title: 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.
abstract: The diuretic ethacrynic acid (EA) has been shown to inhibit signaling by the proinflammatory transcription factor nuclear factor-kappaB (NF-kappaB). 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-kappaB 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-kappaB 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-kappaB 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.
authors.affiliation: Department of Critical Care Medicine, University of Pittsburgh School of MEdicine, 615 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261, USA.
authors.forename: Meaghan E
authors.initials: ME
authors.lastname: Killeen
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Englert
authors.forename: Donna Beer
authors.initials: DB
authors.lastname: Stolz
authors.forename: Mingchen
authors.initials: M
authors.lastname: Song
authors.forename: Yusheng
authors.initials: Y
authors.lastname: Han
authors.forename: Russell L
authors.initials: RL
authors.lastname: Delude
authors.forename: John A
authors.initials: JA
authors.lastname: Kellum
authors.forename: Mitchell P
authors.initials: MP
authors.lastname: Fink
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000818:Animals; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D004976:Ethacrynic Acid / Q000494:pharmacology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D024243:HMGB1 Protein / Q000378:metabolism*; D016753:Interleukin-10 / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D017879:Isothiocyanates; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors*; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D016328:NF-kappa B / Q000502:physiology; D009569:Nitric Oxide / Q000378:metabolism; D010766:Phosphorylation; D011719:Pyrazines / Q000494:pharmacology*; D013454:Sulfoxides; D013861:Thiocyanates / Q000494:pharmacology*; D013871:Thiones; D013876:Thiophenes; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D024243:HMGB1 Protein; D015850:Interleukin-6; D017879:Isothiocyanates; D008070:Lipopolysaccharides; D016328:NF-kappa B; D011719:Pyrazines; D013454:Sulfoxides; D013861:Thiocyanates; D013871:Thiones; D013876:Thiophenes; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D009569:Nitric Oxide; C026209:oltipraz; D004247:DNA; D048049:Extracellular Signal-Regulated MAP Kinases; C016766:sulforaphane; D004976:Ethacrynic Acid