Anandamide inhibits the Wnt/β-catenin signalling pathway in human breast cancer MDA MB 231 cells  Chiara Laezza, Alba D’Alessandro, Simona Paladino, Anna Maria Malfitano, Maria Chiara Proto, Patrizia Gazzerro, Simona Pisanti, Antonietta Santoro, Elena Ciaglia, Maurizio Bifulco  European Journal of Cancer  Volume 48, Issue 16, Pages 3112-3122 (November 2012) DOI: 10.1016/j.ejca.2012.02.062 Copyright © 2012 Elsevier Ltd Terms and Conditions

Fig. 1 Effect of Met-F-AEA on the subcellular localisation of β-catenin in MDA MB 231 cells. (A) Cytoplasmic and nuclear fractions of breast cancer cells were subjected to SDS–PAGE and then immunoblotted with anti-β-catenin anti-Lamin B and anti-tubulin. Tubulin was used as a cytoplasmic protein loading control and Lamin B was used for nuclear protein loading control. Histograms indicated the quantification of signal intensity changes of β-catenin. (∗p<0.05, in comparison with control). (Ctr: DMSO control). Met-F-AEA did not have effects on the subcellular localisation of β-catenin in MCF7 cells. Each experiment was repeated at least three times. The figure shows a representative blot. (B) Time course of treatment with Met-F-AEA in MDA MB 231 cells. Western blotting analysis for phosphorylated GSK-3β(P-GSK3β) total GSK-3β (T-GSK3β), Phosphorylated β-catenin (Pβ-catenin, Ser33/Ser37/Thr41) and β-catenin and using their specific antibodies as specified in Materials and Methods. Membranes were reprobed with Lamin B for nuclear fraction and tubulin for cytosolic fraction as loading controls and as markers for purity of the fractions. (C) Cells were treated with or without 10μM MG132 and 10μM Met-F-AEA for 24h and analysed by western blotting with antibodies against β-catenin. (D) Time course of treatment with Met-F-AEA in cellular lysates of MDA MB 231 cells: western blot analysis for phosphorylated LRP6 (P-LRP6) and total LRP6 (T-LRP6). Immunoreactive bands were quantified using Quantity One programme. The diagrams show quantification of the intensity of bands, calibrated to the intensity of the bands and of Lamin and tubulin bands, expressed as means ± SD (∗p<0.01 compared to control cells). Each experiment was repeated at least 3 times. The figure shows a representative blot. European Journal of Cancer 2012 48, 3112-3122DOI: (10.1016/j.ejca.2012.02.062) Copyright © 2012 Elsevier Ltd Terms and Conditions

Fig. 2 Cellular localisation of β-catenin was also observed by immunofluorescent staining followed by confocal imaging. For this, MDA MB 231 cells grown on coverslips were treated with either DMSO alone or 10μM Met-F-AEA for 12h and with or without SR141716 for the same time, then they were fixed and stained for β-catenin. Bar 10μm. European Journal of Cancer 2012 48, 3112-3122DOI: (10.1016/j.ejca.2012.02.062) Copyright © 2012 Elsevier Ltd Terms and Conditions

Fig. 3 Met-F-AEA inhibited the transcriptional activity of β-catenin/Tcf and the expression of its target genes in MDA MB 231 cells. (A) The cells lines were co-transfected with reporter genes harbouring Tcf-4 binding sites (TOPflash) or a mutant Tcf-binding site (FOPflash), respectively, and β-galactosidase gene. After transfection cells were treated with Met-F-AEA at 10μM with or without SR141716 at 0.1μM as indicated Luciferase activity was determined 24h post-transfection, normalised against values for the corresponding β-galactosidase activity. An equivalent volume of DMSO was used as a vehicle control. Data shown represent mean±SD of three independent observations (∗p<0.01, compared to DMSO control). Met-F-AEA did not have effect on transcriptional activity of β-catenin/Tcf of MCF7 cells. (B) After treatment the total cell lysates were then analysed by Western immunoblot analysis for Cyclin D1, c-Myc and MMP-2levels. Membranes were reprobed with actin as loading control. Densitometry data shown represent fold change compared with control after normalisation with respective loading controls (∗p<0.05compared with control). European Journal of Cancer 2012 48, 3112-3122DOI: (10.1016/j.ejca.2012.02.062) Copyright © 2012 Elsevier Ltd Terms and Conditions

Fig. 4 (A) Time course of Met-F-AEA effect on the expression levels of mesenchymal gene products and of epithelial gene products in MDA MB 231 cells. Total protein lysates derived from cells treated were subjected to western blot analysis for determination of protein expression of the indicated epithelial and mesenchymal gene markers. The results were compared with the expression profiles of the same gene products in untreated cell lysates. Actin was used as an internal control for loading. (B) E-cadherin immunofluorescence in MDA MB 231 cells treated with Met-F-AEA. The arrows display E-cadherin in cell membrane Bar 10μm. European Journal of Cancer 2012 48, 3112-3122DOI: (10.1016/j.ejca.2012.02.062) Copyright © 2012 Elsevier Ltd Terms and Conditions

Fig. 5 Met-F-AEA up-regulated the expression of E-cadherin in MDA MB 231 cells. Time kinetic analysis of E-cadherin, Snail, Slug, Twist and MMP2 mRNA in MDA MB 231 cells treated with Met-F-AEA. Snail, Slug, Twist, MMP2 and E-cadherin transcript levels were determined by quantitative RT-PCR for each time point tested (0–24h). European Journal of Cancer 2012 48, 3112-3122DOI: (10.1016/j.ejca.2012.02.062) Copyright © 2012 Elsevier Ltd Terms and Conditions

Fig. 6 Adriamycin (ADM) induced EMT in MCF7 cells were treated with or without Adriamycin (25μg/mL) for 36h. EMT was examined by phase-contrast microscopy(Zeiss (×100 magnification; bar, 10μm). Met-F-AEA restored the cell–cell contact in Adriamicyn treated cells. European Journal of Cancer 2012 48, 3112-3122DOI: (10.1016/j.ejca.2012.02.062) Copyright © 2012 Elsevier Ltd Terms and Conditions

Fig. 7 Western blotting of expression levels of mesenchymal and of epithelial markers in MCF7cells treated with or without Adriamycin (ADM) for 36h. Met-F-AEA-treated cells prevented the EMT induction by Adriamicyn. Membranes were reprobed with actin as loading control. Densitometry data shown represent fold change compared with control after normalisation with respective loading controls. (∗p<0.05 compared with control). European Journal of Cancer 2012 48, 3112-3122DOI: (10.1016/j.ejca.2012.02.062) Copyright © 2012 Elsevier Ltd Terms and Conditions