Apple polyphenol inhibits colon carcinomametastasis via disrupting Snail binding to focaladhesion kinase

Chia-Hung Hung a,1, Chi-Chou Huang b,c,1, Li-Sung Hsu a,d,Shao-Hsuan Kao a,d,*, Chau-Jong Wang a,d,**a Institute of Biochemistry and Biotechnology, College of Medicine, Chung Shan Medical University, Taichung402, Taiwanb Department of Colorectal Surgery, Chung Shan Medical University Hospital, Taichung 402, Taiwanc School of Medicine, Chung Shan Medical University, Taichung 402, Taiwand Department of Medical Research, Chung Shan Medical University Hospital, Taichung 402, Taiwan

A R T I C L E I N F O

Article history:

Received 8 August 2014

Received in revised form 21 October

2014

Accepted 29 October 2014

Available online

A B S T R A C T

We investigated inhibitory effects of apple polyphenol (AP) in vitro and in vivo. AP signifi-

cantly suppressed migration, invasion, colony formation and adhesion of DLD-1 cells. AP

also reduced expression of tubulin and F-actin, and consequently altered the cytoskeleton

structure. AP reduced expression of focal adhesion kinase (FAK) but not talin, α-actinin and

vinculin. The activation of FAK downstream effectors paxillin, Src, PI3K and Akt were also

inhibited by AP. Moreover, AP inhibited activity but not expression level of FAK-associated

GTPase Rac-1, Cdc42 and Rho A. The inhibition of FAK expression by AP was proteasome-

independent, which was attributed to interfered binding of Snail to a putative E-box on the

FAK promoter. Anti-tumoral and anti-metastatic effects of AP were also demonstrated in

vivo. Collectively, AP significantly inhibited motility of DLD-1 cells via disruption of inter-

action between Snail and the FAK promoter and consequently diminished tumorigenesis

and metastasis of DLD-1 cells.

© 2014 Elsevier Ltd. All rights reserved.

Keywords:

Colon cancer

Metastasis

Apple polyphenol

Focal adhesion kinase

1. Introduction

Colon cancer is one of the most common malignancies in theworld. The highly metastatic capability of colon tumor cells isthe major cause for the mortality of patients with colon cancer(Faerden et al., 2011). Mounting evidence has indicated that in-

travasation and extravasation of tumor cells play pivotal rolesin metastasis, attributing to acquisition of potent cell adhe-sion, motility and colony formation capability for entrance intoand escape from the circulatory system (Gupta & Massague,2006). Recent studies have shown that surgical operations com-bined with chemotherapy may increase quality of life (Hill et al.,2011); however, the effects of the combined therapies on

* Corresponding author. Institute of Biochemistry and Biotechnology, College of Medicine, Chung Shan Medical University, No.110, Sec.1, Jianguo N.Rd., Taichung 402, Taiwan. Tel.: +886 4 24730022 ext. 11681; fax: +886 4 23248110.

E-mail address: kaosh@csmu.edu.tw (S-H. Kao).** Corresponding author. Institute of Biochemistry and Biotechnology, College of Medicine, Chung Shan Medical University, No.110, Sec.

1, Jianguo N.Rd., Taichung 402, Taiwan. Tel.: +886 4 24730022 ext. 11670; fax: +886 4 23248110.E-mail address: wcj@csmu.edu.tw (C.-J. Wang).

1 The authors contributed equally to this work.http://dx.doi.org/10.1016/j.jff.2014.10.0311756-4646/© 2014 Elsevier Ltd. All rights reserved.

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survival improvement of colon cancer patients with metasta-sis are still limited.

Focal adhesion kinase (FAK) has been widely investigatedfor its roles in regulating cytoskeleton organization via mul-tiple pathways (Mitra, Hanson, & Schlaepfer, 2005). Abnormalexpression of FAK has been implicated in tumorigenesis suchas malignant transformation, progression and metastasis (Luo& Guan, 2010). Besides, aberrant FAK expression has also beenreported to associate with poor outcome in cancer patients (Jiet al., 2013). Activation of FAK contributes to conformationalactivation of Src and FAK-associated adhesion proteins in-cluding talin, paxillin, vinculin and α-actinin, and modulatesactivity of Rho-family GTPase (Abbi & Guan, 2002; Mitra et al.,2005). In addition, recent studies show that FAK signaling isinvolved in epithelial–mesenchymal transition (EMT), a re-markable process leading to invasion and metastasis of varioustumors (Avizienyte & Frame, 2005). EMT is a dynamiccytoskeletal change that contributes to loss of cell–cell contactand polarity in epithelial cells accompanied with cytoskel-eton rearrangements and enhancement of cell motility. Thepotent EMT inducers such as Snail, Slug, Twist and ZEB2 havebeen implicated in tumor progression and metastasis (Thiery,2002). Snail and ZEB2 are also reported to affect cell-matrix ad-hesion by modulating integrins and basement membraneproteins (Haraguchi et al., 2008; Nam, Lee, Park, Lee, & Kim,2012). However, the correlation between EMT inducers and celladhesion in tumor metastasis is not completely clarified.

Mounting evidence has shown that native products possessanti-tumoral activity against various malignancies via mul-tiple mechanisms (Hsu et al., 2013; Kaur et al., 2013; Miura et al.,2008; Rizzi et al., 2014). Apple polyphenols are polyphenol-enriched extracts from unripe apples, mainly consisting ofepicatechin and catechin, possessing various medical func-tions in in vivo and in vitro studies (Fujiwara, Nakashima, Sami,& Kanda, 2013; Shoji, Akazome, Kanda, & Ikeda, 2004). Differ-ent apple polyphenol extracts have been demonstrated to exertvarious biological activities against hypersensitivities, obesityand hyperlipidemia (Osada et al., 2006; Sugiyama et al., 2007).Recent studies have also indicated that apple polyphenol ex-tracts inhibit proliferation and induces apoptosis in humancolon cancer cells SW620 and stomach carcinoma cells KATOIII (Gosse et al., 2005; Hibasami, Shohji, Shibuya, Higo, & Kanda,2004).

The aim of this study was to investigate the effects of applepolyphenol (AP) on colon cancer with emphasis on meta-static capability. We hypothesized that AP interfered metastaticcapability of colorectal carcinoma cells via alteration of cell mo-tility. In addition, the roles of FAK in cell motility and metastasisof colorectal carcinoma and correlation between FAK and EMTinducers were also demonstrated using an in vitro cell modeland an in vivo animal model.

2. Materials and methods

2.1. Cell culture and reagents

Human colon cancer cell line DLD-1 and human non-tumorigenic bronchial epithelial cells BEAS-2B were purchasedfrom ATCC and cultured in RPMI-1640 medium and Dulbecco’s

modified Eagle’s medium (DMEM) supplemented with 10% fetalbovine serum (FBS), respectively, and incubated at 37 °C with5% CO2 atmosphere. Cell line characterization was performedaccording to UKCCCR Guidelines every 3 months, including my-coplasma test by PCR and measurement of cell proliferationby counting. AP, extracted from green unripe apple fruits, waspurchased from Asahi Food and Healthcare Co. Ltd (Tokyo,Japan).The composition of AP identified by reversed and normalphase HPLC contains 65.7% procyanidins (13% dimers, 12.3%trimers, 8.7% tetramers, 5.9% pentamers, 4.9% hexamers and20.9% other polymers ), 12.5% flavan-3-ols (2% catechin and10.5% epicatechin), 6.5% other flavonoids and 10.8% non-flavonoids (Shoji et al., 2004; Sugiyama et al., 2007). Matrigelwas purchased from BD Biosciences (San Jose, CA, USA). An-tibodies against focal adhesion kinase (FAK), paxillin, p-paxillin,talin, α-actinin, vinculin, Akt, Src, p-Src, and phosphatidylinositol3-kinase (PI3K) were obtained from Santa Cruz Biotechnol-ogy (Santa Cruz, CA, USA) and Cell Signaling Technology (Beverly,MA, USA). Recombinant protein G agarose beads were pur-chased from Invitrogen (Carlsbad, CA, USA). All the otherreagents were obtained from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Cell viability assay

Cell viability was determined using a MTT assay. Briefly, cellswere seeded at a density of 2 × 105 cells/mL in a 24-well plateand cultured with complete medium (containing 10% FBS) for24 hours (h), and then treated with the complete medium con-taining AP at various concentrations for indicated times. EachAP concentration was performed in triplicate. After the treat-ments, the cells were washed with PBS, and then incubatedwith MTT solution (5 mg/mL; Sigma-Aldrich) for 4 h. After theculture supernatant was removed, isopropanol was added tosolubilize formazan and absorbance at 563 nm of the solu-tion was measured using a spectrophotometer (U-2900, Hitachi,Tokyo, Japan). The percentage of viable cells was estimated bycomparing with untreated cells.

2.3. Immunohistochemical analysis

Ki-67, a proliferating cell nuclear antigen, is highly expressedduring DNA synthesis and widely regarded as a proliferationmarker. The protein level of Ki-67 was determined using thecell proliferation assay kit according to the manufacturer’s in-struction (Millipore, Bedford, MA, USA). Percentage of Ki-67positive cells was calculated by counting the number of positive-stained cells (brown stained) and the total number of cells atfive arbitrarily selected fields from each tumor at 400× mag-nification.

2.4. Cell cycle distribution by flow cytometric analysis

The cell cycle was synchronized by serum-free starvation for16 h and then treated with AP at indicated concentrations for24 h in complete medium. The treated cells were trypsinized,fixed with 70% ethanol, washed with PBS, then incubated withpropidium iodide (50 µg/mL) at room temperature in dark for30 minutes prior to following flow cytometric analysis (BD Bio-sciences, San Jose, CA, USA). Ten thousand cells were counted

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for cell cycle distribution quantitation using CELLQuest Pro soft-ware (version 5.1, BD Biosciences).

2.5. Cell migration and invasion assay

Cells were pretreated with AP for 24 h, harvested, and thenseeded onto 24-well Millicell® Hanging Cell Culture inserts (8 µmpore size, Millipore). A 24-h lung metastatic colon cancer cellculture medium was used as a chemoattractant placed in thelower compartment of the plate and incubated for 12 h. Thecells that migrated to the lower surface of the insert were fixedand stained with Giemsa reagent (Sigma-Aldrich). The stainedcells were photographed and quantified from five random fieldsunder microscopic examination. For the invasion assay, theculture inserts were pre-coated with 100 µL of Matrigel (20× di-lution in PBS) and air-dried overnight. Cells were seeded ontothe coated culture insert and incubated for 18 h. Cells invadedinto the lower surface of the insert were quantitated as de-scribed earlier.

2.6. Cell adhesion assay

Cells were incubated with serial concentrations of AP in com-plete medium for 24 h, transferred into 12-well Matrigel-coated plates (105 cells per well), and then incubated at 37 °Cfor 8 h. After the incubation, non-adherent cells were removedby PBS washing and the attached cells were photographed andquantitated using a cell counter.

2.7. Soft agar assay

Cells suspended in agarose medium (10% FBS and 0.3% agarosein RPMI-1640 medium) containing serial concentrations of APwere plated onto a 6-well plate which was pre-coated with alayer of solidified agarose (10% FBS and 0.6% agarose in RPMImedium), and then incubated at 37 °C in a humidified atmo-sphere containing 5% CO2 for 1 week. At the end of incubation,cell colonies were fixed, stained with crystal violet, and thenphotographed using a Nikon Eclipse TE2000-U microscopeequipped with a Nikon Digital Camera DXM1200 and those colo-nies with size greater than 0.1 mm were counted.

2.8. Immunofluorescence staining

Adherent cells were fixed on the glass slides using 4% ice-cold formaldehyde and reacted with blocking buffer (phosphate-buffered saline (PBS) containing 5% bovine serum albumin (BSA)and 0.5% Triton X-100) for 1 h at room temperature.The reactedcells were incubated with primary antibodies overnight at 4 °C.The bound primary antibodies were detected using Alexa Fluor-labeled secondary antibody (Jackson ImmunoResearchLaboratories, West Grove, PA, USA). Alexa Fluor-conjugated phal-loidin (Cell Signaling) was used for detection of polymerizedF-actin microfilaments. Fluorescence image was acquired byusing a laser scanning confocal microscope system (Zeiss 510meta, Zeiss, Oberkochen, Germany).

2.9. Immunoblotting and GTPase activation assay

Cells were collected by trypsinization and lysed in cell lysisbuffer (PBS containing protease and phosphatase inhibitor cock-

tail, Sigma), centrifuged at 15,000 rpm for 10 min, and then theresulting supernatant was collected and used as crude extract.Protein concentrations were determined by using a BCA proteinassay kit (Pierce Biotechnology, Rockford, IL, USA). Eighty mi-crograms of crude protein were subjected to 12.5% sodiumdodecyl sulfate–polyacrylamide gel and electrophoresed at 120 Vfor 1.5 h. After electrophoresis, the proteins were transferredonto a nitrocellulose membrane (PROTRAN BA85, 0.45 µm;Sigma) by Bio-Rad Scientific Instruments Transphor Unit (Bio-rad Laboratory, Hercules, CA, USA). The transferred membranewas blocked with 1% w/v BSA in PBS followed by 1 h-incuba-tion with primary antibodies and then secondary antibodies.Development was performed by using ECL chemilumines-cence (Supersignal West Dura HRP Detection Kit, PierceBiotechnology, Rockford, IL, USA), and the signals were ac-quired and quantitated by an image analysis system (Fujifilm,Tokyo, Japan).

For the GTPase affinity precipitation experiment, cells werelysed in MLB buffer (Millipore, Bedford, MA, USA) and crudeprotein extracts were obtained as described earlier. After adding20 µg of PBD (Rac/Cdc42-binding domain) and RBD (Rho proteinbinding domain) agarose conjugate beads to 0.5 mL of the crudeextract, the mixture was gently shaken at 4 °C for 60 min. Thereacted beads were collected and washed with MLB buffer, andthen the bound proteins were acquired by boiling the beadswith SDS sample buffer and analyzing by immunoblotting.

2.10. Histopathological examination

The livers were collected, cut into small pieces, fixed in 10%buffered neutral formalin, and embedded in paraffin as de-scribed. Sections were cut at a thickness of 3–5 µm and stainedwith hematoxylin and eosin. The histopathological changes,including cell morphology and cellular lipid vesicles, were ex-amined by light microscopy (400×).

2.11. RNA extraction and RT-PCR

Total RNA was isolated from individual samples using TRIzolreagent (Invitrogen, Carlsbad, CA, USA). The purified RNA wasused as a template to generate first-strand cDNA synthesisusing a GoScript reverse transcription kit (Promega, Madison,WI, USA) The primer sequences used for RT-PCR are listedbelow: FAK, (F)5′-TTG GAG AGC TGA GGT CAT T-3′ and (R)5′-ATA CAC ACA CCA AAC ATC CAT A-3′; GADPH, (F)5′-GTC TTCACT ACC ATG GAG AAG G-3′ and (R) 5′-TCA TGG ATG ACC TTGGCC AG-3′; Snail, (F) 5′-TCT AGG CCC TGG CTG CTA CAA-3′ and(R)5′-ACA TCT GAG TGG GTC TGG AGG TG-3′; Slug, (F)5′-GCCTCC AAA AAG CCA AAC TAC AG-3′ and (R)5′-ACA GTC ATG GGGCTG TAT GC-3′; Twist, (F)5′-GGC GGC CAG GTA CAT CGA CTT-3′ and (R)5′-GCT AGT GGG ACG CGG ACA T- 3′. RT-PCRexperiments were performed in triplicates for each sample.Thecorrect size of the PCR products was confirmed by agarose gelelectrophoresis.

2.12. Transient gene over-expression

For Snail over-expression, DLD-1 cells were seeded at2 × 105 cells/mL in complete medium and cultured to reach 90%confluency, then transfected with control vector pcDNA3.1 or

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pCMV-Tag2B-Snail (a gift from Dr. Li-Sung Hsu, Institute of Bio-chemistry and Biotechnology, College of Medicine, Chung-Shan Medical University) by using T-Pro Non-liposomaltransfection reagent (T-Pro Biotechnology, Taipei City, Taiwan)according to the manufacturer’s instruction.

2.13. Human colon cancer xenograft mouse model

Male Balb/c nude mice aged 5 weeks were purchased from Na-tional Laboratory Animal Center of Taiwan (Taipei City, Taiwan)and maintained under supervision of the Institutional AnimalCare and Use Committee at Chung Shan Medical University.After 1 week maintenance, mice were randomly divided in tothree groups (control, DLD-1, DLD-1+AP), each group con-tained ten mice. Tumor implantation was performed byintraperitoneal injection of DLD-1 cells (2 × 106 in 0.1 PBS) withor without AP treatment (0.7 mg/mL for 24 h). Control mice wereintraperitoneal injected with 0.1 mL PBS as vehicle. After 6weeks, the mice were sacrificed and tumor tissues were excisedand collected for analysis of tumor weight and number. Themetastatic nodules of the visible tumor lesion on the liver wereidentified by histochemical analysis. All the evaluation of his-tological and pathological analysis was provided by theDepartment of Pathology, Chung Shan Medical University Hos-pital.

2.14. Statistical analysis

The data represent the mean ± SD from three independent ex-periments except where indicated.The Student’s t test was usedto analyze the significance of difference. Results with P < 0.05were considered statistically significant.

3. Results

3.1. AP insignificantly affected viability and proliferationof DLD-1 cells

First, we examined the effects of AP on viability and prolif-eration of human colon cancer cells DLD-1. As shown in Fig. 1A,cell viability was insignificantly altered by treatments with aserial concentration of AP (0.1–1.0 mg/mL) for 12, 24 or 48 h ascompared to control. In parallel, neither cell morphology (Fig. 1B)nor expression level of Ki-67 (Fig. 1C) was affected by the APtreatments for 24 h. Furthermore, effects of AP treatments oncell cycle distribution were also investigated, and the resultsrevealed that the AP treatments for 24 h insignificantly af-fected the cell cycle distribution of DLD-1 cells. Taken together,these findings revealed that AP treatments up to 1.0 mg/mLhad no significant effects on cell viability, cell proliferation andcell cycle distribution of DLD-1 cells. However, treated withhigher concentrations of AP (1.5, 2.0 and 3.0 mg/mL) signifi-cantly reduced viability of DLD-1 cells to less than 10% of control(data not shown).

3.2. AP suppressed adhesion, migration and invasion ofDLD-1 cells

Tumors with a malignant phenotype are highly associated withpromoted migration, invasion capability and adhesion. There-

fore, whether AP inhibited the motility and invasiveness ofDLD-1 cells was investigated. As shown in Fig. 2A and B,AP treatments dose-dependently inhibited migration and in-vasiveness of DLD-1 cells, and the inhibition of migrationand invasiveness by 0.5 and 0.7 mg/mL AP was significant(P < 0.01 as compared to control). In addition, growing in ananchorage-independent manner is also an important markerfor metastatic carcinoma (Ward et al., 2013). Therefore, capa-bility of colony formation on soft agar and adhesion toextracellular matrix (ECM) of DLD-1 cells exposed to AP wasalso determined. As shown in Fig. 2C and D, both colonynumbers and adherent cell numbers were significantly reducedin response to AP treatments (0.5 and 0.7 mg/mL, P < 0.01 ascompared to control).

Structure of cytoskeletal components such as tubulin andactin has been known to play an important role in cell adhe-sion and cell motility (Korb et al., 2004). Thus, the effects ofAP on structure of tubulin and actin were further examinedby an immunofluorescence assay using confocal microscopy.As shown in Fig. 2E, tubulin and actin presented well spreadand extended fibrous structure in control culture. Comparingto the control, the spread and extended fibers composing oftubulin and actin were obviously disrupted by AP treat-ments. Taken together, these results revealed that APsignificantly inhibited cell adhesion, migration and invasive-ness and disrupted integrity of microtubules and actinfibers.

3.3. Involvement of FAK signaling in AP-regulatedstructural cytoskeleton and adhesion in DLD-1 cells

Formation of lamellipodia and filopodia comprising actin cy-toskeleton plays a central role in cell adhesion and migration(Yamazaki, Kurisu, & Takenawa, 2005). Besides interferencewith cell adhesion and microtubule integrity, effects of AP onthe structure of F-actin in DLD-1 cell were investigated. Byusing phalloidin staining and a laser confocal microscope,regular architecture and expression level of F-actin were foundto be disrupted and decreased in response to AP treatmentas compared to control (0.7 mg/mL, Fig. 3A). FAK has been re-ported as a key player involved in actin cytoskeletonreconstruction and the consequent regulation of cell adhe-sion and migration (Zhao & Guan, 2009). Therefore, FAK andits associating signaling cascades were investigated next. Ourresults revealed that AP treatment decreased the protein levelof FAK but not the adaptor proteins including talin, α-actinin,and vinculin (Fig. 3B). Further exploration showed that phos-phorylation of FAK downstream targeting of paxillin (Tyr181and Tyr118), Src (Tyr416) and Akt (Ser473) was diminished inDLD-1 cells in response to AP treatment (Fig. 3C). Further-more, involvement of small GTPases associated with FAKsignaling including Rac1, cdc42 and Rho was also investi-gated. Our results showed that the protein levels of Rac1 cdc42and Rho were not influenced by AP treatments (Fig. 3D);however, the activated forms of the three small GTPases, GTP-binding forms, were all significantly diminished in responseto AP treatments (Fig. 3E).These findings revealed that AP treat-ments reduced FAK expression and the downstream signalingactivation.

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3.4. AP disrupted direct binding of Snail to FAK promoterin DLD-1 cells

The protein level of FAK was found decreased in DLD-1 cellsexposed to AP; thus, the underlying mechanism involved inregulation of the FAK level was further investigated. As shown

in Fig. 4A and B, AP treatments decreased protein levels of FAKin a dose-dependent manner and the decrease of FAK was notaffected in the cells pretreated with proteasome inhibitor MG-132 and then treated with AP. Further results showed that mRNAexpression of FAK was dose-dependently reduced in re-sponse to AP treatments, consistent with the decrease of protein

Fig. 1 – Effects of AP treatments on cell viability, cell proliferation and cell cycle distribution of DLD-1 cells. (A) Cells weretreated with indicated concentrations of AP for 12, 24 and 48 h, and then the cell viability was determined by a MTT assayand presented as percentage of normal control. (B) Cells were treated with indicated concentrations of AP for 24 h and thenthe cell morphology was monitored by phase contrast microscopy at 200× magnification. (C) Cells were treated withindicated concentrations of AP for 24 h and subjected to immunohistochemical analysis for Ki-67. Numbers of Ki-67positive cells were obtained from five random fields of view at 200× magnification. The quantitative results were presentedas mean ± SD of three independent experiments. (D) Cells were treated with indicated concentrations of AP for 24 h andsubjected to cell cycle distribution analysis by flow cytometry. Percentage of S phase was indicated.

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levels (Fig. 4C). Previous studies have implicated that FAK isinvolved in metastasis via regulation of cytoskeleton rear-rangement. Snail is also known to control cell-matrix adhesionby modulating cytoskeleton components, integrins and base-ment membrane proteins (Haraguchi et al., 2008). Therefore,the linkage of EMT inducers and decreased FAK expression inresponse to AP was next explored. As shown in Fig. 4D and E,both mRNA and protein levels of Snail, but not Slug or Twist,was decreased in DLD-1 cells with exposure to AP. Further analy-sis showed that a putative E-box binding motif CAGGTG forSnail, Slug and Twist was found located in the proximal pro-moter of the FAK gene (−349 to −344, Fig. 4F). To investigatewhether the EMT inducers bind to the putative E-box bindingmotif on the FAK promoter in epithelial cells, chromatinimmune-precipitation was performed and the results showedthat Snail, Slug and Twist were bound to the motif in both non-tumorigenic epithelial BEAS-2B and DLD-1 cells (Fig. 4G andH). However, in DLD-1 cells, only the binding of Snail to themotif was disrupted in response to AP treatment (0.7 mg/mL,Fig. 4H).Taken together, these findings demonstrated that Snailbound to a putative E-box motif on the FAK promoter in DLD-1cells, which was disrupted by AP treatments.

3.5. AP diminished cell motility of DLD-1 cells via Snail-mediated FAK expression

To confirm the correlation among Snail, FAK expression andcell motility, we transfected a Flag-Snail expression vector intoDLD-1 cells. Our results revealed that total expression level andnuclear translocation of Snail were obviously increased in thecells transfected with Snail as compared to vector (Fig. 5A andB). Snail overexpression restored its binding to FAK promoterin DLD-1 cells treated with AP (Fig. 5C). Moreover, Snailoverexpression also restored the FAK level and its down-stream signaling such as phosphorylation of paxillin (Tyr181and Tyr118), Src(Tyr416) and Akt(Ser473) in DLD-1 cells exposedto AP (Fig. 5D). In parallel, effects of Snail overexpression onmotility of DLD-1 cells were investigated. Our results re-vealed that the adhesion, migration and invasion of DLD-1 cellswere obviously promoted by Snail overexpression (Fig. 5E), andthe adhesion, migration and invasion of DLD-1 cells reducedby AP treatments were all significantly restored by Snailoverexpression (Fig. 5E). Collectively, these findings revealedthat Snail-mediated FAK expression was involved in suppres-sion of DLD-1 cell motility by AP treatments.

Fig. 2 – Effects of AP on adhesion, migration and invasion of DLD-1 cells. (A) Cells were treated with AP for 24 h, seeded ontotranswell inserts, and then incubated for 12 h. (B) Cells were pretreated with AP for 24 h, seeded onto the Matrigel-coatedtranswell inserts, and then incubated for 18 h. Numbers of cells on the lower surface membrane were photographed andcounted for quantitation of invaded cells. (C) Cells were pretreated with AP for 24 h, seeded on soft agar plates, and thenincubated for 1 week. The number of colonies was quantitated. (D) Cells were on a matrix-coated monolayer dish, and thenincubated with AP for 24 h. After washing with PBS, the attached cells were photographed and quantitated. (E) Cells wereseeded on matrix-coated glass slides, incubated with AP for 24 h, and then the actin and tubulin were detected byimmunofluorescence staining. The nuclei were stained with DAPI as shown in blue. Quantitative data were presented asmean ± SD of three independent experiments. * and **, P < 0.01 and P < 0.001 as compared to control, respectively.

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3.6. AP inhibited the tumorigenesis and metastasis ofcolon cancer in vivo

Our findings demonstrated that AP treatments suppressed ad-hesion, migration and invasion of DLD-1 cells in vitro; therefore,in vivo effects of AP treatments on tumorigenesis and metas-tasis caused by DLD-1 cells were further explored. By using axenograft model, our results revealed that implanted DLD-1cells strongly attached to colon tissue and formed numeroustumors in mice (Fig. 6A and B). By contrast, implantation ofAP-treated DLD-1 cells rarely attached to colon tissue andformed fewer number and smaller size of tumors (Fig. 6A andB). Additionally, hepatic and lymphatic metastasis caused by

AP-treated DLD-1 cells was significantly diminished as com-pared to that by untreated cells (Fig. 6C–E). Together, theseresults revealed that AP treatment suppressed tumorigenesisand metastatic capability of DLD-1 cells in vivo.

4. Discussion

The high mortality caused by colon cancer is highly associ-ated with the metastatic capability of the carcinoma cells.In this study, we demonstrated that the apple polyphenolextract AP significantly inhibited adhesion, colony formation,

Fig. 3 – FAK signaling cascades involved in AP-mediated cell adhesion. (A) Cells were incubated with AP (0.7 mg/mL) for24 h, and then the F-actin was detected by immunofluorescence staining. The nuclei were stained with DAPI as shown inblue. (B–D) Cells were incubated with AP at indicated concentrations for 24 h and then lysed for immunodetection of theindicated proteins. β-actin was used as an internal control. (E) Cells were incubated with AP (0.7 mg/mL) for 24 h, and thenlysed for a GTPase affinity precipitation assay and immunodetection of Rac1, Cdc42 and Rho A. Tubulin was used as aninternal control.

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migration and invasiveness of colon cancer cells DLD-1 andconsequently suppressed the metastasis of the colon carci-noma cells. Of importance, our findings indicate that APdiminished the metastatic capability of DLD-1 cells via Snail-mediated down-regulation of FAK expression and thesubsequent inhibition of FAK signaling cascades such as paxillin,Src, Akt, Rho A, Rac-1 and Cdc42.

Mounting evidence has shown that functional compo-nents of dietary food such as fruits and vegetables possess anti-tumor activities through various mechanisms (González-Sarrías,Li, & Seeram, 2012; Zhong, Chiou, Pan, Ho, & Shahidi, 2012).Catechins and epigallocatechin-3-gallate (EGCG) also inhibit mi-gration of human melanoma cells and lung carcinoma cellsCL1-5 via suppression of the NF-κB-COX2 pathway and MMP-2expression, respectively (Deng & Lin, 2011; Singh & Katiyar,2011). Previous studies have shown that catechin andepicatechin, the major components of apple polyphenols, exertanti-migration effects on several human cancer cells. Miura et al.

(2008) have demonstrated that apple polyphenols increased mi-tochondrial membrane permeability, contributing to cytochromeC release, activation of caspase-9 and caspase-3, and the fol-lowing apoptosis of mouse melanoma and mammary tumorcells (Miura et al., 2008). Apple polyphenol fractions also ex-hibited similar anti-tumoral activity to trigger apoptosis ofhuman colon cancer cells SW620 via promotion of caspase-3activity, as well as in vivo diminished aberrant crypt foci for-mation in distal colon (Gosse et al., 2005). Interestingly, ourfindings reveal that the lower dose of AP (<1.0 mg/mL) inhib-ited metastatic capabilities of DLD-1 cells but insignificantlyaltered growth and cell cycle distribution of the cells, whichmay be attributed to biological specificity of individual cell lineand different bioactive compositions.

FAK plays a critical role in cell migration through regula-tion of downstream targets. In response to extracellularstimulation, activated FAK undergoes autophosphorylation, re-cruits Src to form complexes, phosphorylates paxillin, triggers

Fig. 4 – AP reduced Snail expression and binding of Snail to the FAK promoter in DLD-1 cells. (A) Cells were incubated withserial concentrations of AP for 24 h and then the cellular FAK level was determined by immunoblotting. (B) DLD-1 cells weretreated with AP or AP combined with MG132 for 24 h, and then the cellular FAK level was determined by immunoblotting.(C–E) DLD-1 cells were incubated with serial concentrations of AP for 24 h, and lysed for mRNA and protein extraction. ThemRNA and the protein levels were analyzed by RT-PCR and immunoblotting, respectively. (F) A representative diagram ofthe putative E-box on the FAK promoter. (G and H) BEAS-2B and DLD-1 cells were incubated with or without AP (0.7 mg/mL)for 24 h, and then lysed for chromatin immunoprecipitation. The FAK promoter was detected by RT-PCR.

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actin cytoskeleton reorganization and consequently en-hances cell migration (Mitra et al., 2005). It is reported thatblocking the interaction of FAK and paxillin reduced the mi-gration and invasion of mouse embryonic fibroblast cells(Deramaudt et al., 2014). Inhibitory effects of polyphenols ontumor cell migration have been reported to be associated withinhibition of FAK signaling. Resveratrol diminished migra-tion of human breast cancer cells MDA-MB-231 via inhibitionof FAK activities (Azios & Dharmawardhane, 2005). Apigenininhibited migration and invasion of human ovarian cancer cellsA2780 through down-regulation of FAK expression by promot-ing proteasome degradation (Hu, Meng, & Fang, 2008). Moreover,catechin is reported to induce integrin-mediated intestinal cellsurvival signaling, including alteration of cytoskeleton and de-

crease of FAK activity, and consequently inhibits tumorformation in C57BL/6J-Min/+ mice (Weyant, Carothers,Dannenberg, & Bertagnolli, 2001). In line with these observa-tions, our findings revealed that AP decreased FAK expressionand reduced phosphorylation of Src and paxillin, and even-tually suppressed adhesion, migration and invasion of DLD-1cells. Moreover, we demonstrated that AP diminished actin cy-toskeleton reorganization and inhibited activity of Rho A, Rac-1and Cdc42. In contrast to enzymatic activities, protein levelsof Rho A, Rac-1 and Cdc42 were insignificantly affected in DLD-1cells with exposure to AP. Rho small GTPase family memberssuch as Rho A, Rac-1 and Cdc42 play an important role in actincytoskeleton reorganization, involved in cell migration (Hall,2012). Previous investigations have shown that FAK may

Fig. 5 – Involvement of Snail in AP-suppressed FAK expression and AP-reduced motility of DLD-1 cells. (A–D) Cells weretransfected with Flag-Snail expression vector (Snail) or vector only (Vector), treated with or without AP for 24 h, and thenlysed for mRNA and protein extraction. mRNA expression, interaction with FAK promoter and protein level weredetermined by RT-PCR, chromatin immunoprecipitation and immunoblotting, respectively. (E) Cells were transfected withFlag-Snail expression vector (Snail) or vector only (Vector), treated with or without AP for 24 h, and then cell adhesion,migration and invasion were individually analyzed. Quantitative data were presented as average number of adherent,migrated and transmigrated cells obtained from three independent experiments. *, P < 0.01 between the indicated groups.

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Fig. 6 – AP suppressed tumorigenesis and abdominal metastasis of DLD-1 cells in xenograft mice. Cells were pretreatedwithout (DLD-1) or with 0.7 mg/mL AP (DLD-1 + AP) for 24 h, and then injected into the abdomen of Balb/c mice (ten micefor each group). Six weeks after implantation, the mice were sacrificed and tumor growth and metastasis were analyzed.(A) Representative photographs of tumor growth on rectum. (B) Number and total weight of tumor foci from rectum werepresented and quantitated. (C) Representative photographs of metastatic tumor in liver. (D and E) Representative HEstaining of hepatic tissues and lymphatic vessels. Magnification, 40× (left panel) and 100× (right panel). Arrows indicatedthe tumor infiltrate in the liver and the lymphatic vessels.

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activate Rho small GTPases and contributes to formation ofcytoskeletal fibers (Mitra et al., 2005). Chang et al. demon-strated that phosphorylation of β-PIX by FAK resulted inrecruitment of Rac-1 to the focal adhesion site and activatedRac-1 (Chang, Lemmon, Park, & Romer, 2007). In this study, weprovide evidence that AP suppressed FAK expression and ac-tivity of the downstream Rho small GTPases, contributing toinhibition of cytoskeleton reorganization.

The Snail transcript factor family including Snail (Snai-1)and Slug (Snai-2) regulates a wide range of cellular functionssuch as cell survival, apoptosis and epithelial-to-mesenchymaltransition via binding to the E-box located in the promoter regionof the target genes (Nieto, 2002). Recent studies have pro-posed that members of the Snail family play a critical role inmetastasis of human cancers. Miyoshi et al. indicated that ex-pression of Snail was correlated with lymphatic invasion anddistal metastasis in human hepatocellular carcinomas (Miyoshiet al., 2005). In addition, the expression level of Snail and Slugwas found to be associated with advanced stages and metas-tasis in human breast cancer and ovarian cancer (Elloul et al.,2005). Although FAK is reported to be associated with tumormetastasis, the linkage between FAK and Snail family pro-teins has been rarely investigated. Interestingly, we found aputative E-box sequence localized in the promoter region ofFAK, and first demonstrated that Snail directly bound to theE-box and regulated the expression of FAK. Furthermore, ourfindings showed that AP inhibited the interaction between Snailand the E-box on the FAK promoter, contributing to a decreaseof FAK expression and suppression of Rho protein activity, andthe AP-reduced FAK expression was restored by overexpressionof snail. Zhang et al. demonstrated that down regulation of Snailexpression led to a decrease of RhoA activities and loss of cellmigration in breast cancer cells (Zhang et al., 2013). Similarly,our findings showed that AP inhibited Snail expression and RhoA activity. Collectively, these observations indicate that Snailregulates FAK expression through binding to the E-box regionand consequently mediates metastatic properties of DLD-1 cells.However, clinical correlation among FAK, Snail and tumor me-tastasis needs further investigation.

High levels of expression of FAK are associated with lymphnode metastasis in non-small cell lung cancer (Ji et al., 2013),endometrial carcinoma (Zhou et al., 2013), and gastric cancer(Park et al., 2010). Inhibition of FAK activity by a small mo-lecular inhibitor has been reported to attenuate distal metastasisin rats bearing neuroblastoma cells (Megison, Stewart, Nabers,Gillory, & Beierle, 2013). In hepatocellular carcinomas, knock-down of FAK by specific small inhibitory RNA inhibits expressionof MMP-2 and MMP-9 and consequent invasion and metasta-sis (Chen et al., 2010).

In conclusion, the present study provides evidences that APdown-regulates FAK expression via disruption of Snail–FAK pro-moter interaction and inhibits FAK downstream signalingcascades, and consequently diminishes motility of DLD-1 cells.Furthermore, by using a xenograft model, we demonstrate thatAP significantly lowers the number of tumors and reduces theinfiltration of tumor cell into liver and lymph nodes in micebearing DLD-1 cells. These findings indicate that AP sup-presses metastasis of DLD-1 cell in vivo, suggesting that APshould be beneficial to colon cancer treatments via attenua-tion of metastasis.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgement

We would like to thank Dr. Li-Sung Hsu for providing pcDNA3.1and pCMV-Tag2B-Snail plasmid. We are grateful to Dr. BernardA Schwetz for helpful editing of the manuscript.This study wassupported by a grant from Ministry of Science and Technol-ogy (National Science Council), Taiwan (NSC99-2632-B-040-001-MY3).

R E F E R E N C E S

Abbi, S., & Guan, J. L. (2002). Focal adhesion kinase: Proteininteractions and cellular functions. Histology andHistopathology, 17, 1163–1171.

Avizienyte, E., & Frame, M. C. (2005). Src and FAK signallingcontrols adhesion fate and the epithelial-to-mesenchymaltransition. Current Opinion in Cell Biology, 17, 542–547.

Azios, N. G., & Dharmawardhane, S. F. (2005). Resveratrol andestradiol exert disparate effects on cell migration, cell surfaceactin structures, and focal adhesion assembly in MDA-MB-231human breast cancer cells. Neoplasia (New York, N.Y.), 7, 128–140.

Chang, F., Lemmon, C. A., Park, D., & Romer, L. H. (2007). FAKpotentiates Rac1 activation and localization to matrixadhesion sites: A role for betaPIX. Molecular Biology of the Cell,18, 253–264.

Chen, J. S., Huang, X. H., Wang, Q., Chen, X. L., Fu, X. H., Tan, H. X.,Zhang, L. J., Li, W., & Bi, J. (2010). FAK is involved in invasionand metastasis of hepatocellular carcinoma. Clinical andExperimental Metastasis, 27, 71–82.

Deng, Y. T., & Lin, J. K. (2011). EGCG inhibits the invasion of highlyinvasive CL1-5 lung cancer cells through suppressing MMP-2expression via JNK signaling and induces G2/M arrest. Journalof Agricultural and Food Chemistry, 59, 13318–13327.

Deramaudt, T. B., Dujardin, D., Noulet, F., Martin, S., Vauchelles,R., Takeda, K., & Ronde, P. (2014). Altering FAK-paxillininteractions reduces adhesion, migration and invasionprocesses. PLoS ONE, 9, e92059.

Elloul, S., Elstrand, M. B., Nesland, J. M., Trope, C. G., Kvalheim, G.,Goldberg, I., Reich, R., & Davidson, B. (2005). Snail, Slug, andSmad-interacting protein 1 as novel parameters of diseaseaggressiveness in metastatic ovarian and breast carcinoma.Cancer, 103, 1631–1643.

Faerden, A. E., Sjo, O. H., Bukholm, I. R., Andersen, S. N.,Svindland, A., Nesbakken, A., & Bakka, A. (2011). Lymph nodemicrometastases and isolated tumor cells influence survivalin stage I and II colon cancer. Diseases of the Colon and Rectum,54, 200–206.

Fujiwara, K., Nakashima, S., Sami, M., & Kanda, T. (2013). Ninety-day dietary toxicity study of apple polyphenol extracts in Crl:CD (SD) rats. Food and Chemical Toxicology: An InternationalJournal Published for the British Industrial Biological ResearchAssociation, 56, 214–222.

González-Sarrías, A., Li, L., & Seeram, N. P. (2012). Anticancereffects of maple syrup phenolics and extracts onproliferation, apoptosis, and cell cycle arrest of human coloncells. Journal of Functional Foods, 4, 185–196.

90 j o u rna l o f f un c t i ona l f o od s 1 2 ( 2 0 1 5 ) 8 0 – 9 1

Gosse, F., Guyot, S., Roussi, S., Lobstein, A., Fischer, B., Seiler, N., &Raul, F. (2005). Chemopreventive properties of appleprocyanidins on human colon cancer-derived metastaticSW620 cells and in a rat model of colon carcinogenesis.Carcinogenesis, 26, 1291–1295.

Gupta, G. P., & Massague, J. (2006). Cancer metastasis: Building aframework. Cell, 127, 679–695.

Hall, A. (2012). Rho family GTPases. Biochemical SocietyTransactions, 40, 1378–1382.

Haraguchi, M., Okubo, T., Miyashita, Y., Miyamoto, Y., Hayashi, M.,Crotti, T. N., McHugh, K. P., & Ozawa, M. (2008). Snail regulatescell-matrix adhesion by regulation of the expression ofintegrins and basement membrane proteins. The Journal ofBiological Chemistry, 283, 23514–23523.

Hibasami, H., Shohji, T., Shibuya, I., Higo, K., & Kanda, T. (2004).Induction of apoptosis by three types of procyanidin isolatedfrom apple (Rosaceae Malus pumila) in human stomachcancer KATO III cells. International Journal of Molecular Medicine,13, 795–799.

Hill, A. R., McQuellon, R. P., Russell, G. B., Shen, P., Stewart, J. H.,4th, & Levine, E. A. (2011). Survival and quality of lifefollowing cytoreductive surgery plus hyperthermicintraperitoneal chemotherapy for peritoneal carcinomatosisof colonic origin. Annals of Surgical Oncology, 18, 3673–3679.

Hsu, H. Y., Lin, T. Y., Hwang, P. A., Tseng, L. M., Chen, R. H., Tsao,S. M., & Hsu, J. (2013). Fucoidan induces changes in theepithelial to mesenchymal transition and decreasesmetastasis by enhancing ubiquitin-dependent TGFbetareceptor degradation in breast cancer. Carcinogenesis, 34, 874–884.

Hu, X. W., Meng, D., & Fang, J. (2008). Apigenin inhibitedmigration and invasion of human ovarian cancer A2780cells through focal adhesion kinase. Carcinogenesis, 29, 2369–2376.

Ji, H. F., Pang, D., Fu, S. B., Jin, Y., Yao, L., Qi, J. P., & Bai, J. (2013).Overexpression of focal adhesion kinase correlates withincreased lymph node metastasis and poor prognosis in non-small-cell lung cancer. Journal of Cancer Research and ClinicalOncology, 139, 429–435.

Kaur, M., Deep, G., Jain, A. K., Raina, K., Agarwal, C., Wempe, M. F.,& Agarwal, R. (2013). Bitter melon juice activates cellularenergy sensor AMP-activated protein kinase causingapoptotic death of human pancreatic carcinoma cells.Carcinogenesis, 34, 1585–1592.

Korb, T., Schluter, K., Enns, A., Spiegel, H. U., Senninger, N.,Nicolson, G. L., & Haier, J. (2004). Integrity of actin fibers andmicrotubules influences metastatic tumor cell adhesion.Experimental Cell Research, 299, 236–247.

Luo, M., & Guan, J. L. (2010). Focal adhesion kinase: A prominentdeterminant in breast cancer initiation, progression andmetastasis. Cancer Letters, 289, 127–139.

Megison, M. L., Stewart, J. E., Nabers, H. C., Gillory, L. A., & Beierle,E. A. (2013). FAK inhibition decreases cell invasion, migrationand metastasis in MYCN amplified neuroblastoma. Clinicaland Experimental Metastasis, 30, 555–568.

Mitra, S. K., Hanson, D. A., & Schlaepfer, D. D. (2005). Focaladhesion kinase: In command and control of cell motility.Nature Reviews. Molecular Cell Biology, 6, 56–68.

Miura, T., Chiba, M., Kasai, K., Nozaka, H., Nakamura, T., Shoji, T.,Kanda, T., Ohtake, Y., & Sato, T. (2008). Apple procyanidinsinduce tumor cell apoptosis through mitochondrial pathwayactivation of caspase-3. Carcinogenesis, 29, 585–593.

Miyoshi, A., Kitajima, Y., Kido, S., Shimonishi, T., Matsuyama, S.,Kitahara, K., & Miyazaki, K. (2005). Snail accelerates cancerinvasion by upregulating MMP expression and is associatedwith poor prognosis of hepatocellular carcinoma. BritishJournal of Cancer, 92, 252–258.

Nam, E. H., Lee, Y., Park, Y. K., Lee, J. W., & Kim, S. (2012). ZEB2upregulates integrin alpha5 expression through cooperationwith Sp1 to induce invasion during epithelial-mesenchymaltransition of human cancer cells. Carcinogenesis, 33, 563–571.

Nieto, M. A. (2002). The snail superfamily of zinc-fingertranscription factors. Nature Reviews. Molecular Cell Biology, 3,155–166.

Osada, K., Suzuki, T., Kawakami, Y., Senda, M., Kasai, A., Sami, M.,Ohta, Y., Kanda, T., & Ikeda, M. (2006). Dose-dependenthypocholesterolemic actions of dietary apple polyphenol inrats fed cholesterol. Lipids, 41, 133–139.

Park, J. H., Lee, B. L., Yoon, J., Kim, J., Kim, M. A., Yang, H. K., &Kim, W. H. (2010). Focal adhesion kinase (FAK) geneamplification and its clinical implications in gastric cancer.Human Pathology, 41, 1664–1673.

Rizzi, F., Naponelli, V., Silva, A., Modernelli, A., Ramazzina, I.,Bonacini, M., Tardito, S., Gatti, R., Uggeri, J., & Bettuzzi, S.(2014). Polyphenon E (R), a standardized green tea extract,induces endoplasmic reticulum stress, leading to death ofimmortalized PNT1a cells by anoikis and tumorigenic PC3 bynecroptosis. Carcinogenesis, 35, 828–839.

Shoji, T., Akazome, Y., Kanda, T., & Ikeda, M. (2004). Thetoxicology and safety of apple polyphenol extract. Food andChemical Toxicology: An International Journal Published for theBritish Industrial Biological Research Association, 42, 959–967.

Singh, T., & Katiyar, S. K. (2011). Green tea catechins reduceinvasive potential of human melanoma cells by targetingCOX-2, PGE2 receptors and epithelial-to-mesenchymaltransition. PLoS ONE, 6, e25224.

Sugiyama, H., Akazome, Y., Shoji, T., Yamaguchi, A., Yasue, M.,Kanda, T., & Ohtake, Y. (2007). Oligomeric procyanidins inapple polyphenol are main active components for inhibitionof pancreatic lipase and triglyceride absorption. Journal ofAgricultural and Food Chemistry, 55, 4604–4609.

Thiery, J. P. (2002). Epithelial-mesenchymal transitions in tumorprogression. Nature Reviews. Cancer, 2, 442–454.

Ward, K. K., Tancioni, I., Lawson, C., Miller, N. L., Jean, C., Chen, X.L., Uryu, S., Kim, J., Tarin, D., Stupack, D. G., Plaxe, S. C., &Schlaepfer, D. D. (2013). Inhibition of focal adhesion kinase(FAK) activity prevents anchorage-independent ovariancarcinoma cell growth and tumor progression. Clinical andExperimental Metastasis, 30, 579–594.

Weyant, M. J., Carothers, A. M., Dannenberg, A. J., & Bertagnolli,M. M. (2001). (+)-Catechin inhibits intestinal tumor formationand suppresses focal adhesion kinase activation in the Min/+mouse. Cancer Research, 61, 118–125.

Yamazaki, D., Kurisu, S., & Takenawa, T. (2005). Regulation ofcancer cell motility through actin reorganization. CancerScience, 96, 379–386.

Zhang, A., Wang, Q., Han, Z., Hu, W., Xi, L., Gao, Q., Wang, S.,Zhou, J., Xu, G., Meng, L., Chen, G., & Ma, D. (2013). Reducedexpression of Snail decreases breast cancer cell motility bydownregulating the expression and inhibiting the activity ofRhoA GTPase. Oncology Letters, 6, 339–346.

Zhao, J., & Guan, J. L. (2009). Signal transduction by focaladhesion kinase in cancer. Cancer Metastasis Reviews, 28, 35–49.

Zhong, Y., Chiou, Y. S., Pan, M. H., Ho, C. T., & Shahidi, F. (2012).Protective effects of epigallocatechin gallate (EGCG)derivatives on azoxymethane-induced colonic carcinogenesisin mice. Journal of Functional Foods, 4, 323–330.

Zhou, J., Roh, J. W., Bandyopadhyay, S., Chen, Z., Munkarah, A. R.,Hussein, Y., Alosh, B., Jazaerly, T., Hayek, K., Semaan, A., Sood,A. K., & Ali-Fehmi, R. (2013). Overexpression of enhancer ofzeste homolog 2 (EZH2) and focal adhesion kinase (FAK) inhigh grade endometrial carcinoma. Gynecologic Oncology, 128,344–348.

91j o u rna l o f f un c t i ona l f o od s 1 2 ( 2 0 1 5 ) 8 0 – 9 1