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 phosphatidylino sitol 3-kinase (PI 3-kinase), and is structurally related to members of the flavonoid family. Concentrations of LY294002 greater than or equal to 10 m u M were cytostatic, but not cytotoxic, to cultures of the immortalised hum an 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 s teady-state CYP1A1 mRNA. Pretreatment of cultures with greater than or equa l to 10 mu M LY294002 suppressed the TCDD activation of CYP1A1 (IC50 simila r to 10 mu M). Electrophoretic mobility shift assays employing rat liver cy tosol demonstrated that concentrations of LY294002 less than or equal to 40 0 mu M did not transform the AHR into a DNA-binding species. However, the a ddition of LY294002 to cytosol just prior to TCDD addition completely suppr essed AHR transformation by TCDD (IC50 similar to 35 mu M). The PI 3-kinase inhibitor Wortmannin was weakly cytostatic, but not cytotoxic to MCF10A-Ne o cultures at concentrations less than or equal to 500 nM. Exposure of cult ures 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 equ al to 50 mu M and Wortmannin greater than or equal to 10 nM completely supp ressed PI 3 kinase activity. Hence, the ability of LY294002 to suppress TCD D-dependent activation of CYP1A1 is unrelated to PI 3-kinase inhibition. In stead, this activity reflects LY294002 functioning as an AHR antagonist. Fu rthermore, most of the cytostatic activity of LY294002 towards MCF10A-Neo c ells is unrelated to the inhibition of PI 3-kinase. BIOCHEM PHARMACOL 60;5: 635-642, 2000. (C) 2000 Elsevier Science Inc.