title: Tangeretin and its metabolite 4'-hydroxytetramethoxyflavone attenuate EGF-stimulated cell cycle progression in hepatocytes; role of inhibition at the level of mTOR/p70S6K.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  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
  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.
authors.affiliation: The Cell Signalling Laboratory, Leicester School of Pharmacy, De Montfort University, Leicester LE1 9BH, UK.
authors.forename: Z
authors.initials: Z
authors.lastname: Cheng
authors.forename: S
authors.initials: S
authors.lastname: Surichan
authors.forename: K
authors.initials: K
authors.lastname: Ruparelia
authors.forename: R
authors.initials: R
authors.lastname: Arroo
authors.forename: M R
authors.initials: MR
authors.lastname: Boarder
mesh_terms: D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000494:pharmacology; D047309:Flavones / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology*; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D008297:Male; D010766:Phosphorylation / Q000187:drug effects; D011355:Prodrugs; D051381:Rats; D017208:Rats, Wistar; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000187:drug effects / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases / Q000187:drug effects / Q000378:metabolism
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D047309:Flavones; D011355:Prodrugs; C559374:4'-hydroxy-5,6,7,8-tetramethoxyflavone; D004815:Epidermal Growth Factor; C546845:mTOR protein, rat; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases; C059006:tangeretin