Melatonin modulates dysregulated circadian clocks in mice with diethylnitrosamine‐induced hepatocellular carcinoma

Disruption of circadian rhythms, which are regulated by the circadian clock machinery, plays an important role in different long‐term diseases including hepatocellular carcinoma (HCC ). Melatonin has been reported to alleviate promotion and progression of HCC , but the potential contribution of circadian clock modulation is unknown. We investigated the effects of melatonin in mice which received diethylnitrosamine (DEN ) (35 mg/kg body weight ip) once a week for 8 weeks. Melatonin was given at 5 or 10 mg kg^?1d^?1 ip beginning 4 weeks after the onset of DEN administration and ending at the sacrifice time (10, 20, 30, or 40 weeks). Liver expression of Bmal1, Clock, Npas2, Rorα, and Sirt1 increased, whereas Cry1, Per1, Per2, Per3, CK 1ε, Rev‐erbα, and Rev‐erbβ decreased following DEN administration. Melatonin treatment prevented changes in the expression of clock genes, and this effect was accompanied by an upregulation of the MT 1 receptor and reduced levels of the hypoxia‐inducible factors Hif‐1α and Hif‐2α. An increased expression of p21, p53, and PARP 1/2, a higher Bax/Bcl‐2 ratio, and a lower expression of Cyclin D1, CDK 6, HSP 70, HSP 90, and GRP 78 proteins were also observed in melatonin‐treated mice. Melatonin significantly potentiated the suppression of proliferation and cell cycle arrest induced by the synthetic REV ‐ERB agonist SR 9009 in human Hep3B cells, and BMAL 1 knocking down attenuated the pro‐apoptotic and antiproliferative effect of melatonin. Results support a contribution of changes in the circadian clock components to the beneficial effects of melatonin in HCC and highlight the usefulness of strategies modulating the circadian machinery in hepatocarcinogenesis.     

Melatonin receptor‐mediated protection against myocardial ischemia/reperfusion injury: role of SIRT1

Melatonin confers cardioprotective effect against myocardial ischemia/reperfusion (MI/R) injury by reducing oxidative stress. Activation of silent information regulator 1 (SIRT1) signaling also reduces MI/R injury. We hypothesize that melatonin may protect against MI/R injury by activating SIRT1 signaling. This study investigated the protective effect of melatonin treatment on MI/R heart and elucidated its potential mechanisms. Rats were exposed to melatonin treatment in the presence or the absence of the melatonin receptor antagonist luzindole or SIRT1 inhibitor EX527 and then subjected to MI/R operation. Melatonin conferred a cardioprotective effect by improving postischemic cardiac function, decreasing infarct size, reducing apoptotic index, diminishing serum creatine kinase and lactate dehydrogenase release, upregulating SIRT1, Bcl‐2 expression and downregulating Bax, caspase‐3 and cleaved caspase‐3 expression. Melatonin treatment also resulted in reduced myocardium superoxide generation, gp91^phox expression, malondialdehyde level, and increased myocardium superoxide dismutase (SOD) level, which indicate that the MI/R‐induced oxidative stress was significantly attenuated. However, these protective effects were blocked by EX527 or luzindole, indicating that SIRT1 signaling and melatonin receptor may be specifically involved in these effects. In summary, our results demonstrate that melatonin treatment attenuates MI/R injury by reducing oxidative stress damage via activation of SIRT1 signaling in a receptor‐dependent manner.     

Melatonin enhances hyperthermia‐induced apoptotic cell death in human leukemia cells

Melatonin is an endogenous indoleamine with a wide range of biological functions. In addition to modulating circadian rhythms, it plays important roles in the health as an antioxidant. Melatonin has also the ability to induce apoptosis in cancer cells and to enhance the antitumoral activity of chemotherapeutic agents. In this study, the effect of melatonin on hyperthermia‐induced apoptosis was explored using human leukemia cells. The results demonstrate that melatonin greatly improved the cytotoxicity of hyperthermia in U937 cells. The potentiation of cell death was achieved with 1 mmol/L concentrations of the indoleamine but not with concentrations close to physiological levels in blood (1 nmol/L). This effect was associated to an enhancement of the apoptotic response, revealed by an increase in cells with hypodiploid DNA content and activation of multiple caspases (caspase‐2, caspase‐3, caspase‐8, and caspase‐9). Melatonin also increased hyperthermia‐induced Bid activation as well as translocation of Bax from the cytosol to mitochondria and cytochrome c release. Hyperthermia‐provoked apoptosis and potentiation by melatonin were abrogated by a broad‐spectrum caspase inhibitor (z‐VAD‐fmk) as well as by specific inhibitors against caspase‐8 or caspase‐3. In contrast, blocking of the mitochondrial pathway of apoptosis either with a caspase‐9 inhibitor or overexpressing the anti‐apoptotic protein Bcl‐2 (U937/Bcl‐2) reduced the number of apoptotic cells in response to hyperthermia but it was unable to suppress melatonin enhancement. Melatonin appears to modulate the apoptotic response triggered by hyperthermia in a cell type‐specific manner as similar results were observed in HL‐60 but not in K562 or MOLT‐3 cells.     

Melatonin inhibits cholangiocarcinoma and reduces liver injury in Opisthorchis viverrini‐infected and N‐nitrosodimethylamine‐treated hamsters

The human liver fluke Opisthorchis viverrini infection and N‐nitrosodimethylamine (NDMA) administration induce cholangiocarcinoma (CCA) and liver injury in hamsters. Melatonin protects against liver injury and reduces the alteration of mitochondrial structure, mitochondrial membrane potential, and mitochondrial pro‐ and anti‐apoptotic pathways in various cancer types. To investigate the chemopreventive effect of melatonin on CCA genesis and liver injury, hamsters were treated with a combination of O. viverrini infection and NDMA concurrently administered with melatonin (10 mg/kg and 50 mg/kg) for 120 days. Melatonin treatment at 50 mg/kg caused a significant reduction in liver/body weight ratios and decreased tumor volumes leading to an increase in the survival of animals. In the tumorous tissues, the high‐dose melatonin reduced DNA fragmentation and mitochondrial apoptosis by inducing anti‐apoptotic protein (Bcl‐2) in the mitochondrial fraction and down‐regulating cytochrome c, pro‐apoptotic protein (Bax), and caspase‐3 in tumor cytosol. Moreover, a high‐dose melatonin treatment significantly increased mitochondrial antioxidant enzymes and prevented mitochondrial ultrastructure changes in the tumor. Overall, melatonin has potent chemopreventive effects in inhibiting CCA genesis and also reduces liver injury in hamster CCA, which, in part, might involve in the suppression of CCA by reducing tumor mitochondria alteration.     

Melatonin reverses flow shear stress‐induced injury in bone marrow mesenchymal stem cells via activation of AMP‐activated protein kinase signaling

Tissue‐engineered heart valves (TEHVs) are a promising treatment for valvular heart disease, although their application is limited by high flow shear stress (FSS). Melatonin has a wide range of physiological functions and is currently under clinical investigation for expanded applications; moreover, extensive protective effects on the cardiovascular system have been reported. In this study, we investigated the protection conferred by melatonin supplementation against FSS‐induced injury in bone marrow mesenchymal stem cells (BMSCs) and elucidated the potential mechanism in this process. Melatonin markedly reduced BMSC apoptotic death in a concentration‐dependent manner while increasing the levels of transforming growth factor β (TGF‐β), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet‐derived growth factor (PDGF) and B‐cell lymphoma 2 (Bcl2), and decreasing those of Bcl‐2‐associated X protein (Bax), p53 upregulated modulator of apoptosis (PUMA), and caspase 3. Notably, melatonin exerted its protective effects by upregulating the phosphorylation of adenosine monophosphate‐activated protein kinase (AMPK), which promotes acetyl‐CoA carboxylase (ACC) phosphorylation. Further molecular experiments revealed that luzindole, a nonselective antagonist of melatonin receptors, blocked the anti‐FSS injury (anti‐FSSI) effects of melatonin. Inhibition of AMPK by Compound C also counteracted the protective effects of melatonin, suggesting that melatonin reverses FSSI in BMSCs through the AMPK‐dependent pathway. Overall, our findings indicate that melatonin contributes to the amelioration of FSS‐induced BMSC injury by activating melatonin receptors and AMPK/ACC signaling. Our findings may provide a basis for the design of more effective strategies that promote the use of TEHCs in patients.     

Melatonin inhibits apoptosis and improves the developmental potential of vitrified bovine oocytes

Vitrification of oocytes has been shown to be closely associated with increased levels of reactive oxygen species (ROS) and apoptotic events. However, little information is available the effect of melatonin on the ROS levels and apoptotic events in vitrified oocytes. Therefore, we studied the effect of melatonin on ROS and apoptotic events in vitrified bovine oocytes by supplementing vitrification solution or in vitro maturation (IVM) and vitrification solution with 10^?9 m melatonin. We analyzed the ROS, mitochondrial Ca²⁺ (mCa²⁺) and membrane potential (ΔΨm), externalization of phosphatidylserine (PS), caspase‐3 activation, DNA fragmentation, mRNA expression levels of Bax and Bcl2 l1, and developmental potential of vitrified bovine oocytes. Vitrified bovine oocytes exhibited increased levels of ROS, mCa²⁺, Bax mRNA, and caspase‐3 protein and higher rates of PS externalization and DNA fragmentation, and decreased ΔΨm and Bcl2 l1 mRNA expression level. However, melatonin supplementation in vitrification solution or IVM and vitrification solution significantly decreased the levels of ROS, mCa²⁺, Bax mRNA expression, and caspase‐3 protein, and PS externalization and DNA fragmentation rates, and increased the ΔΨm and Bcl2 l1 mRNA expression level in vitrified oocytes, resulting in an increased developmental ability of vitrified bovine oocytes after parthenogenetic activation. The developmental ability of vitrified oocytes with melatonin supplementation in IVM and vitrification solution was similar to that of fresh ones. This study showed that supplementing the IVM and vitrification medium or vitrification medium with 10^?9 m melatonin significantly decreased the ROS level and inhibited apoptotic events of vitrified bovine oocytes, consequently increasing their developmental potential.     

In vitro and in vivo antitumor activity of melatonin receptor agonists

Abstract: Melatonin has been shown to inhibit the proliferation of estrogen receptor α (ERα)‐positive human breast cancer cells in vitro and suppress the growth of carcinogen‐induced mammary tumors in rats. Melatonin’s antiproliferative effect is mediated, at least in part, through the MT1 melatonin receptor and mechanisms involving modulation of the estrogen‐signaling pathway. To develop melatonin analogs with greater therapeutic effects, we have examined the in vitro and in vivo antimitotic activity of two MT1/MT2 melatonin receptor agonists, S23219‐1 and S23478‐1. In our studies, both agonists are quite effective at suppressing the growth of MCF‐7 human breast cancer cells. At a concentration of 10^?6 m , S23219‐1 and S23478‐1 inhibited the growth of MCF‐7 cells by 60% and 73%, respectively. However, S23478‐1 is more effective than melatonin and S23219‐1 at repressing the expression and transactivation of the ERα, and modulating the expression of pancreatic spasmolytic polypeptide (pS2), an estrogen‐regulated gene. The melatonin agonist S23478‐1 exhibited enhanced antitumor potency in the subsequent studies in our animal model. At a dosage of 25 mg/kg/day, S23478‐1 is more efficacious than melatonin at inducing regression of the established N‐nitroso‐N‐methyl‐urea‐induced rat mammary tumors. This dose of S23478‐1 (25 mg/kg/day) generated a significant (P < 0.05) overall regression response of 52%. Furthermore, at this dosage, S23478‐1 is more effective than melatonin at suppressing the estrogen‐signaling pathway and promoting tumor cell apoptosis, significantly increasing the expression of the pro‐apoptotic protein Bax, while decreasing the expression of ERα and the anti‐apoptotic protein Bcl‐2.     

Membrane receptor‐dependent Notch1/Hes1 activation by melatonin protects against myocardial ischemia–reperfusion injury: in vivo and in vitro studies

Melatonin confers profound protective effect against myocardial ischemia–reperfusion injury (MI/RI). Activation of Notch1/Hairy and enhancer of split 1 (Hes1) signaling also ameliorates MI/RI. We hypothesize that melatonin attenuates MI/RI‐induced oxidative damage by activating Notch1/Hes1 signaling pathway with phosphatase and tensin homolog deleted on chromosome 10 (Pten)/Akt acting as the downstream signaling pathway in a melatonin membrane receptor‐dependent manner. Male Sprague Dawley rats were treated with melatonin (10 mg/kg/day) for 4 wk and then subjected to MI/R surgery. Melatonin significantly improved cardiac function and decreased myocardial apoptosis and oxidative damage. Furthermore, in cultured H9C2 cardiomyocytes, melatonin (100 μmol/L) attenuated simulated ischemia–reperfusion (SIR)‐induced myocardial apoptosis and oxidative damage. Both in vivo and in vitro study demonstrated that melatonin treatment increased Notch1, Notch1 intracellular domain (NICD), Hes1, Bcl‐2 expressions, and p‐Akt/Akt ratio and decreased Pten, Bax, and caspase‐3 expressions. However, these protective effects conferred by melatonin were blocked by DAPT (the specific inhibitor of Notch1 signaling), luzindole (the antagonist of melatonin membrane receptors), Notch1 siRNA, or Hes1 siRNA administration. In summary, our study demonstrates that melatonin treatment protects against MI/RI by modulating Notch1/Hes1 signaling in a receptor‐dependent manner and Pten/Akt signaling pathways are key downstream mediators.     

Melatonin prevents human pancreatic carcinoma cell PANC-1-induced human umbilical vein endothelial cell proliferation and migration by inhibiting vascular endothelial growth factor expression

Abstract: Melatonin is an important natural oncostatic agent, and our previous studies have found its inhibitory action on tumor angiogenesis, but the mechanism remains unclear. It is well known that vascular endothelial growth factor (VEGF) plays key roles in tumor angiogenesis and has become an important target for antitumor therapy. Pancreatic cancer is a representative of the most highly vascularized and angiogenic solid tumors, which responds poorly to chemotherapy and radiation. Thus, seeking new treatment strategies targeting which have anti‐angiogenic capability is urgent in clinical practice. In this study, a co‐culture system between human umbilical vein endothelial cells (HUVECs) and pancreatic carcinoma cells (PANC‐1) was used to investigate the direct effect of melatonin on the tumor angiogenesis and its possible action on VEGF expression. We found HUVECs exhibited an increased cell proliferation and cell migration when co‐cultured with PANC‐1 cells, but the process was prevented when melatonin added to the incubation medium. Melatonin at concentrations of 1 μm and 1 mm inhibited the cell proliferation and migration of HUVECs and also decreased both the VEGF protein secreted to the cultured medium and the protein produced by the PANC‐1 cells. In addition, the VEGF mRNA expression was also down‐regulated by melatonin. Taken together, our present study shows that melatonin at pharmacological concentrations inhibited the elevated cell proliferation and cell migration of HUVECs stimulated by co‐culturing them with PANC‐1 cells; this was associated with a suppression of VEGF expression in PANC‐1 cells.     

Melatonin reduces indomethacin‐induced gastric mucosal cell apoptosis by preventing mitochondrial oxidative stress and the activation of mitochondrial pathway of apoptosis

Abstract: Augmentation of gastric mucosal cell apoptosis due to development of oxidative stress is one of the main pathogenic events in the development of nonsteroidal anti‐inflammatory drug (NSAID)‐induced gastropathy. Identification of a nontoxic, anti‐apoptotic molecule is warranted for therapy against NSAID‐induced gastropathy. The objective of the present study was to define the mechanism of the anti‐apoptotic effect of melatonin, a nontoxic molecule which scavenges reactive oxygen species. Using an array of experimental approaches, we have shown that melatonin prevents the development of mitochondrial oxidative stress and activation of mitochondrial pathway of apoptosis induced by indomethacin (a NSAID) in the gastric mucosa. Melatonin inhibits the important steps of indomethacin‐induced activation of mitochondrial pathway of apoptosis such as upregulation of the expression of Bax and Bak, and the downregulation of Bcl‐2 and BclxL. Melatonin also prevents indomethacin‐induced mitochondrial translocation of Bax and prevents the collapse of mitochondrial membrane potential. Moreover, melatonin reduces indomethacin‐mediated activation of caspase‐9 and caspase‐3 by blocking the release of cytochrome c and finally rescues gastric mucosal cells from indomethacin‐induced apoptosis as measured by the TUNEL assay. Histologic studies of gastric mucosa further document that melatonin almost completely protects against gastric damage induced by indomethacin. Thus, melatonin has significant anti‐apoptotic effects to protect gastric mucosa from NSAID‐induced apoptosis and gastropathy, which makes its use as potential therapy against gastric damage during NSAID treatment.     

Melatonin promotes secondary hair follicle development of early postnatal cashmere goat and improves cashmere quantity and quality by enhancing antioxidant capacity and suppressing apoptosis

Development of secondary hair follicles in early postnatal cashmere goats may be adversely affected by reactive oxygen species which cause oxidative stress. Because melatonin is a potent antioxidant and scavenger of free radicals, this study explored the effects of melatonin on secondary hair follicle development and subsequent cashmere production. It was found that the initiation of new secondary follicles in early postnatal Inner Mongolian cashmere goats of both melatonin‐treated and control goats occurred in the first 10 weeks of age. Melatonin promoted the initiation and maturation of secondary follicles and increased their population. Importantly, the beneficial effect of melatonin on secondary follicle population remained throughout life. As a result, melatonin increased cashmere production and improved its quality in terms of reduced fiber diameter. The mechanisms underlying the role of melatonin on secondary follicle development included the enhancement of activities of antioxidant enzymes, for example, superoxide dismutase and glutathione peroxidase (GSH‐Px), elevated total antioxidant capacity, and upregulated anti‐apoptotic Bcl‐2 expression and downregulated expression of the pro‐apoptotic proteins, Bax and caspase‐3. These results reveal that melatonin serves to promote secondary hair follicle development in early postnatal cashmere goats and expands our understanding of melatonin application in cashmere production. Melatonin treatment led to an increase in both the quantity and quality of cashmere fiber. This increased the textile value of the fibers and provided economic benefit.     

Melatonin prevents cell death and mitochondrial dysfunction via a SIRT1‐dependent mechanism during ischemic‐stroke in mice

Silent information regulator 1 (SIRT1), a type of histone deacetylase, is a highly effective therapeutic target for protection against ischemia reperfusion (IR) injury (IRI). Previous studies showed that melatonin preserves SIRT1 expression in neuronal cells of newborn rats after hypoxia–ischemia. However, the definite role of SIRT1 in the protective effect of melatonin against cerebral IRI in adult has not been explored. In this study, the brain of adult mice was subjected to IRI. Prior to this procedure, the mice were given intraperitoneal with or without the SIRT1 inhibitor, EX527. Melatonin conferred a cerebral‐protective effect, as shown by reduced infarct volume, lowered brain edema, and increased neurological scores. The melatonin‐induced upregulation of SIRT1 was also associated with an increase in the anti‐apoptotic factor, Bcl2, and a reduction in the pro‐apoptotic factor Bax. Moreover, melatonin resulted in a well‐preserved mitochondrial membrane potential, mitochondrial Complex I activity, and mitochondrial cytochrome c level while it reduced cytosolic cytochrome c level. However, the melatonin‐elevated mitochondrial function was reversed by EX527 treatment. In summary, our results demonstrate that melatonin treatment attenuates cerebral IRI by reducing IR‐induced mitochondrial dysfunction through the activation of SIRT1 signaling.     

Evidence of the receptor‐mediated influence of melatonin on pancreatic glucagon secretion via the Gαq protein‐coupled and PI3K signaling pathways

Abstract: Melatonin has been shown to modulate glucose metabolism by influencing insulin secretion. Recent investigations have also indicated a regulatory function of melatonin on the pancreatic α‐cells. The present in vitro and in vivo studies evaluated whether melatonin mediates its effects via melatonin receptors and which signaling cascade is involved. Incubation experiments using the glucagon‐producing mouse pancreatic α‐cell line αTC1 clone 9 (αTC1.9) as well as isolated pancreatic islets of rats and mice revealed that melatonin increases glucagon secretion. Preincubation of αTC1.9 cells with the melatonin receptor antagonists luzindole and 4P‐PDOT abolished the glucagon‐stimulatory effect of melatonin. In addition, glucagon secretion was lower in the pancreatic islets of melatonin receptor knockout mice than in the islets of the wild‐type (WT) control animals. Investigations of melatonin receptor knockout mice revealed decreased plasma glucagon concentrations and elevated mRNA expression levels of the hepatic glucagon receptor when compared to WT mice. Furthermore, studies using pertussis toxin, as well as measurements of cAMP concentrations, ruled out the involvement of Gαi‐ and Gαs‐coupled signaling cascades in mediating the glucagon increase induced by melatonin. In contrast, inhibition of phospholipase C in αTC1.9 cells prevented the melatonin‐induced effect, indicating the physiological relevance of the Gαq‐coupled pathway. Our data point to the involvement of the phosphatidylinositol 3‐kinase signaling cascade in mediating melatonin effects in pancreatic α‐cells. In conclusion, these findings provide evidence that the glucagon‐stimulatory effect of melatonin in pancreatic α‐cells is melatonin receptor mediated, thus supporting the concept of melatonin‐modulated and diurnal glucagon release.     

Beneficial effects of melatonin on in vitro bovine embryonic development are mediated by melatonin receptor 1

In the current study, a fundamental question, that is, the mechanisms related to the beneficial effects of melatonin on mammalian embryonic development, was addressed. To examine the potential beneficial effects of melatonin on bovine embryonic development, different concentrations of melatonin (10^?11, 10^?9, 10^?7, 10^?5, 10^?3 m ) were incubated with fertilized embryos. Melatonin in the range of 10^?11 to 10^?5 m significantly promoted embryonic development both in early culture medium (CR1aa +3 mg/mL BSA) and in later culture medium (CR1aa + 6%FBS). The most effective concentrations applied in the current studies were 10^?9 and 10^?7 m . Using quantitative real‐time PCR with immunofluorescence and Western blot assays, the expression of melatonin receptor MT1 and MT2 genes was identified in bovine embryos. Further studies indicate that the beneficial effects of melatonin on bovine embryo development were mediated by the MT1 receptor. This is based on the facts that luzindole, a nonselective MT1 and MT2 antagonist, blocked the effect on melatonin‐induced embryo development, while 4‐P‐PDOT, a selective MT2 antagonist, had little effect. Mechanistic explorations uncovered that melatonin application during bovine embryonic development significantly up‐regulated the expression of antioxidative (Gpx4, SOD1, bcl‐2) and developmentally important genes (SLC2A1, DNMT1A, and DSC2) while down‐regulating expression of pro‐apoptotic genes (P53, BAX, and Caspase‐3). The results obtained from the current studies provide new information regarding the mechanisms by which melatonin promotes bovine embryonic development under both in vitro and in vivo conditions.     

Melatonin promotes sorafenib‐induced apoptosis through synergistic activation of JNK/c‐jun pathway in human hepatocellular carcinoma

Melatonin has been shown to exert anticancer activity on hepatocellular carcinoma (HCC) through its antiproliferative and pro‐apoptotic effect in both experimental and clinical studies, and sorafenib is the only approved drug for the systemic treatment of HCC. Thus, this study was designed to investigate the combined effect of melatonin and sorafenib on proliferation, apoptosis, and its possible mechanism in human HCC. Here, we found that both melatonin and sorafenib resulted in a dose‐dependent growth inhibition of HuH‐7 cells after 48 hours treatment, and the combination of them enhanced the growth inhibition in a synergistic manner. Colony formation assay indicated that co‐treatment of HuH‐7 cells with melatonin and sorafenib significantly decreased the clonogenicity compared to the treatment with single agent. Furthermore, FACS and TUNEL assay confirmed that melatonin synergistically augmented the sorafenib‐induced apoptosis after 48 hours incubation, which was in accordance with the activation of caspase‐3 and the JNK/c‐jun pathway. Inhibition of JNK/c‐jun pathway with its inhibitor SP600125 reversed the phosphorylation of c‐jun and the activation of caspase‐3 induced by co‐treatment of HuH‐7 cells with melatonin and sorafenib in a dose‐dependent manner. Furthermore, SP600125 exhibited protective effect against apoptosis induced by the combination of melatonin and sorafenib. This study demonstrates that melatonin in combination with sorafenib synergistically inhibits proliferation and induces apoptosis in human HCC cells; therefore, supplementation of sorafenib with melatonin may serve as a potential therapeutic choice for advanced HCC.     

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

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

Melatonin treatment induces interplay of apoptosis,autophagy, and senescence in human colorectal cancer cells

In Asia, the incidence of colorectal cancer has been increasing gradually due to a more Westernized lifestyle. The aim of study is to determine the interaction between melatonin‐induced cell death and cellular senescence. We treated HCT116 human colorectal adenocarcinoma cells with 10 μm melatonin and determined the levels of cell death‐related proteins and evaluated cell cycle kinetics. The plasma membrane melatonin receptor, MT1, was significantly decreased by melatonin in a time‐dependent manner, whereas the nuclear receptor, RORα, was increased only after 12 hr treatment. HCT116 cells, which upregulated both pro‐apoptotic Bax and anti‐apoptotic Bcl‐xL in the early response to melatonin treatment, activated autophagic as well as apoptotic machinery within 18 hr. Melatonin decreased the S‐phase population of the cells to 57% of the control at 48 hr, which was concomitant with a reduction in BrdU‐positive cells in the melatonin‐treated cell population. We found not only marked attenuation of E‐ and A‐type cyclins, but also increased expression of p16 and p‐p21. Compared to the cardiotoxicity of Trichostatin A in vitro, single or cumulative melatonin treatment induced insignificant detrimental effects on neonatal cardiomyocytes. We found that 10 μm melatonin activated cell death programs early and induced G1‐phase arrest at the advanced phase. Therefore, we suggest that melatonin is a potential chemotherapeutic agent for treatment of colon cancer, the effects of which are mediated by regulation of both cell death and senescence in cancerous cells with minimized cardiotoxicity.