Isoflavone-mediated inhibition of tyrosine kinase: a novel antiinflammatory approach

Tyrosine kinase (TK)-mediated phosphorylation regulates signal transduction pathways resulting in the expression of a variety of inflammatory genes. Inhibition of TK activity in vivo has been shown to increase survival in a lethal model of murine endotoxemia, suggesting a novel therapeutic approach to inflammation and circulatory shock. We examined the role of TK activity on the expression of the inducible nitric oxide (NO) synthase (iNOS). Under resting conditions, iNOS is not expressed in human cells. In response to various proinflammatory stimuli, however, iNOS expression is upregulated, resulting in high-output NO synthesis. iNOS-derived NO plays a critical role as a cytotoxic effector species and has been implicated in the pathogenesis of many clinical inflammatory conditions, including inflammatory bowel disease, arthritis, transplant rejection, diabetes, and sepsis. We examined the signaling pathways governing iNOS expression in monolayers of DLD-1 cells, a human epithelial cell line derived from an intestinal adenocarcinoma. Induction of iNOS transcription in interferon-gamma-primed cells by treatment with lipopolysaccharide, Salmonella sp., or interleukin-1beta was potently inhibited by pretreatment with genistein, an isoflavone derived from the soy species genistin. Other isoflavones, such as genistin, daidzein, and daidzin, were not inhibitory. TK inhibition by genistein had no effect on the expression or nuclear translocation of the transcription factors interferon regulatory factor-1 and nuclear factor-KB, respectively, both of which have been implicated in transcriptional regulation of the human iNOS gene. Nuclear run-on analysis demonstrated that the effect of genistein on iNOS messenger RNA expression was not at the level of transcription, suggesting that posttranscriptional regulation of iNOS messenger RNA might be TK dependent. Isoflavones, such as genistein, are useful tools to dissect regulatory pathways in vitro and in vivo and may have potential use as novel antiinflammatory therapeutic agents.