Melatonin overcomes gemcitabine resistance in pancreatic ductal adenocarcinoma by abrogating nuclear factor‐κB activation

Constitutive activation and gemcitabine induction of nuclear factor‐κB (NF‐κB) contribute to the aggressive behavior and chemotherapeutic resistance of pancreatic ductal adenocarcinoma (PDAC). Thus, targeting the NF‐κB pathway has proven an insurmountable challenge for PDAC therapy. In this study, we investigated whether the inhibition of NF‐κB signaling pathway by melatonin might lead to tumor suppression and overcome gemcitabine resistance in pancreatic tumors. Our results showed that melatonin inhibited activities of NF‐κB by suppressing IκBα phosphorylation and decreased the expression of NF‐κB response genes in MiaPaCa‐2, AsPc‐1, Panc‐28 cells and gemcitabine resistance MiaPaCa‐2/GR cells. Moreover, melatonin not only inhibited cell proliferation and invasion in a receptor‐independent manner, but also enhanced gemcitabine cytotoxicity at pharmacologic concentrations in these PDAC cells. In vivo, the mice treated with both agents experienced a larger reduction in tumor burden than the single drug‐treated groups in an orthotopic xenograft mouse model. Taken together, these results indicate that melatonin inhibits proliferation and invasion of PDAC cells and overcomes gemcitabine resistance of pancreatic tumors through NF‐κB inhibition. Our findings therefore provide novel preclinical knowledge about melatonin inhibition of NF‐κB in PDAC and suggest that melatonin should be investigated clinically, alone or in combination with gemcitabine for PDAC treatment.     

Corticosterone modulates noradrenaline-induced melatonin synthesis through inhibition of nuclear factor kappa B

In chronically inflamed animals, adrenal hormones exert a positive control on the secretion of melatonin by the pineal gland. In this paper, the mechanism of corticosterone as a modulator of melatonin and N-acetylserotonin (NAS) was determined. Rat pineal glands in culture, stimulated for 5 hr with noradrenaline (10 nm), were previously incubated with corticosterone (1.0 nm-1.0 microm) for 48 hr in the presence or absence of the glucocorticoid receptor (GR) antagonist, mifepristone (1.0 microm), the proteasome inhibitor, N-acetyl-leucinyl-leucinyl-norleucinal-H (ALLN, 12.5 microm) or the antagonist of the nuclear factor kappa B (NFkappaB), pyrrolidinedithiocarbamate (PDTC, 12.5 microm). Corticosterone potentiated noradrenaline-induced melatonin and NAS production in a bell-shaped manner. The increase in NAS (12.9 +/- 2.7, n=6 versus 34.3 +/- 8.3 ng per pineal) and melatonin (16.3 +/- 2.0, n=6 versus 44.3 +/- 12.9 ng per pineal) content induced by 1 microm corticosterone was blocked by mifepristone, and mimicked by ALLN and PDTC. The presence of GRs was shown by [3H]-dexamethasone binding (0.30 +/- 0.09 pmol/mg protein) and corticosterone inhibition of NFkappaB nuclear translocation was demonstrated by electromobility shift assay. Therefore, corticosterone potentiates noradrenaline-induced melatonin and NAS production through GR inhibition of NFkappaB nuclear translocation. To the best of our knowledge, this is the first time that this relevant pathway for passive and acquired immune response is shown to modulate melatonin production in pineal gland.     

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.     

Melatonin inhibits matrix metalloproteinase-9 (MMP-9) activation in the lipopolysaccharide (LPS)-stimulated RAW 264.7 and BV2 cells and a mouse model of meningitis

Abstract: We explored anti‐inflammatory potential of melatonin against the lipopolysaccharide (LPS)‐induced inflammation in vivo and in vitro. RAW 264.7 and BV2 cells were stimulated by LPS, followed by the treatment with melatonin or vehicle at various time intervals. In a mouse model of meningitis induced by LPS, melatonin (5 mg/kg) or vehicle was intravenously injected at 30 min postinsult. The activity of matrix metalloproteinase‐2 (MMP‐2) and metalloproteinase‐9 (MMP‐9) was determined by gelatin zymography. Nuclear factor‐kappa B (NFκB) translocation and binding activity were determined by immunocytochemistry and electrophoretic mobility shift assay (EMSA). Our results showed that either pretreatment or cotreatment with melatonin at 50–500 μm effectively inhibited the LPS‐induced proMMP‐9 activation in the RAW 264.7 and BV2 cells, respectively (P < 0.05). This melatonin‐induced proMMP‐9 inhibition remained effective when treatment was delayed up to 2 and 6 hr postinsult for RAW 264.7 and BV2 cells, respectively (P < 0.05 for both groups). Additionally, melatonin significantly attenuated the rises of circulatory and cerebral MMP‐9 activity, respectively (P < 0.05) and reduced the loss of body weight (P < 0.05) in mice with meningitis. Moreover, melatonin (50 μm ) effectively inhibited nuclear factor‐kappa B (NFκB) translocation and binding activity in the LPS‐treated RAW 264.7 and BV2 cells, respectively (P < 0.05). These results demonstrate direct inhibitory actions of melatonin against postinflammatory NFκB translocation and MMP‐9 activation and highlight its ability to inhibit systemic and cerebral MMP‐9 activation following brain inflammation.     

B‐Lymphocyte stimulator: a new biomarker for multiple myeloma

Multiple myeloma (MM) is a common malignant tumor, characterized by unlimited proliferation of abnormal plasmocytes in bone marrow. Considering the biological function of B‐Lymphocyte stimulator (BLyS) and its receptors in B cell, we examined BLyS and its receptors expression in MM cells. Our studies confirmed that BLyS and its receptors are expressed in MM cells, including KM3, CZ‐1, and primary MM cells, playing an important role in the survival and proliferation of MM cells. Additionally, we provide evidence that BLyS protein is located in the MM cell plasma membrane. We also found that IFN‐γ and IL‐6 can induce BLyS expression on MM cells, while after the treatment of BAY11‐7082, an IkB‐α phosphorylation inhibitor, IFN‐γ induced up regulation of BLyS was completely inhibited, suggesting that nuclear factor κB (NF‐κB) might be involved in the mechanism of the regulation of BLyS levels in response to cytokines. Finally, linear correlation analysis of the Lactate Dehydrogenase concentration and beta 2‐microglobulin level with BLyS, and expressions of BLyS mRNA in MM patients revealed a significant correlation between them (P < 0.01 in all case), showing that BLyS could be a biomarker for the diagnosis and treatment of MM.     

Melatonin inhibits the sphingosine kinase 1/sphingosine‐1‐phosphate signaling pathway in rabbits with fulminant hepatitis of viral origin

The sphingosine kinase (SphK)1/sphingosine‐1‐phosphate (S1P) pathway is involved in multiple biological processes, including liver diseases. This study investigate whether modulation of the SphK1/S1P system associates to the beneficial effects of melatonin in an animal model of acute liver failure (ALF) induced by the rabbit hemorrhagic disease virus (RHDV). Rabbits were experimentally infected with 2 × 10⁴ hemagglutination units of a RHDV isolate and received 20 mg/kg of melatonin at 0, 12, and 24 hr postinfection. Liver mRNA levels, protein concentration, and immunohistochemical labeling for SphK1 increased in RHDV‐infected rabbits. S1P production and protein expression of the S1PR1 receptor were significantly elevated following RHDV infection. These effects were significantly reduced by melatonin. Rabbits also exhibited increased expression of toll‐like receptor (TLR)4, tumor necrosis factor alpha (TNF‐α), interleukin (IL)‐6, nuclear factor‐kappa B (NF‐κB) p50 and p65 subunits, and phosphorylated inhibitor of kappa B (IκB)α. Melatonin administration significantly inhibited those changes and induced a decreased immunoreactivity for RHDV viral VP60 antigen in the liver. Results obtained indicate that the SphK1/S1P system activates in parallel to viral replication and the inflammatory process induced by the virus. Inhibition of the lipid signaling pathway by the indole reveals novel molecular pathways that may account for the protective effect of melatonin in this animal model of ALF, and supports the potential of melatonin as an antiviral agent.     

Enhancement of high glucose‐induced PINK1 expression by melatonin stimulates neuronal cell survival: Involvement of MT2/Akt/NF‐κB pathway

Hyperglycemia is a representative hallmark and risk factor for diabetes mellitus (DM) and is closely linked to DM‐associated neuronal cell death. Previous investigators reported on a genome‐wide association study and showed relationships between DM and melatonin receptor (MT), highlighting the role of MT signaling by assessing melatonin in DM. However, the role of MT signaling in DM pathogenesis is unclear. Therefore, we investigated the role of mitophagy regulators in high glucose‐induced neuronal cell death and the effect of melatonin against high glucose‐induced mitophagy regulators in neuronal cells. In our results, high glucose significantly increased PTEN‐induced putative kinase 1 (PINK1) and LC‐3B expressions; as well it decreased cytochrome c oxidase subunit 4 expression and Mitotracker? fluorescence intensity. Silencing of PINK1 induced mitochondrial reactive oxygen species (ROS) accumulation and mitochondrial membrane potential impairment, increased expressions of cleaved caspases, and increased the number of annexin V‐positive cells. In addition, high glucose‐stimulated melatonin receptor 1B (MTNR1B) mRNA and PINK1 expressions were reversed by ROS scavenger N‐acetyl cysteine pretreatment. Upregulation of PINK1 expression in neuronal cells is suppressed by pretreatment with MT₂ receptor‐specific inhibitor 4‐P‐PDOT. We further showed melatonin stimulated Akt phosphorylation, which was followed by nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) phosphorylation and nuclear translocation. Silencing of PINK1 expression abolished melatonin‐regulated mitochondrial ROS production, cleaved caspase‐3 and caspase‐9 expressions, and the number of annexin V‐positive cells. In conclusion, we have demonstrated the melatonin stimulates PINK1 expression via an MT₂/Akt/NF‐κB pathway, and such stimulation is important for the prevention of neuronal cell apoptosis under high glucose conditions.     

Subfunctionalization of arylalkylamine N‐acetyltransferases in the sea bass Dicentrarchus labrax: two‐ones for one two

Melatonin is an important component of the vertebrates circadian system, synthetized from serotonin by the successive action of the arylalkylamine N‐acetyltransferase (Aanat: serotonin→N‐acetylserotonin) and acetylserotonin‐O‐methyltransferase (Asmt: N‐acetylserotonin→melatonin). Aanat is responsible for the daily rhythm in melatonin production. Teleost fish are unique because they express two Aanat genes, aanat1 and aanat2, mainly expressed in the retina and pineal gland, respectively. In silico analysis indicated that the teleost‐specific whole‐genome duplication generated Aanat1 duplicates (aanat1a and aanat1b); some fish express both of them, while others express either one of the isoforms. Here, we bring the first information on the structure, function, and distribution of Aanat1a and Aanat1b in a teleost, the sea bass Dicentrarchus labrax. Aanat1a and Aanat1b displayed a wide and distinct distribution in the nervous system and peripheral tissues, while Aanat2 appeared as a pineal enzyme. Co‐expression of Aanats with asmt was found in the pineal gland and the three retinal nuclear layers. Enzyme kinetics indicated subtle differences in the affinity and catalytic efficiency of Aanat1a and Aanat1b for indolethylamines and phenylethylamines, respectively. Our data are consistent with the idea that Aanat2 is a pineal enzyme involved in melatonin production, while Aanat1 enzymes have a broader range of functions including melatonin synthesis in the retina, and catabolism of serotonin and dopamine in the retina and other tissues. The data are discussed in light of the recently uncovered roles of N‐acetylserotonin and N‐acetyldopamine as antioxidants, neuroprotectants, and modulators of cell proliferation and enzyme activities.     

A congenic line of the C57BL/6J mouse strain that is proficient in melatonin synthesis

The C57BL/6J (B6) is the most common inbred mouse strain used in biomedical research in the United States. Yet, this strain is notoriously known for being deficient in the biosynthesis of melatonin, an important effector of circadian clocks in the brain and in the retina. Melatonin deficiency in this strain results from nonfunctional alleles of the genes coding 2 key enzymes of the melatonin synthesis pathway: arylalkylamine‐N‐acetyltransferase (Aanat) and N‐acetylserotonin‐O‐methyltransferase (Asmt). By introducing functional alleles of the Aanat and Asmt genes from the melatonin‐proficient CBA/CaJ (CBA) mouse strain to B6, we have generated a B6 congenic line that has acquired the capacity of rhythmic melatonin synthesis. In addition, the melatonin‐dependent rhythm of dopamine release in the retina is restored in the B6 congenic line. Finally, we have partially characterized the Aanat and Asmt genes of the CBA strain and have identified multiple differences between CBA and B6 alleles, including single nucleotide polymorphism and deletion/insertion of DNA segments of various sizes. As an improved model organism with functional components of the melatonin synthesis pathway and melatonin‐dependent circadian regulations, the new line will be useful to researchers studying melatonin physiological functions in a variety of fields including, but not limited to, circadian biology and neuroscience. In particular, the congenic line will be useful to speed up introduction of melatonin production capacity into genetically modified mouse lines of interest such as knockout lines, many of which are on B6 or mixed B6 backgrounds. The melatonin‐proficient B6 congenic line will be widely distributed.     

Phosphorylation of extracellular signal‐regulated kinase 1/2, p38 mitogen‐activated protein kinase and nuclear translocation of nuclear factor‐κB are involved in upregulation of matrix metalloproteinase‐9 by tumour necrosis factor‐α

Background: Upregulation of matrix metalloproteinase‐9 (MMP‐9) induced by tumour necrosis factor‐α (TNF‐α) is reportedly involved in a variety of non‐neoplastic and neoplastic diseases. In this study, we examined which signalling pathways are involved in TNF‐α‐induced MMP‐9 upregulation in cholangiocarcinoma (CC). Methods: We used two CC cell lines: HuCCT‐1 and CCKS‐1. Results: In an ex vivo study using HuCCT‐1 and CCKS‐1 cells, TNF‐α treatment induced MMP‐9 production and activation via interaction with TNF receptor‐1 (TNF‐R1) but not with TNF receptor‐2 (TNF‐R2), shown by zymography, and increased MMP‐9 promoter activity (luciferase assay). As for the signalling pathway, TNF‐α stimulation led to the phosphorylation of extracellular signal‐regulated kinase 1/2 (Erk1/2) and p38 mitogen‐activated protein kinase (p38MAPK) and translocation of nuclear factor κB (NF‐κB) (p65) into the nuclei. Inhibition studies using SB203580 (inhibitor of p38MAPK), U0126 (inhibitor of mitogen‐activated or extracellular signal‐regulated protein kinase 1/2) and MG132 (inhibitor of NF‐κB) showed that the phosphorylation of Erk1/2 and p38MAPK with activation of NF‐κB was closely related to MMP‐9 upregulation in both cell lines. Conclusion: These data suggest that TNF‐α/TNF‐R1 interaction leads to the phosphorylation of Erk1/2 and p38MAPK and nuclear translocation of NF‐κB, which is closely associated with the production and activation of MMP‐9 in cultured CC cells of HuCTT‐1 and CCKS‐1. Upregulation of MMP‐9 with NF‐κB activation may be involved in the tumour invasion of CC.