prevention of helicobacter pylori induced gastric cancers ...€¦ · nal axis into strips of 5 mm...

Research ArticleSee related commentary by Schneider and Peek, p. 253

Prevention of Helicobacter pylori–Induced Gastric Cancersin Gerbils by a DNA Demethylating Agent

Tohru Niwa1, Takeshi Toyoda2, Tetsuya Tsukamoto3, Akiko Mori1, Masae Tatematsu4, andToshikazu Ushijima1

AbstractSuppression of aberrant DNA methylation is a novel approach to cancer prevention, but, so far, the

efficacy of the strategy has not been evaluated in cancers associated with chronic inflammation. Gastric

cancers induced by Helicobacter pylori infection are known to involve aberrant DNA methylation and

associated with severe chronic inflammation in their early stages. Here, we aimed to clarify whether

suppression of aberrant DNAmethylation can preventH. pylori–induced gastric cancers using a Mongolian

gerbil model. Administration of a DNA demethylating agent, 5-aza-20-deoxycytidine (5-aza-dC), to gerbils

(0.125 mg/kg for 50–55 weeks) decreased the incidence of gastric cancers induced by H. pylori infection

andN-methyl-N-nitrosourea (MNU) treatment from 55.2% to 23.3% (P < 0.05). In gastric epithelial cells,

DNA methylation levels of six CpG islands (HE6, HG2, SB1, SB5, SF12, and SH6) decreased to 46% to

68% (P < 0.05) of gerbils without 5-aza-dC treatment. Also, the global DNA methylation level decreased

from 83.0% � 4.5% to 80.3% � 4.4% (mean � SD) by 5-aza-dC treatment (P < 0.05). By 5-aza-dC

treatment, Il1b and Nos2 were downregulated (42% and 58% of gerbils without, respectively) but Tnf was

upregulated (187%), suggesting that 5-aza-dC treatment induced dysregulation of inflammatory responses.

No obvious adverse effect of 5-aza-dC treatment was observed, besides testicular atrophy. These results

showed that 5-aza-dC treatment can preventH. pylori–induced gastric cancers and suggested that removal of

induced DNA methylation and/or suppression of DNA methylation induction can become a target for

prevention of chronic inflammation–associated cancers. Cancer Prev Res; 6(4); 263–70. �2013 AACR.

IntroductionDNA methylation is an epigenetic mechanism for gene

regulation. Methylation of promoter CpG islands (CGIs)consistently suppresses expression of their downstreamgenes (1), and physiologic methylation of retrotransposonsis involved in their transcriptional repression (2). In can-cers, tumor suppressor genes are frequently inactivated byaberrant methylation of their promoter CGIs (3, 4). Suchaberrant methylation is present not only in cancers but alsoin noncancerous tissues exposed to chronic inflammation,such as colonic mucosae with ulcerative colitis, liver tissuesexposed to hepatitis, and gastricmucosae exposed to chron-ic gastritis (5–10). In the case of the stomach, Helicobacter

pylori infection is known to induce severe chronic inflam-mation (11–13) and aberrant methylation in gastric epi-thelial cells (GEC; ref. 14). Accumulation levels of aberrantmethylation in gastric mucosae correlate with risk of gastriccancers (8–10).

Suppression of aberrant methylation is considered asone of the novel targets for cancer chemoprevention (15,16). Traditionally, chemoprevention has used substancesbased on 2 strategies: the anti-initiation and anti-promo-tion/progression strategies (17–19). In the former strategy,blockage of activity of carcinogens that induce genetic orepigenetic alterations and enhancement of repair systemshave been targeted. In the latter strategy, suppression ofproliferation of initiated cells and induction of theirapoptosis have been targeted. However, neither of thesestrategies targeted removal of genetic or epigenetic altera-tions accumulated in the cells, which can be achieved byDNA demethylating agents, such as 5-aza-20-deoxycytidine(5-aza-dC; refs. 20, 21).

The usefulness of DNA demethylating agents in cancerchemoprevention has been shown in several animal mod-els, including intestinal tumors in Apcmin/þ mice (22, 23),prostate tumors in transgenic mice harboring probasinpromoter-driven SV40 antigen (24), 4-(methyl-nitro-samino)-1-(3-pyridyl)-1-butanone–induced mouse lungtumors (25), and 4-nitroquinoline 1-oxide–inducedmouse

Authors' Affiliations: 1Division of Epigenomics, National Cancer CenterResearch Institute; 2Division of Pathology, National Institute of HealthSciences, Tokyo; 3Department of Diagnostic Pathology, Fujita HealthUniversity School ofMedicine, Kutsukake-cho, Toyoake, Aichi; and 4JapanBioassay Research Center, Hadano, Kanagawa, Japan

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

Corresponding Author: Toshikazu Ushijima, National Cancer CenterResearch Institute, 5-1-1-Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. Phone:81-3-3547-5240; Fax: 81-3-5565-1753; E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-12-0369

�2013 American Association for Cancer Research.

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oral tumors (26). Genetic suppression of a maintenanceDNA methyltransferase (Dnmt1) also suppressed tumordevelopment in some of these models (22, 27, 28). How-ever, so far, the efficacy of suppression of aberrant DNAmethylation was not evaluated in chronic inflammation–associated cancers, in which aberrant DNA methylation isheavily involved (29, 30). From this aspect, gastric cancersinduced by H. pylori infection of Mongolian gerbils (Mer-iones unguiculatus) have several advantages. In gerbils, H.pylori infection induces severe chronic inflammation, as inhumans, and promotes gastric cancers initiated byN-meth-yl-N-nitrosourea (MNU; refs. 31, 32). Also, 10 CGIs havealready been established as markers that can be methylatedby H. pylori infection, and a critical role of inflammationtriggered byH. pylori infection, not a direct effect ofH. pylori,inmethylation induction has been shown (14). In contrast,few markers for methylation induction have been isolatedin H. pylori- or Helicobacter felis–infected mice, except Tff2promoter (33).

In this study, using the gerbil model, we aimed to clarifywhether 5-aza-dC treatment can prevent chronic inflam-mation–induced gastric cancers and evaluate its effects onmethylation induction and inflammation triggered by H.pylori infection.

Materials and MethodsAnimals and sample preparation

MaleMongolian gerbils (MGS/Sea) were purchased fromKyudo anddivided into 10 groups (G1–6 in Fig. 1A andG7–10 in Fig. 2A). Gerbils were inoculated withH. pylori [�4�108 colony-forming units (CFU)/gerbil, ATCC 43504;American Type Culture Collection] at 5 weeks of age(34). In a carcinogenicity experiment, 10 ppm of MNU(Sigma-Aldrich) was given in drinking water to gerbils. 5-Aza-dC (125 mg/kg body weight in sterilized PBS; Sigma-Aldrich) was administered to gerbils intraperitoneally twiceper week. The dose was selected from the 3 doses (125, 250,and 500 mg/kg) tested in a preliminary experiment for lackof toxicity. Timing anddurationof the treatments are shownin Figs. 1A and 2A.

The stomach was resected and cut along the greatercurvature. In a carcinogenicity experiment (G1–6), theantral region was fixed in formalin for histologic analysis.From the body region, GECs were isolated by the glandisolation technique (35) and stored in 100% ethanol at�80�C until DNA extraction. The testes, small intestine,liver, and kidneys were resected, and half parts were fixed informalin. The other halves were snap-frozen for DNAextraction. In an experiment to induce H. pylori–triggeredgastritis (G7–10), the antral region was cut into 2 pieces—one was snap-frozen for RNA extraction and the other halfwas fixed in formalin.

In both experiments, samples (tissues or GECs) weredigested with proteinase K, and gDNA was extracted by thestandard phenol/chloroformmethod. RNA of gastric tissuewas isolated using ISOGEN (Nippon Gene). Whole bloodwas obtained from the inferior vena cava, and gDNA was

extracted by aQuickGene DNAWhole Blood Kit (Fujifilm).All the animal experiments were approved by the Commit-tee for Ethics in Animal Experimentation at the NationalCancer Center.

Histological analysisFormalin-fixed tissues were sliced along the longitudi-

nal axis into strips of 5 mm width and embedded inparaffin. Sections of 3 mm thickness were prepared andstained with hematoxylin and eosin. Neoplastic lesions inthe stomach were diagnosed as previously described (36).The size of a gastric cancer was determined as the max-imum diameter of the gastric cancer in the neighboringsections. The degree of infiltration of mononuclear andpolymorphonuclear cells was graded on a 4-point scale(0–3; 0, no or faint; 1, mild; 2, moderate; 3, marked) asdescribed (37).

Luminometric methylation assaygDNA from whole blood was amplified by an illustra

GenomiPhi V2DNAAmplification Kit (GEHealthcare) andused as fully unmethylated DNA. The unmethylated DNAwas methylated by SssI methylase (New England Biolabs)andused as fullymethylatedDNA.A series of standardDNAwas prepared by serial mixing of the unmethylated DNAand the methylated DNA.

Luminometric methylation assay (LUMA) was con-ducted as described (38) with slight modifications. Brief-ly, 3 mg of DNA was digested with 2 pairs of restrictionenzymes (HpaII and EcoRI or MspI and EcoRI) in inde-pendent tubes (all restriction enzymes were purchasedfrom Toyobo). The DNA was purified with a DNA Clean& Concentrator Kit (Zymo Research) and eluted in 40 mLof an annealing buffer (2 mmol/L magnesium acetateand 20 mmol/L Tris-acetate, pH7.6). Using the PSQ 96Pyrosequencing System (Qiagen), 50-CG overhang pro-duced by HpaII (or MspI) and 50-AATT overhang pro-duced by EcoRI was sequenced, and an HpaII/EcoRI (orMspI/EcoRI) signal ratio was determined. An HpaII/MspIvalue was obtained as (HpaII/EcoRI)/(MspI/EcoRI) ineach sample. The HpaII/MspI value was comparedwith those of the standard DNA series, and the globalmethylation level (GML), which is equivalent to thepercentage of methylated DNA in the standard DNA,was determined.

Quantitative methylation-specific PCRDNA digested with BamHI was treated with sodium

bisulfite as described (39) and used as a template forreal-time PCR.With primer sets specific tomethylated CGIs(HE6, HG2, SA9, SB1, SB5, SC3, SD2, SE3, SF12, and SH6;Supplementary Fig. S1) and a B2 repeat sequence, real-timePCR was carried out as described (14). On the basis of thecopy number of sequences measured by real-time PCR,the methylation level was calculated as a percentage of themethylated reference (PMR),whichwas obtained as [(num-ber of methylated fragments of a target CGI in sample)/(number of the B2 repeat in sample)]/[(number of

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Cancer Prev Res; 6(4) April 2013 Cancer Prevention Research264



methylated fragments of a target CGI in SssI-treated DNA)/(number of the B2 repeat in SssI-treated DNA)] � 100.

Gene expression analysesThe number of cDNAmolecules was quantified by quan-

titative reverse transcriptase-PCR (qRT-PCR) using gene-specific primers (Il1b,Nos2, and Tnf) as described (14). Thenumber of cDNAmolecules of a target genewas normalizedto that of Gapdh.

Genomic PCR and sequencingA forward primer (50-AGATTCCTTGATGCCTGGGTGTC-

30) was designed in a region of the mouse Tnf promoterhighly conserved with the human corresponding region.A reverse primer (50-AGATTCCTTGATGCCTGGGTGTC-30) was designed on the gerbil Tnf mRNA sequence(AB177841). The gerbil Tnf promoter was amplified usingthese primers, and the PCR product was directly sequencedwith the same primers. The obtained sequence was

Figure 1. Suppression of DNA methylation by 5-aza-dC treatment. A, design of the carcinogenicity experiment. The number of animals is shownin parentheses. B, global DNA methylation levels analyzed by LUMA in GECs. C, methylation levels of 10 CGIs analyzed by qMSP in GECs.Both preexisting methylation and induction of aberrant methylation were suppressed by 5-aza-dC treatment. Bold horizontal bar, the average.�, P < 0.05; ��, P < 0.01.

Cancer Prevention by a DNA Demethylating Agent

www.aacrjournals.org Cancer Prev Res; 6(4) April 2013 265



registered in GenBank (AB762083.1). A CGI was searchedby a EMBOSS CpG report program (40).

Statistical analysisStatistical analyses were conducted by SPSS 13.0J (SPSS

Japan Inc.). To evaluate significant difference between 2

independent groups of sample data, a Mann–Whitney Utest was used. The difference of the proportion between 2groups was evaluated by Fisher exact test.

ResultsSuppression of H. pylori/MNU-induced gastriccarcinogenesis by 5-aza-dC

To evaluate cancer prevention effects of 5-aza-dC, a carci-nogenicity experiment was carried out (Fig. 1A). Among thegerbils with MNU treatment and H. pylori infection (G5 andG6), 5-aza-dC treatment decreased incidence of gastric can-cers from 55.2% (G5) to 23.3% (G6, P < 0.05; Table 1). Theincidence in G6 was similar to that in MNU-treated gerbilswithout H. pylori infection (G2, 20.7%). There were no sig-nificant differences in the tumor multiplicity and size amongthe groups. These results clearly showed that 5-aza-dC treat-ment suppressed H. pylori/MNU-induced gastric carcinogen-esis in gerbils and suggested that it might have completelyabrogated the cancer promotion effects ofH. pylori infection.Among the MNU-treated gerbils without H. pylori infection(G2 and G3), 5-aza-dC treatment tended to decrease inci-dence of gastric cancers but it was not statistically significant.

Regardless of 5-aza-dC treatment, gerbils with MNUtreatment (G2, G3, G5, and G6) showed low body weightthan the gerbils without MNU treatment (G1 and G4),showing that the body weight loss was dependent uponMNU treatment, not upon 5-aza-dC treatment (Supple-mentary Fig. S2). Survival rates started to decrease from25 weeks of age, and the decrease was dependent uponMNU treatment, not upon 5-aza-dC treatment (Supple-mentary Fig. S3). This showed that the dose of 5-aza-dCused in this study (125 mg/kg body weight) had no obviouseffects on body weight and survival of gerbils.

Reduction of DNA methylation levels in GECs by5-aza-dC

To confirm the demethylating effects of 5-aza-dC in vivo,methylation analyses were conducted in GECs. First, theglobal DNA methylation level was measured by LUMA, inwhich global CCGG methylation was measured by using

Table 1. Suppression of gastric cancers by 5-aza-dC

Adenocarcinoma

GroupEffectivenumber

Well-differ-entiated

Poorly differ-entiated

Multiplicity(mean � SD)

Diameter(mean � SD),mm

Incidence(%) Adenoma Sarcoma

G1. HP(�) þ DW 8 0 0 0 — 0/8 (0) 0 0G2. HP(�) þ MNU þ PBS 29 3 3 1.0 � 0 6.3 � 1.6 6/29 (20.7) 1 2G3. HP(�) þ MNU þ 5-aza-dC 33 0 2 1.0 � 0 3.3 � 3.3 2/33 (6.1) 1 0G4. HP(þ) þ DW 8 0 0 0 — 0/8 (0) 0 0G5. HP(þ) þ MNU þ PBS 29 15 1 1.1 � 0.3 5.7 � 2.7 16/29 (55.2)a 0 2G6. HP(þ) þ MNU þ 5-aza-dC 30 4 3 1.0 � 0 4.8 � 1.7 7/30 (23.3)b 0 2

NOTE: 5-Aza-dC treatment decreased incidence of gastric cancers (adenocarcinomas) from 55.2% (G5) to 23.3% (G6).aP < 0.05 compared with G3.bP < 0.05 compared with G5.

Figure 2. Dysregulation of inflammatory responses by 5-aza-dCtreatment. A, design of the experiment for analysis of gastritis. Thenumber of animals is shown in parentheses. B, expression levels ofinflammation-related genes in gastric tissues containing submucosallayers. Bold horizontal bar indicates the average. C, infiltration score ofpolymorphonuclear cells and mononuclear cells in the stomach.Although the degree of H. pylori–induced infiltration ofpolymorphonuclear andmononuclear cells was not affected by 5-aza-dCtreatment, expression of Il1b and Nos2 was downregulated and that ofTnf was upregulated. Mean and SD are shown. ��, P < 0.01.

Niwa et al.




a combination of methylation-sensitive and -insensitiverestriction enzymes and pyrosequencing. Among thegerbils with MNU treatment and H. pylori infection (G5and G6), 5-aza-dC treatment decreased the global methyl-ation level from 83.0% � 4.5% (mean � SD)% (G5) to80.3% � 4.4% (G6, P < 0.05; Fig. 1B). Among the MNU-treated gerbils without H. pylori infection (G2 and G3), themethylation level was decreased by 5-aza-dC treatment(from 84.4% � 2.3% in G2 to 82.2% � 2.4% in G3, P <0.05). No significant influence of H. pylori infection orMNU treatment was observed. These results indicated that5-aza-dC worked as a DNA demethylating agent in vivo anddecreased global methylation levels in GECs.Next, methylation of 10 CGIs, where H. pylori infection

was previously shown to induce aberrant methylation(Supplementary Fig. S1; refs. 14, 41), was analyzedby quan-titative methylation-specific PCR (qMSP). 5-Aza-dC treat-ment reduced methylation levels in G6 to 46% to 80%of those in G5 for 6 CGIs (HE6, HG2, SB1, SB5, SF12, andSH6; P < 0.05; Fig. 1C). These results showed that 5-aza-dCtreatment suppressed methylation induction by H. pyloriinfection and MNU treatment in GECs. The methylationlevels in G5 were higher than those in G4 whereas thosein G2 were not elevated compared with those in G1,indicating that MNU treatment had an augmenting effecton H. pylori–induced aberrant methylation.

Dysregulation of inflammation-related genes by5-aza-dCAmong 10 inflammation-related genes whose expression

was examined in the stomach, expression of 3 genes (Il1b,Nos2, and Tnf) has been shown to be associated withinduction of methylation in GECs (14, 41). Therefore, weexamined whether 5-aza-dC treatment affected expressionof these 3 genes in the stomach afterH. pylori infection usingH. pylori–infected and -uninfected gerbils without MNUtreatment (Fig. 2A). In theH. pylori–infected gerbils with 5-aza-dC treatment (G10), expression levels of Il1b and Nos2decreased to 42% and 58%, respectively (P < 0.01, respec-tively), of those in H. pylori–infected gerbils without 5-aza-dC treatment (G9; Fig. 2B). In contrast, Tnfwas upregulatedto 187%ofG9 (P < 0.01). These results indicated that 5-aza-dC treatment caused up- and downregulation, namelydysregulation, of inflammation-related genes. As there wasa possibility that upregulation of Tnf was due to demeth-ylation of its promoter CGI, we sequenced its promoterregion but found that there was no CGI (SupplementaryFig. S4). Because methylation of only promoters with CGIscan consistently silence their downstream genes (42), weconsidered that the upregulation of Tnf was unlikely to bedue to demethylation by 5-aza-dC.We also analyzed infiltration of inflammatory cells

in the stomach. In gerbils with H. pylori infection (G9 andG10), 5-aza-dC treatment did not affect infiltration ofmononuclear cells and polymorphonuclear cells (Fig.2C). In gerbils without H. pylori infection (G7 and G8),there was little mononuclear cell and polymorphonuclearcell infiltration, and no effect of 5-aza-dC treatment was

observed. These results showed that 5-aza-dC treatment didnot affect infiltration of inflammatory cells.

Extra-gastric effects of 5-aza-dC treatmentTo evaluate possible adverse effects of 5-aza-dC treat-

ment, we first conducted macroscopic analysis. Althoughmost organs did not show any abnormality, the testes wereprominently atrophic in 5-aza-dC–treated gerbils [0.47 �0.05 (mean� SD) g/2 testes inG3 and0.50�0.05 g/2 testesin G6] compared with gerbils in other groups (1.10 � 0.11g/2 testes in G2 and 1.09 � 0.13 g/2 testes in G5).

We then conducted analysis of histopathologic ab-normalities and global DNA methylation levels in thetestes, small intestine, liver, and kidneys. In the testes of5-aza-dC–treated gerbils, the numbers of spermatozoa andspermatids were markedly decreased regardless of H. pylori

Figure 3. Adverse effects of 5-aza-dC treatment in extra-gastric tissues.A, tissue sections of the testes and small intestine. Bottom, is amagnifiedview of the region in the black rectangle in the top. Numbers ofspermatozoa and spermatids were markedly decreased in the testes of5-aza-dC–treated gerbil. B, global methylation levels in the testes andsmall intestine. Five gerbils in each group were randomly selected, andthe methylation levels were measured by LUMA. Mean and SD areshown. �, P < 0.05. By 5-aza-dC-treatment, the global methylation leveldid not decrease in the testes but did in the small intestine.





infection status (Fig. 3A). Despite the presence of hypos-permatogenesis, there was no significant decrease of theglobal methylation level (Fig. 3B). In contrast, in the smallintestine, the globalmethylation levelwas reducedby10.4%and 5.6% (G3 and G6, respectively Fig. 3B). However, nohistologic changes comparedwith the untreated gerbilswereobserved (Fig. 3A). As for the liver andkidneys, therewerenohistologic abnormalities or reduction of global methylationlevels in G3 and G6 (Supplementary Fig. S5).

DiscussionOur study using a gerbil model showed that 5-aza-dC

treatment suppressed H. pylori/MNU-induced gastric can-cers. This study showed for the first time that chemopre-vention using a DNA demethylating agent is effective forchronic inflammation–associated cancers. As chronicinflammation contributes to about 25% of all cancer cases(43), and aberrant DNAmethylation is frequently observedin tissues exposed to chronic inflammation (29), suppres-sion of aberrant methylation might become an effectivepreventive approach for these types of cancers. This studyalso showed that induction of aberrant methylation is animportantmechanism for gastric carcinogenesis byH. pyloriinfection.

As mechanisms of suppression of gastric cancers by 5-aza-dC, at least 2 modes of action were present. The firstone was the DNA demethylating effect. 5-Aza-dC treat-ment decreased global methylation level in GECs andsuppressed hypermethylation of CGIs. These results sug-gested that 5-aza-dC was capable of removing aberrantDNA methylation induced by H. pylori infection andthereby prevented cancer development. The second onewas the effect on inflammation. It was previously shownthat inflammation triggered byH. pylori infection is pivotalfor aberrant methylation induction, and expression ofinflammation-related genes (Il1b, Nos2, and Tnf) in thestomach is associated with the induction (14, 41). Thepresent study showed that 5-aza-dC treatment dysregu-lated expression of these genes. Therefore, there is a pos-sibility that altered balances among the related cytokinesmight have led to the reduced methylation induction.

Recently, in the stomach of hypergastrinemic INS-GAS mice, inhibition of H. pylori (H. felis)-inducedglobal demethylation by folic acid supplementationwas reported to suppress gastric dysplasia (44). Thedata are seemingly discordant with our data showinga cancer preventive effect by DNA demethylation. How-ever, in the INS-GAS mice, global hypomethylationby H. felis was evident, suggesting that the demethyl-ation plays important roles in the carcinogenesis.Hypermethylation by folic acid might exert the cancerpreventive effect via suppression of global demethyla-tion. In contrast, in our gerbil study, hypermethylationof CGIs by H. pylori infection, rather than global de-methylation, was evident, suggesting that hypermethy-lation of CGIs rather than global demethylation wasthe major mechanism for the carcinogenesis. Thus,

demethylation by 5-aza-dC was considered to haveexerted the preventive effect via suppression of hyper-methylation of CGIs.

No obvious adverse effect of 5-aza-dC treatment wasobserved besides hypospermatogenesis in the testes.Hypospermatogenesis due to 5-aza-dC treatment wasreported in mice (24). As global hypomethylation wasnot detected in the testes of the 5-aza-dC–treated gerbils,the effect was speculated to be independent of its DNAdemethylating activity. However, we cannot exclude thepossibility that decrease of methylation was not detecteddue to immediate elimination of spermatozoa/sperma-tids with decreased DNA methylation. Regardless of themechanism, the presence of this adverse effect precludes5-aza-dC as a chemoprevention agent for the generalpopulation. However, chemoprevention by a DNA de-methylating agent itself still might become a promisingstrategy if a DNA demethylating agent without suchtoxicity is developed.

Demethylating effects by 5-aza-dC were observed in thestomach and the small intestine, but not in the liver andthe kidneys. Specific global demethylation in the stomachand intestine was also observed in the female Apcmin/þ

mice administered a demethylating agent, zebularine(23). Turnover of epithelial cells in these tissues isknown to be very rapid, being 3 to 4 days in mice (45,46). As demethylating effects of 5-aza-dC and zebularineare exerted after their incorporation into gDNA andDNA replication, rapid cell turnover in the stomachand intestine could explain the organ-specific globaldemethylation.

Individuals with a severe epigenetic field defect, incontrast with the general population, can be consideredas a target population for cancer prevention using 5-aza-dC after careful balancing of its preventive and adverseeffects. In the case of gastric cancers, eradication of H.pylori is the primary strategy for prevention (47), but theincidence of gastric cancers remains high, even after H.pylori eradication, especially in persons with intestinalmetaplasia and gastric atrophy (48). Notably, aberrantmethylation in gastric mucosae decreases by H. pylorieradication, but it does not disappear completely(14, 49). The level of the remaining methylation reflectsthe risk of gastric cancers (8, 9). As DNA demethylatingagents are likely to remove such accumulated methyla-tion and suppress gastric cancers development, theseindividuals with a severe epigenetic field defect maybenefit from epigenetic chemoprevention.

In summary, treatment with 5-aza-dC effectively pre-vented gastric cancers induced by H. pylori infection ingerbils, suppressedDNAmethylation inGECs, and induceddysregulation of inflammation. Chemoprevention with aDNA demethylating agent is expected to become an effec-tive strategy for prevention of chronic inflammation–asso-ciated cancers.

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

Niwa et al.




Authors' ContributionsConception and design: T. Niwa, M. Tatematsu, T. UshijimaDevelopment of methodology: T. Niwa, T. TsukamotoAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): T. Niwa, T. ToyodaAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): T. Niwa, T. ToyodaWriting, review, and/or revision of themanuscript: T. Niwa, T. UshijimaAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): A. MoriStudy supervision: M. Tatematsu

AcknowledgmentsThe authors thankMs. R. Shimada (QiagenK.K.) for her technical support

of LUMA.

Grant SupportThis study was supported by Grants-in-Aid for The Third-term Com-

prehensive Cancer Control Strategy from the Ministry of Health, Labour,and Welfare, Japan (T. Ushijima); Grants-in-Aid for Young Scientists(#23701114) from the Japan Society for the Promotion of Science(T. Niwa); andGrants-in-Aid from the Foundation of Promotion of CancerResearch, Japan (T. Niwa).

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 August 29, 2012; revisedDecember 19, 2012; accepted January 8,2013; published online April 4, 2013.

References1. Taby R, Issa JP. Cancer epigenetics. CA Cancer J Clin 2010;60:

376–92.2. Howard G, Eiges R, Gaudet F, Jaenisch R, Eden A. Activation and

transposition of endogenous retroviral elements in hypomethylationinduced tumors in mice. Oncogene 2008;27:404–8.

3. Esteller M. Epigenetics in cancer. N Engl J Med 2008;358:1148–59.4. Jones PA, Baylin SB. The epigenomics of cancer. Cell 2007;128:

683–92.5. Hsieh CJ, Klump B, Holzmann K, Borchard F, Gregor M, Porschen R.

Hypermethylation of the p16INK4a promoter in colectomy specimensof patients with long-standing and extensive ulcerative colitis. CancerRes 1998;58:3942–5.

6. Issa JP, Ahuja N, Toyota M, Bronner MP, Brentnall TA. Acceleratedage-related CpG island methylation in ulcerative colitis. Cancer Res2001;61:3573–7.

7. Kondo Y, Kanai Y, Sakamoto M, Mizokami M, Ueda R, Hirohashi S.Genetic instability and aberrant DNA methylation in chronic hepatitisand cirrhosis–A comprehensive study of loss of heterozygosity andmicrosatellite instability at 39 loci andDNAhypermethylation on 8CpGislands inmicrodissected specimens frompatients with hepatocellularcarcinoma. Hepatology 2000;32:970–9.

8. Maekita T, Nakazawa K, Mihara M, Nakajima T, Yanaoka K, Iguchi M,et al. High levels of aberrant DNA methylation in Helicobacter pylori-infected gastric mucosae and its possible association with gastriccancer risk. Clin Cancer Res 2006;12:989–95.

9. Nakajima T, Maekita T, Oda I, Gotoda T, Yamamoto S, Umemura S,et al. Higher methylation levels in gastric mucosae significantly cor-relatewith higher risk of gastric cancers. Cancer Epidemiol BiomarkersPrev 2006;15:2317–21.

10. Perri F, Cotugno R, Piepoli A, Merla A, Quitadamo M, Gentile A, et al.Aberrant DNA methylation in non-neoplastic gastric mucosa of H.Pylori infected patients and effect of eradication. Am J Gastroenterol2007;102:1361–71.

11. Egan BJ, Holmes K, O'Connor HJ, O'Morain CA. Helicobacter pylorigastritis, the unifying concept for gastric diseases. Helicobacter2007;12 Suppl 2:39–44.

12. Fox JG, Wang TC. Inflammation, atrophy, and gastric cancer. J ClinInvest 2007;117:60–9.

13. Moss SF, Blaser MJ. Mechanisms of disease: inflammation and theorigins of cancer. Nat Clin Pract Oncol 2005;2:90–7.

14. Niwa T, Tsukamoto T, Toyoda T, Mori A, Tanaka H, Maekita T, et al.Inflammatory processes triggered by Helicobacter pylori infectioncause aberrant DNA methylation in gastric epithelial cells. Cancer Res2010;70:1430–40.

15. Blackburn EH, Tlsty TD, Lippman SM. Unprecedented opportunitiesand promise for cancer prevention research. Cancer Prev Res 2010;3:394–402.

16. Issa JP. Cancer prevention: epigenetics steps up to the plate. CancerPrev Res (Phila) 2008;1:219–22.

17. Prevention of cancer in the next millennium: Report of the Chemopre-vention Working Group to the American Association for CancerResearch. Cancer Res 1999;59:4743–58.

18. Greenwald P. Cancer chemoprevention. BMJ 2002;324:714–8.19. Hursting SD, Slaga TJ, Fischer SM, DiGiovanni J, Phang JM. Mech-

anism-based cancer prevention approaches: targets, examples, andthe use of transgenic mice. J Natl Cancer Inst 1999;91:215–25.

20. Issa JP, Kantarjian HM. Targeting DNA methylation. Clin Cancer Res2009;15:3938–46.

21. Yoo CB, Jones PA. Epigenetic therapy of cancer: past, present andfuture. Nat Rev Drug Discov 2006;5:37–50.

22. Laird PW, Jackson-Grusby L, Fazeli A, Dickinson SL, Jung WE, Li E,et al. Suppression of intestinal neoplasia byDNAhypomethylation.Cell1995;81:197–205.

23. Yoo CB, Chuang JC, Byun HM, Egger G, Yang AS, Dubeau L, et al.Long-term epigenetic therapy with oral zebularine has minimal sideeffects and prevents intestinal tumors in mice. Cancer Prev Res2008;1:233–40.

24. McCabe MT, Low JA, Daignault S, Imperiale MJ, Wojno KJ, Day ML.Inhibition of DNA methyltransferase activity prevents tumori-genesis in a mouse model of prostate cancer. Cancer Res 2006;66:385–92.

25. Lantry LE, Zhang Z, Crist KA, Wang Y, Kelloff GJ, Lubet RA, et al. 5-Aza-20-deoxycytidine is chemopreventive in a 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone-induced primary mouse lung tumor model.Carcinogenesis 1999;20:343–6.

26. Tang XH, Albert M, Scognamiglio T, Gudas LJ. A DNAmethyltransfer-ase inhibitor and all-trans retinoic acid reduce oral cavity carcinogen-esis induced by the carcinogen 4-nitroquinoline 1-oxide. Cancer PrevRes 2009;2:1100–10.

27. Baba S, Yamada Y, Hatano Y, Miyazaki Y, Mori H, Shibata T, et al.Global DNA hypomethylation suppresses squamous carcinogenesisin the tongue and esophagus. Cancer Sci 2009;100:1186–91.

28. Belinsky SA, Klinge DM, Stidley CA, Issa JP, Herman JG, March TH,et al. Inhibition of DNAmethylation and histone deacetylation preventsmurine lung cancer. Cancer Res 2003;63:7089–93.

29. Niwa T, Ushijima T. Induction of epigenetic alterations by chronicinflammation and its significance on carcinogenesis. Adv Genet 2010;71:41–56.

30. Ushijima T. Epigenetic field for cancerization. J Biochem Mol Biol2007;40:142–50.

31. Tatematsu M, Tsukamoto T, Mizoshita T. Role of Helicobacter pyloriin gastric carcinogenesis: the origin of gastric cancers and hetero-topic proliferative glands in Mongolian gerbils. Helicobacter 2005;10:97–106.

32. Tsukamoto T, Mizoshita T, Tatematsu M. Animal models of stomachcarcinogenesis. Toxicol Pathol 2007;35:636–48.

33. Peterson AJ, Menheniott TR, O'Connor L, Walduck AK, Fox JG,Kawakami K, et al. Helicobacter pylori infection promotes methylationand silencing of trefoil factor 2, leading to gastric tumor development inmice and humans. Gastroenterology 2010;139:2005–17.

34. Shimizu N, Ikehara Y, Inada K, Nakanishi H, Tsukamoto T, Nozaki K,et al. Eradication diminishes enhancing effects of Helicobacter pyloriinfection on glandular stomach carcinogenesis in Mongolian gerbils.Cancer Res 2000;60:1512–4.





35. Cheng H, Bjerknes M, Amar J. Methods for the determination ofepithelial cell kinetic parameters of human colonic epithelium iso-lated from surgical and biopsy specimens. Gastroenterology1984;86:78–85.

36. Tatematsu M, Yamamoto M, Shimizu N, Yoshikawa A, Fukami H,Kaminishi M, et al. Induction of glandular stomach cancers in Helico-bacter pylori -sensitive Mongolian gerbils treated with N-methyl-N-nitrosourea and N-methyl-N0-nitro-N-nitrosoguanidine in drinkingwater. Jpn J Cancer Res 1998;89:97–104.

37. Toyoda T, Tsukamoto T, Mizoshita T, Nishibe S, Deyama T, TakenakaY, et al. Inhibitory effect of nordihydroguaiaretic acid, a plant lignan, onHelicobacter pylori -associated gastric carcinogenesis in Mongoliangerbils. Cancer Sci 2007;98:1689–95.

38. Karimi M, Johansson S, Ekstrom TJ. Using LUMA: a Luminometric-based assay for global DNA-methylation. Epigenetics 2006;1:45–8.

39. Yamashita S, Takahashi S, McDonell N, Watanabe N, Niwa T,Hosoya K, et al. Methylation silencing of transforming growthfactor-beta receptor type II in rat prostate cancers. Cancer Res2008;68:2112–21.

40. Rice P, Longden I, Bleasby A. EMBOSS: the European MolecularBiology Open Software Suite. Trends Genet 2000;16:276–7.

41. Hur K, Niwa T, Toyoda T, Tsukamoto T, Tatematsu M, Yang HK, et al.Insufficient role of cell proliferation in aberrant DNAmethylation induc-tion and involvement of specific typesof inflammation.Carcinogenesis2011;32:35–41.

42. WeberM, Hellmann I, Stadler MB, Ramos L, Paabo S, RebhanM, et al.Distribution, silencing potential and evolutionary impact of promoterDNA methylation in the human genome. Nat Genet 2007;39:457–66.

43. Hussain SP, Harris CC. Inflammation and cancer: an ancient link withnovel potentials. Int J Cancer 2007;121:2373–80.

44. Gonda TA, Kim YI, Salas MC, Gamble MV, Shibata W, Muthupalani S,et al. Folic acid increases global DNAmethylation and reduces inflam-mation to prevent Helicobacter-associated gastric cancer in mice.Gastroenterology 2012;142:824–33.

45. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmedcell death in situ via specific labeling of nuclear DNA fragmentation.J Cell Biol 1992;119:493–501.

46. Lee ER. Dynamic histology of the antral epithelium in the mousestomach: III. Ultrastructure and renewal of pit cells. Am J Anat1985;172:225–40.

47. Asaka M, Kato M, Takahashi S, Fukuda Y, Sugiyama T, Ota H, et al.Guidelines for the management of Helicobacter pylori infection inJapan: 2009 revised edition. Helicobacter 2010;15:1–20.

48. Kabir S. Effect ofHelicobacter pylori eradication on incidenceof gastriccancer in human and animal models: underlying biochemical andmolecular events. Helicobacter 2009;14:159–71.

49. Nakajima T, Enomoto S, Yamashita S, Ando T, Nakanishi Y, NakazawaK, et al. Persistence of a component of DNA methylation in gastricmucosae after Helicobacter pylori eradication. J Gastroenterol 2010;45:37–44.

Niwa et al.




2013;6:263-270. Cancer Prev Res Tohru Niwa, Takeshi Toyoda, Tetsuya Tsukamoto, et al. Gerbils by a DNA Demethylating Agent

Induced Gastric Cancers in−Helicobacter pyloriPrevention of

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