Signal transduction pathways involved in protective effects of melatonin in C6 glioma cells

IRCCS Centro Neurolesi Bonino-Pulejo, Messina, Italy.
Journal of Pineal Research (Impact Factor: 7.81). 02/2008; 44(1):78-87. DOI: 10.1111/j.1600-079X.2007.00492.x
Source: PubMed

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.

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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. "
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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 . "
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    • "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 [287] [296] [297]. Similar findings were obtained in the suppression of renal inflammation [298]. "
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