Showing papers in "Journal of Pineal Research in 2010"

Melatonin, cardiolipin and mitochondrial bioenergetics in health and disease.

Abstract: Melatonin is a natural occurring compound with well-known antioxidant properties Melatonin is ubiquitously distributed and because of its small size and amphiphilic nature, it is able to reach easily all cellular and subcellular compartments The highest intracellular melatonin concentrations are found in mitochondria, raising the possibility of functional significance for this targeting with involvement in situ in mitochondrial activities Mitochondria, the powerhouse of the cell, are considered to be the most important cellular organelles to contribute to degenerative processes mainly through respiratory chain dysfunction and formation of reactive oxygen species, leading to damage to mitochondrial proteins, lipids and DNA Therefore, protecting mitochondria from oxidative damage could be an effective therapeutic strategy against cellular degenerative processes Many of the beneficial effects of melatonin administration may depend on its effect on mitochondrial physiology Cardiolipin, a phospholipid located at the level of inner mitochondrial membrane is known to be intimately involved in several mitochondrial bioenergetic processes as well as in mitochondrial-dependent steps of apoptosis Alterations to cardiolipin structure, content and acyl chain composition have been associated with mitochondrial dysfunction in multiple tissues in several physiopathological situations and aging Recently, melatonin was reported to protect the mitochondria from oxidative damage by preventing cardiolipin oxidation and this may explain, at least in part, the beneficial effect of this molecule in mitochondrial physiopathology In this review, we discuss the role of melatonin in preventing mitochondrial dysfunction and disease

Melatonin modulates neuroinflammation and oxidative stress in experimental diabetic neuropathy: effects on NF-κB and Nrf2 cascades.

Abstract: Melatonin exhibits an array of biological activities, including antioxidant and anti-inflammatory actions. Diabetic neuropathy is one of the complications of diabetes with a prevalence rate of 50–60%. We have previously reported the protective effect of melatonin in experimental diabetic neuropathy. In this study, we investigated the role of nuclear factor-kappa B (NF-κB) and nuclear erythroid 2-related factor 2 (Nrf2) in melatonin-mediated protection against streptozotocin-induced diabetic neuropathy. Melatonin at doses of 3 and 10 mg/kg was administered daily in seventh and eighth week after diabetes induction. Motor nerve conduction velocity and nerve blood flow were improved in melatonin-treated animals. Melatonin also reduced the elevated expression of NF-κB, IκB-α, and phosphorylated IκB-α. Further, melatonin treatment also reduced the elevated levels of proinflammatory cytokines (TNF-α and IL-6), iNOS and COX-2 in sciatic nerves of animals. The capacity of melatonin to modulate Nrf2 pathway was associated with increased heme oxygenase-1 (HO-1) expression, which strengthens antioxidant defense. This fact was also established by decreased DNA fragmentation (because inhibition of excessive oxidant-induced DNA damage) in the sciatic nerve of melatonin-treated animals. The results of this study suggest that melatonin modulates neuroinflammation by decreasing NF-κB activation cascade and oxidative stress by increasing Nrf2 expression, which might be responsible at least in part, for its neuroprotective effect in diabetic neuropathy.

Melatonin and circadian biology in human cardiovascular disease.

Abstract: Diurnal rhythms influence cardiovascular physiology, i.e. heart rate and blood pressure, and they appear to also modulate the incidence of serious adverse cardiac events. Diurnal variations occur also at the molecular level including changes in gene expression in the heart and blood vessels. Moreover, the risk/benefit ratio of some therapeutic strategies and the concentration of circulating cardiovascular system biomarkers may also vary across the 24-hr light/dark cycle. Synchrony between external and internal diurnal rhythms and harmony among molecular rhythms within the cell are essential for normal organ biology. Diurnal variations in the responsiveness of the cardiovascular system to environmental stimuli are mediated by a complex interplay between extracellular (i.e. neurohumoral factors) and intracellular (i.e. specific genes that are differentially light/dark regulated) mechanisms. Neurohormones, which are particularly relevant to the cardiovascular system, such as melatonin, exhibit a diurnal variation and may play a role in the synchronization of molecular circadian clocks in the peripheral tissue and the suprachiasmatic nucleus. Moreover, mounting evidence reveals that the blood melatonin rhythm has a crucial role in several cardiovascular functions, including daily variations in blood pressure. Melatonin has antioxidant, anti-inflammatory, chronobiotic and, possibly, epigenetic regulatory functions. This article reviews current knowledge related to the biological role of melatonin and its circadian rhythm in cardiovascular disease.

Sirtuins, melatonin and circadian rhythms: building a bridge between aging and cancer

Abstract: Histone deacetylases (HDAC) have been under intense scientific investigation for a number of years. However, only recently the unique class III HDAC, sirtuins, have gained increasing investigational momentum. Originally linked to longevity in yeast, sirtuins and more specifically, SIRT1 have been implicated in numerous biological processes having both protective and/or detrimental effects. SIRT1 appears to play a critical role in the process of carcinogenesis, especially in age-related neoplasms. Similarly, alterations in circadian rhythms as well as production of the pineal hormone melatonin have been linked to aging and cancer risk. Melatonin has been found act as a differentiating agent in some cancer cells and to lower their invasive and metastatic status. In addition, melatonin synthesis and release occurs in a circadian rhythm fashion and it has been linked to the core circadian machinery genes (Clock, Bmal1, Periods, and Cryptochromes). Melatonin has also been associated with chronotherapy, the timely administration of chemotherapy agents to optimize trends in biological cycles. Interestingly, a recent set of studies have linked SIRT1 to the circadian rhythm machinery through direct deacetylation activity as well as through the nicotinamide adenine dinucleotide (NAD(+)) salvage pathway. In this review, we provide evidence for a possible connection between sirtuins, melatonin, and the circadian rhythm circuitry and their implications in aging, chronomodulation, and cancer.

Melatonin inhibits adipogenesis and enhances osteogenesis of human mesenchymal stem cells by suppressing PPARγ expression and enhancing Runx2 expression

Abstract: Adipogenesis and osteogenesis, a reciprocal relationship in bone marrow, are complex processes including proliferation of precursor cells, commitment to the specific lineage, and terminal differentiation. Accumulating evidence from in vitro and in vivo studies suggests that melatonin affects terminal differentiation of osteoblasts and adipocytes, but little is known about the effect of melatonin on the process of adipogenesis and osteogenesis, especially adipogenesis. This study was performed to determine the effect of melatonin on adipogenesis and osteogenesis in human mesenchymal stem cells (hMSCs). Cell proliferation assays demonstrated that melatonin had no apparent effect on the proliferation of hMSCs. When melatonin was added to the adipogenic/osteogenic medium, it directly inhibited adipogenesis and simultaneously promoted osteogenesis of hMSCs in a dose-dependent manner. Furthermore, quantitative RT-PCR demonstrated that melatonin significantly suppressed peroxisome proliferator-activated receptor gamma (PPARγ) expression (day 3, 25% decrease; day 6, 47% decrease), but promoted Runx2 expression (day 3, 87% increase; day 6, 56% increase) in the early stages of adipogenesis and osteogenesis of hMSCs. Moreover, melatonin down-regulated several markers of terminal adipocyte differentiation, including leptin (30%), lipoprotein lipase (LPL, 41%), adiponectin (51%), and adipocyte protein 2 (αP2, 45%). Meanwhile, melatonin up-regulated several markers of osteoblast differentiation, including alkaline phosphatase (110%), osteopontin (218%), and osteocalcin (310%). These results suggest that melatonin directly inhibits hMSCs adipogenic differentiation and significantly enhances hMSCs osteogenic differentiation by suppressing PPARγ expression and enhancing Runx2 expression; this provides further evidence for melatonin as an anti-osteoporosis drug.

Enhanced production of melatonin by ectopic overexpression of human serotonin N-acetyltransferase plays a role in cold resistance in transgenic rice seedlings

Abstract: Serotonin N-acetyltransferase (SNA), a rate-limiting enzyme in melatonin biosynthesis in vertebrates, is responsible for the production of N-acetylserotonin; this molecule is then converted to melatonin by hydroxyindole-O-methyltransferase. We generated transgenic rice plants via expression of the human SNA gene under the constitutive ubiquitin promoter using Agrobacterium-mediated gene transformation. We investigated the role of SNA in the biosynthesis of melatonin and the physiological role of melatonin in rice plants. The integration and expression of the transgene were confirmed in T(1) transgenic rice seedlings by Southern, Northern, and RT-PCR analyses. High SNA-specific enzyme activities were observed in the transgenic rice plants, whereas the wild type revealed a trace level of SNA enzyme activity. The functional expression of SNA protein was closely associated with the elevated synthesis of N-acetylserotonin and melatonin in the transgenic rice plants. Experiments using both exogenous treatment of serotonin and senescent detached leaves, which contain a pool of serotonin, significantly enhanced melatonin biosynthesis, indicating that endogenous serotonin levels play a bottleneck role in the pathway of melatonin biosynthesis. Finally, the transgenic rice seedlings with high levels of melatonin showed elevated chlorophyll synthesis during cold stress, suggesting a role for melatonin in cold-stress resistance.

Melatonin suppresses tumor angiogenesis by inhibiting HIF-1α stabilization under hypoxia

Abstract: Angiogenesis is an important mediator of tumor progression. As tumors expand, diffusion distances from the existing vascular supply increases, resulting in hypoxia in the cancer cells. Sustained expansion of a tumor mass requires new blood vessel formation to provide rapidly proliferating tumor cells with an adequate supply of oxygen and nutrients. The key regulator of hypoxia-induced angiogenesis is the transcription factor known as hypoxia-inducible factor (HIF)-1. HIF-1alpha is stabilized by hypoxia-induced reactive oxygen species (ROS) and enhances the expression of several types of hypoxic genes, including that of the angiogenic activator known as vascular endothelial cell growth factor (VEGF). In this study, we found that melatonin, a small lipophilic molecule secreted primarily by the pineal gland, destabilizes hypoxia-induced HIF-1alpha protein levels in the HCT116 human colon cancer cell line. This destabilization of HIF-1alpha resulted from the antioxidant activity of melatonin against ROS induced by hypoxia. Moreover, under hypoxia, melatonin suppressed HIF-1 transcriptional activity, leading to a decrease in VEGF expression. Melatonin also blocked in vitro tube formation and invasion and migration of human umbilical vein endothelial cells induced by hypoxia-stimulated conditioned media of HCT116 cells. These findings suggest that melatonin could play a pivotal role in tumor suppression via inhibition of HIF-1-mediated angiogenesis.

Visualization of melatonin’s multiple mitochondrial levels of protection against mitochondrial Ca2+-mediated permeability transition and beyond in rat brain astrocytes

Abstract: Melatonin protects cells against various types of oxidative stress-induced apoptosis due primarily to its ability to effectively scavenge pathological and disease condition-augmented generation of mitochondrial reactive oxygen species (mROS). Once produced, mROS indiscriminately damage mitochondrial components and more importantly they crucially activate directly the mitochondrial permeability transition (MPT), one of the critical mechanisms for initiating post mitochondrial apoptotic signaling. Whether or not melatonin targets directly the MPT, however, remains inconclusive, particularly during oxidative stress. This study, thus, investigated this possibility of an 'oxidation free Ca(2+) stress' in the presence of vitamin E after ionomycin exposure as a sole Ca(2+)-mediated MPT in order to exclude melatonin's primary antioxidative effects as well as Ca(2+)-mediated oxidative stress. The studies were carried out using cultured rat brain astrocytes RBA-1. With the application of laser scanning multiple fluorescence imaging microscopy, we visualized for the first time multiple mitochondrial protective effects provided by melatonin during Ca(2+) stress. First, melatonin, due to its primary antioxidative actions, completely prevented mCa(2+)-induced mROS formation during ionomycin exposure. Secondly, when melatonin(')s antioxidative effects were prevented due to the addition of vitamin E, melatonin significantly prevented mCa(2+)-mediated MPT and apoptosis suggesting its direct targeting of the MPT. Surprisingly, in the presence of cyclosporin A, a MPT inhibitor, melatonin reduced further mCa(2+)-mediated apoptosis during ionomycin exposure also suggesting its targeting beyond the MPT. As astrocytes are actively involve in regulating synaptic transmission and neurovascular coupling in the CNS, these multiple mitochondrial layers of protection provided by melatonin against mCa(2+)-and/or mROS-mediated apoptosis in astrocytes may be crucial for future therapeutic prevention and treatment of astrocyte-mediated neurodegenerative diseases in the CNS.

Melatonin or silymarin reduces maneb- and paraquat-induced Parkinson’s disease phenotype in the mouse

Abstract: Oxidative stress is reported as one of the most widely accepted mechanisms of maneb (MB)- and paraquat (PQ)-induced nigrostriatal dopaminergic neurodegeneration leading to the Parkinson's disease (PD) phenotype. The study investigated the effects of silymarin, an antioxidant of plant origin, and melatonin, an indoleamine produced in all species, in MB- and PQ-induced mouse model of PD. The mice were treated intraperitoneally daily with silymarin (40mg/kg) or melatonin (30mg/kg) along with respective controls for 9wk. Subsets of these animals were also treated with MB (30mg/kg) and PQ (10mg/kg), twice a week, for 9wk, 2hr after silymarin/melatonin treatment. Locomotor activities along with striatal dopamine content, tyrosine hydroxylase (TH) immunoreactivity, number of degenerating neurons, lipid peroxidation and nitrite content were estimated. Additionally, mRNA expression of vesicular monoamine transporter, cytochrome P-450 2E1 (CYP2E1), and glutathione-S-transferase A4-4 (GSTA4-4), catalytic activities of CYP2E1 and GSTA4-4 and protein expressions of unphosphorylated and phosphorylated p53 (p53 and P-p53), Bax and caspase 9 were measured in control and MB- and PQ-treated mice with either silymarin or melatonin treatments. Silymarin/melatonin significantly offset MB- and PQ-mediated reductions in locomotor activities, dopamine content, TH immunoreactivity, VMAT 2 mRNA expression and the expression of p53 protein. Silymarin/melatonin attenuated the increases in lipid peroxidation, number of degenerating neurons, nitrite content, mRNA expressions of cytochrome P-450 2E1 (CYP2E1) and GSTA4-4, catalytic activities of CYP2E1 and GST and P-p53, Bax and caspase 9 protein expressions. The results demonstrate that silymarin and melatonin offer nigrostriatal dopaminergic neuroprotection against MB- and PQ-induced PD by the modulation of oxidative stress and apoptotic machinery.

Melatonin effect on plasma adiponectin, leptin, insulin, glucose, triglycerides and cholesterol in normal and high fat–fed rats

Abstract: Melatonin effect on body weight progression, mean levels and 24-hr pattern of circulating adiponectin, leptin, insulin, glucose, triglycerides and cholesterol were examined in rats fed a normal or a high-fat diet. In experiment 1, rats fed a normal diet were divided into two groups: receiving melatonin (25 μg/mL drinking water) or vehicle for 9 wk. In experiment 2, animals were divided into three groups: two fed with a high-fat diet (35% fat) and melatonin (25 μg/mL) or vehicle in drinking water for 11 wk, while a third group was given a normal diet (4% fat). At the end of experiments, groups of eight rats were killed at six different time intervals throughout a 24-hr period. Melatonin administration for 9 wk decreased body weight gain from the 3rd wk on without affecting food intake. A significant reduction in circulating insulin, glucose and triglyceride mean levels and disrupted daily patterns of plasma adiponectin, leptin and insulin were observed after melatonin. In high fat-fed rats, melatonin attenuated body weight increase, hyperglycemia and hyperinsulinemia, as well as the increase in mean plasma adiponectin, leptin, triglycerides and cholesterol levels. The high-fat diet disrupted normal 24-hr patterns of circulating adiponectin, insulin and cholesterol, the effects on insulin and cholesterol being counteracted by melatonin. Nocturnal plasma melatonin concentration in control and obese rats receiving melatonin for 11 wk attained values 21-24-fold greater than controls. The results indicate that melatonin counteracts some of the disrupting effects of diet-induced obesity in rats.

Melatonin treatment normalizes plasma pro‐inflammatory cytokines and nitrosative/oxidative stress in patients suffering from Duchenne muscular dystrophy

Abstract: Duchenne muscular dystrophy (DMD), a lethal disorder characterized by dystrophin absence, courses with chronic inflammation, sarcolemmal damage, and skeletal muscle degeneration. Among the multiple pathogenic mechanisms proposed for DMD, oxidative stress and inflammation are directly involved in the dystrophic process. Unfortunately, there is no current treatment for DMD, and the inflammatory process is an important target for therapies. Based on the antioxidant and anti-inflammatory properties of melatonin, we investigated whether melatonin treatment may reduce the dystrophic process. Ten DMD patients aged 12.8 +/- 0.98 yr, were treated with melatonin (60 mg at 21:00 hr plus 10 mg at 09:00 hr), and plasma levels of lipid peroxidation (LPO), nitrites (NO(x)), interleukin (IL)-1beta, IL-2, IL-6, tumor necrosis factor-alpha, interferon-gamma, and plasma markers of muscle injury, were determined at 3, 6 and 9 months of treatment. Healthy age- and sex-matched subjects were used as controls. The results show a significant increase in LPO, NO(x), and cytokine levels in plasma of DMD patients compared with controls. Melatonin administration reduced these values to control levels at 3 months of treatment, decreasing further 9 months later. In parallel, melatonin also reduced plasma levels of creatine kinase (CK; 50%), lactate dehydrogenase (28%), aspartate aminotransferase (28%), alanine aminotransferase (20%), and myoglobin (13%). These findings strongly support the conclusion that melatonin administration significantly reduced the hyperoxidative and inflammatory process in DMD patients, reducing the muscle degenerative process.

Beneficial effects of melatonin on obesity and lipid profile in young Zucker diabetic fatty rats.

Abstract: The study objective was to investigate the effects of melatonin on obesity and obesity-associated systolic hypertension and dyslipidemia in young male Zucker diabetic fatty (ZDF) rats, an experimental model of the metabolic syndrome. ZDF rats (n = 30) and lean littermates (ZL) (n = 30) were used. At 6 wk of age, both lean and fatty animals were subdivided into three groups (n = 10): naive (N), vehicle-treated (V), and melatonin-treated (M) (10 mg/kg/day) for 6 wk. Vehicle and melatonin were added to the drinking water. Melatonin reduced mean weight gain (51 ± 2/100 g BW) versus N-ZDF group (58 ± 3, P < 0.05) without food intake differences. M-ZDF rats showed an apparent reduction in systolic hypertension that proved not to be statistically significant, and a significant improvement in dyslipidemia, with a reduction in hypertriglyceridemia from 580 ± 40 to 420.6 ± 40.9 mg/dL (P < 0.01). Melatonin raised high-density-lipoprotein (HDL) cholesterol in ZDF (from 81.6 ± 4.9 to 103.1 ± 4.5 mg/dL, P < 0.01) and ZL rats (from 62.8 ± 4.8 to 73.5 ± 4.8 mg/dL, P < 0.05) and significantly reduced low-density-lipoprotein (LDL) cholesterol in ZDF rats from 5.20 ± 0.4 to 4.14 ± 0.3 mg/dL (P < 0.05) but had no effect on total cholesterol levels. To our knowledge, this is the first evidence of a positive effect of melatonin on overweight and lipid pattern of obese Zucker diabetic rats, supporting the proposition that melatonin administration may ameliorate overweight and lipid metabolism in humans. Because these benefits occurred in youth, before advanced metabolic and vascular complications, melatonin might help to prevent cardiovascular disease associated with obesity and dyslipidemia.

Melatonin ameliorates cerulein‐induced pancreatitis by the modulation of nuclear erythroid 2‐related factor 2 and nuclear factor‐kappaB in rats

Abstract: Melatonin exhibits a wide variety of biological effects, including antioxidant and anti-inflammatory functions. Its antioxidant role impedes the etiopathogenesis of pancreatitis, but little is known about the signaling pathway of melatonin in the induction of antioxidant enzymes in acute pancreatitis (AP). The aim of this study was to determine whether melatonin could prevent cerulein-induced AP through nuclear factor erythroid 2-related factor 2 (Nrf2) and curtail inflammation by inhibition of NF-kappaB. AP was induced by two intraperitoneal (i.p.) injections of cerulein at 2 h intervals (50 microg/kg) in Sprague-Dawley rats. Melatonin (10 or 50 mg/kg/daily, i.p.) was administered 24 h before each injection of cerulein. The rats were killed 12 h after the last injection. Acinar cell degeneration, pancreatic edema, and inflammatory infiltration were significantly different in cerulein- and melatonin-treated rats. Melatonin significantly reduced amylase, lipase, MPO, and MDA levels, and increased antioxidant enzyme activities including SOD and GPx, which were decreased in AP (P < 0.05). Melatonin increased the expression of NQO1, HO-1, and SOD2 when compared with the cerulein-induced AP group (P < 0.05). In addition, melatonin increased Nrf2 expression, and reduced expressions of tumor necrosis factor-alpha, IL-1beta, IL-6, IL-8, and iNOS. The elevated nuclear binding of NF-kappaB in the cerulein-induced pancreatitis group was inhibited by melatonin. These results show that melatonin increases antioxidant enzymes and Nrf2 expression, and limits inflammatory mediators in cerulein-induced AP. It is proposed that melatonin may play an important role in oxidative stress via the Nrf2 pathway in parallel with reduction of inflammation by NF-kappaB inhibition.

Melatonin, a novel Sirt1 inhibitor, imparts antiproliferative effects against prostate cancer in vitro in culture and in vivo in TRAMP model.

Abstract: We recently demonstrated that Sirt1, a NAD(+) -dependent histone deacetylase, was overexpressed in prostate cancer (PCa) and its inhibition resulted in a significant antiproliferative response in human PCa cells. Studies have suggested a link between Sirt1 and circadian rhythms, the disruption of which has been linked to cancer. Interestingly, a decreased production of the pineal melatonin has been shown to deregulate the circadian rhythm machinery and increase cancer risk. Furthermore, disruption in melatonin production and circadian rhythmicity has been associated with aging. Here, we challenged our hypothesis that melatonin will impart antiproliferative response against PCa via inhibiting Sirt1. We demonstrated that melatonin significantly inhibited Sirt1 protein and activity in vitro in multiple human PCa cell lines, and melatonin-mediated Sirt1 inhibition was accompanied with a significant decrease in the proliferative potential of PCa cells, but not of normal cells. Forced overexpression of Sirt1 partially rescued the PCa cells from melatonin's antiproliferative effects, suggesting that Sirt1 is a direct target of melatonin. Employing transgenic adenocarcinoma of mouse prostate (TRAMP) mice, we also demonstrated that oral administration of melatonin, at human-achievable doses, significantly inhibited PCa tumorigenesis as shown by decreases in (i) prostate and genitourinary weight, (ii) serum insulin-like growth factor-1 (IGF-1)/IGF-binding protein-3 (IGFBP3) ratio, (iii) mRNA and protein levels of the proliferation markers (PCNA, Ki-67). This anti-PCa response was accompanied with a significant decrease in Sirt1 in TRAMP prostate. Our data identified melatonin as a novel inhibitor of Sirt1 and suggest that melatonin can inhibit PCa growth via Sirt1 inhibition.

Chronic melatonin consumption prevents obesity-related metabolic abnormalities and protects the heart against myocardial ischemia and reperfusion injury in a prediabetic model of diet-induced obesity.

Abstract: Obesity, a major risk factor for ischemic heart disease, is associated with increased oxidative stress and reduced antioxidant status. Melatonin, a potent free radical scavenger and antioxidant, has powerful cardioprotective effects in lean animals but its efficacy in obesity is unknown. We investigated the effects of chronic melatonin administration on the development of the metabolic syndrome as well as ischemia-reperfusion injury in a rat model of diet-induced obesity (DIO). Male Wistar rats received a control diet, a control diet with melatonin, a high-calorie diet, or a high-calorie diet with melatonin (DM). Melatonin (4 mg/kg/day) was administered in the drinking water. After 16 wk, biometric and blood metabolic parameters were measured. Hearts were perfused ex vivo for the evaluation of myocardial function, infarct size (IFS) and biochemical changes [activation of PKB/Akt, ERK, p38 MAPK, AMPK, and glucose transporter (GLUT)-4 expression). The high-calorie diet caused increases in body weight (BW), visceral adiposity, serum insulin and triglycerides (TRIG), with no change in glucose levels. Melatonin treatment reduced the BW gain, visceral adiposity, blood TRIG, serum insulin, homeostatic model assessment index and thiobarbituric acid reactive substances in the DIO group. Melatonin reduced IFS in DIO and control groups and increased percentage recovery of functional performance of DIO hearts. During reperfusion, hearts from melatonin-treated rats had increased activation of PKB/Akt, ERK42/44 and reduced p38 MAPK activation. Chronic melatonin treatment prevented the metabolic abnormalities induced by DIO and protected the heart against ischemia-reperfusion injury. These beneficial effects were associated with activation of the reperfusion injury salvage kinases pathway.

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 induces pro‐apoptotic signaling pathway in human pancreatic carcinoma cells (PANC‐1)

Abstract: Pancreatic cancer is a highly lethal disease with a poor prognosis for long-term survival rate at all stages of invasiveness. It responds poorly to radio- and chemotherapy because the tumor cells are resistant to apoptosis. Melatonin has been reported to inhibit pancreatic cancer growth in experimental studies in animals but the effect of melatonin on cultured human pancreatic carcinoma cells has not been tested. Moreover, we have recently shown that melatonin stimulates production of two major anti-apoptotic heat shock proteins, HSP27 and HSP 90, in pancreatic carcinoma cells. This study investigated the changes in intrinsic pathway of apoptosis at the mitochondrial level and cascade of caspases in human pancreatic carcinoma cells (PANC-1) cells subjected to melatonin and/or luzindole. Melatonin (10⁻⁸ -10⁻¹² m), the nonselective melatonin receptor antagonist, luzindole (10⁻⁸ -10⁻¹² m) or a combination of both agents were added to PANC-1 cell cultures. Cells were harvested, and the cytoplasmic proteins were isolated after 24 and 48 hr of incubation and analyzed employing co-immunoprecipitation and western blot. Administration of melatonin to the PANC-1 cells resulted in the stimulation of Bcl-2/Bax and caspase-9 proteins levels. The strongest signal of these pro-apoptotic factors was observed at the low concentration (10⁻¹² m) of melatonin. Pretreatment with luzindole alone and prior to the addition of melatonin reversed the stimulatory effect of this indoloamine on Bcl-2/Bax and caspase-9 proteins expression in PANC-1 cells. This is the first study to demonstrate a pro-apoptotic effect of low (physiological) concentration of melatonin on the pancreatic carcinoma cells. In conclusion, melatonin induced pro-apoptotic pathways in human pancreatic carcinoma, probably by interaction with the Mel-1 A/B receptors.

Changes in the levels of indoleamine phytochemicals during véraison and ripening of wine grapes

Abstract: Melatonin and serotonin have previously been described in mature wine grapes and finished wines, but the metabolism of these signalling molecules in the development of wine grapes has not previously been investigated. We harvested wine grapes at different stages of development from lag phase through veraison from eight different commercial vineyards representing a diversity of growing conditions, management practices, merlot varietals and localized ecosystems to determine whether different patterns in melatonin and serotonin can be found in wine grapes during seed development and berry maturation. Melatonin was detected in 45% of the fully developed purple, postveraison grapes but only found in 23% of prelag phase samples. However, the actual concentration of melatonin was highest in wine grapes harvested at the early stage of veraison when the seed is developing. Serotonin was not detected in any of the prelag phase grapes but was consistently detected in 30-35% of grapes harvested during the veraison transition at consistent levels of about 8-10 mug/g. Interestingly, the nitrogen storage compound gamma-aminobutyric acid was also found at about 115 mug/g in 77% of early stage green grapes and declined in both prevalence and concentration with ripening. Together, these data are indicative of a potential role for these molecules in the development and maturation of wine grapes.

The mechanism for the neuroprotective effect of melatonin against methamphetamine-induced autophagy

Abstract: Methamphetamine (METH) is a common drug of abuse that induces toxicity in the central nervous system and is connected to neurological disorders such as Parkinson's disease. METH neurotoxicity is induced by reactive oxygen species (ROS) production and apoptosis. Moreover, autophagy is an alternative to cell death and a means for eliminating dysfunctional organelles. In other cases, autophagy can end up in cell death. Nonetheless, it is not clear whether autophagy is also correlated with apoptotic signaling in drug-induced neurotoxicity. Therefore, we hypothesized that METH-generated toxicity associated with initiating the apoptotic signaling cascade can also increase the autophagic phenotype in neuronal cells. Using the SK-N-SH dopaminergic cell line as our model system, we found that METH-induced autophagy by inhibiting dissociation of Bcl-2/Beclin 1 complex and its upstream pathway that thereby led to cell death. We uncovered a novel function for the anti-apoptotic protein Bcl-2, as it played a role in negatively regulating autophagy by blocking an essential protein in the signaling pathway, Beclin 1. Furthermore, Bcl-2 was activated by c-Jun N-terminal kinase 1 (JNK 1), which is upstream of Bcl-2 phosphorylation, to induce Bcl-2/Beclin 1 dissociation. Furthermore, we demonstrated a novel role for melatonin in protecting cells from autophagic cell death triggered by the Bcl-2/Beclin 1 pathway by inhibiting the activation of the JNK 1, Bcl-2 upstream pathway. This study provides information regarding the link between apoptosis and autophagy signaling, which could lead to the development of therapeutic strategies that exploit the neurotoxicity of drugs of abuse.

Melatonin prevents capacitation and apoptotic-like changes of ram spermatozoa and increases fertility rate.

Abstract: We recently demonstrated the presence of melatonin in ram seminal plasma and differences in its concentration in this fluid between the breeding and nonbreeding season. In this study, we investigate the hypothesis that in vitro treatment with melatonin affects ram sperm quality, and that this is reflected in the in vitro fertilization (IVF) results. Semen from nine rams was collected during the nonreproductive season and treated with 1 mum, 10 nm and 100 pm melatonin. Samples were incubated at 39 degrees C and 5% CO2, and motility, viability, capacitation status and phosphatidylserine (PS) translocation were assessed before and after melatonin addition, either 1 or 3 hr of incubation. Fertility rate of the melatonin-treated samples was determined by means of IVF. Although melatonin failed to affect both sperm kinematic parameters and viability, the exposure of ram spermatozoa to melatonin has a direct effect, decreasing capacitation and PS translocation at 1 mum, and increasing short-term capacitation at 100 pm, which caused an increased oocyte fertilization rate following IVF. Furthermore, cleavage rate of oocytes fertilized with 100 pm melatonin-treated spermatozoa was higher than that with 1 mum melatonin and control samples (P < 0.1). These results prove that melatonin has a direct effect on ram spermatozoa in the nonreproductive season, which can be explained, at least in part, by the melatonin capacity as a reactive oxygen species scavenger and antioxidant. These findings might help to select the optimal experimental conditions for IVF and to improve sperm preservation protocols.

Melatonin in traditional Mediterranean diets

Abstract: Compared with other industrialized countries, the lower incidence of chronic-degenerative disorders in Mediterranean populations has been emphasized in recent decades The health-promoting effects arising from Mediterranean dietary habits have been attributed to the large intake of plant foodstuffs rich in bioactive phytochemicals, such as melatonin Recently, it has been suggested that melatonin present in edible plants may improve human health, by virtue of its biological activities and its good bioavailability Plant melatonin, besides contributing to optimize the physiological functions regulated, in humans, by endogenous melatonin, may be involved in nutritional therapy to reduce the risk of cancer, cardiovascular and neurodegenerative diseases in western populations In this view, the presence of melatonin in some Mediterranean foods and beverages adds a new element to the hypothesis of health benefits associated to Mediterranean dietary patterns, although the available data are still preliminary and incomplete

Melatonin protects against isoproterenol-induced myocardial injury in the rat: Antioxidative mechanisms

Abstract: The present study was undertaken to explore the protective effect of melatonin against isoproterenol bitartrate (ISO)-induced myocardial injury in rat. Treatment of rats with ISO increased the level of lipid peroxidation products and decreased the reduced glutathione levels in cardiac tissue indicating that this synthetic catecholamine induces oxidative damage following oxidative stress. Pretreatment of ISO-injected rats with melatonin at a dose of 10 mg/kg body weight, i.p. prevented these changes. Additionally, melatonin also restored the activities and the levels of antioxidant enzymes which were found to be altered by ISO treatment. Treatment of rats with ISO resulted into an increased generation of hydroxyl radicals with melatonin pretreatment significantly reducing their production. Finally, treatment of rats with ISO caused a lowering of systolic pressure with reduced cardiac output and diastolic dysfunction whereas melatonin pretreatment significantly restored many of these parameters to normal. The findings document melatonin's ability to provide cardio protection at a low pharmacological dose. Melatonin has virtually no toxicity which raises the possibility of this indole being a therapeutic treatment for ischemic heart disease.

Synergistic antitumor effect of melatonin with several chemotherapeutic drugs on human Ewing sarcoma cancer cells: potentiation of the extrinsic apoptotic pathway

Abstract: Ewing sarcoma, the second most frequent bone cancer type, affects mainly adolescents, who have a survival of 50% 5 yr after diagnosis. Current treatments include a combination of surgery, radiotherapy and chemotherapy, which present potential serious side effects. Melatonin, a natural molecule without relevant side effects, has been previously shown to induce cytotoxicity in SK-N-MC cells, a Ewing sarcoma cell line. Here, we found that there is a synergy in the antitumor effect when melatonin (50 mum-1 mm) is combined with vincristine at the concentration of 5-10 nm or with ifosfamide at the range of 100 mum-1 mm. This synergism is due to the potentiation of cell death, particularly to the potentiation of apoptosis, i.e., mainly the extrinsic apoptotic pathway. There is a significant increase in the activation of caspase-3, -8, -9 and Bid when melatonin is combined with vincristine or ifosfamide compared to the individual treatments. Finally, there is also a potentiation of the early free radical production, likely dependent on the extrinsic apoptosis pathway activation, when the drugs are combined with melatonin. Other proteins which are related to this pathway including mitogen-activated protein kinase or protein kinase B/Akt are not involved in apoptosis induced by these agents separately or when combined. The results shown here together with the facts that: (i) no relevant side effects have been reported for melatonin and (ii) melatonin has a cytoprotective effect on noncancer cells, opens the door for a new approach in the treatment of the Ewing sarcoma family of tumors.

Determination of the minimal melatonin exposure required to induce osteoblast differentiation from human mesenchymal stem cells and these effects on downstream signaling pathways.

Abstract: The purpose of this study was to determine the critical time periods of melatonin treatment required to induce human mesenchymal stem cells (hAMSCs) into osteoblasts and to determine which osteogenic genes are involved in the process. The study design consisted of adding melatonin for different times (2, 5, 10, 14 or 21 days) toward the end of a 21-day treatment containing osteogenic (OS+) medium or at the beginning of the 21-day treatment and then withdrawn. The results show that a 21-day continuous melatonin treatment was required to induce both alkaline phosphatase (ALP) activity and calcium deposition and these effects were mediated through MT₂Rs. Functional analysis revealed that peak ALP levels induced by melatonin were accompanied by attenuation of melatonin-mediated inhibition of forskolin-induced cAMP accumulation. Immunoprecipitation and western blot analyses, respectively, showed that MT₂R/β-arrestin scaffolds complexed to Gi, MEK1/2 and ERK1/2 formed in these differentiated hAMSCs (i.e., when ALP levels were highest) where ERK1/2 resided primarily in the cytosol. It is hypothesized that these complexes form to modulate the subcellular localization of ERK1/2 to affect osteogenic gene expression. Using real-time RT-PCR, chronic melatonin exposure induced the expression of osteogenic genes RUNX-2, osteocalcin and BMP-2, through MT₂Rs. No melatonin-mediated changes in the mRNA expression of ALP, BMP-6 or in the oxidative enzymes MtTFA, PGC-1α, Polγ, NRF-1, PDH, PDK and LDH occurred. These data show that a continuous 21-day melatonin exposure is required to induce osteoblast differentiation from hAMSCs through the formation of MT₂R/Gi/β-arrestin/MEK/ERK1/2 complexes to induce osteogenesis.

High endogenous melatonin concentrations enhance sperm quality and short-term in vitro exposure to melatonin improves aspects of sperm motility.

Abstract: Although human seminal fluid contains melatonin and spermatozoa reportedly possess membrane melatonin receptors, there are no experimental studies that have ascertained the relationship between melatonin and male infertility. This study evaluated whether urinary 6-sulfatoxymelatonin and urinary total antioxidant capacity correlate with different seminal parameters including sperm concentration, motility and morphology. Also, the in vitro effects of melatonin on human sperm motility were investigated. Semen samples from 52 men who were counselled for infertility were obtained. Sperm concentration was determined using the haemocytometer method, motility kinematic parameters were assessed using a computer-aided semen analysis system, while morphology and vitality were evaluated after Diff-Quick and Eosin-Nigrosin vital staining, respectively. For the quantification of urinary 6-sulfatoxymelatonin, a commercial ELISA kit was used, and urinary total antioxidant capacity was evaluated by means of a colorimetric assay kit. For the in vitro effects of melatonin, samples were incubated for 30min in the presence or absence of 1mm melatonin. Both urinary 6-sulfatoxymelatonin and total antioxidant capacity levels positively correlated with sperm concentration, motility and morphology, as well as negatively correlated with the number of round cells. Additionally, 30-min exposure of sperm to 1mm melatonin improved the percentage of motile and progressively motile cells and decreased the number of static cells, thereby promoting the proportion of rapid cells. Therefore, melatonin improves semen quality, which is important because melatonin supplementation may be potentially used to obtain a successful assisted reproductive technique outcome.

Melatonin and vitamin C increase umbilical blood flow via nitric oxide-dependent mechanisms

Abstract: Inadequate umbilical blood flow leads to intrauterine growth restriction, a major killer in perinatal medicine today. Nitric oxide (NO) is important in the maintenance of umbilical blood flow, and antioxidants increase NO bioavailability. What remains unknown is whether antioxidants can increase umbilical blood flow. Melatonin participates in circadian, seasonal, and reproductive physiology, but has also been reported to act as a potent endogenous antioxidant. We tested the hypothesis that treatment during pregnancy with melatonin increases umbilical blood flow via NO-dependent mechanisms. This was tested in pregnant sheep by investigating in vivo the effects on continuous measurement of umbilical blood flow of melatonin before and after NO blockade with a NO clamp. These effects of melatonin were compared with those of the traditional antioxidant, vitamin C. Under anesthesia, 12 pregnant sheep and their fetuses (0.8 of gestation) were fitted with catheters and a Transonic probe around an umbilical artery, inside the fetal abdomen. Following 5 days of recovery, cardiovascular variables were recorded during fetal i.v. treatment with either melatonin (n=6, 0.5±0.1 μg/kg/min) or vitamin C (n=6, 8.9±0.4 mg/kg/min) before and after fetal NO blockade with the NO clamp. Fetal treatment with melatonin or vitamin C increased umbilical blood flow, independent of changes in fetal arterial blood pressure. Fetal NO blockade prevented the increase in umbilical blood flow induced by melatonin or vitamin C. Antioxidant treatment could be a useful clinical tool to increase or maintain umbilical blood flow in complicated pregnancy.

TLR4 and CD14 receptors expressed in rat pineal gland trigger NFKB pathway.

Abstract: Nuclear factor-kappa B (NFKB), a pivotal player in inflammatory responses, is constitutively expressed in the pineal gland. Corticosterone inhibits pineal NFKB leading to an enhancement of melatonin production, while tumor necrosis factor (TNF) leads to inhibition of Aa-nat transcription and the production of N-acetylserotonin in cultured glands. The reduction in nocturnal melatonin surge favors the mounting of the inflammatory response. Despite these data, there is no clear evidence of the ability of the pineal gland to recognize molecules that signal infection. This study investigated whether the rat pineal gland expresses receptors for lipopolysaccharide (LPS), the endotoxin from the membranes of Gram-negative bacteria, and to establish the mechanism of action of LPS. Here, we show that pineal glands possess both CD14 and toll-like receptor 4 (TLR4), membrane proteins that bind LPS and trigger the NFKB pathway. LPS induced the nuclear translocation of p50/p50 and p50/RELA dimers and the synthesis of TNF. The maximal expression of TNF in cultured glands coincides with an increase in the expression of TNF receptor 1 (TNFR1) in isolated pinealocytes. In addition, LPS inhibited the synthesis of N-acetylserotonin and melatonin. Therefore, the pineal gland transduces Gram-negative endotoxin stimulation by producing TNF and inhibiting melatonin synthesis. Here, we provide evidence to reinforce the idea of an immune-pineal axis, showing that the pineal gland is a constitutive player in the innate immune response.

Melatonin attenuates methamphetamine-induced overexpression of pro-inflammatory cytokines in microglial cell lines.

Abstract: Methamphetamine (METH), the most commonly abused drug, has long been known to induce neurotoxicity. METH causes oxidative stress and inflammation, as well as the overproduction of both reactive oxygen species (ROS) and reactive nitrogen species (RNS). The role of METH-induced brain inflammation remains unclear. Imbroglio activation contributes to the neuronal damage that accompanies injury, disease and inflammation. METH may activate microglia to produce neuroinflammatory molecules. In highly aggressively proliferating immortalized (HAPI) cells, a rat microglial cell line, METH reduced cell viability in a concentration- and time-dependent manner and initiated the expression of interleukin 1beta (IL-1beta), interleukin 6 (IL-6) and tumor necrosis factor alpha. METH also induced the production of both ROS and RNS in microglial cells. Pretreatment with melatonin, a major secretory product of the pineal gland, abolished METH-induced toxicity, suppressed ROS and RNS formation and also had an inhibitory effect on cytotoxic factor gene expression. The expression of cytotoxic factors produced by microglia may contribute to central nervous system degeneration in amphetamine abusers. Melatonin attenuates METH toxicity and inhibits the expression of cytotoxic factor genes associated with ROS and RNS neutralization in HAPI microglia. Thus, melatonin might be one of the neuroprotective agents induced by METH toxicity and/or other immunogens.

The inhibition of apoptosis by melatonin in VSC4.1 motoneurons exposed to oxidative stress, glutamate excitotoxicity, or TNF-α toxicity involves membrane melatonin receptors

Abstract: Loss of motoneurons may underlie some of the deficits in motor function associated with the central nervous system (CNS) injuries and diseases. We tested whether melatonin, a potent antioxidant and free radical scavenger, would prevent motoneuron apoptosis following exposure to toxins and whether this neuroprotection is mediated by melatonin receptors. Exposure of VSC4.1 motoneurons to either 50 microm H(2)O(2), 25 microm glutamate (LGA), or 50 ng/mL tumor necrosis factor-alpha (TNF-alpha) for 24 h caused significant increases in apoptosis, as determined by Wright staining and ApopTag assay. Analyses of mRNA and proteins showed increased expression and activities of stress kinases and cysteine proteases and loss of mitochondrial membrane potential during apoptosis. These insults also caused increases in intracellular free [Ca(2+)] and activities of calpain and caspases. Cells exposed to stress stimuli for 15 min were then treated with 200 nm melatonin. Post-treatment of cells with melatonin attenuated production of reactive oxygen species (ROS) and phosphorylation of p38, MAPK, and JNK1, prevented cell death, and maintained whole-cell membrane potential, indicating functional neuroprotection. Melatonin receptors (MT1 and MT2) were upregulated following treatment with melatonin. To confirm the involvement of MT1 and MT2 in providing neuroprotection, cells were post-treated (20 min) with 10 microm luzindole (melatonin receptor antagonist). Luzindole significantly attenuated melatonin-induced neuroprotection, suggesting that melatonin worked, at least in part, via its receptors to prevent VSC4.1 motoneuron apoptosis. Results suggest that neuroprotection rendered by melatonin to motoneurons is receptor mediated and melatonin may be an effective neuroprotective agent to attenuate motoneuron death in CNS injuries and diseases.

Melatonin synergistically increases resveratrol-induced heme oxygenase-1 expression through the inhibition of ubiquitin-dependent proteasome pathway: a possible role in neuroprotection.

Abstract: Melatonin is an indoleamine secreted by the pineal gland as well as a plant-derived product, and resveratrol (RSV) is a naturally occurring polyphenol synthesized by a variety of plant species; both molecules act as a neuroprotector and antioxidant. Recent studies have demonstrated that RSV reduced the incidence of Alzheimer's disease and stroke, while melatonin supplementation was found to reduce the progression of the cognitive impairment in AD. The heme oxygenase-1 (HO-1) is an inducible and redox-regulated enzyme that provides tissue-specific antioxidant effects. We assessed whether the co-administration of melatonin and RSV shows synergistic effects in terms of their neuroprotective properties through HO-1. RSV significantly increased the expression levels of HO-1 protein in a concentration-dependent manner both in primary cortical neurons and in astrocytes, while melatonin per se did not. Melatonin + RSV showed a synergistic increase in the expression levels of HO-1 protein but not in the HO-1 mRNA level compared to either melatonin or RSV alone, which is mediated by the activation of PI3K-Akt pathway. Treatment of melatonin + RSV significantly attenuated the neurotoxicity induced by H(2) O(2) in primary cortical neurons and also in organotypic hippocampal slice culture. The blockade of HO-1 induction by shRNA attenuated HO-1 induction by melatonin + RSV and hindered the neuroprotective effects against oxidative stress induced by H(2) O(2) . The treatment of MG132 + RSV mimicked the effects of melatonin + RSV, and melatonin + RSV inhibited ubiquitination of HO-1. These data suggest that melatonin potentiates the neuroprotective effect of RSV against oxidative injury, by enhancing HO-1 induction through inhibiting ubiquitination-dependent proteasome pathway, which may provide an effective means to treat neurodegenerative disorders.