Suppression of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated aryl hydrocarbon receptor transformation and CYP1A1 induction by the phosphatidylinositol 3-kinase inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002)

Numerous flavonoids are ligands of the aryl hydrocarbon receptor (AHR) and function as AHR antagonists and/or agonists. LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] is a widely used inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), and is structurally related to members of the flavonoid family. Concentrations of LY294002 greater than or equal to 10 mu M were cytostatic, but not cytotoxic, to cultures of the immortalised human breast epithelial cell line MCF10A-Neo. Treatment of MCF10A-Neo cultures with the AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) stimulated the transcriptional activation of CYP1A1, as monitored by measurements of steady-state CYP1A1 mRNA. Pretreatment of cultures with greater than or equal to 10 mu M LY294002 suppressed the TCDD activation of CYP1A1 (IC50 similar to 10 mu M). Electrophoretic mobility shift assays employing rat liver cytosol demonstrated that concentrations of LY294002 less than or equal to 400 mu M did not transform the AHR into a DNA-binding species. However, the addition of LY294002 to cytosol just prior to TCDD addition completely suppressed AHR transformation by TCDD (IC50 similar to 35 mu M). The PI 3-kinase inhibitor Wortmannin was weakly cytostatic, but not cytotoxic to MCF10A-Neo cultures at concentrations less than or equal to 500 nM. Exposure of cultures to Wortmannin (10-500 nM) did not suppress TCDD activation of CYP1A1. Analyses of the phosphorylation status of Akt-1, an in vivo substrate of PI 3-kinase, demonstrated that concentrations of LY294002 greater than or equal to 50 mu M and Wortmannin greater than or equal to 10 nM completely suppressed PI 3 kinase activity. Hence, the ability of LY294002 to suppress TCDD-dependent activation of CYP1A1 is unrelated to PI 3-kinase inhibition. Instead, this activity reflects LY294002 functioning as an AHR antagonist. Furthermore, most of the cytostatic activity of LY294002 towards MCF10A-Neo cells is unrelated to the inhibition of PI 3-kinase. BIOCHEM PHARMACOL 60;5:635-642, 2000. (C) 2000 Elsevier Science Inc.