Tangeretin and its metabolite 4′‐hydroxytetramethoxyflavone attenuate EGF‐stimulated cell cycle progression in hepatocytes; role of inhibition at the level of mTOR/p70S6K

BACKGROUND AND PURPOSE The mechanisms by which the dietary compound tangeretin has anticancer effects may include acting as a prodrug, forming an antiproliferative product in cancer cells. Here we show that tangeretin also inhibits cell cycle progression in hepatocytes and investigate the role of its primary metabolite 4′‐hydroxy‐5,6,7,8‐tetramethoxyflavone (4′‐OH‐TMF) in this effect. EXPERIMENTAL APPROACH We used epidermal growth factor (EGF)‐stimulated rat hepatocytes, with [ 3 H]‐thymidine incorporation into DNA as an index of progression to S‐phase of the cell cycle, and Western blots for phospho‐proteins involved in the cell signalling cascade. KEY RESULTS Incubation of tangeretin with microsomes expressing CYP1A, or with hepatocytes, generated a primary product we identified as 4′‐OH‐TMF. Low micromolar concentrations of tangeretin or 4′‐OH‐TMF gave a concentration‐dependent inhibition of EGF‐stimulated progression to S‐phase while having little effect on cell viability. To determine whether time for conversion of tangeretin to an active metabolite would enhance the inhibitory effect we used long pre‐incubations; this reduced the inhibitory effect, in parallel with a reduction in the concentration of tangeretin. The EGF‐stimulation of hepatocyte cell cycle progression requires signalling through Akt/mTOR/p70S6K kinase cascades. The tangeretin metabolite 4′‐OH‐TMF selectively inhibited S6K phosphorylation in the absence of significant inhibition of upstream Akt activity, suggesting an effect at the level of mTOR. CONCLUSIONS AND IMPLICATIONS Tangeretin and 4′‐OH‐TMF both inhibit cell cycle progression in primary hepatocytes. The inhibition of p70S6K phosphorylation by 4′‐OH‐TMF raises the possibility that inhibition of the mTOR pathway may contribute to the anticancer influence of a flavonoid‐rich diet.