José L. Mauriz

A review of the molecular aspects of melatonin's anti-inflammatory actions: Recent insights and new perspectives

Abstract:  Melatonin is a highly evolutionary conserved endogenous molecule that is mainly produced by the pineal gland, but also by other nonendocrine organs, of most mammals including man. In the recent years, a variety of anti‐inflammatory and antioxidant effects have been observed when melatonin is applied exogenously under both in vivo and in vitro conditions. A number of studies suggest that this indole may exert its anti‐inflammatory effects through the regulation of different molecular pathways. It has been documented that melatonin inhibits the expression of the isoforms of inducible nitric oxide synthase and cyclooxygenase and limits the production of excessive amounts of nitric oxide, prostanoids, and leukotrienes, as well as other mediators of the inflammatory process such as cytokines, chemokines, and adhesion molecules. Melatonin’s anti‐inflammatory effects are related to the modulation of a number of transcription factors such as nuclear factor kappa B, hypoxia‐inducible factor, nuclear factor erythroid 2‐related factor 2, and others. Melatonin’s effects on the DNA‐binding capacity of transcription factors may be regulated through the inhibition of protein kinases involved in signal transduction, such as mitogen‐activated protein kinases. This review summarizes recent research data focusing on the modulation of the expression of different inflammatory mediators by melatonin and the effects on cell signaling pathways responsible for the indole’s anti‐inflammatory activity. Although there are a numerous published reports that have analyzed melatonin’s anti‐inflammatory properties, further studies are necessary to elucidate its complex regulatory mechanisms in different cellular types and tissues.

Melatonin induces cell cycle arrest and apoptosis in hepatocarcinoma HepG2 cell line

Abstract:  Melatonin reduces proliferation in many different cancer cell lines. However, studies on the oncostatic effects of melatonin in the treatment of hepatocarcinoma are limited. In this study, we examined the effect of melatonin administration on HepG2 human hepatocarcinoma cells, analyzing cell cycle arrest, apoptosis and mitogen‐activated protein kinase (MAPK) signalling pathways. Melatonin was dissolved in the cell culture media in 0.2% dimethyl sulfoxide and administered at different concentrations for 2, 4, 6, 8 and 10 days. Melatonin at concentrations 1000–10,000 μm caused a dose‐ and time‐dependent reduction in cell number. Furthermore, melatonin treatment induced apoptosis with increased caspase‐3 activity and poly(ADP‐ribose) polymerase proteolysis. Proapoptotic effects of melatonin were related to cytosolic cytochrome c release, upregulation of Bax and induction of caspase‐9 activity. Melatonin treatment also resulted in increased caspase‐8 activity, although no significant change was observed in Fas‐L expression. In addition, JNK 1,‐2 and ‐3 and p38, members of the MAPK family, were upregulated by melatonin treatment. Growth inhibition by melatonin altered the percentage or cells in G0–G1 and G2/M phases indicating cell cycle arrest in the G2/M phase. The reduced cell proliferation and alterations of cell cycle were coincident with a significant increase in the expression of p53 and p21 proteins. These novel findings show that melatonin, by inducing cell death and cell cycle arrest, might be useful as adjuvant in hepatocarcinoma therapy.

Inhibition of VEGF expression through blockade of Hif1α and STAT3 signalling mediates the anti-angiogenic effect of melatonin in HepG2 liver cancer cells

Background:Hepatocellular carcinoma (HCC) growth relies on angiogenesis via vascular endothelial growth factor (VEGF) release. Hypoxia within tumour environment leads to intracellular stabilisation of hypoxia inducible factor 1 alpha (Hif1α) and signal transducer and activator of transcription (STAT3). Melatonin induces apoptosis in HCC, and shows anti-angiogenic features in several tumours. In this study, we used human HepG2 liver cancer cells as an in vitro model to investigate the anti-angiogenic effects of melatonin.Methods:HepG2 cells were treated with melatonin under normoxic or CoCl2-induced hypoxia. Gene expression was analysed by RT–qPCR and western blot. Melatonin-induced anti-angiogenic activity was confirmed by in vivo human umbilical vein endothelial cells (HUVECs) tube formation assay. Secreted VEGF was measured by ELISA. Immunofluorescence was performed to analyse Hif1α cellular localisation. Physical interaction between Hif1α and its co-activators was analysed by immunoprecipitation and chromatin immunoprecipitation (ChIP).Results:Melatonin at a pharmacological concentration (1 mM) decreases cellular and secreted VEGF levels, and prevents HUVECs tube formation under hypoxia, associated with a reduction in Hif1α protein expression, nuclear localisation, and transcriptional activity. While hypoxia increases phospho-STAT3, Hif1α, and CBP/p300 recruitment as a transcriptional complex within the VEGF promoter, melatonin 1 mM decreases their physical interaction. Melatonin and the selective STAT3 inhibitor Stattic show a synergic effect on Hif1α, STAT3, and VEGF expression.Conclusion:Melatonin exerts an anti-angiogenic activity in HepG2 cells by interfering with the transcriptional activation of VEGF, via Hif1α and STAT3. Our results provide evidence to consider this indole as a powerful anti-angiogenic agent for HCC treatment.

Effects of Glutamine on Proinflammatory Gene Expression and Activation of Nuclear Factor Kappa B and Signal Transducers and Activators of Transcription in TNBS-induced Colitis

Background: We investigated the effects of glutamine on proinflammatory gene expression and activation of nuclear factor kappa B (NF‐&kgr;B) and signal transducers and activators of transcription (STAT) in a rat model of experimental colitis. Methods: Colitis was induced in male Wistar rats by intracolonic administration of 30 mg of 2,4,6‐trinitrobenzene sulfonic acid (TNBS). Glutamine (25 mg/kg) was given by rectal route daily for 7 days. Results: Glutamine significantly reduced gross damage and histopathological scores and prevented the decrease of anal pressure and the elevated myeloperoxidase activity observed in the colon of animals receiving TNBS. TNBS administration induced a marked increase of vascular cell adhesion molecule (VCAM‐1), inducible nitric oxide synthase (iNOS), and cyclooxygenase‐2 (COX‐2) protein levels. These inflammatory events were associated with increased protein level of NF‐&kgr;B p50 and p65 subunits in the nucleus and significant phosphorylation/degradation of the inhibitor I&kgr;B&agr;. Protein levels of the phosphorylated forms of STAT1, STAT5, and Akt were elevated in animals with colonic damage. All these effects were inhibited by administration of glutamine. Increases in the cytosolic concentration of TBARS and hydroperoxide‐initiated chemiluminescence, markers of oxidative stress, and levels of tumor necrosis factor &agr; (TNF&agr;) and interferon &ggr; (IFN&ggr;) were significantly inhibited at 48 hours of TNBS instillation in glutamine‐treated animals. Conclusions: Inhibition of the expression of proinflammatory mediators that are regulated by the NF‐&kgr;B and STAT signaling pathways contribute to the therapeutical effect of glutamine in the TNBS model of experimental colitis. These effects may be brought about by inhibition of oxidative stress and reduced expression of proinflammatory cytokines.

Melatonin and endoplasmic reticulum stress: relation to autophagy and apoptosis.

Endoplasmic reticulum (ER) is a dynamic organelle that participates in a number of cellular functions by controlling lipid metabolism, calcium stores, and proteostasis. Under stressful situations, the ER environment is compromised, and protein maturation is impaired; this causes misfolded proteins to accumulate and a characteristic stress response named unfolded protein response (UPR). UPR protects cells from stress and contributes to cellular homeostasis re‐establishment; however, during prolonged ER stress, UPR activation promotes cell death. ER stressors can modulate autophagy which in turn, depending of the situation, induces cell survival or death. Interactions of different autophagy‐ and apoptosis‐related proteins and also common signaling pathways have been found, suggesting an interplay between these cellular processes, although their dynamic features are still unknown. A number of pathologies including metabolic, neurodegenerative and cardiovascular diseases, cancer, inflammation, and viral infections are associated with ER stress, leading to a growing interest in targeting components of the UPR as a therapeutic strategy. Melatonin has a variety of antioxidant, anti‐inflammatory, and antitumor effects. As such, it modulates apoptosis and autophagy in cancer cells, neurodegeneration and the development of liver diseases as well as other pathologies. Here, we review the effects of melatonin on the main ER stress mechanisms, focusing on its ability to regulate the autophagic and apoptotic processes. As the number of studies that have analyzed ER stress modulation by this indole remains limited, further research is necessary for a better understanding of the crosstalk between ER stress, autophagy, and apoptosis and to clearly delineate the mechanisms by which melatonin modulates these responses.

Dietary glycine inhibits activation of nuclear factor kappa B and prevents liver injury in hemorrhagic shock in the rat

Abstract We investigated the effects of a glycine-containing diet (5%) on liver injury caused by hemorrhagic shock and resuscitation in rats. Anesthetized rats were bled to a mean arterial blood pressure of 35–40 mm Hg for 1 h and then resuscitated with 60% of shed blood and lactated Ringer’s solution. Feeding the rats glycine significantly reduced mortality, the elevation of plasma transaminase levels and hepatic necrosis. The increase in plasma TNFα and nitric oxide (NO) was also blunted by glycine feeding. Hemorrhagic shock resulted in oxidative stress (significant elevations in TBARS and in the oxidized/reduced glutathione ratio) and was accompanied by a reduced activity of the antioxidant enzymes Mn- and Cu,Zn-superoxide dismutase, glutathione peroxidase and catalase, overexpression of inducible NO synthase (iNOS), and activation of nuclear factor kappa B (NF-κB). Glycine ameliorated oxidative stress and the impairment in antioxidant enzyme activities, inhibited NF-κB activation, and prevented expression of iNOS. Dietary glycine blocks activation of different mediators involved in the pathophysiology of liver injury after shock.

Melatonin prevents oxidative stress and changes in antioxidant enzyme expression and activity in the liver of aging rats

Abstract:  This study compared the effects of melatonin supplementation on markers of oxidative stress, and on the activity and expression of antioxidant enzymes in the liver of young (3‐month‐old) and aging (24‐month‐old) rats. Animals were supplemented with melatonin in the drinking water (20 mg/L) for 4 wk. Liver concentration of thiobarbituric‐reactive substances (TBARS), as an index of lipid peroxidation, and the oxidized to reduced glutathione ratio significantly increased in aged rats (+58%), while values did not significantly differ from the young in aged animals receiving melatonin. Significant decreases in the liver activities of Cu,Zn‐superoxide dismutase (SOD) (−25%), cytosolic (−21%) and mitochondrial (−40%) glutathione peroxidase (GPx), and catalase (CAT) (−34%) were found in aged rats. Melatonin abolished these changes and also prevented the reduction of Cu,Zn‐SOD (−33%), cytosolic GPx (−30%), and mitochondrial GPx (−47%) liver protein content as measured by Western blot. Reductions in Cu,Zn‐SOD mRNA (−39%), and GPx mRNA (−86%) levels induced by aging were also abolished by melatonin. In summary, our data indicate that melatonin treatment abrogates oxidative stress in the liver of aged rats, and that prevention of the decreased activity of CAT and the downregulation of Cu,Zn‐SOD and GPx gene expression contribute to this effect.

Inhibition of matrix metalloproteinase‐9 and nuclear factor kappa B contribute to melatonin prevention of motility and invasiveness in HepG2 liver cancer cells

Hepatocellular carcinoma (HCC) is one of the most lethal human cancers worldwide because of its high incidence and its metastatic potential. Extracellular matrix degradation by matrix metalloproteinases (MMPs) has been connected with cancer cell invasion, and it has been suggested that inhibition of MMPs by synthetic and natural inhibitors may be of great importance in the HCC therapies. Melatonin, the main product of the pineal gland, exerts antiproliferative, proapoptotic, and antiangiogenic properties in HepG2 human hepatocellular cells, and exhibits anti‐invasive and antimetastatic activities by suppressing the enzymatic activity of MMP‐9 in different tumor types. However, the underlying mechanism of anti‐invasive activity in HCC models has not been fully elucidated. Here, we demonstrate that 1 mm melatonin dosage reduced in IL‐1β‐induced HepG2 cells MMP‐9 gelatinase activity and inhibited cell invasion and motility through downregulation of MMP‐9 gene expression and upregulation of the MMP‐9‐specific inhibitor tissue inhibitor of metalloproteinases (TIMP)‐1. No significant changes were observed in the expression and activity of MMP‐2, the other proteinase implicated in matrix collagen degradation, and its tissue inhibitor, TIMP‐2. Also, melatonin significantly suppressed IL‐1β‐induced nuclear factor‐kappaB (NF‐κB) translocation and transcriptional activity. In summary, we demonstrate that melatonin modulates motility and invasiveness of HepG2 cell in vitro through a molecular mechanism that involves TIMP‐1 upregulation and attenuation of MMP‐9 expression and activity via NF‐κB signal pathway inhibition.

Melatonin modulation of intracellular signaling pathways in hepatocarcinoma HepG2 cell line: role of the MT1 receptor.

Abstract:  Melatonin reduces proliferation in many different cancer cell lines. However, studies on the oncostatic effects of melatonin in hepatocarcinoma are limited. We have previously demonstrated that melatonin administration induces cycle arrest, apoptosis, and changes in the expression of its specific receptors in HepG2 human hepatocarcinoma cells. In this study, we used the receptor antagonist luzindole to assess the contribution of MT1 melatonin membrane receptor to melatonin effects on cell viability, mitogen‐activated protein kinase (MAPKs) activation, and cAMP levels. Additionally, effects of MT1 inhibition on mRNA levels of cytosolic quinone reductase type‐2 (NQO2) receptor and nuclear retinoic acid‐related orphan receptor alpha (RORα) were tested. Melatonin, at 1000 and 2500 μm, significantly reduced cell viability. Pre‐incubation with luzindole partially inhibited the effects of melatonin on cell viability. Melatonin at 2500 μm significantly reduced cAMP levels, and this effect was partially blocked by luzindole. Both melatonin concentrations increased the expression of phosphorylated p38, ERK, and JNK. ERK activation was completely abolished in the presence of luzindole. NQO2 but not RORα mRNA level significantly increased in luzindole‐treated cells. Results obtained provide evidence that the melatonin effects on cell viability and proliferation in HepG2 cells are partially mediated through the MT1 membrane receptor, which seems to be related also with melatonin modulation of cAMP and ERK activation. This study also highlights a possible interplay between MT1 and NQO2 melatonin receptors in liver cancer cells.

Caspase inhibition does not protect against liver damage in hemorrhagic shock

This study was aimed to determine whether administration of an inhibitor of caspase-3 protects hepatocellular function in rats with hemorrhagic shock and whether caspases are important pharmacological targets in attenuating liver injury induced by hemorrhagic shock and resuscitation. Male adult rats were subjected to hemorrhagic shock by bleeding to a mean arterial blood pressure of 35-40 mmHg for 1 h and were then resuscitation with 60% shed blood and lactated Ringers solution. A subgroup of animals was injected i.v. with 2 mg/kg caspase inhibitor, Z-DEVD-FMK, prior to blood withdrawal. Fas ligand expression was markedly elevated and caspase-3 activity increased by 3-fold in hemorrhagic untreated rats. The increase in caspase-3 activity was prevented by administration of Z-DEVD-FMK prior to shock and resuscitation. Poly (adenosine diphosphate ribose) polymerase proteolysis was reduced in rats treated with the caspase-3 inhibitor compared with hemorrhagic untreated animals. Plasma aspartate aminotransferase and alanine aminotransferase values showed a significant increase at 6 h of shock in untreated animals (+360% and +515% as compared with sham-operated animals, respectively). Administration of the caspase-3 inhibitor did not prevent the increase in plasma transaminases. The cytosolic concentration of thiobarbituric acid-reactive substances (TBARS) and the oxidized:reduced glutathione ratio increased in the animals with hemorrhagic shock (+94% and +170%, respectively). These parameters were not significantly modified by pretreatment with Z-DEVD-FMK. It appears that caspase inhibition does not attenuate hepatocellular depression and liver injury induced by hemorrhagic shock and resuscitation.