YTAAP-12760; No.

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Toxicology and Applied Pharmacology xxx (2013) xxxxxx

Contents lists available at SciVerse ScienceDirect

Toxicology and Applied Pharmacology

journal homepage: www.elsevier.com/locate/ytaap

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Molecular Oncology and Epigenetics Laboratory, The First Afliated Hospital of Chongqing Medical University, Chongqing, China Department of Pharmacology, Chongqing Medical University, Chongqing, China

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Hongzhong Li a, Jing Huang a, Bing Yang a, Tingxiu Xiang a, Xuedong Yin a, Weiyan Peng a, Wei Cheng a, Jingyuan Wan b, Fuling Luo b, Hongyuan Li a, Guosheng Ren a,

Mangiferin exerts antitumor activity in breast cancer cells by regulating matrix metalloproteinases, epithelial to mesenchymal transition, and -catenin signaling pathway

Keywords: Mangiferin Breast cancer Matrix metalloproteinase Epithelialmesenchymal transition -Catenin

Article history: Received 28 February 2013 Revised 26 April 2013 Accepted 14 May 2013 Available online xxxx

Although mangiferin which is a naturally occurring glucosylxanthone has exhibited promising anticancer activities, the detailed molecular mechanism of mangiferin on cancers still remains enigmatic. In this study, the anticancer activity of mangiferin was evaluated in breast cancer cell line-based in vitro and in vivo models. We showed that mangiferin treatment resulted in decreased cell viability and suppression of metastatic potential in breast cancer cells. Further mechanistic investigation revealed that mangiferin induced decreased matrix metalloproteinase (MMP)-7 and -9, and reversal of epithelialmesenchymal transition (EMT). Moreover, it was demonstrated that mangiferin signicantly inhibited the activation of -catenin pathway. Subsequent experiments showed that inhibiting -catenin pathway might play a central role in mangiferin-induced anticancer activity through modulation of MMP-7 and -9, and EMT. Consistent with these ndings in vitro, the antitumor potential was also veried in mangiferin-treated MDA-MB-231 xenograft mice where signicantly decreased tumor volume, weight and proliferation, and increased apoptosis were obtained, with lower expression of MMP-7 and -9, vimentin and active -catenin, and higher expression of E-cadherin. Taken together, our study suggests that mangiferin might be used as an effective chemopreventive agent against breast cancer. 2013 Published by Elsevier Inc.

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Introduction

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in worldwide females, accounting for 23% of the total cancer cases and 14% of the cancer deaths (DeSantis et al., 2011; Siegel et al., 2011). As it is widely accepted that carcinogenesis is a complex and multi-stage process, breast cancer prevention by the use of pharmacological agents, especially naturally occurring dietary substances has been considered as a practical approach to reduce the incidence of breast cancer. It has been revealed that many dietary materials such as soy isoavone genistein, olive oil phenols, grape polyphenols, axseed lignan and extracts from green tea or blueberry exert inhibitory effects on the growth and/or metastasis of breast cancer cells (Adams et al., 2010; Casaburi et al., 2013;

Abbreviations: MMP, matrix metalloproteinase; EMT, epithelialmesenchymal transition; MET, mesenchymalepithelial transition; ER, estrogen receptor; GSK-3, glycogen synthase kinase 3; DMSO, dimethyl sulfoxide; PI, propidium iodide; IHC, immunohistochemistry; SP, streptavidin-peroxidase; SD, standard deviation; ECM, extracellular matrix. Corresponding author at: Molecular Oncology and Epigenetics Laboratory, The First Afliated Hospital of Chongqing Medical University, No. 1 Youyi Road, Yuzhong District, Chongqing 400016, China. Fax: +86 2389012305. E-mail address: rgs726@163.com (G. Ren). 0041-008X/$ see front matter 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.taap.2013.05.011

Castillo-Pichardo and Dharmawardhane, 2012; Saggar et al., 2010; Ullah et al., 2011; Wu and Butler, 2011). Moreover, since effective therapeutic drugs are limited and drug resistance occurs frequently, these materials have also attracted more attentions in the exploration of effective anti-cancer drugs. Mangiferin is a naturally occurring glucosylxanthone and exists in several folk medicines and food such as mango which is one of the most popular, nutritionally rich tropical fruits. Mangiferin has been shown to exert many benecial biological activities including antioxidant, hypolipidemic, anti-inammatory, neuroprotective, immunomodulatory and hepatoprotective effects (Biradar et al., 2012; Das et al., 2012; Garcia et al., 2003; Guo et al., 2011; Marquez et al., 2012). Recent studies have demonstrated that mangiferin exhibits anticancer activity against a variety of cancers such as lung, cervical and breast cancers. Although it was reported that immunosuppressive effect or cell cycle modulation might be involved in the antitumor activity of mangiferin, the detailed mechanisms by which mangiferin inhibits cell growth and metastasis of breast cancer have not been fully elucidated (du Plessis-Stoman et al., 2011; Garcia-Rivera et al., 2011; Rajendran et al., 2008; Yao et al., 2010). In our study, we report the inhibitory effect of mangiferin on breast cancer cells in both in vitro and in vivo models. We identify that mangiferin inhibits cell proliferation and suppresses the migration and invasion of breast cancer cells, which may be related

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Please cite this article as: Li, H., et al., Mangiferin exerts antitumor activity in breast cancer cells by regulating matrix metalloproteinases, epithelial to mesenchymal transition, and -catenin signaling pathway, Toxicol. Appl. Pharmacol. (2013), http://dx.doi.org/10.1016/j.taap.2013.05.011

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to the modulatory effect of mangiferin on MMP-7 and -9, epithelial mesenchymal transition (EMT) and -catenin pathway. These ndings suggest that mangiferin might be a very promising candidate for breast cancer intervention and prevention. Materials and methods Reagents and cell lines. Mangiferin (C19H18O11, MW: 422.34, purity: 95%, shown in Fig. 1A) determined by HPLC as previously described was purchased from Sigma Chemical Co. (Sigma, St. Louis, MO, USA) (Zhang et al., 2012). Human estrogen receptor (ER)-negative breast cancer cell lines (MDA-MB-231 and BT-549), and ER-positive breast cancer cell lines (MCF-7 and T47D) were obtained from American Type Culture Collection (ATCC, Rockville, MD), and cultured in RPMI-1640 supplemented with 10% FBS and 1% penicillin/streptomycin. All the cells were incubated at 37 C in humidied atmosphere containing 5% CO2. Cell viability assay. Cells seeded into a 96-well plate at 4000 cells/well were treated with 100 l medium plus DMSO (vehicle control, nal concentration b 0.5%) or mangiferin (nal concentration: 75, 150 and 300 M) and incubated for 48 h. At the end of the drug exposure duration, cell viability was measured according to the protocol of CCK-8 (KeyGEN Biotech, Nanjing, China). All plates had control wells containing medium without cells to obtain a value for background spectrometric absorbance which was subtracted from the test sample readings. Data were expressed as ratios of treated to control cells, mean SD for three replications.

Flow cytometry analysis of cell apoptosis. For apoptosis analysis, Annexin V-FITC/propidium iodide (PI) staining (KeyGEN Biotech, Nanjing, China) was performed by Elite ESP ow cytometry according to the manufacturer's guidelines. In vitro scratch assay. The cancer cells were cultured in 6-well plates and grown in medium containing 10% FBS to nearly conuent cell monolayer, then carefully scratched using a plastic pipette tip to draw a linear wound in the cell monolayer of each well. The monolayer was washed twice with PBS to remove debris or the detached cells from the monolayer. Cancer cells were exposed to serum-free medium with or without different concentrations of mangiferin (12.5, 25 and 50 M) for 12 h. Wound closure was recorded and photographed using a microscope with Leica cameras.

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Fig. 1. Mangiferin inhibits cell proliferation and induces apoptosis in breast cancer cells. (A) Chemical structure of mangiferin. (B) Breast cancer cell lines were treated with various concentrations of mangiferin for 48 h, cell viability was measured by CCK-8 kit. Data were expressed as ratios of treated cells to control cells. (C) Cell apoptosis in MDA-MB-231 and MCF-7 cells induced by mangiferin was examined by ow cytometry analysis of Annexin V-FITC/PI staining. Numbers inside dot plots indicates the percentages of apoptotic MDA-MB-231 cells. All the experiments were performed thrice in triplicate. Mean SD, *p b 0.05 and **p b 0.01, vs. control group.

Please cite this article as: Li, H., et al., Mangiferin exerts antitumor activity in breast cancer cells by regulating matrix metalloproteinases, epithelial to mesenchymal transition, and -catenin signaling pathway, Toxicol. Appl. Pharmacol. (2013), http://dx.doi.org/10.1016/j.taap.2013.05.011

Cell migration/invasion assays. The cancer cell migration/invasion assays were conducted with transwell membranes (8 m pore size, 24-well plate, BD Biosciences, Billerica, MA). In migration assay, breast cancer cells were trypsinized, washed, and suspended in medium without FBS. To the lower wells of the chambers, migration-inducing medium (with 10% FBS) was added. Upper wells were lled with cells (20,000 cells/well) in serum-free medium containing various concentrations of mangiferin (12.5, 25 and 50 M) or vehicle solvent (DMSO). After 24 h, the top surface of the chambers was scraped using a cotton swab, and the cells on the lower surface of the membranes were xed for 15 min with methanol and then stained with Giemsa solution. Evaluation of completed transmigration was performed under

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the microscope, and random elds were scanned (four elds per lter) for the presence of cells at the lower membrane side only. For cell invasion assay, transwell membranes were rstly coated with 100 l Matrigel Matrix (1 mg/ml, BD Biosciences), and subsequent procedures were performed according to the same protocol in the migration assay. Western blotting. Cell lysate was prepared according to the method described by the protein extract kit (Active Motif Company, Carlsbad, USA). Protein concentrations were determined by BCA protein assay kit (Pierce Biotechnology Inc., Rockford, USA). Cell lysate was analyzed for Western blot analysis using MMP-2, -7 and -9, E-cadherin, vimentin, snail, slug, total and phosphor(p)-GSK-3, total and active -catenin, plus -actin (Cell Signaling Technology, Inc., Danvers, MA or Abcam Inc., Cambridge, MA). Antibody binding was visualized with an ECL chemiluminescence system and short exposure of the membrane to X-ray lms (Kodak, Japan). ELISA. The levels of MMP-2, -7 and -9 in cell culture supernatants were detected using ELISA kits (R&D Systems, Inc. Minneapolis, MN) according to the manufacturer's instructions. Briey, standards and samples were pipetted into the wells coated with specic antibodies for human MMP-2, -7 or -9, and incubated for 2 h at room temperature. A 200 l conjugate was added and incubated for 1 h on

the shaker after washing the wells for 4 times. Then 50 l of stop solu- 149 tion was placed in each well, followed by 200 l substrate solution 150 added. The intensity was measured at 450 nm within 30 min. 151 Indirect immunouorescence analysis. For the immunouorescence experiments, cells were prepared and analyzed under a uorescence microscope (Leica DM IRB) following the procedures described previously (Cole et al., 2009). Briey, cells were incubated with primary antibody against E-cadherin or vimentin (Cell Signaling Technology, Inc., Danvers, MA), and then incubated with DyLight 549 or DyLight 488 (Cwbiotech, Beijing, China) secondary antibody against rabbit IgG. Cells were then counterstained with DAPI and imaged with the uorescence microscope. Dual-luciferase reporter assay. TOPFlash (4 TCF binding sites) luciferase reporter was used as reported previously to elucidate the effect of mangiferin on the activation of -catenin signaling pathway (Wang et al., 2012). Reporter activity was analyzed by the dual-luciferase reporter assay system (Promega, Madison, WI) and normalized to the control Renilla.
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Fig. 2. Mangiferin inhibits breast cancer cell migration and invasion. (A) The wound healing assay. Conuent monolayers of MDA-MB-231 or BT-549 cells were scarred, and the cells were treated for 12 h with different concentrations (12.5, 25 and 50 M) of mangiferin or the control solvent (DMSO). The migration length of the cells in the denuded zone was quantied over a 12 h period by inverted microscopy. The left gure shows the wound healing assay in MDA-MB-231 cells, white lines indicate the scraped zone. The transwell migration assay (B) and the Matrigel invasion assay (C). After 24 h of incubation with or without mangiferin, cells that migrated to the lower chamber or invaded through the Matrigel were xed, stained, and counted using light microscopy. Random elds were scanned (four elds per lter) for the presence of cells on the lower side of the membrane. The left gures show cell migration assay and invasion assay in MDA-MB-231 cells respectively. All the experiments were performed thrice in triplicate. Mean SD, *p b 0.05 and **p b 0.01, vs. control group.

Please cite this article as: Li, H., et al., Mangiferin exerts antitumor activity in breast cancer cells by regulating matrix metalloproteinases, epithelial to mesenchymal transition, and -catenin signaling pathway, Toxicol. Appl. Pharmacol. (2013), http://dx.doi.org/10.1016/j.taap.2013.05.011

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Adenovirus infection. The adenovirus expressing -catenin, scrambled siRNA or siRNA -catenin (gift from Doctor Tong-chuan He, University of Chicago) was used following the procedure described previously (Luo et al., 2007). In addition to the expression of transgenes, Ad--

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catenin, Ad-scrambled siRNA and Ad-si -catenin also expressed RFP as a marker for monitoring transfection efciency. An analogous adenovirus expressing only RFP (Ad-RFP) was used as a control, and expression efciency was assessed by real-time PCR, Western blotting, and functional assays of -catenin signaling pathway. Animal experiments. All the animal studies were approved by the Animal Ethics Committee of Chongqing Medical University. 5-Week old severe combined immunodeciency (SCID) hairless female mice were purchased (Institute of Laboratory Animal Science, Chinese Academy of Medical Science, Beijing, China) and randomly divided into two groups of 9 mice each. All the mice were housed according to the

national and institutional guidelines for humane animal care. At 6 weeks of age, the mice were perorally (p.o.) gavaged with either 100 l water control or mangiferin (100 mg/kg weight). the animals were gavaged daily for the duration of the experiment. At 7 weeks of age, the mice were injected subcutaneously with MDA-MB-231 cells (2 106) in Matrigel on the right rear anks. Body weights were monitored weekly as an indicator of overall health. Tumor diameter was measured every week, and tumor volumes were calculated with the formula: tumor volume (mm3) = 0.5 length (mm) width2 (mm2). At the end of 5 weeks of gavage treatment, the mice were euthanized via CO2 asphyxiation. Tumors were then removed, weighed, and sent for immunohistochemistry (IHC) analysis.

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Fig. 3. Down-regulation of MMP-7 and -9 in breast cancer cells treated with mangiferin. (A) Breast cancer cells were treated with various concentrations of mangiferin for 48 h, the expression of MMP-2, -7 and -9 in cells was then measured by Western blotting. -Actin was used as an internal loading control. The blots shown are representative of six independent experiments. Mean SD, **p b 0.01, vs. control group. (B) The expression of MMP-2, -7 and -9 in cell supernatants collected from untreated or mangiferin-treated cells was detected by ELISA. The experiments were performed thrice in triplicate. Mean SD, *p b 0.05 and **p b 0.01, vs. control group.

Please cite this article as: Li, H., et al., Mangiferin exerts antitumor activity in breast cancer cells by regulating matrix metalloproteinases, epithelial to mesenchymal transition, and -catenin signaling pathway, Toxicol. Appl. Pharmacol. (2013), http://dx.doi.org/10.1016/j.taap.2013.05.011

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Please cite this article as: Li, H., et al., Mangiferin exerts antitumor activity in breast cancer cells by regulating matrix metalloproteinases, epithelial to mesenchymal transition, and -catenin signaling pathway, Toxicol. Appl. Pharmacol. (2013), http://dx.doi.org/10.1016/j.taap.2013.05.011

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Fig. 4. The reversal effect of mangiferin on EMT in breast cancer cells. (A) Morphological differences between control cells and cells treated with mangiferin (50 M) for 48 h (original magnication, 400). (B) Breast cancer cells were treated with different concentrations of mangiferin for 48 h, the effect of mangiferin on the expression of EMT markers was then detected by Western blotting. -Actin was used as an internal loading control. The blots shown are representative of six independent experiments. Mean SD, *p b 0.05 and **p b 0.01, vs. control group. (C) Immunostaining shows the up-regulation of E-cadherin and down-regulation of vimentin in breast cancer cells treated with mangiferin (50 M) for 48 h (original magnication, 400). Nucleus is stained with DAPI (blue), E-cadherin with DyLight 549 (red) and vimentin with DyLight 488 (green). The experiments were performed thrice in triplicate. Mean SD, *p b 0.05 and **p b 0.01, vs. control group. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.) 5

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Immunohistochemistry. Tumor tissues were xed in 4% formaldehyde solution (pH 7.0) and subsequently embedded in parafn. Immunohistochemical studies were performed using the standard streptavidin peroxidase (SP) method with the UltraSensitive TM SP Kit (Maixin-Bio, Fujian, China) according to the manufacturer's instructions. Tumor specimens were stained using Ki-67 antibody (Maixin-Bio, Fujian, China) for cell proliferation and cleaved caspase 3 antibody (Cell Signaling Technology, Inc., Danvers, MA) for apoptosis. Negative control was performed by replacing the primary antibody with PBS. Immunostained

slides were blindly evaluated by a trained pathologist under a transmis- 202 sion light microscope. 203

Statistical analysis. The data were presented as the mean values standard deviation (SD). Values were compared to controls with either Student's t-test or one-way ANOVA using Prism GraphPad 4 software (GraphPad Software, Inc.). Differences were considered signicant when the p values were 0.05.

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Fig. 5. Mangiferin inhibits the activation of -catenin pathway in breast cancer cells. (A) Breast cancer cells were treated with various concentrations of mangiferin for 48 h, the effects of mangiferin on the expression of total or p-GSK-3, and total or active -catenin were then measured by Western blotting. -Actin was used as an internal loading control. The blots shown are representative of six independent experiments. Mean SD, **p b 0.01, vs. control group. (B) Breast cancer cells were treated with various concentrations of mangiferin for 48 h, the effect of mangiferin on the activation of -catenin signaling pathway was then assessed by dual-luciferase reporter assay. Mean SD, n = 9, **p b 0.01, vs. control group.

Please cite this article as: Li, H., et al., Mangiferin exerts antitumor activity in breast cancer cells by regulating matrix metalloproteinases, epithelial to mesenchymal transition, and -catenin signaling pathway, Toxicol. Appl. Pharmacol. (2013), http://dx.doi.org/10.1016/j.taap.2013.05.011

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Results Mangiferin inhibits breast cancer cell proliferation As shown in Fig. 1B, we observed that mangiferin inhibited the cell proliferation of breast cancer cell lines including MDAMB-231, BT-549, MCF-7 and T47D in a dose-dependent manner. To clarify whether the decreased cell proliferation was due to the induction of cell apoptosis, ow cytometry for the apoptosis analysis was further performed. Following treatment with mangiferin (75, 150 and 300 M) for 48 h, signicantly increased apoptosis was found when breast cancer cells were treated with 300 M mangiferin, which may partly contribute to the decreased cell viability (Fig. 1C).

Mangiferin suppresses the metastatic potential of breast cancer cells In this study, high-metastatic cell lines MDA-MB-231 and BT-549 were utilized to evaluate the effect of mangiferin on cancer cell migration and invasion. Due to the fact that mangiferin could inhibit cell viability at the concentrations over 50 M (the IC50 in MDA-MB-231 or BT-549 cells are 298.6 and 273.8 M respectively, data not shown), the related experiments including wound-healing assay, transwell migration and invasion assays were performed at the concentrations of 12.5, 25 and 50 M. As shown in Figs. 2AB, in both the wound-healing and transwell migration assays, mangiferin dose-dependently inhibited cancer cell migration. Moreover, mangiferin treatment signicantly reduced the number of cells which could invade through the Matrigel-coated insert membranes (Fig. 2C).

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Fig. 6. Inactivation of -catenin signaling pathway is responsible for the anti-tumor activity of mangiferin in breast cancer cells. (A) Cell proliferation assay. Breast cancer cells transfected with Ad-RFP, Ad-scrambled siRNA, Ad-si -catenin or Ad--catenin were treated with or without mangiferin (150 M) for 48 h, cell viability was measured by CCK-8 kit. Data were expressed as ratios of treated cells to control cells. (B) and (C) The effect of -catenin on metastatic potential of breast cancer cells treated with or without mangiferin (25 M) was measured by both cell migration and invasion assays. The experiments above were performed thrice in triplicate. Mean SD, **p b 0.01. (D) Cells transfected with Ad-RFP, Ad-scrambled siRNA, Ad-si -catenin or Ad--catenin were treated with or without mangiferin (25 M) for 48 h, the expression of related proteins in breast cancer cells was then measured by Western blotting. -Actin was used as an internal loading control. The blots shown are representative of six independent experiments. Mean SD, **p b 0.01.

Please cite this article as: Li, H., et al., Mangiferin exerts antitumor activity in breast cancer cells by regulating matrix metalloproteinases, epithelial to mesenchymal transition, and -catenin signaling pathway, Toxicol. Appl. Pharmacol. (2013), http://dx.doi.org/10.1016/j.taap.2013.05.011

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Please cite this article as: Li, H., et al., Mangiferin exerts antitumor activity in breast cancer cells by regulating matrix metalloproteinases, epithelial to mesenchymal transition, and -catenin signaling pathway, Toxicol. Appl. Pharmacol. (2013), http://dx.doi.org/10.1016/j.taap.2013.05.011

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Down-regulation of MMP-7 and -9 by mangiferin Matrix metalloproteinases (MMPs), functioning in the remodeling of extracellular matrix (ECM), exert a great role in tumor invasion and metastasis. Since MMP-2, -7 and -9 were reported to be involved in breast cancer metastasis by numerous studies, the expression of MMP-2, -7 and -9 was detected by Western blotting and ELISA in our study (Beeghly-Fadiel et al., 2009; Jezierska and Motyl, 2009; Rydlova et al., 2008; Sullu et al., 2011). As depicted in Figs. 3A and B, MMP-7 and -9 were found down-regulated by mangiferin, while the expression of MMP-2 wasn't signicantly affected by mangiferin. These results strongly suggest that down-regulation of MMP-7 and -9 might be involved in the anti-metastatic activity of mangiferin. Mangiferin induces reversal of EMT in breast cancer cells The most comprehensive theory describing how initially quiescent tumor cells acquire metastatic capability is the EMT (Gomes et al., 2011). We next utilized two mesenchymal-like breast cancer cell lines, MDA-MB-231 and BT-549, to evaluate the effect of mangiferin on EMT. As shown in Fig. 4A, a signicant change from a spindle, broblast-like shape with migratory protrusions to characteristic cobblestone-like epithelial morphology was observed in mangiferintreated cancer cells. Consistent with these morphological changes, increased expression of epithelial marker E-cadherin and decreased mesenchymal markers such as vimentin, snail and slug were demonstrated in mangiferin-treated cancer cells (Figs. 4B and C). Collectively, these observations support that mangiferin induces an effective switch from mesenchymal to epithelial phenotype of breast cancer cells. Inactivation of -catenin pathway is involved in the anti-tumor activity of mangiferin

had no detectable toxicity since there were no differences in body weight between control and treated groups, without any side effects observed. Signicantly decreased proliferation (Ki-67 staining) and increased apoptosis (cleaved caspase 3 staining) were detected in the tumor specimens of mangiferin-treated mice (Fig. 7C). Furthermore, lower expression of MMP-7 and -9, vimentin and active -catenin, and higher expression of E-cadherin were obtained in mangiferintreated group by Western blotting (Fig. 7D). These results are in full agreement with our ndings in vitro, showing that mangiferin possesses a marked antitumor activity against breast cancer. Discussion Many studies about mangiferin focus on its antioxidant activity, for four aromatic hydroxyl groups which determine its strong antioxidant properties in the molecule of mangiferin. Although reports about the antitumor activity of mangiferin are limited, mangiferin was found to exert immunosuppressive antitumor effect on MDAMB-231 breast cancer cells, chemopreventive and chemotherapeutic effects on bowel and lung carcinogenesis, and inhibitory activity on cell proliferation and cycle of promyelocytic leukemic HL-60 cells (Garcia-Rivera et al., 2011; Rajendran et al., 2008; Yao et al., 2010; Yoshimi et al., 2001). Additionally, it was reported that mangiferin in combination with oxaliplatin favored apoptotic cell death and thereby improved the efcacy of oxaliplatin in vitro, and norathyriol as a metabolite of mangiferin suppressed UV-induced skin cancer (du Plessis-Stoman et al., 2011; Li et al., 2012). In our study, we showed that mangiferin could effectively inhibit cell proliferation of breast cancer cells, while only higher dose of mangiferin induced signicant apoptosis, suggesting that the results of the viability assay could be largely attributed to the inhibition of proliferation. Furthermore, it was demonstrated that mangiferin markedly suppressed the metastatic potential of breast cancer cells by wound healing, transwell migration and invasion assays. Taken together, these ndings strongly showed that mangiferin possessed multiple antitumor activities. Since mounting evidence has implied the great role of MMPs in the process of tumor invasion and metastasis, MMPs have always been regarded as potential therapeutic targets for cancers (Roy et al., 2009; Rydlova et al., 2008). MMP-7 and -9 which are important members of MMP family exert great effects on promoting cancer development. MMP-7 was found involved in the invasion of breast cancer through its collaboration with indicators of invasion, and elimination of MMP-7 in MDA-MB-231 cells was associated with low invasiveness and slow tumor growth (Jiang et al., 2005; Mylona et al., 2005). Similarly, a number of studies suggested that MMP-9 promoted metastasis, and might be associated with poor prognosis of breast cancer (Ranogajec et al., 2012; Scorilas et al., 2001). It was reported that mangiferin exerted anti-photoaging activity in UVB-irradiated hairless mice by regulating MMP-9 expression (Kim et al., 2012). In addition, mangiferin was found to selectively inhibit the expression of MMP-9 in PMA-stimulated human astroglioma cells without affecting other MMPs such as MMP-1, -2, -3, and -14 (Jung et al., 2012). In our study, it was demonstrated that mangiferin signicantly suppressed the expression of MMP-7 and -9 in breast cancer cells, suggesting that inhibition of MMP-7 and -9 might be involved in the anti-metastatic potential of mangiferin. EMT is a process characterized by loss of cell adhesion, repression of E-cadherin expression, and increased cell motility. Accumulating studies have suggested that EMT is closely linked to metastatic propensity of cancers, and implied in cancer stem cell transformation,

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In vivo antitumor activity of mangiferin MDA-MB-231 xenograft model was employed to evaluate the antitumor potential of mangiferin in vivo. Mangiferin was orally initiated one week prior to tumor cell injection and then continued until the end of the experiment. As shown in Figs. 7A and B, smaller tumor volumes and lower tumor weights were observed in mangiferin-treated mice as compared with control mice. Oral administration of mangiferin

Fig. 7. The anti-tumor activity of mangiferin in MDA-MB-231 breast cancer model. (A) and (B) Tumor volume and weight were measured in different groups. (C) Ki-67 staining for cell proliferation and cleaved caspase 3 staining for apoptosis were evaluated by immunohistochemistry (original magnication, 400). The number of stained and unstained cells was counted to generate the percentage of positive cells in each group. (D) The expression of MMP-7 and -9, E-cadherin, vimentin and active -catenin in the tumor tissues was measured by Western-blotting. All the data were presented as the mean SD, n = 9, *p b 0.05 and **p b 0.01, vs. control group.

Please cite this article as: Li, H., et al., Mangiferin exerts antitumor activity in breast cancer cells by regulating matrix metalloproteinases, epithelial to mesenchymal transition, and -catenin signaling pathway, Toxicol. Appl. Pharmacol. (2013), http://dx.doi.org/10.1016/j.taap.2013.05.011

Aberrant activation of -catenin is often implicated in the proliferation and metastasis of breast cancer. -Catenin can be phosphorylated and degraded by GSK-3. However, various signals can inhibit GSK-3-mediated phosphorylation of -catenin, allowing -catenin to translocate into the nucleus to perform a variety of functions (Clevers and Nusse, 2012). In the present study, it was revealed by Western blotting that active -catenin and inactive GSK-3 (p-GSK-3) was down-regulated by mangiferin (Fig. 5A). Consistently, the inhibitory effect of mangiferin on -catenin pathway activation was also observed by dual-luciferase reporter assay (Fig. 5B). Furthermore, over-expression of -catenin by adenovirus system improved the malignant potential of breast cancer cells and reversed the antitumor activity of mangiferin, while abrogating -catenin by Ad-si -catenin led to the inhibition of cell proliferation and suppression of metastatic potential in breast cancer cells (Figs. 6AC). More importantly, we found out that regulating -catenin signaling pathway could result in the modulation of MMP-7, -9 and snail, which strongly suggested that inhibition of -catenin pathway might play a key role in mangiferin-induced antitumor activity in breast cancer cells (Fig. 6D).

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Please cite this article as: Li, H., et al., Mangiferin exerts antitumor activity in breast cancer cells by regulating matrix metalloproteinases, epithelial to mesenchymal transition, and -catenin signaling pathway, Toxicol. Appl. Pharmacol. (2013), http://dx.doi.org/10.1016/j.taap.2013.05.011

The authors thank Doctor Tong-chuan He (Molecular Oncology Laboratory, the University of Chicago Medical Center) for providing adenoviral expression systems. This study was supported by grants from the National Natural Science Foundation of China (No. 81102007 and No. 31171243).

Acknowledgments

The authors declare that there are no conicts of interest.

Conict of interest statement

drug resistance, immunosuppression and poorer prognosis for types of human cancers. Therefore, pharmacologic inhibition of EMT or induction of mesenchymalepithelial transition (MET) may be instrumental for cancer prevention and treatment (Dave et al., 2012; Foroni et al., 2012; Voulgari and Pintzas, 2009). Some bioactive food components such as curcumin, grape seed proanthocyanidins and epigallocatechin-3 gallate have been reported to reverse EMT in various cancers (Chen et al., 2011; Huang et al., 2013; Prasad and Katiyar, 2012). In the present study, we veried the reversal effect of mangiferin on EMT in metastatic breast cancer cell lines. These data severely indicated that inhibition of EMT might be another mechanism involved in the antitumor activity of bioactive materials in dietary food. -Catenin, a key factor in the Wnt signaling pathway, has been linked to various disease pathologies, including a critical role in carcinogenesis. Besides being closely related to cell proliferation, aberrant -catenin was reported to be associated with increased MMP expression, and some MMP members such as MMP-1, -7, -9 and -26 were the down-stream targets of -catenin signaling pathway (Brabletz et al., 2000; Crawford et al., 1999; Ingraham et al., 2011; Marchenko et al., 2004; Paul and Dey, 2008). Moreover, a number of investigations revealed that -catenin signaling pathway exerted an essential effect on EMT, while inhibiting this way could cause a reversal of EMT (Guarino et al., 2007; Sanchez-Tillo et al., 2011; Zhao et al., 2011). We hereby demonstrated that mangiferin signicantly inhibited the activation of -catenin pathway. Further experiments supported that inhibiting -catenin played a key role in mangiferin-induced anticancer activities through modulating cell proliferation, MMPs and EMT. To conclude, our results dramatically indicate that mangiferin exhibits signicant effects on inhibition of cell proliferation and metastatic ability in breast cancer cells through modulating MMPs, EMT and -catenin pathway. Since mangiferin occurs naturally in mango which is one of the most popular fruits in the world, our ndings indicate that dietary intake of mangoes might be a useful supplement for prevention of breast cancer.

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Please cite this article as: Li, H., et al., Mangiferin exerts antitumor activity in breast cancer cells by regulating matrix metalloproteinases, epithelial to mesenchymal transition, and -catenin signaling pathway, Toxicol. Appl. Pharmacol. (2013), http://dx.doi.org/10.1016/j.taap.2013.05.011

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