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- Toshihiro Tsuneyoshi1, Hideya Terunuma2,
Rie Tomita1, Midori Ohta1, Saori Suzuki=
1,
Masanori Goto1, Hidetaka Yamada3 and Haruhiko
Sugimura3
1: Shizuoka Inst. of Sci. and Technol., Fukuroi
437-8555,
2: Ibaraki Clin=
ic,
Hitachinaka 312-0062, =
3:
Hamamatsu
Univ. School of=
Med.,
Hamamatsu 431-3192, Japan
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2
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- DNA methylatio=
n is a
general epigenetic gene alteration accompanied by aging or
carcinogenesis. In a
previous paper, we showed that green tea administration significantly
suppressed DNA methylation of mice estrogen receptor gene. Recently, green tea catechi=
n was
shown to inhibit DNA methyltransferase (Dnmt1), suppress DNA methyla=
tion
and re-expresse mRNA and protein of 4 genes in various human cancer =
cell
lines.
We
demonstrate, here, that DNA methylation of human collagen type I gen=
es
(COL1A1 and COL1A2) promoter region is significantly suppressed in v=
ivo
by the administration of green tea.
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3
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-  =
;
Epigenetic gene silencing through DNA methylation is thought =
to
be an important step of tumourigenesis and aging (1)(Fig.1, 2). CpG island methylation of t=
he
estrogen receptor (ER) gene and some other genes increases as a dire=
ct
function of age in normal human colonic mucosa (2).  =
; &n=
bsp;
.
- &nb=
sp;
Collagen is a key material of bone and decrease of collagen
production reduces the bone density and causes osteoporosis, a typic=
al
symptom of aging.
Methylation of collagen gene is suggested to play an important
role of the age-dependent degenerative alteration in human periodont=
al
ligament tissues (3).
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4
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- ・Adds methyl group to the 5-th position of cytosine. (=
Dnmt,
SAM)
- ・Epigenetic gene alteration accompanying tumourigenesis or aging.
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5
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6
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- Rec=
ently,
epigallocatechin gallate (EGCg), the green tea natural polyphenol
antioxidant, “catechin”, was shown to be anti-carcinogen=
ic
through the antifolate activity like methotrexate (MTX)(4). We think that E=
GCg
might have an anti-aging activity through the inhibitory effect on D=
NA
methylation.  =
; &n=
bsp;
. &nb=
sp;
In
a previous paper, we showed that green tea administration significan=
tly suppressed
DNA methylation of mice ER gene, using our original quantitative
detection method of DNA methylation (5). Recently, EGCg was proved t=
o inhibit
Dnmt1, suppress DNA methylation and re-express mRNA and protein of 4
genes in human cancer cell lines (6).  =
; &n=
bsp;  =
;
.
We
report, here, that green tea administration significantly reduces the
DNA methylation in human collagen genes (COL1A1 and COL1A2) promoter
region in vivo, using our previously reported method.  =
; &n=
bsp;
.
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7
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- Administr=
ation
of green tea  =
; &n=
bsp;  =
;
.
Seven
volunteers were administered commercially PET bottled green tea of 2=
000
ml a day for two weeks. In
control experiment, they were prohibited to drink any beverage which
might contain catechins also for two weeks.  =
; &n=
bsp;  =
;
.
Sample preparation  =
; &n=
bsp;  =
;
.
&nbs=
p;
Genomic DNA was extracted from oral mucosa samples of
volunteers. Extracted =
DNA
was digested with methylation sensitive restriction enzyme, Hpa II,
mixed with control DNA and applied to the PCR amplification with pri=
mers
for COL1A1 (Fig.3) and COL1A2 (Fig.4). After electrophoresing PCR
products with agarose gel, the fluorescent intensity of ethidium bro=
mide
stained band of target and control DNA was obtained by
densitometry. The
methylation rate was calculated from the intensity ratio of digested=
DNA
to that of non-digested DNA (Fig.5)
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10
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-  =
;
The methylation rate of COL1A1 and COL1A2 calculated from the
gels such as Fig.6 are shown in Fig.7 and Fig.8. Methylation rate of the oral
tissue after two weeks administration of green tea is significantly
lower than that after two weeks prohibition of any catechin containi=
ng
food or drink in both COL1A1 and COL1A2.  =
; &n=
bsp;  =
;
.
-  =
;
As Fang et al. reported that catechins inhibit human Dnmt1 and
suppress DNA methylation in vitro (6)(Fig.10), green tea administrat=
ion
is also shown here to inhibit human DNA methylation of collagen gene=
s in
vivo. Type I collagen =
is the
major component of bone and skin, and the content decrease is a func=
tion
of age and leads to bone fracture or wrinkles. Bone fracture especially pl=
ays an
important trigger role of becoming bed-ridden and resulting death of
elderly people through osteoporosis.
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13
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14
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-  =
;
However, the molecular basis for the age-dependent reduction =
in
collagen synthesis is not fully understood. The decrease of colla=
gen is
highly responsible for the bone fracture, osteoporosis, and it is al=
so
responsible for skin elasticity, and its degradation leads to wrinkl=
es
that accompany aging. One pathway of aging could be inhibited by
catechins through the inhibition of DNA methylation by inhibiting the
Dnmt1.
-  =
;
We speculate that age-dependent increase of DNA methylation i=
n COL1A1
and COL1A2 decreases the mRNA expression and protein expression of
collagen not only in the periodontal ligament but also in bone and s=
kin
of whole body, and human Dnmt1 inhibitors like 5-azacytidine, zebula=
rine
or EGCg will reverse the increase of methylation and decrease of
expression, and consequently contribute to the anti-aging and longev=
ity.
-  =
;
This is the first report of green tea inhibition of the human=
DNA
methylation in vivo, to our knowledge.
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- (1) Feinberg AP, Proc. Natl. Acad. Sci. USA, 98, 392, 2001.
- (2) Ahuja N et al., Cancer Res., 58, 5489, 1998.
- (3) Takatsu M et al., Mech. Ageing Dev., 110, 37, 1999.
- (4) Navarro-Peran E et al., Cancer Res. 65, 2059, 2005.
- (5) Yamada H et al., Proc. 2001 ICOS Health & Benefits, 268,
2001.
Yamada H et al.=
, J.
Food Agric. & Environ., 3, 73, 2005
- (6) Fang MZ et al., Cancer Res., 63, 7563, 2003.
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