Effect of 4-trifluoromethyl derivatives of salicylate on nuclear factor kappaB-dependent transcription in human astrocytoma cells.
1. The effect of two derivatives of salicylate, 2-hydroxy-4-trifluoromethylbenzoic acid (HTB) and 2-acetoxy-4-trifluoromethylbenzoic acid (triflusal), on the expression of several proteins displaying pro-inflammatory activities the regulation of which is associated to the transcription factor NF-kappaB, was assayed in the human astrocytoma cell line 1321N1. 2. Tumour necrosis factor-alpha (TNF-alpha) activated NF-kappaB as judged from both the appearance of kappaB-binding activity in the nuclear extracts, the degradation of IkappaB proteins in the cell lysates, and the activation of IkappaB kinases using an immunocomplex kinase assay with glutathione S-transferase (GST)-IkappaB proteins as substrates. 3. HTB up to 3 mM did not inhibit the nuclear translocation of NK-kappaB/Rel proteins as judged from electrophoretic mobility-shift assays; however, HTB inhibited the degradation of IkappaBbeta without significantly affecting the degradation of both IkappaBalpha and IkappaBepsilon. 4. In keeping with their inhibitory effect on IkappaBbeta degradation in the cell lysates, both HTB and triflusal inhibited the phosphorylation of GST-IkappaBbeta elicited by TNF-alpha, without affecting the phosphorylation of GST-IkappaBalpha. 5. The effect of both HTB and triflusal on kappaB-dependent trans-activation was studied by assaying the expression of both cyclo-oxygenase-2 (COX-2) and vascular cell adhesion molecule-1 (VCAM-1). HTB and triflusal inhibited in a dose-dependent manner the expression of COX-2 and VCAM-1 mRNA and the induction of COX-2 protein at therapeutically relevant concentrations. 6. These findings show the complexity of the biochemical mechanisms underlying the activation of NF-kappaB in the different cell types and extend the anti-inflammatory effects of HTB and triflusal to neural cells.
Hernández
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Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas, 47005-Valladolid, Spain.
de Arriba
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Merlos
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Fuentes
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Crespo
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Nieto
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D001254:Astrocytoma / Q000378:metabolism*; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000378:metabolism*; D051546:Cyclooxygenase 2; D018076:DNA, Complementary / Q000096:biosynthesis; D004586:Electrophoresis; D006801:Humans; D007202:Indicators and Reagents; D007527:Isoenzymes / Q000096:biosynthesis; D008565:Membrane Proteins; D016328:NF-kappa B / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000096:biosynthesis; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012459:Salicylates / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D019010:Vascular Cell Adhesion Molecule-1 / Q000096:biosynthesis
D018076:DNA, Complementary; D007202:Indicators and Reagents; D007527:Isoenzymes; D008565:Membrane Proteins; D016328:NF-kappa B; D009363:Neoplasm Proteins; D012459:Salicylates; D019010:Vascular Cell Adhesion Molecule-1; C016167:triflusal; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases