Chinese Journal of Cancer

窑Original Article窑 
Polymorphic catechol­ ­methyltransferase gene, soy
isoflavone intake and breast cancer in postmenopausal
women: a case­control study 
Qiong Wang 1 , Yuan­Ping Wang 1 , Jia­Yuan Li 1 , Ping Yuan 1 , Fei Yang 2 , Hui Li 3 
1 
Department of Epidemiology, West China School of Public Health, Sichuan University, Chengdu, Sichuan 610041, P. R. China;  2  Chengdu 
Municipal Center for Disease Control & Prevention, Chengdu, Sichuan 610021, P. R. China;  3  Sichuan Cancer Hospital, Chengdu, Sichuan 
610041, P. R. China

揖Abstract铱 Background and Objective:

Methods:

Results:

Conclusions:

Key words: 

Breast cancer is generally known as a kind of  estrogen, but the single nucleotide polymorphism G­A in the 
estrogen­dependent tumor. The adverse estrogen  codon 158 of  gene can decrease the activity of 
metabolite 4­OHE 2  and its oxidation product E 2­3,4
  quinone  encoded  enzyme [2] . In addition, previous studies 
can bind to DNA to form adducts, which finally leads to  found that the soy isoflavones at a low dose could decrease 
breast carcinogenesis [1] . The catechol­  ­methyltransferase  the expression level of  mRNA in MCF­7 breast 
(  ) is a key enzyme in the metabolic detoxification of  cancer cells, and the activity of synthesized COMT enzyme 
was decreased and the methylation of 4­OHE 2  was 
reduced [3,4] , which suggests that the soy isoflavones can 
Correspondence to: Jia­Yuan Li; Tel: +86­28­85501604;  interfere the synthesis and activity of metabolic enzyme of 
Email: lijiayuan73@163.com 
endogenous estrogen. Although recent studies found that 
This paper was translated from Chinese into English  by  Medical 
soy isoflavones can prevent osteoporosis and 
Translation and edited by Wei Liu on 2010­04­28. 
cardio­cerebrovascular diseases in postmenopausal women, 
Received: 2009­12­01; Accepted: 2010­02­01 

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 Chinese Journal of Cancer 

their effect on breast cancer remains unclear. Because soy 

isoflavones at a certain dose and  mutational 
genotype can both decrease the activity of  enzyme,  The genetic polymorphism G­A in the codon 158 of 
it is inferred that they may synergistically increase the risk  COMT gene was determined. The genomic DNA was 
for breast cancer in postmenopausal women. However, to  extracted from each blood sample to perform the 
date, the population studies on the interaction between plant  allele­specific polymerase chain reaction (AS­PCR) in tubes 
estrogen and  gene in Asian women have not been  A and B, respectively, using the following primers: the 
reported. This study was to investigate the relationships  common forward primer added into tubes A and B was 5 爷
between  genetic polymorphism, soy food intake and  ­TCCAAGTTCCCCTCTCTC­3 爷 ; reverse primer 1 (wild 
breast cancer through an age­matched case­control study.  COMTG) added into tube A was 5爷 ­GCACACCTTGTCC 
TTCAC­3 爷 ; reverse primer 2 (mutant COMTA) added into 
tube B was 5爷 ­GCACACCTTGTCCTTCAT­3爷 . The length 
of AS­PCR product was 258 bp. In addition, the quality 
control was performed in 35 random samples (10%) through 
the backward amplification sequencing using the following 
primers: forward primer was 5爷 ­AGCGGATGGTGGATT 
Between May 2007 and July 2009, 176 newly admitted  TCG­3 爷; reverse primer was 5 爷 ­TCTTTAGGGTTCTGGGA 
female patients, who were histologically diagnosed with  TGAC­3爷 . The length of amplified product was 303 bp. The 
primary breast cancer at Sichuan Cancer Hospital, were  sequencing results of genetic polymorphism in the codon 
sequentially enrolled. Other endocrine diseases were  158 of  gene were intercompared to the assessment 
excluded in all these patients. In the same period, 176  results of electrophoresis of AS­PCR products. 
healthy women with physical examinationin in the 
Department of physical Examination in Chengdu Municipal 
Center for Disease Control and Prevention were recruited as 
controls. The patients and controls were matched by age.  The data were input into the database created by 
Moreover, radiation exposure, endocrine­related diseases  Epidata3.0 and analyzed using the Epi Info 2002 software. 
and other tumors were excluded in the control group. All  The coincident degree between the genotype frequencies in 
subjects had lived in Sichuan province for more than 20  control group and Hardy­Weinberg equilibrium was analyzed 
years, and they ranged in age from 40 to 65 years. The  using the Chi­square test. The trend Chi­square test was 
menopause in all subjects was not caused by drugs or  used to analyze the dose­effect relationships between 
treatment. All subjects voluntarily participated in the study,  susceptible genotype, soy isoflavone intake grade 
and accepted the questionnaire survey and laboratory  and breast cancer risk, and 琢 = 0.05 was set as level of 
detection.  significance. After adjustment of related risk factors, the 
logistic regression was used to estimate exposure odd ratio 
(OR) between  polymorphic genotype, soy isoflavone 
intake or their interaction and breast cancer. In the analysis 
In all subjects, the unified questionnaires were used to  of interaction,  ­HH and  ­HL were low risk 
collect demography­related information and breast  genotypes, and  ­LL was high risk genotype. The 
cancer­related risk factors, including age, height, weight,  median intake of soy isoflavones in control group was used 
occupation, educational level, drinking history, smoking  as a cutoff value for the division of high exposure and low 
history, menstruation, childbearing history, lactation,  exposure. 
estrogenic drugs administration, family history, disease 
history, and so on. At the same time, the food­frequency 
questionnaire was used to collect the long­term intake 
information of soy food (bean curd, soybean milk and hard 
bean curd). According to the data of soy isoflavone content 
in soy food reported in references [5­7] , the daily intake of soy 
food was converted into the daily intake of soy isoflavones  The electrophoresis results of amplified products are 
(mg/d). EDTA anticoagulative tubes were used to collect  showed in Figure 1, and the results of backward sequencing 
peripheral venous blood (5 mL from each subject), which  are showed in Figure 2. In Figure 1, the genotype was 
was kept at  ­20益 for further detection.  identified as the wild homozygous genotype (GG, 
­HH) if the target band only occurred in tube A, 

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Figure 1 Electrophoresis of allele鄄specific polymerase

100 bp
chain reaction (AS鄄PCR) products of COMT codon 158
200 bp
300 bp
G寅A genotypes
400 bp PCR were performed in tube A with wild鄄 type primers and in tube B with
500 bp mutant primer pairs, targeting a 258 bp DNA molecule. From left to right,
600 bp
the two bands in lane A and lane B are heterozygous genotype (GA, COMT鄄
HL); the only one band in lane B is mutant homozygous genotype (AA,
COMT鄄 LL), and the only one band in lane A is homozygous wild鄄 type (GG,
COMT鄄 HH).
B A B A B A Marker
GA genotype AA genotype GG genotype

COMT鄄 GG genotype

COMT鄄 GA genotype

Figure 2 Sequencing of COMT

codon 158 G寅A genotypes
Arrows show the COMT codon 158 G 寅A
genotypes, as homozygous wild鄄 type (GG,
COMT鄄 HH), heterozygous genotype (GA,
COMT鄄 HL) and mutant homozygous genotype
(AA, COMT鄄LL) from the top to bottom.

COMT鄄 AA genotype

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 Chinese Journal of Cancer 

mutant homozygous genotype (AA,  ­LL) if only 

occurred in tube B, and heterozygous genotype (GA, 
­HL) if occurred in both tubes A and B. In Figure 2, 
the genotypes in the codon 158 of  gene were 
indicated by the arrows, as wild homozygous genotype (GG, 
In control group, the genotype frequencies were 
­HH), heterozygous genotype (GA,  ­HL), and 
coincident with Hardy­Weinberg equilibrium (字2  = 0.31,  = 
mutant homozygous genotype (AA,  ­LL) from top to 
0.58). Compared with  ­HH as the low risk reference 
bottom. The sequencing results of genotypes in the codon 
genotype, the OR (95% CI) of  ­LL susceptible 
158 of  gene were intercompared to the assessment 
genotype was 3.14 (1.48­6.66). There was a linear trend 
results of electrophoresis of AS­PCR products, and the 
between the genotype frequencies and breast cancer risk 
coincidence rate was 100%. 
(trend 字2  =9.91,  = 0.007). The intakes of soy isoflavones 
were (15.56 依 13.45) mg/d in case group and (19.57 依  
16.27) mg/d in control group, and the median (25% quartile 
­75% quartile) intakes were 10.65 (6.12­22.44) mg/d and 
16.26 (10.67­23.64) mg/d, respectively. The exposure 
grades of soy isoflavones were divided according to the 
In control group, the age ranged from 44 to 77 years  quartiles in control group. Using the lowest soy isoflavone 
with a median of 56 years. In case group, the age ranged  intake (< 10.67 mg/d) as reference grade, other exposure 
from 42 to 77 years with a median of 55 years. The results  grades were all negatively correlated with the risk of breast 
of univariate non­conditional logistic regression showed that,  cancer in a dose­dependent manner (trend 字2  = 28.26,  < 
between case group and control group, there were no  0.001). In order to analyze the interaction between the soy 
significant distribution differences of menarche age,  isoflavone intake and  susceptible genotype, the 
menopausal age, history of benign breast diseases  median exposure value (16.26 mg/d) of soy isoflavone 
(hyperplasia of mammary glands), smoking and drinking  intake in control group was used as a cutoff value, and the 
habit (  >0.05) (data not shown). The risk factors of breast  subjects were divided into high exposure group and low 
caner are listed in Table 1.  exposure group (Table 2). 

Selected risk Cancer patients Cancer鄄 free OR (95% CI) b

factor (No.) subjects (No.) The stratified analysis results suggested that there was 
BMI (kg/m2) no significant relationship between only exposure to high 
< 24 106 126 1.00
risk genotype (  ­LL) and the risk of breast caner, and 
逸 24 70 50 1.66 (1.07-2.60)
there was a negative correlation between high exposure to 
Age at first birth (years)
< 30 166 154 1.00
soy isoflavones alone and the risk of breast caner. In 
逸 30 10 22 0.42 (0.19-0.92) addition, the results also showed that there was no 
No. of abortiona significant correlation between breast cancer and the 
0 62 68 1.00 interaction of  ­LL genotype and high soy isoflavone 
1 or 2 84 102 0.90 (0.58-1.42) intake (逸 16.26 mg/d) (Table 3). 
逸 3 30 6 5.48 (2.14-14.06)
Oral contraceptive use (years)
臆 0.5 150 162 1.00
> 0.5 26 14 2.01 (1.01-3.99)
Time of breast feeding (months)
Breast cancer­related data in China are limited. 
臆 3 30 54 1.00
According to the data from big cities, such as Shanghai and 
> 3 146 122 2.15 (1.30-3.58)
Mastitis
Beijing, the onset age of breast cancer trends to scatter 
No 160 170 1.00 rather than centralize, the morbidity is increasing in women 
Yes 16 6 2.83 (1.08-7.42) under age of 39 or above age of 60 [8] . However, the related 
BMI, body mass index. aAbortion includes natural abortion and induced abortion,
risk factors remain unclear. In postmenopausal women, the 
with 0 as the reference. bOR and 95% CI were calculated by univariate synthesis of estrogen in the ovaries is reduced, but the 
nonconditional logistic regression. estrogen could be in­situ synthesized in breast tissue

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Cancer patients Cancer鄄 free subjects OR (95% CI) a 字2 for trend

Factor
[No. (%)] [No. (%)] (P)
Genotype 9.91 (0.007)
HHb 80(45.5) 96(54.5) 1.00
HL 62(35.2) 66(37.5) 1.43(0.87-2.37)
LL 34(19.3) 14(8.0) 3.14(1.48-6.66)
Soy isoflavone intake (mg/d) 28.26 (<0.001)
< 10.67 88(50.0) 42(23.9) 1.00
10.67-16.25 24(13.6) 46(26.1) 0.28(0.14-0.56)
16.26-23.63 26(14.8) 44(25.0) 0.28(0.14-0.54)
逸 23.64 38(21.6) 44(25.0) 0.32(0.20-0.71)
a
OR and 95% CI were calculated by multivariable nonconditional logistic regression, adjusted for BMI, time of breast feeding, oral contraceptive use, number of abortion,
age at first birth and mastitis. b References.

Isoflavone level Genotypea Cancer patients / cancer鄄 free subjects OR (95% CI) b
Low (< 16.26 mg/d) HH+HL 88/80 1.00
Low (< 16.26 mg/d) LL 24/8 2.20 (0.87-5.52)
High (逸 16.26 mg/d) HH+HL 54/82 0.52 (0.31-0.86)
High (逸 16.26 mg/d) LL 10/6 1.66 (0.52-5.24)
a
Low鄄 risk genotype: HH+HL; high鄄 risk genotype: LL.
b
OR and 95% CI were calculated by multivariable nonconditional logistic regression, adjusted for BMI, time of breast feeding, oral contraceptive use, number of abortion,
age at first birth and mastitis.

through the route outside gonads [9] , suggesting that the  finally completed in 51 women), Cheng  . [14]  found that 
estrogen level in postmenopausal women is easily affected  there were no significant differences of peripheral estrogen 
by exogenous estrogen. In recent years, a part of  level and the expressions of estrogen receptor and cell 
premenopausal women took the soy isoflavone extract  proliferation markers between intervention group and control 
instead of estrogen to prevent osteoporosis and  group. In the present study, it was found that the intake of 
cardiovascular diseases, and their intake of soy isoflavones  soy isoflavones might decrease the risk of breast cancer in 
is increasing correspondingly. Currently, there is no  postmenopausal women in a dose­dependent manner (trend
consensus about the influence of soy isoflavones on breast  字2  = 28.26,  < 0.001). 
cancer. It was found that genistein (Gen) and daidzein  Another kind of factor affecting the risk of breast cancer 
(Dai), two kinds of soy isoflavones, could inhibit cell  is the amount and activity of estrogen­metabolizing enzyme. 
proliferation through inhibiting the activity of epidermal  The activity of  , a key enzyme in estrogen 
growth factor receptor of specific tyrosine protein kinase [10] ,  metabolism, is affected by its polymorphic genotypes. The 
induce or accelerate cell apoptosis through regulating the  activity of  enzyme encoded by mutant homozygous 
expression of apoptosis­related genes (Bax, Bcl­2 and  genotype  ­LL is reduced to 1/3­1/4 of that encoded 
TNFR, etc) [11] , and reduce oxidative damage of DNA via  by wild homozygous genotype  ­ HH [2] , which leads to 
exerting antioxidant action [12] , which consequently play an  the decrease of anticancer substance 2MeO­E 2  and the 
antitumor role. On the other hand, in vitro and in vivo  accumulation of gene toxicant 4­OHE 2  and its oxidation 
experiments also showed that Gen at a low dose (< 10 product E 2­3,4   quinone. Therefore, it is considered that the 
滋mol/L)
  could stimulate the proliferation of  genotype  ­LL could increase the risk of breast 
estrogen­dependent breast cancer cells [3] , suggesting that  cancer. However, the conclusions in previous 
soy isoflavones might exert cancer­promoting effect. A  epidemiological studies are still in dispute. Lavigne  . [15] 
case­control study in Shanghai showed that the soy food  found that carrying  ­LL genotype could increase the 
intake in adolescent females decreases their risk of breast  risk of breast cancer in postmenopausal women. Thompson 
cancer in adulthood [13] . However, through a prospective  . [16]  found that carrying  ­L allele could significantly 
double­blind randomized controlled trial in which the purified  decrease the risk of breast cancer in postmenopausal 
soy isoflavones was supplemented in diet for 3 months in  women. But, in Korean women, Yim  . [17]  found that 
60 healthy postmenopausal Swedish women (the trial was  there was no association between  genotype and the

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