Melatonin ameliorates Aβ42‐induced alteration of βAPP‐processing secretases via the melatonin receptor through the Pin1/GSK3β/NF‐κB pathway in SH‐SY5Y cells

Melatonin is involved in the physiological regulation of the β‐amyloid precursor protein (βAPP)‐cleaving secretases which are responsible for generation of the neurotoxic amyloid beta (Aβ) peptide, one of the hallmarks of Alzheimer's disease (AD) pathology. In this study, we aimed to determine the underlying mechanisms of this regulation under pathological conditions. We establish that melatonin prevents Aβ₄₂‐induced downregulation of a disintegrin and metalloproteinase domain‐containing protein 10 (ADAM10) as well as upregulation of β‐site APP‐cleaving enzyme 1 (BACE1) and presenilin 1 (PS1) in SH‐SY5Y cell cultures. We also demonstrate that the intrinsic mechanisms of the observed effects occurred via regulation of nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) and glycogen synthase kinase (GSK)‐3β as melatonin reversed Aβ₄₂‐induced upregulation and nuclear translocation of NF‐κBp65 as well as activation of GSK3β via its receptor activation. Furthermore, specific blocking of the NF‐κB and GSK3β pathways partially abrogated the Aβ₄₂‐induced reduction in the BACE1 and PS1 levels. In addition, GSK3β blockage affected α‐secretase cleavage and modulated nuclear translocation of NF‐κB. Importantly, our study for the first time shows that peptidyl‐prolyl cis‐trans isomerase NIMA‐interacting 1 (Pin1) is a crucial target of melatonin. The compromised levels and/or genetic variation of Pin1 are associated with age‐dependent tau and Aβ pathologies and neuronal degeneration. Interestingly, melatonin alleviated the Aβ₄₂‐induced reduction of nuclear Pin1 levels and preserved the functional integrity of this isomerase. Our findings illustrate that melatonin attenuates Aβ₄₂‐induced alterations of βAPP‐cleaving secretases possibly via the Pin1/GSK3β/NF‐κB pathway.     

Melatonin synergizes the chemotherapeutic effect of 5‐fluorouracil in colon cancer by suppressing PI3K/AKT and NF‐κB/iNOS signaling pathways

5‐Fluorouracil (5‐FU) is one of the most commonly used chemotherapeutic agents in colon cancer treatment, but has a narrow therapeutic index limited by its toxicity. Melatonin exerts antitumor activity in various cancers, but it has never been combined with 5‐FU as an anticolon cancer treatment to improve the chemotherapeutic effect of 5‐FU. In this study, we assessed such combinational use in colon cancer and investigated whether melatonin could synergize the antitumor effect of 5‐FU. We found that melatonin significantly enhanced the 5‐FU‐mediated inhibition of cell proliferation, colony formation, cell migration and invasion in colon cancer cells. We also found that melatonin synergized with 5‐FU to promote the activation of the caspase/PARP‐dependent apoptosis pathway and induce cell cycle arrest. Further mechanism study demonstrated that melatonin synergized the antitumor effect of 5‐FU by targeting the PI3K/AKT and NF‐κB/inducible nitric oxide synthase (iNOS) signaling. Melatonin in combination with 5‐FU markedly suppressed the phosphorylation of PI3K, AKT, IKKα, IκBα, and p65 proteins, promoted the translocation of NF‐κB p50/p65 from the nuclei to cytoplasm, abrogated their binding to the iNOS promoter, and thereby enhanced the inhibition of iNOS signaling. In addition, pretreatment with a PI3K‐ or iNOS‐specific inhibitor synergized the antitumor effects of 5‐FU and melatonin. Finally, we verified in a xenograft mouse model that melatonin and 5‐FU exerted synergistic antitumor effect by inhibiting the AKT and iNOS signaling pathways. Collectively, our study demonstrated that melatonin synergized the chemotherapeutic effect of 5‐FU in colon cancer through simultaneous suppression of multiple signaling pathways.     

Melatonin attenuates TNF‐α and IL‐1β expression in synovial fibroblasts and diminishes cartilage degradation: Implications for the treatment of rheumatoid arthritis

The hormone melatonin has many properties, including antioxidant, anti‐inflammatory, and immunomodulatory effects. Melatonin has been demonstrated to be beneficial in several inflammatory autoimmune diseases, but its effects in rheumatoid arthritis (RA) remain controversial. We sought to determine how melatonin regulates inflammation in RA. We found that melatonin dose‐dependently inhibits tumor necrosis factor‐α (TNF‐α) and interleukin (IL)‐1β expression through the PI3K/AKT, ERK, and NF‐κB signaling pathways. We also identified that melatonin inhibits TNF‐α and IL‐1β production by upregulating miR‐3150a‐3p expression. Synovial tissue specimens from RA patients and culture of human rheumatoid fibroblast‐like synoviocytes confirmed that the MT1 receptor is needed for the anti‐inflammatory activities of melatonin. Importantly, melatonin also significantly reduced paw swelling, cartilage degradation, and bone erosion in the collagen‐induced arthritis mouse model. Our results indicate that melatonin ameliorates RA by inhibiting TNF‐α and IL‐1β production through downregulation of the PI3K/AKT, ERK, NF‐κB signaling pathways, as well as miR‐3150a‐3p overexpression. The role of melatonin as an adjuvant treatment in patients with RA deserves further clinical studies.     

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 prevents Drp1‐mediated mitochondrial fission in diabetic hearts through SIRT1‐PGC1α pathway

Myocardial contractile dysfunction is associated with an increase in mitochondrial fission in patients with diabetes. However, whether mitochondrial fission directly promotes diabetes‐induced cardiac dysfunction is still unknown. Melatonin exerts a substantial influence on the regulation of mitochondrial fission/fusion. This study investigated whether melatonin protects against diabetes‐induced cardiac dysfunction via regulation of mitochondrial fission/fusion and explored its underlying mechanisms. Here, we show that melatonin prevented diabetes‐induced cardiac dysfunction by inhibiting dynamin‐related protein 1 (Drp1)‐mediated mitochondrial fission. Melatonin treatment decreased Drp1 expression, inhibited mitochondrial fragmentation, suppressed oxidative stress, reduced cardiomyocyte apoptosis, improved mitochondrial function and cardiac function in streptozotocin (STZ )‐induced diabetic mice, but not in SIRT 1^?/? diabetic mice. In high glucose‐exposed H9c2 cells, melatonin treatment increased the expression of SIRT 1 and PGC ‐1α and inhibited Drp1‐mediated mitochondrial fission and mitochondria‐derived superoxide production. In contrast, SIRT 1 or PGC ‐1α siRNA knockdown blunted the inhibitory effects of melatonin on Drp1 expression and mitochondrial fission. These data indicated that melatonin exerted its cardioprotective effects by reducing Drp1‐mediated mitochondrial fission in a SIRT 1/PGC ‐1α‐dependent manner. Moreover, chromatin immunoprecipitation analysis revealed that PGC ‐1α directly regulated the expression of Drp1 by binding to its promoter. Inhibition of mitochondrial fission with Drp1 inhibitor mdivi‐1 suppressed oxidative stress, alleviated mitochondrial dysfunction and cardiac dysfunction in diabetic mice. These findings show that melatonin attenuates the development of diabetes‐induced cardiac dysfunction by preventing mitochondrial fission through SIRT 1‐PGC 1α pathway, which negatively regulates the expression of Drp1 directly. Inhibition of mitochondrial fission may be a potential target for delaying cardiac complications in patients with diabetes.     

Melatonin reduces inflammatory response in human intestinal epithelial cells stimulated by interleukin‐1β

Melatonin is the main secretory product of the pineal gland, and it is involved in the regulation of periodic events. A melatonin production independent of the photoperiod is typical of the gut. However, the local physiological role of melatonin at the intestinal tract is poorly characterized. In this study, we evaluated the anti‐inflammatory activities of melatonin in an in vitro model of inflamed intestinal epithelium. To this purpose, we assessed different parameters usually associated with intestinal inflammation using IL‐1β‐stimulated Caco‐2 cells. Differentiated monolayers of Caco‐2 cells were preincubated with melatonin (1 nmol/L‐50 μmol/L) and then exposed to IL‐1β. After each treatment, different inflammatory mediators, DNA‐breakage, and global DNA methylation status were assayed. To evaluate the involvement of melatonin membrane receptors, we also exposed differentiated monolayers to melatonin in the presence of luzindole, a MT1 and MT2 antagonist. Our results showed that melatonin, at concentrations similar to those obtained in the lumen gut after ingestion of dietary supplements for the treatment of sleep disorders, was able to attenuate the inflammatory response induced by IL‐1β. Anti‐inflammatory effects were expressed as both a decrease of the levels of inflammatory mediators, including IL‐6, IL‐8, COX‐2, and NO, and a reduced increase in paracellular permeability. Moreover, the protection was associated with a reduced NF‐κB activation and a prevention of DNA demethylation. Conversely, luzindole did not reverse the melatonin inhibition of stimulated‐IL‐6 release. In conclusion, our findings suggest that melatonin, through a local action, can modulate inflammatory processes at the intestinal level, offering new opportunities for a multimodal management of IBD.     

Melatonin ameliorates vascular endothelial dysfunction,inflammation, and atherosclerosis by suppressing the TLR4/NF‐κB system in high‐fat‐fed rabbits

Vascular endothelial dysfunction (VED) and inflammation contribute to the initiation and progression of atherosclerosis. Melatonin (MLT) normalizes lipid profile, improves endothelial function, and possesses anti‐inflammatory properties. However, the precise mechanisms are still unclear. This study investigated whether MLT could ameliorate VED, inflammation, and atherosclerosis by suppressing the Toll‐like receptor 4 (TLR4)/nuclear factor kappa B (NF‐κB) system in high‐fat‐fed rabbits. Rabbits were randomly divided into three groups that received a standard diet (control group), high‐cholesterol diet (atherosclerosis group), or high‐cholesterol diet plus 10 mg/kg/day MLT (MLT group) for 12 wk. After treatment, high‐fat diet significantly increased serum lipid and inflammatory markers in rabbits in atherosclerosis group compared with that in control group. In addition, high‐fat diet also induced VED and typical atherosclerotic plaque formation and increased intima/media thickness ratio, which were significantly improved by MLT therapy as demonstrated in MLT group. Histological and immunoblot analysis further showed that high‐fat diet enhanced the expressions of TLR4, myeloid differentiation primary response protein (MyD88), and NF‐κB p65, but decreased inhibitor of NF‐κB (IκB) expression. By contrast, MLT therapy decreased the expressions of TLR4, MyD88, and NF‐κB p65 and increased IκB expression. This study has demonstrated that MLT ameliorates lipid metabolism, VED, and inflammation and inhibits the progression of atherosclerosis in high‐fat‐fed rabbits. Moreover, our study indicates for the first time that suppression of the TLR4/NF‐κB system in local vasculature with atherosclerotic damage is important for the protective effects of MLT.     

Melatonin alleviates adipose inflammation through elevating α‐ketoglutarate and diverting adipose‐derived exosomes to macrophages in mice

Obesity is associated with macrophage infiltration and metabolic inflammation, both of which promote metabolic disease progression. Melatonin is reported to possess anti‐inflammatory properties by inhibiting inflammatory response of adipocytes and macrophages activation. However, the effects of melatonin on the communication between adipocytes and macrophages during adipose inflammation remain elusive. Here, we demonstrated melatonin alleviated inflammation and elevated α‐ketoglutarate (αKG) level in adipose tissue of obese mice. Mitochondrial isocitrate dehydrogenase 2 (Idh2) mRNA level was also elevated by melatonin in adipocytes leading to increase αKG level. Further analysis revealed αKG was the target for melatonin inhibition of adipose inflammation. Moreover, sirtuin 1 (Sirt1) physically interacted with IDH2 and formed a complex to increase the circadian amplitude of Idh2 and αKG content in melatonin‐inhibited adipose inflammation. Notably, melatonin promoted exosomes secretion from adipocyte and increased adipose‐derived exosomal αKG level. Our results also confirmed that melatonin alleviated adipocyte inflammation and increased ratio of M2 to M1 macrophages by transporting of exosomal αKG to macrophages and promoting TET‐mediated DNA demethylation. Furthermore, exosomal αKG attenuated signal transducers and activators of transduction‐3 (STAT3)/NF‐κB signal by its receptor oxoglutarate receptor 1 (OXGR1) in adipocytes. Melatonin also attenuated adipose inflammation and deceased macrophage number in chronic jet‐lag mice. In summary, our results demonstrate melatonin alleviates metabolic inflammation by increasing cellular and exosomal αKG level in adipose tissue. Our data reveal a novel function of melatonin on adipocytes and macrophages communication, suggesting a new potential therapy for melatonin to prevent and treat obesity caused systemic inflammatory disease.     

α‐thalassaemia masked by β gene defects and a new polyadenylation site mutation on the α2‐globin gene

We report three examples of chronic anaemia involving complex combinations of α‐ and β‐globin gene defects. The first case had a potential Hb H disease caused by the classic SEA/RW deletions masked by Hb E [β26(B8)Glu→Lys] in the homozygous state. The second had an unusual Hb H disease caused by compound heterozygosity for two different α2 polyadenylation site mutations masked by a β‐thalassaemia heterozygosity. The third had an intermediate α‐thalassaemia with considerable anaemia caused by an as yet unknown polyadenylation site (AATAAA>AATAAC) mutation in combination with a common RW deletion masked by a common Hb C [β6(A3)Glu→Lys] heterozygosity. Diagnostic methods, genotype/phenotype correlations and the chance of overlooking these combinations during risk assessment in a multiethnic society are discussed.     

MTNR1B loss promotes chordoma recurrence by abrogating melatonin‐mediated β‐catenin signaling repression

Chordoma is an extremely rare malignant bone tumor with a high rate of relapse. While cancer stem cells (CSCs) are closely associated with tumor recurrence, which depend on its capacity to self‐renew and induce chemo‐/radioresistance, whether and how CSCs participate in chordoma recurrence remains unclear. The current study found that tumor cells in recurrent chordoma displayed more dedifferentiated CSC‐like properties than those in corresponding primary tumor tissues. Meanwhile, MTNR1B deletion along with melatonin receptor 1B (MTNR1B) down‐regulation was observed in recurrent chordoma. Further investigation revealed that activation of Gαi2 by MTNR1B upon melatonin stimulation could inhibit SRC kinase activity via recruiting CSK and SRC, increasing SRC Y530 phosphorylation, and decreasing SRC Y419 phosphorylation. This subsequently suppressed β‐catenin signaling and stemness via decreasing β‐catenin p‐Y86/Y333/Y654. However, MTNR1B loss in chordoma mediated increased CSC properties, chemoresistance, and tumor progression by releasing melatonin's repression of β‐catenin signaling. Clinically, MTNR1B deletion was found to correlate with patients’ survival. Together, our study establishes a novel convergence between melatonin and β‐catenin signaling pathways and reveals the significance of this cross talk in chordoma recurrence. Besides, we propose that MTNR1B is a potential biomarker for prediction of chordoma prognosis and selection of treatment options, and chordoma patients might benefit from targeting MTNR1B/Gαi2/SRC/β‐catenin axis.     

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

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

Melatonin potentiates the antiproliferative and pro‐apoptotic effects of ursolic acid in colon cancer cells by modulating multiple signaling pathways

Ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid, is largely distributed in medical herbs and edible plants. Melatonin is an indoleamine compound produced in the pineal gland and also a plant‐derived product. Both UA and melatonin have been shown to inhibit cancer cell growth in numerous studies, but they have never been combined altogether as an anticolon cancer treatment. In this study, we investigated whether the association between UA and melatonin leads to an enhanced antiproliferative and pro‐apoptotic activities in colon cancer SW480 and LoVo cells. We found that combined treatment with UA and melatonin significantly enhanced inhibition of cell viability and migration, promoted changes in cell morphology and spreading, and increased induction of apoptosis, thereby potentiating the effects of UA alone in colon cancer cells. Moreover, we found that the enhanced effects of UA and melatonin combination are mediated through simultaneous modulation of cytochrome c/caspase, MMP9/COX‐2, and p300/NF‐κB signaling pathways. Combined treatment with UA and melatonin triggered the release of cytochrome c from the mitochondrial intermembrane space into the cytosol, induced cleavage of caspase and PARP proteins, enhanced inhibition of MMP9 and COX‐2 expression, promoted p300 and NF‐κB translocation from cell nuclei to cytoplasm, and abrogated NF‐κB binding and p300 recruitment to COX‐2 promoter in colon cancer cells. These results, therefore, demonstrated that melatonin potentiated the antiproliferative and pro‐apoptotic effects of UA in colon cancer cells by modulating multiple signaling pathways and suggest that such a combinational treatment might potentially become an effective way in colon cancer therapy.