5-Aza-2′-deoxycytidine stimulates inducible nitric oxide synthase induction in C6 astrocytoma cells

• Published in: Brain research 2004-02, Vol.998 (1), p.83-90
• Main Authors: Cvetkovic, I., Popadic, D., Vuckovic, O., Harhaji, Lj, Miljkovic, Dj, Trajkovic, V.
• Format: Article
• Language: English
• Subjects: 5-Aza-2′-deoxycytidine; Animals; Animals, Newborn; Antimetabolites, Antineoplastic - pharmacology; Astrocyte; Astrocytes - drug effects; Astrocytoma - pathology; Azacitidine - analogs & derivatives; Azacitidine - pharmacology; Biological and medical sciences; Blotting, Northern; Cells, Cultured; Decitabine; DNA Methylation - drug effects; DNA Modification Methylases - antagonists & inhibitors; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Induction - drug effects; Fundamental and applied biological sciences. Psychology; Gene Expression - drug effects; iNOS; Interferon Regulatory Factor-1; Interferons - pharmacology; Interleukin-1 - pharmacology; Isolated neuron and nerve. Neuroglia; Mitogen-Activated Protein Kinase 1 - metabolism; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases - metabolism; Nitric oxide; Nitric Oxide Synthase - biosynthesis; Phosphoproteins - genetics; Phosphoproteins - metabolism; Rats; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA, Messenger - biosynthesis; Time Factors; Tumor Necrosis Factor-alpha - pharmacology; Vertebrates: nervous system and sense organs
• ISSN: 0006-8993; 1872-6240
• DOI: 10.1016/j.brainres.2003.11.014

The influence of a nucleoside analog 5-aza-2′-deoxycytidine (5-AzadC) on inducible nitric oxide synthase (iNOS)-dependent nitric oxide (NO) production in various rat cell types was investigated. In C6 astrocytoma cell line and primary astrocytes, 5-AzadC enhanced proinflammatory cytokine (IFN-γ, TNF-α, IL-1)-triggered NO synthesis in a time- and dose-dependent manner. In contrast, 5-AzadC did not potentiate NO production in IFN-γ-stimulated macrophages, fibroblasts, or endothelial cells. Blockade of transcription or translation in C6 cells abolished the observed effect, suggesting the iNOS gene expression, rather than its catalytic activity, as a target for the drug action. Accordingly, 5-AzadC upregulated IFN-γ-induced expression of iNOS mRNA in C6 astrocytes. The effect of 5-AzadC on astrocyte NO release was blocked by the inhibitor of p44/42 mitogen activated protein kinase-dependent signaling. Finally, the observed stimulatory effect of 5-AzadC on iNOS expression was apparently independent of DNA demethylation, as DNA digestion with methylation-sensitive restriction enzyme HpaII showed that 5-AzadC failed to demethylate cellular DNA in conditions used for iNOS induction.