Relationship between CDX2 gene methylation and dietary factors in gastric cancer patients
Yasuhito Yuasa1,6,
Hiromi Nagasaki1,
Yoshimitsu Akiyama1,
Hidekazu Sakai1,
Tomoko Nakajima1,
Yasuo Ohkura2,
Touichirou Takizawa3,
Morio Koike3,
Masao Tani4,
Takehisa Iwai4,
Kenichi Sugihara4,
Kazue Imai5 and
Kei Nakachi5
1 Department of Molecular Oncology, Tokyo Medical and Dental University, Tokyo, Japan, 2 Department of Surgical and Molecular Pathology, Dokkyo University School of Medicine, Tochigi, Japan, 3 Department of Pathology, Tokyo Medical and Dental University, Tokyo, Japan, 4 Department of Surgery, Tokyo Medical and Dental University, Tokyo, Japan and 5 Department of Radiobiology/Molecular Epidemiology, Radiation Effects Research Foundation, Hiroshima, Japan
6 To whom correspondence should be addressed Email: yuasa.monc{at}tmd.ac.jp
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Abstract
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Epigenetic gene silencing through DNA methylation is one of the important steps in the mechanism underlying tumorigenesis, including in the stomach. Past lifestyle factors of cancer patients, such as intake of vegetables, are very important in affecting gastric carcinogenesis. However, the relationship between DNA methylation and past dietary habits in cancer patients remains largely unknown. The CDX2 homeobox transcription factor plays a key role in intestinal development, but CDX2 is also expressed in most of the intestinal metaplasia and part of the carcinomas of the stomach. We analyzed the methylation status of the CDX2 5' CpG island in gastric cancer cell lines by methylation-specific PCR (MSP), and then CDX2 mRNA was found to be activated after 5-aza-2'-deoxycytidine treatment of the methylation-positive cells. We further examined the methylation status of CDX2 in primary gastric carcinomas by MSP and compared it with the past lifestyle of the patients, including dietary habits. Methylation of CDX2 was found in 20 (34.5%) of the 58 male patients and one (6.7%) of the 15 female patients. Since the methylation frequency was low in the female patients, the analysis was performed only on the male cases. CDX2 methylation was correlated with the decreased intake of green tea and cruciferous vegetables, and also with full or overeating habits. These findings are consistent with epidemiological observations on gastric cancer. We also analyzed the methylation status of p16/INK4a and hMLH1, but their frequencies were not associated with dietary factors or other lifestyle factors. Thus, diet could be an important factor determining the methylation status of genes such as CDX2 and the resultant aberrant expression of genes involved in carcinogenesis.
Abbreviations: MSP, methylation-specific PCR; RTPCR, reverse transcriptionpolymerase chain reaction
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Introduction
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Gastric cancer is the second most frequent cause of death from cancer in both sexes in the world (1). The precise mechanism underlying gastric carcinogenesis is not fully understood yet. However, several environmental factors, such as Helicobacter pylori infection, excessive intake of salt and low intake of vegetables and fruits, have been linked with gastric carcinogenesis (24).
Since alterations of gene functions in cancer cannot be explained by only the mutational rate, there should be a non-structural mechanism. Some cancers show hypermethylation of CpG islands in gene promoters, resulting in loss of gene function. Patterns of DNA methylation can be inherited when cells divide. This epigenetic process, as an alternative to mutations, inhibits tumor suppressor gene function (5,6).
Dietary factors are important determinants of cancer risk, including that of gastric cancer (2). Certain dietary factors and other lifestyle factors are associated with variations in DNA methylation, and these variations might underlie gastric carcinogenesis. For example, the incidence of hypermethylation of p16/INK4a (hereafter p16) in lung cancer is significantly higher in cigarette smokers than in those who have never smoked (7). Consequently, p16 expression is silenced, resulting in the progression of carcinogenesis. The prevalence of promoter hypermethylation of six genes, such as APC, p14ARF, p16 and hMLH1, was higher in colorectal cancers derived from patients with a low folate/high alcohol intake than in ones with a high folate/low alcohol intake, but the differences were not statistically significant (8).
Several genes are aberrantly methylated in human primary cancers, including that of the stomach (9). p16 and hMLH1 have been extensively examined in gastric cancer (1012). p16 inhibits G1 cyclin-dependent kinases and hence induces cell-cycle arrest, and has a tumor-suppressor gene function (13,14). Epigenetic inactivation of hMLH1 due to promoter methylation is strongly associated with microsatellite instability and seems to be a significant event in the development of gastric cancer (1012).
Human CDX2 is a member of the caudal-related homeobox gene family (15,16). The expression of the rodent Cdx2 homeobox gene is intestine-specific, and occurs from the early embryo to the adult stage (17), and thus it is likely that Cdx2 plays roles in both the establishment and maintenance of the intestinal epithelial phenotype. On the other hand, the ectopic expression of CDX2 has been related to intestinal metaplasia formation in the stomach. First, the CDX2 protein is not expressed in the normal stomach, but is highly expressed in nearly all of the intestinal metaplasia of the stomach (1820). Secondly, when Cdx2 expression was directed to the gastric mucosae in transgenic mice using cis-regulatory elements of gastric mucosa-specific genes, ectopic Cdx2 expression induced gastric intestinal metaplasia in the mice (21,22). Gastric cancer is histologically classified into two main types, intestinal and diffuse (23). Intestinal type gastric cancers are thought to develop from intestinal metaplasia, while diffuse type ones may mainly develop from the normal mucosae (16). CDX2 expression is stronger in intestinal type than in diffuse type gastric cancers (18).
We analyzed here the methylation status of p16 and hMLH1 in gastric cancers. Furthermore, since CDX2 expression is lower in gastric cancers than in intestinal metaplasia (18), we also analyzed methylation of the CDX2 5' CpG island, and compared them with the dietary habits, specifically those which have previously been reported as risk or preventive factors of gastric cancer in epidemiological observation.
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Materials and methods
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Cell culture and drug treatment
Human gastric cancer cell lines GT3TKB and MKN74 were grown in Dulbecco's modified minimum essential medium and RPMI1640 medium, respectively, supplemented with 10% fetal bovine serum. A human colon cancer cell line, RKO, was cultured in Eagle's minimum essential medium containing 10% fetal bovine serum. For demethylation studies, cells were treated daily with 5 µM 5-aza-2'-deoxycytidine (Sigma, St Louis, MO) for 3 days.
RNA extraction and reverse transcriptionpolymerase chain reaction (RTPCR)
Total RNA was isolated using TRIZOL reagent (Invitrogen, Carlsbad, CA). To synthesize the RTPCR template, we used 2 µg of total RNA and reverse-transcribed it using a Superscript kit (Invitrogen). The primers used for CDX2 amplification were described previously (24). We amplified with multiple cycle numbers (2835 cycles) to obtain semi-quantitative differences in the expression level. As an internal control for RTPCR analysis, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts were amplified for 19 cycles from the same cDNA samples (24).
Methylation analyses by the methylation-specific PCR (MSP) procedure and bisulfite DNA sequencing
We extracted genomic DNA from cultured cells or paraffin-embedded tissues by the phenolchloroform method, and then carried out bisulfite modification and the MSP procedure as described previously (25). The primer sequences of CDX2 for the unmethylated reaction were 5'-GAAGTTGTTGGTTTGGGGTTTTGTAT-3' (sense) and 5'-CCCACAATACTCCACTAACTCCTCACA-3' (antisense), and for the methylated reaction 5'-CGTCGGTTTGGGGTTTCGTAC-3' (sense) and 5'-GATACTCCGCTAACTCCTCGCG-3' (antisense), according to the GenBank sequence (AL591024). The PCR reaction for CDX2 was performed for 35 cycles in a 25-µl mixture containing bisulfite-modified DNA (
50 ng), 2.5 µl of 10x PCR buffer, 1.25 µl of 25 mM dNTP, 10 pmol of each primer and 1 U of JumpStart Red Taq polymerase (Sigma). Each PCR cycle consisted of 95°C for 30 s, 58°C for 30 s and 72°C for 30 s, followed by final extension at 72°C for 5 min. The MSP primers and conditions used for p16 and hMLH1 were described previously (25,26). The PCR products were electrophoresed in 2.5% agarose gels. All the MSP procedures were repeated more than twice.
Bisulfite DNA sequencing of the CDX2 5' CpG island was performed as described previously (27). The primer sequences for amplification were 5'-GAAGTTTTTAATTATTGGTGTTTGTGTT-3' (sense) and 5'-AAACCTCACCATACTACCTAAAAACC-3' (antisense).
Immunohistochemistry
Immunohistochemical analysis of the CDX2 protein was performed as described previously (18). Monoclonal antibody to the CDX2 protein (Bio Genex, San Ramon, CA) was diluted at 1:100.
Study population
Cancer tissue specimens were collected from 73 consecutive patients with primary gastric carcinoma in an affiliated hospital of the Tokyo Medical and Dental University, and Saitama Cancer Center Hospital during 20002002. Informed consent was obtained from all patients, and the study was approved by the appropriate institutional review committee. A self-administered questionnaire was used in this study to assess their lifestyle before cancer onset, covering disease history, familial history of cancer, medication, cigarette smoking, alcohol consumption, physical activity, intake frequencies of selected food groups and food items, daily consumption of tea (green tea, oolong tea and black tea), regularity of sleep and meals, eating quantity, bowel motion, height and body weight. Food groups were beef, pork, chicken, ham/sausage/bacon, grilled meat, all meat, grilled fish, salted/dried/other processed fish products, pickled vegetables, green leaf vegetables, yellow color vegetables, cruciferous vegetables, all vegetables, fruits and probiotics-fermented milk. Intake frequencies of these food groups were categorized into not eating, 12 times/month, 12 times/week, 34 times/week, almost every day and almost every meal. Eating quantity was categorized as full or overeating, eating in moderation and consciously under-eating. Most lifestyle factors in this questionnaire were selected from those which have previously been reported as risk or preventive factors of gastric and colon cancers in epidemiological observation.
Tumors were reviewed by a pathologist and microdissected prior to DNA extraction. Histological classification was performed according to the general rules established by the Japanese Gastric Cancer Association (28) and the Laurén's classification (23).
Statistical analysis
The promoter methylation status of specific genes, clinicopathological parameters and lifestyle variables in the patients were computed. Differences in frequency by methylation status were tested using Fisher's exact test, and differences in mean values were tested using t test. Association between the methylation status and dietary variables was also analyzed by non-parametric test (MannWhitney U test). We further studied association by categorical regression analysis with optimal scaling using alternating least squares (29). In this analysis, intake frequencies of food groups and eating quantity were dichotomous:
twice/week versus
3 times/week for vegetable groups,
twice/month versus
once/week for meat and fish groups,
4 times/week versus
5 times/week for fruits and
6 cups/day versus
7 cups/day for green tea and full/over versus moderate/light for eating quantity. The statistical software used was SPSS software (version 11.0).
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Results
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Epigenetic silencing of CDX2 in a gastric cancer cell line
With a semi-quantitative RTPCR assay to measure the CDX2 mRNA level, GT3TKB did not express any CDX2 mRNA, whereas MKN74 expressed it abundantly (Figure 1A). We then used the demethylating agent 5-aza-2'-deoxycytidine to study the epigenetic status of CDX2 in these cell lines. CDX2 was re-expressed in GT3TKB cells with this treatment (Figure 1A).

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Fig. 1. CDX2 expression and methylation status in gastric cancer cell lines. (A) The CDX2 expression level was examined by RTPCR in two cell lines with (lanes +) or without (lanes ) treatment with 5-aza-2'-deoxycytidine. GAPDH expression was used as an internal loading control for the RTPCR and H2O (no cDNA added). (B) Schematic representation of the CDX2 gene promoter region. A box indicates exon 1, including coding (black) and non-coding (white) regions. Vertical bars show CpG sites. Arrows indicate the regions analyzed by MSP and bisulfite sequencing (BS). (C) MSP analysis of the CpG island of CDX2 in two gastric cancer cell lines and peripheral blood lymphocytes (lane PBL). PCR products recognizing unmethylated (lanes U) and methylated (lanes M) CpG sites were analyzed in 2.5% agarose gels and stained with ethidium bromide. (D) Sodium bisulfite DNA sequencing of the CDX2 CpG island in gastric cancer cell lines and normal stomach mucosa. Each horizontal row of squares represents analysis, in a single clone of bisulfite-treated DNA, of 24 CpG sites contained in the region shown. Solid and open squares represent methylated and unmethylated CpG sites, respectively.
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We have identified CpG islands associated with the CDX2 5' region (Figure 1B) and thus the methylation status of CDX2 was studied by MSP and sodium bisulfite DNA sequencing. GT3TKB cells only exhibit a methylation signal, but MKN74 cells do not exhibit any (Figure 1C), consistent with the CDX2 expression levels in these cells. The sodium bisulfite DNA sequencing of CDX2 in these cell lines confirmed the methylation status, i.e. CDX2 expression-negative GT3TKB cells show densely methylated clones, while expression-positive MKN74 cells and normal gastric mucosa have unmethylated clones (Figure 1D).
Methylation status of CDX2, p16 and hMLH1 in primary gastric carcinomas
Methylation of CDX2, as determined by MSP, was frequent in primary gastric carcinomas, i.e. there were 21 positive cases among 73 (28.8%) total cases. Representative examples of gel analysis of MSP are shown in Figure 2. CDX2 protein expression was also analyzed in gastric carcinomas by immunohistochemistry (Figure 3 and Table I). As shown in Table I, negative or partial CDX2 expression was more frequently observed in diffuse type (28/32, 87.5%) than in intestinal type (20/35, 57.1%) gastric cancers (P < 0.01), which is consistent with the previous data (18). The CDX2 methylation frequencies in gastric cancers with negative or partial expression were similar between the intestinal (40%) and diffuse types (35.7%). On the other hand, the overall methylation frequency in cancers with negative or partial CDX2 expression (18/48, 37.5%) was significantly higher than that with positive expression (2/19, 10.5%) (P = 0.03), suggesting that methylation of the CDX2 gene is important for gene silencing in primary gastric cancers. When we analyzed the CDX2 methylation status by MSP in five normal and five intestinal metaplastic tissues of the stomach from independent patients, we found no methylation in any samples (Table I). Since the normal gastric mucosae did not reveal CDX2 gene methylation, strong CDX2 expression in intestinal metaplasia compared with the normal mucosae is not attributable to demethylation but possibly to aberrant transcriptional activation.

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Fig. 2. Representative examples of MSP analyses of CDX2, p16 and hMLH1 in primary gastric cancer tissues (Ca), normal gastric mucosae (N), and peripheral blood lymphocytes (PBL). A colorectal cancer cell line, RKO, was used as a methylation-positive control. Lanes: U, unmethylated alleles; M, methylated alleles; H2O, no DNA added.
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Fig. 3. Immunohistochemical staining of CDX2 in representative cancers. (A) A methylation-negative intestinal type gastric carcinoma showed nuclear CDX2 staining. (B) A diffuse type gastric carcinoma did not express the CDX2 protein, while adjacent intestinal metaplastic cells (right side) expressed CDX2. The microdissected cancer portion (Ca2) of this sample exhibited CDX2 gene methylation (Figure 2). Original magnification x200.
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The methylation status of the p16 and hMLH1 genes was also examined by MSP (Figure 2). Nine of 66 (13.6%) and seven of 67 (10.4%) gastric cancers exhibited MSP methylation signals, respectively.
The relationship between methylation frequencies of CDX2, p16 and hMLH1 and clinicopathological parameters
Clinicopathological characteristics of study patients by the methylation status of CDX2, p16 and hMLH1 are shown in Table II. The methylation of CDX2 was significantly more frequent in males (20/58, 34.5%) than in females (1/15, 6.7%) (P = 0.03). p16 methylation was more frequently found in younger patients and larger cancers (P = 0.04), and was more common in diffuse type than in intestinal type gastric carcinomas (P = 0.02). In contrast, there was no statistically significant correlation between hMLH1 methylation and clinicopathological parameters (Table II).
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Table II. Clinicopathological characteristics of study patients according to the methylation status of CDX2, p16 and hMLH1
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The relationship between methylation frequencies of CDX2, p16 and hMLH1, and epidemiological parameters in male patients
Since CDX2 methylation was found only in one female cancer case, the following epidemiological analyses were only performed for male patients. Methylation of CDX2, p16 and hMLH1 was found in 20/58 (34.5%), 8/53 (15.1%) and 5/55 (9.1%) male patients, respectively; those revealing methylation in any of these genes were 23/53 (43.4%). None of the epidemiological variables revealed statistical significance in relation to the methylation status of CDX2, p16, hMLH1 and any of the three genes, except beef intake and p16 methylation (Table III). Because the methylation frequency of hMLH1 was low and was not associated with epidemiological variables, its data are not shown in Table III. Since dietary factors are closely interrelated, we further conducted categorical regression analyses of clinical and epidemiological variables, and methylation in male gastric cancer patients. A significant association was found between eating quantity or the intake of green tea and methylation of CDX2, and between eating quantity and methylation of any of the three genes (Table III). Increased methylation frequency of CDX2 was significantly associated with full or overeating habits, adjusting for confounding variables (P = 0.02). On the other hand, increased daily consumption of green tea (7 or more cups/day) showed a significant association with decreased methylation frequency of CDX2 after adjustment (P = 0.02). These epidemiological factors also revealed a close association with methylation frequency of any of CDX2, p16 and hMLH1 genes (P = 0.02 and 0.06 for eating quantity and intake of green tea, respectively).
When we analyzed the association between the methylation status and dietary variables by non-parametric test, increased intake of cruciferous vegetables was significantly associated with decreased frequency of CDX2 methylation (P = 0.03). Distinct distribution of patients with the methylated and unmethylated CDX2 is demonstrated for the intake of cruciferous vegetables (Figure 4).

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Fig. 4. Frequencies of the presence (closed bars) or absence (open bars) of CDX2 methylation in gastric cancers stratified as to intake of cruciferous vegetables.
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Both green tea and cruciferous vegetables were inversely associated with CDX2 methylation. Then we further analyzed the relationship between CDX2 methylation and combination of these two factors. We found a stronger association in the combination (Table IV), i.e. the patients drinking more green tea or consuming cruciferous vegetables more often showed a lower methylation frequency than the patients drinking less green tea and consuming cruciferous vegetables less frequently.
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Discussion
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We reported here that cultured gastric cancer cells exhibiting no CDX2 expression showed methylation of the CDX2 CpG island, while cultured gastric cancer cells exhibiting high CDX2 expression did not show methylation. Moreover, treatment of cultured cells with the demethylating agent 5-aza-2'-deoxycytidine activated CDX2 expression. The data indicate that CDX2 expression is silenced on methylation of the CpG island associated with the CDX2 gene promoter region.
CDX2 gene methylation was found in 21 of the 73 (28.8%) primary gastric cancers. The methylation frequency in cancers with negative or partial CDX2 expression was significantly higher than that with positive expression, and five CDX2 expression-positive intestinal metaplastic tissues revealed no CDX2 gene methylation as shown in Table I. Therefore, CDX2 expression may be silenced with gene methylation in primary gastric cancers, even though there may be also other unknown mechanisms. Because intestinal type gastric cancers are thought to develop through intestinal metaplasia (CDX2 expression-positive) (16,1820), there may be more CDX2 expression-positive cancers in this type. On the contrary, many diffuse type gastric cancers may develop from the normal mucosa (CDX2 expression-negative) (16), and thus it is reasonable that there were more CDX2 expression-negative or -partial cancers in the diffuse type. The CDX2 methylation frequencies were similar between the intestinal and diffuse type gastric cancers even in cancers with negative or partial expression. One reason might be that some gastric cancers develop as the intestinal type and then progress to the diffuse type with time (30).
The methylation frequency of CDX2 did not show any significant relationship to pathologic characteristics, which may cause a bias in further analyses. We then analyzed the association between CDX2 methylation and selected lifestyle factors known to be risky or preventive for gastric and colon cancers. The epidemiological analyses were carried out only on males, because CDX2 methylation was found in only one female case. Univariate analysis revealed that none of the lifestyle variables was significantly associated with the methylation frequencies of CDX2, p16, hMLH1 and any of the three genes except for beef intake and p16. However, multivariate analysis revealed significant differences between eating quantity and the methylation frequency of CDX2 or any of the three genes, and also between the intake of green tea and CDX2 methylation.
Full or overeating was associated with an increased frequency of methylation in CDX2 and any of the three genes (P = 0.02 and P = 0.02, respectively). Although a few epidemiological studies have investigated eating quantity and gastric cancer, full eating is a consistent risk factor in the etiology of gastric cancer (3133). One plausible interpretation may be that the gastric mucosa is physically damaged by repeated compulsory expansion of the gastric lining upon full eating, possibly resulting in an increased sensitivity of the cells to various exogenous compounds in food, which may include those promoting DNA methylation.
Among the 15 food groups and selected food items, an increased intake of green tea was independently and significantly associated with the CDX2 methylation frequency (P = 0.02) and methylation in any of CDX2, p16 and hMLH1 (borderline significance, P = 0.06), after adjusting for confounding lifestyle and clinical variables. The effects of green tea seem to be dose-dependent: the CDX2 methylation frequencies, 10/25 (40%), 7/18 (39%), 2/8 (25%), and 0/6 (0%) in three or less, four to six, seven to nine and ten cups or more a day, respectively.
Green tea contains several polyphenolic compounds, such as epigallocatechin gallate (EGCG). Significant inhibitory effects of EGCG or green tea extracts on carcinogenesis of rodents in various organs including the stomach have been demonstrated in many studies (34,35). Most epidemiological studies in Japan revealed cancer-preventive effects of drinking green tea (3639). Furthermore, it was reported recently that EGCG dose-dependently inhibited DNA methyltransferase activity in several cancer cells, resulting in the reactivation of methylation-silenced genes (p16, retinoic acid receptor ß and hMLH1) (40). Taken together, our findings imply a novel mechanism of green tea in cancer prevention, i.e. inhibition of methylation of selected genes involved in gastric carcinogenesis.
Although we analyzed the association between dietary factors and DNA methylation using the categorical regression model, this analysis may overlook some factors due to a small number of study subjects. Therefore, we further reinvestigated the association by examining overall differences in the distribution of patients with methylated or unmethylated CDX2 on the intake of food groups, using the non-parametric test, and found that the intake frequency of cruciferous vegetables in patients with unmethylated CDX2 was distributed in higher categories than that in patients with methylated CDX2 (P = 0.03, Figure 4). Cruciferous vegetables have been reported to be anticarcinogenic in a number of epidemiological and laboratory studies. Particularly, the active compounds in cruciferous vegetables, such as arylalkyl isothiocyanates (and their glucosinolate precursors) and indole-3-carbinol, have been extensively investigated, and the roles of isothiocyanates were presumed to suppress activating enzymes and induce detoxifying ones of carcinogens (41,42). There were several reports suggesting other cancer-preventive mechanisms of isothiocyanates, for example, dose-dependent inhibition of DNA methylation in nitrosomethylbenzylamine-induced esophageal tumorigenesis of rats (43), and inhibition of Helicobacter pylori and benzo[a]pyrene-induced stomach tumors in mice (44). Our findings provide evidence in humans supporting the cancer-preventive effects of cruciferous vegetables through the inhibition of DNA methylation.
We observed large differences in the methylation frequencies of genes examined, particularly CDX2, between male and female patients. Unexpectedly, gender comparison in dietary factors including eating quantity, and intake of green tea and cruciferous vegetables did not reveal any significant difference. However, male patients included more cases with advanced age at diagnosis (>60 years) than female ones (P = 0.06), and the intestinal type was more frequently found in male patients than in female ones (P < 0.05), implying a gender difference in the etiology of gastric cancer. These are in consistence with previous reports on gender difference in age at diagnosis and frequencies of gastric carcinoma and intestinal metaplasia (45,46). Thus, the gender difference in methylation frequency might be ascribed to gender-specific host factors, such as estrogens, not to lifestyle factors.
In this study, the methylation of CDX2 and other genes involved in gastric carcinogenesis was investigated in relation to the clinicopathological and selected lifestyle factors of gastric cancer patients. We therein hypothesized that some of the lifestyle factors, particularly dietary ones, which have been reported to be risky or preventive for gastric cancer in epidemiological observation, may influence the development of gastric cancer through methylation of the selected genes. We for the first time found the inverse association of CDX2 methylation with the intake of green tea and cruciferous vegetables, which have previously been suggested only by in vitro or animal studies, although a further study with an increased number of study patients should be required. Our findings may thus advance the chemoprevention of gastric cancer from a view of inhibiting gene methylation.
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Acknowledgments
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This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Drs Stephen B.Baylin, James G.Hermann and Satoshi Miyake for helpful discussion, and Yoshiko Ozawa for assistance in the preparation of this manuscript.
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Received July 22, 2004;
revised September 5, 2004;
accepted September 29, 2004.