Original Contribution

Biochanin A and prunetin improve epithelial barrier functionin intestinal CaCo-2 cells via downregulation of ERK, NF-κB,and tyrosine phosphorylation

Stefanie Piegholdt a, Kathrin Pallauf a, Tuba Esatbeyoglu a, Nancy Speck b, Karina Reiss b,Lars Ruddigkeit c, Achim Stocker c, Patricia Huebbe a, Gerald Rimbach a,n

a Institute of Human Nutrition and Food Science, Christian-Albrechts-University Kiel, D-24118 Kiel, Germanyb Department of Dermatology and Allergology, University Hospital Schleswig-Holstein, Campus Kiel, D-24105 Kiel, Germanyc Department of Chemistry and Biochemistry, University of Bern, CH-3012 Bern, Switzerland

a r t i c l e i n f o

Article history:Received 23 November 2013Received in revised form14 February 2014Accepted 25 February 2014Available online 12 March 2014

Keywords:IsoflavonesBiochanin APrunetinIntestinal epithelial barrierTyrosine phosphorylationERKNF-κB

a b s t r a c t

The single-layered gut epithelium represents the primary line of defense against environmentalstressors; thereby monolayer integrity and tightness are essentially required to maintain gut healthand function. To date only a few plant-derived phytochemicals have been described as affectingintestinal barrier function. We investigated the impact of 28 secondary plant compounds on the barrierfunction of intestinal epithelial CaCo-2/TC-7 cells via transepithelial electrical resistance (TEER)measurements. Apart from genistein, the compounds that had the biggest effect in the TEER measure-ments were biochanin A and prunetin. These isoflavones improved barrier tightness by 36 and 60%,respectively, compared to the untreated control. Furthermore, both isoflavones significantly attenuatedTNFα-dependent barrier disruption, thereby maintaining a high barrier resistance comparable tononstressed cells. In docking analyses exploring the putative interaction with the tyrosine kinase EGFR,these novel modulators of barrier tightness showed very similar values compared to the known tyrosinekinase inhibitor genistein. Both biochanin A and prunetin were also identified as potent reducers ofNF-κB and ERK activation, zonula occludens 1 tyrosine phosphorylation, and metalloproteinase-mediatedshedding activity, which may account for the barrier-improving ability of these isoflavones.

& 2014 Elsevier Inc. All rights reserved.

The intestinal epithelial barrier is mediated by junctionalcomplexes between adjacent cells including tight junction (TJ),adherence junction (AJ) and gap junction proteins as well asdesmosomes. These junctional complexes comprise membrane-spanning proteins such as occludin (OCLN) [1], claudins (CLDNs)[2], junctional adhesion molecules (JAMs) [3], E-cadherin (CDH1)[4] and desmosomes [5] as well as the scaffolding proteins zonulaoccludens 1 (ZO-1) [6] and β-catenin (β-CTNN) [7]. Thereby ZO-1plays a central role in maintaining the barrier integrity byorchestrating the localization of junctional proteins and theirinteraction with the actomyosin cytoskeleton [6]. Moreover, thephosphorylation status of junctional proteins affects barrier

tightness. Whereas phosphorylation of serine/threonine residuescan ameliorate [8] or deteriorate [9] barrier function, tyrosinephosphorylation strongly impairs barrier function [10,11].

Deteriorated barrier function is a hallmark of inflammatorydisorders [12,13] and the proinflammatory tumor necrosis factor α(TNFα) is a central cytokine involved in acute [14] and chronicinflammation [15]. TNFα activates the NF-κB pathway via bindingto the TNFα receptor (TNFR1) [16] and accounts for reduced ZO-1[17] and OCLN [18] protein levels. Furthermore, TNFα hindersproper localization of junctional proteins [17] and activates matrixmetalloproteinases that cleave extracellular matrix and junctionalproteins [19,20]. All these alterations contribute to a TNFα-dependent loss of transepithelial electrical resistance (TEER) andincrease in monolayer permeability in vitro and in vivo [12,21].

Food-derived secondary plant compounds can easily access theintestinal epithelium via chyme and may contribute to themodification of the intestinal barrier function by modulation ofthe junctional complex. Daily uptake of secondary plant com-pounds is 1.5 g or higher, depending on the composition of thefood. However, only a minority of studies has examined intestinalbarrier-modulating effects of dietary phytochemicals and only a

Contents lists available at ScienceDirect

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

Free Radical Biology and Medicine

http://dx.doi.org/10.1016/j.freeradbiomed.2014.02.0250891-5849/& 2014 Elsevier Inc. All rights reserved.

Abbreviations: ADAM, a disintegrin and metalloproteinase; AJ, adherence junc-tion; bio A, biochanin A; CDH1, E-cadherin; CLDN, claudin; β-CTNN, β-catenin;genis, genistein; NF-κB, nuclear factor κB; OCLN, occludin; prun, prunetin; sTNFR1,soluble TNFα receptor 1; TEER, transepithelial electrical resistance; TJ, tightjunction; TNFα, tumor necrosis factor α; TNFR1, TNFα receptor 1; ZO-1, zonulaoccludens 1

n Corresponding author. Fax: þ49 431 880 2628.E-mail address: rimbach@foodsci.uni-kiel.de (G. Rimbach).

Free Radical Biology and Medicine 70 (2014) 255–264

few substances have been described as improving epithelialbarrier integrity and attenuating intestinal inflammation in vitroand in vivo. Interestingly, these barrier-improving plant bioactivessuch as chrysin [22], curcumin [23], genistein [24], kaempferol[25], and quercetin [26] exhibit a polyphenolic structure.

The aim of this study was to identify secondary plant com-pounds that may promote barrier function under normal andinflammatory conditions and to investigate potential underlyingmechanisms. Twenty-eight different plant compounds, the major-ity with a basic polyphenolic structure (see Fig. 1), were system-atically screened in a CaCo-2/TC-7 cell model with regard to theireffect on monolayer integrity. A large variety of polyphenols fromdifferent polyphenolic subgroups was chosen to study the mainsecondary plant metabolites that are consumed in the daily diet byvarious population groups. Isoflavones and their metabolitesfound in soy, kudzu, and beans, as well as curcumin, the principalcurcuminoid in turmeric, are widely consumed in Asia [27,28].Hydroxytyrosol is found in olive oil, which is part of a Mediterra-nean diet [29]. Sulforaphane, anthocyanins, procyanidins, and theremaining selected flavonoids (Fig. 1) are found in fruits such asberries, apples, grapes, and citrus fruits, but also in cocoa beans,tea, and herbs, and are consumed daily with a normal European/Western or Mediterranean diet [30].

We identified the isoflavones biochanin A and prunetin asimproving TEER and preventing TNFα-dependent disruptionof barrier integrity. Biochanin A and prunetin inhibit TNFα-dependent transactivation of the proinflammatory NF-κB pathwayin CaCo-2 cells and reduce tyrosine phosphorylation of ZO-1.

Material and methods

Test compounds and inhibitors

Genistein and quercetin were obtained from Carl Roth (Karls-ruhe, Germany). Biochanin A, prunetin, daidzein, catechin, luteo-lin, naringenin, p-ethylphenol, and sulforaphane were purchasedfrom Sigma–Aldrich Chemie GmbH (Taufkirchen, Germany).8-Hydroxydaidzein was purchased from CHESS Fine Organics(Mannheim, Germany), curcumin was from Aquanova AG (Darm-stadt, Germany), and equol, isorhamnetin, and the anthocyaninswere from Extrasynthese (Genay, France). Glycitein, formononetin,orobol, pratensein, and santal were obtained from Plantech UK(Reading, UK). Genistein-7β-glucuronide was purchased fromToronto Research Chemicals (North York, ON, Canada). The pro-cyanidins B1–B5 were isolated and provided as described inprevious publications [31,32]. Hydroxytyrosol was obtained fromSeprox Biotech (Madrid, Spain). The MEK1 inhibitor PD98059 wasobtained from Enzo Life Sciences GmbH (Loerrach, Germany).Compounds and inhibitors were dissolved in dimethyl sulfoxide(DMSO; Carl Roth) or in sterile-filtered double-distilled water(procyanidins) and stored as stock solutions at �80 and �201C.The tyrosine kinase inhibitor genistein served as a positive controlin TEER and immunoprecipitation experiments.

Cell culture

Human colon carcinoma epithelial CaCo-2/TC-7 cells weremaintained in Dulbecco’s modified Eagle medium (DMEM) con-taining 4.5 g/L glucose, 4 mmol/L L-glutamine, 1 mmol/L sodiumpyruvate, 100 U/ml penicillin, 100 mg/ml streptomycin, and 20%(v/v) fetal calf serum (FCS). Cells were grown in 5% CO2/95% air at371C in a humidified atmosphere until 90–95% confluency. Forsubculturing, cells were detached with 0.05% trypsin and 0.02% EDTAin Ca and Mg-free phosphate-buffered saline (PBS). Cell culturereagents were purchased from PAA Laboratories GmbH (Coelbe,Germany). Cell culture plastic labware was obtained from Sarstedt

(Nuembrecht, Germany) except when otherwise mentioned. CaCo-2cells spontaneously start to differentiate after reaching confluenceand develop a polarized small intestinal enterocyte-like phenotype[33,34]. For our experiments, cells were stimulated with the highestnontoxic concentrations of the test compounds (Table 1) or the ERKinhibitor PD98059 (25 mmol/L) as assessed with the neutral red assay[35]. The highest applied concentration of the vehicle DMSO (0.1%)had no significant effect on cell viability and was used as the control.All test compounds were administered to the apical compartment(simulating the luminal side of the cells), excluding TNFα andepidermal growth factor (EGF), which were added to the basolateralcompartment (according to the serosal route).

Cytotoxicity measurement

Cytotoxicity was evaluated via the neutral red assay as describedpreviously [35]. In brief, CaCo-2 cells were precultured for 24 h andsubsequently treated with increasing concentrations of the testcompounds (1–100 mmol/L) or the inhibitors (1–50 mmol/L) for48 h. Ethanol (10% (v/v)) was used as a positive control. Then theculture medium was replaced with medium containing 50 mg/mlneutral red (Carl Roth) and incubated for 2 h. Neutral red wasextracted with neutral red extraction buffer (50% ethanol, 49%double-distilled water, 1% glacial acetic acid (v/v)) for 15 min on ashaking platform. Neutral red absorbance was measured in amicroplate reader at 540 nm against blank wells containing extrac-tion buffer only. Viability of the compound-treated cells wascalculated as percentage absorbance of the vehicle-treated cells.Noncytotoxic concentrations of the test compounds or the inhibi-tors were used for all experiments.

Determination of barrier function

TEER measures electrical conductivity of cell monolayers grownon permeable filters (Corning, Inc., Corning, NY, USA). The TEERcorrelates with the differentiation state of CaCo-2 cells, indicatingmonolayer barrier tightness [36]. TEER was measured with aMillicell ERS-2 V-Ohm Meter (Merck KGaA, Darmstadt, Germany)and calculated as follows: (resistance of treated cells (Ω) �resistance of blank well (Ω)) � effective membrane area (1.12 cm2)normalized to the control (untreated cells). The cells were seededat high density onto permeable filters, precultured for 4 days,and then incubated with the test compounds for a further 5 days.The TEER measurement started at the beginning of the testcompound supplementation to document the development ofbarrier tightness. Recombinant TNFα (rhTNFα; 100 ng/ml; Immu-notools, Friesoythe, Germany) was added on the last day to inducebarrier disruption. After 24 h, TEER was measured again toevaluate the test compound-mediated protection against TNFα-induced reduction of barrier tightness.

Transient transfection and luciferase reporter gene assay

CaCo-2 cells were grown in plastic dishes for 24 h. Cells weretransiently cotransfected with an expression vector containing anNF-κB enhancer element fused with the firefly luciferase gene(pNFκB-Luc; Clontech Laboratories, Mountain View, CA, USA) and anormalization vector (phRL-TK-Luc; Promega GmbH, Mannheim,Germany) containing the renilla luciferase gene. The mock vectorpTAL-Luc (Clontech) lacking the NF-κB enhancer element servedas a negative control. Transfection was performed using theJetPEI transfection reagent (Polyplus Transfection, Illkirch, France)according to the manufacturer’s instructions. After 24 h of trans-fection, the medium was replaced with fresh medium containingbiochanin A, prunetin, or PD98059. The cells were incubated withthe test substances for 20 h and subsequently TNFα (100 ng/ml)

S. Piegholdt et al. / Free Radical Biology and Medicine 70 (2014) 255–264256

genistein orobol

daidzein

biochanin A

formononetin

santal

pratensein

curcumin 3-hydroxy-tyrosol sulforaphane

equol8-hydroxydaidzein

p-ethylphenol

naringenin

catechin

isorhamnetin

malvidin-3-O-glucoside

luteolin(-)-epicatechin-4β-6-(-)-epicatechin ( B5)

(-)-epicatechin-4β-8-(+)-catechin (B1)

(-)-epicatechin-4β-8-(-)-epicatechin (B2)

(+)-catechin-4α-8-(+)-catechin (B3)

(+)-catechin-4α-8-(-)-epicatechin (B4)

quercetin

isoflavonoids

isoflavonoid-metabolites

anthocyanins

flavanol

flavanone

flavone

dimeric procyanidins

others

cyanidin-3-O-glucoside

genistein-7β-glucuronide

glycitein

prunetin

flavonols

Fig. 1. Chemical structures of the secondary plant compounds screened for intestinal epithelial barrier modulation.

S. Piegholdt et al. / Free Radical Biology and Medicine 70 (2014) 255–264 257

was applied for 4 h before lysis. The luciferase activities weremeasured using the Dual-Luciferase Reporter DLR Assay System(Promega) according to the manufacturer’s protocol in a TecanInfinite200 luminescence microplate reader (Tecan DeutschlandGmbH, Crailsheim, Germany). The firefly luciferase activity wasnormalized to the renilla luciferase activity.

ADAM activity

Soluble TNFR1 was determined as an indicator for ADAM17-dependent shedding activity in CaCo-2 cells. Cells were grown toconfluency in six-well plates and were allowed to differentiate inthe presence of biochanin A or prunetin for 5 days. The mediumwas replaced every other day. To avoid interference with thesoluble TNFR1 enzyme-linked immunosorbent assay system, FCScontent was reduced to 5% 24 h before collection of cell culturesupernatants. Supernatants were analyzed for sTNFR1 concentra-tion using the human sTNFR1 DuoSet ELISA Kit (R&D Systems)according to the manufacturer’s protocol in a Biotek ELx800absorbance microplate reader (Biotek Germany, Bad Friedrichshall,Germany).

Immunoprecipitation of proteins with phosphotyrosineand phosphothreonine residues

Cells were precultured in six-well plates for 4 days and wereallowed to differentiate in the presence of genistein, biochanin A,prunetin, or PD98059 for a further 5 days. The medium wasreplaced every other day. Subsequently, the cells were starved inFCS-deprived DMEM for 1 h in the presence of test substances.For stimulation of tyrosine phosphorylation, hydrogen peroxide

(H2O2; 250 mmol/L) was added for 1 h [37,38]. To examine levels ofthreonine phosphorylation, cells were incubated with phytochemicalsonly. Cells were washed twice with ice-cold Tris-buffer (20 mmol/L, pH 7.4). Cell lysates were prepared by adding prechilled NP-40lysis buffer (1% Nonidet P-40 Substitute (v/v), 50 mM Tris, pH 7.5,150 mM NaCl, 5 mM EDTA, supplemented with protease inhibitorsfrom Sigma–Aldrich and phosphatase inhibitors from RocheApplied Science, Mannheim, Germany). Lysates were incubatedon ice on a shaking platform for 30 min, then homogenized threetimes, and subsequently centrifuged at 18,000 g (41C, 20 min).Protein concentrations were determined using the BCA proteinassay (Thermo Fisher Scientific–Germany GmbH, Schwerte, Ger-many) and 1.5 mg of protein was subjected to immunoprecipita-tion. In brief, lysates were precleared with 15 ml of protein G–Sepharose beads (GE Healthcare by VWR International GmbH,Darmstadt, Germany) on a rotating wheel for 30 min at 41C.Cleared samples were incubated with 3 mg of a phosphotyrosineor 6 mg of a phosphothreonine antibody (Santa Cruz Biotechnology,Heidelberg, Germany) for 3 h. Then 30 ml of beads were added andthe samples were incubated for another hour. Samples werewashed five times with NP-40 buffer and heated with loadingbuffer. Samples were stored at �801C until separation by Westernblotting on a 4–20% ready-to-use gel (Bio-Rad, Munich, Germany).

Western blotting

For (a) CLDN1, OCLN, CDH1, β-CTNN, ZO-1, TNFR1, andglycerin-aldehyde-3-phosphate dehydrogenase (GAPDH) and(b) phosphorylated extracellular signal-regulated kinase (p-ERK)and p-c-SrcY530 detection CaCo-2 cells were allowed to differ-entiate in the presence of genistein, biochanin A, or prunetin for

Table 1Effects of various plant metabolites on the transepithelial electrical resistance (TEER) of CaCo-2 cells.

Plant bioactive ΔTEER (% control; mean7SEM)

Type of compound Name of compound μmol/L

Isoflavonoid Genistein 50 157713n

Isoflavonoid Biochanin A 50 136712n

Isoflavonoid Prunetin 50 16078n

Isoflavonoid Daidzein 50 11479Isoflavonoid Formononetin 50 9276Isoflavonoid Orobol 10 10178Isoflavonoid Pratensein 50 11575Isoflavonoid Santal 10 12676Isoflavonoid Glycitein 50 87710Isoflavonoid metabolite Genistein-7β-glucuronide 50 10779

Isoflavonoid metabolite 8-Hydroxydaidzein 50 10373Isoflavonoid metabolite Equol 50 10474Isoflavonoid metabolite p-Ethylphenol 50 11173n

Flavonol Quercetin 25 125714Flavonol Isorhamnetin 25 118715Flavanol Catechin 50 121712Flavanone Naringenin 50 115723Flavone Luteolin 10 118718Anthocyanin Cyanidin-3-O-glucoside 50 10477Anthocyanin Malvidin-3-O-glucoside 50 117715Dimeric procyanidin (�)-Epicatechin-4β-8-(þ)-catechin (B1) 50 111711

Dimeric procyanidin (�)-Epicatechin-4β-8-(�)-epicatechin (B2) 50 11078

Dimeric procyanidin (þ)-Catechin-4α-8-(þ)-catechin (B3) 50 116715

Dimeric procyanidin (þ)-Catechin-4α-8-(�)-epicatechin (B4) 50 12279

Dimeric procyanidin (�)-Epicatechin-4β-6-(�)-epicatechin (B5) 50 105712

Curcuminoid Curcumin 10 12879n

Phenylethanol Hydroxytyrosol 10 151710n

Isothiocyanate Sulforaphane 5 9877

Cells were allowed to differentiate on permeable filters for 5 days in the presence of secondary plant metabolites at the indicated concentrations. Barrier function wasmeasured over time via TEER and increase in TEER values was normalized to the control cells (%).

n po0.05; significant difference from control.

S. Piegholdt et al. / Free Radical Biology and Medicine 70 (2014) 255–264258

5 days. For p-ERK, cells were subsequently serum-starved for 18 hin the presence of plant bioactives and stimulated with recombinantEGF (10 ng/ml; Immunotools) for 15 min. Cells were washed twicewith ice-cold (a) PBS or (b) Tris buffer and lysed in RIPA buffer(50 mmol/L Tris, 150 mmol/L NaCl, 0.5% sodium deoxycholate (v/v),0.1% SDS (w/v), 1% NP-40 (v/v), pH 7.4) containing proteinase andphosphatase inhibitors. Protein concentrations were determined bythe BCA protein assay. A quantity of 30 mg of each sample was heatedwith loading buffer and separated (a) on a 4–20% ready-to-use gel or(b) on a 12% polyacrylamide gel. Then samples were transferred ontoa polyvinylidene difluoride membrane (Bio-Rad) and blocked with(a) 5% (w/v) skimmed milk or (b) bovine serum albumin dissolved inTris-buffered salineþ0.05% (v/v) Tween 20 for 2 h. Membranes wereprobed overnight with the respective antibody against CLDN1, OCLN,CDH1, β-CTNN, ZO-1, TNFR1, p-ERK, p-c-SrcY530, or GAPDH (SantaCruz), followed by the corresponding secondary antibody (anti-rabbit(Bio-Rad) for OCLN, ZO-1; anti-mouse (Bio-Rad) for CLDN1, CDH1,p-ERK, p-c-SrcY530, TNFR1; anti-goat (Santa Cruz) for β-CTNN,GAPDH) for 1 h at room temperature. Bands were visualized withECL substrate (Thermo Fisher Scientific) in a ChemiDoc XRS systemusing Quantity One software (version 4.6.3; Bio-Rad). Density ana-lyses were performed with Image Lab software (version 4.1; Bio-Rad).

Computational analyses

Affinity values for predicted binding intensities were calculatedwith AutoDock Vina software [39] (Molecular Graphics Laboratory,La Jolla, CA, USA). Cubic grids were centered so as to fully cover therespective active site (x¼17, y¼34, and z¼89). Before docking,water molecules and the oxime inhibitor of the EGF receptor(EGFR) kinase cocrystal structure (3BEL.pdb) were removed fromthe model [40,41]. Total cubic grid dimensions of 15 Å �15 Å �16 Å were defined with a grid spacing of 1.0 Å using theAutoDockTools graphical user interface. Calculations were carriedout at an exhaustiveness parameter of 8. The TanimotoCombo-Score for biochanin A and prunetin compared with genistein wascalculated by computational alignment via ROCS analysis [42]using the OpenEye Scientific Software (Santa Fe, NM, USA). Imageswere generated using the pyMOL software (Schroedinger, Mann-heim, Germany).

Statistical analyses

Values are given as the mean7SEM except when otherwisementioned. Data were analyzed for normality of distribution(Kolmogorov–Smirnov). In the case of normally distributed data,mean comparison was carried out using a two-tailed Student'st-test. Otherwise, the nonparametric Mann–Whitney-U testwas used. Statistical analysis was performed with SPSS (version19; SPSS, Inc., Munich, Germany). Significance was assumed atpo0.05.

Results

Plant-derived compounds including biochanin A and prunetinmodulate intestinal barrier integrity

Twenty-eight different plant compounds were screened formodulation of the intestinal barrier function in our cell culturemodel, of which 6 significantly increased CaCo-2 monolayerintegrity as measured by TEER (Table 1). Three isoflavonoids(genistein, biochanin A, and prunetin), the genistein metabolitep-ethylphenol, curcumin, and hydroxytyrosol increased TEERvalues by 11–60% compared to the control. To the best of ourknowledge both biochanin A and prunetin could be identified as

TEER-modulating substances for the first time, whereas curcuminis already known for its potential barrier-modulating abilities [43].As the concentration of hydroxytyrosol is comparably low in thediet [29] and p-ethylphenol increased TEER by only 11%, wefurther focused on the highly active isoflavones biochanin A andprunetin. As both compounds are structurally related to genistein,which has already been described as increasing monolayer integ-rity, we studied the potential underlying mechanisms of biochaninA- and prunetin-mediated barrier improvement and comparedthese results with genistein-mediated effects.

Biochanin A and prunetin decrease TNFα-dependent barrierdisruption and NF-κB activation

Incubation of intact CaCo-2 monolayers with rhTNFα resultedin marked disruption of barrier integrity and TEER loss by 25%.Biochanin A, prunetin, and genistein significantly rescued TNFα-induced integrity loss (Fig. 2A). As TNFα can lead to increasedshedding by metalloproteinases and thereby deteriorate barrierintegrity we studied whether these flavonoids affected cellularsupernatant concentration of sTNFR1. sTNFR1 concentrations wereshown to correlate with the activities of metalloproteinases suchas ADAM17 [44], and our results indicate that genistein, biochaninA, and prunetin reduce metalloproteinase activity (Fig. 2B). How-ever, Western blotting of TNFR1 in CaCo-2 cell lysates suggestedthat genistein also reduced TNFR1 expression, which may havecontributed to the decreased sTNFR1 concentration of supernatantcollected from genistein-treated cells (Fig. 2C). Total protein levelsof the junctional proteins CLDN1, CDH1, OCLN, and ZO-1 were notmodulated by isoflavone treatment in the presence or absence ofTNFα (data not shown). Interestingly, TNFα-induced NF-κB trans-activation was inhibited by biochanin A and prunetin, but not bygenistein (Fig. 2D).

Genistein, biochanin A, and prunetin reduce tyrosine phosphorylationof junctional protein ZO-1

The levels of phosphorylated junctional proteins (ZO-1, CLDN1,CDH1, β-CTNN, and OCLN) were determined in phosphotyrosineand phosphothreonine immunoprecipitates of CaCo-2 cell lysates.Tyrosine phosphorylation was induced by serum starvation andH2O2, whereas threonine phosphorylation was investigated underbasal conditions. Biochanin A and prunetin, similar to genistein,significantly reduced tyrosine phosphorylation of ZO-1 (Fig. 3A),whereas tyrosine phosphorylation of other junctional proteins aswell as threonine phosphorylation remained unchanged (data notshown). The protein kinase c-Src has been described as increasingtyrosine phosphorylation of TJ and AJ proteins playing a major rolein the impairment of barrier integrity [45,46]. Inactivation of c-Srcvia phosphorylation of Y530 was, however, not influenced byisoflavonoid treatment (data not shown). A rigid docking analysisexploring the binding affinities of genistein, biochanin A, andprunetin to the tyrosine kinase domain of the EGFR was per-formed. The interaction of EGFR with the highly potent synthetictyrosine kinase inhibitor POX served as a starting template and aspositive control for the docking calculations (PDB ID: 3BEL; [40,41]).The obtained binding affinities qualitatively follow the pattern of theexperimental results: POX4prunetin, biochanin A, genistein4cyani-din-3-O-glucoside, 8-hydroxydaidzein, glycitein ≫ procyanidin B5(Fig. 3B). In this series, the relatively large procyanidin B5 moleculewas shown to lack binding to EGFR owing to steric hindrance. Incontrast, biochanin A and prunetin showed strong binding to the EGFRtyrosine kinase (ATP-binding) pocket (Fig. 3B and C) with affinityscores very similar to that of the tyrosine kinase inhibitor genistein[47]. The plant bioactives glycitein, 8-hydroxydaidzein, cyanidin-3-O-glucoside, and procyanidin B5 were selected as representatives of

S. Piegholdt et al. / Free Radical Biology and Medicine 70 (2014) 255–264 259

different polyphenolic subgroups lacking TEER-enhancing activity andthus serving as negative controls. The relative binding affinities ofthese compounds to the tyrosine kinase domain of EGFR weresignificantly lower than those of genistein, biochanin A, and prunetin.

Biochanin A and prunetin affect ERK phosphorylation

The activation of ERK through phosphorylation can be triggeredby EGF [48] and is associated with reduced intestinal barrierfunction. Whereas genistein treatment did not decrease ERKactivation in our experiments, biochanin A and prunetin preventedTNFα- and EGF-stimulated ERK phosphorylation (Fig. 4A). Asbiochanin A and prunetin (in contrast to genistein) also inhibitedNF-κB transactivation, we hypothesized that NF-κB inhibition maybe connected to reduced ERK activation. We therefore studied theeffects of PD98059, an inhibitor of the ERK-activating kinase MEK1,on TEER, NF-κB transactivation, and ZO-1 tyrosine phosphoryla-tion in CaCo-2 cells. Similar to biochanin A and prunetin, PD98059addition to the cells significantly increased TEER (Fig. 4B), inhib-ited TNFα-dependent NF-κB transactivation (Fig. 4C), and reduced

tyrosine phosphorylation of ZO-1 (Fig. 4D) compared to untreatedcontrol cells.

Discussion

While searching for secondary plant metabolites that mayimprove epithelial barrier function, we identified the flavonoidsbiochanin A and prunetin, curcumin, and hydroxytyrosol aspositive modulators of TEER in CaCo-2 cells (Table 1). Interestingly,biochanin A and prunetin share a high structural similarity withgenistein, which also improved TEER in our model. Althoughgenistein has been described as protecting the intestinal epithe-lium in vivo [24], we could not find literature on biochanin A andprunetin in the context of improving epithelial barrier function.TNFα is one of the main cytokines inducing the proinflammatoryNF-κB transactivation pathway [22,49] and is also involved inintestinal inflammatory processes [50,51] by reducing TJ and AJprotein levels [17,52], promoting internalization of junctionalproteins in cytosolic vacuolar storage compartments [53,54] and

-30

-25

-20

-15

-10

-5

0

5

bio A prun

+++ +

*

*

*re

lativ

e TE

ER v

alue

s(%

)

rhTNFα

450

500

550

600

650

700

**

*

sTN

FR1

(pg/

ml)

**

*

0.0

0.4

0.8

1.2

2.0

2.4

rela

tive

luci

fera

se a

ctiv

ity (N

FkB

/phR

L-TK

)

+++ +rhTNFα

GAPDH

TNFR1

control genis

control

control

genis

genis

bio A

bio A

prun

prun

mock+

control bio A prungenis

1.6

Fig. 2. Genistein, biochanin A, and prunetin prevent TNFα-dependent decrease in the transepithelial electrical resistance (TEER) of CaCo-2 cells through different cellularmechanisms. (A) Cells were allowed to differentiate on permeable filters for 5 days in the presence of the indicated test compounds (50 mmol/L). At day 5 TNFα (100 ng/ml)was added to the basolateral chamber for 24 h to induce barrier integrity loss. The TEER was measured before and after TNFα treatment. Values were normalized to theuntreated control (%). Bars represent the means7SEM of three individual wells. (B) ADAM17-dependent shedding activity as measured by ELISA of sTNFR1 in thesupernatant of differentiated cells treated with the indicated test compounds. Bars represent the means7SEM of three independent experiments. (C) Western blot of TNFR1in CaCo-2 whole-cell extracts after differentiation in the presence of the indicated test compounds. A representative blot of three independent experiments is shown. GAPDHserved as the loading control. (D) NF-κB transactivation in the NF-κB reporter gene assay. Biochanin A and prunetin inhibited the TNFα-induced NF-κB transactivation butgenistein did not. The bars represent the ratio of the NF-κB-activated firefly luciferase activity normalized to renilla luciferase activity7SEM from three independentexperiments performed in duplicate. *po0.05; significant difference compared to the control.

S. Piegholdt et al. / Free Radical Biology and Medicine 70 (2014) 255–264260

Energy values for the docking interaction between EGFR tyrosine kinase and isoflavones, POX or control compounds.

distance from best modeProtein compound mode affinity(kcal/mol) RMSD l.b. RMSD u.b.

PDB ID:3BEL

ZO -1

control genis bio A prunH2O2 + + + +

IP: phospho-tyrosine

genistein (1) biochanin A (1) prunetin (1)

genistein (2) biochanin A (2) prunetin (2)

Fig. 3. Involvement of tyrosine phosphorylation in isoflavonoid-mediated effects on CaCo-2 cells. (A) Tyrosine phosphorylation of ZO-1 after treatment with the indicatedisoflavonoids was determined by immunoprecipitation (IP) with an antibody against phosphotyrosine followed byWestern blotting with a ZO-1 antibody. Cells were allowedto differentiate for 5 days in the presence of genistein, biochanin A, or prunetin (50 mmol/L). Then the cells were serum-starved for 1 h and subsequently treated with H2O2

(250 mmol/L) for 1 h to induce tyrosine phosphorylation before harvesting the cellular protein fraction. Similar results were obtained in four independent experiments.(B and C) Rigid docking analyses of genistein, biochanin A, and prunetin to the tyrosine kinase domain of the EGFR. EGFR tyrosine kinase interacting with the tyrosine kinaseinhibitor POX served as a template (PDB ID: 3BEL [49]). (B) Affinity values for predicted binding intensities of genistein, biochanin A, and prunetin within the EGFR tyrosinekinase pocket. The synthetic tyrosine kinase inhibitor POX served as positive control; glycitein, 8-hydroxydaidzein, cyanidin-3-O-glucoside, and procyanidin B5 served asnegative controls. (C) Potential binding positions of genistein, biochanin A, and prunetin within the tyrosine kinase domain of the EGFR. The numbers below the imagescorrespond to the affinity calculation mode in (B). IP, immunoprecipitation; RMSD, root mean square deviation; l.b., lower bound; u.p., upper bound; POX, 4-amino-6-{[1-(3-fluorobenzyl)-1H-indazol-5-yl]amino}pyrimidine-5-carbaldehyde O-(2 methoxy-ethyl)oxime.

S. Piegholdt et al. / Free Radical Biology and Medicine 70 (2014) 255–264 261

cytoskeletal contraction via induction of MLC-phosphorylation [55].As TNFα impairs barrier function, we studied whether genistein,biochanin A, and prunetin could prevent TNFα-mediated TEER loss.Because the isoflavonoids did not affect the protein levels of CLDN1,CDH1, OCLN, and ZO-1, we expected mechanisms other than theprotein expression of junction proteins to be involved. Metallopro-teinases cleave junctional proteins, extracellular matrix proteins, andcytokines, thereby contributing to impaired epithelial integrity andbarrier [56,57]. Moreover, ADAM17 plays a critical role in epithelialhomeostasis by activating EGFR signaling and ERK activation [58,59].Genistein, biochanin A, and prunetin reduced sTNFR1 concentration.In the case of genistein, the lowered sTNFR1 concentration may beexplained by lowered TNFR1 protein levels. However, TNFR1 expres-sion appeared unchanged in cells treated with biochanin A andprunetin, thus indicating that these two flavonoids reduce ADAM17-mediated shedding. This effect could contribute to the observeddecrease in ERK activation after prunetin and biochanin A treatmentand could explain why genistein did not affect p-ERK generation.Thus, it seems that, although improving barrier tightness to similarextents (Table 1), genistein as opposed to prunetin and biochanin Adoes so by different mechanisms. As previously described by Romierand colleagues [22], genistein did not affect cytokine-induced NF-κBactivation. In contrast, we could show that biochanin A and prunetinreduced TNFα-induced NF-κB transactivation by more than 70%compared to the control in the CaCo-2 cell culture model, indicatingstrong interdependence of structure and specific function. In contrastto genistein, both biochanin A and prunetin possess a methoxygroup, which may contribute to their inhibitory function on NF-κBtransactivation. Having shown that the levels of TJ and AJ proteins

did not change in response to isoflavone treatment, we hypothesizedthat our flavonoids may alter the phosphorylation status of theseproteins. Tyrosine phosphorylation of TJ and AJ proteins impairsintestinal epithelial barrier function [10,11]. Interestingly, in ourexperiments, phosphorylation levels of ZO-1 tyrosine residues werelikewise reduced upon biochanin A, prunetin, and genistein treat-ment. The scaffolding ZO-1 plays a central role in junctional proteincomplexes and is important for the interaction with the cytoskeleton[6,60]. Correct relocalization of TJ and AJ proteins to the junctionalcomplex within the membranes may also contribute to the con-servation of barrier resistance. As genistein is a potent inhibitor oftyrosine kinase activity, we carried out rigid-docking analyses for thetyrosine kinase domain of the EGFR with the goal to qualitativelycompare the binding affinities of the putative ligand complexes bycomputational methods. We obtained highly similar binding scoresfor biochanin A and prunetin compared with genistein. This impliesthat biochanin A and prunetin may represent physiologically activetyrosine kinase inhibitors. Stereoscopic alignments of docked genis-tein, biochanin A, and prunetin within the EGFR tyrosine kinasedomain are shown in Fig. 3C, illustrating the high positionalconservation of the binding modes. Furthermore, this is in agreementwith the relatively high shape similarity of these three isoflavonoids(TanimotoComboScore of 1.889). The highly active tyrosine kinaseinhibitor POX served as validation tool of the docking. Redocking ofPOX to EGFR resulted in the highest binding intensity score anda binding mode highly similar to the one found in the cocrystalstructure. Although the flavonoids could competitively inhibit ZO-1phosphorylating kinases, improved barrier function might alsoresult from decreased EGFR signaling. The activation of the EGFR

0.0

0.4

0.8

1.2

1.6

2.0

2.4

2.8

NFk

B/p

hRL-

TK (x

-fold

con

trol

)

PD98059controlrhTNF ++

*

0

50

100

150

200

250

300

350

400

control PD98059

incr

ease

in T

EER

(% c

ontr

ol)

*

control PD98059H2O2

IP: phospho-tyrosine

ZO-1+ +

p-ERK

GAPDH

rhEGFbio A

prun

+ +gen

is

++co

ntrol

Fig. 4. Effect of ERK activation on TEER, NF-κB transactivation, and tyrosine phosphorylation of ZO-1 in CaCo-2 cells. (A) ERK activation was determined by Western blottingin CaCo-2 whole-cell extracts. The cells were allowed to differentiate in the presence of genistein, biochanin A, and prunetin (50 mmol/L); serum-starved for 18 h; andsubsequently treated with EGF (10 ng/ml) for 15 min before harvest. The blot shown is representative of three independent experiments. GAPDH served as the loadingcontrol. (B) Effect of the MEK1 inhibitor PD98059 on TEER of CaCo-2 cells. Cells were allowed to differentiate in the presence of PD98059 (25 mmol/L) for 4 days. The increasein TEER was normalized to the untreated control (%). The bars represent the means7SEM of four individual wells. (C) NF-κB transactivation in the NF-κB reporter geneassay. PD98059 (25 mmol/L) inhibited the TNFα-induced NF-κB transactivation. The bars represent the ratio of the NF-κB-activated firefly luciferase activity normalized torenilla luciferase activity7SEM from three independent experiments performed in duplicate. (D) The PD98059-induced phosphorylation of ZO-1 tyrosine residues wasdetermined by immunoprecipitation of anti-phosphotyrosine followed by Western blotting. Cells were allowed to differentiate for 5 days in the presence of PD98059(25 mmol/L), followed by serum starvation for 1 h and subsequent H2O2 treatment (250 mmol/L) for 1 h. The blot shown is representative of four independent wells. *po0.05;significant difference from untreated control.

S. Piegholdt et al. / Free Radical Biology and Medicine 70 (2014) 255–264262

downstream target ERK has been described as reducing the intestinalbarrier integrity by inhibiting epithelial cell differentiation [61]. ERKis activated upon phosphorylation of threonine and tyrosine residues[62]; therefore alterations in tyrosine kinase activity may affect ERKsignaling. Interestingly, phospho-ERK was decreased in cells that hadbeen treated with biochanin A or prunetin. Similar to biochanin Aand prunetin, ERK inhibition with PD98059 (an inhibitor of the ERKupstream regulator MEK) increased TEER values and reduced tyr-osine phosphorylation of ZO-1 in our model. The ERK inhibitor alsoreduced cytokine-induced NF-κB transactivation. These findingspoint to the notion that biochanin A and prunetin improve barrierfunction via downregulation of ERK and NF-κB. Although genistein,biochanin A, and prunetin lowered ZO-1 phosphorylation andincreased TEER values, the mechanisms strengthening the epithelialbarrier seem to be different. Because, in contrast to biochanin A,prunetin, and the ERK inhibitor PD98059, genistein showed no effecton ERK and NF-κB activation, it seems that genistein protects theepithelial barrier from TNFα-induced damage without altering NF-κB-dependent transcription. In recent publications it has beenreported that genistein increased Nrf2-dependent gene expressionvia activation of the ERK pathway [63,64]. Because we stimulatedERK to show its inhibition by biochanin A and prunetin treatment, itis likely that we were not able to detect the putative ERK activationby genistein. Nevertheless, this study also points to the notion thatgenistein, biochanin A, and prunetin act differently on ERK.

NF-κB-driven inflammation, barrier loss, and increased paracel-lular permeability play central roles in acute intestinal infectionsand chronic inflammatory bowel diseases [13,65]. Identification ofpotent inhibitors of epithelial tyrosine kinase and NF-κB activitysuch as biochanin A and prunetin may help to develop strategies forprevention and also treatment of intestinal disorders. Dietary intakeof biochanin A and prunetin through soy, mangifera fruit, kudzuand yam bean based food products may improve barrier functionand enterocyte resistance to pathogens and xenobiotics in vivo andthus help to reduce acute infections. In contrast to numerous othertarget tissues, the intestinal epithelium can be directly reached andaffected by isoflavonoids at reasonably high concentrations, as theyare easily provided with food. Therefore the cell culture-basedresults obtained within this study may indicate physiological andpotentially reproducible effects in vivo. Further studies are requiredto prove the newly discovered barrier-modulating effects of biocha-nin A and prunetin also in vivo.

Conclusion

Biochanin A and prunetin prevented TNFα-induced decline inbarrier integrity possibly through suppression of the NF-κB path-way. This may be caused via ERK inhibition because phospho-ERKwas decreased upon biochanin A and prunetin treatment and theERK inhibitor PD98059 mimicked the effects of these flavonoids onNF-κB, tyrosine phosphorylation of ZO-1 and TEER.

Acknowledgments

Karina Reiss and Gerald Rimbach are supported by the DFGCluster of Excellence “Inflammation at Interfaces.” Patricia Huebbe,Karina Reiss and Gerald Rimbach are supported by the DFG ResearchTraining Group 1743 “Genes, Environment and Inflammation.”

References

[1] Furuse, M.; Itoh, M.; Hirase, T.; Nagafuchi, A.; Yonemura, S.; Tsukita, S. Directassociation of occludin with ZO-1 and its possible involvement in thelocalization of occludin at tight junctions. J. Cell Biol. 127:1617–1626; 1994.

[2] Tsukita, S.; Yamazaki, Y.; Katsuno, T.; Tamura, A. Tight junction-based epithelialmicroenvironment and cell proliferation. Oncogene 27:6930–6938; 2008.

[3] Bazzoni, G.; Martinez-Estrada, O. M.; Mueller, F.; Nelboeck, P.; Schmid, G.;Bartfai, T.; Dejana, E.; Brockhaus, M. Homophilic interaction of junctionaladhesion molecule. J. Biol. Chem. 275:30970–30976; 2000.

[4] Escaffit, F.; Perreault, N.; Jean, D.; Francoeur, C.; Herring, E.; Rancourt, C.;Rivard, N.; Vachon, P. H.; Pare, F.; Boucher, M. P.; Auclair, J.; Beaulieu, J. F.Repressed E-cadherin expression in the lower crypt of human small intestine:a cell marker of functional relevance. Exp. Cell Res. 302:206–220; 2005.

[5] Sumigray, K. D.; Lechler, T. Desmoplakin controls microvilli length but not celladhesion or keratin organization in the intestinal epithelium. Mol. Biol. Cell23:792–799; 2012.

[6] Van Itallie, C. M.; Fanning, A. S.; Bridges, A.; Anderson, J. M. ZO-1 stabilizes thetight junction solute barrier through coupling to the perijunctional cytoske-leton. Mol. Biol. Cell 20:3930–3940; 2009.

[7] Piedra, J.; Martinez, D.; Castano, J.; Miravet, S.; Dunach, M.; de Herreros, A. G.Regulation of beta-catenin structure and activity by tyrosine phosphorylation.J. Biol. Chem. 276:20436–20443; 2001.

[8] Alema, S.; Salvatore, A. M. p120 catenin and phosphorylation: mechanismsand traits of an unresolved issue. Biochim. Biophys. Acta 1773:47–58; 2007.

[9] Goldblum, S. E.; Rai, U.; Tripathi, A.; Thakar, M.; De Leo, L.; Di Toro, N.; Not, T.;Ramachandran, R.; Puche, A. C.; Hollenberg, M. D.; Fasano, A. The active Zotdomain (aa 288–293) increases ZO-1 and myosin 1C serine/threonine phos-phorylation, alters interaction between ZO-1 and its binding partners, andinduces tight junction disassembly through proteinase activated receptor2 activation. FASEB J. 25:144–158; 2011.

[10] Roura, S.; Miravet, S.; Piedra, J.; Garcia de Herreros, A.; Dunach, M. Regulationof E-cadherin/catenin association by tyrosine phosphorylation. J. Biol. Chem.274:36734–36740; 1999.

[11] Elias, B. C.; Suzuki, T.; Seth, A.; Giorgianni, F.; Kale, G.; Shen, L.; Turner, J. R.;Naren, A.; Desiderio, D. M.; Rao, R. Phosphorylation of Tyr-398 and Tyr-402 inoccludin prevents its interaction with ZO-1 and destabilizes its assembly at thetight junctions. J. Biol. Chem. 284:1559–1569; 2009.

[12] Zeissig, S.; Burgel, N.; Gunzel, D.; Richter, J.; Mankertz, J.; Wahnschaffe, U.;Kroesen, A. J.; Zeitz, M.; Fromm, M.; Schulzke, J. D. Changes in expression anddistribution of claudin 2, 5 and 8 lead to discontinuous tight junctions andbarrier dysfunction in active Crohn's disease. Gut 56:61–72; 2007.

[13] Schmitz, H.; Barmeyer, C.; Fromm, M.; Runkel, N.; Foss, H. D.; Bentzel, C. J.;Riecken, E. O.; Schulzke, J. D. Altered tight junction structure contributes to theimpaired epithelial barrier function in ulcerative colitis. Gastroenterology116:301–309; 1999.

[14] Lu, R.; Liu, X.; Wu, S.; Xia, Y.; Zhang, Y. G.; Petrof, E. O.; Claud, E. C.; Sun, J.Consistent activation of the beta-catenin pathway by Salmonella type-threesecretion effector protein AvrA in chronically infected intestine. Am. J. Physiol.Gastrointest. Liver Physiol. 303:G1113–G1125; 2012.

[15] Schulzke, J. D.; Bojarski, C.; Zeissig, S.; Heller, F.; Gitter, A. H.; Fromm, M.Disrupted barrier function through epithelial cell apoptosis. Ann. N. Y. Acad. Sci.1072:288–299; 2006.

[16] Piguet, P. F.; Vesin, C.; Guo, J.; Donati, Y.; Barazzone, C. TNF-induced enterocyteapoptosis in mice is mediated by the TNF receptor 1 and does not require p53.Eur. J. Immunol. 28:3499–3505; 1998.

[17] Ma, T. Y.; Iwamoto, G. K.; Hoa, N. T.; Akotia, V.; Pedram, A.; Boivin, M. A.; Said,H. M. TNF-alpha-induced increase in intestinal epithelial tight junctionpermeability requires NF-kappa B activation. Am. J. Physiol. Gastrointest. LiverPhysiol. 286:G367–G376; 2004.

[18] Poritz, L. S.; Harris 3rd L. R.; Kelly, A. A.; Koltun, W. A. Increase in the tight junctionprotein claudin-1 in intestinal inflammation. Dig. Dis. Sci. 56:2802–2809; 2011.

[19] Bojarski, C.; Weiske, J.; Schoneberg, T.; Schroder, W.; Mankertz, J.; Schulzke, J.D.; Florian, P.; Fromm, M.; Tauber, R.; Huber, O. The specific fates of tightjunction proteins in apoptotic epithelial cells. J. Cell Sci. 117:2097–2107; 2004.

[20] Marchiando, A. M.; Shen, L.; Graham, W. V.; Edelblum, K. L.; Duckworth, C. A.;Guan, Y.; Montrose, M. H.; Turner, J. R.; Watson, A. J. The epithelial barrier ismaintained by in vivo tight junction expansion during pathologic intestinalepithelial shedding. Gastroenterology 140:1208–1218; 2011.

[21] Gitter, A. H.; Bendfeldt, K.; Schmitz, H.; Schulzke, J. D.; Bentzel, C. J.; Fromm, M.Epithelial barrier defects in HT-29/B6 colonic cell monolayers induced bytumor necrosis factor-alpha. Ann. N. Y. Acad. Sci. 915:193–203; 2000.

[22] Romier, B.; Van De Walle, J.; During, A.; Larondelle, Y.; Schneider, Y. J.Modulation of signalling nuclear factor-kappaB activation pathway by poly-phenols in human intestinal Caco-2 cells. Br. J. Nutr. 100:542–551; 2008.

[23] Song, W. B.; Wang, Y. Y.; Meng, F. S.; Zhang, Q. H.; Zeng, J. Y.; Xiao, L. P.; Yu, X. P.;Peng, D. D.; Su, L.; Xiao, B.; Zhang, Z. S. Curcumin protects intestinal mucosal barrierfunction of rat enteritis via activation of MKP-1 and attenuation of p38 and NF-kappaB activation. PLoS One 5:e12969; 2010.

[24] Sato, Y.; Itagaki, S.; Oikawa, S.; Ogura, J.; Kobayashi, M.; Hirano, T.; Sugawara, M.;Iseki, K. Protective effect of soy isoflavone genistein on ischemia–reperfusion in therat small intestine. Biol. Pharm. Bull. 34:1448–1454; 2011.

[25] Suzuki, T.; Tanabe, S.; Hara, H. Kaempferol enhances intestinal barrier functionthrough the cytoskeletal association and expression of tight junction proteinsin Caco-2 cells. J. Nutr. 141:87–94; 2011.

[26] Suzuki, T.; Hara, H. Quercetin enhances intestinal barrier function through theassembly of zonula [corrected] occludens-2, occludin, and claudin-1 and theexpression of claudin-4 in Caco-2 cells. J. Nutr. 139:965–974; 2009.

[27] Nakamura, Y.; K. A.; Yoshii, K.; Tsumura, Y.; Ishimitsu, S.; Tonogai, Y. Contentand composition of isoflavonoids in mature or immature beans and beansprouts consumed in Japan. J. Health Sci. 47:394–406; 2011.

S. Piegholdt et al. / Free Radical Biology and Medicine 70 (2014) 255–264 263

[28] Dulbecco, P.; Savarino, V. Therapeutic potential of curcumin in digestivediseases. World J. Gastroenterol. 19:9256–9270; 2013.

[29] Romero, C.; Brenes, M. Analysis of total contents of hydroxytyrosol and tyrosolin olive oils. J. Agric. Food Chem. 60:9017–9022; 2012.

[30] Cooper, A. J.; Forouhi, N. G.; Ye, Z.; Buijsse, B.; Arriola, L.; Balkau, B.; Barricarte, A.;Beulens, J. W.; Boeing, H.; Buchner, F. L.; Dahm, C. C.; de Lauzon-Guillain, B.;Fagherazzi, G.; Franks, P. W.; Gonzalez, C.; Grioni, S.; Kaaks, R.; Key, T. J.; Masala, G.;Navarro, C.; Nilsson, P.; Overvad, K.; Panico, S.; Ramon Quiros, J.; Rolandsson, O.;Roswall, N.; Sacerdote, C.; Sanchez, M. J.; Slimani, N.; Sluijs, I.; Spijkerman, A. M.;Teucher, B.; Tjonneland, A.; Tumino, R.; Sharp, S. J.; Langenberg, C.; Feskens, E. J.;Riboli, E.; Wareham, N. J. Fruit and vegetable intake and type 2 diabetes: EPIC-InterAct prospective study and meta-analysis. Eur. J. Clin. Nutr. 66:1082–1092; 2012.

[31] Esatbeyoglu, T.; Winterhalter, P. Preparation of dimeric procyanidins B1, B2,B5, and B7 from a polymeric procyanidin fraction of black chokeberry (Aroniamelanocarpa). J. Agric. Food Chem. 58:5147–5153; 2010.

[32] Esatbeyoglu, T.; Wray, V.; Winterhalter, P. Dimeric procyanidins: screening forB1 to B8 and semisynthetic preparation of B3, B4, B6, And B8 from a polymericprocyanidin fraction of white willow bark (Salix alba). J. Agric. Food Chem.58:7820–7830; 2010.

[33] Hara, A.; Hibi, T.; Yoshioka, M.; Toda, K.; Watanabe, N.; Hayashi, A.; Iwao, Y.;Saito, H.; Watanabe, T.; Tsuchiya, M. Changes of proliferative activity andphenotypes in spontaneous differentiation of a colon cancer cell line. Jpn. J.Cancer Res. 84:625–632; 1993.

[34] Gres, M. C.; Julian, B.; Bourrie, M.; Meunier, V.; Roques, C.; Berger, M.; Boulenc, X.;Berger, Y.; Fabre, G. Correlation between oral drug absorption in humans, andapparent drug permeability in TC-7 cells, a human epithelial intestinal cell line:comparison with the parental Caco-2 cell line. Pharm. Res. 15:726–733; 1998.

[35] Ernst, I. M.; Wagner, A. E.; Schuemann, C.; Storm, N.; Hoppner, W.; Doring, F.;Stocker, A.; Rimbach, G. Allyl-, butyl- and phenylethyl-isothiocyanate activateNrf2 in cultured fibroblasts. Pharmacol. Res. 63:233–240; 2011.

[36] Grasset, E.; Pinto, M.; Dussaulx, E.; Zweibaum, A.; Desjeux, J. F. Epithelialproperties of human colonic carcinoma cell line Caco-2: electrical parameters.Am. J. Physiol. 247:C260–C267; 1984.

[37] Barchowsky, A.; Williams, M. E.; Benz, C. C.; Chepenik, K. P. Oxidant-sensitiveprotein phosphorylation in endothelial cells. Free Radic. Biol. Med. 16:771–777;1994.

[38] Vepa, S.; Scribner, W. M.; Natarajan, V. Activation of protein phosphorylationby oxidants in vascular endothelial cells: identification of tyrosine phosphor-ylation of caveolin. Free Radic. Biol. Med. 22:25–35; 1997.

[39] Trott, O.; Olson, A. J. AutoDock Vina: improving the speed and accuracy ofdocking with a new scoring function, efficient optimization, and multithread-ing. J. Comput. Chem. 31:455–461; 2010.

[40] Abad, M. C. X.G.; Neeper, M.P.; Struble, G.T.; Gaul, M.D.; Connolly, P.J. X-raystructure of EGFR in complex with oxime inhibitor. URL: ⟨http://www.rcsb.org/pdb/explore/explore.do?pdbId=3BEL⟩ (22.11.2013); 2008.

[41] Xu, G.; Searle, L. L.; Hughes, T. V.; Beck, A. K.; Connolly, P. J.; Abad, M. C.;Neeper, M. P.; Struble, G. T.; Springer, B. A.; Emanuel, S. L.; Gruninger, R. H.;Pandey, N.; Adams, M.; Moreno-Mazza, S.; Fuentes-Pesquera, A. R.; Middleton,S. A.; Greenberger, L. M. Discovery of novel 4-amino-6-arylaminopyrimidine-5-carbaldehyde oximes as dual inhibitors of EGFR and ErbB-2 protein tyrosinekinases. Bioorg. Med. Chem. Lett. 18:3495–3499; 2008.

[42] Tresadern, G.; Bemporad, D.; Howe, T. A comparison of ligand based virtualscreening methods and application to corticotropin releasing factor 1 receptor.J. Mol. Graphics Modell. 27:860–870; 2009.

[43] Wang, N.; Wang, G.; Hao, J.; Ma, J.; Wang, Y.; Jiang, X.; Jiang, H. Curcuminameliorates hydrogen peroxide-induced epithelial barrier disruption by upre-gulating heme oxygenase-1 expression in human intestinal epithelial cells.Dig. Dis. Sci. 57:1792–1801; 2012.

[44] Bell, J. H.; Herrera, A. H.; Li, Y.; Walcheck, B. Role of ADAM17 in the ectodomainshedding of TNF-alpha and its receptors by neutrophils and macrophages. J.Leukocyte Biol. 82:173–176; 2007.

[45] Basuroy, S.; Sheth, P.; Kuppuswamy, D.; Balasubramanian, S.; Ray, R. M.; Rao, R. K.Expression of kinase-inactive c-Src delays oxidative stress-induced disassemblyand accelerates calcium-mediated reassembly of tight junctions in the Caco-2 cellmonolayer. J. Biol. Chem. 278:11916–11924; 2003.

[46] Kale, G.; Naren, A. P.; Sheth, P.; Rao, R. K. Tyrosine phosphorylation of occludinattenuates its interactions with ZO-1, ZO-2, and ZO-3. Biochem. Biophys. Res.Commun. 302:324–329; 2003.

[47] Akiyama, T.; Ishida, J.; Nakagawa, S.; Ogawara, H.; Watanabe, S.; Itoh, N.;Shibuya, M.; Fukami, Y. Genistein, a specific inhibitor of tyrosine-specificprotein kinases. J. Biol. Chem. 262:5592–5595; 1987.

[48] Edelblum, K. L.; Goettel, J. A.; Koyama, T.; McElroy, S. J.; Yan, F.; Polk, D. B.TNFR1 promotes tumor necrosis factor-mediated mouse colon epithelial cellsurvival through RAF activation of NF-kappaB. J. Biol. Chem. 283:29485–29494;2008.

[49] Carlsen, H.; Alexander, G.; Austenaa, L. M.; Ebihara, K.; Blomhoff, R. Molecularimaging of the transcription factor NF-kappaB, a primary regulator of stressresponse. Mutat. Res. 551:199–211; 2004.

[50] Shin, H. S.; Zhao, Z.; Satsu, H.; Totsuka, M.; Shimizu, M. Synergistic effect oftumor necrosis factor-alpha and hydrogen peroxide on the induction of IL-8production in human intestinal Caco-2 cells. Inflammation 34:440–447; 2011.

[51] Pavlick, K. P.; Laroux, F. S.; Fuseler, J.; Wolf, R. E.; Gray, L.; Hoffman, J.; Grisham, M. B.Role of reactive metabolites of oxygen and nitrogen in inflammatory bowel disease.Free Radic. Biol. Med. 33:311–322; 2002.

[52] He, F.; Peng, J.; Deng, X. L.; Yang, L. F.; Camara, A. D.; Omran, A.; Wang, G. L.;Wu, L. W.; Zhang, C. L.; Yin, F. Mechanisms of tumor necrosis factor-alpha-induced leaks in intestine epithelial barrier. Cytokine 59:264–272; 2012.

[53] Ivanov, A. I.; Nusrat, A.; Parkos, C. A. Endocytosis of epithelial apical junctionalproteins by a clathrin-mediated pathway into a unique storage compartment.Mol. Biol. Cell 15:176–188; 2004.

[54] Bruewer, M.; Utech, M.; Ivanov, A. I.; Hopkins, A. M.; Parkos, C. A.; Nusrat, A.Interferon-gamma induces internalization of epithelial tight junction proteinsvia a macropinocytosis-like process. FASEB J. 19:923–933; 2005.

[55] Ma, T. Y.; Boivin, M. A.; Ye, D.; Pedram, A.; Said, H. M. Mechanism ofTNF-{alpha} modulation of Caco-2 intestinal epithelial tight junction barrier:role of myosin light-chain kinase protein expression. Am. J. Physiol. Gastro-intest. Liver Physiol. 288:G422–G430; 2005.

[56] Chalaris, A.; Adam, N.; Sina, C.; Rosenstiel, P.; Lehmann-Koch, J.; Schirmacher, P.;Hartmann, D.; Cichy, J.; Gavrilova, O.; Schreiber, S.; Jostock, T.; Matthews, V.;Hasler, R.; Becker, C.; Neurath, M. F.; Reiss, K.; Saftig, P.; Scheller, J.; Rose-John, S.Critical role of the disintegrin metalloprotease ADAM17 for intestinal inflamma-tion and regeneration in mice. J. Exp. Med. 207:1617–1624; 2010.

[57] Reiss, K.; Saftig, P. The "a disintegrin and metalloprotease" (ADAM) family ofsheddases: physiological and cellular functions. Semin. Cell Dev. Biol. 20:126–-137; 2009.

[58] Maretzky, T.; Reiss, K.; Ludwig, A.; Buchholz, J.; Scholz, F.; Proksch, E.; deStrooper, B.; Hartmann, D.; Saftig, P. ADAM10 mediates E-cadherin sheddingand regulates epithelial cell–cell adhesion, migration, and beta-catenintranslocation. Proc. Natl. Acad. Sci. USA 102:9182–9187; 2005.

[59] Blobel, C. P. ADAMs: key components in EGFR signalling and development. Nat.Rev. Mol. Cell Biol., 6; 2005; 32–43; 2005.

[60] Fanning, A. S.; Little, B. P.; Rahner, C.; Utepbergenov, D.; Walther, Z.; Anderson, J. M.The unique-5 and -6 motifs of ZO-1 regulate tight junction strand localization andscaffolding properties. Mol. Biol. Cell 18:721–731; 2007.

[61] Lemieux, E.; Boucher, M. J.; Mongrain, S.; Boudreau, F.; Asselin, C.; Rivard, N.Constitutive activation of the MEK/ERK pathway inhibits intestinal epithelialcell differentiation. Am. J. Physiol. Gastrointest. Liver Physiol. 301:G719–G730;2011.

[62] Toledano-Katchalski, H.; Kraut, J.; Sines, T.; Granot-Attas, S.; Shohat, G.;Gil-Henn, H.; Yung, Y.; Elson, A. Protein tyrosine phosphatase epsilon inhibitssignaling by mitogen-activated protein kinases. Mol. Cancer Res. 1:541–550;2003.

[63] Zhai, X.; Lin, M.; Zhang, F.; Hu, Y.; Xu, X.; Li, Y.; Liu, K.; Ma, X.; Tian, X.; Yao, J.Dietary flavonoid genistein induces Nrf2 and phase II detoxification geneexpression via ERKs and PKC pathways and protects against oxidative stress inCaco-2 cells. Mol. Nutr. Food Res. 57:249–259; 2013.

[64] Borras, C.; Gambini, J.; Gomez-Cabrera, M. C.; Sastre, J.; Pallardo, F. V.; Mann, G. E.;Vina, J. Genistein, a soy isoflavone, up-regulates expression of antioxidant genes:involvement of estrogen receptors, ERK1/2, and NFkappaB. FASEB J. 20:2136–2138;2006.

[65] Rosenshine, I.; Donnenberg, M. S.; Kaper, J. B.; Finlay, B. B. Signal transductionbetween enteropathogenic Escherichia coli (EPEC) and epithelial cells: EPECinduces tyrosine phosphorylation of host cell proteins to initiate cytoskeletalrearrangement and bacterial uptake. EMBO J. 11:3551–3560; 1992.

S. Piegholdt et al. / Free Radical Biology and Medicine 70 (2014) 255–264264