The phase 2 enzyme inducers ethacrynic acid, DL-sulforaphane, and oltipraz inhibit lipopolysaccharide-induced high-mobility group box 1 secretion by RAW 264.7 cells

The diuretic ethacrynic acid ( EA) has been shown to inhibit signaling by the proinflammatory transcription factor nuclear factor-kappa B ( NF-kappa B). Accordingly, we sought to determine whether this compound is capable of inhibiting the release of cytokines [ interleukin ( IL)- 6 and IL- 10] and NO from RAW 264.7 murine macrophage-like cells stimulated with lipopolysaccharide ( LPS). Additionally, we sought to determine whether EA can inhibit secretion of high-mobility group box 1 ( HMGB1), a nuclear protein that is secreted by immunostimulated macrophages and functions in the extracellular milieu as a proinflammatory mediator. In a concentration- dependent manner, EA inhibited secretion of IL- 6, IL- 10, nitric oxide, and HMGB1. As expected, EA inhibited NF-kappa B DNA binding in LPS-stimulated RAW 264.7 cells. Treating these cells with pyrrolidine dithiocarbamate, SN50 ( amino acid sequence AAVALLPAVLLALLAPVQRKRQKLMP) or 5-( thien-3-yl)-3-aminothiophene-2-carboxamide ( SC-514) also inhibited LPS-induced NF-kappa B DNA binding, but these compounds failed to inhibit LPS-induced HMGB1 secretion. These findings suggested that inhibition of HMGB1 secretion by EA might occur via a mechanism unrelated to the NF-kappa B signaling pathway. Because EA is an electrophilic compound that is known to be capable of inducing expression of so-called phase 2 proteins, we sought to determine whether two other phase 2 enzyme inducers, oltipraz and DL-sulforaphane, also are capable of inhibiting HMGB1 release from immunostimulated macrophages. Incubating RAW 264.7 cells with either oltipraz or DL-sulforaphane inhibited LPS- induced HMGB1 secretion. Moreover, both EA and DL-sulforaphane inhibited relocalization of nuclear HMGB1 into the cytoplasm of LPS-stimulated RAW 264.7 cells. These data suggest that phase 2 inducers may exert anti-inflammatory effects by inhibiting secretion of the cytokine-like nuclear protein HMGB1.