3-methylquercetin and baicalein

Functional activation of beta-catenin/T-cell factor (Tcf) signaling has been implicated in human carcinogenesis. We identified the inhibitory effect of various polyphenolic flavonoid compounds against beta-catenin/Tcf signaling in beta-catenin-activated cells. Genistein, kaempferol, isorhamnentin, and baicalein inhibited the transcriptional activity of beta-catenin/Tcf in HEK293 cells transiently transfected with a constitutively active mutant beta-catenin gene. To investigate the inhibitory mechanism, electrophoresis mobility shift assay, immunoprecipitation, and Western blot experiments were performed. The shift assay showed that the binding of Tcf complexes with its specific DNA-binding sites was suppressed by four kinds of flavonoids. Immunoprecipitation analysis also showed that the binding of beta-catenin to Tcf-4 was also disrupted by these flavonoids. Western blot analysis showed a decreased level of beta-catenin in nucleus caused by genistein. Genistein also decreased phosphorylation of Akt and GSK3 beta. Taken together, these results suggest that the polyphenolic flavonoids genistein, kaempferol, isorhamnentin, and baicalein are negative regulators of beta-catenin/Tcf signaling and their inhibitory mechanism is related to the decreased binding of beta-catenin/Tcf complexes to consensus DNA.

Topics: Axin Protein; beta Catenin; Blotting, Western; Cell Line; Cell Line, Tumor; Cyclin D1; Cytoskeletal Proteins; Flavanones; Flavonoids; Flavonols; Gene Expression; Genistein; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Kaempferols; Mutation; Phosphorylation; Protein Binding; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Quercetin; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; TCF Transcription Factors

An effective, accurate and reliable HPLC with UV detection method was developed and validated for quantitation of six components: baicalin, berberine hydrochloride, quercetin, kaempferol, isorhamnetin and baicalein in intestinal perfusate using rotundin as an internal standard. The chromatographic separation was performed on a Welchrom-C(18) column (250 x 4.6 mm i.d. with 5.0 microm particle size) with a mobile phase consisting of acetonitrile, water, phosphoric acid and triethylamine (30:70:0.2:0.1,v/v) at a flow rate of 1.0 mL/min and a UV detection at 270 nm. The method had a chromatographic run time of 30 min and excellent linear behavior over the investigated concentration ranges observed with the values of r higher than 0.99 for all the analytes. The lower limit of quantification of the analytical method was 0.09 microg/mL for berberine hydrochloride, quercetin, kaempferol and baicalein and 0.18 microg/mL for baicalin and isorhamnetin. The intra- and inter-day precisions measured at three concentration levels were all less than 10% for all analytes. The bias ranged from -6.91 to 4.33%. The validated method has been successfully applied to investigate the rat intestine absorption profiles of baicalin, berberine hydrochloride, quercetin, kaempferol, isorhamnetin and baicalein.

Topics: Animals; Berberine; Chromatography, High Pressure Liquid; Flavanones; Flavonoids; Flavonols; Intestinal Absorption; Kaempferols; Male; Quercetin; Rats; Rats, Wistar; Sensitivity and Specificity