Flavonoids are plant-derived polyphenolic molecules that have potential biological effects including

Flavonoids are plant-derived polyphenolic molecules that have potential biological effects including anti-oxidative anti-inflammatory anti-viral and anti-tumoral effects. expression. In addition this flavonoid shows anti-tumoral effects on BX-795 colon cancer cells (SW480 DLD-1 and HCT116) whereas exerting no significant effect on non-tumor colon cell (IEC-18) suggesting a specific effect in tumor cells effects of the flavonoid quercetin as a negative modulator of the Wnt/β-catenin signaling pathway in embryos (15). We also observed that quercetin shows high and nonspecific toxicity. Our previous data have shown that isoquercitrin which is derived from quercetin affects the proliferation of glioblastoma cells with lower toxicity (16). These anti-proliferative effects were accompanied by changes in β-catenin cellular localization suggesting that Wnt/β-catenin signaling might be altered by this flavonoid (16). Hence we conducted a series of assays in embryos to investigate whether isoquercitrin has an effect on Wnt/β-catenin signaling. The use of allows a fast and clear functional reading on the role of small molecules in this signaling pathway (11 15 16 In addition we monitored cell growth death migration and toxicity of colon cancer cells upon isoquercitrin treatment. Taken together our data indicate that isoquercitrin acts as an inhibitor of Wnt/β-catenin in embryo experiments (and thus should be further investigated as a potential anti-tumoral agent. EXPERIMENTAL PROCEDURES Embryo Manipulations Adult frogs (Nasco Inc.) were stimulated with 1000 IU human chorionic gonadotropin (Ferring Pharmaceuticals Kiel Germany). embryos were obtained by fertilization and staged according to Nieuwkoop and Farber (17). We treated the embryos with flavonoids and performed the embryo manipulations according to Amado (15). Histological Analysis For histological staining embryos were BX-795 fixed in Bouin’s fixative (Sigma-Aldrich) dehydrated embedded in Paraplast Plus (Sigma-Aldrich) sectioned at 7 μm dewaxed and stained with hematoxylin and eosin as described by Reis (18). In Situ Hybridization embryos were fixed in MEMFA (MOPS EGTA MgSO4 and formaldehyde buffer; final concentrations: 100 mm MOPS (pH 7.4) 2 mm EGTA 1 mm MgSO4 3.7% (v/v) formaldehyde) at 4 °C overnight and then dehydrated in a methanol series (25 50 75 and 100%). Whole-mount hybridization was performed according to Abreu (19) with modifications suggested by Reversade and De Robertis (20) for embryos were treated with a bleaching solution (2.5% 20× SSC 5 formamide 4 H2O2 in H2O). Luciferase Assay Four-cell-stage embryos were injected into the ventral or dorsal marginal zone with 300 pg of luciferase reporter plasmid (S01234-Luc) and 50 pg of TK-(21). Four-cell-stage embryos were treated with flavonoids at 75 and 150 μm whereas controls were treated with 1% DMSO.2 When embryos reached the 32-cell stage they were treated with 0.3 m LiCl in 0.1× Barth for 15 min and then thoroughly washed in 0.1× Barth. After LiCl treatment embryos were treated again with flavonoids and the control embryos were treated with 1% DMSO until stage 10.5. Then the flavonoids or DMSO were removed and the embryos were cultured until stage 32. Axis phenotypes were scored by the dorsal-anterior index (DAI) (21). Western Blotting Analysis Lysate samples from flavonoid HCT-116-treated cells at 75 and Ednra 150 μm BX-795 were BX-795 harvested in a sample buffer (0.02 mmol/liter dithiothreitol; 1.38 mmol/liter sodium dodecyl sulfate; 125 mmol/liter Tris-HCl pH 6.8; and 20% glycerol). Protein was quantified by the Lowry method and 10 mg of the total lysate was loaded in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electroblotted and transferred to a polyvinylidene fluoride membrane (Hybond-PTM Amersham Biosciences S?o Paulo Brazil). Membranes were pre-blocked in 5% nonfat dry milk in Tris-buffered saline 0.001% Tween 20 for 1 h and then incubated overnight with anti-cyclin D1 (1:2000 Cell Signaling) anti-PCNA (1:2000 Cell Signaling) anti-cleaved caspase-3 (1:500 Cell Signaling) anti-??catenin (1:1000 Sigma) anti-lamin (1:1000 Cell Signaling) and anti-α-tubulin (1:5000 Sigma) primary antibodies previously diluted in Tris-buffered.