms of melatonin's effect. Melatonin inhibited RA-FLS proliferation in a dose-dependent manner. It reduced proliferation of passage 2 FLSs by 25% at 10 microm and by nearly 40% at 100 microm concentrations. The inhibitory effect of melatonin on RA-FLS proliferation was also observed in passages 4 and 6. Melatonin upregulated the expression levels of P21 and P27 dose-dependently (24 hr), induced the phosphorylation of extracellular signal-regulated protein kinase (ERK) time-dependently (10 microm), but did not affect phosphorylation of P38 in RA-FLSs. In addition, the expression of P21 and P27 triggered by melatonin was inhibited by the pretreatment of the ERK inhibitor, PD98059 (10 microm). The anti-proliferative action of melatonin in RA-FLSs was also blocked by PD98059. Taken together, these results suggest that melatonin exerts the inhibitory effect of the proliferation of RA-FLSs through the activation of P21 and P27 mediated by ERK. Hence we suggest that melatonin could be used as a therapeutic agent for the treatment of RA.

Melatonin inhibits NO‐induced cGMP accumulation in porcine coronary arteries

Recent studies in our laboratory indicate that melatonin modulates vascular tone by interfering with nitric oxide (NO) signaling in coronary arteries. Here we tested the hypothesis that melatonin impairs NO‐induced vasorelaxation by inhibiting the NO‐induced increase in cGMP levels in coronary arteries. In organ bath studies, sodium nitroprusside (SNP) (10−9‐10−5 M) caused relaxation of isolated porcine coronary artery rings. Incubation of the tissues with melatonin (10−7 M) caused a rightward shift in the SNP concentration‐response curve. In the presence of the phosphodiesterase inhibitor, zaprinast (10−5 M), the inhibitory effect of melatonin on SNP‐induced vasorelaxation was abolished. Melatonin (10−7 M) had no effect on basal cGMP levels but markedly inhibited the SNP‐induced increase in cGMP. The effect of melatonin on cGMP accumulation was significantly diminished by incubation of the tissues with either zaprinast (10−5 M) or the MT2‐receptor antagonist, 4P‐PDOT (10−7 M). The expression of MT2‐receptors in coronary arteries was confirmed by immunoblot analysis. Taken together, these results suggest that the inhibitory effect of melatonin on NO signaling in coronary arteries is most likely due to impaired cGMP accumulation in response to NO. The inhibitory effect of melatonin is mediated by MT2‐receptors, perhaps via a mechanism that is coupled to increased phosphodiesterase activity.