The transition from vegetative to reproductive growth is a key developmental event in a plant's life cycle. The process is mediated by a combination of phytohormones, including melatonin (MT) and strigolactone (SL). Here, the Arabidopsis mutants, d14-1 and max4-1, which are compromised with respect to either SL synthesis or signaling, were shown to flower earlier than wild types. The tissue MT content in both mutants was higher than in wild types, as a result of the up-regulation of various genes encoding enzymes involved in MT synthesis. The abundance in the mutants of transcripts derived from each of the genes SPLs, AP1, and SOC1 was reduced with exogenously supplied MT, while FLC was induced. Plants exposed to a high concentration of MT did not flower earlier than wild types. The tissue MT content of a mutant unable to synthesize caffeic acid O-methyltransferase was less than that of wild type and flowered earlier than did wild types. The suggestion is that the flowering time of Arabidopsis is altered if the tissue content of MT is either higher than ~ 8 ng/g F.W, or lower than ~ 0.9 ng/g. Within this range, SL acts to determine flowering time by its regulation of SPL genes. The application of exogenous SL reduces tissue MT content. The flowering time of the flc-3 mutant was unaffected by exogenously supplying either MT or/and SL. It is proposed that MT acts downstream of SL to activate FLC, inducing a delay to flowering if its concentration lies outside a certain range.
Exercise-induced physiological hypertrophy provides protection against cardiovascular disease, whereas disease-induced pathological hypertrophy leads to heart failure. Emerging evidence suggests pleiotropic roles of melatonin in cardiac disease; however, the effects of melatonin on physiological vs pathological cardiac hypertrophy remain unknown. Using swimming-induced physiological hypertrophy and pressure overload-induced pathological hypertrophy models, we found that melatonin treatment significantly improved pathological hypertrophic responses accompanied by alleviated oxidative stress in myocardium but did not affect physiological cardiac hypertrophy and oxidative stress levels. As an important mediator of melatonin, the retinoid-related orphan nuclear receptor-alpha (ROR alpha) was significantly decreased in human and murine pathological hypertrophic cardiomyocytes, but not in swimming-induced physiological hypertrophic murine hearts. In vivo and in vitro loss-of-function experiments indicated that ROR alpha deficiency significantly aggravated pathological cardiac hypertrophy, and notably weakened the anti-hypertrophic effects of melatonin. Mechanistically, ROR alpha mediated the cardioprotection of melatonin in pathological hypertrophy mainly by transactivation of manganese-dependent superoxide dismutase (MnSOD) via binding to the ROR alpha response element located in the promoter region of the MnSOD gene. Furthermore, MnSOD overexpression reversed the pro-hypertrophic effects of ROR alpha deficiency, while MnSOD silencing abolished the anti-hypertrophic effects of ROR alpha overexpression in pathological cardiac hypertrophy. Collectively, our findings provide the first evidence that melatonin exerts an anti-hypertrophic effect on pathological but not physiological cardiac hypertrophy via alleviating oxidative stress through transactivation of the antioxidant enzyme MnSOD in a ROR alpha-dependent manner.
Brain insulin resistance, induced by neuroinflammation and oxidative stress, contributes to neurodegeneration, that is, processes that are associated with A beta accumulation and TAU hyperphosphorylation. Here, we tested the effect of chronic administration of melatonin (MLT) on brain insulin resistance and cognition deficits caused by a high-fat diet (HFD) in aged rats. Results showed that MLT supplementation attenuated peripheral insulin resistance and lowered hippocampal oxidative stress levels. Activated microglia and astrocytes and hippocampal levels of TNF-alpha in HFD-fed rats were reduced by MLT treatment. Melatonin also prevented HFD-induced increases in beta-amyloid (A beta) accumulation and TAU phosphorylation in the hippocampus. In addition, impairments of brain insulin signaling elicited by long-term HFD were restored by MLT treatment, as confirmed by ex vivo insulin stimulation. Importantly, MLT reversed HFD-induced cognitive decline as measured by a water maze test, normalized hippocampal LTP and restored CREB activity and BDNF levels as well as cholinergic neuronal activity in the hippocampus. Collectively, these findings indicate that MLT may exhibit substantial protective effects on cognition, via restoration of brain insulin signaling.
Histone deacetylase 9 functions as an oncogene in a variety of cancers, but its role on non-small cell lung cancer (NSCLC) has not been reported. Melatonin was proven to possess anticancer actions, whereas its effect on NSCLC and underlying mechanisms remains poorly understood. In this study, 337 patients with complete clinicopathologic characteristics who underwent NSCLC surgery were recruited for the study. We found that NSCLC patients with high HDAC9 expression were correlated with worse overall survival and poor prognosis. HDAC9 knockdown significantly reduced NSCLC cell growth and induced apoptosis both in vivo and in vitro. Melatonin application also markedly inhibited cell proliferation, metastasis, and invasion and promoted apoptosis in NSCLC cells. Moreover, RNA-seq, real-time quantitative polymerase chain reaction, and western blot analyses showed that melatonin treatment decreased the HDAC9 level in NSCLC cells. A mechanistic study revealed that HDAC9 knockdown further enhanced the anticancer activities of melatonin treatment, whereas HDAC9 overexpression partially reversed the melatonin's anticancer effects. Additionally, the in vivo study found melatonin exerted anti-proliferative and pro-apoptotic effects on xenograft tumors which were also strengthened by HDAC9 knockdown. These results indicated that HDAC9 downregulation mediated the anti-NSCLC actions of melatonin, and targeting HDAC9 may be the novel therapeutic strategy for NSCLC.
Melatonin has been shown to reduce oxidative stress and mitigate hypercoagulability. We hypothesized that maternally administered melatonin may reduce placental oxidative stress and hypercoagulability associated with exposure to intrauterine inflammation (IUI) and consequently improve fetoplacental blood flow and fetal sequelae. Mice were randomized to the following groups: control (C), melatonin (M), lipopolysaccharide (LPS; a model of IUI) (L), and LPS with melatonin (ML). The expression of antioxidant mediators in the placenta was significantly decreased, while that of pro-inflammatory mediators was significantly increased in L compared to C and ML. The systolic/diastolic ratio, resistance index, and pulsatility index in uterine artery (UtA) and umbilical artery (UA) were significantly increased in L compared with other groups when analyzed by Doppler ultrasonography. The expression of antioxidant mediators in the placenta was significantly decreased, while that of pro-inflammatory mediators was significantly increased in L compared to C and ML. Vascular endothelial damage and thrombi formation, as evidenced by fibrin deposits, were similarly increased in L compared to other groups. Maternal pretreatment with melatonin appears to modulate maternal placental malperfusion, fetal cardiovascular compromise, and fetal neuroinflammation induced by IUI through its antioxidant properties.
Disruption of circadian time structure and suppression of circadian nocturnal melatonin (MLT) production by exposure to dim light at night (dLAN), as occurs with night shift work and/or disturbed sleep-wake cycles, is associated with a significantly increased risk of breast cancer and resistance to tamoxifen and doxorubicin. Melatonin inhibition of human breast cancer chemoresistance involves mechanisms including suppression of tumor metabolism and inhibition of kinases and transcription factors which are often activated in drug-resistant breast cancer. Signal transducer and activator of transcription 3 (STAT3), frequently overexpressed and activated in paclitaxel (PTX)-resistant breast cancer, promotes the expression of DNA methyltransferase one (DNMT1) to epigenetically suppress the transcription of tumor suppressor Aplasia Ras homolog one (ARHI) which can sequester STAT3 in the cytoplasm to block PTX resistance. We demonstrate that breast tumor xenografts in rats exposed to dLAN and circadian MLT disrupted express elevated levels of phosphorylated and acetylated STAT3, increased DNMT1, but reduced sirtuin 1 (SIRT1) and ARHI. Furthermore, MLT and/or SIRT1 administration blocked/reversed interleukin 6 (IL-6)-induced acetylation of STAT3 and its methylation of ARH1 to increase ARH1 mRNA expression in MCF-7 breast cancer cells. Finally, analyses of the I-SPY 1 trial demonstrate that elevated MT1 receptor expression is significantly correlated with pathologic complete response following neo-adjuvant therapy in breast cancer patients. This is the first study to demonstrate circadian disruption of MLT by dLAN driving intrinsic resistance to PTX via epigenetic mechanisms increasing STAT3 expression and that MLT administration can reestablish sensitivity of breast tumors to PTX and drive tumor regression.
Melanogenesis is a key parameter of differentiation in melanocytes and melanoma cells; therefore, search for factors regulating this pathway are strongly desired. Herein, we investigated the effects of melatonin, a ubiquitous physiological mediator that is found throughout animals and plants. In mammals, the pineal gland secretes this indoleamine into the blood circulation to exert an extensive repertoire of biological activities. Our in vitro assessment indicates an oncostatic capacity of melatonin in time-dependent manner (24, 48, 72 hours) in highly pigmented MNT-1 melanoma cells. The similar pattern of regulation regarding cell viability was observed in amelanotic Sk-Mel-28 cells. Subsequently, MNT-1 cells were tested for the first time for evaluation of melanin/melatonin interaction. Thus primary, electron paramagnetic resonance (EPR) spectroscopy demonstrated that melatonin reduced melanin content. Artificially induced disturbances of melanogenesis by selected inhibitors (N-phenylthiourea or kojic acid) were slightly antagonized by melatonin. Additionally, analysis using transmission electron microscopy has shown that melatonin, particularly at higher dose of 10(-3) mol/L, triggered the appearance of premelanosomes (stage I-II of melanosome) and MNT-1 cells synthesize de novo endogenous melatonin shown by LC-MS. In conclusion, these studies show a melanogenic-like function of melatonin suggesting it as an advantageous agent for treatment of pigmentary disorders.
Disruption of circadian rhythm by means of shift work has been associated with cardiovascular disease in humans. However, causality and underlying mechanisms have not yet been established. In this study, we exposed hyperlipidemic APOE*3-Leiden.CETP mice to either regular light-dark cycles, weekly 6 hours phase advances or delays, or weekly alternating light-dark cycles (12 hours shifts), as a well-established model for shift work. We found that mice exposed to 15 weeks of alternating light-dark cycles displayed a striking increase in atherosclerosis, with an approximately twofold increase in lesion size and severity, while mice exposed to phase advances and delays showed a milder circadian disruption and no significant effect on atherosclerosis development. We observed a higher lesion macrophage content in mice exposed to alternating light-dark cycles without obvious changes in plasma lipids, suggesting involvement of the immune system. Moreover, while no changes in the number or activation status of circulating monocytes and other immune cells were observed, we identified increased markers for inflammation, oxidative stress, and chemoattraction in the vessel wall. Altogether, this is the first study to show that circadian disruption by shifting light-dark cycles directly aggravates atherosclerosis development.
Melatonin has been previously shown to prevent nonalcoholic fatty liver disease (NAFLD), yet the underlying mechanisms are poorly understood. Here, we identified a previously unknown regulatory action of melatonin on apoptosis signal-regulating kinase 1 (ASK1) signaling pathway in the pathogenesis and development of NAFLD. Although melatonin administration did not alter food intake, it significantly alleviated fatty liver phenotypes, including the body weight gain, insulin resistance, hepatic lipid accumulation, steatohepatitis, and fibrosis in a high-fat diet (HFD)-induced NAFLD mouse model (in vivo). The protection of melatonin against NAFLD was not affected by inactivation of Kupffer cell in this model. In NAFLD mice liver, ASK1 signal cascade was substantially activated, evidence by the enhancement of total ASK1, phospho-ASK1, phospho-MKK3/6, phospho-p38, phospho-MKK4/7, and phospho-JNK. Melatonin treatment significantly suppressed the ASK1 upregulation and the phosphorylation of ASK1, MKK3/6, MKK4/7, p38, and JNK. Mechanistically, we found that lipid stress triggered the interaction between ASK1 and TNF receptor-associated factors (TRAFs), including TRAF1, TRAF2, and TRAF6, which resulted in ASK1 deubiquitination and thereby increased ASK1 protein stability. Melatonin did not alter ASK1 mRNA level; however, it activated a scaffold protein beta-arrestin-1 and enabled it to bind to ASK1, which antagonized the TRAFs-mediated ASK1 deubiquitination, and thus reduced ASK1 protein stability. Consistent with these findings, knockout of beta-arrestin-1 in mice partly abolished the protection of melatonin against NAFLD. Taken together, our results for the first time demonstrate that melatonin safeguards against NAFLD by eliminating ASK1 activation via inhibiting TRAFs-mediated ASK1 deubiquitination and stabilization in a beta-arrestin-1 dependent manner.
Fan, C., Pan, Y., Yang, Y., Di, S., Jiang, S., Ma, Z., Li, T., Zhang, Z., Li, W., Li, X., Reiter, R. J. and Yan, X. (2015), HDAC1 inhibition by melatonin leads to suppression of lung adenocarcinoma cells via induction of oxidative stress and activation of apoptotic pathways. J. Pineal Res., 59: 321-333. The above article, first published online in Wiley Online Library (wileyonlinelibrary.com) on 16 July 2015, has been retracted by agreement between the authors, the journal Editor-in-Chief Gianluca Tosini, and John Wiley & Sons Ltd. Since the publication of the above article, it has been brought to the attention of the authors that there are serious errors in the reporting of the data, which renders the conclusions invalid. The authors apologize for any inconvenience the publication of this work may have caused to readers.
Abnormal proliferation and motility of retinal pigment epithelial cells leads to proliferative vitreoretinopathy (PVR). Melatonin is a known effective antitumour and anti-invasive agent, but whether it affects the formation and underlying mechanisms of PVR remains unclear. In this study, the results of the MTT assay, colony formation and propidium iodide (PI) staining with flow cytometry revealed that melatonin dose dependently inhibited epidermal growth factor (EGF)-induced proliferation of human ARPE-19 cells. Furthermore, melatonin reduced EGF-induced motility by suppressing cathepsin S (CTSS) expression. Pretreatment with ZFL (a CTSS inhibitor) or overexpression of CTSS (pCMV-CTSS) significantly inhibited EGF-induced cell motility when combined with melatonin. Epidermal growth factor induced the phosphorylation of AKT(S473)/mTOR (S2448) and transcription factor (c-Jun/Sp1) signaling pathways. Pretreatment of LY294002 (a PI3K inhibitor) or rapamycin (an mTOR inhibitor) markedly reduced EGF-induced motility and p-AKT/p-mTOR/c-Jun/Sp1 expression when combined with melatonin. Taken together, these data indicate that melatonin inhibited EGF-induced proliferation and motility of human ARPE-19 cells by activating the AKT/mTOR pathway, which is dependent on CTSS modulation of c-Jun/Sp1 signalling. Melatonin may be a promising therapeutic drug against PVR.
Homeobox genes generally encode transcription factors involved in regulating developmental processes. In the pineal gland, a brain structure devoted to nocturnal melatonin synthesis, a number of homeobox genes are also expressed postnatally; among these is the LIM homeobox 4 gene (Lhx4). We here report that Lhx4 is specifically expressed in the postnatal pineal gland of rats and humans. Circadian analyses revealed a fourfold rhythm in Lhx4 expression in the rat pineal gland, with rhythmic expression detectable from postnatal day 10. Pineal Lhx4 expression was confirmed to be positively driven by adrenergic signaling, as evidenced by in vivo modulation of Lhx4 expression by pharmacological (isoprenaline injection) and surgical (superior cervical ganglionectomy) interventions. In cultured pinealocytes, Lhx4 expression was upregulated by cyclic AMP, a second messenger of norepinephrine. By use of RNAscope technology, Lhx4 transcripts were found to be exclusively localized in melatonin-synthesizing pinealocytes. This prompted us to investigate the possible role of Lhx4 in regulation of melatonin-producing enzymes. By use of siRNA technology, we knocked down Lhx4 by 95% in cultured pinealocytes; this caused a reduction in transcripts encoding the melatonin-producing enzyme arylalkylamine N-acetyl transferase (Aanat). Screening the transcriptome of siRNA-treated pinealocytes by RNAseq revealed a significant impact of Lhx4 on the phototransduction pathway and on transcripts involved in development of the nervous system and photoreceptors. These data suggest that rhythmic expression of Lhx4 in the pineal gland is controlled via an adrenergic-cyclic AMP mechanism and that Lhx4 acts to promote nocturnal melatonin synthesis.
Previous studies confirmed that melatonin regulates Runx2 expression but the mechanism is unclear. There is a direct interaction between Runx2 and the vitamin D receptor (VDR). Herein, we observed a direct interaction between melatonin and the VDR but not Runx2 using isothermal titration calorimetry. Furthermore, this direct binding was detected only in the C-terminal ligand binding domain (LBD) of the VDR but not in the N-terminal DNA-binding domain (DBD) or the hinge region. Spectrophotometry indicated that melatonin and vitamin D3 (VD3) had similar uptake rates, but melatonin's uptake was significantly inhibited by VD3 until the concentration of melatonin was obviously higher than that of VD3 in a preosteoblastic cell line MC3T3-E1. GST pull-down and yeast two-hybrid assay showed that the interactive smallest fragments were on the 319-379 position of Runx2 and the N-terminus 110-amino acid DBD of the VDR. Electrophoretic mobility shift assay (EMSA) demonstrated that Runx2 facilitated the affinity between the VDR and its specific DNA substrate, which was further documented by a fluorescent EMSA assay where Cy3 labeled Runx2 co-localized with the VDR-DNA complex. Another fluorescent EMSA assay confirmed that the binding of the VDR to Runx2 was significantly enhanced with an increasing concentrations of the VDR, especially in the presence of melatonin; it was further documented using a co-immunoprecipitation assay that this direct interaction was markedly enhanced by melatonin treatment in the MC3T3-E1 cells. Thus, the VDR is a novel melatonin-binding nuclear receptor, and melatonin indirectly regulates Runx2 when it directly binds to the LBD and the DBD of the VDR, respectively.
Characterization of the melatonin (MLT) biosynthesis pathway in plants is still limited. Additionally, a metabolomic analysis of MLT biosynthesis in plants is still a challenge due to analyte structural and chemical diversity, low analyte abundances, and plant matrix complexities. Herein, a sensitive liquid chromatography-mass spectrometry (LC-MS) method enabling the simultaneous determination of seven plant MLT biosynthetic metabolites was developed. In the proposed strategy, the targeted metabolites, which included tryptophan (Trp), tryptamine (TAM), 5-hydroxytryptophan (5HTP), serotonin (5HT), N-acetylserotonin (NAS), 5-methoxytryptamine (5MT), and MLT, were purified from plant extracts using a one-step dispersive solid-phase extraction (DSPE). The samples were then chemically labeled with dansyl chloride (DNS-Cl), followed by analysis using LC-MS. The limit of detection (LOD) values ranged from 0.03 to 1.36 pg/mL and presented a 22- to 469-fold decrease when compared to the unlabeled metabolites. Due to the high sensitivity of the proposed method, the consumption of plant materials was reduced to 10 mg FW. Ultimately, the established method was utilized to examine the distributions of MLT and its intermediates in rice shoots and roots with or without cadmium (Cd) stress. The results suggested that under normal condition, MLT may also be generated via a Trp/TAM/5HT/5MT/MLT path (Pathway II) in addition to the previously reported Trp/TAM/5HT/NAS/MLT path (Pathway I), although Pathway I was shown to be dominant. During Cd stress, MLT was also shown to be produced through these two pathways, with Pathway II shown to be dominant in rice shoots and roots.