Clinical Cancer Therapy: Clinical Cancer Researchclincancerres.aacrjournals.org/content/clincanres/17/3/598.full.pdf · Cancer Therapy: Clinical ... angiogenesis, and Wnt pathway

Cancer Therapy: Clinical

Modulation of Genetic and Epigenetic Biomarkers of Colorectal Cancerin Humans by Black Raspberries: A Phase I Pilot Study

Li-Shu Wang1, Mark Arnold2, Yi-Wen Huang3, Christine Sardo1, Claire Seguin1, Edward Martin2,Tim H.-M. Huang3, Ken Riedl4, Steven Schwartz4, Wendy Frankel5, Dennis Pearl6,Yiqing Xu7, John Winston III8, Guang-Yu Yang9, and Gary Stoner1

AbstractPurpose: This study evaluated the effects of black raspberries (BRBs) on biomarkers of tumor

development in the human colon and rectum including methylation of relevant tumor suppressor genes,

cell proliferation, apoptosis, angiogenesis, and expression of Wnt pathway genes.

Experimental Design: Biopsies of adjacent normal tissues and colorectal adenocarcinomas were taken

from 20 patients before and after oral consumption of BRB powder (60 g/d) for 1–9 weeks. Methylation

status of promoter regions of five tumor suppressor genes was quantified. Protein expression of DNA

methyltransferase 1 (DNMT1) and genes associated with cell proliferation, apoptosis, angiogenesis, and

Wnt signaling were measured.

Results: The methylation of three Wnt inhibitors, SFRP2, SFRP5, and WIF1, upstream genes in Wnt

pathway, and PAX6a, a developmental regulator, was modulated in a protective direction by BRBs in

normal tissues and in colorectal tumors only in patients who received BRB treatment for an average of

4 weeks, but not in all 20 patients with 1–9 weeks of BRB treatment. This was associated with decreased

expression of DNMT1. BRBs modulated expression of genes associated with Wnt pathway, proliferation,

apoptosis, and angiogenesis in a protective direction.

Conclusions: These data provide evidence of the ability of BRBs to demethylate tumor suppressor genes

and to modulate other biomarkers of tumor development in the human colon and rectum. While

demethylation of genes did not occur in colorectal tissues from all treated patients, the positive results

with the secondary endpoints suggest that additional studies of BRBs for the prevention of colorectal cancer

in humans now appear warranted. Clin Cancer Res; 17(3); 598–610. �2010 AACR.

Introduction

Colorectal cancer is the third most common cancer inboth men and women in the United States (1). Anestimated 49,920 deaths from colorectal cancer wereexpected to occur in 2009, accounting for 9% of all cancer

deaths. Mortality rates from colorectal cancer havedeclined in both men and women over the past twodecades, a reflection of declining rates in incidence andan increase in early screening for the disease. The 5-yearsurvival from this disease is 64% and continues to declineto 57% at 10 years after diagnosis. For persons withdistant metastases at diagnosis, the 5-year survival is only10%. Therefore, the prevention of colorectal cancerremains an important goal, and chemoprevention isone approach to achieve this goal.

Our laboratory has shown that the administration of BRBpowder at 2.5%, 5%, and 10% of a synthetic diet to azox-ymethane (AOM)-treated Fischer 344 (F-344) rats resulted inan average 42%, 45%, and 71% (P < 0.05 for all groups)decline, respectively, in tumor number relative to the AOM-only group in a 36-week assay (2). We also found significantreductions in urinary levels of the oxidative DNA adduct,8-hydroxy-20-deoxyguanosine (8-OHdG) in these samegroups, suggesting the ability of berries to reduce oxidativestress. The mechanism(s) by which BRBs reduced AOM-induced tumors in rat colonwere not investigated. However,in studies using a rat model of nitrosamine-induced esopha-geal squamous cell carcinoma (SCC), BRBs were found toreduce proliferation, inflammation, and angiogenesis, and

Authors' Affiliations: 1Department of Internal Medicine, ComprehensiveCancer Center, College of Medicine, 2Department of Surgery, 3HumanCancer Genetics Program, Comprehensive Cancer Center, 4Departmentof Food Science and Technology, 5Department of Pathology, 6Departmentof Statistics, The Ohio State University, Columbus, Ohio; 7Department ofMedicine, Maimonides Medical Center, Brooklyn, New York; 8Departmentof Surgery, University of Texas Health Science Center, San Antonio,Texas; and 9Department of Pathology, Northwestern University, FeinbergSchool of Medicine, Chicago, Illinois

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

L-S. Wang and M. Arnold contributed equally to the study.

Corresponding Author: Gary D. Stoner, Department of Internal Medicine,Ohio State University Comprehensive Cancer Center, 2001 Polaris Pkwy,Columbus, OH 43240. Phone: (614)293–3268, Fax: (614)293–2690; E-mail:[email protected] or Li-Shu Wang, Comprehensive Cancer Center,Ohio State University, 2001 Polaris Pkwy, Columbus, OH 43240, Phone:(614)293–2692, Fax: (614)293–2690; E-mail: [email protected],

doi: 10.1158/1078-0432.CCR-10-1260

�2010 American Association for Cancer Research.

ClinicalCancer

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stimulate apoptosis and differentiation of premalignant cellsand tissues, resulting in reduced tumor development. Genesassociatedwith these cellular functionswere alsoprotectivelymodulated by BRB diets (3).DNA methylation in mammalian cells is regulated by a

family of highly related DNA methyltransferase enzymes(DNMT1,DNMT3a, andDNMT3b) thatmediate the transferof methyl groups from S-adenosylmethionine to the 5 posi-tion of cytosine bases in the dinucleotide sequence CpG.DNMT1 functions as the maintenance DNAmethyltransfer-ase in mammalian cells and is responsible for accuratelyreplicating genomic DNA methylation patterns during the Sphase of the cell cycle (4). In contrast, de novomethylation ofDNA is believed to be performed byDNMT3a andDNMT3benzymes that possess both maintenance and de novo DNAmethylation activities (5). Both groupsof enzymes, however,have been shown to exhibit some level of bothmaintenanceanddenovomethylation in vitro, suggesting that this classifica-tionoftheDNMTsmaybeoversimplified(6).Confirmingtheimportance of DNA methylation in tumorigenesis, studieshave shown all 3 DNMTs to be overly expressed in severaltumor types, including tumors of the colon and rectum,bladder, and kidney (7). When DNMT1 and DNMT3b areknocked out in colon cancer cell lines, methylation of tumorsuppressorgenessuchasp16 isalmostentirelyeliminatedandthe gene is re-expressed (8). Inhibition of DNMTs, therefore,may lead to demethylation and reactivation of the silencedgenes. Aberrant methylation of tumor suppressor genes byDNMTs may be a promising target for chemoprevention.DNMT inhibitors are currently being developed as potentialtherapeutic agents for cancer (9, 10).Aberrant Wnt/b-catenin (Wnt) pathway signaling occurs

in about 85% of sporadic colorectal cancers and is due

principally to mutations in the Apc gene (11). Recent find-ings have demonstrated an important role of the Wntpathway in cell proliferation, differentiation, and stem cellmaintenance. Disruption of Wnt signaling due to dysregu-lated gene methylation is also a common event in humancolorectal cancer, for example, frequent methylation of thenegativeWnt regulators, secreted frizzled-related proteins 2and 5 (SFRP2 and SFRP5), and Wnt inhibitory factor 1(WIF1) (11). Methylation of SFRP-gene promoter regionshas beendetected in aberrant crypt foci (ACF) andpersists inprimary colon cancer cells (12). Re-expression of SFRPs incolon cancer cells blocks Wnt signaling and results inapoptosis (12). Importantly, these events occur in coloncancer cells that exhibit b-catenin mutations, a gene in thedownstream Wnt pathway (12). Silencing of p16, alsoknown as CDKN2A, a cell cycle regulator, and PAX6a, adevelopment regulator, in colorectal cancer is reported tobe associated with promoter hypermethylation (13, 14).Unlike mutational changes in DNA bases, the potentialreversibility of DNA methylation patterns of tumor sup-pressor genes suggests that these are a viable target for theprevention and/or treatment of colorectal cancer.

The goal of this study was to investigate the effects ofshort-term treatment with dietary BRBs on promotermethylation of the Wnt inhibitors, SFRP2, SFRP5, andWIF1, as well as p16 and PAX6a, and onmodulating DNMTfamily enzymes in human colorectal cancer tissues. Theeffects of the berries on protein expression of downstreamgenes in the Wnt pathway; that is, b-catenin, E-cadherin,and c-Myc, and on biomarkers of cell proliferation (Ki-67),apoptosis (TUNEL), and angiogenesis (CD-105) were alsoevaluated. Our results indicate that BRBs, administeredorally on a daily basis for an average of approximately4 weeks, led to the protective modulation of multipleepigenetic and cellular biomarkers in human colorectaltissues. These effects likely occurred through local absorp-tion of berry bioactives into colorectal tissues.

Materials and Methods

Clinical trialThe present trial to evaluate the effects of BRBs on bio-

markers of colorectal cancer development was approved bythe Institutional Review Boards of the Ohio State UniversityComprehensive Cancer Center and the University of Texas,San Antonio. Written informed consent was obtained fromall patients before participation in the trial. Most of thepatients accrued to this trial were individuals who werediagnosed with colorectal cancer elsewhere and sent toOhioState University James Cancer Hospital for surgery. There-fore, in order to be eligible for this trial, they had to agree toundergo a second colonoscopy or sigmoidoscopy (depend-ing upon tumor location) in order to obtain baseline color-ectal tumor and adjacent normal tissues for the study.

A. Inclusion criteria. Patients accrued to the trialhad one of the following: (i) Patients with a patho-logical diagnosis of early-stage colorectal cancer

Translational Relevance

We are developing strategies for the chemopreventionof colorectal cancer using freeze-dried berries. The pre-sent trial was conducted to determine, in a small cohortof colorectal cancer patients, if short-term treatmentwith freeze-dried black raspberries (BRBs) would resultin modulating biomarkers of tumor development.Twenty colorectal cancer patients were administered60 g/d of BRBs orally for 1–9weeks. Biopsies of adjacentnormal and colorectal tumor tissues were taken beforeand after the berry treatment. BRBs were found toprotectively modulate biomarkers of cell proliferation,apoptosis, angiogenesis, and Wnt pathway in tumorand adjacent normal specimens. BRBs caused demethy-lation of the promoter regions of relevant tumor sup-pressor genes in adjacent normal tissues and colorectaltumors, and this effect was dependent upon total dose.These data, along with our preclinical data demonstrat-ing the ability of BRBs to prevent tumor development inthe rat colon and mouse small intestine, provide morerationale for the further evaluation of BRBs for theprevention of colorectal cancer in humans.

Gene Demethylation by Berries in Colorectal Cancer

www.aacrjournals.org Clin Cancer Res; 17(3) February 1, 2011 599




(primary or recurrent), who had a primary lesion inthe colon or rectum, and were considered as candi-dates for elective colorectal surgery. (ii) Patientswho had a diagnosis of stage IV colorectal cancerwith metastatic lesions in the liver or abdomen(carcinomatosis) were also eligible, provided thatthe primary lesion was present in the colon orrectum, and a surgery to resect the colorectal lesionwas planned. (iii) Patients with a colon polyp, orpathological diagnosis of adenoma, not removableby initial colonoscopy or sigmoidoscopy, due tosize or other reasons (such as receiving anticoagula-tion treatment), who required a repeat colonoscopyor sigmoidoscopy or surgery for removal. (iv)Patients who were considered to have a high like-lihood for colorectal malignancy by the discretionof the surgeon, with histories such as anemia, rectalbleeding, weight loss, bowel movement habitchanges, X-ray evidence of colorectal mass, etc. wereconsidered eligible. (v) Patients with rectal cancerswere eligible prior to proceeding with chemoradia-tion or surgery if they did not have obstructivelesions or obstructive symptoms.

B. Exclusion criteria. Patients excluded from the trialhad one of the following: (i) Clinical symptoms ofobstruction or bleeding and considered for immedi-ate surgery. (ii) Patients with rectal cancer or obstruc-tive lesions who were considered candidates forimmediate neoadjuvant chemoradiation treatmentor surgery. (iii) Patients currently receiving che-motherapy or radiation therapy (must be > 4 weekssince last chemotherapy treatment and > 3 weeksafter radiation treatment). (iv) Patients who tooknonsteroidal anti-inflammatory drugs (NSAIDs)and could not be taken off the medication due totheir clinical condition. (v) Pregnant or lactatingpatients. (vi) Patients with uncontrolled, uncompen-sated cardiac, pulmonary or hepatic diseases, oruncontrolled infectious diseases or diabetes mellitus.

Black raspberriesFresh frozen BRBs (Jewel variety, 2004 harvest) were

purchased from an Ohio farm and shipped frozen toVan Drunen Farms in Momence, Illinois for freeze-dryingas described (15). Nutrient analysis and packing of BRBsare detailed in Supplementary Methods.

Administration of black raspberriesOne dose, 20 g, of freeze-dried berry powder was mixed

with approximately 100 mL of water and consumed orally3 times a day (60 g/d total) for 1–9 weeks. Berry powderwas consumed regularly at 3 times throughout the day,6 hours apart. Sixty grams of berry powder approximates1.3 lbs of fresh BRBs per day and is equivalent to a rodentdiet of �7% berry powder (16). BRB powder was foundto be chemopreventive in the rat colon when provided inthe diet at concentrations of 2.5% to 10% (3).

Evaluations before and during berry treatmentInitially, all patients were given a physical examination

during which their medical/surgical history was obtained,and laboratory tests for lactic dehydrogenase (LDH), car-cinoembryonic antigen (CEA), complete blood count(CBC), and a complete metabolic panel were completed.They then participated in a 24-hour verbal food recall toestablish consumption patterns of phenol-rich foods,including berry products, before treatment with berrypowder. During berry treatment, patients were evaluatedonce per week for compliance and any adverse event(s).Patients were asked to avoid consumption of berry typesother than black raspberry powder during the trial.

Tissue and urine collectionAll patients accrued to the trial had a diagnosis of color-

ectal cancer before entry into the trial. Because tissue takenfor initial diagnosis was not available for study, it wasnecessary to obtain additional tissue specimens beforetreatment of the patients with berries. Initially, patientssigned a consent form, after which they discontinued use ofNSAIDs. After approximately 5 days, 3 biopsies each ofadjacent normal (<2 cm from tumors) and tumor tissueswere taken from patients with either rectal or colon cancer.One-half of the each biopsy was placed in 10% bufferedformalin and the other half was frozen in liquid nitrogen.Patients began taking BRB powder approximately 24 hoursafter the tissue biopsies were taken and daily until �12–36hours before surgery to remove the tumor. At surgery, anadditional 3 biopsies each of adjacent normal and tumortissues were taken from each patient and placed in bufferedformalin or frozen as described for the pretreatment biop-sies. All tissue specimens were classified histopathologi-cally as either normal or tumor byDr.W. Frankel, amedicalpathologist. All tumors were adenocarcinomas.

Pre- and posttreatment urine specimens (�50 mL each)were collected at baseline and after berry treatment. Toensure the stability of berry anthocyanins, the specimenswere stored at�80�C after adding 5% trifluoroacetic acid toreduce the pH to acidity.

Measurement of berry anthocyanins in colorectaltissues and urine

Sample preparation, and HPLC–MS/MS analysis andquantification of anthocyanins (17) are detailed in Sup-plementary Methods.

Analysis of DNA methylation

A. DNA extraction and bisulfite conversion. Adja-cent normal and tumor tissues were used for extrac-tion of genomic DNA as described in SupplementaryMethods.

B. MassARRAY. High-throughput MassARRAY wasused toquantifymethylation levels of theCpG islandsof p16, PAX6a, SFRP2, SFRP5, andWIF1 as described(18) and detailed in Supplementary Methods.

Wang et al.

Clin Cancer Res; 17(3) February 1, 2011 Clinical Cancer Research600




C. Pyrosequencing. The LINE-1 repetitive elementbisulfite/pyrosequencing assay was used to estimateglobal methylation (19) as detailed in Supplemen-tary Methods.

Immunohistochemical staining and computer-assisted image analysisImmunohistochemical staining procedures and quanti-

fication of staining of b-catenin, E-cadherin, c-Myc, Ki-67,TUNEL, p16, CD105, or DNMT1 are detailed in Supple-mentary Methods.

StatisticsMethylation and immunohistochemical staining data

were quantified by determining pre and post berry treat-ment, and the percent change of each variable from base-line. Differences in mean of pre and post, percent change,and anthocyanin levels were compared by Student’s t-test.All analyses were two-sided, and a P value of less than 0.05was considered to be significant. Linear regression was usedto determine the correlation between the percent change ofmethylation and the percent change of DNMT1 expression,berry dose, and age.

Results

Patient characteristicsTwenty patients were included into the trial and their

characteristics are summarized in Table 1. Seventeenpatients were accrued at the Ohio State University and 3were accrued at the University of Texas, San Antonio.Seventeen of the 20 patients were male. The average ageof the study populationwas 59 years. Six patients had coloncancer (30%) and the other 14 (70%) had rectal cancer.Two patients had metastatic disease.

Berry treatment and compliancePatients were treated with BRB powder orally for 1–9

weeks (Table 1). Patient compliance to berry treatment wasexcellent with each patient consuming >90% of the stipu-lated daily doses, based upon self-reporting and return ofempty bags. The berry powder was generally well toleratedwith 7 patients reporting mild disturbances of the gastro-intestinal tract; that is, diarrhea and constipation, thatresolved within 2–3 days. Patient LDH, CBC, CEA, andmetabolic profiles were not altered by BRB treatment dur-ing the study.

Analysis of anthocyanins in urine and colorectaltissueAnthocyanins were not present in the urine of any

patient before berry treatment. All 4 anthocyanins weredetected in the urine of all 20 patients after berry treatment(Supplementary Table S1). The amounts of the 4 antho-cyanins in the urine of all patients ranged from 56.2 to1822.1 pmol/mL. The 4 anthocyanins were also detected incolorectal tissues from 18 of the 20 patients, however, the

amounts were much lower than those in the urine, rangingfrom 1.7 to 2011.5 fmol/mg tissue (SupplementaryTable S1). The levels of total anthocyanins in adjacentnormal tissues versus tumor tissues were 299.9 � 754.9and 55.4 � 60.8 fmol/mg tissue (mean � S.D.), respec-tively. Anthocyanin levels in adjacent normal tissueswere not significantly different from those in tumor tissues(P ¼ 0.21).

Effect of BRBs on promoter demethylation of tumorsuppressor genes and DNMT1 protein expression

Preliminary analysis of combined methylation datafrom adjacent normal and colorectal tumor tissues fromall 20 patients indicated that BRB treatment for different

Table 1. Characteristics of colorectal cancerpatients in this study

No. ofpatients (%)

GenderFemale 3 (15)Male 17 (85)

Age<45 2 (10)45–60 8 (40)>60 10 (50)Range 32–82

Tumor locationTransverse, descending,and sigmoid colon

3 (15)

Cecum and ascending colon 3 (15)Rectum 14 (70)

Metastatic diseaseLymph node involved 2 (10)No evidence 18 (90)

Berry treatment (wks)<2 7 (35)2–4 4 (20)>4 9 (45)Range 1–9

Berry doses (20 g/dose, 3 doses/d)<50 8 (40)50–85 8 (40)>85 4 (20)Range 10–189

Compliance (%)80–90 1 (5)90–100 8 (40)100 11 (55)

Toxicities reportedDiarrhea 3 (15)Constipation 4 (20)None 13 (65)

Improved bowel movements 15 (75)






periods of time (1–9 weeks; average ¼ 3 weeks) failed toproduce significant effects on promoter methylation ofSFRP2, PAX6a, p16, SFRP5, and WIF1, and on LINE-1repetitive element in colorectal tumor (adenocarcinoma)tissues (Fig. 1). In fact, the only significant effectsobserved were decreased SFRP2 and SFRP5 promotermethylation in adjacent normal tissues. It is apparentfrom Figure 1, that the effects of berry treatment onpromoter methylation of genes in both adjacent normaland tumor tissues were quite variable from one patient toanother.

Significant differences in promoter methylation of thesegenes were observed, however, when the patients weredivided into two groups based upon: (a) the total numberof berry doses taken and, (b) the extent of changes inDNMT1 protein expression. The data from analyses ofadjacent normal tissues are shown in Figure 2. N/H refersto adjacent normal tissues taken from patients who hadreceived an average of 83 berry doses (�4 weeks of treat-

ment). N/L refers to adjacent normal tissues taken frompatients who had received an average of 52 berry doses(�2 weeks of treatment). Figure 2 shows significantdecreases in percent methylation change from baselineof the SFRP2 and PAX6a tumor suppressor genes, as wellas DNMT1 protein expression, in N/H tissues compared toN/L tissues. The box labeled "All" indicates that the reduc-tion in methylation of all 5 tumor suppressor genes(SFRP2, PAX6a, p16, SFRP5, and WIF1) combined wassignificant in the N/H group versus the N/L group.

Figure 3 illustrates data from analyses of colorectaladenocarcinomas from patients who had received differentdoses of berries. T/H refers to adenocarcinomas taken frompatients who had received an average of 85 berry doses(�4 weeks of treatment). T/L refers to adenocarcinomastaken from patients who had received an average of53 berry doses (�2 weeks of treatment). Significantdecreases in percent methylation change from baselineof the SFRP2, PAX6a, and WIF1 genes, as well as DNMT1

p16C

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Figure 1. Effects of BRBs onpromoter methylation of SFRP2,PAX6a, p16, SFRP5, and WIF1,and on LINE-1 repetitive elementin adjacent normal tissues andcolorectal adenocarcinomas from20 patients before and after 1–9weeks of berry treatment.*P < 0.05.

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protein expression, were observed in T/H group tumorsversus T/L group tumors. When promoter methylation datafrom all 5 suppressor genes were combined, the T/H tumordata were significantly different from the T/L tumor data.These results suggest that the degree of change in promotermethylation of tumor suppressor genes and in DNMT1protein expression was influenced by the total dose of BRBpowder consumed.

In the heat-maps in Figures 2B and 3B, green andred colors depict decreased and increased methylation,respectively. When comparing these figures, adjacent nor-mal colorectal tissues generally responded more favorablyto berry-induced promoter demethylation than colorectaladenocarcinomas as evidenced by the relative amountof green color. Further, there was a positive correlationbetween changes in DNMT1 protein expression and

Figure 2. Effects of BRBs onpromoter methylation of SFRP2,PAX6a, p16, SFRP5, and WIF1,and on DNMT1 protein expressionin adjacent normal tissues from 20patients treated for an average ofeither 4 or 2 weeks with BRBs.A, N/H refers to adjacent normaltissue taken from patients whohad received an average of 83berry doses (�4 weeks). N/L refersto adjacent normal tissue takenfrom patients who had received anaverage of 52 berry doses(�2 weeks). Significant decreasesfrom baseline in promotermethylation of SFRP2, PAX6a,and all 5 genes combined(combination of changes fromSFRP2, PAX6a, p16, SFRP5, andWIF1) are seen in N/H versus N/Lgroups (P < 0.05). This isassociated with decreasedDNMT1 protein expression, andlonger berry treatment. B, heat-map of methylation changes frombaseline of SFRP2, PAX6a, p16,SFRP5, and WIF1, and proteinexpression change of DNMT1in adjacent normal colorectaltissues.

SFRP2 PAX6a

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P = 0.001 P = 0.028 P = 0.064

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promoter methylation (Fig. 4A, P ¼ 0.006, R2 ¼ 0.181). Ingeneral, treatment with BRBs for �4 weeks (high dose) butnot �2 weeks (low dose) led to decreases in promotermethylation in both adjacent normal and adenocarcinomatissues as indicated in Figure 4B (P ¼ 0.035, R2 ¼ 0.118).Berry treatment did not cause significant changes frombaseline in global methylation in either adjacent normaltissues or colorectal tumors from high and low berry dosegroups as measured by the LINE-1 repetitive elementbisulfite/pyrosequencing assay (Figure 4C). There wereno significant differences in the age of patients treated witheither high- or low-dose berries (Supplementary Fig. S1A)and berry-induced promoter methylation changes were notage dependent (Supplementary Fig. S1B).

Differential responses of colon and rectal tissues toBRB treatmentBecause the exposure of colon tissues to BRB compounds

administered orally might differ from that of rectal tissues,we determined if adjacent normal colon and colon tumorsmight respond differently to the demethylation effects ofberry treatment than adjacent normal rectum and rectaltumors. Figure 4D depicts the percent change in methyla-tion from baseline of all 5 tumor suppressor genes (SFRP2,PAX6a, p16, SFRP5, and WIF1) combined in adjacentnormal colon and rectal tissues from the high-dose(N/H) and low-dose (N/L) treatment groups. When adja-cent normal tissues from the two dose groups were com-pared, only the methylation changes in rectal tissues in theN/H versus N/L treatment groups were significantly differ-ent (Figure 4D, P ¼ 0.011). Similar results were obtainedwith tumor specimens in that methylation changes in rectaltumors in the T/H group were significantly different fromthe T/L group (Figure 4E, P < 0.0001). Interestingly, T/Lgroup tumors located in the colon responded more favor-ably to BRBs than those in the rectum (Fig. 4E).

Effect of BRBs on Wnt signaling, cell proliferation,apoptosis, angiogenesis, and cell cycle regulationQuantitative immunohistochemistry was used to mea-

sure the expression of three proteins associated with theWnt pathway (b-catenin, E-cadherin, c-Myc) as well asproteins associated with cell proliferation (Ki-67), apop-tosis (TUNEL), angiogenesis (CD105), and cell cycleregulation (p16) in adjacent normal tissues and color-ectal tumors. Representative staining of these proteins isshown in Figure 5A. Combined data from all 20 patientsbefore and after 1–9 weeks of BRB treatment indicatedthat the berries protectively modulated b-catenin, Ki-67,TUNEL, CD105, and DNMT1 in colorectal tumor tissues,

and CD105 and DNMT1 in adjacent normal tissues(Fig. 5B).

When BRBs were evaluated for their effects on thesetissue biomarkers as a function of total dose, they werealso found to be effective. In adjacent normal tissues fromthe high-dose (N/H; �4 weeks treatment) berry group,berry treatment led to a decrease in b-catenin expressionand an increase in E-cadherin expression when comparedto the low-dose (N/L; �2 weeks treatment) group (Fig. 6A,P < 0.05). This was accompanied by a decrease in Ki-67protein expression and an increase in TUNEL staining inthe N/H as compared to the N/L group (P < 0.05). CD105was decreased and p16 was increased in both N/H and N/Lgroups but the differences between groups were not sig-nificant (P > 0.05).

In colorectal adenocarcinomas, BRB treatment led tosignificant decreases in percent protein expression changefrom baseline of b-catenin, Ki-67, and CD105, andincreases in the expression of TUNEL and p16 in T/Htissues versus T/L tissues (Fig. 6B). Again, these data suggestthat the responses to BRBs are related to the cumulativeberry dose.

Discussion

The results from this study indicate that treatment ofcolorectal cancer patients with BRBs led to positive mod-ulation of both genetic and epigenetic biomarkers incolorectal adenocarcinomas and also adjacent normaltissues. Immmunohistochemical data from analysis ofbiomarkers such as Ki-67, TUNEL, b-catenin, CD105,and DNMT1 indicated that these markers were modu-lated protectively in tissues from all 20 patients after BRBtreatment. In contrast, positive modulation of the DNAmethylation markers occurred only in tissues frompatients who were treated with BRBs for an average of�4 weeks (high dose), suggesting that berry treatmenthad to occur for longer periods of time to be effective. Thereason for these differences might be related to thefact that whole tissue specimens, containing multiplecell types, were used for the methylation studies whereasthe immunohistochemical data were obtained largelyfrom analysis of the epithelium. Therefore, if the effectsof berries are more pronounced in the epithelium thanin other tissues, such as stroma, then one might expectto detect berry effects more readily using the immuno-histochemical markers. It is also possible that the inhibi-tion of DNA methyltransferases such as DNMT1 mightoccur only after prolonged treatment with berries. Theseobservations suggest that BRBs may well be effective in

Figure 3. Effects of BRBs on promoter methylation of SFRP2, PAX6a, p16, SFRP5, and WIF1, and on DNMT1 protein expression in colorectaladenocarcinomas from 20 patients treated for an average of either 4 or 2 weeks with BRBs. A, T/H refers to adenocarcinomas taken from patients who hadreceived an average of 85 berry doses (�4 weeks). T/L refers to adenocarcinomas taken from patients who had received an average of 53 berry doses (�2weeks). Significant decreases from baseline in promoter methylation of SFRP2, PAX6a, WIF1, and all 5 genes combined are seen in T/H versus T/L groups (P <0.05). This is associated with decreased DNMT1 protein expression and longer berry treatment. B, heat-map of methylation changes from baseline of SFRP2,PAX6a, p16, SFRP5, and WIF1, and protein expression change of DNMT1 in colorectal adenocarcinomas.






modulating promoter methylation of tumor suppressorgenes in long-term phase II and III clinical trials where theberries would be administered for months to years. Weshould also point out that, although we made equalattempts to recruit men and women to our trial, we foundthat men were more likely to participate. Nevertheless,

more effort will be made to reduce the gender imbalancein future trials.

Results from the present study suggest that BRBs mayhave potential for inhibition of DNA methylation inaddition to their many other mechanisms of action (20).BRBs elicit minimal or no toxicity when administered

Promoter methylation % change from baseline

DN

MT

1 pr

otei

n %

cha

nge

from

bas

elin

e

T/H

T/L

N/H

N/L

P = 0.006

R2 = 0.181

A C LINE-1

% c

hang

e fr

om b

asel

ine

N/H N/L N/H+N/L T/H T/L T/H+T/L

P = 0.303 P = 0.215

Promoter methylation % change from baseline

Ber

ry d

ose T/H

T/L

N/H

N/L

P = 0.035

R2 = 0.118

B

+1 −1

−

−

−

−

0 +2

−4−2

N/H – RN/H – C N/L – RN/L – C

P = 0.105 P = 0.656

P = 0.790 P = 0.025

P = 0.011

P < 0.0001

Met

hyla

tion

% c

hang

e fr

om b

asel

ine

Met

hyla

tion

% c

hang

e fr

om b

asel

ine

D

E

T/H – RT/H – C T/L – RT/L – C

−190 +7 +3

−9−11 +5

+37

−

−−

−

−

−

Figure 4.Correlation of methylation change with DNMT1 change or total berry dose and differential responses of adenocarcinomas from the colon and rectumto BRB treatment. A, berry-induced methylation changes are positively correlated with changes in DNMT1 protein expression. B, berry-induced methylationchanges are negatively correlated with total berry dose. C, overall, BRB treatment did not affect global methylation as measured by LINE-1 repetitiveelement bisulfite/pyrosequencing assay. Percent change from baseline of all 5 genes combined (SFRP2, PAX6a, p16, SFRP5, andWIF1) in D, adjacent normaltissues and E, in both colon and rectal tumors. Colon group: combining data from descending, transverse, and ascending colon. Significant differencesof mean percent change in promoter methylation are observed in the following comparisons: N/H – R vs. N/L – R, T/H – R vs. T/L – R, and T/L – C vs. T/L – R.C ¼ colon, R ¼ rectum.

Wang et al.





orally to humans at high daily doses making them attrac-tive for chemoprevention. Further, we recently showed thatthe oral administration of BRB powder (60 g/d-–equivalentto about 1.3 lbs of fresh berries) in patients with familialadenomatous polyposis for 9 months produced onlyminor gastrointestinal disturbances in some patients thatwere resolved in 2–3 days (21).Although global DNA hypomethylation is closely linked

to chromatin restructuring and nuclear disorganization incancer cells leading to chromosomal instability (22), theeffects of global DNA hypomethylation in animals havebeen controversial. For example, in contrast to the overallinhibition of intestinal tumorigenesis in these animals,hypomethylation caused the development of multifocal

liver tumors. These results clearly demonstrate the oppos-ing effects of DNA hypomethylation on intestinal and livercarcinogenesis (23). In humans, a study examining globalmethylation in cancer cell lines, including breast, centralnervous system, colon, leukemia, liver, lung, ovary,and prostate showed that 85% of tested cell lines (51/60) were globally hypomethylated (24). Interestingly, thesame study demonstrated that global methylation in color-ectal cancers is highly variable with increased, no change,or decreased global methylation when comparing color-ectal carcinomas with their adjacent tissues; global hypo-methylation is partially reversed in cancers withmicrosatellite instability that may reflect alternative pro-gression pathways in cancer. Therefore, the concept of

Figure 5. Effects of BRBconsumption on expression ofproteins (b-catenin, E-cadherin,and c-Myc) downstream of theWnt pathway as well asbiomarkers of cell proliferation(Ki-67), apoptosis (TUNEL), andangiogenesis (CD105) in adjacentnormal tissues and colorectaladenocarcinomas from 20patients before and aftertreatment with BRBs for 1–9weeks. A, representative stainingof b-catenin, E-cadherin, c-Myc,Ki-67, TUNEL, p16, and CD105and B, dot-line plots ofquantitative staining of theseproteins. *P < 0.05.

Representative stainingA

Ki-67

N

T

TUNEL p16 CD105-catenin c-MycE-cadherin

-catenin

E-cadherin

c-Myc

Ki-67

TUNEL

p16

CD105

DNMT1

B

Pre PostN

Pre PostT

Pre PostN

Pre PostT

% s

tain

ing

% s

tain

ing

% s

tain

ing

% s

tain

ing

DNMT1

*

*

*

*

*

*

* *






global DNA hypomethylation in cancers might be toosimplified; alterations of global DNA methylation patternsin carcinogenesis are organ and tumor type specific. DNAmethylation inhibitors cause hypomethylation in promo-ters and global repeat sequences, and yet they exert ther-apeutic activities.

Further, based on Issa (9), although demethylation oftumor suppressor genes may have a beneficial effect,decreased methylation of oncogenes could lead to theirreactivation and a subsequent adverse effect. It has beenshown however, that hypomethylation agents elicit ther-apeutic effects that may be due to the fact that tumors aremore dependent upon gene silencing to maintain theirphenotype than normal adult cells. Thus, the overall effectof decreasing methylation appears to be positive. In addi-tion, methylation is age dependent, and age-related methy-lation appears to be gene and tissue specific. However, themethylation of some genes, for example, p16 and SFRP2,and genes associated with DNA repair, is not affected byaging; rather their methylation levels are increased withcancer progression. This is especially true of colorectalcancer (25).

In the present study, BRBs showed differential effects onpromoter methylation of SFRP2, SFRP5, WIF1, p16, andPAX6a genes. For example, while BRBs reduced methyla-tion of the SFRP2 and PAX6a genes in the N/H versus N/Lgroups (Fig. 2), they had no effect on methylation of theother 3 genes in these same groups. Thus, it would appearthat BRBs exhibit some selectivity in their effects on methy-lation of different genes; however, the data are preliminaryand the mechanism(s) for this effect are unknown.

The effects of BRBs on gene demethylation are likelythrough the localized absorption of berry active com-pounds into colorectal tissues. This is evidenced by thedetection, albeit at low levels, of BRB anthocyanins incolorectal tissues following berry treatment. The metabo-lites of cyanidin-based anthocyanins include protocate-chuic acid, the predominant degradation product incultured cells, and both 2,4-dihydroxybenzoic acid and2,4,6-trihydroxybenzoic acid (26). This same studyreported that 50 mM of protocatechuic acid exerted anti-oxidant activity but not the other 2 metabolites. We aredetermining if these metabolites can be detected in bloodfrom animals and humans. The large patient variation in

-catenin (P = 0.030) E-cadherin (P = 0.042) c-Myc (P = 0.110)

Ki-67 (P = 0.045) TUNEL (P = 0.024) p16 (P = 0.229) CD105 (P = 0.531)

B Adenocarcinoma

-catenin (P = 0.011) E-cadherin (P = 0.340) c-Myc (P = 0.413)

Ki-67 (P = 0.034) TUNEL (P = 0.006) p16 (P = 0.004) CD105 (P = 0.043)

A Adjacent normal

% c

hang

e fr

om

base

line

% c

hang

e fr

om

base

line

% c

hang

e fr

om

base

line

% c

hang

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om

base

line

T/H T/L T/H T/L T/H T/L

T/H T/L T/H T/L T/H T/L T/H T/L

N/H N/L N/H N/L N/H N/L

N/H N/LN/H N/L N/H N/L N/H N/L

–37

+11

+123–40

+20–13

–42

+8+233

–4+250

+96

–36 –9

–60

–15 –51–33

+24+3

–20

–2 +417

+42 +555

+23

–70

–36

Figure 6. Effects of BRBconsumption on proteinexpression of b-catenin,E-cadherin, and c-Mycdownstream of the Wnt signalingpathway as well as biomarkers ofcell proliferation (Ki-67), apoptosis(TUNEL), and angiogenesis(CD105) in A, adjacent normaltissues and B, adenocarcinomasfrom 20 colorectal cancer patientswho had consumed BRBs for anaverage of either 4 or 2 weeks. Inadjacent normal tissues, nuclearb-catenin and Ki-67 staining aredecreased significantly in the N/Hgroup versus the N/L group,whereas, E-cadherin and TUNELstaining are significantly increasedin N/H versus N/L. Inadenocarcinomas, b-catenin,Ki-67, TUNEL, p16, and CD105are protectively modulated to agreater extent in tumors from theT/H group than those in theT/L group.

Wang et al.





the amounts of anthocyanins (1.7–2011.5 fmol/mg)detected in colorectal tissues is likely due to the fact thatthe patients took their last berry dose at different times (12–36 hours) before surgery. Therefore, the biomarker datacould not be correlated with anthocyanin levels. Pharma-cokinetic studies indicate that BRB anthocyanins reachpeak levels in the blood and urine at 1 and 4 hours,respectively, after oral administration of berry powder(27). Therefore, onemight expect large differences in color-ectal tissue levels in patients who consumed berries as longas 12–36 hours before surgery.The "field effect" model of carcinogenesis suggests that

cells adjacent to tumors harbor at least some of the geneticalterations present in the tumors themselves (28). Indeed,epigenetic alterations in adjacent normal tissues have beenassociated with cancer progression in the human colon andbreast (29, 30). As indicated in Figures 1, 2, 5, and 6,however, BRBs caused demethylation of tumor suppressorgenes and protectively modulated biomarkers of cell pro-liferation and apoptosis in adjacent "normal" colorectaltissues that could well have restored these tissues to ahigher degree of "normalcy."In conclusion, our results suggest that BRBs protectively

modulate both genetic and epigenetic biomarkers in tissuesfrom colorectal cancer patients. The berries appeared todemethylate tumor suppressor genes (SFRP2 and WIF1)upstream, and protectively modulate the expression ofgenes (b-catenin, E-cadherin) downstream, of the Wntpathway. Gene demethylation was likely a result of inhibi-tion of DNMT1, although other methyltransferase enzymesmight also have been affected. Colon and rectal tissuesshowed differential responses to berry treatment; in parti-cular, rectal adenocarcinomas from patients who weretreated with BRBs for a short period of time (�2 weeks)had reduced responses. This suggests that longer termtreatment of colorectal cancer patients with berries maybe beneficial. It also suggests that BRBs might be moreeffective in patients with rectal tumors if a method were

developed for localized delivery of the berries to rectaltissues.

While the data are supportive of the ability of BRBs todemethylate tumor suppressor genes, the fact that theprimary endpoint; that is, gene demethylation in tissuesfrom all 20 treated patients was, by in large, not positivemeans that the data need to be interpreted with caution.Furthermore, given that methylation is not a validatedendpoint in colorectal cancer chemoprevention, one hasto be circumspect in extrapolating these results. Althoughwe do not recommend berries for cancer treatment, cer-tainly not in lieu of standard methods of therapy, theymight be considered for use in combination with othertherapeutic modalities, such as chemotherapy, immu-notherapy, radiotherapy, and/or surgery. Alternatively, itwould be interesting to determine if the dietary consump-tion of BRBs on a regular basis following therapy forcolorectal cancer reduces the risk for recurrent disease.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank SaraOlivarri for her assistance in accruing patients to the trial atthe University of Texas, San Antonio, and we also thank ComprehensiveCancer Center Tissue Procurement Shared Resource at Ohio State Universityin handling specimens for this study. Finally, we thank all patients for theirparticipation in this trial.

Grant Support

This research was supported by USDA grant 38903-03560, and NCIgrants CA103180 and CA148818.

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 May 11, 2010; revised October 28, 2010; accepted November23, 2010; published OnlineFirst December 1, 2010.

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2011;17:598-610. Published OnlineFirst December 1, 2010.Clin Cancer Res Li-Shu Wang, Mark Arnold, Yi-Wen Huang, et al. I Pilot Study

PhaseColorectal Cancer in Humans by Black Raspberries: A Modulation of Genetic and Epigenetic Biomarkers of

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