Prevention of Colitis-Associated Colorectal Cancer with 8 ......Research Article Prevention of Colitis-Associated Colorectal Cancer with 8-Hydroxydeoxyguanosine Chan Young Ock1, Eun-Hee

Research Article

Prevention of Colitis-Associated Colorectal Cancerwith 8-Hydroxydeoxyguanosine

Chan Young Ock1, Eun-Hee Kim1, Hua Hong1, Kyung Sook Hong1, Young-Min Han1, Ki-Seok Choi1,Ki-Baik Hahm1,2, and Myung-Hee Chung3

AbstractColitis-associated cancer (CAC) is one of clear examples of inflammation–carcinogenesis sequence, by

which the strict control of colitis with potent anti-inflammatory or antioxidative agent offers the chance of

cancer prevention. Supported with the facts that Rac1 binds and activates STAT3, which are significantly

upregulated in inflammatory bowel disease (IBD) as well as CAC, but 8-hydroxydeoxyguanosine (8-oxo-

7,8-dihydrodeoxyguanosine or 8-OHdG) paradoxically can block Rac1 activation and subsequent NADPH

oxidase (NOX) inactivation in various inflammation models, we hypothesized that attenuated Rac1–

STAT3 and COX–NF-kB pathway by exogenous 8-OHdG administration may ameliorate inflammatory

signaling in dextran sodium sulfate (DSS)-induced colitis and can prevent CAC. Before commencing

carcinogenesis model, we checked whether exogenous 8-OHdG can alleviate IBD, for which interleukin

(IL)-10 knockout mice were designed to ingest 5% DSS for 1 week, and 8-OHdG is given through

intraperitoneal route daily. 8-OHdG treatment groups significantly reduced pathologic grade of DSS-

induced colitis as well as various inflammatory mediators such as TNF-a, IL-6, COX-2, and iNOS in a

dose-dependent manner. To document the cancer prevention effects of 8-OHdG, mice were injected

azoxymethane followed by drinking 2.5% DSS for 1 week, after which 8-OHdG–containing diets were

given for 20 weeks. As results, mice that consumed 8-OHdG–containing diet significantly reduced both

tumor incidence and multiplicity. Rac1 activity and phosphorylated STAT3 level were significantly

attenuated in the 8-OHdG–treated group. Significantly decreased levels of malondialdehyde, monocyte

chemotactic protein-1, matrix metalloproteinasess, COX-2, NOX4, and b-catenin nuclear accumulation

were responsible for cancer prevention effects of exogenous 8-OHdG. In conclusion, we clearly showed

cancer-preventive effect of exogenous 8-OHdG against CAC. Cancer Prev Res; 4(9); 1507–21.�2011 AACR.

Introduction

Ulcerative colitis and Crohn’s disease are a form ofchronic inflammatory bowel disease (IBD) that usuallytakes a clinical course of repeated bouts of inflammationand remission. IBD can not only cause problematicsequeale that needed extensive surgical procedures but alsoincreases risk for the development of colitis-associated

colorectal cancer (CAC), also known as colitic cancer(1, 2). In contrast to "adenoma–carcinoma sequence" ofsporadic colorectal cancer (CRC), CAC is one of well-known examples of "inflammation–dysplasia–carcinomasequence" that reversely implies that modulating coloninflammation by efficacious anti-inflammatory treatmentat early phase of disease may more likely inhibit subse-quent carcinogenesis (3, 4). However, it seems to be verydifficult to control the activity of disease continuously witheach or combination of anti-inflammatory agents suchas 5-aminosalicylic acid or other immunosuppressivedrugs such as glucocorticoids and 6-mercaptopurine (5).Although biologics have been reported to be effective ineither induction of remission or treatment of complica-tions as well as the preventive strategy of colitic cancer (4),there remains room for improvement and newer agentshould be investigated.

Pathogenesis of CAC includes complex factors of inflam-mation-initiated or -promotedmutagenesis process. That is,mucosal inflammation is provokedby releasing chemokinesto recruit and activate inflammatory cells (6, 7) and turningthe arachidonic acid cascade on mediation by upregulationof COX-2 and downregulation of 15-hydroprostaglandin

Authors' Affiliations: 1Laboratory of Translational Medicine, GachonUniversity Lee Gil Ya Cancer and Diabetes Institute; 2Department ofGastroenterology, Gachon Graduate School of Medicine, Gil MedicalCenter, Incheon; and 3Department of Pharmacology, Seoul National Uni-versity College of Medicine, Seoul, Republic of Korea

Note: Supplementary data for this article are available at Cancer Preven-tion Research Online (http://cancerprevres.aacrjournals.org/).

Corresponding Author: Ki-Baik Hahm, Laboratory of Translational Med-icine, Gachon University Lee Gil Ya Cancer and Diabetes Institute, andDepartment of Gastroenterology, Gachon Graduate School of Medicine,7-45 Songdo-dong, Yeonsu-gu, Incheon 406-840, Republic of Korea.Phone: 82-32-899-6055; Fax: 82-32-899-6054;E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-11-0161

�2011 American Association for Cancer Research.

CancerPreventionResearch

www.aacrjournals.org 1507

Research. on April 22, 2021. © 2011 American Association for Cancercancerpreventionresearch.aacrjournals.org Downloaded from

Published OnlineFirst July 6, 2011; DOI: 10.1158/1940-6207.CAPR-11-0161

http://cancerpreventionresearch.aacrjournals.org/

dehydrogenase (PGDH; refs. 8, 9). Nuclear factor kappaB(NF-kB) is known to play a main role in these early stepsof inflammation (10, 11), but it can also provoke other

inflammatory cascades such as IL-6/JAK/STAT3 signalingpathways to destroy adjacent tissues by activating matrixmetalloproteinase (MMP) complexes (12, 13) and to turn

A1 7th day

DSS 5%

Normal

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Tap water

+ 8-OHdG 50 mg/kg

daily ip injection of 8-OHdG

DSS 5%

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+ 8-OHdG 100 mg/kg

Sacrifice

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* P < 0.05

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dex

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B

DSS 5%

Figure 1. Efficacy of exogenous 8-OHdG on DSS-induced acute colitis. A, overview of experimental protocol of DSS-induced colitis. Top, 5 groups,normal control, 5% DSS-induced colitis group (DSS), 50 mg/kg, 100 mg/kg, and 150 mg/kg 8-OHdG i.p. injection group. Bottom, the changes of body weight,colon length, and incidence of hematochezia according to groups. Bar represents mean � SD. B, the changes of pathologic index according to groups.Bar represents mean � SD.

Ock et al.

Cancer Prev Res; 4(9) September 2011 Cancer Prevention Research1508




on mutagenesis process even in normal crypts (14, 15).When mutagenesis occurs in intestinal epithelium at thevery first time, APC–b-catenin pathway is disturbed by lossof APC function that normally degrades b-catenin, whicheventually causes cell proliferation and confers resistance toapoptosis. Increased levels of b-cateninmay translocate into

nucleus and act as transcription factor with Tcf/Lef complex,turning normal gland into dysplastic gland (16–18). Fol-lowing these mutagenesis sequences, mutations of K-Rasand p53 as well as microsatellite instability pathway, causedloss of heterozygosity and progressed more up to aggressivenature.

CNormal DSS 50 100 150

8-OHdG (mg/kg)

COX-2

iNOS

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D

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Figure 1. (Continued ) C, Western blot of COX-2, iNOS, and HO-1 according to group. Three representative cases of 6 mice per group were presented.Bottom, the expression levels of the ratio of COX-2 to b-actin, iNOS to b-actin, and HO-1 to b-actin were measured by densitometry. Bar represents mean �SD. D, ELISA levels of mucosal TNF-a and IL-6 according to groups. Bar represents mean � SD.

Prevention of Colitic Cancer with 8-OHdG

www.aacrjournals.org Cancer Prev Res; 4(9) September 2011 1509




Although 8-hydroxydeoxyguanosine (8-oxo-7,8-dihy-drodeoxyguanosine or 8-OHdG) has been acknowledgedas a sensitive marker for oxidative stress, a potent muta-gen or a toxic product, we previously published that 8-OHdG specifically inhibits Rac1/2, the members of smallGTPase family that play a crucial role of generatingreactive oxygen species (ROS) by activating NADPH oxi-dase (NOX) complex, subsequently regulating redox-sen-sitive NF-kB pathway, in not only in vitromacrophage celllines but also animal model of lipopolysaccharide (LPS)-induced septicemia and ovalbumin-sensitized allergicreaction as well as water immersion–restraint stress-induced gastritis (19–22). In addition, Simon andcolleagues (23) showed that Rac1 directly binds andactivates STAT3 and the inhibition of Rac1 by exogenoustreatment with 8-OHdG affects STAT3 signal pathway,which may regulate the genes associated with inflamma-tion–dysplasia–carcinoma sequence.

All of these backgrounds led us to investigate the hypoth-esis that exogenous administration of 8-OHdG might havethe preventive effect on the animal model of colitic cancerbased on its potent anti-inflammatory and antioxidativeactions (19–22). Using dextran sodium sulfate (DSS)-induced colitis and 2-stage colon cancer model first estab-lished by Tanaka and colleagues (24), in which coloncancer is initiated by azoxymethane (AOM) and promotedby DSS ingestion, we studied whether exogenous 8-OHdGis effective in ameliorating colitis, after which long-termadministration of 8-OHdG–containing diet can preventcolitis-associated cancer in interleukin (IL)-10 knockoutmice to prove the potential role of exogenous 8-OHdG as anatural terminator or means of negative feedback ofinflammation in chronic inflammation–carcinogenesissequence.

Materials and Methods

MaterialsAll chemicals and reagents were purchased from Sigma-

Aldrich otherwise specified. Primary antibodies forWesternblotting and immunohistochemistry were purchased asfollows: NOX4, b-actin, b-catenin, and STAT3 from SantaCruz Biotechnology, iNOS from BD Biosciences, HO-1from Enzo Life Sciences, COX-2 from Thermo Scientific,15-PGDH from Cayman Chemical, phosphorylatedSTAT3Tyr705 from Cell Signaling Technology, and F4/80from AbD Serotec.

Breeding of IL-10 knockout miceTwo pairs of breeding IL-10 knockout mice (�/�) and

genotyping primers were purchased from Jackson Labora-tory. Background of IL-10�/� mice was C57BL/6J. Femaleand male IL-10�/� mice were mated, and all offspring ofmice were considered as homozygote after genotyping. Atotal 70 of 5-week-old specific pathogen-free (SPF)IL-10�/� mice were fed a sterilized commercial pellet diet(DH Biolink), given sterile water ad libitum, and housed inan air-conditioned room under a 12-hour light/dark cycle.

Animals were handled in an accredited animal facility inaccordance with Association for Assessment and Accred-itation of Laboratory Animal Care International (AAALACInternational) guidelines under the facility named CACU(The Center of Animal Care and Use) of Gachon UniversityLee Gil Ya Cancer and Diabetes Institute after InstitutionalReview Board approval.

Animal model of DSS-induced colitisA total of 30 mice were divided into 5 groups, 6 mice

per group, respectively; a nontreated control group (nor-mal), 5% DSS (molecular weight ¼ 36,000–50,000; MPBiomedicals) in tap water ingestion for 1 week (DSS), andDSS with daily intraperitoneal (i.p.) injection of 8-OHdG50, 100, and 150 mg/kg groups. Powers of 8-OHdG wasdissolved in PBS and mice of the normal group and theDSS group were injected with PBS i.p. as a negativecontrol. Clinical phenotypes such as hematochezia andrectal prolapse were investigated and charted daily. Therewere no mortalities observed in all groups. After 7 days ofDSS ingestion, all mice were killed and colons wereremoved, opened longitudinally, and rinsed with PBS.The lengths of colon were measured, and isolated tissueswere subjected to a histologic examination and extractionof protein.

Animal model of DSS-induced colitis and 2-stagecolitic cancer

The remaining 40 mice were divided into 4 groups, 10mice per group, respectively; a nontreated control group(normal), AOM 10 mg/kg injection followed by 1 cycle of2.5% DSS ingestion for 1 week after which mice were fedwith normal AIN-76 diet group (AOM þ DSS), and AIN-76–containing 8-OHdG 0.03% diet (8-OHdG 0.03%) or 8-OHdG 0.06% diet group (8-OHdG 0.06%). Mice of thenormal group were injected with PBS i.p. and drank tapwater. A total of 4 mice were dead during 2.5% DSSingestion: 1 in the AOM þ DSS group, 2 in the 8-OHdG0.03% group, and 1 in the 8-OHdG 0.06% group, respec-tively. Daily consumption of 8-OHdG per animal wasestimated approximately 50 mg/kg/d for the 8-OHdG0.03% group, and 100 mg/kg/day for the 8-OHdG0.06% group, respectively. After 20 weeks of diet, all micewere killed and colons were removed, opened longitudin-ally, and rinsed with PBS. The number and size of grosspolypoid tumors were charted. Isolated tissues from distalone-third of colon were subjected to a histologic examina-tion and mucosal scratches of tissues from proximal two-third part were subjected to immediate freezing for theextraction of RNA and protein.

Histopathologic evaluationIsolated tissues for histologic examination were spread

onto a plastic sheet, fixed in 3.7% formalin for 5 hours, andprepared for paraffin tissue slides (25). The paraffin sectionswere stainedwithhematoxylinand eosin (H&E)or saved forimmunohistochemical staining. Pathologic index wasgraded according to criteria as shown in Supplementary

Ock et al.





Table S1. Colitis-associated colon neoplasmswere analyzedmicroscopically and diagnosed as low-grade dysplasia,high-grade dysplasia, and adenocarcinoma. Tumor inci-dence was calculated as the number of tumor-bearing mice

dividedby the total numberof survivalmice,whereas tumornumber was calculated as the total number of tumorsdivided by the number of tumor-bearing mice. Tumor sizewas calculated as the sum of size of tumors divided by the

Figure 2. Establishment of animalmodel of 2-stage colitic cancerinduced by AOM þ DSS. A,overview of experimental protocolof AOM þ DSS model. B, left,gross appearances of therepresentative case of groups.Colon tumors, polypoid masseswere observed mostly in distalone-third part of colon in allgroups, but the number of tumorsin proximal two-third part of colonin 8-OhdG–containing diet groupswas decreased than that in AOMþ DSS group. Right, microscopicphotographs (�50) of therepresentative case of groups.Despite adenocarcinomaformation in the AOM þ DSSgroup, the 8-OHdG 0.03% and8-OHdG 0.06% groups showedhigh-grade dysplasia andadenoma formation. C, tumorincidence and tumor size ofproximal two-third part and distalone-third part were counted. Barrepresents mean � SD.

A Kill

Normal dietNormal

–2 –1 0 20 (wk)

AOM

Normal dietAOM+DSS

AOM10 mg/kg

8-OHdG 0.03%8-OHdG 0.03% DSS2.5%

DSS2.5%

8-OHdG 0.06%8-OHdG 0.06% DSS2.5%

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+ 8-OHdG 0.03%

+ 8-OHdG 0.06%

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size

(m

m)

††

AOM+DSS

§ P < 0.05 (vs. Normal group), † P < 0.05 (vs. AOM+DSS group)






number of tumor-bearing mice, and percentage of mucosalsurface with tumor was grossly estimated. Pathologic dataand slides were blindly reviewed by 2 independent gastro-intestinal specialist (K.B. Hahm and Y.J. Kim) and the firstauthor (C.Y. Ock).

Cytokine array and ELISARaybio mouse cytokine antibody array 3 were purchased

from RayBiotech and carried out strictly according to man-ufacturer’s instruction. Three hundred micrograms of arepresentative case of each group was used for this assay.ELISA kits for mouse TNF-a, mouse IL-6 (R&D Systems),mouse monocyte chemotactic protein-1 (MCP-1; Invitro-

gen), andprostaglandinE2 (PGE2; Cayman)were purchasedandused strictly according to themanufacturer’s instruction.

Reverse transcriptase PCR, Western blots,electrophoretic mobility gel shift assay

This assay was carried out as previously described (19).The sequences of primers are listed in SupplementaryTable S2.

Rac1 activity assayThis assay was carried out as previously described (19).

Rac1 activity was analyzed by determining the amount ofGTP-boundRac1pulleddownby interactingGST–PAK–PBD

A

Figure 3. Changes of inflammatory cytokines, chemokines, and MMPs according to groups. A, mouse cytokine antibody array was carried out with arepresentative case of 8 mice per group. Top, array membranes according to groups. Bottom, the density value of each test sample was measured bydensitometry, and 22 representative cytokines of a total of 62 were graphed.

Ock et al.





(p21 activated kinase–protein binding domain) with Rac1using the Rac1 activation assay (cytoskeleton), according tothe manufacturer’s instructions. Four hundred microgramsof a representative case of each group was used for this assay.

Immunohistochemistry andimmunohistofluorescence

Immunohistochemistry with anti-F4/80 antibody andanti-b-catenin antibody was carried out as previously

C AOM + DSS

MMP-2

N – 8-OHdG (%)

0.03 0.06

MMP-3

MMP-9

GAPDH

MMP-11

§ P < 0.05 (vs. Normal group), † P < 0.05 (vs. AOM + DSS group)


Rela

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Normal – 8-OHdG 0.03% 8-OHdG 0.06% Normal – 8-OHdG 0.03% 8-OHdG 0.06%

BNormal AOM+DSS 8-OHdG 0.03% 8-OHdG 0.06%

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tein

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/

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/80

(+

) ce

lls in

×2

00

hp

f

* P < 0.05

Figure 3. (Continued ) B, Top, immunostaining of F4/80. The microscopic photographs (�200) of the representative case of groups. Bottom, Left, theF4/80-positive cells of groups were counted in �200 hpf. Bar represents mean � SD. Bottom, Right, the tissue levels of MCP-1 according to groupswere measured to confirm cytokine array result. All cases of 8 mice per group were used for this assay. Bar represents mean � SD. C, the mRNAlevels of MMP-2, MMP-3, MMP-9, and MMP-11 were assayed by reverse transcriptase PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)was used as a loading control. Left, the expressions of MMPs of a representative case of 8 were presented. Right, the expression levels of the ratio ofMMPs to GAPDH were measured by densitometry. Bar represents mean � SD.






described (4). For immunohistofluorescence study, thesections were deparaffinized and blocked with 2.5% goatserum for 30 minutes, incubated for 12 hours at 4�C withantibody against NOX4 (1:100 dilution), and finally incu-bated for 1 hour with Alexa Fluor 488 goat anti-rabbitimmunoglobulin G (Invitrogen) in the presence of 40,6-diamidino-2-phenylindole (DAPI; Invitrogen). The slideswere mounted with Prolong Gold anti-fade agent (Invitro-gen) and inspected using a confocal microscope (LSM700;Carl Zeiss).

Total malondialdehyde assayMDA-586 kit was purchased fromOxis International and

used according to the manufacturer’s instructions. To mea-sure total level of malondialdehyde (MDA), isolated tissueswere incubated with PBS (pH 2.0) for 80 minutes at 60�Cbefore carrying out this assay.

Statistical analysisThe data are presented as means � SD. The Tukey test or

the Student t for unpaired results was used to evaluatedifferences between more than 3 groups or between 2groups, respectively. Differences were considered to besignificant for values of P < 0.05.

Result

The ameliorating efficacy of exogenous 8-OHdG onDSS-induced colitis

We used IL-10�/�mice, IBD-pronemice (26), to provokeintense colitis in the SPF environment of our animal facility.As shown in Figure 1A, 5% DSS administration provokedsignificant levels of colitis as reflected with gross findingsthat the body weight of mice and the length of colon weresignificantly decreased accompanied with significantly


A

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*P < 0.05

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Normal8

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sue)

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Free probe N – 0.03% 0.06% Comp

8-OHdG

NF BNF-κB

Figure 4. Changes of COX-2/PGDH/PGE2 and NF-kB pathway according to groups. A, total levels of MDA were measured by thiobarbituric acidreactive substances assay. All cases of 8 mice per group were used for this assay. Bar represents mean � SD. B, immunoblots of COX-2 and PGDH.Top, Western blots were probed with anti-COX-2, anti-PGDH, and anti-b-actin antibodies. Four representative cases of 8 mice per group were presented.Bottom, the expression levels of the ratio of COX-2 to b-actin and PGDH to b-actin were measured by densitometry. Bar represents mean � SD.C, the tissue levels of PGE2 according to groups were assayed by ELISA. Bar represents mean � SD. D, the NF-kB consensus oligomer-binding activities ofnuclear extracts of a representative case of 8 mice per group were assayed by electrophoretic mobility gel shift assay in the presence or absence of an excessof cold probe (Comp).

Ock et al.





increased analbleedings.However, i.p. injectionof 8-OHdGleads to increases in the length of colon as well as main-tenance of body weight in spite of DSS administration (P <0.05).Onpathologic analysis, DSS administrationdistortedglandular formation and recruited inflammatory cells espe-cially in submucosal layer, leading to mucosal destruction,whereas 8-OHdG treatment significantly attenuated thesepathologic indices as shown in Figure 1B. In addition, theinflammatory mediators such as COX-2 and iNOS wereincreased in 5%DSS-induced colitis comparedwith normalcontrol but significantly decreased in the 8-OHdG injectedgroup in a dose-dependent manner. The expression level ofHO-1was increased in theDSSgroup, speculated tobe apartof host defense mechanism, but they were further increasedwith 8-OHdG treatment (Fig. 1C). The levels of mucosalTNF-a and IL-6, crucial cytokines engaged in colitis initia-tion and propagation, were far increased in the DSS-admi-nistered group, but their levels were significantly decreasedwith 8-OHdG treatment (Fig. 1D).

The preventive effect of 8-OHdG–containing diet oncolitic cancer induced by AOM þ DSSIL-10�/� mice injected with AOM followed by 2.5% DSS

in drinking water for 1 week developed colorectal tumors at20th week after DSS ingestion (Fig. 2A). Most tumors wereconcentrated in distal one-third part where we found avariety of stages of cancer progression such as dysplasia,adenoma, and adenocarcinoma in histologic examination(Fig. 2B). Mice fed with 8-OHdG–containing diet 0.03% or0.06% decreased either incidence or size of tumor espe-cially in proximal two-third part of colon (Fig. 2C). Theexact numbers of incidence, number, and size of tumors aswell as percentage of mucosal surface with tumor weresummarized in Supplementary Table S3. In addition tothese antitumorigenic outcomes of 8-OHdG evaluated withthe numbers and sizes of colitic tumor, carcinogenesisprogress was trapped in adenoma in the 8-OhdG–treatedgroup compared with high incidence of adenocarcinoma inthe AOM þ DSS group.

Attenuated footprint of inflammation on coliticcancer administered with 8-OHdG–containing dietTo understand what kinds of cytokine were increased in

colitic tumors of the AOM þ DSS group compared withnormal mice, we carried out mouse cytokine antibodyarray. As shown in Figure 3A, abundant cytokines suchas series of IL, TNF, as well as chemokines such as MCP-1,P-selectin, and RANTES were increased in the representa-tive case of the AOM þ DSS group, of which levels weredecreased in the representative cases of the 8-OHdG 0.03%and 8-OHdG 0.06% groups, respectively (Fig. 3A). Becausethese cytokines and chemokines were essential in chemo-taxis including macrophages, we carried out immunohis-tochemical staining with anti-F4/80 antibody, a well-known marker for macrophage. The number of cellsexpressing F4/80 was increased in the AOM þ DSS groupcompared with the normal group, but chronic consump-tion of 8-OHdG–containing diet decreased the recruit-

ment of F4/80-positive cells. Similar to cytokine array,MCP-1 levels upregulated in the AOM þ DSS group weresignificantly decreased with 8-OHdG in a dose-dependentmanner (Fig. 3B). On the basis of array results, wechecked the expression of MMPs, well known to beengaged in invasion as well as metastasis of colitic cancer(13). Figure 3C showed that the mRNA levels of MMP-2,MMP-3, MMP-9, and MMP-11 were increased in the AOMþ DSS group, but significantly decreased with 8-OHdG0.03% and 8-OHdG 0.06% administration. The mRNAlevels of other series of MMPs such as MMP-7, MMP-13,TIMP-1, TIMP-2, MT-MMP1, and MT-MMP2 were mea-sured, but there were no significant changes betweengroups (data not shown).

Attenuated prostaglandin pathway with 8-OHdG–containing diet

Increased inflammatory foot prints as shown in Figure 3were reflected with increased status of oxidative stress,leading to increased lipid peroxidation in colonic mucosa.Figure 4A showed the results of MDA to reflect lipidperoxidation. As anticipated, AOM þ DSS induced signifi-cant levels of MDA formation, whereas their levels weresignificantly decreased with 8-OHdG administration.Because many have reported that increased expressionand activity of COX-2 led to the activation of arachidonicpathway, mainly, PGE2 upregulation, in inflammatorybowel disease as well as colitic cancer, whereas decreasedexpression and activity of PGDH were found in CRC, wechecked expression levels of COX-2 and PGDH accordingto groups. As expected, the significantly increased expres-sions of COX-2 and decrements in PGDH expressions wereobserved in the AOM þ DSS group (Fig. 4B). The expres-sion levels of COX-2 were significantly reduced in the 8-OHdG 0.03% and 8-OHdG 0.06% groups compared withthe AOM þ DSS group, but there were no changes in theexpression levels of PGDH between those groups. To checkthe changes of COX-2 activity, we measured tissue levels ofPGE2 using homogenates of isolated colon. PGE2 was sig-nificantly increased in the AOM þ DSS group, of whichlevels were significantly attenuated in the 8-OHdG 0.03%and 8-OHdG0.06%groups comparedwith the AOMþDSSgroup (Fig. 4C). As NF-kB signal pathway was intimatelyinvolved in upregulation of COX-2 (27), we checked theDNA binding activity of NF-kB, which was markedly ele-vated in the AOMþDSS group, but its DNA bindings weresignificantly decreased in the 8-OHdG 0.03% and 8-OHdG0.06% groups, in a dose-dependent manner (Fig. 4D).

Attenuated Rac1–STAT3 pathway with resultant NOX4inhibition with 8-OHdG

Previously, we have reported that the biological target ofexogenously administered 8-OHdG would be Rac1, thesmall GTPase, as it also has guanine base that could inter-rupt GTP binding (19, 28). Although best evaluation mightbe to document 3-dimensional analysis of binding site aswell as more sophisticated designs to confirm biologicalinteraction between 8-OHdG and the target site, in the






current study, we hypothesized that if the main target of 8-OHdG for chemopreventive effect of theAOMþDSSmodelis to inhibit Rac1 binding, 8-OHdG could inactivate STAT3,a well-known significant transcription factor in initiationand progression of colitic cancer as well as sporadic CRC

(29, 30). As expected, the activity of Rac1 was far increasedin the AOM þ DSS group, but interestingly, dramaticallydecreased in the 8-OHdG 0.03% and 8-OHdG 0.06%groups, in a dose-dependent manner (Fig. 5A). Becauseactive form of Rac1 can directly bind to STAT3 (21, 23),

A

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0.03% 0.06%

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IB : α-Rac1

IB STAT3

IP : PAK-PBD

IB : α-STAT3

Whole lysateIB : α-Rac1

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GAPDHGAPDH

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ty

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Re

lativ

e d

en

sit

(NO

X4

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ctin

)

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AOM+DSSAOM+DSS* P < 0.05

Figure 5. Changes of Rac1–STAT3 pathway according to groups. A, Rac1 activities of a representative case of 8 mice per group were assayed byimmunoprecipitation (IP) using PAK–PBD beads that specifically precipitate with Rac1–GTP, the active form of Rac1. IB, immunoblotting. Activated Rac1 alsobound with STAT3. The levels of Rac1 and STAT3 in whole lysate were presented as a loading control. B, Top, Western blots were probed with anti-phospho-STAT3 (p-STAT3) and anti-STAT3 antibodies. Four representative cases of 8 mice per group were presented. Bottom, the expression levels of the ratio ofp-STAT3 to STAT3 were measured by densitometry. Bar represents mean� SD. C, changes of NOXs according to groups. The mRNA levels of NOX1, NOX2,and NOX4 were assayed by reverse transcriptase PCR. glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Top, theexpressions of NOXs of a representative case of 8 were presented. Bottom, the expression levels of the ratio of NOX4 to GAPDH were measured bydensitometry. Bar represents mean� SD. D, Top, Western blots were probed with anti-NOX4 and anti-b-actin antibodies. Four representative cases of 8 miceper group were presented. Bottom, the expression levels of the ratio of NOX4 to b-actin were measured by densitometry. Bar represents mean � SD.

Ock et al.





we carried out further immunoprecipitation assay, immu-noprecipitated with PAK–PBD beads, which specificallybinds to active form of Rac1, and then immunoblottedwith a-STAT3 antibody. AOM þ DSS induced significantactivationof STAT3as shown inFigure 5A. In this condition,significantly blocked interaction of Rac1–STAT3 was notedin 8-OhdG–treated group in a dose-dependent manner.Because the binding of Rac1–STAT3 might lead to STAT3activation, we further checked phosphorylation statusof STAT3. The ratio of phopho-STAT3 to STAT3 wasincreased in the AOM þ DSS group but significantlyblocked in the 8-OHdG 0.06% group (Fig. 5B). We alsoassayed the DNA-binding activity of STAT3, which wasincreased in the AOM þ DSS group but decreased in the8-OHdG 0.03% and 8-OHdG 0.06% groups as well asnonlabeled competitor group (data not shown). There-fore, the inhibition of Rac1 activity leading to blockingRac1–STAT3 binding might be the main mechanism of

action of 8-OHdG on chemoprevention of murine modelof colitic cancer.

We previously reported that regulating Rac1 activity by 8-OHdGmight affect not only production of ROS in an acutephase but also upregulation of other subsets of NOX togenerate ROS (19). Moreover, several investigators pub-lished that STAT3 pathway directly upregulated transcrip-tion of NOX1 and NOX4 in vitro (31, 32). Therefore, wehypothesized that the level of NOX would be changedduring carcinogenesis of animal model of colitic cancerinduced by AOMþDSS. First, we checked the mRNA levelsof various subsets of NOX. Figure 5C showed that themRNA level of NOX1 was decreased in the AOM þ DSSgroup compared with the normal group, but there were nochanges in the mRNA levels of NOX2 between groups.NOX3 was not detected in all groups, although Raw264.7,mouse macrophage cells, clearly expresses NOX3 (data notshown). Interestingly, the mRNA levels of NOX4 were far

Figure 5. (Continued ) E, Top,immunohistofluorescence ofNOX4 with DAPI staining wascarried out and a representativecase of the normal, AOM þ DSS,and 8-OHdG 0.06% groups werepresented with H&E staining(�50). The aberrantly increasedexpression of NOX4was observedin the AOM þ DSS group,especially inside adenocarcinomalesions. In the 8-OHdG 0.06%group, much weak expression ofNOX4 was observed inprecancerous lesions. Bottom, thedensities of NOX4 expression (515nm) and DAPI (460 nm) weremeasured by a densitometer.Three independent regions ofeach slide were observed, and 4slides of 8 mice per group wereused for this experiment.

E

*P < 0.05

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increased in the AOM þ DSS group compared with thenormal group, and 8-OHdG–containing diet reduced itsexpression significantly in a dose-dependent manner. Theprotein levels of NOX4 were expressed in a manner similarto that of mRNA levels (Fig. 5D). Moreover, NOX4 washighly expressed inside and around cancer tissues, but thenormal group and the 8-OHdG 0.06% group had lowprofiles of NOX4 expression (Fig. 5E).

Attenuated formation of b-catenin–accumulated cryptwith 8-OHdG–containing diet

Because there was no significant difference in apoptosisbetween group (see Supplementary Fig. S1) and b-catenin–accumulated crypt (BCAC) is the useful marker for dys-plastic lesions or aberrant crypt foci (ACF; ref. 17), theexpression of b-catenin was visualized with immunohisto-

chemical staining. As shown in Figure 6A, the coloniccrypts of the AOM þ DSS group were intensively stainedwith b-catenin, more prominently in nucleus than in thecrypts of the normal group whose patterns of staining werevery weak and noted in the membrane and cytoplasm. Thenumber of BCACs was significantly decreased in the 8-OHdG 0.06% group compared with the AOM þ DSSgroup. Moreover, the whole expression levels of b-cateninwere also significantly decreased in 8-OHdG 0.06%(Fig. 6B).

Discussion

Similar results as shown in our previous issues (4, 25)that the prevention of CACwith either infliximab, biologicscomposed of TNF-a antibody or omeprazole, one of

* P < 0.05

* P < 0.05

A

8-OHdG 8-OHdG

B AOM+DSS

β-Catenin

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ensi

ty(β

-cate

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Num

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r of

BC

AC

6

4

2

0

AOM+DSS

Normal – 8-OHdG 0.03% 8-OHdG 0.06%Figure 6. Assessment ofb-catenin expressions and BCACaccording to groups. A,immunostaining of b-catenin. Left,prominent BCAC was seen in theAOM þ DSS group that showedenhanced expression of b-catenininvolved in the nucleus of aberrantcolonocytes. Right, the number ofBCACs of groups was counted.Bar represents mean � SD. B,immunoblot of b-catenin. Top,Western blots were probed withanti-b-catenin and anti-b-actinantibodies. Four representativecases of 8 mice per group werepresented. Bottom, theexpression levels of the ratio ofb-catenin to b-actin weremeasured by densitometry. Barrepresents mean � SD.

Ock et al.





proton pump inhibitors capable of inhibiting TNF-a, wecan add more evidence showing that the application of apotent antioxidative and anti-inflammatory agent, 8-OHdG in the current study, can be an effective strategyto prevent inflammation-based carcinogenesis. Althoughthe clinical significance of 8-OHdG as biomarker for oxi-dative stress has been well studied and associated withinflammation, mutagenesis, and carcinogenesis because itis easily detected in sera or urine of patients by its mem-brane permeability (33–35) because 8-OHdG might incor-porate into genomic DNA, causing transversion mutation(36, 37), our group has clearly reported that paradoxicallyexogenous 8-OHdG exerted anti-inflammatory and anti-oxidant action in various kinds of inflammation-basedanimal model as well as in vitro experiment (19–22).Because exogenous 8-OHdG is not salvaged into genomicDNA, as it is to be neither degraded into 8-oxo-7,8-dihy-droguanine nor phosphorylated into 8-oxo-7,8-dihydro-guanine triphosphate and it indeed never does seem toincorporate into nucleus compared with deoxyguanosinecontrol (38, 39), 8-OHdG can be a novel drug candidate.The current study might be the first publication provingthat long-term efficacy of exogenous 8-OHdG administra-tion in the prevention of colitis-associated carcinogenesisand inflammatory bowel disease. Although acute inflam-mation that persists for short term mediates host defenseagainst infections, chronic inflammation that lasts forlonger periods can predispose the host to various chronicillnesses, especially cancer, confirming a cross-link betweeninflammation and cancer.Therefore, the strategy to cover the control of both acute

and chronic inflammation seems to be themore efficaciousway for preventing the journey to inflammation-associatedcarcinogenesis, for which the following mechanisms inaddition to relieving action against DSS-induced acutecolitis should be incorporated as the cancer-preventingelixir of exogenous 8-OHdG; first, the regulation of tran-scription factors NF-kB and STAT3, 2 major pathways forinflammation-based carcinogenesis as well as inflamma-tion; second, potent anti-inflammatory action exerted inthe forefront of colitic cancer development; third, theregulation of the most gene products linked to survival,proliferation, and mutagenesis; fourth, relieving oxidativestress; and finally, strict control of Wnt signaling includingb-catenin accumulation and nuclear translocation. Success-fully, 8-OHdG fulfilled all of these qualifications for theprevention of CAC.In detail, Rac1 activity itself is also implicated in cancer

progression, such as cytoskeleton rearrangement, cellmigration, cell growth, and apoptosis (40, 41). In thisstudy, we focused on STAT3 activation, NOX induction,and subsequent ROS generation by active form of Rac1 incolitis-associated cancer model. The activity of Rac1 isknown to be a crucial organizer of NOX complex that isresponsible for producing ROS, all of which in turn lead toa variety of inflammatory cascades (42). Previously, wereported that increased Rac1 activitymay directly organizedNOX2, a housekeeping gene in phagocytes, generating ROS

in a very acute phase of treatment with LPS, but prolongedmaintained ROS increased the expressions of other subsetsof NOX, such as NOX1 and NOX4, in a murine macro-phage cell line (19). Although the close correlation of Rac1with NOX1 and NOX2 has been clearly clarified, therelationship of Rac1 with other subsets of NOX has notyet been proven. Recent report (43) showed that theexpression levels of NOX1 and NOX4 were increased inhuman sample of colon cancers as well as animal model ofcarcinogen-induced colon cancers. In this current study,the expression level of NOX4 was dramatically increasedbut the mRNA level of NOX1 paradoxically seemed to bedecreased by AOM treatment followed by DSS ingestion.Exogenous 8-OHdG significantly blocked NOX4 upregula-tion (Fig. 5). On the basis of a previous report by Maneaand colleagues (31) that the transcription of NOX4 wasincreased directly by STAT3 pathway in smooth musclecells and decreased Rac1 activity by 8-OHdG furthermitigated STAT3 pathway by us (19), it might be possiblethat activated Rac1–STAT3 pathway directly induces thetranscription of NOX4, generating more potent ROS,leading to activation of redox-sensitive transcription fac-tor, NF-kB, and activating prostaglandin pathways(Fig. 4), all of which were known to be critically andprincipally involved in "inflammation–dysplasia–carci-noma sequence." Conclusively, the cancer-preventiveoutcome of exogenous 8-OHdG also can be explainedwith blocking this "inflammation–dysplasia–carcinoma"sequence. Because the limited success of current treat-ments of most advanced common malignancies high-lights the importance of cancer prevention, moreefforts should be paid in cancer arising from inflamma-tion basis as seen in our study.

Next focus relating to 8-OHdG and cancer preventionshould be "COX." Between 2 forms of COX, COX-1 con-stitutively expressed in normal tissues and served as a"housekeeper" of mucosal integrity and COX-2 served asan immediate early response gene that is highly inducibleby neoplastic and inflammatory stimuli. Increased levels ofCOX-2 mRNA and proteins are found in premalignantlesions and malignant tissues of colitic cancer, making itan attractive therapeutic target (44). Chemoprevention ofCRC is already possible with celecoxib, although it is stillnot the ultimate drug of choice, especially because of thecardiovascular risk associated with COX-2 inhibitors, bywhich agent targeted for prevention of colon cancer shouldexert inhibition of COX-2 but maintenance of PGDH,tumor suppressor gene related to COX-2 (45, 46). Exogen-ous 8-OHdGwas very efficient in either suppressing COX-2expression or decreasing mucosal levels of PGE2 (Fig. 4Band C). Although the levels of PGDH were significantlydecreased in AOM þ DSS tumor-promoting conditioncompared with normal, clear preservation of PGDH wasnot seen in 8-OHdG treatment in spite of strong suppres-sion of COX-2.

As much as colonocytes, cancer-associated fibroblasts(CAF) represent the predominant cell type of the neoplasticstroma of colon cancer, although their exact biology and






functional specificity for cancer pathogenesis remainunclear. Mueller and colleagues (47) showed that primaryCAFs from colorectal liver metastases express severalinflammatory, tumor-enhancing factors, including IL-6and MCP-1, both molecules intensely induced by TNF-a.Although not documented about detailed nature of expres-sing cell in this study, 8-OHdG imposed significantdecreases in MCP-1 levels as well as inhibiting macrophageinfiltration (Fig. 3A and B). In addition to MCP-1, severalgrowth and angiogenic factors as well as matrix-degradingproteases, such as MMP-2, MMP-3, MMP-9, and MMP-11,are involved in general tumorigenesis, invasion, andmetas-tasis as well as colon cancer. MMPs can be released inresponse to proinflammatory cytokines such as TNF-a andIL-1b and play an important role in the process of tissueremodeling and destruction, especially MMP-9 is the mostabundantly expressed protease in inflamed tissues of colon.Also, MMPs, including MMP-2 and MMP-9, are expressedin human and animal cancers (48, 49). Exogenous 8-OHdG imposed significant inhibition of these tumorigenicactions of MMPs on colitic cancer. Finally, inactivatingaction of b-catenin accumulation was imposed with 8-OHdG. ACFs are now frequently used as effective surrogatebiomarkers of CRCs, but the real preneoplastic or precan-cerous nature of ACFs in rodents and human still remainsinconclusive (50). Instead of these obscuring implicationsof ACFs, early appearing BCACs have been described in enface preparations of colonic mucosa and are suggested to bepremalignant in much higher fidelity than in ACFs. His-tologic observation showed that BCACs exhibit cellulardysplasia, higher cellular proliferation, and more likelyto progress to malignant transformation and this is whyBCAC is acknowledged as an intermediate biomarker forcolon carcinogenesis with higher fidelity (17). Therefore,

significant decreases in BCAC with 8-OHdG as shown inFigure 6A might be the one of the kernel evidence support-ing cancer-preventing action of 8-OHdG. In addition, wefound that there was no difference in apoptosis as evi-denced by terminal deoxynucleotidyl transferase–mediateddUTP nick end labeling (TUNEL) staining (see Supplemen-tary Fig. S1).

In summary, the significance of our study is that we weresuccessful in not only documenting the cancer-preventiveeffect of CAC with exogenous 8-OHdG, formerly recog-nized as the product of oxidative stress, but also elucidatingthe core pharmacologic actions of 8-OHdG, paradoxicallyantioxidative and anti-inflammatory (19–22) actions suchas the regulation of Rac1–STAT3, NOX4, b-catenin, MMP,and COX-2, key mediators reported to be principallyengaged in colon carcinogenesis.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgment

We sincerely thank Dr. Shinichi Kato (Kyoto Pharmaceutical Univ.) whocommented and shared information on the expressions of NOX in animalmodel.

Grant Support

This study was supported by a National Research Foundation grant of theKorean government (2010-0002052).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 30, 2011; revised May 23, 2011; accepted June 7, 2011;published OnlineFirst July 6, 2011.

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2011;4:1507-1521. Published OnlineFirst July 6, 2011.Cancer Prev Res Chan Young Ock, Eun-Hee Kim, Hua Hong, et al. 8-HydroxydeoxyguanosinePrevention of Colitis-Associated Colorectal Cancer with

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Prevention of Colitis-Associated Colorectal Cancer with 8 ......Research Article Prevention of Colitis-Associated Colorectal Cancer with 8-Hydroxydeoxyguanosine Chan Young Ock1, Eun-Hee