Involvement of autophagy in melatonin‐induced cytotoxicity in glioma‐initiating cells

Glioblastoma‐initiating cells (GICs) represent a stem cell‐like subpopulation within malignant glioblastomas responsible for tumor development, progression, therapeutic resistance, and tumor relapse. Thus, eradication of this subpopulation is essential to achieve stable, long‐lasting remission. We have previously reported that melatonin decreases cell proliferation of glioblastoma cells both in vitro and in vivo and synergistically increases effectiveness of drugs in glioblastoma cells and also in GICs. In this study, we evaluated the effect of the indolamine alone in GICs and found that melatonin treatment reduces GICs proliferation and induces a decrease in self‐renewal and clonogenic ability accompanied by a reduction in the expression of stem cell markers. Moreover, our results also indicate that melatonin treatment, by modulating stem cell properties, induces cell death with ultrastructural features of autophagy. Thus, data reported here reinforce the therapeutic potential of melatonin as a treatment of malignant glioblastoma both by inhibiting tumor bulk proliferation or killing GICs, and simultaneously enhancing the effect of chemotherapy.     

Crystal structure and functional mapping of human ASMT,the last enzyme of the melatonin synthesis pathway

Abstract: Melatonin is a synchronizer of many physiological processes. Abnormal melatonin signaling is associated with human disorders related to sleep, metabolism, and neurodevelopment. Here, we present the X‐ray crystal structure of human N‐acetyl serotonin methyltransferase (ASMT), the last enzyme of the melatonin biosynthesis pathway. The polypeptide chain of ASMT consists of a C‐terminal domain, which is typical of other SAM‐dependent O‐methyltransferases, and an N‐terminal domain, which intertwines several helices with another monomer to form the physiologically active dimer. Using radioenzymology, we analyzed 20 nonsynonymous variants identified through the 1000 genomes project and in patients with neuropsychiatric disorders. We found that the majority of these mutations reduced or abolished ASMT activity including one relatively frequent polymorphism in the Han Chinese population (N17K, rs17149149). Overall, we estimate that the allelic frequency of ASMT deleterious mutations ranges from 0.66% in Europe to 2.97% in Asia. Mapping of the variants on to the 3‐dimensional structure clarifies why some are harmful and provides a structural basis for understanding melatonin deficiency in humans.     

Melatonin overcomes apoptosis resistance in human hepatocellular carcinoma by targeting Survivin and XIAP

Apoptosis resistance in hepatocellular carcinoma (HCC) is one of the most significant factors for hepatocarcinogenesis and tumor progression, and leads to resistance to conventional chemotherapy. It is well known that inhibitor of apoptosis proteins (IAPs) play key roles in apoptosis resistance, it has become an important target for antitumor therapy. In this study, we examined if melatonin, the main secretory product of the pineal gland, targeted IAPs, leading to the inhibition of apoptosis resistance. To accomplish this, we first observed that four members of IAPs (cIAP‐1, cIAP‐2, Survivin, and XIAP) were overexpressed in human HCC tissue. Interestingly, melatonin significantly inhibited the growth of HepG2 and SMMC‐7721 cells and promoted apoptosis along with the downregulation of Survivin and XIAP, but had no effect on the expression of cIAP‐1 and cIAP‐2. These data suggest that the inhibition of Survivin and XIAP by melatonin may play an important part in reversing apoptosis resistance. Notably, cIAP‐1, Survivin and XIAP were significantly associated with the coexpression of COX‐2 in human HCC specimens. Melatonin also reduced the expression of COX‐2 and inhibited AKT activation in HepG2 and SMMC‐7721 cells. Inhibition of COX‐2 activity with the selective inhibitor, NS398, and inhibition of AKT activation using the PI3K inhibitor, LY294002, in tumor cells confirmed that melatonin‐induced apoptosis was COX‐2/PI3K/AKT‐dependent, suggesting that the COX‐2/PI3K/AKT pathway plays a role in melatonin inhibition of IAPs. Taken together, these results suggest that melatonin overcomes apoptosis resistance by the suppressing Survivin and XIAP via the COX‐2/PI3K/AKT pathway in HCC cells.     

Same molecule but different expression: aging and sepsis trigger NLRP3 inflammasome activation,a target of melatonin

The connection between the innate immune system, clock genes, and mitochondrial bioenergetics was analyzed during aging and sepsis in mouse heart. Our results suggest that the sole NF‐κB activation does not explain the inflammatory process underlying aging; the former also triggers the NLRP3 inflammasome that enhances caspase‐1‐dependent maturation of IL‐1β. In this way, aged mice enter into a vicious cycle as IL‐1β further activates the NF‐κB/NLRP3 inflammasome link. The origin of NF‐κB activation was related to the age‐dependent Bmal1/Clock/RORα/Rev‐Erbα loop disruption, which lowers NAD⁺ levels, reducing the SIRT1 deacetylase ability to inactivate NF‐κB. Consequently, NF‐κB binding to DNA increases, raising the formation of proinflammatory mediators and inducing mitochondrial impairment. The cycle is then closed with the subsequent NLRP3 inflammasome activation. This paired contribution of the innate immune pathways serves as a catalyst to magnify the response to sepsis in aged compared with young mice. Melatonin administration blunted the septic response, reducing inflammation and oxidative stress, and enhancing mitochondrial function at the levels of nonseptic aged mice, but it did not counteract the age‐related inflammation. Together, our results suggest that, although with different strengths, chronoinflammaging constitutes the biochemical substrate of aging and sepsis, and identifies the NLRP3 inflammasome as a new molecular target for melatonin, providing a rationale for its use in NLRP3‐dependent diseases.     

Additional benefit of combined therapy with melatonin and apoptotic adipose‐derived mesenchymal stem cell against sepsis‐induced kidney injury

This study tested whether combined therapy with melatonin and apoptotic adipose‐derived mesenchymal stem cells (A‐ADMSCs) offered additional benefit in ameliorating sepsis‐induced acute kidney injury. Adult male Sprague–Dawley rats (n = 65) were randomized equally into five groups: Sham controls (SC), sepsis induced by cecal‐ligation and puncture (CLP), CLP‐melatonin, CLP‐A‐ADMSC, and CLP‐melatonin‐A‐ADMSC. Circulating TNF‐α level at post‐CLP 6 hr was highest in CLP and lowest in SC groups, higher in CLP‐melatonin than in CLP‐A‐ADMSC and CLP‐melatonin‐A‐ADMSC groups (all P < 0.001). Immune reactivity as reflected in the number of splenic helper‐, cytoxic‐, and regulatory‐T cells at post‐CLP 72 hr exhibited the same pattern as that of circulating TNF‐α among all groups (P < 0.001). The histological scoring of kidney injury and the number of F4/80+ and CD14+ cells in kidney were highest in CLP and lowest in SC groups, higher in CLP‐melatonin than in CLP‐A‐ADMSC and CLP‐melatonin‐A‐ADMSC groups, and higher in CLP‐A‐ADMSC than in CLP‐melatonin‐A‐ADMSC groups (all P < 0.001). Changes in protein expressions of inflammatory (RANTES, TNF‐1α, NF‐κB, MMP‐9, MIP‐1, IL‐1β), apoptotic (cleaved caspase 3 and PARP, mitochondrial Bax), fibrotic (Smad3, TGF‐β) markers, reactive‐oxygen‐species (NOX‐1, NOX‐2), and oxidative stress displayed a pattern identical to that of kidney injury score among the five groups (all P < 0.001). Expressions of antioxidants (GR+, GPx+, HO‐1, NQO‐1+) were lowest in SC group and highest in CLP‐melatonin‐A‐ADMSC group, lower in CLP than in CLP‐melatonin and CLP‐A‐ADMSC groups, and lower in CLP‐melatonin‐ than in CLP‐A‐ADMSC‐tretaed animals (all P < 0.001). In conclusion, combined treatment with melatonin and A‐ADMSC was superior to A‐ADMSC alone in protecting the kidneys from sepsis‐induced injury.     

The protective effect of melatonin on smoke‐induced vascular injury in rats and humans: a randomized controlled trial

Smoking is one of the most harmful lifestyles in the world. Very few studies have investigated the effects of melatonin in smoke‐induced vascular injury. This study was designed to investigate whether melatonin could protect rats and humans from smoke‐induced vascular injury. 32 male rats and a double‐blind randomized controlled trial (RCT) containing 63 participants formed the subjects of this study. In rats, 10 mg/kg of melatonin was intraperitoneally injected. Blood samples and abdominal artery were harvested two weeks later. Melatonin decreased the expression of platelet endothelial cell adhesion molecule‐1 (CD31), intercellular adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1) and endothelin‐1 (ET‐1) compared with the smoke exposed group (P < 0.05), whereas endothelial nitric oxide synthase (eNOS), nuclear erythroid 2‐related factor 2 (Nrf2), NAD(P)H quinone oxidoreductase 1 (NQO‐1), catalytic glutamate cysteine ligase (GCLC) and heme oxygenase‐1 (HO‐1) recovered markedly (P < 0.05). In humans, 3 mg/day of melatonin was taken orally by the participants. Blood samples were drawn at baseline and after two weeks of treatment. Compared with the oral placebo group, melatonin decreased the concentration of fibrinogen (Fbg) (P = 0.04) and free fatty acids (FFA) (P = 0.04) in smokers, along with the decreased expression of ICAM‐1, VCAM‐1 and ET‐1 (P = 0.004, P = 0.001, P < 0.0001, respectively). In contrast, Nrf2 and HO‐1 expression were markedly increased (P = 0.0001, P = 0.0049, respectively) after smokers took melatonin orally. In summary, our present data suggest that melatonin could ameliorate smoke‐induced vascular injury.