Time-dependent modulation of thioredoxin reductase activity might contribute to sulforaphane-mediated inhibition of NF-κB binding to DNA

The chemopreventive agent sulforaphane (SFN) exerts anti-inflammatory activity by thiol-dependent inhibition of nuclear factor kappa B (NF-kappa B) DNA binding. To further analyze the underlying mechanisms, we focused on the thioredoxin/thioredoxin reductase (TrxR) system as a key redox mechanism regulating NF-KB DNA binding. Using cultured Raw 264.7 mouse macrophages as a model, 1-chloro-2,4-dinitrobenzene (CDNB), a known inhibitor of TrxR, was identified as an inhibitor of lipopolysaccharide (LPS)-mediated nitric oxide (NO) production and of NF-KB DNA binding. CDNB and SFN acted synergistically with respect to inhibition of LPS-induced NO release, and we consequently identified SFN as a novel inhibitor of TrxR enzymatic activity in vitro. Short-term treatment of Raw macrophages with SFN or CDNB resulted in the inhibition of TrxR activity in vivo with half-maximal inhibitory concentration of 25.0 +/- 3.5 mu M and 9.4 +/- 3.7 mu M, respectively, whereas after a 24(..)h treatment with 25 mu M SFN, TrxR activity was > 1.5-fold elevated. In additional experiments, we could exclude that inhibition of trans-activating activity of NF-KB contributed to the reduced expression of pro-inflammatory proteins by SFN, based on transient transfection experiments with a (kappa B)(2)-chloramphenicol acetyltransferase construct and a lack of inhibition of. protein kinase A activity. These findings further emphasize the importance of redox modulation or thiol reactivity for the regulation of NF-kappa B-dependent transcription by SFN.