Melatonin antagonizes hypoxia‐mediated glioblastoma cell migration and invasion via inhibition of HIF‐1α

Hypoxia is a crucial factor in tumor aggressiveness and resistance to therapy, especially in glioblastoma. Our previous results have shown that melatonin exerts antimigratory and anti‐invasive action in glioblastoma cells under normoxia. However, the effect of melatonin on migration and invasion of glioblastoma cells under hypoxic condition remains poorly understood. Here, we show that melatonin strongly reduced hypoxia‐mediated invasion and migration of U251 and U87 glioblastoma cells. In addition, we found that melatonin significantly blocked HIF‐1α protein expression and suppressed the expression of downstream target genes, matrix metalloproteinase 2 (MMP‐2) and vascular endothelial growth factor (VEGF). Furthermore, melatonin destabilized hypoxia‐induced HIF‐1α protein via its antioxidant activity against ROS produced by glioblastoma cells in response to hypoxia. Along with this, HIF‐1α silencing by small interfering RNA markedly inhibited glioblastoma cell migration and invasion, and this appeared to be associated with MMP‐2 and VEGF under hypoxia. Taken together, our findings suggest that melatonin suppresses hypoxia‐induced glioblastoma cell migration and invasion via inhibition of HIF‐1α. Considering the fact that overexpression of the HIF‐1α protein is often detected in glioblastoma multiforme, melatonin may prove to be a potent therapeutic agent for this tumor.     

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 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 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.     

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.     

Increased levels of monokine induced by interferon‐γ (Mig) in the vitreous of patients with diabetic retinopathy

Aim To determine the intravitreous concentration of monokine induced by interferon‐γ (Mig) in patients with diabetic retinopathy (DR) and the relation between Mig and vascular endothelial growth factor (VEGF). Research design and methods Vitreous samples were obtained at the time of vitrectomy from 41 eyes of 38 DR patients (30 with active DR and 11 with inactive DR) and from 15 eyes of 15 non‐diabetic patients who had macular disease (control subjects). Human Mig and VEGF were quantified using a FACS Caliber® flow cytometer. Results The vitreous concentration of Mig was increased significantly in patients with both active and inactive DR [148.0 (31.6–997.2; median range) and 82.3 (25.7–347.7) pg/ml, respectively] compared with control subjects [21.0 (5.2–74.3) pg/ml; P < 0.0001 and P < 0.001, respectively]. In DR patients, a significant (P < 0.01) correlation was observed between vitreous concentrations of Mig and VEGF. Conclusion Our results suggest that Mig may play an important role in the pathogenesis of DR and works in consort with VEGF in the progression of pathological angiogenesis in DR.     

The inhibition of TNF‐α‐induced leucocyte apoptosis by melatonin involves membrane receptor MT1/MT2 interaction

The pro‐apoptotic signalling cascades induced by tumour necrosis factor‐alpha (TNF‐α) have been intensively studied in multiple cellular systems. So far, it is known that TNF‐α can simultaneously activate survival and apoptotic cell death responses. The balance between these signals determines the ultimate response of the cell to TNF‐α. Moreover, emerging evidence suggests that melatonin may be involved in the protection of different cell types against apoptosis. Thus, the objective of this study was to evaluate the effect of melatonin on TNF‐α‐induced apoptosis in human leucocytes. Cells were treated with TNF‐α alone or in the presence of cycloheximide (CHX), which promotes caspase‐8 activation by eliminating the endogenous caspase‐8 inhibitor, c‐FLIP. Treatment with TNF‐α/CHX led to apoptotic cell death, as ascertained by annexin V/propidium iodide (PI) staining. Likewise, in the presence of CHX, TNF‐α stimulation produced cFLIP down‐regulation and subsequent caspase‐8 activation, thus directly triggering caspase‐3 activation and causing Bid truncation and subsequent caspase‐9 activation. Conversely, pre‐incubation of cells with melatonin inhibited TNF‐α‐/CHX‐evoked leucocyte apoptosis. Similarly, pretreatment of leucocytes with melatonin increased cFLIP protein levels, thereby preventing TNF‐α‐/CHX‐mediated caspase processing. Blockade of melatonin membrane receptor MT1/MT2 or extracellular signal‐regulated kinase (ERK) pathway with luzindole or PD98059, respectively, abolished the inhibitory effects of melatonin on leucocyte apoptosis evoked by TNF‐α/CHX. In conclusion, the model proposed by these findings is that the MT1/MT2 receptors, which are under the positive control of melatonin, trigger an ERK‐dependent signalling cascade that interferes with the anti‐apoptotic protein cFLIP modulating the cell life/death balance of human leucocytes.     

Amyloid‐β neurotoxicity in organotypic culture is attenuated by melatonin: involvement of GSK‐3β, tau and neuroinflammation

Abstract: Alzheimer’s disease (AD) is a progressive neurodegenerative disorder marked by accumulation of extracellular deposits of amyloid‐β (Aβ) peptide in brain regions that are important for memory and cognition. The buildup of Aβ aggregates in the AD is followed by the formation of intracellular neurofibrillary tangles and activation of neuroinflammatory reactions. The present study investigated whether melatonin possesses a neuroprotective effect against Aβ‐induced toxicity. For this purpose, organotypic hippocampal slices were cultured and exposed to 25 μm of Aβ_25–35 in the absence or in the presence of melatonin (25, 50, or 100 μm ). In addition, the authors have investigated the involvement of GSK‐3β, tau protein, astroglial, and microglial activation, and cytokine levels in the melatonin protection against Aβ‐induced neurotoxicity. Melatonin prevented the cell damage in hippocampus induced by the exposure to Aβ_25–35. In addition, melatonin significantly reduced the activation of GSK‐3β, the phosphorylation of tau protein, the glial activation and the Aβ‐induced increase of TNF‐α and IL‐6 levels. On the basis of these findings, we speculate that melatonin may provide an effective therapeutic strategy for AD, by attenuating Aβ‐induced phosphorylation of tau protein, and preventing GSK‐3β activation and neuroinflammation.     

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.     

Melatonin suppresses activation of hepatic stellate cells through RORα‐mediated inhibition of 5‐lipoxygenase

Liver fibrosis is scar tissue resulting from an uncontrolled wound‐healing process in response to chronic liver injury. Liver damage generates an inflammatory reaction that activates hepatic stellate cells (HSC) that transdifferentiate from quiescent cells that control retinol metabolism to proliferative and migratory myofibroblasts that produce excessive amounts of extracellular matrix proteins, in particular collagen 1a1 (COL1A1). Although liver fibrosis is reversible, no effective drug therapy is available to prevent or reverse HSC activation. Melatonin has potent hepatoprotective properties in a variety of acute and chronic liver injury models and suppresses liver fibrosis. However, it remains unclear whether melatonin acts indirectly or directly on HSC to prevent liver fibrosis. Here, we studied the effect of melatonin on culture‐activated rat HSC. Melatonin dose‐dependently suppressed the expression of HSC activation markers Col1a1 and alpha‐smooth muscle actin (αSMA, Acta2), as well as HSC proliferation and loss of lipid droplets. The nuclear melatonin sensor retinoic acid receptor‐related orphan receptor‐alpha (RORα/Nr1f1) was expressed in quiescent and activated HSC, while the membranous melatonin receptors (Mtrn1a and Mtrn1b) were not. The synthetic RORα agonist SR1078 more potently suppressed Col1a1 and αSma expression, HSC proliferation, and lipid droplet loss, while the RORα antagonist SR1001 blocked the antifibrotic features of melatonin. Melatonin and SR1078 inhibited the expression of Alox5, encoding 5‐lipoxygenase (5‐LO). The pharmacological 5‐LO inhibitor AA861 reduced Acta2 and Col1a1 expression in activated HSC. We conclude that melatonin directly suppresses HSC activation via RORα‐mediated inhibition of Alox5 expression, which provides novel drug targets to treat liver fibrosis.     

Melatonin ameliorates amyloid beta‐induced memory deficits,tau hyperphosphorylation and neurodegeneration via PI3/Akt/GSk3β pathway in the mouse hippocampus

Alzheimer's disease (AD) is the most prevalent age‐related neurodegenerative disease, pathologically characterized by the accumulation of amyloid beta (Aβ) aggregation in the brain, and is considered to be the primary cause of cognitive dysfunction. Aβ aggregates lead to synaptic disorder, tau hyperphosphorylation, and neurodegeneration. In this study, the underlying neuroprotective mechanism of melatonin against Aβ_1‐42‐induced neurotoxicity was investigated in the mice hippocampus. Intracerebroventricular (i.c.v.) Aβ_1‐42‐injection triggered memory impairment, synaptic disorder, hyperphosphorylation of tau protein, and neurodegeneration in the mice hippocampus. After 24 hr of Aβ_1‐42 injection, the mice were treated with melatonin (10 mg/kg, intraperitonially) for 3 wks, reversed the Aβ_1‐42‐induced synaptic disorder via increasing the level of presyanptic (Synaptophysin and SNAP‐25) and postsynaptic protein [PSD95, p‐GluR1 (Ser845), SNAP23, and p‐CREB (Ser133)], respectively, and attenuated the Aβ_1‐42‐induced memory impairment. Chronic melatonin treatment attenuated the hyperphosphorylation of tau protein via PI3K/Akt/GSK3β signaling by activating the p‐PI3K, p‐Akt (Ser 473) and p‐GSK3β (Ser9) in the Aβ_1‐42‐treated mice. Furthermore, melatonin decreased Aβ_1‐42‐induced apoptosis through decreasing the overexpression of caspase‐9, caspase‐3, and PARP‐1 level. Additionally, the evaluation of immunohistochemical analysis of caspase‐3, Fluorojade‐B, and Nissl staining indicated that melatonin prevented neurodegeneration in Aβ_1‐42‐treated mice. Our results demonstrated that melatonin has neuroprotective effect against Aβ_1‐42‐induced neurotoxicity through decreasing memory impairment, synaptic disorder, tau hyperphosphorylation, and neurodegeneration via PI3K/Akt/GSK3β signaling in the Aβ_1‐42‐treated mouse model of AD. On the basis of these results, we suggest that melatonin could be an effective, promising, and safe neuroprotective candidate for the treatment of progressive neurodegenerative disorders, such as AD.