The chemopreventive agent sulforaphane (SFN) exerts anti-inflammatory activity by thiol-dependent inhibition of nuclear factor κB (NF-κB) DNA binding. To further analyze the underlying mechanisms, we focused on the thioredoxin/thioredoxin reductase (TrxR) system as a key redox mechanism regulating NF-κB 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-κB 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 activityin 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 µM and 9.4 ± 3.7 µM, respectively, whereas after a 24-h treatment with 25 µM SFN, TrxR activity was >1.5-fold elevated. In additional experiments, we could exclude that inhibition of trans-activating activity of NF-κB contributed to the reduced expression of pro-inflammatory proteins by SFN, based on transient transfection experiments with a (κB)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-κB-dependent transcription by SFN.
Antioxid. Redox Signal. 7, 1601–1611.
