Melatonin and verteporfin synergistically suppress the growth and stemness of head and neck squamous cell carcinoma through the regulation of mitochondrial dynamics

The prevalence of head and neck squamous cell carcinoma (HNSCC) has continued to rise for decades. However, drug resistance to chemotherapeutics and relapse, mediated by cancer stem cells (CSCs), remains a significant impediment in clinical oncology to achieve successful treatment. Therefore, we focused on analyzing CSCs in HNSCC and demonstrated the effect of melatonin (Mel) and verteporfin (VP) on SCC-25 cells. HNSCC CSCs were enriched in the reactive oxygen species-low state and in sphere-forming cultures. Combination treatment with Mel and VP decreased HNSCC viability and increased apoptosis without causing significant damage to normal cells. Sphere-forming ability and stem cell population were reduced by co-treatment with Mel and VP, while mitochondrial ROS level was increased by the treatment. Furthermore, the expression of mitophagy markers, parkin and PINK1, was significantly decreased in the co-treated cells. Mel and VP induced mitochondrial depolarization and inhibited mitochondrial function. Parkin/TOM20 was localized near the nucleus and formed clusters of mitochondria in the cells after treatment. Moreover, Mel and VP downregulated the expression of markers involved in epithelial-mesenchymal transition and metastasis. The migration capacity of cells was significantly decreased by co-treatment with Mel and VP, accompanied by the down-regulation of MMP-2 and MMP-9 expression. Taken together, these results indicate that co-treatment with Mel and VP induces mitochondrial dysfunction, resulting in the apoptosis of CSCs. Mel and VP could thus be further investigated as potential therapies for HNSCC through their action on CSCs.     

Detection of recombinant and endogenous mouse melatonin receptors by monoclonal antibodies targeting the C‐terminal domain

Melatonin receptors play important roles in the regulation of circadian and seasonal rhythms, sleep, retinal functions, the immune system, depression, and type 2 diabetes development. Melatonin receptors are approved drug targets for insomnia, non‐24‐hour sleep‐wake disorders, and major depressive disorders. In mammals, two melatonin receptors (MTRs) exist, MT₁ and MT₂, belonging to the G protein‐coupled receptor (GPCR) superfamily. Similar to most other GPCRs, reliable antibodies recognizing melatonin receptors proved to be difficult to obtain. Here, we describe the development of the first monoclonal antibodies (mABs) for mouse MT₁ and MT₂. Purified antibodies were extensively characterized for specific reactivity with mouse, rat, and human MT₁ and MT₂ by Western blot, immunoprecipitation, immunofluorescence, and proximity ligation assay. Several mABs were specific for either mouse MT₁ or MT₂. None of the mABs cross‐reacted with rat MTRs, and some were able to react with human MTRs. The specificity of the selected mABs was validated by immunofluorescence microscopy in three established locations (retina, suprachiasmatic nuclei, pituitary gland) for MTR expression in mice using MTR‐KO mice as control. MT₂ expression was not detected in mouse insulinoma MIN6 cells or pancreatic beta‐cells. Collectively, we report the first monoclonal antibodies recognizing recombinant and native mouse melatonin receptors that will be valuable tools for future studies.     

Anticonvulsant effect of melatonin through ATP-sensitive channels in mice

Melatonin is a neurohormone secreted principally by the pineal gland. This molecule has various pharmacological properties including improving immune system, prevent cancer, anti-aging, and anti-oxidant effects. The anticonvulsant effects of melatonin have been proved by previous studies. Adenosine triphosphate (ATP)-sensitive potassium (K_ATP) channels are considered as an important target in the seizure modulation. The aim of the present study was to investigate the anticonvulsant effect of melatonin in pentylenetetrazole (PTZ)-induced seizures in mice, focusing on its ability to regulate K_ATP channels. Acute intraperitoneal administration of melatonin (40 and 80 mg/kg) increased clonic seizure threshold induced by intravenous administration of PTZ. Melatonin (40 and 80 mg/kg) increased the latency of clonic seizure and reduced its frequency in mice receiving an intraperitoneal injection of PTZ. Administration of glibenclamide, a K_ATP channels blocker, before intravenous injection of PTZ reduced melatonin anticonvulsant effect. Diazoxide and cromakalim, as K_ATP channels openers, increased antiseizure effect of melatonin in PTZ model of seizures. These findings suggest that the antiseizure effect of melatonin probably is gained through increasing the opening of K_ATP channels.     

The effects of melatonin on signaling pathways and molecules involved in glioma

Glioblastoma is one of the most common brain tumors with high invasion and malignancy. Despite extensive research in this area and the use of new and advanced therapies, the survival rate in this disease is very low. In addition, resistance to treatment has also been observed in this disease. One of the reasons for rapid progression and failure in treatment for this disease is the presence of a class of cells with high proliferation and high differentiation, a class called glioblastoma stem-like cells shown as being the source of glioblastoma tumors. It has been reported that several oncogenes are expressed in this disease. One important issue in recognizing the pathogenesis of this disease, and which could improve the treatment process, is the identification of involved oncogenes as well as molecules that affect the reduction of the expression of these oncogenes. Melatonin regulates the biological rhythm and inhibits the proliferation of malignant glioma cells due to antioxidant and anti-apoptotic effects. Melatonin has been considered in biological processes and in signaling pathways involved in the development of glioma. The aim of this review is to investigate the effects of melatonin on signaling pathways and molecules involved in the progression of glioma.     

基于层次分析法（AHP）的保健食品原料评价体系构建及分析

目的建立保健食品原料评价体系(Functional Food Crude Materials Evaluation System,FUFMES),为保健食品原料目录排名提供科学依据与技术保障。方法首先,利用文献调研和多轮专家访谈方法筛选FUFMES的指标并确定其层级关系;第二,使用层次分析法(Analytic Hierarchy Process,AHP)计算指标权重,具体方法是依据专家打分构建判断矩阵,利用R语言进行一致性检验与最大特征根检验,得出各级指标权重;第三,使用极值法计算原料的单个指标值;第四,利用线性加权综合法得到每种原料的评价指数并据此进行排名;最后,将获得的分析结果与专家评价结果进行比较。结果 FUFMES包括6个一级指标、39个二级指标、11个三级指标。利用FUFMES对9种保健食品原料进行评价,获得的评价指数依次是:西洋参(0.49)、人参(0.48)、银杏叶(0.21)、灵芝孢子粉(0.08)、鱼油(0.06)、螺旋藻(0.03)、辅酶Q10(0.02)、褪黑素(0.01)、大蒜油(-0.03)。基于该评价指数的排名结果与专家评价结果显示了较高一致性。结论构建了科学、完整的FUFMES,FUFMES将成为保健食品原料目录评价与排名的有力工具,为推进保健食品原料备案制提供科学依据与技术保障。     

Melatonin treatment improves primary progressive multiple sclerosis: a case report

We describe the case of a female patient who, at the age of 28, was diagnosed with symptoms of primary progressive multiple sclerosis (PPMS). Glucocorticoid treatment was immediately initiated. The disease and the demyelinating lesions progressed during the following 9 years reaching Expanded Disability Status Scale (EDSS) 8.0 (patient essentially restricted to bed, a chair or perambulated in a wheelchair). At this point, the patient began taking melatonin at doses ranging from 50 to 300 mg per day. Melatonin was her only treatment for the next 4 years; during this interval, her EDSS progressively recovered to 6.0 (the person needs intermittent or unilateral constant assistance such as cane, crutch, or brace to walk 100 meters with or without resting). This long‐lasting improvement is likely due to melatonin usage since it is related in time and because of its exceptionally long duration.     

Comparative physiological and proteomic analyses reveal the actions of melatonin in the reduction of oxidative stress in Bermuda grass (Cynodon dactylon (L). Pers.)

The fact of melatonin as an important antioxidant in animals led plant researchers to speculate that melatonin also acts in the similar manner in plants. Although melatonin has significant effects on alleviating stress‐triggered reactive oxygen species (ROS), the involvement of melatonin in direct oxidative stress and the underlying physiological and molecular mechanisms remain unclear in plants. In this study, we found that exogenous melatonin significantly alleviated hydrogen peroxide (H₂O₂)‐modulated plant growth, cell damage, and ROS accumulation in Bermuda grass. Additionally, 76 proteins significantly influenced by melatonin during mock or H₂O₂ treatment were identified by gel‐free proteomics using iTRAQ (isobaric tags for relative and absolute quantitation). Metabolic pathway analysis showed that several pathways were markedly enhanced by melatonin and H₂O₂ treatments, including polyamine metabolism, ribosome pathway, major carbohydrate metabolism, photosynthesis, redox, and amino acid metabolism. Taken together, this study provides more comprehensive insights into the physiological and molecular mechanisms of melatonin in Bermuda grass responses to direct oxidative stress. This may relate to the activation of antioxidants, modulation of metabolic pathways, and extensive proteome reprograming.     

Reduced silent information regulator 1 signaling exacerbates myocardial ischemia–reperfusion injury in type 2 diabetic rats and the protective effect of melatonin

Diabetes mellitus (DM) increases myocardial oxidative stress and endoplasmic reticulum (ER) stress. Melatonin confers cardioprotective effect by suppressing oxidative damage. However, the effect and mechanism of melatonin on myocardial ischemia–reperfusion (MI/R) injury in type 2 diabetic state are still unknown. In this study, we developed high‐fat diet‐fed streptozotocin (HFD‐STZ) rat, a well‐known type 2 diabetic model, to evaluate the effect of melatonin on MI/R injury with a focus on silent information regulator 1 (SIRT1) signaling, oxidative stress, and PERK/eIF2α/ATF4‐mediated ER stress. HFD‐STZ treated rats were exposed to melatonin treatment in the presence or the absence of sirtinol (a SIRT1 inhibitor) and subjected to MI/R surgery. Compared with nondiabetic animals, type 2 diabetic rats exhibited significantly decreased myocardial SIRT1 signaling, increased apoptosis, enhanced oxidative stress, and ER stress. Additionally, further reduced SIRT1 signaling, aggravated oxidative damage, and ER stress were found in diabetic animals subjected to MI/R surgery. Melatonin markedly reduced MI/R injury by improving cardiac functional recovery and decreasing myocardial apoptosis in type 2 diabetic animals. Melatonin treatment up‐regulated SIRT1 expression, reduced oxidative damage, and suppressed PERK/eIF2α/ATF4 signaling. However, these effects were all attenuated by SIRT1 inhibition. Melatonin also protected high glucose/high fat cultured H9C2 cardiomyocytes against simulated ischemia–reperfusion injury‐induced ER stress by activating SIRT1 signaling while SIRT1 siRNA blunted this action. Taken together, our study demonstrates that reduced cardiac SIRT1 signaling in type 2 diabetic state aggravates MI/R injury. Melatonin ameliorates reperfusion‐induced oxidative stress and ER stress via activation of SIRT1 signaling, thus reducing MI/R damage and improving cardiac function.     

Melatonin improves bone mineral density at the femoral neck in postmenopausal women with osteopenia: a randomized controlled trial

Melatonin is known for its regulation of circadian rhythm. Recently, studies have shown that melatonin may have a positive effect on the skeleton. By increasing age, the melatonin levels decrease, which may lead to a further imbalanced bone remodeling. We aimed to investigate whether treatment with melatonin could improve bone mass and integrity in humans. In a double‐blind RCT, we randomized 81 postmenopausal osteopenic women to 1‐yr nightly treatment with melatonin 1 mg (N = 20), 3 mg (N = 20), or placebo (N = 41). At baseline and after 1‐yr treatment, we measured bone mineral density (BMD) by dual X‐ray absorptiometry, quantitative computed tomography (QCT), and high‐resolution peripheral QCT (HR‐pQCT) and determined calciotropic hormones and bone markers. Mean age of the study subjects was 63 (range 56–73) yr. Compared to placebo, femoral neck BMD increased by 1.4% in response to melatonin (P < 0.05) in a dose‐dependent manner (P < 0.01), as BMD increased by 0.5% in the 1 mg/day group (P = 0.55) and by 2.3% (P < 0.01) in the 3 mg/day group. In the melatonin group, trabecular thickness in tibia increased by 2.2% (P = 0.04), and volumetric bone mineral density (vBMD) in the spine, by 3.6% (P = 0.04) in the 3 mg/day. Treatment did not significantly affect BMD at other sites or levels of bone turnover markers; however, 24‐hr urinary calcium was decreased in response to melatonin by 12.2% (P = 0.02). In conclusion, 1‐yr treatment with melatonin increased BMD at femoral neck in a dose‐dependent manner, while high‐dose melatonin increased vBMD in the spine. Further studies are needed to assess the mechanisms of action and whether the positive effect of nighttime melatonin will protect against fractures.     

Melatonin compensates silencing of heat shock protein 70 and suppresses ultraviolet radiation‐induced inflammation in human skin ex vivo and cultured keratinocytes

Melatonin, a lipophilic compound synthesized and released from the pineal gland, effectively acts against ultraviolet radiation (UVR), one of the main inducers of epidermal damage, skin cancer, inflammation, and DNA photo damage. One of the common known stress protein induced by UVR is heat shock protein 70 (Hsp70), highly expressed in human keratinocytes, providing cellular resistance to such stressors. Here, using human full‐thickness skin and normal human epidermal keratinocytes (NHEK), we investigated the interaction of melatonin and Hsp70 toward UVR‐induced inflammatory and apoptotic responses. The following observations were made: (i) UVR upregulated Hsp70 gene expression in human epidermis while melatonin significantly inverted this effect, (ii) similar patterns of regulation were observed within Hsp70 protein level, and (iii) mechanistic studies involving silencing of Hsp70 RNA (Hsp70 siRNA) showed prominent decrease of IκB‐α (an inhibitor of NF‐κB) and enhanced gene expression of pro‐inflammatory cytokines (IL‐1β, IL‐6, Casp‐1) and pro‐apoptotic protein (Casp‐3) in NHEK. Parallel investigation using melatonin (10^?3 m ) significantly inverted these responses regardless depletion of Hsp70 RNA suggesting a compensatory action of this compound in the defense mechanisms. Our findings combined with data reported so far thus enrich existing knowledge about the potent anti‐apoptotic and anti‐inflammatory action of melatonin.