Molecular deficiency (ies) in MT1 melatonin signaling pathway underlies the melatonin‐unresponsive phenotype in MDA‐MB‐231 human breast cancer cells

Melatonin has been shown repeatedly to inhibit the growth of human breast tumor cells in vitro and in vivo. Its antiproliferative effects have been well studied in MCF‐7 human breast cancer cells and several other estrogen receptor α (ERα)‐positive human breast cancer cell lines. However, the MDA‐MB‐231 breast cancer cell line, an ERα‐negative cell line widely used in breast cancer research, has been shown to be unresponsive to melatonin's growth‐suppressive effect in vitro. Here, we examined the effect of melatonin on the cell proliferation of several ERα‐negative breast cancer cell lines including MDA‐MB‐231, BT‐20, and SK‐BR‐3 cells. Although the MT1 G‐protein‐coupled receptor is expressed in all three cell lines, melatonin significantly suppressed the proliferation of SK‐BR‐3 cells without having any significant effect on the growth of MDA‐MB‐231 and BT‐20 cells. We confirmed that the MT1‐associated Gα proteins are expressed in MDA‐MB‐231 cells. Further studies demonstrated that the melatonin unresponsiveness in MDA‐MB‐231 cells may be caused by aberrant signaling downstream of the Gαi proteins, resulting in differential regulation of ERK1/2 activity.     

Overexpression of melatonin membrane receptors increases calcium‐binding proteins and protects VSC4.1 motoneurons from glutamate toxicity through multiple mechanisms

Abstract: Melatonin has shown particular promise as a neuroprotective agent to prevent motoneuron death in animal models of both amyotrophic lateral sclerosis (ALS) and spinal cord injuries (SCI). However, an understanding of the roles of endogenous melatonin receptors including MT1, MT2, and orphan G‐protein receptor 50 (GPR50) in neuroprotection is lacking. To address this deficiency, we utilized plasmids for transfection and overexpression of individual melatonin receptors in the ventral spinal cord 4.1 (VSC4.1) motoneuron cell line. Receptor‐mediated cytoprotection following exposure to glutamate at a toxic level (25 μm ) was determined by assessing cell viability, apoptosis, and intracellular free Ca²⁺ levels. Our findings indicate a novel role for MT1 and MT2 for increasing expression of the calcium‐binding proteins calbindin D28K and parvalbumin. Increased levels of calbindin D28K and parvalbumin in VSC4.1 cells overexpressing MT1 and MT2 were associated with cytoprotective effects including inhibition of proapoptotic signaling, downregulation of inflammatory factors, and expression of prosurvival markers. Interestingly, the neuroprotective effects conferred by overexpression of MT1 and/or MT2 were also associated with increases in the estrogen receptor β (ERβ): estrogen receptor α (ERα) ratio and upregulation of angiogenic factors. GPR50 did not exhibit cytoprotective effects. To further confirm the involvement of the melatonin receptors, we silenced both MT1 and MT2 in VSC4.1 cells using RNA interference technology. Knockdown of MT1 and MT2 led to an increase in glutamate toxicity, which was only partially reversed by melatonin treatment. Taken together, our findings suggest that the neuroprotection against glutamate toxicity exhibited by melatonin may depend on MT1 and MT2 but not GPR50.     

Expression of melatonin receptor MT1 in cells of human invasive ductal breast carcinoma

In humans, two main types of membrane melatonin receptors have been identified, MT1 and MT2. Expression of MT1 in neoplastic cells seems to increase the efficacy of melatonin's oncostatic activity. The purpose of this study was to determine the distribution and the intensity of MT1 expression in breast cancer cells and to correlate it with clinicopathological factors. Immunohistochemical studies (IHC) were conducted on 190 cases of invasive ductal breast carcinomas (IDC) and molecular studies were performed on 29 cases of frozen tumor fragments and selected breast cancer cell lines. Most of the studied tumors manifested a membranous/cytoplasmic IHC expression of MT1. In IDC, the MT1 expression was higher than in fibrocystic breast disease. MT1 expression was higher in estrogen receptor positive (ER+) and HER2 positive (HER2+) tumors. Triple negative tumors (TN) manifested the lowest MT1 expression level. The lowest MT1 protein expression level was noted in the TN breast cancer cell line MDA‐MB‐231 compared with ER+ cell lines MCF‐7 and SK‐BR‐3. MT1 mRNA expression was negatively correlated with the malignancy grade of the studied IDC cases. Moreover, higher MT1 expression was associated with patients' longer overall survival (OS) in the group of ER+ breast cancers and treated with tamoxifen. Multivariate analysis indicated that MT1 was an independent prognostic factor in the ER+ tumors for OS and event‐free survival in the ER+ tumors. The results of this study may point to a potential prognostic and therapeutic significance of MT1 in IDC.     

Inverse agonist exposure enhances ligand binding and G protein activation of the human MT1 melatonin receptor, but leads to receptor down-regulation

Melatonin binds and activates G protein-coupled melatonin receptors. The density and affinity of the endogenous melatonin receptors change throughout the 24-hr day, and the exposure of recombinant melatonin receptors to melatonin often results in desensitization of the receptors. Receptor density, G protein activation and expression level were analyzed in CHO cell lines stably expressing the human MT1 receptors after 1 or 72 hr of exposure to melatonin (agonist, 10 nm) and luzindole (antagonist/inverse agonist, 10 microm). The 72-hr exposure to luzindole significantly increased the apparent receptor density in cell lines with both high and low MT1 receptor expression levels (MT1(high) and MT1(low) cells, respectively). In the constitutively active MT1(high) cells, luzindole pretreatment also stimulated the functional response to melatonin in [(35)S]GTPgammaS binding assays, whereas melatonin pretreatment attenuated the functional response at both time points. Receptor ELISA was used to analyze the cell membrane and total expression level of the MT1 receptor in intact and permeabilized cells, respectively. Luzindole pretreatment decreased the total cellular level of MT1 receptor in the MT1(high) cells at both time points but increased the cell surface expression of MT1 receptor at 72 hr. Melatonin significantly decreased MT1 receptor cell surface expression only in MT1(high) cells after a 1-hr treatment. These results indicate that melatonin treatment desensitizes MT1 receptors, whereas luzindole increases ligand binding and G-protein activation. Luzindole also stimulates downregulation of the MT1 receptor protein, interfering with the synthesis and/or degradation of the receptor.     

Placental melatonin system is present throughout pregnancy and regulates villous trophoblast differentiation

Melatonin is highly produced in the placenta where it protects against molecular damage and cellular dysfunction arising from hypoxia/re‐oxygenation‐induced oxidative stress as observed in primary cultures of syncytiotrophoblast. However, little is known about melatonin and its receptors in the human placenta throughout pregnancy and their role in villous trophoblast development. The purpose of this study was to determine melatonin‐synthesizing enzymes, arylalkylamine N‐acetyltransferase (AANAT) and hydroxyindole O‐methyltransferase (HIOMT), and melatonin receptors (MT1 and MT2) expression throughout pregnancy as well as the role of melatonin and its receptors in villous trophoblast syncytialization. Our data show that the melatonin generating system is expressed throughout pregnancy (from week 7 to term) in placental tissues. AANAT and HIOMT show maximal expression at the 3rd trimester of pregnancy. MT1 receptor expression is maximal at the 1st trimester compared to the 2nd and 3rd trimesters, while MT2 receptor expression does not change significantly during pregnancy. Moreover, during primary villous cytotrophoblast syncytialization, MT1 receptor expression increases, while MT2 receptor expression decreases. Treatment of primary villous cytotrophoblast with an increasing concentration of melatonin (10 pm –1 mm ) increases the fusion index (syncytium formation; 21% augmentation at 1 mm melatonin vs. vehicle) and β‐hCG secretion (121% augmentation at 1 mm melatonin vs. vehicle). This effect of melatonin appears to be mediated via its MT1 and MT2 receptors. In sum, melatonin machinery (synthetizing enzymes and receptors) is expressed in human placenta throughout pregnancy and promotes syncytium formation, suggesting an essential role of this indolamine in placental function and pregnancy well‐being.     

Impaired mouse mammary gland growth and development is mediated by melatonin and its MT1 G protein-coupled receptor via repression of ERα, Akt1, and Stat5

To determine whether melatonin, via its MT(1) G protein-coupled receptor, impacts mouse mammary gland development, we generated a mouse mammary tumor virus (MMTV)-MT1-Flag-mammary gland over-expressing (MT1-mOE) transgenic mouse. Increased expression of the MT(1) -Flag transgene was observed in the mammary glands of pubescent MT1-mOE transgenic female mice, with further significant increases during pregnancy and lactation. Mammary gland whole mounts from MT1-mOE mice showed significant reductions in ductal growth, ductal branching, and terminal end bud formation. Elevated MT(1) receptor expression in pregnant and lactating female MT1-mOE mice was associated with reduced lobulo-alveolar development, inhibition of mammary epithelial cell proliferation, and significant reductions in body weights of suckling pups. Elevated MT(1) expression in pregnant and lactating MT1-mOE mice correlated with reduced mammary gland expression of Akt1, phospho-Stat5, Wnt4, estrogen receptor alpha, progesterone receptors A and B, and milk proteins β-casein and whey acidic protein. Estrogen- and progesterone-stimulated mammary gland development was repressed by elevated MT(1) receptor expression and exogenous melatonin administration. These studies demonstrate that the MT(1) melatonin receptor and its ligand melatonin play an important regulatory role in mammary gland development and lactation in mice through both growth suppression and alteration of developmental paradigms.     

Melatonin promotes sleep in mice by inhibiting orexin neurons in the perifornical lateral hypothalamus

Melatonin promotes sleep. However, the underlying mechanisms are unknown. Orexin neurons in the perifornical lateral hypothalamus (PFH ) are pivotal for wake promotion. Does melatonin promote sleep by inhibiting orexin neurons? We used C57BL /6J mice and designed 4 experiments to address this question. Experiment 1 used double‐labeled immunofluorescence and examined the presence of melatonin receptors on orexin neurons. Second, mice, implanted with bilateral guides targeted toward PFH and sleep‐recording electrodes, were infused with melatonin (500 pmole/50 nL/side) at dark onset (onset of active period), and spontaneous bouts of sleep‐wakefulness were examined. Third, mice, implanted with bilateral guides into the PFH , were infused with melatonin (500 pmole/50 nL/side) at dark onset and euthanized 2 hours later, to examine the activation of orexin neurons using c‐Fos expression in orexin neurons. Fourth, mice, implanted with PFH bilateral guides and sleep‐recording electrodes, were infused with melatonin receptor antagonist, luzindole (10 pmol/50 nL/side), at light onset (onset of sleep period), and spontaneous bouts of sleep‐wakefulness were examined. Our results suggest that orexin neurons express MT 1, but not MT 2 receptors. Melatonin infusion into the PFH , at dark onset, site‐specifically and significantly increased NREM sleep (43.7%, P  = .003) and reduced wakefulness (12.3%, P  = .013). Local melatonin infusion at dark onset inhibited orexin neurons as evident by a significant reduction (66%, P  = .0004) in the number of orexin neurons expressing c‐Fos. Finally, luzindole infusion‐induced blockade of melatonin receptors in PFH at sleep onset significantly increased wakefulness (44.1%, P  = .015). Based on these results, we suggest that melatonin may act via the MT 1 receptors to inhibit orexin neurons and promote sleep.     

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 impedes Tet1‐dependent mGluR5 promoter demethylation to relieve pain

Melatonin (N‐acetyl‐5‐methoxytryptamine)/MT2 receptor‐dependent epigenetic modification represents a novel pathway in the treatment of neuropathic pain. Because spinal ten‐eleven translocation methylcytosine dioxygenase 1 (Tet1)‐dependent epigenetic demethylation has recently been linked to pain hypersensitivity, we hypothesized that melatonin/MT2‐dependent analgesia involves spinal Tet1‐dependent demethylation. Here, we showed that spinal Tet1 gene transfer by intrathecal delivery of Tet1‐encoding vectors to naïve rats produced profound and long‐lasting nociceptive hypersensitivity. In addition, enhanced Tet1 expression, Tet1‐metabotropic glutamate receptor subtype 5 (mGluR5) promoter coupling, demethylation at the mGluR5 promoter, and mGluR5 expression in dorsal horn neurons were observed. Rats subjected to spinal nerve ligation and intraplantar complete Freund's adjuvant injection displayed tactile allodynia and behavioral hyperalgesia associated with similar changes in the dorsal horn. Notably, intrathecal melatonin injection reversed the protein expression, protein‐promoter coupling, promoter demethylation, and pain hypersensitivity induced by Tet1 gene transfer, spinal nerve ligation, and intraplantar complete Freund's adjuvant injection. All the effects caused by melatonin were blocked by pretreatment with a MT2 receptor‐selective antagonist. In conclusion, melatonin relieves pain by impeding Tet1‐dependent demethylation of mGluR5 in dorsal horn neurons through the MT2 receptor. Our findings link melatonin/MT2 signaling to Tet1‐dependent epigenetic demethylation of nociceptive genes for the first time and suggest melatonin as a promising therapy for the treatment of pain.     

Inhibiting MT2‐TFE3‐dependent autophagy enhances melatonin‐induced apoptosis in tongue squamous cell carcinoma

Autophagy modulation is a potential therapeutic strategy for tongue squamous cell carcinoma (TSCC). Melatonin possesses significant anticarcinogenic activity. However, whether melatonin induces autophagy and its roles in cell death in TSCC are unclear. Herein, we show that melatonin induced significant apoptosis in the TSCC cell line Cal27. Apart from the induction of apoptosis, we demonstrated that melatonin‐induced autophagic flux in Cal27 cells as evidenced by the formation of GFP‐LC3 puncta, and the upregulation of LC3‐II and downregulation of SQSTM1/P62. Moreover, pharmacological or genetic blockage of autophagy enhanced melatonin‐induced apoptosis, indicating a cytoprotective role of autophagy in melatonin‐treated Cal27 cells. Mechanistically, melatonin induced TFE3^(Ser321) dephosphorylation, subsequently activated TFE3 nuclear translocation, and increased TFE3 reporter activity, which contributed to the expression of autophagy‐related genes and lysosomal biogenesis. Luzindole, a melatonin membrane receptor blocker, or MT2‐siRNA partially blocked the ability of melatonin to promote mTORC1/TFE3 signaling. Furthermore, we verified in a xenograft mouse model that melatonin with hydroxychloroquine or TFE3‐siRNA exerted a synergistic antitumor effect by inhibiting autophagy. Importantly, TFE3 expression positively correlated with TSCC development and poor prognosis in patients. Collectively, we demonstrated that the melatonin‐induced increase in TFE3‐dependent autophagy is mediated through the melatonin membrane receptor in TSCC. These data also suggest that blocking melatonin membrane receptor‐TFE3‐dependent autophagy to enhance the activity of melatonin warrants further attention as a treatment strategy for TSCC.     

Abnormal melatonin receptor 1B expression in osteoblasts from girls with adolescent idiopathic scoliosis

Abstract: Melatonin signaling dysfunction has been associated with the etiology of adolescent idiopathic scoliosis (AIS). Genetic analysis has also associated the occurrence of AIS with the MT2 gene. Thus, we determined whether there is abnormality in the protein expression of melatonin receptors (MT) in AIS osteoblasts. In this study, we recruited 11 girls with severe AIS and eight normal subjects for intraoperative bone biopsies. MT1 and MT2 receptor protein expressions in the isolated osteoblasts were detected. Also, cell proliferation assay using different melatonin concentrations (0, 10^?9, 10^?5, 10^?4 m ) was carried out. The results showed that both MT1 and MT2 receptors are expressed in osteoblasts of the controls. While MT1 receptors were expressed in osteoblasts of all AIS subjects, osteoblasts of only 7 of 11 AIS showed expression of MT2 receptors. Melatonin stimulated control osteoblasts to proliferate. However, proliferation of AIS osteoblasts without expression of MT2 receptor, after treatment with melatonin, was minimal when compared with control and AIS osteoblasts with MT2 receptor expression. The proliferation of AIS osteoblasts with MT2 receptor was greater than those without. This is the first report demonstrating a difference between AIS and normal osteoblasts in the protein expression of MT2 receptor. The results suggest that there is a possible functional effect of MT2 receptor on osteoblast proliferation. AIS osteoblasts without expression of MT2 receptor showed the lowest percentage of viable cells after melatonin treatment. This possibly indicates the modulating role of melatonin through MT2 receptor on the proliferation of osteoblasts.     

Melatonin improves uterine-conceptus interaction via regulation of SIRT1 during early pregnancy

Melatonin has been shown to improve in vitro fertilization and offspring survival after bacterial infection, but its role in regulating maternal-fetal communication during early pregnancy has not been investigated. Results of this study demonstrated expression of abundant melatonin receptors in conceptus and endometrium during early pregnancy. In gilts, expression of melatonin receptor 1A (MTNR1A or MT1) and melatonin receptor 1B (MTNR1B or MT2) increased in trophectoderm (Tr) and uterine luminal epithelium (LE) with advancing days during early pregnancy in a different manner. Melatonin increased proliferation and migration of porcine trophectoderm (pTr) cell, the percent pTr cells in the G2 phase of the cell cycle, and the expression of implantation-related genes by pTr cells and endometrial luminal epithelium (pLE). Melatonin also attenuated the production of LPS-induced pro-inflammatory cytokines and tunicamycin-induced endoplasmic reticulum (ER) stress-sensing proteins. The expression of sirtuin 1 (SIRT1) as a potential target of melatonin increased between Days 9 and 14 of gestation. Co-treatment with SIRT1 inhibitor EX527 and melatonin restored cell-cell interactions through PI3K and MAPK signaling. Knockdown of SIRT1 decreased the expression of implantation-related genes, as well as migration of pTr and pLE cells. The expression of microRNAs regulated by SIRT1 was suppressed in response to melatonin. Furthermore, melatonin significantly increased lipopolysaccharide (LPS)-reduced fertilization and embryogenesis in zebrafish model. These results suggest that melatonin may improve the uterine-conceptus interactions via the regulation of SIRT1 during early pregnancy.     

MT1 and MT2 melatonin receptors are expressed in nonoverlapping neuronal populations

Melatonin (MLT) exerts its physiological effects principally through two high‐affinity membrane receptors MT1 and MT2. Understanding the exact mechanism of MLT action necessitates the use of highly selective agonists/antagonists to stimulate/inhibit a given MLT receptor. The respective distribution of MT1 and MT2 within the CNS and elsewhere is controversial, and here we used a “knock‐in” strategy replacing MT1 or MT2 coding sequences with a LacZ reporter. The data show striking differences in the distribution of MT1 and MT2 receptors in the mouse brain: whereas the MT1 subtype was expressed in very few structures (notably including the suprachiasmatic nucleus and pars tuberalis), MT2 subtype receptors were identified within numerous brain regions including the olfactory bulb, forebrain, hippocampus, amygdala and superior colliculus. Co‐expression of the two subtypes was observed in very few structures, and even within these areas they were rarely present in the same individual cell. In conclusion, the expression and distribution of MT2 receptors are much more widespread than previously thought, and there is virtually no correspondence between MT1 and MT2 cellular expression. The precise phenotyping of cells/neurons containing MT1 or MT2 receptor subtypes opens new perspectives for the characterization of links between MLT brain targets, MLT actions and specific MLT receptor subtypes.     

Melatonin regulates the activities of ovary and delays the fertility decline in female animals via MT1/AMPK pathway

Female fertility irreversibly declines with aging, and this is primarily associated with the decreased quality and quantity of oocytes. To evaluate whether a long‐term of melatonin treatment would improve the fertility of aged mice, different concentrations of melatonin (10^?3, 10^?5, 10^?7 mol/L) were supplemented into drinking water. Melatonin treatments improved the litter sizes of mice at the age of 24 weeks. Mice treated with 10^?5 mol/L melatonin had the largest litter size among other concentrations. At this optimal concentration, melatonin not only significantly increased the total number of oocytes but also their quality, having more oocytes with normal morphology that could generate more blastocyst after in vitro fertilization in melatonin (10^?5 mol/L)‐treated group than that in the controls. When these blastocysts were transferred to recipients, the litter size was also significantly larger in melatonin treated mice than that in controls. The increases in TAOC and SOD level and decreases in MDA were detected in ovaries and uterus from melatonin‐treated mice compared to the controls. Melatonin reduced ROS level and maintained mitochondrial membrane potential in the oocytes cultured in vitro. Mechanistically studies revealed that the beneficial effects of melatonin on oocytes were mediated by MT1 receptor and AMPK pathway. Thereafter, MT1 knocking out (MT1‐KO) were generated and shown significantly reduced number of oocytes and litter size. The expression of SIRT1, C‐myc, and CHOP were downregulated in the ovary of MT1‐KO mice, but SIRT1 and p‐NF‐kB protein level were elevated in response to disturbed redox balance. The results have convincingly proven that melatonin administration delays ovary aging and improves fertility in mice via MT1/AMPK pathway.