Background Activation from the Wnt pathway is known to promote tumorigenesis and tumor metastasis and Rabbit Polyclonal to DRP1. targeting Wnt pathway inhibition has emerged as a stylish approach for controlling tumor invasion and metastasis. such as blueberry cranberry and red cabbage [9 10 Anthocyanins have been linked to a number of intracellular functions including cellular redox status modification free radical scavenging activity and chelation of metals [11]. In addition anthocyanins (delphinidin-3 5 cyanidin-3 5 petunidin-3 5 delphinidin-3-glucoside: malvdin-3 5 peonidin-3 5 cyanidin-3-glucoside: petunidin-3-glucoside: peonidin-3-glucoside: malvidin-3-glucoside?=?27:63:8.27:1:2.21:2.21:6.7:1.25:5.72:1.25) isolated from Pulliat fruits show anti-invasive effects and apoptotic effects in human hepato-carcinoma cells [12 13 They also exhibit cancer-preventive effects that occur through their abilities to interfere with the cell signaling pathway [8]. Previous experiments have shown that anthocyanins induce cell cycle blockage at G1/G0 and G2/M phases and regulate the extracellular regulated kinase (ERK) c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways Bosentan in several malignancy types [14-16]. In addition anthocyanins have been shown to inhibit the activation of transcription factors such as nuclear factor-κB (NF-κB) and activator protein-1 (AP1) [17]. In this study we analyzed downstream signals of AMPK to search for naturally originating novel modulators of the AMPK/GSK3β/β-catenin pathway to control malignancy cell proliferation and metastasis. We found that anthocyanins activated GSK3β thereby decreasing β-catenin and that AMPK was an upstream regulator of GSK3β/β-catenin pathway. This information holds promise for therapeutic modulation of GSK3β/β-catenin-pathway-dependent invasiveness in cancer cells. Methods Cell culture and Bosentan reagents The Hep3B hepato-carcinoma cell line was purchased from the American Type Culture Collection (Manassas VA) and was cultured in Dulbecco’s altered Eagle’s medium with 10% fetal bovine serum (Gibco Grand Island NY). Insulin-like growth factor (IGF)-1 3 5 5 bromide (MTT) and Hoechst 33342 were obtained from Sigma (St Louis MO). Compound C and 6-bromoindirubin-3′-oxime (BIO) were purchased from Calbiochem (San Diego CA). Monoclonal antibodies specific for p-AMPK (Thr172) AMPKα1 p-GSK3β (Ser9) GSK3β β-catenin Ang-1 VEGF and MMP-9 were purchased from Cell signaling Technology (Beverly MA USA). CD31 antibody was purchased from Abcam (Cambridge UK) and β-actin antibody was obtained from Sigma (St Louis MO). Isolation of anthocyanins from Meoru Anthocyanins were conducted by Won Sup Lee’s group at Gyeongsang National University School of Medicine. The herb with voucher specimen number KNKA200506031111 was deposited in the Korea national arboretum. Fruit of Meoru was collected in the middle of September 2007 at Jiri mountain in Korea freeze-dried and stored in dark glass containers at ?20°C until required for analysis. Anthocyanins pigments were extracted by maceration of the fruits (100?g) in methanol containing 0.1% HCl at 5°C for 24?h. The extraction procedure was repeated three times. After concentration under reduced pressure (Rotavapor R-124 Buchi Switzerland) the extract was diluted with distilled water (100?ml) and partitioned against ethyl acetate (100?ml). The water layer made up of the pigments was concentrated to 50?ml. Bosentan The concentrate was purified according to established procedures by means of ethyl acetate/water partitioning and adsorption chromatography on a bed of Amberlite XAD-7 (Sigma Yongin South Korea) [18]. Cell proliferation measurements Hep3B cells seeded on 96-well microplates at 4?×?103 cells per well were incubated with the anthocyanins at the indicated concentrations for 48?h. Following incubation with the anthocyanins the medium was removed and the cells Bosentan were then incubated with 100?μl MTT solution (2?mg/ml MTT in phosphate-buffered saline (PBS)) for 4?h. The samples were then solubilized in dimethyl sulfoxide and the purple formazan dye converted from MTT by viable cells was quantified by absorbance Bosentan at 560?nm. Apoptosis detection Apoptosis was measured using an FITC-Annexin V apoptosis detection kit (BD Pharmingen? San Diego CA) or Hoechst 33342 chromatin staining dye. For Annexin V/propidium iodide staining after treatment with anthocyanins cells were harvested by trypsinization washed with ice-cold PBS and suspended in a binding buffer at a density of 1 1?×?106 cells/ml. Cells were stained with Annexin V-fluorescein isothiocyanate.