DNA methylation status is inversely correlated with green tea intake and physical
activity in gastric cancer patients
Yasuhito Yuasa1*, Hiromi Nagasaki1, Yoshimitsu Akiyama1, Yutaka Hashimoto1, Touichirou Takizawa2, Kazuyuki Kojima3,
Tatsuyuki Kawano3, Kenichi Sugihara3, Kazue Imai4and Kei Nakachi4
1Department of Molecular Oncology, Tokyo Medical and Dental University, Tokyo, Japan
2Department of Pathology, Tokyo Medical and Dental University, Tokyo, Japan
3Department of Surgery, Tokyo Medical and Dental University, Tokyo, Japan
4Department of Radiobiology/Molecular Epidemiology, Radiation Effects Research Foundation, Hiroshima, Japan
Epigenetic silencing of genes by aberrant DNA methylation is rec-
ognized as a crucial component of the mechanism underlying tu-
morigenesis. However, the relationship between DNA methylation
and the past lifestyle in cancer patients remains largely unknown.
We examined the methylation statuses of 6 tumor-related genes,
CDX2 (homeobox transcription factor), BMP-2 (bone morphoge-
netic protein 2), p16 (INK4A), CACNA2D3 (calcium channel-
related), GATA-5 (transcription factor) and ER (estrogen recep-
tor), in 106 primary gastric carcinomas by methylation-specific
PCR and compared them with the past lifestyles of the patients.
The methylation frequencies of the genes were 23.6, 21.7, 9.4, 32.4,
40.8 and 59.1%, respectively. Significant association was found
between a decreased intake of green tea and methylation of CDX2
and BMP-2. More physical activity was correlated with a lower
methylation frequency of CACNA2D3. Of these 6 genes, the meth-
ylation statuses of CDX2, BMP-2 and p16 revealed a significant
interrelationship and those of CACNA2D3, GATA-5 and ER did
likewise. Thus, some epidemiological factors, such as green tea
intake, could be important as to determination of the methylation
statuses of selected genes and may influence the development of
cancer, including that of the stomach.
' 2008 Wiley-Liss, Inc.
Key words: DNA methylation; green tea; gastric cancer; CDX2;
Epigenetic changes, particularly methylation of cytosine in
CpG dinucleotides in gene promoters, are found in almost every
type of human neoplasm and are associated with transcriptional
gene silencing.1,2Such promoter hypermethylation is as common
as the disruption of tumor-suppressor genes in human cancer by
mutation. Unlike irreversible genetic changes, epigenetic changes
are thought to possibly be reversible by the environment, diet or
pharmacological intervention. For example, monozygotic twins
are considered genetically identical and are thus ideal for studying
the effects of environmental and dietary factors on human health
and diseases. In a study of a large cohort of identical twins, the
patterns of DNA methylation across the genome were found to be
very similar in young monozygotic twins in several cell types, but
in older twins the patterns diverged.3This strongly suggests that 1
or more environmental factors affect individuals throughout life,
modifying gene expression through epigenetic mechanisms that
have important implications for health.
Dietary factors are important determinants of cancer risk.4
Aberrant DNA methylation is associated with dietary factors and
other lifestyle factors and may underlie carcinogenesis. The preva-
lence of promoter hypermethylation of 6 genes, such as APC,
p14ARF, p16/INK4a (hereafter p16) and hMLH1, was higher in col-
orectal cancers derived from patients with a low folate/high alco-
hol intake than in ones with a high folate/low alcohol intake, but
the differences were not statistically significant.5The incidences
of hypermethylation of D17S5 and p16 in lung cancer are signifi-
cantly higher in cigarette smokers than in those who have never
smoked.6–8However, the relationship between DNA methylation
and the past lifestyle in cancer patients remains largely unknown.
In 2000, gastric cancer was the second most frequent cause of
cancer death worldwide.9Infection with Helicobacter pylori is a
strong risk factor for gastric cancer but is not a sufficient cause for
its development.10Epidemiological studies have strongly sug-
gested that the risk may be increased with a high intake of salt and
salt-preserved foods and decreased with a high intake of fruit and
vegetables.11The aberrant methylation of many genes has been
reported in gastric cancer.12–14We previously reported that CDX2
methylation in men was correlated with a decreased intake of
green tea, suggesting that diet could be an important factor deter-
mining the methylation status of genes such as CDX2 and the re-
sultant aberrant expression of genes involved in carcinogenesis.15
However, these effects may not be universal but gene-specific,
and female patients have not been examined. Thus, we analyzed
the methylation states of 6 genes in more gastric cancer patients.
Five of the 6 genes, that is, CDX2,15BMP2,16p16,17CAC-
NA2D318and GATA5,19were often methylated in gastric cancers
but rarely in noncancerous epithelia. We, then, compared the rela-
tionship between DNA methylation and the past lifestyle in cancer
patients including female ones.
Material and methods
Cancer tissue specimens were collected from 106 consecutive
patients with primary gastric carcinoma in a hospital affiliated to
Tokyo Medical and Dental University during 2000–2005.
Informed consent was obtained from all patients, and the study
was approved by the institutional review committee of Tokyo
Medical and Dental University. A self-administered questionnaire
was used in this study to assess the lifestyle before cancer onset,
covering the disease history, familial history of cancer, medica-
tion, 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), regular-
ity of sleep and meals, eating quantity, bowel motion, height and
body weight. The food groups were beef, pork, chicken, ham/sau-
sage/bacon, grilled meat, all meat, grilled fish, salted/dried/other
processed fish products, pickled vegetables, green leaf vegetables,
yellow colored vegetables, cruciferous vegetables, all vegetables,
fruits and probiotics-fermented milk. The intake frequencies of
these food groups were categorized into not eaten, 1–2 times/
month, 1–2 times/week, 3–4 times/week, almost every day and
almost every meal. Most lifestyle factors in this questionnaire
were selected from those which had previously been reported to
be risk or preventive factors for gastric and colon cancers on epi-
Grant sponsor: Ministry of Education, Culture, Sports, Science, and
Technology, Japan; Grant number: 17015013.
*Correspondence to: Department of Molecular Oncology, Graduate
School of Medicine and Dentistry, Tokyo Medical and Dental University,
1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.
Fax: 181-3-5803-0125. E-mail: firstname.lastname@example.org
Received 18 July 2008; Accepted after revision 1 December 2008
Published online 18 December 2008 in Wiley InterScience (www.
Int. J. Cancer: 124, 2677–2682 (2009)
' 2008 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
This work was supported by Grant-in-Aid for Scientific
Research on Priority Areas-Cancer 17015013 from the Ministry of
Education, Culture, Sports, Science, and Technology, Japan (Y.
Yuasa). The authors thank Ms. Y. Ozawa for assistance in the
preparation of the manuscript.
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