Effect of 4-trifluoromethyl derivatives of salicylate on nuclear factor κB-dependent transcription in human astrocytoma cells

Abstract

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-κB, was assayed in the human astrocytoma cell line 1321N1. Tumour necrosis factor-α (TNF-α) activated NF-κB as judged from both the appearance of κB-binding activity in the nuclear extracts, the degradation of IκB proteins in the cell lysates, and the activation of IκB kinases using an immunocomplex kinase assay with glutathione S-transferase (GST)-IκB proteins as substrates. HTB up to 3 mM did not inhibit the nuclear translocation of NK-κB/Rel proteins as judged from electrophoretic mobility-shift assays; however, HTB inhibited the degradation of IκBβ without significantly affecting the degradation of both IκBα and IκBε. In keeping with their inhibitory effect on IκBβ degradation in the cell lysates, both HTB and triflusal inhibited the phosphorylation of GST-IκBβ elicited by TNF-α, without affecting the phosphorylation of GST-IκBα. The effect of both HTB and triflusal on κB-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. These findings show the complexity of the biochemical mechanisms underlying the activation of NF-κB in the different cell types and extend the anti-inflammatory effects of HTB and triflusal to neural cells.