Anticonvulsant activity of melatonin and its success in ameliorating epileptic comorbidity-like symptoms in zebrafish.

Epilepsy is one of common neurological disorders, greatly distresses the well-being of the sufferers. Melatonin has been used in clinical anti-epileptic studies, but its effect on epileptic comorbidities is unknown, and the underlying mechanism needs further investigation. Herein, by generating PTZ-induced zebrafish seizure model, we carried out interdisciplinary research using neurobehavioral assays, bioelectrical detection, molecular biology, and network pharmacology to investigate the activity of melatonin as well as its pharmacological mechanisms. We found melatonin suppressed seizure-like behavior by using zebrafish regular locomotor assays. Zebrafish freezing and bursting activity assays revealed the ameliorative effect of melatonin on comorbidity-like symptoms. The preliminary screening results of neurobehavioral assays were further verified by the expression of key genes involved in neuronal activity, neurodevelopment, depression and anxiety, as well as electrical signal recording from the midbrain of zebrafish. Subsequently, network pharmacology was introduced to identify potential targets of melatonin and its pathways. Real-time qPCR and protein-protein interaction (PPI) were conducted to confirm the underlying mechanisms associated with glutathione metabolism. We also found that melatonin receptors were involved in this process, which were regulated in response to melatonin exposure before PTZ treatment. The antagonists of melatonin receptors affected anticonvulsant activity of melatonin. Overall, current study revealed the considerable ameliorative effects of melatonin on seizure and epileptic comorbidity-like symptoms and unveiled the underlying mechanism. This study provides an animal model for the clinical application of melatonin in the treatment of epilepsy and its comorbidities.

Characterization of the various functional pathways elicited by synthetic agonists or antagonists at the melatonin MT1 and MT2 receptors.

Melatonin is a neurohormone that translates the circadian rhythm to the peripheral organs through a series of binding sites identified as G protein-coupled receptors MT1 and MT2. Due to minute amounts of receptor proteins in target organs, the main tool of studies of the melatoninergic system is recombinant expression of the receptors in cellular hosts. Although a number of studies exist on these receptors, studies of several signaling pathways using a large number of melatoninergic compounds are rather limited. We chose to fill this gap to better describe a panel of compounds that have been only partially characterized in terms of functionality. First, we characterized HEK cells expressing MT1 or MT2, and several signaling routes with melatonin itself to validate the approach: GTPγS, cAMP production, internalization, ß-arrestin recruitment, and cell morphology changes (CellKey ® ). Second, we chose 21 compounds from our large melatoninergic chemical library and characterized them using this panel of signaling pathways. Notably, antagonists were infrequent, and their functionality depended largely on the pathway studied. This will permit redefining the availability of molecular tools that can be used to better understand the in situ activity and roles of these receptors.

Fetal liver mesenchymal stem cells restore ovarian function in premature ovarian insufficiency by targeting MT1.

BACKGROUND: With the development of regenerative medicine and tissue engineering technology, almost all stem cell therapy is efficacious for the treatment of premature ovarian failure (POF) or premature ovarian insufficiency (POI) animal models, whereas little stem cell therapy has been practiced in clinical settings. The underlying molecular mechanism and safety of stem cell treatment in POI are not fully understood. In this study, we explored whether fetal mesenchymal stem cells (fMSCs) from the liver restore ovarian function and whether melatonin membrane receptor 1 (MT1) acts as a regulator for treating POI disease. METHODS: We designed an in vivo model (chemotherapy-induced ovary damage) and an in vitro model (human ovarian granulosa cells (hGCs)) to understand the efficacy and molecular cues of fMSC treatment of POI. Follicle development was observed by H&E staining. The concentration of sex hormones in serum (E2, AMH, and FSH) and the concentration of oxidative and antioxidative metabolites and the enzymes MDA, SOD, CAT, LDH, GR, and GPx were measured by ELISA. Flow cytometry (FACS) was employed to detect the percentages of ROS and proliferation rates. mRNA and protein expression of antiapoptotic genes (SURVIVIN and BCL2), apoptotic genes (CASPASE-3 and CASPASE-9), and MT1 and its downstream genes (JNK1, PCNA, AMPK) were tested by qPCR and western blotting. MT1 siRNA and related antagonists were used to assess the mechanism. RESULTS: fMSC treatment prevented cyclophosphamide (CTX)-induced follicle loss and recovered sex hormone levels. Additionally, fMSCs significantly decreased oxidative damage, increased oxidative protection, improved antiapoptotic effects, and inhibited apoptotic genes in vivo and in vitro. Furthermore, fMSCs also upregulated MT1, JNK1, PCNA, and AMPK at the mRNA and protein levels. With MT1 knockdown or antagonist treatment in normal hGCs, the protein expression of JNK1, PCNA, and AMPK and the percentage of proliferation were impaired. CONCLUSIONS: fMSCs might play a crucial role in mediating follicular development in the POI mouse model and stimulating the activity of POI hGCs by targeting MT1.

Melatonin protects rabbit spermatozoa from cryo-damage via decreasing oxidative stress.

Mammalian spermatozoa are highly susceptible to reactive oxygen species (ROS) stress. The aim of the present study was to investigate whether and how melatonin protects rabbit spermatozoa against ROS stress during cryopreservation. Semen was diluted with Tris-citrate-glucose extender in presence of different concentrations of melatonin. It was observed that addition of 0.1 mM melatonin significantly improved spermatozoa motility, membrane integrity, acrosome integrity, mitochondrial membrane potential as well as AMP-activated protein kinase (AMPK) phosphorylation. Meanwhile, the lipid peroxidation (LPO), ROS levels and apoptosis of post-thaw spermatozoa were reduced in presence of melatonin. Interestingly, when fresh spermatozoa were incubated with 100 µM H2O2, addition of 0.1 mM melatonin significantly decreased the oxidative damage compared to the H2O2 treatment, whereas addition of luzindole, an MT1 receptor inhibitor, decrease the effect of melatonin in spermatozoa. It was observed that the glutathione (GSH) content and activities of glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT) were significantly increased with addition of melatonin during cryopreservation. In conclusion, addition of melatonin to the freezing extender protects rabbit spermatozoa against ROS attack by enhancing AMPK phosphorylation for increasing the antioxidative defense.

Melatonin reduces oxidative damage and upregulates heat shock protein 90 expression in cryopreserved human semen.

Sperm cells can be damaged during the semen cryopreservation process, decreasing their fertilizing ability. Physical damage and oxidative stress may occur during the freeze-thawing process. Antioxidants such as the native antioxidant melatonin can potentially improve cryopreservation outcomes. In this study, we added melatonin to cryoprotectant to examine its effect on frozen-thawed human sperm. We found that adding 0.1mM melatonin to cryoprotectant significantly increased sperm viability (24.80 ± 0.46% vs. 20.97 ± 1.27%, P < 0.05) and membrane integrity (P < 0.05), and decreased intracellular reactive oxygen species and lipid peroxidation damage. Furthermore, mRNA levels of the transcription factor NF-E2-related factor-2 and its downstream genes were significantly increased. Resistance to oxidative stress was enhanced and expression of the antiapoptotic gene Bcl-2 was increased by inclusion of 0.1mM melatonin in the cryoprotectant. Moreover, 0.1mM melatonin upregulated the expression of heat shock protein 90 (HSP90), which confers resistance to stressors in frozen-thawed sperm. Results obtained upon addition of inhibitors of melatonin receptors (luzindole and 4-P-PDOT) and an HSP90 inhibitor (geldanamycin) in the cryoprotectant demonstrated that melatonin promoted HSP90 translation via the melatonin receptor MT1 and increased adenosine triphosphate levels, thus increasing the viability of thawed sperm.

Melatonin Synergizes the Chemotherapeutic Effect of Cisplatin in Ovarian Cancer Cells Independently of MT1 Melatonin Receptors.

BACKGROUND/AIM: Melatonin (MLT), through the interaction with membrane melatonin receptors MT1, can improve the effectiveness of cytostatic agents, including cisplatin (CP). The aim of this study was to examine the synergistic effect of MLT and CP in three cell lines: IOSE 364, SK-OV-3 and OVCAR-3, as well as to assess the role of MT1 receptors in this mechanism. MATERIALS AND METHODS: Using the SRB assay we investigated the effect of different concentrations of CP and MLT on cell viability. Tests, using luzindole - MT1 inhibitor, allowed us to assess the potential involvement of MT1 in the mechanism of MLT action. RESULTS: MLT at certain concentrations demonstrated a synergistic effect in combination with CP. The addition of luzindole did not affect the action of MLT in combination with CP. CONCLUSION: In summary, the synergistic effect of MLT with CP seems to be independent of membrane MT1 receptors.

Melatonin receptors as therapeutic targets in the suprachiasmatic nucleus.

INTRODUCTION: Disorders of rhythmicity can cause a variety of pathologies and are known to impair processes involved in metabolism, as well as in cardiovascular disease and cancer. Developing strategies to treat or prevent such diseases is a new challenge for medicine. Rhythms depend on a complex multi-oscillatory circadian network which, in mammals, is hierarchically organized with the suprachiasmatic nuclei (SCN) as master clock. The SCN, thus form an ideal structure for target discovery in circadian pathologies. AREAS COVERED: The development of strategies to treat or prevent disorders of rhythmicity is a new challenge for medicine. Several pharmacological approaches have been suggested, but until now, it has been mostly melatonin (MTL) or MTL-agonists which have demonstrated usefulness in modulating clock activities in vivo. A great number of structurally different MTL receptor ligands have been developed, some of which are already approved and marketed as drugs. The MTL receptor involved in phase-shifting circadian rhythms (chronobiotic effect) is the MT1 subtype. EXPERT OPINION: As the two receptor subtypes for MTL may have divergent functions, the development of highly selective MT1 and MT2 agonists (and antagonists) is key for the discovery of novel therapeutic agents in specifically defined circadian pathologies. The identification of cells expressing the different MTL receptor subtypes should also permit a better understanding of MLT physiology/pharmacology.

Melatonin Upregulates the Activity of AMPK and Attenuates Lipid Accumulation in Alcohol-induced Rats.

AIMS: Melatonin is supposed to be an effective hepatoprotective agent. The effects and mechanisms of melatonin on alcoholic fatty liver (AFL) have not been well explored. The aim of this study was to investigate the preventive and therapeutic effects of melatonin on alcohol-induced fatty liver rats. METHODS: The AFL rats were induced by intragastric infusion of alcohol plus a high-fat diet for 6 weeks, and melatonin (10, 20, 40 mg/kg) was administered by gastric perfusion. We also established fatty acid overload cell model in HepG2 cells to investigate the effect of melatonin on AMP-activated protein kinase (AMPK) activity. RESULTS: The results showed that melatonin (20 and 40 mg/kg) administration significantly reduced alcohol-induced hepatic steatosis with lowering activities of serum alanine aminotransferase, aspartate aminotransferase and levels of serum and hepatic triglyceride. The activity of superoxide dismutase was increased and the content of malondialdehyde was decreased in liver homogenates of rats treated with melatonin. Melatonin increased the phosphorylation of AMPK in the liver tissues of alcohol-induced rats as well. Additionally, in vitro studies showed that melatonin increased the expression of melatonin1A receptor (MT1R), whereas luzindole, a receptor antagonist of melatonin, had no effect on its expression. In addition, melatonin reduced the levels of adenosine 3',5'-cyclic monophosphate (cAMP) and increased the phosphorylation of AMPK, and melatonin treatment could markedly reverse these effects. CONCLUSION: In conclusion, melatonin could protect against liver injury caused by alcohol gastric perfusion. The effect may be related to alleviating lipid peroxidation and upregulating the activity of AMPK mediated by MT1R signaling pathway.

Role of melatonin, melatonin receptors and STAT3 in the cardioprotective effect of chronic and moderate consumption of red wine.

We have recently discovered that melatonin, given acutely and directly to the isolated heart at the concentration found in wine, confers cardioprotection against ischemia-reperfusion (I/R). However, whether the presence of melatonin in wine contributes to the cardioprotective effect of chronic and moderate consumption of wine and its signalling mechanisms of protection are unknown. We therefore used both in vivo and in vitro models of I/R to investigate whether the presence of melatonin in red wine may contribute to the cardioprotective effect of chronic and moderate consumption of red wine. Wistar rats and C57black6 mice (WT) received drinking water supplemented daily with a moderate amount of red wine or melatonin given at the concentration found in the red wine. Rats were also pretreated with luzindole, a specific inhibitor of melatonin receptors 1 and 2 (2.3 mg/kg/day, intraperitoneally) or prazosin, a specific inhibitor of melatonin receptor type 3 (2.5 mg/kg/day, intraperitoneally). After 14 days, hearts were subjected to I/R in vivo or ex vivo. Red wine reduced the infarct size in both rats and WT mice (p < 0.001). Luzindole did not affect wine-induced cardioprotection, while prazosin reduced the infarct sparing effect of red wine (p < 0.05). Furthermore, red wine or melatonin failed to protect tumor necrosis factor alpha (TNF) receptor 2 knockout or cardiomyocyte specific signal transducer and activator of transcription 3 (STAT3) deficient mice (n.s. vs. control). Our novel findings suggest that the presence of melatonin in red wine contributes to the cardioprotective effect of chronic and moderate consumption of red wine against lethal I/R injuries. This effect is most likely mediated, at least in part, via melatonin receptor 3 and the activation of TNF and STAT3, both key players of the prosurvival and well described SAFE pathway.

Epigenetic mechanisms of melatonin action in human SH-SY5Y neuroblastoma cells.

We have shown that melatonin induces histone hyperacetylation in vitro and in vivo. To clarify the mechanisms involved, we have now investigated its effects on histone acetylation and signaling pathways in human SH-SY5Y neuroblastoma cells, which express melatonin MT1 receptors. Melatonin caused significant concentration-dependent increases in both histone H3 and H4 acetylation. Blockade of melatonin receptors with luzindole abolished the histone hyperacetylating effect of melatonin, whereas inhibition of MAPK-ERK by PD98059 attenuated but did not block this effect. Melatonin treatment for 24-h decreased the levels of phospho-ERK1/2, but significantly increased Akt phosphorylation and protein expression of the histone acetyltransferase, p300. These findings suggest that the epigenetic effects of melatonin in SH-SY5Y cells are mediated by G protein-coupled MT1 melatonin receptors. Furthermore, upregulation of the histone acetyltransferase/transcriptional co-activator p300, along with phosphorylation of Akt, which is essential for p300 activation, appear to be key mechanisms underlying the epigenetic effects of melatonin.

The MT2 receptor stimulates axonogenesis and enhances synaptic transmission by activating Akt signaling.

The MT2 receptor is a principal type of G protein-coupled receptor that mainly mediates the effects of melatonin. Deficits of melatonin/MT2 signaling have been found in many neurological disorders, including Alzheimer's disease, the most common cause of dementia in the elderly, suggesting that preservation of the MT2 receptor may be beneficial to these neurological disorders. However, direct evidence linking the MT2 receptor to cognition-related synaptic plasticity remains to be established. Here, we report that the MT2 receptor, but not the MT1 receptor, is essential for axonogenesis both in vitro and in vivo. We find that axon formation is retarded in MT2 receptor knockout mice, MT2-shRNA electroporated brain slices or primary neurons treated with an MT2 receptor selective antagonist. Activation of the MT2 receptor promotes axonogenesis that is associated with an enhancement in excitatory synaptic transmission in central neurons. The signaling components downstream of the MT2 receptor consist of the Akt/GSK-3ß/CRMP-2 cascade. The MT2 receptor C-terminal motif binds to Akt directly. Either inhibition of the MT2 receptor or disruption of MT2 receptor-Akt binding reduces axonogenesis and synaptic transmission. Our data suggest that the MT2 receptor activates Akt/GSK-3ß/CRMP-2 signaling and is necessary and sufficient to mediate functional axonogenesis and synaptic formation in central neurons.

Regulation of L1 expression and retrotransposition by melatonin and its receptor: implications for cancer risk associated with light exposure at night.

Expression of long interspersed element-1 (L1) is upregulated in many human malignancies. L1 can introduce genomic instability via insertional mutagenesis and DNA double-strand breaks, both of which may promote cancer. Light exposure at night, a recently recognized carcinogen, is associated with an increased risk of cancer in shift workers. We report that melatonin receptor 1 inhibits mobilization of L1 in cultured cells through downregulation of L1 mRNA and ORF1 protein. The addition of melatonin receptor antagonists abolishes the MT1 effect on retrotransposition in a dose-dependent manner. Furthermore, melatonin-rich, but not melatonin-poor, human blood collected at different times during the circadian cycle suppresses endogenous L1 mRNA during in situ perfusion of tissue-isolated xenografts of human cancer. Supplementation of human blood with exogenous melatonin or melatonin receptor antagonist during the in situ perfusion establishes a receptor-mediated action of melatonin on L1 expression. Combined tissue culture and in vivo data support that environmental light exposure of the host regulates expression of L1 elements in tumors. Our data imply that light-induced suppression of melatonin production in shift workers may increase L1-induced genomic instability in their genomes and suggest a possible connection between L1 activity and increased incidence of cancer associated with circadian disruption.

Agomelatine but not melatonin improves fatigue perception: a longitudinal proof-of-concept study.

Chronic Fatigue Syndrome (CFS) represents a disabling condition characterized by persistent mental and physical fatigue, bodily discomfort and cognitive difficulties. To date the neural bases of CFS are poorly understood; however, mono-aminergic abnormalities, sleep-wake cycle changes and prefrontal dysfunctions are all thought to play a role in the development and maintenance of this condition. Here we explored in a group of 62 CFS subjects the impact on fatigue levels of agomelatine, an antidepressant with agonist activity at melatonin receptors (MT1 and MT2) and antagonist activity at serotoninergic 2C receptors (5HT2C). To tease out the relative effects of MT-agonism and 5HT2C antagonism on fatigue, we compared agomelatine 50mg u.i.d. with sustained release melatonin 10mg u.i.d. in the first 12-week-long phase of the study, and then switched all melatonin-treated subjects to agomelatine in the second 12-week-long phase of the study. Agomelatine treatment, but not melatonin, was associated with a significant reduction of perceived fatigue and an increase in perceived quality of life. Moreover the switch from melatonin to agomelatine was associated with a reduction of fatigue levels. Agomelatine was well tolerated by all enrolled subjects. Our data, albeit preliminary, suggest that agomelatine treatment could represent a novel useful approach to the clinical care of subjects with CFS.

Melatonin receptors mediate improvements of survival in a model of polymicrobial sepsis.

OBJECTIVES: Melatonin has been demonstrated to improve survival after experimental sepsis via antioxidant effects. Yet, recent evidence suggests that this protective capacity may also rely on melatonin receptor activation. Therefore, the present study was designed to investigate whether selective melatonin receptor-agonist ramelteon may influence survival and immune response in a model of polymicrobial sepsis in rats, wild-type and melatonin receptor MT1/MT2 double knockout mice. DESIGN: Prospective, randomized, controlled study. SETTING: University research laboratory. SUBJECTS: Male Sprague-Dawley rats (200-250 g) and male C3H/HeN wild-type and MT1/MT2 receptor knockout mice (20-22 g). INTERVENTIONS: Animals underwent cecal ligation and incision and remained anesthetized for evaluation of survival for 12 hours (rats: n = 15 per group) or 15 hours (mice: n = 10 per group). Analysis of immune response by means of enzyme-linked immunosorbent assay was performed before and 5 hours after cecal ligation and incision (rats only; n = 5 per group). After induction of sepsis, animals were treated IV with vehicle, different doses of melatonin (rats: 0.01/0.1/1.0/10 mg/kg; mice: 1.0 mg/kg), ramelteon, melatonin receptor-antagonist luzindole, ramelteon + luzindole, or melatonin + luzindole (each 1.0 mg/kg). Sham controls underwent laparotomy but not cecal ligation and incision. MEASUREMENTS AND MAIN RESULTS: Compared with vehicle, administration of ramelteon or melatonin significantly improved median survival time in rats (sepsis/melatonin [0.1 mg/kg], 554 min, [1.0 mg/kg] 570 min, [10 mg/kg] 579 min; sepsis/ramelteon, 468 min; each p < 0.001 vs sepsis/vehicle, 303 min) and wild-type mice (sepsis/melatonin, 781 min; sepsis/ramelteon, 701 min; both p < 0.001 vs sepsis/vehicle, 435 min). This effect was completely antagonized by coadministration of luzindole in all groups. Melatonin, ramelteon, or luzindole had no significant effect on survival time in knockout mice. Significantly elevated concentrations of tumor necrosis factor-α, interleukin-6, and interleukin-10 were observed 5 hours after cecal ligation and incision in rats (p < 0.05 vs baseline and corresponding sham); neither ramelteon nor melatonin treatment significantly affected immune response. CONCLUSIONS: Melatonin receptors mediate improvements of survival after polymicrobial sepsis in rats and mice; this effect appears to be independent from major alterations of cytokine release.

Expression of functional melatonin MT(1) 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.

Inhibition of breast cancer cell invasion by melatonin is mediated through regulation of the p38 mitogen-activated protein kinase signaling pathway.

INTRODUCTION: The pineal gland hormone, melatonin, has been shown by numerous studies to inhibit the proliferation of estrogen receptor α (ERα)-positive breast cancer cell lines. Here, we investigated the role of melatonin in the regulation of breast cancer cell invasion. METHODS: Three invasive MCF-7 breast cancer cell clones - MCF-7/6, MCF-7/Her2.1, and MCF-7/CXCR4 cells - were employed in these studies. All three cell lines exhibited elevated phosphorylation of the ERK1/2 and p38 mitogen-activated protein kinase (MAPK) as determined by Western blot analysis. The effect of melatonin on the invasive potential of these human breast cancer cells was examined by matrigel invasion chamber assays. The expression and proteinase activity of two matrix metalloproteinases (MMPs), MMP-2 and MMP-9, were analyzed by Western blot analysis and gelatin zymography, respectively. RESULTS: Melatonin (10-9 M) significantly suppressed the invasive potential of MCF-7/6 and MCF-7/Her2.1 cells as measured by matrigel invasion chamber assays, and significantly repressed the proteinase activity of MMP-2 and MMP-9. In MCF-7/CXCR4 cells, melatonin significantly inhibited stromal-derived factor-1 (SDF-1/CXCL12) induced cell invasion and activity of MMP-9. Elevated expression of the MT1 melatonin receptor further enhanced, while luzindole, an MT1/MT2 antagonist, abrogated melatonin's anti-invasive effect, suggesting that melatonin's effect on invasion is mediated, principally, through the MT1 receptor. Furthermore, melatonin repressed the phosphorylation of p38 MAPK in MCF-7/Her2.1 cells and blocked stromal-derived factor-1 (SDF-1) induced p38 phosphorylation in MCF-7/CXCR4 cells. SB230580, a p38 inhibitor, was able to mimic, while transfection of the cells with a constitutively-active MKK6b construct blocked melatonin's effect on cell invasion, suggesting that the anti-invasive action of melatonin is mediated through the p38 pathway. CONCLUSIONS: Melatonin exerts an inhibitory effect on breast cancer cell invasion through down-regulation of the p38 pathway, and inhibition of MMP-2 and MMP-9 expression and activity.

Melatonin antagonizes the intrinsic pathway of apoptosis via mitochondrial targeting of Bcl-2.

We have recently shown that melatonin antagonizes damage-induced apoptosis by interaction with the MT-1/MT-2 plasma membrane receptors. Here, we show that melatonin interferes with the intrinsic pathway of apoptosis at the mitochondrial level. In response to an apoptogenic stimulus, melatonin allows mitochondrial translocation of the pro-apoptotic protein Bax, but it impairs its activation/dimerization The downstream apoptotic events, i.e. cytochrome c release, caspase 9 and 3 activation and nuclear vesiculation are equally impaired, indicating that melatonin interferes with Bax activation within mitochondria. Interestingly, we found that melatonin induces a strong re-localization of Bcl-2, the main Bax antagonist to mitochondria, suggesting that Bax activation may in fact be antagonized by Bcl-2 at the mitochondrial level. Indeed, we inhibit the melatonin anti-apoptotic effect (i) by silencing Bcl-2 with small interfering RNAs, or with small-molecular inhibitors targeted at the BH3 binding pocket in Bcl-2 (i.e. the one interacting with Bax); and (ii) by inhibiting melatonin-induced Bcl-2 mitochondrial re-localization with the MT1/MT2 receptor antagonist luzindole. This evidence provides a mechanism that may explain how melatonin through interaction with the MT1/MT2 receptors, elicits a pathway that interferes with the Bcl-2 family, thus modulating the cell life/death balance.