Signal transduction pathways involved in protective effects of melatonin in C6 glioma cells
ABSTRACT Melatonin (N-acetyl-5-methoxytryptamine), an indole hormone, is the chief secretory product of the pineal gland and is an efficient free radical scavenger and antioxidant, both in vitro and in vivo. The role of melatonin as an immunomodulator is, in some cases, contradictory. Although melatonin is reported to influence a variety of inflammatory and immune responses, evidence supporting its effects on important glioma cells-derived mediators is incomplete. We studied, in rat glioma cell line (C6), the role of melatonin (100 microm-1 mm) in the regulation of the expression of nitric oxide synthase (NOS) caused by incubation with lipopolysaccharide (LPS)/interferon (IFN)-gamma (1 microg/mL and 100 U/mL, respectively) and defined the mode of melatonin's action. Treatment with LPS/IFN-gamma for 24 hr elicited the induction of inducible (iNOS) activity as determined by nitrite and nitrate (NO(x)) accumulation in the culture medium. Preincubation with melatonin abrogated the mixed cytokines-mediated induction of iNOS. The effect of melatonin was concentration-dependent. Moreover, Western blot analysis showed that melatonin inhibited LPS/IFN-gamma-induced expression of COX-2 protein, but not that of constitutive cyclooxygenase. Inhibition of iNOS and COX-2 expression was associated with inhibition of activation of the transcription factor nuclear factor kappa B (NF-kappaB). The ability of melatonin to inhibit NF-kappaB activation was further confirmed by studies on the degradation of the inhibitor of NF-kappaB, IkappaB-alpha. Increased production of lipid peroxidation products using thiobarbituric acid assay were found in cellular contents from activated cultures. Lipid peroxidation was decreased by melatonin treatment in a concentration-dependent manner. Moreover, several genes having roles in heat-shock response were downregulated in melatonin-treated cells, such as 70 proteins, reflecting the reduced oxidative stress in these cells. The mechanisms underlying in vitro the neuroprotective properties of melatonin involve modulation of transcription factors and consequent altered gene expression, resulting in downregulation of inflammation.
- SourceAvailable from: Pradip Kumar Kamat
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- "Nitrite estimation was done in the culture medium according to Esposito et al. (2008). Briefly, the nitrite concentration in the samples was measured by the Griess reaction, by adding 100 ll of Griess reagent [0.1 % (w/v) napthylethylenediamine HCl and 1 % (w/v) sulfanilamide in 5 % (v/v) phosphoric acid (vol. "
ABSTRACT: Rotenone causes cytotoxicity in astrocytic cell culture by glial activation, which is linked to free radical generation. The present study is an investigation to explore whether rotenone could also cause cellular toxicity in mouse neuroblastoma cells (Neuro-2a) under treatment similar to astroglial cells. The effect of rotenone (0.1, 1, and 10 μM) on mitochondrial dehydrogenase enzyme activity by MTT reduction assay, PI uptake, total reactive oxygen species (ROS)/superoxide levels, nitrite levels, extent of DNA damage (by comet assay), and nuclear morphological alteration by Hoechst staining was studied. Caspase-3 and Ca(2+)/calmodulin-dependent protein kinase II (CaMKIIα) gene expression was determined to evaluate the apoptotic cell death and calcium kinase, respectively. Calcium level was estimated fluorometrically using fura-2A stain. Rotenone decreased mitochondrial dehydrogenase enzyme activity and generated ROS, superoxide, and nitrite. Rotenone treatment impaired cell intactness and nuclear morphology as depicted by PI uptake and chromosomal condensation of Neuro-2a cells, respectively. In addition, rotenone resulted in increased intracellular Ca(+2) level, caspase-3, and CaMKIIα expression. Furthermore, co-exposure of melatonin (300 μM), an antioxidant to cell culture, significantly suppressed the rotenone-induced decreased mitochondrial dehydrogenase enzyme activity, elevated ROS and RNS. However, melatonin was found ineffective to counteract rotenone-induced increased PI uptake, altered morphological changes, DNA damage, elevated Ca(+2), and increased expression of caspase-3 and CaMKIIα. The study indicates that intracellular calcium rather than oxidative stress is a major factor for rotenone-induced apoptosis in neuronal cells.Archives of Toxicology 04/2012; 86(9):1387-97. DOI:10.1007/s00204-012-0853-z
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- "Recently, by using computer graphics applications, it has been described that melatonin has an excellent steric and electronic effect that is complementary with COX and, therefore, it seems possible that melatonin might bind to the active site of COX-1 and COX-2, modulating the activity of this enzyme (De la Rocha et al, 2007). Anti-inflammatory actions of melatonin, in which this indolamine specifically prevents the activation of the COX-2 enzyme, have also been described in vitro in cultures of C6 glioma cells (Esposito et al, 2008) and in macrophages (Mayo et al, 2005). Melatonin at physiological (1 nM) doses reduced aromatase mRNA steady-state levels both under basal conditions and when aromatase mRNA expression was stimulated by adding PGE 2 . "
ABSTRACT: Melatonin reduces the development of breast cancer interfering with oestrogen-signalling pathways, and also inhibits aromatase activity and expression. Our objective was to study the promoters through which melatonin modifies aromatase expression, evaluate the ability of melatonin to regulate cyclooxygenases and assess whether the effects of melatonin are related to its effects on intracellular cAMP, in MCF-7 cells. Total aromatase mRNA, aromatase mRNA promoter regions and cyclooxygenases mRNA expression were determined by real-time RT-PCR. PGE(2) and cAMP were measured by kits. Melatonin downregulated the gene expression of the two major specific aromatase promoter regions, pII and pI.3, and also that of the aromatase promoter region pI.4. Melatonin 1 nM was able to counteract the stimulatory effect of tetradecanoyl phorbol acetate on PGE(2) production and inhibit COX-2 and COX-1 mRNA expression. Melatonin 1 nM elicited a parallel time-dependent decrease in both cyclic AMP formation and aromatase mRNA expression. This study shows that melatonin inhibits aromatase activity and expression by regulating the gene expression of specific aromatase promoter regions. A possible mechanism for these effects would be the regulation by melatonin of intracellular cAMP levels, mediated by an inhibition of cyclooxygenase activity and expression.British Journal of Cancer 09/2009; 101(9):1613-9. DOI:10.1038/sj.bjc.6605336
The Open Physiology Journal 04/2008; 1(1):1-22. DOI:10.2174/1874360900801010001
- "Some further contributions to the defense in favor of oxidatively, nitrosatively or otherwise challenged cells seem to be related to signaling pathways and transcription factors. In murine macrophages, C6 glioma cells and skeletal muscle, downregulation of inducible NO synthase and, in glioma cells, cyclooxygenase-2, was shown to be associated with prevention of NFB activation   . Similar findings were obtained in the suppression of renal inflammation . "