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β-catenin signalling pathway in human breast cancer MDA MB 231 cells
European Journal of Cancer (2012) 48, 3112– 3122
Available at www.sciencedirect.com
journal homepage: www.ejcancer.info
Anandamide inhibits the Wnt/b-catenin signalling pathway in human breast cancer MDA MB 231 cells Chiara Laezza a,b,⇑, Alba D’Alessandro c, Simona Paladino b, Anna Maria Malfitano c, Maria Chiara Proto c, Patrizia Gazzerro c, Simona Pisanti c, Antonietta Santoro c, Elena Ciaglia c, Maurizio Bifulco c, Endocannabinoid Research Group a
Institute of Endocrinology and Experimental Oncology, IEOS CNR, Via Pansini 5, 80131 Naples, Italy Department of Biology and Cellular and Molecular Pathology, University of Naples Federico II, Via Pansini, 80131 Naples, Italy c Department of Pharmaceutical and Biomedical Sciences, University of Salerno, Via Ponte don Melillo, 84084 Fisciano, Salerno, Italy b
Available online 14 March 2012
KEYWORDS Anandamide CB1 receptor Wnt/b-catenin pathway Epithelial-mesenchymal transition (EMT) E-cadherin
We previously showed that methyl-F-anandamide, a stable analogue of the anandamide, inhibited the growth and the progression of cultured human breast cancer cells. As accumulating evidences indicate that the constitutive activation of the canonical Wnt pathway in human breast cancer may highlight a key role for aberrant activation of the b-catenin–TCF cascade and tumour progression, we studied the anandamide effect on the key elements of Wnt pathway in breast cancer cells. In this study we described that the treatment of human breast cancer cells, MDA MB 231 cells, with methyl-F-anandamide reduced protein levels of b-catenin in the cytoplasmic and nuclear fractions inhibiting the transcriptional activation of T Cell Factor (TCF) responsive element (marker for b-catenin signalling). The anandamide treatment resulted in up-regulation of epithelial markers, like E-cadherin with a concomitant decrease in protein levels of mesenchymal markers, including vimentin and Snail1. We, furthermore, observed that the induction of experimental epithelial-mesenchymal transition by exposure to adriamycin in MCF7 human breast cancer cell line was inhibited by anandamide treatment. In the present study we reported a novel anticancer effect of anandamide involving the inhibition of epithelial-mesenchymal transition, a process triggered during progression of cancer to invasive state. Ó 2012 Elsevier Ltd. All rights reserved.
Abstract
1. Introduction ⇑ Corresponding author at: Institute of Endocrinology and Experi-
mental Oncology, IEOS CNR, Via Pansini 5, 80131 Naples, Italy. E-mail addresses: [email protected], [email protected] (C. Laezza). 0959-8049/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.ejca.2012.02.062
The endocannabinoid system, comprising the cannabinoid CB1 and CB2 receptors, the endogenous ligands, endocannabinoids and the enzymes that regulate their biosynthesis and degradation, is a ubiquitous signalling
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system involved in the control of cell fate. The endocannabinoids (anandamide, AEA and 2-arachidonoyglycerol, 2AG), agonists of CB1 receptors, have been shown to inhibit the growth of tumour cells in culture and animal models by modulating key cell signalling pathways.1–3 We have shown that a metabolically stable anandamide analogue, 2-methyl-20 -F-anandamide (Met-F-AEA) by cannabinoid CB1 receptors activation arrested the growth of K-ras-dependent tumours.4,5 In addition, we described that Met-F-AEA treatment inhibited vascular endothelial cell migration by decreasing the expression and the activity of metalloprotease-2, MMP-2, a proteolytic enzyme involved in tissue remodelling during angiogenesis and metastasis6 and it reduced the cell migration of the highly invasive and metastatic breast cancer cell lines, MDA MB 231.7 In this study, we investigated the effect of CB1 receptor activation by Met-F-AEA on the Wnt/b-catenin signalling pathway. This pathway is highly regulated and it has important functions in development, tissue homeostasis and regeneration. Activation of the canonical Wnt pathway involves the stabilisation of b-catenin through the binding of Wnt ligands to cell surface receptors: Frizzled (Fz) family receptors and lowdensity lipoprotein receptor (LDLR)-related protein 5 (LRP5) and LRP6. In the absence of Wnt ligands, b-catenin is phosphorylated by a multiprotein degradation complex, which marks it for ubiquitination and degradation by the proteasome. In the presence of appropriate Wnt ligands, b-catenin is stabilised in the cytoplasm, promoting its translocation to the nucleus.8–11 In the nucleus, b-catenin interacts with T-Cell Factor (TCF)/Lef transcription factors and activates cell transcription of target genes, including cyclin D1,12 c-Myc13 and metalloproteases that promote cell proliferation, differentiation and tissue development.14 In the mammary tissues, Wnt signalling plays an important role in stem cell self-renewal and mammary gland development.15 Compelling evidence indicates that when the Wnt/b-catenin pathway is aberrantly activated, it may lead to mammary carcinogenesis.16 In this study we examined the effect of the CB1 receptor activation by Met-F-AEA on modulation of the components of the Wnt/b-catenin signalling pathway, more, we showed that anandamide treatment inhibited epithelial-mesenchymal transition (EMT)17–19 involved in progression of cancers to invasive state
2. Materials and methods 2.1. Materials Met-F-AEA (2-methyl-20 -F-anandamide) was purchased from Sigma. The selective CB1 antagonist, SR141716, was kindly provided by Sanofi-Aventis (Montpellier, France). Doxorudicin hydrochloride (Adriamycin) and MG132 was from SIGMA, anti-b-catenin, anti-E-cadherin, anti N-cadherin, anti-vimentin, anti-phospho-GSK3b, anti-GSK3b
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and anti-Cytokeratin 18, anti-fibronectin, were purchased from Santa Cruz Biotechnology, anti-phospho (Ser33/Ser37/Thr41)-b-catenin, anti-phospho LRP6 and anti-LRP6 were from Cell Signalling. 2.2. Cell culture MDA MB 231, an invasive human breast carcinoma cell line, was grown in RPMI 1640 medium (Gibco BRL Life technologies, MD) supplemented with 10% inactivated foetal bovine serum (FBS) and 2 mM L-glutamine. MCF7, a non invasive human breast cancer cell line, was grown in DMEM medium supplemented with 10% inactivated FBS and 2 mM l-glutamine. Cells were cultured at 37 °C in a humidified 5% CO2 atmosphere. 2.3. Subcellular fractionation Cells were resuspended in a hypotonic buffer [10 mmol/ L Tris–HCl (pH 7.5), 25 mmol/L KCl, 2 mmol/L magnesium acetate, 1 mmol/L DTT, 0.5 mmol/L phenylmethylsulphomyl fluoride (PMSF), protease inhibitor cocktails]. After incubating on ice for 10 min, cell membranes were disrupted by 10 passes through a 23-gauge needle, and nuclear isolation was monitored under a microscope. After centrifugation for 5 min at 1000g, the supernatant was saved as the ‘cytoplasmic fraction’. The nuclear pellet was washed once with the hypotonic buffer and lysed with hypertonic buffer (hypotonic buffer plus 400 mmol/L KCl and 20% glycerol). The lysates were then centrifuged at 12,000g for 5 min, and the supernatant was collected as the ‘nuclear fraction’ and frozen at 80 °C. Total cell lysates and cytoplasmic or nuclear fractions were separated by SDS–PAGE, transferred to nitrocellulose membranes, and then probed with various primary antibodies to determine the expression of the signalling proteins. 2.4. Quantitative real-time PCR Total RNA was extracted from cultures using TRIzol reagent according to the manufacturer’s instructions. cDNA synthesis was achieved with 2 lg of total RNA by Moloney Murine leukaemia viruses (M-MuLV) reverse transcriptase. The efficiency of the used primers was evaluated by calculating the linear regression of Ct data points obtained with a series of different cDNA dilutions and inferring the efficiency from the slope of the line. We used the primers that gave efficiency close to 100%. The used primer sequences were the following: E-cadherin F 50 -TCG ACA CCC GAT TCA AAG TGG-30 and R 50 -TTC CAG AAA CGG AGG CCT GAT-30 ; MMP2 F 50 -ACA TCA AGG GCA TTC AGG AG-30 and R 50 -GCC TCG TAT ACC GCA TCA AT-30 ; Snail1 F 50 -AAG GAC CCC ACA TCC TTC TC-30 and R 50 -GGA GCT TCC CAG TGA GTC TG-30 ; Slug F50 -CGCCTCCAAAAAG CCAAAC-30 and R 50 -CGGTAGTCCACACAGT-
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GATG-30 ; Twist F 50 -GGAGTCCGCAGTCTTAC GAG-30 and R 50 TCTGGAGGACCTGGTAGAGG-30 ; b-actin F 50 -GCGTGACATCAAAGAGAAG-30 and bactin R 50 -ACTGTGTTGGCATAGAGG-30 . Real-time PCR was performed using So Fast Eva Green Super mix (Biorad) according to the manufacturer’s instructions in an iQ5 real-time PCR detection system (Bio-Rad). Fluorescence was determined at the last step of every cycle. Real-time PCR assay was done under the following universal conditions: 2 min at 50 °C, 10 min at 95 °C, 50 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min. Relative quantification was performed using the DDCt method. 2.5. b-Catenin/T-cell factor-lymphoid enhancer factor-1 transcription reporter assay The nuclear activity of endogenous b-catenin in MDA MB 231 and MCF7 cells was analysed by using the TOPflash/FOPflash reporter system (Upstate, Charlottesville, VA). Cells grown in 24-well dishes were transfected with either the TOPflash or FOPflash plasmid using LipofectAMINEPlus reagent. To normalise transfection efficiency in the reporter assays, cells were cotransfected with b-gal plasmid, which contains a functional b-gal luciferase gene cloned downstream of a herpes simplex virus thymidine kinase promoter (Promega, Madison, WI). Four hours after transfection, the culture medium was replaced by DMEM supplemented with 10% FBS in the presence or absence of drugs. Fortyeight hours after transfection, cells were washed with PBS and then lysed with the reporter lysis buffer (Promega). Luciferase activities in cell lysates were measured with the Promega Dual-Luciferase Reporter System and normalised to control b-gal luciferase signal. 2.6. Immunofluorescence Cells were plated on cover slips; when they reached 80% confluence, they were treated with Met-F-AEA (10 lM). After the incubation with various drugs and SR141716 at 0.1 lM, the cells were washed twice with PBS, fixed in 3.7% paraformaldehyde in PBS for 20 min and followed by two washes in 50 mM NH4Cl for 10 min. Permeabilisation was achieved by incubating the fixed cells in 0.1% Triton -100 in PBS for 5 min at room temperature. The cells were then blocked in final dilution buffer (FDB) (1 mM MgCl2, 1 mM CaCl2, 5% foetal calf serum and 2% BSA in PBS) for 30 min at room temperature. All primary and secondary antibody incubations were performed in FDB buffer. Cells were then incubated with specific antibodies for 1 h at room temperature and for the detection of b-catenin we used a goat anti-mouse IgG labelled with green fluorescent Alexa Fluor 488, while donkey anti-mouse IgG labelled red-fluorescent Alexa Fluor 594 was used for the
detection of E-cadherin. The relevant antibodies were then added at the dilutions recommended by the manufacturers. Cover slips were mounted on 50% glycerol in PBS and examined by using a Zeiss Laser Scanning Confocal Microscope. 2.7. Western blotting analysis After 24 h of incubation, cells were washed twice with PBS and re-suspended in lysis buffer (Hepes 50 mM, NaCl 150 mM, ethylene diamine tetra-acetic acid (EDTA) 50 mM, NaF 100 mM, Na ortovanadate 2 mM, glycerol, Na4P2O7 10 mM, 10% triton pH 7.5) and passed through a 23 gauge needle 10 times before centrifugation at 12000g at 4 °C. Aliquots of the cellular lysates (40 lg of protein) were boiled for 5 min and electrophoresed on SDS–polyacrylamide gel, transferred to nitrocellulose membranes and incubated with antibodies. The blots were blocked in PBS containing 0.1% Tween 20 and 5% non-fat dry milk for 1 h at room temperature. The filters were then probed overnight with primary specific antibodies. Immunodetection of specific proteins was carried out with horseradish peroxidase-conjugated donkey antimouse or anti-rabbit IgG (Biorad, Hercules, CA), using the enhanced chemiluminescence (ECL) system (Amersham, Buckinghamshire, United Kingdom). 2.8. Densitometric and statistical analysis The intensities of bands obtained from Western blots and RT-PCR were estimated with Alpha ImagerTm2200 (Alpha Innotech Corporation, United States of America). All the measurements were made in triplicate and all values are represented as mean ± SD. Comparison between groups was done using Student’s unpaired t test. In all statistical comparisons, p < 0.05 was used to indicate a significant difference. 3. Results 3.1. Anandamide decreased stabilisation and nuclear translocation of b-catenin Previously we observed that the CB1 receptor activation by Met-F AEA at 10 lM inhibited cell proliferation after 24 h of breast cancer cells in both ER-positive/luminal epithelial-like MCF7 cells and ER-negative/mesenchymal-like MDA MB 231 cells.20,21 As b-catenin/TCF transcriptional activity is known to regulate proliferative activities of breast epithelial cells, we first investigated the effects of methyl-F-anandamide (Met-F-AEA), a stable analogue of anandamide, on the expression level and subcellular localisation of b-catenin in MDA MB 231 and MCF7 cells. Subcellular fractionation analysis of cellular lysates of MDA MB 231 revealed that the majority of b-catenin was accumulated in the nuclear fractions.
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Treatment with the agonist of the CB1 receptor at 10 lM for 24 h reduced the expression levels of b-catenin protein and its accumulation in the nuclei of MDA MB 231 cells (Fig. 1A). Cells treated with an antagonist of CB1 receptor, SR141617 at 0.1 lM did not reveal any change of b-catenin localisation, instead the co-treatment with Met-F-AEA recovered the b-catenin subcellular localisation (Fig. 1A), suggesting that the Met-F-AEA effect was mediated by CB1 receptors. On the other hand, Met-F-AEA had not obvious effects on the intracellular level or nuclear translocation of b-catenin in MCF7 cells (Fig. 1A). 3.2. Anandamide induced the degradation of b-catenin Next we investigated the effects of Met-F-AEA on the expression and subcellular localisation of this transcription factor in MDA MB 231 cells. Western blot analysis (Fig. 1B) showed that the expression levels of b-catenin decreased in the nuclear fraction after 1 h of treatment while it increased in the cytosolic fraction at the same time point. At 3 and 6 h time points the expression of
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nuclear b-catenin was decreased compared to cytosolic b-catenin. After 24 h of treatment the protein levels of total as well as of cytoplasmic and nuclear b-catenin protein were significantly decreased. (Fig. 1B). These observations were further checked and verified in breast cancer cells using immunofluorescence staining (Fig. 2). Magnified cells inside the box clearly show the reduced staining of nuclear b-catenin after 12 h of treatment with Met-F-AEA compared to untreated cells. On the contrary the cells treated with CB1 receptor antagonist SR141716 alone or in combination with anandamide analogue showed the same profile of expression of nuclear b-catenin observed in untreated cells. Since nuclear accumulation of b-catenin is inversely correlated with phosphorylation at certain key residues of b-catenin (Ser33/Ser37/Thr41), we tested the effect of Met-FAEA on the levels of b-catenin phosphorylation at these sites. Western blot analysis revealed that treatment of cells with the CB1 receptor agonist increased the phosphorylation of b-catenin at Ser33/Ser37/Thr41 in a time-dependent manner (Fig. 1B). As the b-catenin phosphorylation occurs in a multiprotein complex
Fig. 1. Effect of Met-F-AEA on the subcellular localisation of b-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-b-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 b-catenin. (*p < 0.05, in comparison with control). (Ctr: DMSO control). Met-F-AEA did not have effects on the subcellular localisation of b-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-3b(P-GSK3b) total GSK-3b (T-GSK3b), Phosphorylated b-catenin (Pb-catenin, Ser33/Ser37/Thr41) and b-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 10 lM MG132 and 10 lM Met-F-AEA for 24 h and analysed by western blotting with antibodies against b-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.
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Fig. 2. Cellular localisation of b-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 10 lM Met-F-AEA for 12 h and with or without SR141716 for the same time, then they were fixed and stained for b-catenin. Bar 10 lm.
containing axin, GSK3 a/b, we also investigated the effect of Met-F-AEA treatment of MDA MB 231 cells on the expression levels and the phosphorylation state of GSK-3b. This kinase is known to target b-catenin for proteasomal degradation via combined phosphorylation at key residues of b-catenin.22 As shown in Fig. 1B, the phosphorylation of GSK3b was significantly increased upon the treatment with CB1 receptor agonist and it was attenuated after 24 h in MDA MB 231 cells. Moreover, because LRP6 is an essential Wnt co-receptor for the Wnt/b-catenin signalling pathway, and LRP6 phosphorylation is critical for Wnt/b-catenin signalling activation induced by Wnt proteins,23–25 we examined LRP6 expression and phosphorylation after methyl-F-anandamide treatment. As shown in Fig. 1D we found that both the total cellular level of endogenous LRP6 and its phosphorylation status were greatly decreased in treated cells in comparison to control cells. Finally, in order to examine the involvement of the proteasome in the anandamide-mediated b-catenin downregulation, we used a proteasome inhibitor, MG132, in MDA MB 231 cells treated with the CB1 receptor agonist. As shown in Fig. 1C, the treatment of these cells with anandamide analogue consistently led to a decrease in the cytosolic b-catenin level while the co-treatment with MG132 was able to abrogate this effect.
3.3. Anandamide inhibited the TCF promoter activity b-catenin acts to regulate the transcription of genes through the binding of a complex of b-catenin and T Cell Factor (TCF) family of transcription factors to specific promoter elements. The decrease of nuclear b-catenin by Met-F-AEA treatment suggested that b-catenin nuclear signalling might have been attenuated. So, we next evaluated the effect of Met-F-AEA on the transcriptional activities of b-catenin in MDA MB 231 cells by using the TOPflash/FOPflash reporter system. The TOPflash luciferase reporter plasmid contains three copies of the consensus T-cell factor (TCF) binding sites upstream of the luciferase gene, whereas its negative control version (FOPflash) carries mutations at these binding sites. The ratios of the luciferase activities of the TOPflash versus FOPflash were considered a measure of the relative transcriptional activity of the b-catenin/TCF complexes. We investigated the effect of CB1 receptor activation on TCF4 activity. As shown in Fig. 3A, Met-F-AEA-treatment for 24 h reduced luciferase activity (TOPFlash) in MDA MB 231 cells. In the cells treated with anandamide analogue in the presence of antagonist of CB1 receptor the TCF4 activity was recovered while the antagonist alone did not have any effect (Fig. 3A). b-catenin is known to relieve the inhibition of TCF/lymphoid enhancer
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factor by repressors leading to transcriptional activation of target genes, such as c-Myc, metalloproteinase-2 (MMP-2) and cyclin D1. Next, we determined the effect of Met-F-AEA treatment on the protein levels of c-myc, cyclin D1 and MMP-2 in the same breast cancer cell line. As expected, Met-F-AEA treatment caused a significant decrease in the cyclin D1, c-Myc and MMP-2 protein levels in MDA MB 231 cells (Fig. 3B). On the contrary, in MCF7 cells we did not observe changes in the transcriptional activity of TOPflash luciferase reporter plasmid (Fig. 3A). 3.4. Anandamide upregulated E-cadherin protein expression in MDA MB 231 cancer cells Accumulating evidence points to a critical role of epithelial-mesenchymal transition (EMT) like events
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during tumour progression and malignant transformation, endowing the incipient cancer cell with invasive and metastatic properties. Several oncogenic pathways (Wnt/b-catenin, Notch signalling, Ras and PI3K/Akt) induce EMT and a critical molecular event is the downregulation of the cell adhesion molecule E-cadherin.26 MDA MB 231 cells, a highly invasive ‘basal’ type and oestrogen-independent fibroblastic human breast cancer cell line,27 undergo autocrine Wnt signalling which downregulate E-cadherin expression at cell–cell borders and translocate b-catenin to the nuclear compartment and at the same time are characterised by increased levels of Snail1.28,29 Unlike MDA MB 231 cells, the MCF-7 cell line is a well accepted representative of oestrogen receptor-positive ‘luminal’ type breast cancer that it exhibits epithelial phenotype with high expression of E-cadherin and Cytokeratin 18, while the expression of
Fig. 3. Met-F-AEA inhibited the transcriptional activity of b-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 b-galactosidase gene. After transfection cells were treated with Met-F-AEA at 10 lM with or without SR141716 at 0.1 lM as indicated Luciferase activity was determined 24 h post-transfection, normalised against values for the corresponding b-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 b-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).
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mesenchymal markers vimentin, fibronectin and N-cadherin is not detectable in MCF-7 cells27 (Fig. 4A). Initially we studied whether anandamide administration affected EMT by modulation of expression levels of E-cadherin. As shown in Fig. 4A, the exposure of MDA MB 231 cells to 10 lM Met-F-AEA at several times resulted in an upregulation of E-cadherin expression in a time dependent manner. This result was checked by immunofluorescence staining in MDA MB 231 breast cancer cells. Cells treated with Met-F-AEA enhanced the levels of epithelial biomarker, E-cadherin, which is clearly evident by the strong intensity of fluorescence staining to the cellular membrane compared to untreated controls (Fig. 4B). As shown in Fig. 4A the anandamide-mediated induction of E-cadherin expression in this cell line was accompanied by a marked decrease in mesenchymal markers (vimentin, fibronectin and N-cadherin) (Fig. 4A). Moreover, it significantly induced the expression of Cytokeratin 18 in MDA MB 231 cells, where the basal levels of the above epithelial gene products are low. The MCF7 cells have undetectable vimentin (Fig. 4A) expression and their low basal fibronectin levels remained unchanged after the anandamide treatment. Furthermore, because Wnt signals have been reported to promote migration and EMT in breast cancer cells through stabilisation of Snail128 and
up-regulation of the transcription factors Slug and Twist29,30 we analysed the effect of anandamide treatment on mRNA levels of these transcriptional factors. We also observed the mRNA levels of the E-cadherin and MMP2 in MDA MB 231 cells by quantitative RT-PCR analysis. In the cells treated with anandamide analogue at several times we observed that the mRNA levels of Snail, Slug, Twist and MMP2 were decreased instead the mRNA levels of the E-cadherin were increased in treated cells compared with control cells (Fig. 5). 3.5. Reversal of doxorubicin-induced EMT by Anandamide in MCF7 cells In order to confirm anandamide-mediated inhibition of epithelial-mesenchymal transition (EMT) we used an experimental system involving Adriamycin at 6 ng/ml and MCF7 cells. Exposure of MCF7 cells to Adriamicyn for 36 h induced EMT as described by Li et al.31 We observed that DMSO-treated control MCF7 cells exhibited round and well-packed cobblestone appearance, a morphological feature of epithelial cells (Fig. 6). Morphology of the MCF7 cells was altered after 36 h of exposure to Adriamicyn with a large fraction of cells exhibiting mesenchymal phenotype characterised by spindle-shaped
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 10 lm.
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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 (0–24 h).
morphology with cell scattering and loss of cell–cell contact (Fig. 6). These phenomena were associated with decreased expression of E-cadherin and Cytokeratin 18 and up-regulation of N-cadherin, vimentin and Fibronectin (Fig. 7). The loss of epithelial markers, including E-cadherin, and a corresponding increase in mesenchymal markers, such as vimentin, N-cadherin are the critical events signalling the loss of the epithelial phenotype and commencement of mesenchymalisation.32Furthermore, we observed that the anandamide administration at 10 lM for 36 h alone did not have an appreciable effect on MCF7 morphology whereas the Adriamicyn-induced EMT was inhibited in the presence of anandamide with restoration of cell–cell contact (Fig. 6). Next, we analysed by immunoblotting cellular lysates from anandamidetreated cells in the presence or not of Adriamycin to confirm reversal of EMT by anandamide. As shown in Fig. 7, exposure of MCF7 cells to Adriamicyn resulted in downregulation of E-cadherin protein (80% decrease compared with DMSO treated control) and Cytokeratin 18, and in induction of vimentin (2-fold induction compared with DMSO-treated control). On the contrary in the cells cotreated with anandamide we observed the recovery of
epithelial proteins and the loss of the mesenchymal markers (Fig. 7). 4. Discussion Our studies in vitro showed that Met-F-AEA at 10 lM inhibited the growth of adenocarcinoma breast cancer cells, MDA MB 231 cells. These data in vitro are further corroborated by our animal study showing that supplement therapy could inhibit the breast tumour development in nude mice. In addition, we observed a much lower degree of breast tumour metastasis in mice treated with Met-F-AEA.5 Various studies have shown that alterations in the b-catenin pathway may contribute to progression of breast cancer. Oncogenic activation of the Wnt/b-catenin-signalling pathway has been reported in the abnormal accumulation of b-catenin and nuclear translocation of b-catenin, where it binds to transcription factor TCF/lymphoid enhancer factor (LEF) and consequently activates a cluster of genes that ultimately establish the oncogenic phenotype such as c-Myc, MMP-2 and cyclin D1. Furthermore, increased b-catenin activity was found to be significantly correlated with
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Fig. 6. Adriamycin (ADM) induced EMT in MCF7 cells were treated with or without Adriamycin (25 lg/mL) for 36 h. EMT was examined by phase-contrast microscopy(Zeiss (100 magnification; bar, 10 lm). Met-F-AEA restored the cell–cell contact in Adriamicyn treated cells.
Fig. 7. Western blotting of expression levels of mesenchymal and of epithelial markers in MCF7cells treated with or without Adriamycin (ADM) for 36 h. 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).
the poor prognosis of breast cancer patients.12 Consistent with these clinical data, numerous animal studies have shown that aberrant activation of the Wnt/b-catenin signalling, either by overexpression of canonical Wnt proteins or by direct stabilisation of b-catenin, can lead to mammary tumourigenesis.8–11 Recently it has been shown that the constitutive Wnt pathway activation, as evidenced by accumulation of active form of b-catenin in the nuclei, was only observed in a few
invasive breast carcinoma cell lines, including MDA MB 231 cells.33 In this study we observed that Met-FAEA treatment decreased the protein level of b-catenin in nuclei of treated cells thus inhibiting the transcription of proliferation-associated genes. In most cases intracellular accumulation of b-catenin is prevented by glycogen synthetase kinase 3 beta(GSK-b)-dependent phosphorylation of b-catenin and subsequent ubiquitination and degradation through the proteosome.34 Interference with
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b-catenin degradation such as occurs during activation of the canonical Wnt signalling pathway leads to accumulation of dephosphorylated (DP)-b-catenin that translocates to the nucleus and functions as a transcription factor in a complex with TCF/LEF-1 to activate genes such as c-Myc, cyclin-D1 and Snail1, involved in cell survival, proliferation and migration. GSK3a/b is known to be regulated by several signalling pathways, including PI3K/Akt, MEK/ERK, and Wnt signalling pathway.35 Among them the Wnt/GSK3b pathway is most extensively studied. In the presence of Wnt signalling, casein kinase I (CKI) and GSK3b become inactivated, leading to cytoplasmic, and subsequently nuclear accumulation of b-catenin; this transcriptional factor in the nucleus forms complexes with members of the lymphoid enhancer factor/T-cell factor (LEF/TCF) family of transcription factors to activate transcription.8,9 In the absence of Wnt signalling, b-catenin is constitutively phosphorylated by GSK3a/band CKI, at serine and threonine residues in the N-terminal region, resulting in ubiquitination and subsequent proteosomal degradation.11 The CB1receptor activation by Met-F-AEA markedly induced phosphorylation GSK3b, with the consequent decrease of the intracellular accumulation and nuclear translocation of b-catenin. As the inhibitor of proteosomal MG123 inhibited the degradation of bcatenin induced by Met-F-AEA we can suggest that the activation of CB1 receptor could activate the degradation of b-catenin. The suppression of this transcriptional factor caused a decrease in b-catenin transcriptional activities, suppressing the expression of its target genes such as cyclin D1, c-myc and MMP2 inhibiting in turn the cell proliferation. Moreover we observed that the methyl-F-anandamide induced degradation of the LRP6 receptor and it inhibited its phosphorylation suggesting that this anandamide analogue is an inhibitor of Wnt/b-catenin signalling pathway in MDA MB 231 cells. In MCF7 cells although the expression of b-catenin is detectable, this protein is localised mainly in the nucleus where its transcriptional activity is not changed upon anandamide treatment, suggesting that Wnt/b-catenin signalling might not be the major player in determining the oncogenic phenotype of this breast cancer cell line. The Wnt signalling pathway mediates a wide variety of processes, including cell proliferation, migration, differentiation, adhesion and death.36 Furthermore, Wnt signals have been reported to promote migration and EMT in breast cancer cells through stabilisation of Snail37and up-regulation of the transcription factors Slug and Twist38,39 which are both known to be transcriptional repressors of E-cadherin. The EMT is essential for normal physiological processes such as embryonic development, tissue remodelling and wound healing. Moreover, the EMT assumes a central place in the pathogenesis of aggressive cancers.26,27 Here we observed for the first time that Met-F-AEA inhibited
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EMT in MDA MB 231 cells. More importantly, the CB1 activation by Met-F-AEA elicited E-cadherin induction and suppression of mesenchymal markers as vimentin, N-cadherin and fibronectin in MDA MB 231 cells. The E-cadherin is regarded as a tumour suppressor because of its role in maintenance of epithelial phenotype and it is frequently downregulated during cancer progression and correlates with poor prognosis.40,41 We observed that Met-F-AEA administration suppressed expression of Snail1, Slug and Twist and increased transcription of E-cadherin. More we described that Met-FAEA treatment clearly decreased vimentin42 protein level in MDA MB 231 cells and during experimental EMT in MCF7 cells. We found that the Adriamicyntreated MCF7 cells showed morphological alterations from epithelial to mesenchymal phenotype in association with downregulation of E-cadherin concomitant with gain of vimentin, N-cadherin and fibronectin protein levels. The co-treatment with Met-F-Anandamide in MCF7 cells prevented the EMT induction by Adriamicyn. In conclusion, this study is the first published report to document the inhibition of EMT by Met-F-AEA that is characterised by the upregulation of E-cadherin, the downregulation of mesenchymal markers such as vimentin and fibronectin and suppression of transcriptional repressors of E-cadherin such as Snail1, Slug and Twist. In summary, the outcome of this study suggests that the endo/cannabinoids have the ability to block or inhibit the invasive potential of breast cancer cells and the suggested anti-invasion effect of this anandamide analogue is mediated through the inactivation of the b-catenin. Overall our results revealed the ability of this endo/cannabinoid to restore epithelial morphology by the induction of E-cadherin in metastatic breast cancer cells. Conflict of interest statement None declared. Acknowledgments This study was supported by AssociazioneEducazione e RicercaMedicaSalernitana (ERMES). S.P. and E.C. were supported by fellowships from FIRC (Italian Foundation for Cancer Research). A.M. was supported by a FondazioneItalianaSclerosiMultipla (FISM) fellowship. References 1. Bifulco M, Malfitano AM, Pisanti S, Laezza C. Endocannabinoids in endocrine and related tumours. Endocr Relat Cancer 2008;15:391–408. 2. Bifulco M, Laezza C, Pisanti S, Gazzerro P. Cannabinoids and cancer: pros and cons of an antitumour strategy. Br J Pharmacol 2006;148:123–35. 3. Mackie K, Stella N. Cannabinoid receptors and endocannabinoids: evidence for new players. AAPS J 2006;8:E298–306.
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4. Bifulco M, Laezza C, Portella G, et al. Control by the endogenous cannabinoid system of rasoncogene-dependent tumor growth. FASEB J 2001;15:2745–7. 5. Portella G, Laezza C, Laccetti G, et al. Inhibitory effects of cannabinoid CB1 receptor stimulation on tumor growth and metastatic spreading. Actions on signals involved in angiogenesis and metastasis. FASEB J 2003;17:1771–3. 6. Pisanti S, Borselli C, Gazzerro P, Laezza C, Bifulco M. Antiangiogenetic activity of the endocannabinoidanandamide.Correlation to its tumour-suppressor efficacy. J Cell Physiol 2007;211:495–503. 7. Grimaldi C, Pisanti S, Laezza C, et al. Anandamide inhibits adhesion and migration of breast cancer cells. Exp Cell Res 2006;312:363–73. 8. Clevers H. Wnt/beta-catenin signaling in development and disease. Cell 2006;127:469–80. 9. Karim R, Tse G, Putti T, Scolyer R, Lee S. The significance of the Wnt pathway in the pathology of human cancers. Pathology 2004;36:120–8. 10. Huber AH, Weis WI. The structure of the beta-catenin/E-cadherin complex and the molecular basis of diverse ligand recognition by beta-catenin. Cell 2001;105:391–402. 11. Brown AM. Wnt signaling in breast cancer: have we come full circle? Breast Cancer Res 2001;3:351–5. 12. Lin SY, Xia W, Wang JC, et al. b-Catenin, a novel prognostic marker for breast cancer: its roles in cyclin D1 expression and cancer progression. Proc Natl Acad Sci USA 2000;97:4262–6. 13. He TC, Sparks AB, Rago C, Hermeking, et al. Identification of cMYC as a target of the APC pathway. Science 1998;281:1509–12. 14. Behrens J. Control of beta-catenin signaling in tumor development. Ann NY Acad Sci 2000;910:21–33. 15. Turashvili G, Bouchal J, Burkadze G, Kolar Z. Wnt signaling pathway in mammary gland development and carcinogenesis. Pathobiology 2006;73:213–23. 16. Michaelson JS, Leder P. b-Catenin is a downstream effector of Wnt-mediated tumorigenesis in the mammary gland. Oncogene 2001;20:5093–9. 17. Hugo H, Ackland ML, Blick T, et al. Epithelial-mesenchymal and mesenchymal-epithelial transitions in carcinoma progression. J Cell Physiol 2007;213:374–83. 18. Tomaskovic-Crook E, Thompson EW, Thiery JP. Epithelial to mesenchymal transition and breast cancer. Breast Cancer Res 2009;11:213. 19. Hollier BG, Evans K, Mani SA. The epithelial-to-mesenchymal transition and cancer stem cells: a coalition against cancer therapies. J Mammary Gland Biol Neoplasia 2009;14:29–43. 20. Laezza C, Pisanti S, Crescenzi E, Bifulco M. Anandamide inhibits Cdk2 and activates Chk1leading to cell cycle arrest in human breast cancer cells. FEBS Lett 2006;580:5082–6076. 21. De Petrocellis L, Melck D, Palmisano A, et al. The endogenous cannabinoid anandamide inhibits human breast cancer cell proliferation Proc. Natl Acad Sci USA 1998;95:8375–80. 22. Liu C, Li Y, Semenov M, et al. Control of beta-catenin phosphorylation/degradation by a dual kinase mechanism. Cell 2002;108:837–47. 23. Tamai K, Zeng X, Liu C, Zhang X, Harada Y, et al. A mechanism for Wnt co-receptor activation. Mol Cell 2004;13:149–56.
24. Zeng X, Tamai K, Doble B, et al. A dual-kinase mechanism for Wntco-receptor phosphorylation and activation. Nature 2005;438:873–7. 25. Mi K, Dolan PJ, Johnson GV. The low density lipoprotein receptor related protein 6 interacts with glycogen synthase kinase 3 and attenuates activity. J Biol Chem 2006;281:4787–94. 26. Larue L, Bellacosa A. Epithelial–mesenchymal transition in development and cancer: role of phosphatidylinositol 30 kinase/ AKT pathways. Oncogene 2005;24:7443–54. 27. Blick T, Widodo E, Hugo H, et al. Epithelial mesenchymal transition traits in human breast cancer cell lines. Clin Exp Metastasis 2008;25:629–42. 28. Yook JI, Li XY, Ota I, et al. A Wnt-Axin2-3b cascade regulates Snail1 activity in breast cancer cells. Nat Cell Biol 2006;8:1398–406. 29. Conacci-Sorrell M, Simcha I, Ben-Yedidia T, et al. Autoregulation of E-cadherin expression by cadherin-cadherin interactions: the roles of b-catenin signaling, slug, and MAPK. J Cell Biol 2003;163:847–57. 30. Howe LR, Watanabe O, Leonard J, Brown AM. Twist is upregulated in response to Wnt1 and inhibits mouse mammary cell differentiation. Cancer Res 2003;63:1906–13. 31. Li QQ, Xu JD, Wang WJ, et al. Twist1-mediated Adriamycininduced epithelial–mesenchymal transition relates to multidrug resistance and invasive potential in breast cancer cells. Clin Cancer Res 2009;15:2657–65. 32. Lee JM, Dedhar S, Kalluri R, Thompson EW. The epithelial– mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol 2006;172:973–81. 33. Jo M, Lester RD, Montel V, et al. Reversibility of epithelial– mesenchymal transition (EMT) induced in breast cancer cells by activation of urokinase receptor-dependent cell signaling. J Biol Chem 2009;284, 22825–2283. 34. Henderson BR, Fagotto F. The ins and outs of APC and betacatenin nuclear transport. EMBO Rep 2002;9:834–9. 35. Rayasam GV, Tulasi VK, Sodhi R, Davis JA, Ray A. A glycogen synthase kinase 3: more than a namesake. Br J Pharmacol 2009;156:885–98. 36. Dihlmann S, von Knebel DM. Wnt/b-catenin-pathway as a molecular target for future anti-cancer therapeutics. Int J Cancer 2005;113:515–24. 37. Yook JI, Li XY, Ota I, et al. A Wnt-Axin2-3b cascade regulates Snail1 activity in breast cancer cells. Nat Cell Biol 2006;8:1398–406. 38. Conacci-Sorrell M, Simcha I, Ben-Yedidia T, et al. Autoregulation of E-cadherin expression by cadherin-cadherin interactions: the roles of b-catenin signaling, slug, and MAPK. J Cell Biol 2003;163:847–57. 39. Howe LR, Watanabe O, Leonard J, Brown AM. Twist is upregulated in response to Wnt1 and inhibits mouse mammary cell differentiation. Cancer Res 2003;63:1906–13. 40. Hazan RB, Qiao R, Keren R, Badano I, Suyama K. Cadherin switch in tumor progression. Ann NY Acad Sci 2004;1014:155–83. 41. Batlle E, Sancho E, Franci C, et al. The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat Cell Biol 2000;2:84–9. 42. Ivaska J, Pallari HM, Nevo J, Eriksson JE. Novel functions of vimentin in cell adhesion, migration, and signaling. Exp Cell Res 2007;313:2050–62.
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journal homepage: www.ejcancer.info
Anandamide inhibits the Wnt/b-catenin signalling pathway in human breast cancer MDA MB 231 cells Chiara Laezza a,b,⇑, Alba D’Alessandro c, Simona Paladino b, Anna Maria Malfitano c, Maria Chiara Proto c, Patrizia Gazzerro c, Simona Pisanti c, Antonietta Santoro c, Elena Ciaglia c, Maurizio Bifulco c, Endocannabinoid Research Group a
Institute of Endocrinology and Experimental Oncology, IEOS CNR, Via Pansini 5, 80131 Naples, Italy Department of Biology and Cellular and Molecular Pathology, University of Naples Federico II, Via Pansini, 80131 Naples, Italy c Department of Pharmaceutical and Biomedical Sciences, University of Salerno, Via Ponte don Melillo, 84084 Fisciano, Salerno, Italy b
Available online 14 March 2012
KEYWORDS Anandamide CB1 receptor Wnt/b-catenin pathway Epithelial-mesenchymal transition (EMT) E-cadherin
We previously showed that methyl-F-anandamide, a stable analogue of the anandamide, inhibited the growth and the progression of cultured human breast cancer cells. As accumulating evidences indicate that the constitutive activation of the canonical Wnt pathway in human breast cancer may highlight a key role for aberrant activation of the b-catenin–TCF cascade and tumour progression, we studied the anandamide effect on the key elements of Wnt pathway in breast cancer cells. In this study we described that the treatment of human breast cancer cells, MDA MB 231 cells, with methyl-F-anandamide reduced protein levels of b-catenin in the cytoplasmic and nuclear fractions inhibiting the transcriptional activation of T Cell Factor (TCF) responsive element (marker for b-catenin signalling). The anandamide treatment resulted in up-regulation of epithelial markers, like E-cadherin with a concomitant decrease in protein levels of mesenchymal markers, including vimentin and Snail1. We, furthermore, observed that the induction of experimental epithelial-mesenchymal transition by exposure to adriamycin in MCF7 human breast cancer cell line was inhibited by anandamide treatment. In the present study we reported a novel anticancer effect of anandamide involving the inhibition of epithelial-mesenchymal transition, a process triggered during progression of cancer to invasive state. Ó 2012 Elsevier Ltd. All rights reserved.
Abstract
1. Introduction ⇑ Corresponding author at: Institute of Endocrinology and Experi-
mental Oncology, IEOS CNR, Via Pansini 5, 80131 Naples, Italy. E-mail addresses: [email protected], [email protected] (C. Laezza). 0959-8049/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.ejca.2012.02.062
The endocannabinoid system, comprising the cannabinoid CB1 and CB2 receptors, the endogenous ligands, endocannabinoids and the enzymes that regulate their biosynthesis and degradation, is a ubiquitous signalling
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system involved in the control of cell fate. The endocannabinoids (anandamide, AEA and 2-arachidonoyglycerol, 2AG), agonists of CB1 receptors, have been shown to inhibit the growth of tumour cells in culture and animal models by modulating key cell signalling pathways.1–3 We have shown that a metabolically stable anandamide analogue, 2-methyl-20 -F-anandamide (Met-F-AEA) by cannabinoid CB1 receptors activation arrested the growth of K-ras-dependent tumours.4,5 In addition, we described that Met-F-AEA treatment inhibited vascular endothelial cell migration by decreasing the expression and the activity of metalloprotease-2, MMP-2, a proteolytic enzyme involved in tissue remodelling during angiogenesis and metastasis6 and it reduced the cell migration of the highly invasive and metastatic breast cancer cell lines, MDA MB 231.7 In this study, we investigated the effect of CB1 receptor activation by Met-F-AEA on the Wnt/b-catenin signalling pathway. This pathway is highly regulated and it has important functions in development, tissue homeostasis and regeneration. Activation of the canonical Wnt pathway involves the stabilisation of b-catenin through the binding of Wnt ligands to cell surface receptors: Frizzled (Fz) family receptors and lowdensity lipoprotein receptor (LDLR)-related protein 5 (LRP5) and LRP6. In the absence of Wnt ligands, b-catenin is phosphorylated by a multiprotein degradation complex, which marks it for ubiquitination and degradation by the proteasome. In the presence of appropriate Wnt ligands, b-catenin is stabilised in the cytoplasm, promoting its translocation to the nucleus.8–11 In the nucleus, b-catenin interacts with T-Cell Factor (TCF)/Lef transcription factors and activates cell transcription of target genes, including cyclin D1,12 c-Myc13 and metalloproteases that promote cell proliferation, differentiation and tissue development.14 In the mammary tissues, Wnt signalling plays an important role in stem cell self-renewal and mammary gland development.15 Compelling evidence indicates that when the Wnt/b-catenin pathway is aberrantly activated, it may lead to mammary carcinogenesis.16 In this study we examined the effect of the CB1 receptor activation by Met-F-AEA on modulation of the components of the Wnt/b-catenin signalling pathway, more, we showed that anandamide treatment inhibited epithelial-mesenchymal transition (EMT)17–19 involved in progression of cancers to invasive state
2. Materials and methods 2.1. Materials Met-F-AEA (2-methyl-20 -F-anandamide) was purchased from Sigma. The selective CB1 antagonist, SR141716, was kindly provided by Sanofi-Aventis (Montpellier, France). Doxorudicin hydrochloride (Adriamycin) and MG132 was from SIGMA, anti-b-catenin, anti-E-cadherin, anti N-cadherin, anti-vimentin, anti-phospho-GSK3b, anti-GSK3b
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and anti-Cytokeratin 18, anti-fibronectin, were purchased from Santa Cruz Biotechnology, anti-phospho (Ser33/Ser37/Thr41)-b-catenin, anti-phospho LRP6 and anti-LRP6 were from Cell Signalling. 2.2. Cell culture MDA MB 231, an invasive human breast carcinoma cell line, was grown in RPMI 1640 medium (Gibco BRL Life technologies, MD) supplemented with 10% inactivated foetal bovine serum (FBS) and 2 mM L-glutamine. MCF7, a non invasive human breast cancer cell line, was grown in DMEM medium supplemented with 10% inactivated FBS and 2 mM l-glutamine. Cells were cultured at 37 °C in a humidified 5% CO2 atmosphere. 2.3. Subcellular fractionation Cells were resuspended in a hypotonic buffer [10 mmol/ L Tris–HCl (pH 7.5), 25 mmol/L KCl, 2 mmol/L magnesium acetate, 1 mmol/L DTT, 0.5 mmol/L phenylmethylsulphomyl fluoride (PMSF), protease inhibitor cocktails]. After incubating on ice for 10 min, cell membranes were disrupted by 10 passes through a 23-gauge needle, and nuclear isolation was monitored under a microscope. After centrifugation for 5 min at 1000g, the supernatant was saved as the ‘cytoplasmic fraction’. The nuclear pellet was washed once with the hypotonic buffer and lysed with hypertonic buffer (hypotonic buffer plus 400 mmol/L KCl and 20% glycerol). The lysates were then centrifuged at 12,000g for 5 min, and the supernatant was collected as the ‘nuclear fraction’ and frozen at 80 °C. Total cell lysates and cytoplasmic or nuclear fractions were separated by SDS–PAGE, transferred to nitrocellulose membranes, and then probed with various primary antibodies to determine the expression of the signalling proteins. 2.4. Quantitative real-time PCR Total RNA was extracted from cultures using TRIzol reagent according to the manufacturer’s instructions. cDNA synthesis was achieved with 2 lg of total RNA by Moloney Murine leukaemia viruses (M-MuLV) reverse transcriptase. The efficiency of the used primers was evaluated by calculating the linear regression of Ct data points obtained with a series of different cDNA dilutions and inferring the efficiency from the slope of the line. We used the primers that gave efficiency close to 100%. The used primer sequences were the following: E-cadherin F 50 -TCG ACA CCC GAT TCA AAG TGG-30 and R 50 -TTC CAG AAA CGG AGG CCT GAT-30 ; MMP2 F 50 -ACA TCA AGG GCA TTC AGG AG-30 and R 50 -GCC TCG TAT ACC GCA TCA AT-30 ; Snail1 F 50 -AAG GAC CCC ACA TCC TTC TC-30 and R 50 -GGA GCT TCC CAG TGA GTC TG-30 ; Slug F50 -CGCCTCCAAAAAG CCAAAC-30 and R 50 -CGGTAGTCCACACAGT-
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GATG-30 ; Twist F 50 -GGAGTCCGCAGTCTTAC GAG-30 and R 50 TCTGGAGGACCTGGTAGAGG-30 ; b-actin F 50 -GCGTGACATCAAAGAGAAG-30 and bactin R 50 -ACTGTGTTGGCATAGAGG-30 . Real-time PCR was performed using So Fast Eva Green Super mix (Biorad) according to the manufacturer’s instructions in an iQ5 real-time PCR detection system (Bio-Rad). Fluorescence was determined at the last step of every cycle. Real-time PCR assay was done under the following universal conditions: 2 min at 50 °C, 10 min at 95 °C, 50 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min. Relative quantification was performed using the DDCt method. 2.5. b-Catenin/T-cell factor-lymphoid enhancer factor-1 transcription reporter assay The nuclear activity of endogenous b-catenin in MDA MB 231 and MCF7 cells was analysed by using the TOPflash/FOPflash reporter system (Upstate, Charlottesville, VA). Cells grown in 24-well dishes were transfected with either the TOPflash or FOPflash plasmid using LipofectAMINEPlus reagent. To normalise transfection efficiency in the reporter assays, cells were cotransfected with b-gal plasmid, which contains a functional b-gal luciferase gene cloned downstream of a herpes simplex virus thymidine kinase promoter (Promega, Madison, WI). Four hours after transfection, the culture medium was replaced by DMEM supplemented with 10% FBS in the presence or absence of drugs. Fortyeight hours after transfection, cells were washed with PBS and then lysed with the reporter lysis buffer (Promega). Luciferase activities in cell lysates were measured with the Promega Dual-Luciferase Reporter System and normalised to control b-gal luciferase signal. 2.6. Immunofluorescence Cells were plated on cover slips; when they reached 80% confluence, they were treated with Met-F-AEA (10 lM). After the incubation with various drugs and SR141716 at 0.1 lM, the cells were washed twice with PBS, fixed in 3.7% paraformaldehyde in PBS for 20 min and followed by two washes in 50 mM NH4Cl for 10 min. Permeabilisation was achieved by incubating the fixed cells in 0.1% Triton -100 in PBS for 5 min at room temperature. The cells were then blocked in final dilution buffer (FDB) (1 mM MgCl2, 1 mM CaCl2, 5% foetal calf serum and 2% BSA in PBS) for 30 min at room temperature. All primary and secondary antibody incubations were performed in FDB buffer. Cells were then incubated with specific antibodies for 1 h at room temperature and for the detection of b-catenin we used a goat anti-mouse IgG labelled with green fluorescent Alexa Fluor 488, while donkey anti-mouse IgG labelled red-fluorescent Alexa Fluor 594 was used for the
detection of E-cadherin. The relevant antibodies were then added at the dilutions recommended by the manufacturers. Cover slips were mounted on 50% glycerol in PBS and examined by using a Zeiss Laser Scanning Confocal Microscope. 2.7. Western blotting analysis After 24 h of incubation, cells were washed twice with PBS and re-suspended in lysis buffer (Hepes 50 mM, NaCl 150 mM, ethylene diamine tetra-acetic acid (EDTA) 50 mM, NaF 100 mM, Na ortovanadate 2 mM, glycerol, Na4P2O7 10 mM, 10% triton pH 7.5) and passed through a 23 gauge needle 10 times before centrifugation at 12000g at 4 °C. Aliquots of the cellular lysates (40 lg of protein) were boiled for 5 min and electrophoresed on SDS–polyacrylamide gel, transferred to nitrocellulose membranes and incubated with antibodies. The blots were blocked in PBS containing 0.1% Tween 20 and 5% non-fat dry milk for 1 h at room temperature. The filters were then probed overnight with primary specific antibodies. Immunodetection of specific proteins was carried out with horseradish peroxidase-conjugated donkey antimouse or anti-rabbit IgG (Biorad, Hercules, CA), using the enhanced chemiluminescence (ECL) system (Amersham, Buckinghamshire, United Kingdom). 2.8. Densitometric and statistical analysis The intensities of bands obtained from Western blots and RT-PCR were estimated with Alpha ImagerTm2200 (Alpha Innotech Corporation, United States of America). All the measurements were made in triplicate and all values are represented as mean ± SD. Comparison between groups was done using Student’s unpaired t test. In all statistical comparisons, p < 0.05 was used to indicate a significant difference. 3. Results 3.1. Anandamide decreased stabilisation and nuclear translocation of b-catenin Previously we observed that the CB1 receptor activation by Met-F AEA at 10 lM inhibited cell proliferation after 24 h of breast cancer cells in both ER-positive/luminal epithelial-like MCF7 cells and ER-negative/mesenchymal-like MDA MB 231 cells.20,21 As b-catenin/TCF transcriptional activity is known to regulate proliferative activities of breast epithelial cells, we first investigated the effects of methyl-F-anandamide (Met-F-AEA), a stable analogue of anandamide, on the expression level and subcellular localisation of b-catenin in MDA MB 231 and MCF7 cells. Subcellular fractionation analysis of cellular lysates of MDA MB 231 revealed that the majority of b-catenin was accumulated in the nuclear fractions.
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Treatment with the agonist of the CB1 receptor at 10 lM for 24 h reduced the expression levels of b-catenin protein and its accumulation in the nuclei of MDA MB 231 cells (Fig. 1A). Cells treated with an antagonist of CB1 receptor, SR141617 at 0.1 lM did not reveal any change of b-catenin localisation, instead the co-treatment with Met-F-AEA recovered the b-catenin subcellular localisation (Fig. 1A), suggesting that the Met-F-AEA effect was mediated by CB1 receptors. On the other hand, Met-F-AEA had not obvious effects on the intracellular level or nuclear translocation of b-catenin in MCF7 cells (Fig. 1A). 3.2. Anandamide induced the degradation of b-catenin Next we investigated the effects of Met-F-AEA on the expression and subcellular localisation of this transcription factor in MDA MB 231 cells. Western blot analysis (Fig. 1B) showed that the expression levels of b-catenin decreased in the nuclear fraction after 1 h of treatment while it increased in the cytosolic fraction at the same time point. At 3 and 6 h time points the expression of
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nuclear b-catenin was decreased compared to cytosolic b-catenin. After 24 h of treatment the protein levels of total as well as of cytoplasmic and nuclear b-catenin protein were significantly decreased. (Fig. 1B). These observations were further checked and verified in breast cancer cells using immunofluorescence staining (Fig. 2). Magnified cells inside the box clearly show the reduced staining of nuclear b-catenin after 12 h of treatment with Met-F-AEA compared to untreated cells. On the contrary the cells treated with CB1 receptor antagonist SR141716 alone or in combination with anandamide analogue showed the same profile of expression of nuclear b-catenin observed in untreated cells. Since nuclear accumulation of b-catenin is inversely correlated with phosphorylation at certain key residues of b-catenin (Ser33/Ser37/Thr41), we tested the effect of Met-FAEA on the levels of b-catenin phosphorylation at these sites. Western blot analysis revealed that treatment of cells with the CB1 receptor agonist increased the phosphorylation of b-catenin at Ser33/Ser37/Thr41 in a time-dependent manner (Fig. 1B). As the b-catenin phosphorylation occurs in a multiprotein complex
Fig. 1. Effect of Met-F-AEA on the subcellular localisation of b-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-b-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 b-catenin. (*p < 0.05, in comparison with control). (Ctr: DMSO control). Met-F-AEA did not have effects on the subcellular localisation of b-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-3b(P-GSK3b) total GSK-3b (T-GSK3b), Phosphorylated b-catenin (Pb-catenin, Ser33/Ser37/Thr41) and b-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 10 lM MG132 and 10 lM Met-F-AEA for 24 h and analysed by western blotting with antibodies against b-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.
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Fig. 2. Cellular localisation of b-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 10 lM Met-F-AEA for 12 h and with or without SR141716 for the same time, then they were fixed and stained for b-catenin. Bar 10 lm.
containing axin, GSK3 a/b, we also investigated the effect of Met-F-AEA treatment of MDA MB 231 cells on the expression levels and the phosphorylation state of GSK-3b. This kinase is known to target b-catenin for proteasomal degradation via combined phosphorylation at key residues of b-catenin.22 As shown in Fig. 1B, the phosphorylation of GSK3b was significantly increased upon the treatment with CB1 receptor agonist and it was attenuated after 24 h in MDA MB 231 cells. Moreover, because LRP6 is an essential Wnt co-receptor for the Wnt/b-catenin signalling pathway, and LRP6 phosphorylation is critical for Wnt/b-catenin signalling activation induced by Wnt proteins,23–25 we examined LRP6 expression and phosphorylation after methyl-F-anandamide treatment. As shown in Fig. 1D we found that both the total cellular level of endogenous LRP6 and its phosphorylation status were greatly decreased in treated cells in comparison to control cells. Finally, in order to examine the involvement of the proteasome in the anandamide-mediated b-catenin downregulation, we used a proteasome inhibitor, MG132, in MDA MB 231 cells treated with the CB1 receptor agonist. As shown in Fig. 1C, the treatment of these cells with anandamide analogue consistently led to a decrease in the cytosolic b-catenin level while the co-treatment with MG132 was able to abrogate this effect.
3.3. Anandamide inhibited the TCF promoter activity b-catenin acts to regulate the transcription of genes through the binding of a complex of b-catenin and T Cell Factor (TCF) family of transcription factors to specific promoter elements. The decrease of nuclear b-catenin by Met-F-AEA treatment suggested that b-catenin nuclear signalling might have been attenuated. So, we next evaluated the effect of Met-F-AEA on the transcriptional activities of b-catenin in MDA MB 231 cells by using the TOPflash/FOPflash reporter system. The TOPflash luciferase reporter plasmid contains three copies of the consensus T-cell factor (TCF) binding sites upstream of the luciferase gene, whereas its negative control version (FOPflash) carries mutations at these binding sites. The ratios of the luciferase activities of the TOPflash versus FOPflash were considered a measure of the relative transcriptional activity of the b-catenin/TCF complexes. We investigated the effect of CB1 receptor activation on TCF4 activity. As shown in Fig. 3A, Met-F-AEA-treatment for 24 h reduced luciferase activity (TOPFlash) in MDA MB 231 cells. In the cells treated with anandamide analogue in the presence of antagonist of CB1 receptor the TCF4 activity was recovered while the antagonist alone did not have any effect (Fig. 3A). b-catenin is known to relieve the inhibition of TCF/lymphoid enhancer
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factor by repressors leading to transcriptional activation of target genes, such as c-Myc, metalloproteinase-2 (MMP-2) and cyclin D1. Next, we determined the effect of Met-F-AEA treatment on the protein levels of c-myc, cyclin D1 and MMP-2 in the same breast cancer cell line. As expected, Met-F-AEA treatment caused a significant decrease in the cyclin D1, c-Myc and MMP-2 protein levels in MDA MB 231 cells (Fig. 3B). On the contrary, in MCF7 cells we did not observe changes in the transcriptional activity of TOPflash luciferase reporter plasmid (Fig. 3A). 3.4. Anandamide upregulated E-cadherin protein expression in MDA MB 231 cancer cells Accumulating evidence points to a critical role of epithelial-mesenchymal transition (EMT) like events
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during tumour progression and malignant transformation, endowing the incipient cancer cell with invasive and metastatic properties. Several oncogenic pathways (Wnt/b-catenin, Notch signalling, Ras and PI3K/Akt) induce EMT and a critical molecular event is the downregulation of the cell adhesion molecule E-cadherin.26 MDA MB 231 cells, a highly invasive ‘basal’ type and oestrogen-independent fibroblastic human breast cancer cell line,27 undergo autocrine Wnt signalling which downregulate E-cadherin expression at cell–cell borders and translocate b-catenin to the nuclear compartment and at the same time are characterised by increased levels of Snail1.28,29 Unlike MDA MB 231 cells, the MCF-7 cell line is a well accepted representative of oestrogen receptor-positive ‘luminal’ type breast cancer that it exhibits epithelial phenotype with high expression of E-cadherin and Cytokeratin 18, while the expression of
Fig. 3. Met-F-AEA inhibited the transcriptional activity of b-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 b-galactosidase gene. After transfection cells were treated with Met-F-AEA at 10 lM with or without SR141716 at 0.1 lM as indicated Luciferase activity was determined 24 h post-transfection, normalised against values for the corresponding b-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 b-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).
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mesenchymal markers vimentin, fibronectin and N-cadherin is not detectable in MCF-7 cells27 (Fig. 4A). Initially we studied whether anandamide administration affected EMT by modulation of expression levels of E-cadherin. As shown in Fig. 4A, the exposure of MDA MB 231 cells to 10 lM Met-F-AEA at several times resulted in an upregulation of E-cadherin expression in a time dependent manner. This result was checked by immunofluorescence staining in MDA MB 231 breast cancer cells. Cells treated with Met-F-AEA enhanced the levels of epithelial biomarker, E-cadherin, which is clearly evident by the strong intensity of fluorescence staining to the cellular membrane compared to untreated controls (Fig. 4B). As shown in Fig. 4A the anandamide-mediated induction of E-cadherin expression in this cell line was accompanied by a marked decrease in mesenchymal markers (vimentin, fibronectin and N-cadherin) (Fig. 4A). Moreover, it significantly induced the expression of Cytokeratin 18 in MDA MB 231 cells, where the basal levels of the above epithelial gene products are low. The MCF7 cells have undetectable vimentin (Fig. 4A) expression and their low basal fibronectin levels remained unchanged after the anandamide treatment. Furthermore, because Wnt signals have been reported to promote migration and EMT in breast cancer cells through stabilisation of Snail128 and
up-regulation of the transcription factors Slug and Twist29,30 we analysed the effect of anandamide treatment on mRNA levels of these transcriptional factors. We also observed the mRNA levels of the E-cadherin and MMP2 in MDA MB 231 cells by quantitative RT-PCR analysis. In the cells treated with anandamide analogue at several times we observed that the mRNA levels of Snail, Slug, Twist and MMP2 were decreased instead the mRNA levels of the E-cadherin were increased in treated cells compared with control cells (Fig. 5). 3.5. Reversal of doxorubicin-induced EMT by Anandamide in MCF7 cells In order to confirm anandamide-mediated inhibition of epithelial-mesenchymal transition (EMT) we used an experimental system involving Adriamycin at 6 ng/ml and MCF7 cells. Exposure of MCF7 cells to Adriamicyn for 36 h induced EMT as described by Li et al.31 We observed that DMSO-treated control MCF7 cells exhibited round and well-packed cobblestone appearance, a morphological feature of epithelial cells (Fig. 6). Morphology of the MCF7 cells was altered after 36 h of exposure to Adriamicyn with a large fraction of cells exhibiting mesenchymal phenotype characterised by spindle-shaped
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 10 lm.
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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 (0–24 h).
morphology with cell scattering and loss of cell–cell contact (Fig. 6). These phenomena were associated with decreased expression of E-cadherin and Cytokeratin 18 and up-regulation of N-cadherin, vimentin and Fibronectin (Fig. 7). The loss of epithelial markers, including E-cadherin, and a corresponding increase in mesenchymal markers, such as vimentin, N-cadherin are the critical events signalling the loss of the epithelial phenotype and commencement of mesenchymalisation.32Furthermore, we observed that the anandamide administration at 10 lM for 36 h alone did not have an appreciable effect on MCF7 morphology whereas the Adriamicyn-induced EMT was inhibited in the presence of anandamide with restoration of cell–cell contact (Fig. 6). Next, we analysed by immunoblotting cellular lysates from anandamidetreated cells in the presence or not of Adriamycin to confirm reversal of EMT by anandamide. As shown in Fig. 7, exposure of MCF7 cells to Adriamicyn resulted in downregulation of E-cadherin protein (80% decrease compared with DMSO treated control) and Cytokeratin 18, and in induction of vimentin (2-fold induction compared with DMSO-treated control). On the contrary in the cells cotreated with anandamide we observed the recovery of
epithelial proteins and the loss of the mesenchymal markers (Fig. 7). 4. Discussion Our studies in vitro showed that Met-F-AEA at 10 lM inhibited the growth of adenocarcinoma breast cancer cells, MDA MB 231 cells. These data in vitro are further corroborated by our animal study showing that supplement therapy could inhibit the breast tumour development in nude mice. In addition, we observed a much lower degree of breast tumour metastasis in mice treated with Met-F-AEA.5 Various studies have shown that alterations in the b-catenin pathway may contribute to progression of breast cancer. Oncogenic activation of the Wnt/b-catenin-signalling pathway has been reported in the abnormal accumulation of b-catenin and nuclear translocation of b-catenin, where it binds to transcription factor TCF/lymphoid enhancer factor (LEF) and consequently activates a cluster of genes that ultimately establish the oncogenic phenotype such as c-Myc, MMP-2 and cyclin D1. Furthermore, increased b-catenin activity was found to be significantly correlated with
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Fig. 6. Adriamycin (ADM) induced EMT in MCF7 cells were treated with or without Adriamycin (25 lg/mL) for 36 h. EMT was examined by phase-contrast microscopy(Zeiss (100 magnification; bar, 10 lm). Met-F-AEA restored the cell–cell contact in Adriamicyn treated cells.
Fig. 7. Western blotting of expression levels of mesenchymal and of epithelial markers in MCF7cells treated with or without Adriamycin (ADM) for 36 h. 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).
the poor prognosis of breast cancer patients.12 Consistent with these clinical data, numerous animal studies have shown that aberrant activation of the Wnt/b-catenin signalling, either by overexpression of canonical Wnt proteins or by direct stabilisation of b-catenin, can lead to mammary tumourigenesis.8–11 Recently it has been shown that the constitutive Wnt pathway activation, as evidenced by accumulation of active form of b-catenin in the nuclei, was only observed in a few
invasive breast carcinoma cell lines, including MDA MB 231 cells.33 In this study we observed that Met-FAEA treatment decreased the protein level of b-catenin in nuclei of treated cells thus inhibiting the transcription of proliferation-associated genes. In most cases intracellular accumulation of b-catenin is prevented by glycogen synthetase kinase 3 beta(GSK-b)-dependent phosphorylation of b-catenin and subsequent ubiquitination and degradation through the proteosome.34 Interference with
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b-catenin degradation such as occurs during activation of the canonical Wnt signalling pathway leads to accumulation of dephosphorylated (DP)-b-catenin that translocates to the nucleus and functions as a transcription factor in a complex with TCF/LEF-1 to activate genes such as c-Myc, cyclin-D1 and Snail1, involved in cell survival, proliferation and migration. GSK3a/b is known to be regulated by several signalling pathways, including PI3K/Akt, MEK/ERK, and Wnt signalling pathway.35 Among them the Wnt/GSK3b pathway is most extensively studied. In the presence of Wnt signalling, casein kinase I (CKI) and GSK3b become inactivated, leading to cytoplasmic, and subsequently nuclear accumulation of b-catenin; this transcriptional factor in the nucleus forms complexes with members of the lymphoid enhancer factor/T-cell factor (LEF/TCF) family of transcription factors to activate transcription.8,9 In the absence of Wnt signalling, b-catenin is constitutively phosphorylated by GSK3a/band CKI, at serine and threonine residues in the N-terminal region, resulting in ubiquitination and subsequent proteosomal degradation.11 The CB1receptor activation by Met-F-AEA markedly induced phosphorylation GSK3b, with the consequent decrease of the intracellular accumulation and nuclear translocation of b-catenin. As the inhibitor of proteosomal MG123 inhibited the degradation of bcatenin induced by Met-F-AEA we can suggest that the activation of CB1 receptor could activate the degradation of b-catenin. The suppression of this transcriptional factor caused a decrease in b-catenin transcriptional activities, suppressing the expression of its target genes such as cyclin D1, c-myc and MMP2 inhibiting in turn the cell proliferation. Moreover we observed that the methyl-F-anandamide induced degradation of the LRP6 receptor and it inhibited its phosphorylation suggesting that this anandamide analogue is an inhibitor of Wnt/b-catenin signalling pathway in MDA MB 231 cells. In MCF7 cells although the expression of b-catenin is detectable, this protein is localised mainly in the nucleus where its transcriptional activity is not changed upon anandamide treatment, suggesting that Wnt/b-catenin signalling might not be the major player in determining the oncogenic phenotype of this breast cancer cell line. The Wnt signalling pathway mediates a wide variety of processes, including cell proliferation, migration, differentiation, adhesion and death.36 Furthermore, Wnt signals have been reported to promote migration and EMT in breast cancer cells through stabilisation of Snail37and up-regulation of the transcription factors Slug and Twist38,39 which are both known to be transcriptional repressors of E-cadherin. The EMT is essential for normal physiological processes such as embryonic development, tissue remodelling and wound healing. Moreover, the EMT assumes a central place in the pathogenesis of aggressive cancers.26,27 Here we observed for the first time that Met-F-AEA inhibited
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EMT in MDA MB 231 cells. More importantly, the CB1 activation by Met-F-AEA elicited E-cadherin induction and suppression of mesenchymal markers as vimentin, N-cadherin and fibronectin in MDA MB 231 cells. The E-cadherin is regarded as a tumour suppressor because of its role in maintenance of epithelial phenotype and it is frequently downregulated during cancer progression and correlates with poor prognosis.40,41 We observed that Met-F-AEA administration suppressed expression of Snail1, Slug and Twist and increased transcription of E-cadherin. More we described that Met-FAEA treatment clearly decreased vimentin42 protein level in MDA MB 231 cells and during experimental EMT in MCF7 cells. We found that the Adriamicyntreated MCF7 cells showed morphological alterations from epithelial to mesenchymal phenotype in association with downregulation of E-cadherin concomitant with gain of vimentin, N-cadherin and fibronectin protein levels. The co-treatment with Met-F-Anandamide in MCF7 cells prevented the EMT induction by Adriamicyn. In conclusion, this study is the first published report to document the inhibition of EMT by Met-F-AEA that is characterised by the upregulation of E-cadherin, the downregulation of mesenchymal markers such as vimentin and fibronectin and suppression of transcriptional repressors of E-cadherin such as Snail1, Slug and Twist. In summary, the outcome of this study suggests that the endo/cannabinoids have the ability to block or inhibit the invasive potential of breast cancer cells and the suggested anti-invasion effect of this anandamide analogue is mediated through the inactivation of the b-catenin. Overall our results revealed the ability of this endo/cannabinoid to restore epithelial morphology by the induction of E-cadherin in metastatic breast cancer cells. Conflict of interest statement None declared. Acknowledgments This study was supported by AssociazioneEducazione e RicercaMedicaSalernitana (ERMES). S.P. and E.C. were supported by fellowships from FIRC (Italian Foundation for Cancer Research). A.M. was supported by a FondazioneItalianaSclerosiMultipla (FISM) fellowship. References 1. Bifulco M, Malfitano AM, Pisanti S, Laezza C. Endocannabinoids in endocrine and related tumours. Endocr Relat Cancer 2008;15:391–408. 2. Bifulco M, Laezza C, Pisanti S, Gazzerro P. Cannabinoids and cancer: pros and cons of an antitumour strategy. Br J Pharmacol 2006;148:123–35. 3. Mackie K, Stella N. Cannabinoid receptors and endocannabinoids: evidence for new players. AAPS J 2006;8:E298–306.
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