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. 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 prevent 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. yielded the crude 4-demethyltangeretin. Purification by column chromatography [SiO2, dichloromethane with an increasing gradient of ethyl acetate (20C50%)] yielded 4-demethyltangeretin as an off-white Nutlin-3 powder (0.032 g, 67%). The structure (see Physique 1) was established by 1H and 13C-NMR spectra recorded on a 400 MHz super-conducting Bruker Spectrometer (Karlsruhe, Germany) at 30C. Thin layer chromatography was performed on aluminium linens precoated Nutlin-3 with silica gel 60f254 (Merck, Darmstadt, Philippines) observed under UV light (450 nm). Mass spectra were recorded on a Micromass Quattro II low resolution triple quadrupole mass spectrometer (EPSRC National Mass Spectrometry Support Centre, Swansea, UK). Physique 1 The HPLC information of tangeretin after incubation with microsomes conveying CYP1A1, CYP1A2 and CYP1W1 and with hepatocytes. (A) The structures of tangeretin and 4-hydroxy-5,6,7,8-tetramethoxyflavone (4-OH-TMF). (W) Upper trace: extracts … Statistical assessments Data were analysed by GraphPad prism using anova followed by Bonferroni’s post test. Results Initial experiments identified a major metabolic product of the incubation of tangeretin with specific CYP enzymes. Physique 1B (upper panel) shows HPLC information following the incubation (30 min) of tangeretin with microsomes conveying CYP1A1, CYP1A2 or CYP1W1 enzymes, compared with control microsomes and a tangeretin standard. Incubation with CYP1-conveying microsomes led to an incubation time-dependent reduced tangeretin peak and the appearance of peaks with a shorter retention time. The rank order of rate of loss of area under the tangeretin peak was CYP1A1>CYP1A2>>>CYP1W1. Two major peaks from metabolites were seen; the one that formed first and eluted closest to tangeretin was characterized as 4-OH-TMF, following the work of Nielsen and co-workers (Nielsen and used along Rabbit Polyclonal to PPP1R7 with tangeretin in the work described here. We also incubated tangeretin with hepatocytes to investigate whether 4-OH-TMF is usually formed. Nutlin-3 We found (Physique 1C) that after the 24 h incubation there was a Nutlin-3 peak on the HPLC trace, which co-eluted with 4-OH-TMF, consistent with the formation of this metabolite within hepatocytes. We tested tangeretin and 4-OH-TMF for effects on the cell cycle progression of primary hepatocytes using [3H]-thymidine incorporation into DNA as an index of progression to S-phase. We have previously shown that under the conditions of culture used here the maximum stimulant effect of EGF (3 nM) is usually observed when [3H]-thymidine is usually present for the last 4 h of a 24 h activation period, suggesting that the onset of the S-phase occurs 20 h after exposure to EGF. As seen in Physique 2 (A,W) the unstimulated hepatocytes showed a low level of [3H]-thymidine incorporation into DNA, with a substantial increase when EGF was present. The [3H]-thymidine response to EGF was inhibited when tangeretin was added to the cells 15 min before EGF and was then present for the duration of a 24 h activation period (Physique 2A). This inhibition was concentration dependent, with the onset between 1 and 3 M tangeretin and an IC50 of about 5 M; there was some.