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

J Pharmacol Exp Ther. 2006 Mar;316(3):1070-9. doi: 10.1124/jpet.105.092841. Epub 2005 Dec 28.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Active Transport, Cell Nucleus / drug effects
  • Animals
  • Cells, Cultured
  • DNA / metabolism
  • Enzyme Induction / drug effects
  • Ethacrynic Acid / pharmacology*
  • Extracellular Signal-Regulated MAP Kinases / metabolism
  • HMGB1 Protein / metabolism*
  • Interleukin-10 / metabolism
  • Interleukin-6 / metabolism
  • Isothiocyanates
  • Lipopolysaccharides / antagonists & inhibitors*
  • Macrophages / drug effects
  • Macrophages / metabolism*
  • Mice
  • NF-kappa B / physiology
  • Nitric Oxide / metabolism
  • Phosphorylation
  • Pyrazines / pharmacology*
  • Sulfoxides
  • Thiocyanates / pharmacology*
  • Thiones
  • Thiophenes
  • Tumor Necrosis Factor-alpha / metabolism


  • HMGB1 Protein
  • Interleukin-6
  • Isothiocyanates
  • Lipopolysaccharides
  • NF-kappa B
  • Pyrazines
  • Sulfoxides
  • Thiocyanates
  • Thiones
  • Thiophenes
  • Tumor Necrosis Factor-alpha
  • Interleukin-10
  • Nitric Oxide
  • oltipraz
  • DNA
  • Extracellular Signal-Regulated MAP Kinases
  • sulforaphane
  • Ethacrynic Acid