mangiferin chemoprevention breast cancer b-catenin

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Toxicology and Applied Pharmacology xxx (2013) xxx–xxx

YTAAP-12760; No. of pages: 11; 4C:

Contents lists available at SciVerse ScienceDirect

Toxicology and Applied Pharmacology

j ourna l homepage: www.e lsev ie r .com/ locate /ytaap

Mangiferin exerts antitumor activity in breast cancer cells by regulatingmatrix metalloproteinases, epithelial to mesenchymal transition, andβ-catenin signaling pathway

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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,⁎a Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing, Chinab Department of Pharmacology, Chongqing Medical University, Chongqing, China

Abbreviations: MMP, matrix metalloproteinase; Etransition; MET, mesenchymal–epithelial transition; Eglycogen synthase kinase 3β; DMSO, dimethyl sulfoxidemunohistochemistry; SP, streptavidin-peroxidase; SD, scellular matrix.⁎ Corresponding author at: Molecular Oncology and E

Affiliated Hospital of Chongqing Medical University, No. 1Chongqing 400016, China. Fax: +86 2389012305.

E-mail address: [email protected] (G. Ren).

0041-008X/$ – see front matter © 2013 Published by Elhttp://dx.doi.org/10.1016/j.taap.2013.05.011

Please cite this article as: Li, H., et al.,Mangifeto mesenchymal transition, and β-catenin si

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

Keywords:MangiferinBreast cancerMatrix metalloproteinaseEpithelial–mesenchymal transitionβ-Catenin

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CTED PAlthough mangiferin which is a naturally occurring glucosylxanthone has exhibited promising anticancer activ-

ities, the detailed molecular mechanism of mangiferin on cancers still remains enigmatic. In this study, the anti-cancer activity of mangiferin was evaluated in breast cancer cell line-based in vitro and in vivo models. Weshowed that mangiferin treatment resulted in decreased cell viability and suppression of metastatic potentialin breast cancer cells. Further mechanistic investigation revealed that mangiferin induced decreased matrixmetalloproteinase (MMP)-7 and -9, and reversal of epithelial–mesenchymal transition (EMT). Moreover, itwas demonstrated that mangiferin significantly inhibited the activation of β-catenin pathway. Subsequent ex-periments showed that inhibiting β-catenin pathwaymight play a central role inmangiferin-induced anticanceractivity through modulation of MMP-7 and -9, and EMT. Consistent with these findings in vitro, the antitumorpotential was also verified in mangiferin-treated MDA-MB-231 xenograft mice where significantly decreasedtumor volume, weight and proliferation, and increased apoptosis were obtained, with lower expression ofMMP-7 and -9, vimentin and active β-catenin, and higher expression of E-cadherin. Taken together, our studysuggests that mangiferin might be used as an effective chemopreventive agent against breast cancer.

© 2013 Published by Elsevier Inc.

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RRIntroduction

Breast cancer is the most frequently diagnosed cancer and theleading cause of cancer death in worldwide females, accounting for23% of the total cancer cases and 14% of the cancer deaths (DeSantiset al., 2011; Siegel et al., 2011). As it is widely accepted that carcino-genesis is a complex and multi-stage process, breast cancer preven-tion by the use of pharmacological agents, especially naturallyoccurring dietary substances has been considered as a practical ap-proach to reduce the incidence of breast cancer. It has been revealedthat many dietary materials such as soy isoflavone genistein, olive oilphenols, grape polyphenols, flaxseed lignan and extracts from greentea or blueberry exert inhibitory effects on the growth and/or metas-tasis of breast cancer cells (Adams et al., 2010; Casaburi et al., 2013;

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MT, epithelial–mesenchymalR, estrogen receptor; GSK-3β,; PI, propidium iodide; IHC, im-tandard deviation; ECM, extra-

pigenetics Laboratory, The FirstYouyi Road, Yuzhong District,

sevier Inc.

rin exerts antitumor activity ingnaling pathway, Toxicol. App

Castillo-Pichardo and Dharmawardhane, 2012; Saggar et al., 2010;Ullah et al., 2011; Wu and Butler, 2011). Moreover, since effectivetherapeutic drugs are limited and drug resistance occurs frequently,these materials have also attracted more attentions in the explorationof effective anti-cancer drugs.

Mangiferin is a naturally occurring glucosylxanthone and exists inseveral folk medicines and food such as mango which is one of themost popular, nutritionally rich tropical fruits. Mangiferin has beenshown to exert many beneficial biological activities including anti-oxidant, hypolipidemic, anti-inflammatory, neuroprotective, immuno-modulatory and hepatoprotective effects (Biradar et al., 2012; Daset al., 2012; Garcia et al., 2003; Guo et al., 2011; Marquez et al., 2012).Recent studies have demonstrated that mangiferin exhibits anticanceractivity against a variety of cancers such as lung, cervical and breastcancers. Although it was reported that immunosuppressive effect orcell cycle modulation might be involved in the antitumor activity ofmangiferin, the detailed mechanisms by which mangiferin inhibitscell growth andmetastasis of breast cancer have not been fully elucidat-ed (du Plessis-Stoman et al., 2011; Garcia-Rivera et al., 2011; Rajendranet al., 2008; Yao et al., 2010). In our study,we report the inhibitory effectof mangiferin on breast cancer cells in both in vitro and in vivo models.We identify that mangiferin inhibits cell proliferation and suppressesthe migration and invasion of breast cancer cells, which may be related

breast cancer cells by regulatingmatrixmetalloproteinases, epitheliall. Pharmacol. (2013), http://dx.doi.org/10.1016/j.taap.2013.05.011

http://dx.doi.org/10.1016/j.taap.2013.05.011

mailto:[email protected]


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to the modulatory effect of mangiferin on MMP-7 and -9, epithelial–mesenchymal transition (EMT) and β-catenin pathway. These findingssuggest that mangiferin might be a very promising candidate for breastcancer 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 describedwas purchased from Sigma Chemical Co. (Sigma, St. Louis, MO, USA)(Zhang et al., 2012). Human estrogen receptor (ER)-negative breastcancer cell lines (MDA-MB-231 and BT-549), and ER-positive breast can-cer cell lines (MCF-7 and T47D) were obtained from American TypeCulture Collection (ATCC, Rockville, MD), and cultured in RPMI-1640supplemented with 10% FBS and 1% penicillin/streptomycin. All the cellswere incubated at 37 °C in humidified atmosphere containing 5% CO2.

Cell viability assay. Cells seeded into a 96-well plate at 4000 cells/wellwere treated with 100 μl medium plus DMSO (vehicle control, finalconcentration b0.5%) or mangiferin (final concentration: 75, 150and 300 μM) and incubated for 48 h. At the end of the drug exposureduration, cell viability was measured according to the protocol ofCCK-8 (KeyGEN Biotech, Nanjing, China). All plates had controlwells containing medium without cells to obtain a value for back-ground spectrometric absorbance which was subtracted from thetest sample readings. Data were expressed as ratios of treated to con-trol cells, mean ± SD for three replications.

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Fig. 1. Mangiferin inhibits cell proliferation and induces apoptosis in breast cancer cells. (A)concentrations of mangiferin for 48 h, cell viability was measured by CCK-8 kit. Data were exMCF-7 cells induced by mangiferin was examined by flow cytometry analysis of AnnexinMDA-MB-231 cells. All the experiments were performed thrice in triplicate. Mean ± SD, *p

Please cite this article as: Li, H., et al.,Mangiferin exerts antitumor activity into mesenchymal transition, and β-catenin signaling pathway, Toxicol. App

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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 flow cytometry accordingto the manufacturer's guidelines.

In vitro scratch assay. The cancer cells were cultured in 6-well platesand grown in medium containing 10% FBS to nearly confluent cellmonolayer, then carefully scratched using a plastic pipette tip todraw a linear “wound” in the cell monolayer of each well. The mono-layer was washed twice with PBS to remove debris or the detachedcells from the monolayer. Cancer cells were exposed to serum-freemedium with or without different concentrations of mangiferin(12.5, 25 and 50 μM) for 12 h. Wound closure was recorded andphotographed using a microscope with Leica cameras.

Cell migration/invasion assays. The cancer cell migration/invasion as-says were conducted with transwell membranes (8 μm pore size,24-well plate, BD Biosciences, Billerica, MA). In migration assay, breastcancer cells were trypsinized, washed, and suspended inmediumwith-out FBS. To the lower wells of the chambers, migration-inducing medi-um (with 10% FBS) was added. Upper wells were filled with cells(20,000 cells/well) in serum-free medium containing various con-centrations of mangiferin (12.5, 25 and 50 μM) or vehicle solvent(DMSO). After 24 h, the top surface of the chambers was scrapedusing a cotton swab, and the cells on the lower surface of themembraneswere fixed for 15 min with methanol and then stained with Giemsasolution. Evaluation of completed transmigration was performed under

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Chemical structure of mangiferin. (B) Breast cancer cell lines were treated with variouspressed as ratios of treated cells to control cells. (C) Cell apoptosis in MDA-MB-231 andV-FITC/PI staining. Numbers inside dot plots indicates the percentages of apoptoticb 0.05 and **p b 0.01, vs. control group.



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the microscope, and random fields were scanned (four fields perfilter) for the presence of cells at the lower membrane side only. Forcell invasion assay, transwell membranes were firstly coated with100 μl Matrigel Matrix (1 mg/ml, BD Biosciences), and subsequent pro-cedureswere performed according to the same protocol in themigrationassay.

Western blotting. Cell lysate was prepared according to the methoddescribed by the protein extract kit (Active Motif Company, Carlsbad,USA). Protein concentrations were determined by BCA protein assaykit (Pierce Biotechnology Inc., Rockford, USA). Cell lysate was ana-lyzed for Western blot analysis using MMP-2, -7 and -9, E-cadherin,vimentin, snail, slug, total and phosphor(p)-GSK-3β, total and activeβ-catenin, plus β-actin (Cell Signaling Technology, Inc., Danvers, MAor Abcam Inc., Cambridge, MA). Antibody binding was visualizedwith an ECL chemiluminescence system and short exposure of themembrane to X-ray films (Kodak, Japan).

ELISA. The levels of MMP-2, -7 and -9 in cell culture supernatantswere detected using ELISA kits (R&D Systems, Inc. Minneapolis,MN) according to the manufacturer's instructions. Briefly, standardsand samples were pipetted into the wells coated with specificantibodies for human MMP-2, -7 or -9, and incubated for 2 h at roomtemperature. A 200 μl conjugate was added and incubated for 1 h on

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Fig. 2.Mangiferin inhibits breast cancer cellmigration and invasion. (A) Thewound healing asstreated for 12 hwith different concentrations (12.5, 25 and 50 μM)ofmangiferin or the controla 12 h period by invertedmicroscopy. The left figure shows the wound healing assay in MDA-Mthe Matrigel invasion assay (C). After 24 h of incubation with or without mangiferin, cells thatcounted using light microscopy. Random fieldswere scanned (four fields per filter) for the presand invasion assay in MDA-MB-231 cells respectively. All the experiments were performed th


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the shaker after washing the wells for 4 times. Then 50 μl of stop solu-tion was placed in each well, followed by 200 μl substrate solutionadded. The intensity was measured at 450 nm within 30 min.

Indirect immunofluorescence analysis. For the immunofluorescence ex-periments, cells were prepared and analyzed under a fluorescence mi-croscope (Leica DM IRB) following the procedures described previously(Cole et al., 2009). Briefly, cells were incubated with primary antibodyagainst 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 thencounterstainedwith DAPI and imagedwith the fluorescencemicroscope.

Dual-luciferase reporter assay. TOPFlash (4 × TCF binding sites) lucif-erase reporter was used as reported previously to elucidate the effectof mangiferin on the activation of β-catenin signaling pathway (Wanget al., 2012). Reporter activity was analyzed by the dual-luciferase re-porter assay system (Promega, Madison, WI) and normalized to thecontrol Renilla.

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

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ay. Confluentmonolayers ofMDA-MB-231 or BT-549 cells were scarred, and the cells weresolvent (DMSO). Themigration length of the cells in the denuded zonewas quantified overB-231 cells, white lines indicate the scraped zone. The transwell migration assay (B) andmigrated to the lower chamber or invaded through the Matrigel were fixed, stained, andence of cells on the lower side of themembrane. The left figures show cell migration assayrice in triplicate. Mean ± SD, *p b 0.05 and **p b 0.01, vs. control group.



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catenin, Ad-scrambled siRNA and Ad-si β-catenin also expressed RFP asa marker for monitoring transfection efficiency. An analogous adenovi-rus expressing only RFP (Ad-RFP) was used as a control, and expressionefficiency was assessed by real-time PCR, Western blotting, and func-tional assays of β-catenin signaling pathway.

Animal experiments. All the animal studies were approved by the Ani-mal Ethics Committee of ChongqingMedical University. 5-Week old se-vere combined immunodeficiency (SCID) hairless female mice werepurchased (Institute of Laboratory Animal Science, Chinese Academyof Medical Science, Beijing, China) and randomly divided into twogroups of 9 mice each. All the mice were housed according to the

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Fig. 3. Down-regulation of MMP-7 and -9 in breast cancer cells treated with mangiferin. (A)expression of MMP-2, -7 and -9 in cells was then measured by Western blotting. β-Actin wpendent experiments. Mean ± SD, **p b 0.01, vs. control group. (B) The expression of MMPwas detected by ELISA. The experiments were performed thrice in triplicate. Mean ± SD, *


national and institutional guidelines for humane animal care. At6 weeks of age, the mice were perorally (p.o.) gavaged with either100 μl water control or mangiferin (100 mg/kg weight). the animalswere gavaged daily for the duration of the experiment. At 7 weeks ofage, the mice were injected subcutaneously with MDA-MB-231 cells(2 × 106) in Matrigel on the right rear flanks. Body weights were mon-itored weekly as an indicator of overall health. Tumor diameter wasmeasured everyweek, and tumor volumeswere calculatedwith the for-mula: tumor volume (mm3) = 0.5 × length (mm) × width2 (mm2). Atthe end of 5 weeks of gavage treatment, the mice were euthanized viaCO2 asphyxiation. Tumors were then removed, weighed, and sent forimmunohistochemistry (IHC) analysis.

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Breast cancer cells were treated with various concentrations of mangiferin for 48 h, theas used as an internal loading control. The blots shown are representative of six inde--2, -7 and -9 in cell supernatants collected from untreated or mangiferin-treated cellsp b 0.05 and **p b 0.01, vs. control group.



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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 μM) for 48 h (original magnification, 400×). (B) Breast cancer cells were treatedwith different concentrations of mangiferin for 48 h, the effect of mangiferin on the expression of EMT markers was then detected by Western blotting. β-Actin was used as an internal loading control. The blots shown are representative ofsix 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 μM) for48 h (original magnification, 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 figure legend, the reader is referred to the web version of this article.)

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Immunohistochemistry. Tumor tissues were fixed in 4% formaldehydesolution (pH 7.0) and subsequently embedded in paraffin. Immunohis-tochemical 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. Tumorspecimens were stained using Ki-67 antibody (Maixin-Bio, Fujian,China) for cell proliferation and cleaved caspase 3 antibody (Cell Signal-ing Technology, Inc., Danvers, MA) for apoptosis. Negative control wasperformed by replacing the primary antibodywith PBS. Immunostained

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Fig. 5. Mangiferin inhibits the activation of β-catenin pathway in breast cancer cells. (A) Beffects of mangiferin on the expression of total or p-GSK-3β, and total or active β-catenin weThe blots shown are representative of six independent experiments. Mean ± SD, **p b 0.01mangiferin for 48 h, the effect of mangiferin on the activation of β-catenin signaling pathwaycontrol group.


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

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

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reast cancer cells were treated with various concentrations of mangiferin for 48 h, there then measured by Western blotting. β-Actin was used as an internal loading control., vs. control group. (B) Breast cancer cells were treated with various concentrations ofwas then assessed by dual-luciferase reporter assay. Mean ± SD, n = 9, **p b 0.01, vs.



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Results

Mangiferin inhibits breast cancer cell proliferation

As shown in Fig. 1B, we observed that mangiferin inhibitedthe cell proliferation of breast cancer cell lines including MDA-MB-231, BT-549, MCF-7 and T47D in a dose-dependent manner.To clarify whether the decreased cell proliferation was due to theinduction of cell apoptosis, flow cytometry for the apoptosis anal-ysis was further performed. Following treatment with mangiferin(75, 150 and 300 μM) for 48 h, significantly increased apoptosiswas found when breast cancer cells were treated with 300 μMmangiferin, which may partly contribute to the decreased cell via-bility (Fig. 1C).

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Fig. 6. Inactivation of β-catenin signaling pathway is responsible for the anti-tumor activitransfected with Ad-RFP, Ad-scrambled siRNA, Ad-si β-catenin or Ad-β-catenin were treCCK-8 kit. Data were expressed as ratios of treated cells to control cells. (B) and (C) The efmangiferin (25 μM) was measured by both cell migration and invasion assays. The expetransfected with Ad-RFP, Ad-scrambled siRNA, Ad-si β-catenin or Ad-β-catenin were treatbreast cancer cells was then measured by Western blotting. β-Actin was used as an internaMean ± SD, **p b 0.01.


Mangiferin suppresses the metastatic potential of breast cancer cells

In this study, high-metastatic cell lines MDA-MB-231 and BT-549were utilized to evaluate the effect ofmangiferin on cancer cellmigrationand invasion. Due to the fact thatmangiferin could inhibit cell viability atthe concentrations over 50 μM(the IC50 inMDA-MB-231 or BT-549 cellsare 298.6 and 273.8 μMrespectively, data not shown), the related exper-iments includingwound-healing assay, transwellmigration and invasionassays were performed at the concentrations of 12.5, 25 and 50 μM. Asshown in Figs. 2A–B, in both thewound-healing and transwell migrationassays, mangiferin dose-dependently inhibited cancer cell migration.Moreover, mangiferin treatment significantly reduced the number ofcells which could invade through theMatrigel-coated insert membranes(Fig. 2C).

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ty of mangiferin in breast cancer cells. (A) Cell proliferation assay. Breast cancer cellsated with or without mangiferin (150 μM) for 48 h, cell viability was measured byfect of β-catenin on metastatic potential of breast cancer cells treated with or withoutriments above were performed thrice in triplicate. Mean ± SD, **p b 0.01. (D) Cellsed with or without mangiferin (25 μM) for 48 h, the expression of related proteins inl loading control. The blots shown are representative of six independent experiments.



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Please cite this article as: Li, H., et al.,Mangiferin exerts antitumor activity in breast cancer cells by regulatingmatrixmetalloproteinases, epithelialto mesenchymal transition, and β-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 remodelingof extracellular matrix (ECM), exert a great role in tumor invasion andmetastasis. Since MMP-2, -7 and -9 were reported to be involved inbreast cancer metastasis by numerous studies, the expression ofMMP-2, -7 and -9 was detected by Western blotting and ELISA inour study (Beeghly-Fadiel et al., 2009; Jezierska and Motyl, 2009;Rydlova et al., 2008; Sullu et al., 2011). As depicted in Figs. 3A andB, MMP-7 and -9 were found down-regulated by mangiferin, whilethe expression of MMP-2 wasn't significantly affected by mangiferin.These results strongly suggest that down-regulation of MMP-7 and -9might 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 quiescenttumor cells acquire metastatic capability is the EMT (Gomes et al.,2011). We next utilized two mesenchymal-like breast cancer celllines, MDA-MB-231 and BT-549, to evaluate the effect of mangiferinon EMT. As shown in Fig. 4A, a significant change from a spindle,fibroblast-like shape with migratory protrusions to characteristiccobblestone-like epithelial morphology was observed in mangiferin-treated cancer cells. Consistent with these morphological changes, in-creased expression of epithelial marker E-cadherin and decreasedmes-enchymal markers such as vimentin, snail and slug were demonstratedin mangiferin-treated cancer cells (Figs. 4B and C). Collectively, theseobservations support that mangiferin induces an effective switch frommesenchymal to epithelial phenotype of breast cancer cells.

Inactivation of β-catenin pathway is involved in the anti-tumor activityof mangiferin

Aberrant activation of β-catenin is often implicated in the prolifera-tion and metastasis of breast cancer. β-Catenin can be phosphorylatedand degraded by GSK-3β. However, various signals can inhibitGSK-3β-mediated phosphorylation of β-catenin, allowing β-catenin totranslocate into the nucleus to perform a variety of functions (Cleversand Nusse, 2012). In the present study, it was revealed by Westernblotting that active β-catenin and inactive GSK-3β (p-GSK-3β) wasdown-regulated by mangiferin (Fig. 5A). Consistently, the inhibitoryeffect of mangiferin on β-catenin pathway activation was also ob-served by dual-luciferase reporter assay (Fig. 5B). Furthermore,over-expression of β-catenin by adenovirus system improved the ma-lignant potential of breast cancer cells and reversed the antitumor activ-ity of mangiferin, while abrogating β-catenin by Ad-si β-catenin led tothe inhibition of cell proliferation and suppression of metastatic poten-tial in breast cancer cells (Figs. 6A–C). More importantly, we found outthat regulatingβ-catenin signaling pathway could result in themodula-tion of MMP-7, -9 and snail, which strongly suggested that inhibition ofβ-catenin pathway might play a key role in mangiferin-inducedantitumor activity in breast cancer cells (Fig. 6D).

In vivo antitumor activity of mangiferin

MDA-MB-231 xenograft model was employed to evaluate theantitumor potential of mangiferin in vivo. Mangiferin was orally initiat-ed one week prior to tumor cell injection and then continued until theend of the experiment. As shown in Figs. 7A and B, smaller tumor vol-umes and lower tumor weights were observed in mangiferin-treatedmice 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 (cell proliferation and cleaved caspase 3 staining for apoptosis were evaluated by immunohiswas counted to generate the percentage of positive cells in each group. (D) The expressionmeasured by Western-blotting. All the data were presented as the mean ± SD, n = 9, *p b


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had no detectable toxicity since there were no differences in bodyweight between control and treated groups, without any side effectsobserved. Significantly decreased proliferation (Ki-67 staining) and in-creased apoptosis (cleaved caspase 3 staining) were detected in thetumor specimens of mangiferin-treated mice (Fig. 7C). Furthermore,lower expression of MMP-7 and -9, vimentin and active β-catenin,and higher expression of E-cadherin were obtained in mangiferin-treated group by Western blotting (Fig. 7D). These results are in fullagreement with our findings in vitro, showing that mangiferin pos-sesses 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 antiox-idant properties in the molecule of mangiferin. Although reportsabout the antitumor activity of mangiferin are limited, mangiferinwas found to exert immunosuppressive antitumor effect on MDA-MB-231 breast cancer cells, chemopreventive and chemotherapeuticeffects on bowel and lung carcinogenesis, and inhibitory activity oncell 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 mangiferinin combination with oxaliplatin favored apoptotic cell death andthereby improved the efficacy of oxaliplatin in vitro, and norathyriolas a metabolite of mangiferin suppressed UV-induced skin cancer(du Plessis-Stoman et al., 2011; Li et al., 2012). In our study, weshowed that mangiferin could effectively inhibit cell proliferation ofbreast cancer cells, while only higher dose of mangiferin induced sig-nificant apoptosis, suggesting that the results of the viability assaycould be largely attributed to the inhibition of proliferation. Further-more, it was demonstrated that mangiferin markedly suppressedthe metastatic potential of breast cancer cells by wound healing,transwell migration and invasion assays. Taken together, these find-ings strongly showed that mangiferin possessed multiple antitumoractivities.

Since mounting evidence has implied the great role of MMPs inthe process of tumor invasion and metastasis, MMPs have alwaysbeen regarded as potential therapeutic targets for cancers (Roy etal., 2009; Rydlova et al., 2008). MMP-7 and -9 which are importantmembers of MMP family exert great effects on promoting cancer de-velopment. MMP-7 was found involved in the invasion of breastcancer through its collaboration with indicators of invasion, and elim-ination of MMP-7 in MDA-MB-231 cells was associated with low in-vasiveness and slow tumor growth (Jiang et al., 2005; Mylona et al.,2005). Similarly, a number of studies suggested that MMP-9 promot-ed metastasis, and might be associated with poor prognosis of breastcancer (Ranogajec et al., 2012; Scorilas et al., 2001). It was reportedthat mangiferin exerted anti-photoaging activity in UVB-irradiatedhairless mice by regulating MMP-9 expression (Kim et al., 2012). Inaddition, mangiferin was found to selectively inhibit the expressionof MMP-9 in PMA-stimulated human astroglioma cells withoutaffecting other MMPs such as MMP-1, -2, -3, and -14 (Jung et al.,2012). In our study, it was demonstrated that mangiferin significantlysuppressed the expression of MMP-7 and -9 in breast cancer cells,suggesting that inhibition of MMP-7 and -9 might be involved inthe anti-metastatic potential of mangiferin.

EMT is a process characterized by loss of cell adhesion, repressionof E-cadherin expression, and increased cell motility. Accumulatingstudies have suggested that EMT is closely linked to metastatic pro-pensity of cancers, and implied in cancer stem cell transformation,

B) Tumor volume and weight were measured in different groups. (C) Ki-67 staining fortochemistry (original magnification, ×400). The number of stained and unstained cellsof MMP-7 and -9, E-cadherin, vimentin and active β-catenin in the tumor tissues was0.05 and **p b 0.01, vs. control group.



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drug resistance, immunosuppression and poorer prognosis fortypes of human cancers. Therefore, pharmacologic inhibition of EMTor induction of mesenchymal–epithelial transition (MET) may be in-strumental for cancer prevention and treatment (Dave et al., 2012;Foroni et al., 2012; Voulgari and Pintzas, 2009). Some bioactive foodcomponents such as curcumin, grape seed proanthocyanidins andepigallocatechin-3 gallate have been reported to reverse EMT in var-ious cancers (Chen et al., 2011; Huang et al., 2013; Prasad and Katiyar,2012). In the present study, we verified the reversal effect ofmangiferin on EMT in metastatic breast cancer cell lines. These dataseverely indicated that inhibition of EMT might be another mecha-nism involved in the antitumor activity of bioactive materials in die-tary food.

β-Catenin, a key factor in the Wnt signaling pathway, has beenlinked to various disease pathologies, including a critical role in carci-nogenesis. Besides being closely related to cell proliferation, aberrantβ-catenin was reported to be associated with increased MMP expres-sion, and some MMP members such as MMP-1, -7, -9 and -26 werethe down-stream targets of β-catenin signaling pathway (Brabletzet al., 2000; Crawford et al., 1999; Ingraham et al., 2011; Marchenkoet al., 2004; Paul and Dey, 2008). Moreover, a number of investiga-tions revealed that β-catenin signaling pathway exerted an essentialeffect 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). Wehereby demonstrated that mangiferin significantly inhibited theactivation of β-catenin pathway. Further experiments supported thatinhibiting β-catenin played a key role inmangiferin-induced anticanceractivities through modulating cell proliferation, MMPs and EMT.

To conclude, our results dramatically indicate that mangiferin ex-hibits significant effects on inhibition of cell proliferation and meta-static ability in breast cancer cells through modulating MMPs, EMTand β-catenin pathway. Since mangiferin occurs naturally in mangowhich is one of the most popular fruits in the world, our findings in-dicate that dietary intake of mangoes might be a useful supplementfor prevention of breast cancer.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

The authors thank Doctor Tong-chuan He (Molecular OncologyLaboratory, the University of Chicago Medical Center) for providing ad-enoviral expression systems. This study was supported by grants fromthe National Natural Science Foundation of China (No. 81102007 andNo. 31171243).

References

Adams, L.S., Phung, S., Yee, N., Seeram, N.P., Li, L., Chen, S., 2010. Blueberry phytochem-icals inhibit growth and metastatic potential of MDA-MB-231 breast cancer cellsthrough modulation of the phosphatidylinositol 3-kinase pathway. Cancer Res.70, 3594–3605.

Beeghly-Fadiel, A., Shu, X.O., Long, J., Li, C., Cai, Q., Cai, H., Gao, Y.T., Zheng, W., 2009.Genetic polymorphisms in the MMP-7 gene and breast cancer survival. Int. J. Cancer124, 208–214.

Biradar, S.M., Joshi, H., Chheda, T.K., 2012. Neuropharmacological effect of mangiferinon brain cholinesterase and brain biogenic amines in the management ofAlzheimer's disease. Eur. J. Pharmacol. 683, 140–147.

Brabletz, T., Jung, A., Dag, S., Reu, S., Kirchner, T., 2000. β-Catenin induces invasivegrowth by activating matrix metalloproteinases in colorectal carcinoma. Verh.Dtsch. Ges. Pathol. 84, 175–181.

Casaburi, I., Puoci, F., Chimento, A., Sirianni, R., Ruggiero, C., Avena, P., Pezzi, V., 2013.Potential of olive oil phenols as chemopreventive and therapeutic agents againstcancer: a review of in vitro studies. Mol. Nutr. Food Res. 57, 71–83.

Castillo-Pichardo, L., Dharmawardhane, S.F., 2012. Grape polyphenols inhibit Akt/mammalian target of rapamycin signaling and potentiate the effects of gefitinibin breast cancer. Nutr. Cancer 64, 1058–1069.


ED P

RO

OF

Chen, P.N., Chu, S.C., Kuo, W.H., Chou, M.Y., Lin, J.K., Hsieh, Y.S., 2011. Epigallocatechin-3 gallate inhibits invasion, epithelial–mesenchymal transition, and tumor growthin oral cancer cells. J. Agric. Food Chem. 59, 3836–3844.

Clevers, H., Nusse, R., 2012. Wnt/β-catenin signaling and disease. Cell 149, 1192–1205.Cole, L., Anderson, M., Antin, P.B., Limesand, S.W., 2009. One process for pancreatic

beta-cell coalescence into islets involves an epithelial–mesenchymal transition.J. Endocrinol. 203, 19–31.

Crawford, H.C., Fingleton, B.M., Rudolph-Owen, L.A., Goss, K.J., Rubinfeld, B., Polakis, P.,Matrisian, L.M., 1999. The metalloproteinase matrilysin is a target of beta-catenintransactivation in intestinal tumors. Oncogene 18, 2883–2891.

Das, J., Ghosh, J., Roy, A., Sil, P.C., 2012. Mangiferin exerts hepatoprotective activityagainst D-galactosamine induced acute toxicity and oxidative/nitrosative stressvia Nrf2-NFkappaB pathways. Toxicol. Appl. Pharmacol. 260, 35–47.

Dave, B., Mittal, V., Tan, N.M., Chang, J.C., 2012. Epithelial–mesenchymal transition,cancer stem cells and treatment resistance. Breast Cancer Res. 14, 202.

DeSantis, C., Siegel, R., Bandi, P., Jemal, A., 2011. Breast cancer statistics, 2011. CA CancerJ. Clin. 61, 409–418.

du Plessis-Stoman, D., du Preez, J., van de Venter, M., 2011. Combination treatmentwith oxaliplatin and mangiferin causes increased apoptosis and downregulationof NFkappaB in cancer cell lines. Afr. J. Tradit. Complement. Altern. Med. 8,177–184.

Foroni, C., Broggini, M., Generali, D., Damia, G., 2012. Epithelial–mesenchymal transi-tion and breast cancer: role, molecular mechanisms and clinical impact. CancerTreat. Rev. 38, 689–697.

Garcia, D., Leiro, J., Delgado, R., Sanmartin, M.L., Ubeira, F.M., 2003. Mangifera indica L.extract (Vimang) and mangiferin modulate mouse humoral immune responses.Phytother Res. 17, 1182–1187.

Garcia-Rivera, D., Delgado, R., Bougarne, N., Haegeman, G., Berghe, W.V., 2011. Gallicacid indanone and mangiferin xanthone are strong determinants of immunosup-pressive anti-tumour effects of Mangifera indica L. bark in MDA-MB231 breastcancer cells. Cancer Lett. 305, 21–31.

Gomes, L.R., Terra, L.F., Sogayar, M.C., Labriola, L., 2011. Epithelial–mesenchymal tran-sition: implications in cancer progression and metastasis. Curr. Pharm. Biotechnol.12, 1881–1890.

Guarino, M., Rubino, B., Ballabio, G., 2007. The role of epithelial–mesenchymal transi-tion in cancer pathology. Pathology 39, 305–318.

Guo, F., Huang, C., Liao, X., Wang, Y., He, Y., Feng, R., Li, Y., Sun, C., 2011. Beneficialeffects of mangiferin on hyperlipidemia in high-fat-fed hamsters. Mol. Nutr. FoodRes. 55, 1809–1818.

Huang, T., Chen, Z., Fang, L., 2013. Curcumin inhibits LPS-induced EMT throughdownregulation of NF-kappaB-Snail signaling in breast cancer cells. Oncol. Rep.29, 117–124.

Ingraham, C.A., Park, G.C., Makarenkova, H.P., Crossin, K.L., 2011. Matrixmetalloproteinase (MMP)-9 induced by Wnt signaling increases the proliferationand migration of embryonic neural stem cells at low O2 levels. J. Biol. Chem. 286,17649–17657.

Jezierska, A., Motyl, T., 2009. Matrix metalloproteinase-2 involvement in breast cancerprogression: a mini-review. Med. Sci. Monit. 15, RA32–RA40.

Jiang, W.G., Davies, G., Martin, T.A., Parr, C., Watkins, G., Mason, M.D., Mokbel, K.,Mansel, R.E., 2005. Targeting matrilysin and its impact on tumor growth invivo: the potential implications in breast cancer therapy. Clin. Cancer Res. 11,6012–6019.

Jung, J.S., Jung, K., Kim, D.H., Kim, H.S., 2012. Selective inhibition of MMP-9 gene ex-pression by mangiferin in PMA-stimulated human astroglioma cells: involvementof PI3K/Akt and MAPK signaling pathways. Pharmacol. Res. 66, 95–103.

Kim, H.S., Song, J.H., Youn, U.J., Hyun, J.W., Jeong, W.S., Lee, M.Y., Choi, H.J., Lee, H.K.,Chae, S., 2012. Inhibition of UVB-induced wrinkle formation and MMP-9 expres-sion by mangiferin isolated from Anemarrhena asphodeloides. Eur. J. Pharmacol.689, 38–44.

Li, J., Malakhova, M., Mottamal, M., Reddy, K., Kurinov, I., Carper, A., Langfald, A., Oi, N.,Kim, M.O., Zhu, F., Sosa, C.P., Zhou, K., Bode, A.M., Dong, Z., 2012. Norathyriol sup-presses skin cancers induced by solar ultraviolet radiation by targeting ERK ki-nases. Cancer Res. 72, 260–270.

Luo, J., Deng, Z.L., Luo, X., Tang, N., Song, W.X., Chen, J., Sharff, K.A., Luu, H.H., Haydon,R.C., Kinzler, K.W., Vogelstein, B., He, T.C., 2007. A protocol for rapid generationof recombinant adenoviruses using the AdEasy system. Nat. Protoc. 2, 1236–1247.

Marchenko, N.D., Marchenko, G.N., Weinreb, R.N., Lindsey, J.D., Kyshtoobayeva, A.,Crawford, H.C., Strongin, A.Y., 2004. Beta-catenin regulates the gene of MMP-26,a novel metalloproteinase expressed both in carcinomas and normal epithelialcells. Int. J. Biochem. Cell Biol. 36, 942–956.

Marquez, L., Garcia-Bueno, B., Madrigal, J.L., Leza, J.C., 2012. Mangiferin decreases in-flammation and oxidative damage in rat brain after stress. Eur. J. Nutr. 51, 729–739.

Mylona, E., Kapranou, A., Mavrommatis, J., Markaki, S., Keramopoulos, A., Nakopoulou,L., 2005. The multifunctional role of the immunohistochemical expression of MMP-7in invasive breast cancer. APMIS 113, 246–255.

Paul, S., Dey, A., 2008. Wnt signaling and cancer development: therapeutic implication.Neoplasma 55, 165–176.

Prasad, R., Katiyar, S.K., 2012. Grape seed proanthocyanidins inhibit migration poten-tial of pancreatic cancer cells by promoting mesenchymal-to-epithelial transitionand targeting NF-kappaB. Cancer Lett.

Rajendran, P., Ekambaram, G., Magesh, V., Sakthisekaran, D., 2008. Chemopreventiveefficacy of mangiferin against benzo(a)pyrene induced lung carcinogenesis in ex-perimental animals. Environ. Toxicol. Pharmacol. 26, 278–282.

Ranogajec, I., Jakic-Razumovic, J., Puzovic, V., Gabrilovac, J., 2012. Prognostic value ofmatrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) andaminopeptidase N/CD13 in breast cancer patients. Med. Oncol. 29, 561–569.


http://refhub.elsevier.com/S0041-008X(13)00209-3/rf0005




































































































496497498499500501502503504505506507508509510511512513514515516517518

519520521522523524525526527528529530531532533534535536Q4537538539540541

543


Roy, R., Yang, J., Moses, M.A., 2009. Matrix metalloproteinases as novel biomarkers andpotential therapeutic targets in human cancer. J. Clin. Oncol. 27, 5287–5297.

Rydlova, M., Holubec Jr., L., Ludvikova Jr., M., Kalfert, D., Franekova, J., Povysil, C.,Ludvikova, M., 2008. Biological activity and clinical implications of the matrixmetalloproteinases. Anticancer. Res. 28, 1389–1397.

Saggar, J.K., Chen, J., Corey, P., Thompson, L.U., 2010. Dietary flaxseed lignan or oil com-bined with tamoxifen treatment affects MCF-7 tumor growth through estrogenreceptor- and growth factor-signaling pathways. Mol. Nutr. Food Res. 54, 415–425.

Sanchez-Tillo, E., de Barrios, O., Siles, L., Cuatrecasas, M., Castells, A., Postigo, A., 2011.β-Catenin/TCF4 complex induces the epithelial-to-mesenchymal transition(EMT)-activator ZEB1 to regulate tumor invasiveness. Proc. Natl. Acad. Sci. U. S. A.108, 19204–19209.

Scorilas, A., Karameris, A., Arnogiannaki, N., Ardavanis, A., Bassilopoulos, P., Trangas, T., Talieri,M., 2001. Overexpression of matrix-metalloproteinase-9 in human breast cancer: a po-tential favourable indicator in node-negative patients. Br. J. Cancer 84, 1488–1496.

Siegel, R., Ward, E., Brawley, O., Jemal, A., 2011. Cancer statistics, 2011: the impact ofeliminating socioeconomic and racial disparities on premature cancer deaths. CACancer J. Clin. 61, 212–236.

Sullu, Y., Demirag, G.G., Yildirim, A., Karagoz, F., Kandemir, B., 2011. Matrixmetalloproteinase-2 (MMP-2) and MMP-9 expression in invasive ductal carcino-ma of the breast. Pathol. Res. Pract. 207, 747–753.

Ullah, M.F., Ahmad, A., Zubair, H., Khan, H.Y., Wang, Z., Sarkar, F.H., Hadi, S.M., 2011. Soyisoflavone genistein induces cell death in breast cancer cells through mobilization

UNCO

RRECT

542


OF

of endogenous copper ions and generation of reactive oxygen species. Mol. Nutr.Food Res. 55, 553–559.

Voulgari, A., Pintzas, A., 2009. Epithelial–mesenchymal transition in cancer metastasis:mechanisms, markers and strategies to overcome drug resistance in the clinic.Biochim. Biophys. Acta 1796, 75–90.

Wang, S., Kang, W., Go, M.Y., Tong, J.H., Li, L., Zhang, N., Tao, Q., Li, X., To, K.F., Sung, J.J.,Yu, J., 2012. Dapper homolog 1 is a novel tumor suppressor in gastric cancerthrough inhibiting the nuclear factor-kappaB signaling pathway. Mol. Med. 18,1402–1411.

Wu, A.H., Butler, L.M., 2011. Green tea and breast cancer. Mol. Nutr. Food Res. 55,921–930.

Yao, Y.B., Peng, Z.G., Liu, Z.F., Yang, J., Luo, J., 2010. Effects of mangiferin on cell cyclestatus and CDC2/Cyclin B1 expression of HL-60 cells. Zhong Yao Cai 33, 81–85.

Yoshimi, N., Matsunaga, K., Katayama, M., Yamada, Y., Kuno, T., Qiao, Z., Hara, A.,Yamahara, J., Mori, H., 2001. The inhibitory effects of mangiferin, a naturally occur-ring glucosylxanthone, in bowel carcinogenesis of male F344 rats. Cancer Lett. 163,163–170.

Zhang, X., Su, B., Li, J., Li, Y., Lu, D., Zhu, K., Pei, H., Zhao, M., 2012. Analysis by RP-HPLC ofmangiferin component correlation between medicinal loranthus and their mangohost trees. J. Chromatogr. Sci.

Zhao, J.H., Luo, Y., Jiang, Y.G., He, D.L., Wu, C.T., 2011. Knockdown of beta-cateninthrough shRNA cause a reversal of EMT and metastatic phenotypes induced byHIF-1alpha. Cancer Invest. 29, 377–382.

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