grapeseedextractefﬁcacyagainstazoxymethane-induced colon … · window (10–15 years),...

Research ArticleSee related commentaries by Katiyar and Athar, p. 617 and by Kong et al., p. 622

Grape Seed Extract Efficacy against Azoxymethane-InducedColon Tumorigenesis in A/J Mice: Interlinking miRNA withCytokine Signaling and Inflammation

Molly M. Derry1, Komal Raina1, Velmurugan Balaiya1, Anil K. Jain1, Sangeeta Shrotriya1, Kendra M. Huber2,Natalie J. Serkova2,3, Rajesh Agarwal1,3, and Chapla Agarwal1,3

AbstractColorectal cancer (CRC) is the second leading causeof cancer-associateddeaths, suggesting that additional

strategies are needed to prevent/control this malignancy. As CRC growth and progression involve a large

window (10–15 years), chemopreventive intervention could be a practical/translational strategy. Azox-

ymethane (AOM)-induced colon tumorigenesis in mice resembles human CRC in terms of progression of

ACF to polyps, adenoma, and carcinomas and associated molecular mechanisms. Accordingly, herein we

investigatedgrape seedextract (GSE)efficacyagainstAOM-inducedcolon tumorigenesis inA/Jmice.GSEwas

fed in diet at 0.25% or 0.5% (w/w) dose starting 2 weeks after last AOM injection for 18 or 28 weeks. Our

results showed that GSE feeding significantly decreases colon tumor multiplicity and overall tumor size. In

biomarker analysis, GSE showed significant antiproliferative and pro-apoptotic activities. Detailed mech-

anistic studies highlighted that GSE strongly modulates cytokines/interleukins and miRNA expression

profiles as well as miRNA processing machinery associated with alterations in NF-kB, b-catenin, andmitogen-activated protein kinase (MAPK) signaling. Additional studies using immunohistochemical anal-

yses found that indeedGSE inhibits NF-kB activation and decreases the expression of its downstream targets

(COX-2, iNOS,VEGF) related to inflammatory signaling,downregulatesb-cateninsignalinganddecreases itstarget gene c-myc, and reduces phosphorylated extracellular signal—regulated kinase (ERK)1/2 levels.

Together, these finding suggested that inflammation, proliferation, and apoptosis are targeted by GSE to

prevent CRC. In summary, this study for the first time shows alterations in the expression of miRNAs and

cytokinesbyGSE in its efficacyagainstAOM-inducedcolon tumorigenesis inA/Jmouse sporadicCRCmodel,

supporting its translationalpotential inCRCchemoprevention.CancerPrevRes; 6(7); 625–33.�2013AACR.

IntroductionColorectal cancer (CRC) is the second leading (both

genders combined) cause of cancer-related deaths and thethird most frequently diagnosed malignancy in the UnitedStates (1). Adoption of Western and sedentary lifestylesincrease CRC incidence worldwide, with most (�75%)CRCs developing sporadically (2). Despite efforts, compli-ance with screening recommendations is low and notenough to halt cancer growth/progression and preventassociatedmortality (2). Traditional CRC chemopreventive

agents, such as nonsteroidal anti-inflammatory drugs(NSAID), exert gastrointestinal side effects, suggesting thatadditional approaches are needed to manage CRC (3, 4).Use of natural products with limited or no toxicity is onesuch approach to prevent, suppress, or reverse the carcino-genesis process (5). In this regard, several studies haveindicated that one third of all cancer deaths in United Statescould be prevented through dietmodification and that highconsumption of fruits and vegetables or their bioactivecomponents could significantly decrease CRC (5–7). Grapeseed extract (GSE) is one such natural agent, rich inproanthocyanidins, whichhas shown cancer chemopreven-tive and anti-cancer efficacy in various preclinical cancermodels including CRC (6, 8, 9). Importantly, clinical stud-ies have shown that active components of GSE are bioavail-able and well-tolerated in humans (10). However, to ourknowledge, GSE has not been examined inmousemodel ofsporadic CRC. Considering that the sporadic CRC patientpopulation represents the largest clinical population ofdiagnosed CRCs, we aimed to evaluate GSE efficacy againsta chemical carcinogen azoxymethane (AOM)-inducedcolon tumorigenesis in A/J mouse model that mimicsgeneration of sporadic neoplastic lesions as observed in

Authors' Affiliations: 1Department of Pharmaceutical Sciences, SkaggsSchool of Pharmacy, 2Department of Anesthesiology, and 3University ofColorado Cancer Center, University of Colorado Anschutz Medical Cam-pus, Aurora, Colorado

Note: V. Balaiya and A.K. Jain contributed equally to this work.

Corresponding Author: Chapla Agarwal, Department of PharmaceuticalSciences, Skaggs School of Pharmacy and Pharmaceutical Sciences,University of Colorado Anschutz Medical Campus, 12850 East MontviewBlvd, C238, Room V20-2117, Aurora, CO 80045. Phone: 303-724-4057;Fax: 303-724-7266; E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-13-0053

�2013 American Association for Cancer Research.

CancerPreventionResearch

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Published OnlineFirst May 2, 2013; DOI: 10.1158/1940-6207.CAPR-13-0053

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the human colon (11). Furthermore, we also conductedmechanistic studies interlinking miRNA expression withcytokine signaling and inflammation in overall efficacy ofGSE against colon tumorigenesis.

Materials and MethodsReagents

GSE-standardized preparation was a gift from KikkomanCorp. As reported earlier (12), the analyzed composition ofGSE is 89.3% procyanidins, 6.6% monomeric flavanols,2.24%moisture content, 1.06%ofprotein, and0.8%of ash.AOM was purchased from Sigma and dissolved in saline.Primary antibodies include, anti-PCNA (proliferating cellnuclear antigen) from Dako; anti-iNOS, anti-VEGF, anti-Ago2 from Abcam; anti-COX-2, anti-C-myc, anti-b-cateninfrom Santa Cruz Biotechnology; anti-phospho-ERK1/2 andanti-NF-kB/p65 from Cell Signaling; and anti-mouse andanti-rabbit horseradish peroxidase (HRP) secondary anti-bodies from Invitrogen and Cell Signaling, respectively.Mouse cytokine antibody array was from RayBiotech.miRNA was isolated using Qiagen miRNeasy Kit, amplifiedby Qiagen RT2 miRNA qPCR kit, and quantified usingmiRNA Cancer PCR array (Qiagen).

Animals and treatmentsAnimal experiment was done under institutional guide-

lines using approved IACUC protocol. Animals were main-tained under standard conditions with free access to waterand food. GSE was mixed in AIN-76A powder diet (DyetsInc.) at 0.25% and 0.5% (w/w). Five-week-old male A/Jmice (from Jackson Laboratory) were fed AIN-76A powderdiet for 2 weeks (to acclimatize them to new diet) and thendivided into 5 groups: (i) negative control group (n ¼ 20)on AIN-76A diet; (i) positive control group (AOM, n¼ 35),injected with 5 mg/kg AOM intraperitoneally (i.p.) once aweek for 6 weeks, and on AIN-76A diet; (iii) AOMþ 0.25%GSE group (n ¼ 35), GSE supplemented diet was started 2weeks after last AOM injection and continued for 18 weeks(n¼ 25) or 28weeks (n¼ 10); (iv) AOMþ 0.5%GSE group(n ¼ 35), GSE supplemented diet was started 2 weeks afterlast AOM injection and continued for 18 weeks (n¼ 25) or28 weeks (n ¼ 10); and (v) 0.5% GSE group, GSE supple-mented diet was started at 5 weeks of mice age and con-tinued for remainder of study. The selection of 2 GSE dosesfor current study was based on our published studieswherein these GSE doses showed a dose-dependent che-mopreventive effect against AOM-induced aberrant cryptfoci formation in F344 rats and strong efficacy against smallintestine tumorigenesis inAPCmin/þmousemodels (8, 9).Similarly, the selection of AOM-induced colon tumorigen-esis experimental protocol in A/J mice in present study wasbased on our and others recent studies showingmeasurableto strong colon tumor numbers for agent chemopreventiveefficacy studies (5, 13). Body weight and diet consumptionwere recorded weekly. At the end of the study, at 33 and 43weeks of age, mice were sacrificed, entire colon excisedstarting from ileocecal junction to anal verge and cut openlongitudinally along main axis and gently flushed with ice-

cold PBS, divided into 3 equal sections (proximal, medial,and distal), tumors counted, and tumor diameters mea-sured with digital calipers under dissecting microscope.Colon tissues and/or tumors were either fixed flat in for-malin and embedded in paraffin, snap-frozen in liquidnitrogen, or stored in Qiagen RNAlater.

Anatomic MRIAnatomic gadolinium-enhanced T1-weighted MRI was

also used to noninvasively assess colon tumor progressionin mice. Bruker multi slice multi echo (MSME) T1-scanswere conducted at a Bruker 4.7 Tesla PharmaScan (BrukerMedical) following a bolus injection of 0.1mmol/kgMulti-Hance via a tail catheter on anesthetized mice (2% isoflur-ane). A mouse volume transmitter/receiver coil (36-mmdiameter) was used for all MRI studies using flowing para-meters: field of view (FOV)¼ 4cm, slice thickness¼ 1 mm,number of slices 16 (coronal) and 40 (axial), TR/TE¼ 725/11 ms, number of averages 2, matrix size ¼ 256 � 256, flipangle¼180. Total acquisition timewas 6.5minutes for eachplane. All imaging acquisition and analysis was conductedusing Bruker ParaVision software (at the Animal ImagingSharedResources,University ofColoradoAnschutzMedicalCampus).

Mouse cytokine expressionTissue lysates of colonic mucosa with tumors from ran-

domly selected animals in different groups were applied toMouse Cytokine Antibody Array. Expression of variouscytokine molecules was analyzed in duplicate on the mem-branes, which were scanned and quantified by ImageJ, anddensitometric data analyzed using antibody array analysistool.

Mouse miRNA expressionmiRNA isolation was done using Qiagen miRNeasy Kit,

starting with 20 mg of mouse colonic mucosa with tumors.Isolated miRNA was used for miRNA arrays using QiagenRT2 miRNA qPCR kit using Applied Biosciences Fast-Realtime PCR 7500. Replicates were conducted and the averageDCt value of each miRNA assay was calculated for eachgroup. The DDCt was calculated for eachmiRNA between¼DCt (AOMþ 0.5%GSE)�DCt (AOM), then the fold changewas calculated for each miRNA.

Immunohistochemistry and statistical analysesParaffin-embedded sections (5 mm) of distal colon were

subjected to standard immunohistochemical (IHC) proce-dures described previously (8). Dilutions of primary anti-bodies used were anti-PCNA (1:250), anti-iNOS (1:100),anti-COX-2 (1:50), anti-phosho-ERK1/2 (1:100), anti-Ago2 (1:100), anti-b-catenin (1:50), anti-NF-kB/p65(1:50), anti-VEGF (1:100), and anti-C-myc (1:25). Negativestaining controls were used for each protein. Apoptotic cellswere detected by DeadEnd Colorimetric TUNEL system(Promega; ref. 8). Microscopic analyses were conductedusing Zeiss Axioscope 2 microscope; photomicrographscaptured by Carl Zeiss AxioCam MrC5 camera with

Derry et al.

Cancer Prev Res; 6(7) July 2013 Cancer Prevention Research626




Axiovision Rel 4.5 software. Quantification of IHC dataincludes counts from both colonic mucosa and tumorstaining and is shown as mean � SEM from 10 fields persection in each group. Statistically significant differencebetween AOM and AOM þ GSE groups was analyzed fortumor multiplicity, tumor size, and protein expressionusing one-way ANOVA (Sigma Stat 2.03, Jandel Scientific).P � 0.05 was considered significant.

ResultsDietary GSE feeding suppresses AOM-induced colontumorigenesis (MRI study)GSE feeding did not show considerable difference in diet

consumption and body weight gain profiles between AOM-and AOM þ GSE–fed mice during entire study (data notshown). Real-time assessment of colon tumor formationwas conducted in negative control, AOM, and AOMþ 0.5%GSEmice at 32weeks of age using CE-MRI (Fig. 1A).We didnot detect any tumors/other lesions or abnormalities innegative control mice, which were further confirmed uponnecropsy (conducted 1 week post-MRI at 33 weeks of miceage; Fig. 1A, left). In AOMgroup, 6 tumors were detected ongadolinium-enhanced T1-MRI images and 12 tumors wereeventually found upon necropsy (Fig. 1A, middle). Mice in

AOM þ 0.50% GSE group showed 3 tumors on MRIwhereas necropsy identified 4 tumors (Fig. 1A, right). Com-paring the number of tumors between AOM and AOM þ0.5% GSE groups, GSE treatment resulted in 50% to 67%inhibition as identified by gadolinium-enhanced T1-MRIand necropsy, respectively.

Dietary GSE feeding suppresses AOM-inducedtumorigenesis in both colon and small intestine

Figure 1B summarizes the effect of GSE feeding on AOM-induced tumor formation in both colon and small intestinefollowing necropsy. Whereas AOM group showed 8.56 �0.58 colon tumors/mouse at 33weeks of age,GSE treatmentpost-AOM exposure significantly reduced colon tumorigen-esis in a dose-dependent manner; specifically, there was a46% (P < 0.001) and 55% (P < 0.001) inhibition in thenumber of colon tumors in AOMþ 0.25%GSE and AOMþ0.5% GSE groups (Fig. 1B), respectively. Significant inhibi-tion in colon tumorigenesis by GSE feeding was alsoobserved at 43 weeks of mouse age; however, the 2 dosesshowed comparable efficacy (Fig. 1B). GSE feeding alsosignificantly decreased the number of small intestinetumors compared with AOM group (Fig. 1B); however,again there was no GSE dose–response effect.

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Figure 1. Chemopreventive efficacyof GSE against AOM-inducedcolon tumorigenesis in A/J mice.The details of the experimentalprotocol are outlined in Materialsand Methods. A, real-timenoninvasive assessment of tumornumbers and sizes formed afterGSE feeding in AOM-exposed A/Jmice using noninvasivegadolinium-enhanced T1-MRI.Representative coronal and axialT1-weighted MRI images arepresented for untreated negativecontrol (left, coronal image), AOMuntreated (middle, coronal), andAOM þ 0.5% GSE–fed mice (right,axial). Effect of GSE feeding on (B)tumor multiplicity in colon andsmall intestine and (C) tumor size incolon, in AOM-induced colontumorigenesis in A/J mice.Columns represent mean � SEM(error bars) in each group. �, P �0.05; #, P� 0.01; $, P� 0.001. NC,negative control.

GSE Efficacy in Colon Cancer Prevention

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As tumor incidence is greater in distal region of the colonin AOM mouse model, GSE effect on tumor formation indifferent regions of the colonwas also analyzed. At 33weeksof age, AOM group showed 2.97� 0.3 colon tumors in thedistal region/mouse, whereas AOMþ 0.5% GSE group had1.66 � 0.2 tumors, indicating a 44% (P < 0.001) reductionin tumor formation in the distal regionbyGSE feeding (datanot shown). This trend was also observed in other regions(proximal and medial) of the colon and also at 43 weeks ofmouse age (data not shown); 0.25% and 0.5% GSE dosesshowed comparable efficacy. Additional analysis of colontumorigenesis study data showed that GSE feeding alsoreduces the overall tumor size (Fig. 1C). Importantly, whilethere was a significant dose-dependent decrease in thenumber of tumors in the size range of 2 to 3 mm, therewere absolutely no tumors larger than 3 mm in AOM þGSE–fed groups compared with AOM alone (Fig. 1C).

Dietary GSE feeding decreases proliferation butinduces apoptosis in its efficacy against AOM-inducedcolon tumorigenesis

CRC growth and progression to advanced stages is asso-ciated with enhanced cell proliferation and evasion ofapoptosis (11), and therefore the agentswhich could inhibitproliferation and induce apoptosis could play an importantrole in controlling CRC growth and progression (5). As wefound strong efficacy of GSE in preventing AOM-inducedcolon tumorigenesis, tissue samples were next analyzed forproliferation and apoptotic indices. Qualitative examina-tion of PCNA-stained sections showed a decrease in PCNA-positive cells in AOM þ GSE–fed groups compared withAOMalone (Fig. 2A). Further quantification showed17%�2%PCNA-positive cells in AOMþ 0.25%andAOMþ 0.5%GSE–fed groups ofmice comparedwith 47%� 5%, inAOMalone (Fig. 2A), accounting for 65% (P < 0.001) decrease inproliferation. Regarding in vivo apoptosis, GSE feeding onAOM-induced colon tumorigenesis, qualitative microscop-ic examination showed increased number of TUNEL-posi-tive cells in AOM þ GSE–fed groups compared with AOMalone (Fig. 2B). The number of apoptotic cells was about5% (Fig. 2B, P < 0.001) in both AOM þ GSE–fed groupscompared with 1.7% � 0.43% in AOM alone, accountingfor a 3-fold (P < 0.001) increase (Fig. 2B).

Dietary GSE feeding alters cytokine and miRNAexpression profiles as well as miRNA processingmachinery in its efficacy against AOM-induced colontumorigenesis

The mixture of host-derived cytokines produced in thetumormicroenvironment plays an important role in cancerprogression (14) where cancer cells use cytokines to pro-mote growth, diminish apoptosis, and stimulate both inva-sion and metastasis (14). Thus, management of cytokineequilibrium could be an important strategy for both pre-vention and treatment of various malignancies includingCRC (14). To examine whether GSE efficacy against AOM-induced colon tumorigenesis involves altered expression ofvarious cytokines, we used inflammatory cytokine antibody

array analysis on colonic mucosa with tumors from AOMand AOM þ 0.5% GSE groups (Fig. 3A). Our results indi-cated thatGSE feeding causes an alteration in the expressionof various cytokines/interleukins involved in innate immu-nity, proliferation, and apoptotic cell death. Specifically,GSE up regulated the levels of B-lymphocyte chemoattrac-tant (BLC; �8-fold), granulocyte macrophage colony-stim-ulating factor (GM-CSF; �10-fold), chemokine CCL1 (I-309; �9-fold), interleukin (IL)-1a (�10-fold), IL-23 (�20-fold), and M-CSF (�9-fold) but downregulated IL-1ra(�26-fold), IL-2 (�9-fold), and IL-27 (�8-fold) levels com-pared with AOM alone (Fig. 3A).

miRNAs are small noncoding RNAswhich are implicatedin cell-cycle regulation, cell growth and differentiation,stress response, and apoptosis (15). In addition, miRNAsalso play an essential role during carcinogenesis and theirexpression patterns have been now correlated with diseaseprognosis in patients with cancer (15). Furthermore,

Figure 2. Dietary GSE feeding decreases proliferation but inducesapoptosis in its efficacy against AOM-induced colon tumorigenesis.Antiproliferative (A) and pro-apoptotic effects (B) of GSE feeding oncolon/tumor tissue from AOM-induced colon tumorigenesis in A/J mice.IHCstaining for PCNAwasbasedonDABstaining asdetailed inMaterialsand Methods. Proliferation index was calculated as the number ofpositive cells � 100/total number of cells counted under �400magnifications in 10 randomly selected areas in each sample and shownas mean � SEM (error bars) for each group. Apoptosis was analyzed byTUNEL staining in colon/tumor tissues as detailed in Materials andMethods. Apoptotic index was calculated as the number of TUNEL-positive cells � 100/total number of cells counted under �400magnifications in 10 randomly selected areas in each sample and shownas mean� SEM (error bars) for each group. $, P� 0.001. Representativepictographs are depicted at �400. NC, negative control.

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miRNA knockdown or re-expression induces drug sensitiv-ity, impedes proliferation, and reduces invasion of cancercells (15). In view of above summarized critical roles ofmiRNAs, next we assessed whether their altered expressionalso contributes toGSE efficacy against AOM-induced colontumorigenesis. A comparison of miRNA expression profilesof the tissue from AOM and AOM þ 0.5% GSE groupsshowed that GSE feeding causes an up regulation of Snord68 (�6,500-fold), miR-19a (�2,400-fold), miR-20a(�1,000-fold), and miR-let7a (�1,200-fold; Fig. 3B) but-down regulates miR-205 (�400-fold), miR-135b (�100-fold), miR-196a (�24-fold), miR-21 (�25-fold), miR-148a(�21-fold), and miR-103 (�9-fold; Fig. 3B). Furthermore,IHC analysis revealed about 6.5-fold induction in the levelof Ago2, which is involved in miRNA processing (16), inAOM þ 0.5% GSE group compared with AOM alone,indicating an increased RNA-induced gene silencing inresponse to GSE feeding (Fig. 3C).

On the basis of an extensive literature search for thepossible role of above identified various cytokines/inter-leukins andmiRNAs with altered expression following GSEtreatment and the associated cross-talks among them, wewere able to identify a pattern in their modulation asdiscussed in detail in the Discussion (14, 17, 18). Specif-ically, we found that GSEmediates its efficacy against AOM-induced colon tumorigenesis by possibly modulating NF-kB and its downstream targets related to inflammatorysignaling, b-catenin signaling, and mitogen-activated pro-tein kinase (MAPK) pathway. Accordingly, next we inves-tigated their possible roles in GSE efficacy against AOM-induced colon tumorigenesis.

Dietary GSE feeding inhibits NF-kB activation anddecreases expression of associated molecules in itsefficacy against AOM-induced colon tumorigenesis

Transcription factor NF-kB mediates the transcription ofvarious genes which are associated with tumor initiation,promotion, progression, metastasis, as well as inflamma-tion, including those for COX-2, iNOS, and VEGF (19). Inaddition, inflammation is associated with CRC develop-ment, and chronic inflammation increasesCRC risk (20). Inaddition, experimental and epidemiologic evidences indi-cate an upregulation of COX-2, iNOS, and VEGF (down-stream target genes of NF-kB) in various malignancies,including human CRCs (21). Together, on the basis ofthis information and our array data analyses suggestingthe involvement of NF-kB and its downstream inflam-matory molecules in GSE efficacy, IHC analyses weredone to examine their expression. Our results showedthat GSE feeding indeed interferes with NF-kB activity, asobserved by a significant decrease in nuclear expression ofNF-kB/p65 in the distal colon/tumor tissues by both GSEdoses (Fig. 4A). Next we examined COX-2 and iNOSlevels (Fig. 4B and C). AOM alone treated mice showeda significant increase in COX-2 and iNOS immunoreac-tivity scores [�10-fold (P < 0.001) and �2.5-fold (P <0.001), respectively] in the distal colon/tumor tissues,compared with untreated negative controls. Importantly,mice in the AOM þ GSE–fed groups exhibited a signif-icant downregulation of COX-2 and iNOS protein levelsin the distal colon/tumor tissues (Fig. 4B and C). Simi-larly, a decrease in VEGF immunoreactivity scores (Fig.4D) was also observed in the distal colon/tumor tissuesfrom the mice in AOM þ GSE–fed groups compared withAOM alone.

Dietary GSE feeding modulates b-catenin and MAPKpathways in its efficacy against AOM-induced colontumorigenesis

Our array data analyses also suggested a possible involve-ment of b-catenin and MAPK signaling in GSE efficacyagainst AOM-induced colon tumorigenesis, and according-ly, next we focused on these 2 pathways. b-Catenin isimportant in the development of CRC and inappropriatestabilization, translocation and activation of b-cateninoccur in sporadic and familial CRC resulting in downstream

Figure 3. Dietary GSE feeding alters cytokine and miRNA expressionprofiles as well as miRNA processing machinery in its efficacy againstAOM-induced colon tumorigenesis. Alteration in (A) cytokine and (B)miRNA expression profiles and (C) Ago2 expression in colonic mucosawith tumors from AOM-induced colon tumorigenesis in A/J mice. Thearrays were done as detailed in Materials and Methods. IHC staining forAgo2 was based on DAB staining as detailed in Materials and Methods.Representative pictographs are depicted at �400. Immunoreactivity(represented by cytosolic intensity of brown staining) was scored as 0 (nostaining), þ1 (weak), þ2 (moderate), þ3 (strong), and þ4 (very strong).Columns represent mean� SEM (error bars) in each group. $, P� 0.001.NC, negative control.






modulation of oncogenic target genes such as c-myc andVEGF (22). IHC results for b-catenin staining revealed asignificant dose-dependent reduction (P < 0.001) in thenuclear expression of b-catenin in the distal colon/tumortissues of AOMþGSE–fed groups compared to AOM alone(Fig. 5A). Following the above results and further examin-ing the downstream b-catenin pathway targets, we assessedthe expression of c-myc (22). The quantification of IHCstaining for c-myc in distal colon/tumor tissues revealed asignificant (P<0.001) dose-dependent reduction inAOMþGSE groups compared with AOM alone (Fig. 5B). The RAS/RAF/MEK/MAPK signaling cascade plays an important rolein tumorigenesis including CRC (23), and accordingly, wealso investigated the role of this pathway in GSE efficacyagainst AOM-induced colon tumorigenesis by assessing theexpression of extracellular signal–regulated kinase 1/2(ERK1/2), which is phosphorylated in response to RASactivation (23). Microscopic examination of distal colon/tumor tissue sections from all groups revealed a significantreduction (94%–97%, P < 0.001) in the phosphorylation ofERK1/2 (Fig. 5C) inAOMþGSE–fed groups comparedwithAOM alone.

DiscussionThis is the first study reporting that long-term dietary

feeding of GSE (0.25% and 0.5% w/w in AIN-76A diet) for18 and 28 weeks following exposure to AOM results in asignificant decrease in AOM-induced colon tumor multi-plicity. More importantly, GSE feeding caused a significantreduction in colon tumor size in a dose-dependentmanner.Furthermore, GSE feeding did not show any effect on foodconsumption and body weight gain profiles, which is con-sistentwith previous anti-cancer efficacy studieswithGSE inother cancer models (6). In the present study, we alsoestablished feasibility of noninvasive real-time examinationof colon lesions by gadolinium-enhanced MRI, whichallowed the visualization of colon tumors in AOM mousemodel with 75% to 81% sensitivity. This, somewhat low,sensitivity of MRI (as compared with necropsy results) canbe attributed to nonoptimal experimental conditions dur-ing tumor visualization (such as small size lesions andbowel movements), which could possibly be improved infuture by altering image contrast as reported for otherstudies (24). Biomarker analyses in colonic mucosa withtumors indicated that GSE feeding decreases proliferation

Figure 4. Dietary GSE feeding inhibits NF-kB activation and decreases associated molecules in its efficacy against AOM-induced colon tumorigenesis. IHCstaining for (A) NF-kB/p65 (B) COX-2, (C), iNOS, and (D) VEGFwas based on DAB staining as detailed inMaterials andMethods. Representative pictographsare depicted at �400. Percent positive cells were calculated as number of brown nuclei � 100/total number of cells counted under �400 magnificationsin 10 randomly selected areas in each sample and shown as mean � SEM (error bars) for each group. Immunoreactivity (represented by cytosolicintensity of brown staining) was scored as 0 (no staining), þ1 (weak), þ2 (moderate), þ3 (strong), and þ4 (very strong). Columns represent mean � SEM(error bars) in each group. $, P � 0.001. NC, negative control.

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but induces apoptosis in its chemopreventive efficacy. Wealso focused our efforts on identifying the targets and path-ways modulated by GSE in its colon cancer chemopreven-tive efficacy by using cytokine and miRNA arrays. Ourresults and their analyses showed that a wide number ofcytokines/ILs and miRNAs were significantly modulated byGSE which were associated with alterations in NF-kB,

b-catenin, and MAPK signaling, suggesting that inflamma-tion, proliferation, and apoptosis are targeted by GSE in itschemopreventive efficacy.However, unlike previous studieswith GSE by us and others showing a dose-dependentcancer chemopreventive and anti-cancer efficacy in variouscancer models including AOM-induced ACF in rats (8), inpresent study, we observed comparable efficacy of GSE oncolon tumor numbers and most of the associated mecha-nistic pathways. However, GSE did affect colon tumor sizesas well as b-catenin and C-myc expression in colon tumortissues in a dose-response manner. Together, these findingssuggest that possibly a lower dose of GSE is optimum for itsefficacy in AOM-induced colon tumorigenesis in A/J miceand that additional proliferation markers should be exam-ined in future to further support b-catenin and C-mycfindings.

Further analysis of array data showed that GSE decreasesIL-2, which is an important finding because activation ofNF-kB, a transcription factor associated with inflammation,leads to IL-2 production (25). GSE also significantly down-regulated the levels of IL-27 and IL-1a, which are known tomodify NF-kB signaling (26–28). Similarly, miRNA arrayanalysis showed that GSE upregulates miR-19a, which islinked to NF-kB regulation (29). miR-20a is known toinhibit hypoxia-inducible factor (HIF)-1a pathway, whichplays a major role in the survival of cancer cells in tumormicroenvironment; importantly, GSE upregulatedmiR-20asuggesting its activity in inhibiting HIF-1a pathway and itsdownstream target VEGF (30, 31). Similarly, miRNA arrayresults showed that GSE causes a dramatic downregulationof miR-205, which targets VEGF (32) that is also transcrip-tionally regulated by NF-kB. Consistent with these observa-tions, our results showed that GSE decreases the expressionof nuclear NF-kB p65 subunit as well as those of COX-2,iNOS, and VEGF. These results are important as overexpres-sion of proinflammatory molecules, such as iNOS andCOX-2, is observed in AOM-induced CRCs, and selectiveCOX-2 inhibitors have been shown to prevent diseaseprogression (33, 34). In addition, decreased expression ofthe pro-angiogenic factor VEGF suggests that GSE mightalso interference in tumor angiogenesis; a strong dose-dependent decrease in colon tumor size by GSE supportsthis notion. Together, these results suggested thatGSE exertsits efficacy against colon tumorigenesis, at least in part, bymodulating NF-kB signaling and its downstream transcrip-tional targets COX-2, iNOS, and VEGF, which are implicat-ed in inflammation, tumor promotion, progression, andmetastasis of CRCs (19, 35, 36).

b-Catenin signaling is extensively studied for its criticalrole inhumanCRCgrowth andprogression (22), suggestingthat the agents targeting this pathway would be useful inCRC control. In our study, we found that GSE causes anincrease in M-CSF, which is linked to b-catenin signaling(37). In addition, GSE also significantly decreased miR-135b, which is also linked to b-catenin pathway (38).Moreover, GSE upregulated let-7a, which is implicated ininhibiting the expression of the c-myc oncogene, a down-stream targetofb-catenin (39).Consistentwith these results,

Figure 5. Dietary GSE feeding modulates b-catenin and MAPK pathwaysin its efficacy against AOM-induced colon tumorigenesis. IHC staining for(A) b-catenin, (B) C-myc, and (C) phospho-ERK1/2 was based on DABstaining as detailed in Materials and Methods. Representative DAB-stained tissue specimens from AOM control and AOM þ 0.5% GSE–fedgroups showing brown-colored positive cells or cytosolic staining aredepicted at �400 magnifications. Percentage of positive cells wascalculated as number of cells with brown cytoplasmic or nuclei staining�100/total number of cells counted under �400 magnifications in 10randomly selected areas in each sample and shown as mean � SEM(error bars) for each group. Immunoreactivity (represented by intensity ofbrown staining) was scored as 0 (no staining),þ1 (weak),þ2 (moderate),þ3 (strong), andþ4 (very strong). Error bars indicate�SEM. $,P� 0.001.NC, negative control.






GSE significantly downregulated b-catenin protein expres-sion, which was also associated with the decreased expres-sion of its downstream target C-myc (22), suggesting theirpossible role in GSE efficacy against colon tumorigenesis.

Cytokine/IL array findings also showed upregulation ofBLC, CCL1/I-309, and IL-1a but downregulation of IL-1raand IL-27 by GSE; all these molecules are involved inregulating the MAPK pathway (40–43). Furthermore, inmiRNA array analysis, GSE showed an upregulation of let-7a which is known to inhibit MAPK pathway (44). We alsofound that GSE decreases miR-205, which is upregulated insomemalignancies and known to interact with both MAPKand NOTCH pathways (45). Notably, MAPK signalingcascade is known toplay an important role in tumorigenesisby modulating cell growth, differentiation, proliferation,apoptosis, and migration (23). GSE also decreased miR-103, which is involved in oncogenic KRAS signaling and isupregulated in CRC cells (46). Our IHC results clearlyshowed that GSE causes a strong decrease in MAPK/ERK1/2 phosphorylation which is consistent with our cyto-kine/IL and miRNA array profiles. Together, these resultssuggested that GSE exerts its efficacy against colon tumor-igenesis, at least in part, by also modulating KRAS/MAPKsignaling.

In addition to the miRNAs described above and theirassociation with signaling pathways, GSE also modulatedthe expression of several additional miRNAs, which areimplicated in CRCs. For example, GSE downregulatedmiR-135b that is upregulated in CRC tissue compared withthe normal surrounding tissue and has been specificallyshown to target the APC gene involved in CRCs (47, 48).Similarly, GSE decreased miR-196a, the high levels of thismiRNA exert a pro-oncogenic effect in CRC cells (49).Likewise, the ability of GSE to decrease miR-21 is of clinicalsignificance because its increased levels correlate withincreased stage of clinical CRCs (47, 50). Importantly, in

this study, we also observed predominate global upregula-tion of miRNAs by GSE, coupled with downregulation ofoncogenic miRNAs and upregulation of tumor-suppressivemiRNAs. Accordingly, we also investigated a key proteininvolved in miRNA homeostasis (16), namely Ago2, whichbinds to the mRNA and represses translation or transloca-tion. Colon tissue analysis revealed significant upregulationof Ago2protein in colons of AOMþGSE–fed groups, whichfurther supports our miRNA array results. Together, this isthe first study showing alteration inmiRNAs expression andcytokine signaling by GSE in its chemopreventive efficacyagainst AOM-induced colon tumorigenesis in A/J mousesporadic CRC model.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. Derry, V. Balaiya, R. Agarwal, C. AgarwalDevelopment of methodology: M. Derry, V. Balaiya, A. Jain, S. Shrotriya,N.J. SerkovaAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): M. Derry, K. Huber, N.J. SerkovaAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): M. Derry, K. Raina, V. Balaiya, N.J.Serkova, R. AgarwalWriting, review, and/or revisionof themanuscript:M.Derry, K. Raina, V.Balaiya, N.J. Serkova, R. AgarwalAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): M. Derry, R. Agarwal, C. AgarwalStudy supervision: R. Agarwal, C. Agarwal

Grant SupportThis work was supported by NIH R01 AT003623, NCI Cancer Center P30

CA046934, and NCRR CTSA UL1 RR025780.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 14, 2013; revised April 5, 2013; accepted April 18, 2013;published OnlineFirst May 2, 2013.

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2013;6:625-633. Published OnlineFirst May 2, 2013.Cancer Prev Res Molly M. Derry, Komal Raina, Velmurugan Balaiya, et al. Signaling and InflammationTumorigenesis in A/J Mice: Interlinking miRNA with Cytokine Grape Seed Extract Efficacy against Azoxymethane-Induced Colon

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