Black Raspberries Protectively Regulate Methylation of Wnt ... · Research Article Black...

Research Article

Black Raspberries Protectively Regulate Methylation of WntPathway Genes in Precancerous Colon Tissue

Li-Shu Wang1, Chieh-Ti Kuo1, Tim H.-M. Huang4, Martha Yearsley5, Kiyoko Oshima2, Gary D. Stoner1,Jianhua Yu6, John F. Lechner1, and Yi-Wen Huang3

AbstractUlcerative colitis is frequently an intermediate step to colon cancer. The interleukin-10 knockoutmouse is

a genetic model of this progression. We report that knockout mice fed 5% black raspberries (BRB) had

significantly less colonic ulceration as compared with knockout mice that consumed the control diet.

Dysfunction of the Wnt signaling pathway is a key event in ulcerative colitis–associated colon carcino-

genesis. Therefore, we investigated the effects of BRBs on the Wnt pathway and found that the BRB-fed

knockout mice exhibited a significantly lower level of b-catenin nuclear translocation. We followed-up this

observation by evaluating the effect of BRBs on selected Wnt pathway antagonists. The mRNA expression

levels of wif1, sox17, and qkiwere diminished in the knockout mice, whereas they were expressed at normal

levels in knockoutmice thatwere fed BRBs. The lowermRNA expression of these genes in the colon from the

knockout mice correlated with hypermethylation of their promoter regions; BRBs decreased their promoter

methylation and increased mRNA expression of these genes. This hypomethylation was associated with

elevated protein expression of key proteins/enzymes that augment methylation, for example, dnmt3b,

hdac1, hdac2, andmbd2 in the knockout mice; in addition, BRBs decreased the protein expression of these

proteins/enzymes. The knockout mouse model recapitulates what occurs in human ulcerative colitis.

Promoter methylation of CDH1 and SFRP1 was significantly higher in human ulcerative colitis tissues

compared with their adjacent normal tissues. In conclusion, our results suggest that BRBs inhibit colonic

ulceration and, ultimately, colon cancer partly through inhibiting aberrant epigenetic events that dysre-

gulate Wnt signaling. Cancer Prev Res; 6(12); 1317–27. �2013 AACR.

IntroductionThe Wnt signaling pathway promotes increased nuclear

localization of b-catenin protein when associated with T-cell factor (TCF) and other proteins, and participates ininitiating the transcriptionof cancer-associated genes. In thenormal colon epithelial cells, much of the b-catenin isbound to E-cadherin at the cellular membrane where itparticipates in cellular adhesion and differentiation pro-cesses. b-Catenin binds with APC, GSK3b, axin2, and CK1aproteins to prime its destruction by proteasome degrada-tion and these processes keepb-catenin activity in check (1).

Dysfunction ofWnt signaling that occurs inmore than 85%of sporadic colon cancer is primarily caused bymutation ofthe APC gene (2, 3). Ulcerative colitis is a precancerousdisease that is caused by chronic inflammation. Ten percentof patients with ulcerative colitis progress to colon cancerwithin 20 years (4). Dysfunction of the Wnt to b-cateninsignaling pathway is a key event in the genesis of ulcerativecolitis–associated colon carcinogenesis (3). However, APCmutations are rarely observed in these cases; instead, Wntsignaling dysfunction in ulcerative colitis–associated coloncancer is strongly associated with epigenetic silencing ofnegative regulators of the pathway (3, 5, 6).

Although genetic mutations are generally irreversible,epigenetic alterations can be reversed by exogenous agents(7, 8), including some constituents of foods (9). We (10)have reported that black raspberries (BRB), which are rich inprotective antioxidant and anti-inflammatory compoundssuch as anthocyanins and ellagitannins, inhibit colon car-cinogenesis in animal models. In addition, we recentlyreported that a 5% BRB diet reduced colonic injury in amouse model in which ulcerative colitis was induced bydextran sodium sulfate (DSS; ref. 11). This protective effectwas associated with reduced levels of cytokines TNF andinterleukin-1b (IL-1b), as well as COX-2 and prostaglandinE2 (PGE2) in colonic tissues (11). COX-2 is target of Wntsignaling (12), and we recently reported that patients with

Authors' Affiliations: 1Division of Hematology andOncology, DepartmentofMedicine, Departments of 2Pathology, and 3Obstetrics andGynecology,Medical College of Wisconsin, Milwaukee, Wisconsin; 4Department ofMolecular Medicine, Cancer Therapy & Research Center, University ofTexas Health Science Center, San Antonio, Texas; 5Department of Pathol-ogy; and 6Comprehensive Cancer Center, The Ohio State University,Columbus, Ohio

Note:Supplementary data for this article are available atCancer PreventionResearch Online (http://cancerprevres.aacrjournals.org/).

Corresponding Author: Li-ShuWang, Division of Hematology and Oncol-ogy, Department of Medicine, Medical College of Wisconsin, 8701Watertown Plank Rd, TBRC, Room C4930, Milwaukee, WI 53226. Phone:414-955-2827; Fax: 414-955-6059; E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-13-0077

�2013 American Association for Cancer Research.

CancerPreventionResearch

www.aacrjournals.org 1317

Cancer Research. on February 23, 2020. © 2013 American Association forcancerpreventionresearch.aacrjournals.org Downloaded from

Published OnlineFirst October 15, 2013; DOI: 10.1158/1940-6207.CAPR-13-0077

http://cancerpreventionresearch.aacrjournals.org/

colon cancer who consumed BRBs exhibited hypomethy-lated inhibitor genes Wnt signaling pathway (13). Thus,BRBs affect carcinogenesis partially by regulating the level ofDNAmethylation of selected genes. For this study, we haveused the IL-10 knockout mouse because it is a geneticanimal model that recapitulates the genesis of humanulcerative colitis and ulcerative colitis–initiated coloncancer (14). We recently reported that the DNA (cyto-sine-5)-methyltransferase 1 (DNMT1) protein was reducedin colon tumor tissue collected from patients that con-sumed BRBs (13). We studied the effect of BRBs on othermethylation-controlling enzymes and proteins. TheDNMT3b protein was evaluated because it is involved inde novo DNA methylation, and has been shown to beoverexpressed in colorectal cancer (15). In addition, weassessed the methylation binding domain 2 (MBD2), his-tone deacetylase 1 (HDAC1), and HDAC2, all of which areinvolved in gene silencing via chromatin condensation (16,17), and we measured the relative levels of mRNA expres-sion of the cognate genes.

Potential effects of the 5% BRB diet on the methylationstatus andmRNA expression of the selected regulator genes,wif1, sox17, dkk2, dkk3, qki, and wnt3a, of the Wnt signalingpathway in colonic tissues from IL-10 knockout mice werealso examined. Finally, we assessed the state of methylationof selected Wnt signaling regulatory genes in human ulcer-ative colitis specimens. It has been previously reported thatthe WNT regulatory genes, APC, APC2, SFRP1, and SFRP2are hypermethylated in human ulcerative colitis specimens(18, 19). In the current study, we extended this databaseby evaluating cadherin-1 (CDH1; also called E-cadherin),WIF1, and WNT3A in colonic tissues from healthy donoras well as paired adjacent normal and ulcerative colitistissues from patients with ulcerative colitis.

Overall, our results lend credence to the supposition thatincluding significant quantities of BRBs in the diet mayreduce the risk of patients with ulcerative colitis to developcolon cancer. In addition, our data suggest that part of themechanism of prevention by BRBs is by antagonizing thedevelopment of inappropriate epigenetic events at the onsetof cancer development.

Materials and MethodsAnimals, berry treatment, and colon preparation

All protocols were carried out in accordance with insti-tutional guidelines for animal care procedures as dictated bytheOhio State University Animal Care andUse Committee.Wild-type (WT) and knockout male mice were purchasedfrom The Jackson Laboratory when they were 3 to 4 weeksold. The animals were placed in the protocol 1 week afterthey arrived from the vendor, and the study was of 8 weeksduration.WTmice were fed a control diet (n¼ 5) or controldiet supplemented with 5% BRBs (n ¼ 5), and knockoutmice were fed either control diet (n ¼ 15) or control dietsupplemented with 5% BRBs (n¼ 15). The control diet wasthe American Institute of Nutrition synthetic diet 76A (AIN-76A; Dyets Inc). The preparation of BRB powder is detailed

by Montrose and colleagues (11), and is also provided inSupplementary Materials; this is the same batch of BRBpowder used by Montrose and colleagues (11). The micewere sacrificed by CO2 asphyxiation and full-length colonswere removed, flushed with cold saline, and opened lon-gitudinally. Theywere then Swiss-rolled, formalin-fixed andparaffin-embedded (FFPE), and stained with hematoxylinand eosin (H&E). The stained colonic tissues were exam-ined according to standard pathologic criteria to confirmboth normalcy and to demark regions of colonic ulceration.

Evaluation of colonic ulcerationFFPE colons were stained by H&E for colonic ulceration

analysis. The entire colon was viewed under �200 magni-fication (high-power view). The percentage of ulcerationwas calculated as the involved tissue in high power areasover total high power areas of the entire length of the colon.Areas of ulceration in the mucosa and submucosa werecounted separately. Colons from all mice on this studywere evaluated for colonic ulceration in a blinded mannerby M. Yearsley.

Immunohistochemical staining and computer-assistedimage analysis

Paraffin-embedded colonic tissues from themicewere cutinto 4-mm sections and placed on slides. Staining proce-dures, antibody information forDNMT3B,MBD2,HDAC1,HDAC2, and b-catenin, and procedures for computer-assisted image analysis are detailed in SupplementaryMaterials.

Human colonic tissuesForty-eight normal colon specimens from healthy

donors, as well as 24 paired ulcerative colitis specimensand their adjacent normal tissues were obtained from theCooperative Human Tissue Network (CHTN). The speci-mens were obtained and used in accordance with thedictates of the Institutional Review Board of the MedicalCollege of Wisconsin (Milwaukee, WI).

Real-time PCRmRNA was extracted from paraffin-embedded entire

mouse colon tissues using the RecoverAll Total NucleicAcid Isolation Kit for FFPE (Ambion). Two microgramsof total RNA per sample was reverse transcribed usingSuperScript III RT (Invitrogen). Quantitative PCR (qPCR)procedures and primer information is provided in Supple-mentary Materials.

PyrosequencingParaffin-embedded entiremouse colon tissues from5WT

mice fed control diet, 5 WTmice fed BRB diet, 10 knockoutmice fed control diet, and 10 knockout mice fed BRB dietwere cut into 10-mm sections for DNA extraction. Approx-imately, 10 mg of each frozen human colonic tissue spec-imen, representing true normal (N), ulcerative colitis, oradjacent normal-appearing tissue (AN), was used for DNAextraction. Both mouse and human colonic DNAs were

Wang et al.

Cancer Prev Res; 6(12) December 2013 Cancer Prevention Research1318




extracted using PicoPure DNA kit (MDS Analytical Tech-nologies). Extracted DNA was purified using the QIAquickPCR Purification Kit (Qiagen). Of note, 500 ng of extractedDNAwasbisulfite-convertedusing the EZDNAMethylationKit (Zymo Research). A pyrosequencing system (Qiagen)was used to quantify methylated CpGs as described previ-ously (20). Specified primer sequences in the promoterregion of the evaluated genes are listed in SupplementaryMaterials.

Statistical analysisData from evaluation of colonic ulceration, immunohis-

tochemical staining, real-time PCR, and promoter methyl-ation determined by pyrosequencing were compared by theStudent t test. All analyses were two-sided, and a P value lessthan 0.05 was considered to be significant.

ResultsDietary BRBs reduce colon ulceration in IL-10knockout miceRepresentative H&E staining of colonic tissues from WT

mice that consumed control orBRBdiet, andknockoutmiceon either control or BRB diet are shown in Fig. 1A–D, top tobottom, respectively. Figure 1E depicts quantified results.The data show that knockout mice that consumed dietaryBRBs exhibited significantly less ulceration in colonicmucosa and submucosa than did the knockout mice oncontrol diet.

Effect of BRBs on b-cateninThe Wnt signaling cascade causes an increase in b-catenin

protein translocation into the nucleus (1, 21). Representativestaining of b-catenin protein inWTmice fed either control orBRB diet is depicted in Fig. 2A and B, respectively, whereas itsappearance in the knockout mice is seen in Fig. 2C (controldiet) and Fig. 2D (BRB diet). Quantification of the immu-nohistochemical staining is depicted in Fig. 2E. The percent-ageof stainednucleiwithin colonic tissues collected fromWTmice thatwere fed either control or BRB-containing diets wasstatistically identical to that in adjacent normal-appearingtissues fromknockoutmice fed controlorBRBdiet.However,twiceasmanycellswithin theulcerative colitis tissue fromtheknockoutmice exhibited strong nuclear staining. In contrast,nuclear staining in the ulcerative colitis tissue from theknockout mice that consumed BRBs was not significantlydifferent from the adjacent normal tissue. ThemRNA expres-sion level of b-catenin was found to be elevated in theknockout mice, but significantly less in the knockout micethat consumed BRBs (Fig. 2F).

BRBs modulate expression of Wnt signaling pathwayregulatorsmRNA expression of the Wnt signaling pathway negative

regulators wif1, sox17, and qki in knockout mice fed controlor 5% BRB diets was measured using real-time PCR. Allthese genes were expressed at lower levels in knockout micethat were fed the control diet as comparedwith theWTmice

on either control or berry diet, and BRBs maintained theirexpression at control levels in the knockout mice (Fig. 3A–C, respectively). In addition, we evaluated the effectsof BRBs on mRNA expression of dkk2 and dkk3 (Fig. 3Dand E). Neither of these genes was downregulated in theknockout mice; however, animals on the BRB diet exhibit-ed significantly higher mRNA expression levels of dkk2and dkk3. Interestingly, BRBs decreased mRNA expressionof wnt3a in the knockout mice (Fig. 3F).

We used pyrosequencing to evaluate promoter methyla-tion of the same six genes, wif1, sox17, qki, dkk2, dkk3, andwnt3a, and the data are shown in Fig. 4A–F. In knockoutmice on control diet, wif1, sox17, qki, and dkk2 were rela-tively hypermethylated than in WT mice on control orBRB diet, and BRBs maintained the percentage of methyl-ation at control levels (Fig. 4A–D, respectively). In contrast,the level of methylation of dkk3 was not increased in theknockout mice, and BRBs had no effect on its methylationlevel (Fig. 4E).

In addition, we evaluated the promoter of Wnt ligandgene wnt3a.Wnt3a has been reported to be one of the mostupregulated genes in the inflamed human colon (21).Therefore, we anticipated that the gene would be relativelyhypomethylated in the knockout mice, and that BRBswould cause the level of methylation to be maintained atthe control level. Instead, we found that wnt3a was signif-icantly hypermethylated in the knockout mice, and BRBscause the level of methylation to be maintained at controllevels (Fig. 4F). As mentioned earlier, mRNA expression ofwnt3awas decreased by BRBs in the knockoutmice (Fig. 3F)although the berries decreased promoter methylation ofwnt3a (Fig. 4F). These results suggest that othermechanismsare involved in the regulation of wnt3a expression.

Immunohistochemical quantification of DNMT3B,MBD2, HDAC1, and HDAC2

DNMT1 is chiefly responsible for maintaining DNAmethylation homeostasis, whereas DNMT3B, HDAC1,HDAC2, and MBD2 are involved in de novo DNA hyper-methylation and chromatinmethylation, and in convertingeuchromatin into heterochromatin (15–17, 22, 23). All ofthese enzymes have been shown to be overexpressed inseveral tumor types, including colorectal cancer (16). Asnoted previously (13), biopsies collected from humanpatients with colorectal cancer after they had consumedBRBs for an average of 4 weeks had lower levels of DNMT1protein. Therefore, we quantified the levels of methylation-regulating proteins in the knockout mice to see whetherBRBs affect their expression, and whether this might beassociatedwith their ability to correct aberrantmethylation.Immunohistochemical staining of DNMT3B, MBD2,HDAC1, and HDAC2 is depicted in Fig. 5E. The levels ofall enzymes were significantly elevated in the ulcerativecolitis samples from the knockout mice. However, speci-mens from the BRB-treated knockout mice exhibited levelsthat were statistically comparable with the normal colonfrom WT mice on control or berry diet (Fig. 5A–D). ThemRNA expression of dnmt3b, mbd2, hdac1, and hdac2 is

Black Raspberries Regulate Wnt Pathway Regulatory Genes

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depicted in Supplementary Fig. S1. Knockout animals onthe BRB diet exhibited significantly decreased expression ofall of these mRNAs.

Promoter methylation of the Wnt signaling pathwaynegative regulator genes is elevated in ulcerative colitisspecimens from patients

We and other researchers (8, 13) have shown that thelevel of methylation in the promoter regions of the Wntsignaling pathway negative regulatory genes SFRP2, SFRP5,and WIF1 are elevated in human colon tumor specimens.Dhir and colleagues (18) and You and colleagues (19)noted that APC, APC2, SFRP1, SFRP4, SFRP5, and DKK1

also become epigenetically silenced during the transitionfrom normal to ulcerative colitis to human colon cancer.Therefore, we evaluated whether CDH1, SFRP1,WIF1, andAPC are hypermethylated in ulcerative colitis tissues. Thelevels of methylation of CDH1 and SFRP1were found to besignificantly higher in the ulcerative lesionswhen comparedwith adjacent normal specimens (Fig. 6A and B, respective-ly). Interestingly, we did not observe significant differencesin promoter methylation between tissues from healthydonors and the adjacent normal-appearing tissues frompatients with ulcerative colitis for APC, WNT3A, and WIF1(Fig. 6C–E, respectively). These levels correlated withincreases of more than 50% in 14 of 24 patients for CDH1

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Figure 1. Dietary supplementationwith 5% BRBs decreased coloniculceration in IL-10 knockout (KO)mice. Representative H&E-stainedsections of colon tissues (�200): A,WT mouse fed control diet; B, WTmouse fed BRB diet; C, knockoutmouse fed control diet; and D,knockout mouse that consumedBRBs. Note that the level ofulceration is reduced. In (C), regulararrows indicate the areaofmucosalulceration; the superficialulceration is in the mucosa.Dashed-arrows indicate the area ofsubmucosal ulceration; ulcerationextends to the submucosa. Bothmucosal and submucosalulcerations are indicators of injury.In (D), arrows indicate the area ofpast injury with the crypts dropoutand the surface epitheliumrecovered. The amount ofinflammatory cells reflects thenormal state. E, percentages ofcolon ulceration in IL-10 knockoutmice fed control or BRB diet. Notethat the level of mucosal andsubmucosal ulceration wassignificantly less in the IL-10knockout mice that consumedBRBs. �, P < 0.05.

Wang et al.





(Fig. 6A) and in 6 of 24 patients for SFRP1 (Fig. 6B). Thepercentagemethylation value for theAPC genewas very lowin all specimens, and there was no significant differenceamong the specimens (Fig. 6C). However, when theinflamed tissues were compared with the adjacent nor-mal-appearing tissue collected from the same patient, 9 of24 lesions had a more than 50% increase in the level ofhypermethylation of APC (Fig. 6C). The data for WNT3aalso showed that the hypermethylation level of the genein the ulcerative colitis tissues significantly exceeded that

measured in the corresponding normal-appearing adjacentregion, and a more than 50% increase in methylation wasfound in 5 of 24 patients (Fig. 6D). The methylation levelsof WIF1 were not significantly altered in ulcerative colitiscompared with normal tissues from healthy donors andadjacent normal specimens (Fig. 6E).

DiscussionIn the absence of a Wnt ligand, the b-catenin protein is

primarily either bound to E-cadherin or within a complex

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Figure 2. Dietary BRBs decreasedb-catenin nuclear localization incolons from IL-10 knockout (KO)mice. Representative b-catenin–stained sections of colon tissues(�200): A, WT mouse fed controldiet; B, WT mouse fed BRB diet; C,knockout mouse fed control diet;and D, knockout mouse thatconsumed BRBs. BRBssignificantly decreased b-cateninnuclear staining in tissues ofulcerative colitis (E) and b-cateninmRNA expression in the entirecolon (F) collected from IL-10knockout mice. �, P < 0.05.






that augments its destruction by proteasome degradation(1). In normal cells, the binding of a Wnt ligand to Dishev-eled initiates the canonical Wnt signaling pathway, whichresults in the transcription of Wnt-specific genes that reg-ulate cell-fate decisions. There is considerable regulation ofthe activity of the pathway through extracellular and intra-cellular controlling proteins. Dysfunction of this pathway isa formative event in the genesis of sporadic colon cancer,primarily by mutation or occasional hypermethylation ofthe APC gene (1). However, the genetic animal model usedherein recapitulates the genesis of ulcerative colitis–associ-ated colon cancer (14), and the molecular mechanismseems to be primarily epigenetic in nature (3, 5, 6).

b-Catenin–mediated signaling is a key regulator ofepithelial proliferative responses, and it increases inrepair responses in human chronic ulcerative colitis(24). Activation of b-catenin signaling increases in accor-dance with the degree of mucosal inflammation inhuman ulcerative colitis and one of the mechanisms thatcorrelates with the genesis and maintenance of colitis is

inappropriate nuclear accumulation of b-catenin (24).These observations are similar to those found in animalmodels of ulcerative colitis. For example, staining ofphosphorylated-b-catenin increases in colitic and dys-plastic colon of IL-10 knockout mice and is, therefore,suggested to be a biomarker of colitis-induced neoplastictransformation (24). In DSS-induced ulcerative colitisand cancer in mice, the b-catenin nuclear/cytoplasmictranslocation is an early event in the development ofdysplastic lesions (25). On the basis of these observa-tions, increased activation of b-catenin signaling can leadto oncogenic transformation of colonic epithelium.

Wnt3a, a Wnt ligand gene, is associated with the regula-tion of Wnt pathway and repair, and it has been shown thatactivation ofwnt3a signaling stimulates intestinal epithelialrepair by promoting c-Myc–regulated gene expression (26).Furthermore, wnt3a is one of themost upregulated genes sofar known in inflamed human colon (19). In the currentstudy, we showed that BRBs decreased promoter methyla-tion of wnt3a (Fig. 3F) and, interestingly, the berries also

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Figure 3. Dietary BRBs protectivelymodulate mRNA expression ofgenes in theWnt signaling pathwayin the entire colon collected fromIL-10 knockout (KO) mice. mRNAexpressionof (A)wif1, (B) sox17, (C)qki, (D) dkk2, (E) dkk3, and (F)wnt3a. �, P < 0.05.

Wang et al.





decreased mRNA expression of wnt3a (Fig. 4F) in colonsfrom knockout mice, suggesting that they decrease activa-tion of wnt3a signaling. These results agree with the obser-vation that BRBs decreased b-catenin nuclear localization(Fig. 2); BRBs effectively inhibited the translocation of thistranscription-regulating cofactor. In addition, our resultssuggest other mechanisms are involved in regulating theexpression of wnt3a.Mesalamine is a mainstay therapeutic agent in human

chronic ulcerative colitis; partly through decreasing b-cate-nin activation which, in turn, decreases colitis-induceddysplasia and colitis-associated cancer (24). Our findingssuggest that BRBs inhibit colonic ulceration and ultimatelycolon cancer partly through inhibiting aberrant epigeneticevents and b-catenin nuclear translocation which thencorrect dysregulated Wnt signaling.

Expression of b-catenin mRNA was found to be signifi-cantly elevated in knockout mice, and was reduced byberries in these mice. Upregulation of the gene in colontissue of the knockout mice was unexpected. The mecha-nism causing increased b-cateninmRNA production and itsdownregulation by BRBs is unknown; however as discussedfurther, we speculate that it could be associated with thehypermethylation of qki. Alternatively, it has been shownthat Disheveled–KSRP complex stabilizes b-catenin mRNA(27).

Dhir and colleagues (18) noted that the Wnt pathwaynegative-regulatory genes, APC, APC2, SFRP1, SFRP2,SFRP4, SFRP5, and DKK1 become epigenetically down-regulated during the transition from normal human colonto ulcerative colitis to colon cancer. We investigated theregulatory genes wif1, sox17, dkk2, dkk3, and qki in the IL-10

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Figure 4. Regulation of promotermethylation of Wnt signalingpathway genes by BRBs in wholecolon specimens collected from IL-10 knockout (KO) mice. Promotermethylation of (A) wif1, (B) sox17,(C) qki, (D) dkk2, (E) dkk3, and (F)wnt3a. �, P < 0.05.






knockoutmice to determinewhether BRBs affect their levelsof transcription and DNA methylation. Wif1, dkk2, anddkk3 are extracellular antagonists of Wnt ligands (28, 29),whereas sox17 augments degradation of the TCF–b-cate-nin complex (29, 30). The RNA-binding protein genequaking (qki) was included in these investigationsbecause its loss of expression is a common feature inhuman colon cancer. In addition, this gene controls thelevels of b-catenin, presumably through interaction withits cognate protein with a response element in the b-cate-

nin 30-untranslated region (UTR; ref. 31). Wif1, sox17,and qki exhibited lower mRNA expression in the colons ofthe knockout mice, and ingested BRBs maintained theirexpressions at normal levels (Fig. 3). In addition, BRBsmaintained DNAmethylation homeostasis of several Wntpathway negative regulatory genes in the knockout mice(Fig. 4). Moreover, we found that expression of proteinsinvolved in DNA methylation was decreased by BRBs inthe knockout mice (Fig. 5). Thus, our data imply that onemechanism whereby BRBs inhibit colonic ulceration

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Figure 5. BRBs decreased proteinexpression of DNMT3B, MBD2,HDAC1, and HDAC2. Histogramsof DNMT3B (A), MBD2 (B), HDAC1(C), and HDAC2(D) are thequantification plots obtained fromimmunohistochemical staining.Expression of all of these proteinswas elevated in tissues withulcerative colitis in comparisonwith adjacent normal (AN) tissuesfrom IL-10 knockout (KO) micefed control diet (knockout), andBRBs decreased their levels intissues with ulcerative colitis.E, representative immunohisto-chemical staining (�200) ofDNMT3B, MBD2, HDAC1, andHDAC2 of specimens from WTmice fed control or BRB diet andIL-10 KO mice fed control or BRBdiet. �, P < 0.05.

Wang et al.





occurs by preventing epigenetic dysregulation of theWnt signaling pathway, in part by deterring DNA hyper-methylation, inappropriate histone modifications, andcondensation of chromatin. Further research is required

to ascertain whether BRBs actually inhibit methylation orrapidly remove methyl groups.

The results for dkk2 and dkk3 were different. The expres-sion of dkk2 mRNA in colon tissues of knockout mice fed

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Figure 6. Promoter methylation ofWnt signaling pathway regulatorsin normal colon tissue (N) from 48healthy donors, and in pairedtissues with ulcerative colitis (UC)and adjacent normal tissue (AN)from 24 patients with ulcerativecolitis. A, CDH1; B, SFRP1; C,APC; D, WNT3A; and E, WIF1.�, P < 0.05.






control diet was not significantly different from coloncollected from WT mice fed either control or BRB diet (Fig.3D). However, dkk2 was relatively hypermethylated inthe knockout mice fed control diet, and BRBs decreasedthe DNA methylation of dkk2 in the knockout mice (Fig.4D). In contrast, transcription of dkk2 in knockout micethat consumed BRBs was significantly upregulated to levelsthat were greater than noted for the control mice (Fig. 3D).Thus, within an inflammatory environment, BRBs bothantagonized the hypermethylation of this gene while aug-menting its expression. As noted for dkk2, BRBs specificallystimulated expression of dkk3 in colon collected from theknockout mice. These results suggest a novel mechanismthat makes BRBs effective in stimulating expression ofWnt signaling negative regulatory genes that remains to beelucidated.

IL-10 is known to regulate growth of commensal flora inthe gut and, thus, it also maintains gut homeostasis (32).We have evidence that dietary BRBs increase the abundanceof Lactobacillus, Coriobacteriales, Bifidobacteriales, Bacteroides,and Clostridiales in F344 rats (unpublished data). The Bray–Curtis analysis showed a strong time-dependent change inbacterial diversity of the microbiota in response to dietarytreatment with BRBs (unpublished data). It is possible thatBRBsmay also have effects on themicrobial flora ofWT andknockout mice. We speculate that if BRBs exhibit effects onthe flora, this could potentially lead to less inflammationas witnessed in the knockout mice. We are currently eval-uating the effects of dietary BRBs on gut flora inWT and theknockout mice.

The IL-10 knockout mouse model is a genetic animalmodel that recapitulates the genesis of human colon cancerthat originates within an ulcerative colitis lesion (14). Ourcurrent study shows that proper governance of the Wntsignaling pathway is maintained by BRBs by sustainingcorrect methylation patterns and expression of Wnt path-way negative-regulatory genes. Cancer chemoprevention by

affecting histone modification and/or DNA methylationand DNMT1, DNMT3b, HDAC1, HDAC2, and MBD2 isa common observation. Indeed, Schnekenburger and Die-derich (9) recently listed 52 dietary foods and constituentsthat exhibit these activities. We have now shown thatingestion of BRBs prevent the onset of their aberrant actionsin the knockout mice. One question that remains, however,is whether BRBs prevent upregulation of DNMT1,DNMT3b, HDAC1, HDAC2, and MBD2, or cause theirdownregulation, or both. Another question is whether theyaffect expression of these enzymes/proteins directly or indi-rectly. Further research is ongoing to clarify these questions.

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

Authors' ContributionsConception and design: L.-S. Wang, T.H.M. Huang, J. YuDevelopment of methodology: J. Yu, Y.-W. HuangAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): C.-T. KuoAnalysis and interpretation of data (e.g., statistical analysis, bio-statistics, computational analysis): L.-S. Wang, M. Yearsley, K. Oshima,G.D. Stoner, J.F. Lechner, Y.-W. HuangWriting, review, and/or revision of themanuscript: L.-S.Wang, G.D. Stoner,J.F. LechnerAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): C.-T. Kuo, Y.-W. HuangStudy supervision: L.-S. Wang

Grant SupportThisworkwas financially supported by theNIH (grant no. R01CA148818

to L.-S. Wang). L.-S.Wangwas also supported by the ACS (RSG-13-138-01—CNE). T.H.M. Huang was supported by the National Cancer Institute (NCI;grant nos. R01 CA069065, U54 CA113001, P50 CA134254, R01 ES017594,and U01 ES019482). G.D. Stoner was the recipient of an NCI grant (R01CA103180) as was J. Yu (R01 CA155521).

The costs of publication of this article were defrayed in part by thepayment 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 28, 2013; revised August 13, 2013; accepted August 29,2013; published OnlineFirst October 15, 2013.

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2013;6:1317-1327. Published OnlineFirst October 15, 2013.Cancer Prev Res Li-Shu Wang, Chieh-Ti Kuo, Tim H.-M. Huang, et al. Pathway Genes in Precancerous Colon TissueBlack Raspberries Protectively Regulate Methylation of Wnt

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