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Certain polyphenols inhibit the activity of aromatase, a critical enzyme in estrogen synthesis that is coded by the CYP19A1 gene. Consumption of polyphenol-rich foods and beverages, thus, may interact with CYP19A1 genetic polymorphisms in the development of endometrial cancer. We tested this hypothesis in a population-based case-control study of 1,204 endometrial cancer cases and 1,212 controls. Dietary information was obtained using a validated food frequency questionnaire. Genotypes of CYP19A1 at rs28566535, rs1065779, rs752760, rs700519 and rs1870050 were available for 1,042 cases and 1,035 controls. Unconditional logistic regression models were used to calculate odds ratios (ORs) and their 95% confidence intervals (95% CI) after adjusting for potential confounding factors. Higher intake of soy foods and tea consumption were both inversely associated with the risk of endometrial cancer with ORs of 0.8 (95% CI: 0.6,1.0) for the highest versus the lowest tertiles intake of soy and 0.8 (95% CI: 06,0.9) for ever tea consumption. The association of SNPs rs1065779, rs752760, and rs1870050 with endometrial cancer was modified by tea consumption (P for interaction < 0.05) but not by soy isoflavone intake. Our findings suggest that tea polyphenols may modify the effect of CYP19A1 genetic polymorphisms on the development of endometrial cancer.
The incidence rate of endometrial cancer in Asian countries, such as China, is substantially lower than that in Western countries, and incidence of the disease has been found to increase when Asian women emigrate to the US (1). One possible explanation is that certain lifestyle factors, particularly diets commonly consumed in Asian countries that include soy and tea, foods and beverages rich in certain polyphenols, may confer some protection against endometrial cancer (2, 3).
Animal and cell culture studies have suggested that dietary polyphenols may have a preventive effect on cancers (4, 5). Furthermore, certain polyphenols, such as isoflavones from soy foods, are classified as phytoestrogens because they have a structure similar to steroid hormones and weak estrogenic or anti-estrogenic activity. Evidence from many in vitro and in vivo observations suggests that phytoestrogens may reduce the risk of hormone-related cancers (2, 6), possibly by interfering with the synthesis, metabolism, and signal transduction of steroid hormones (2, 7, 8). Epidemiological studies have suggested that polyphenols from soy foods and tea may reduce the risk of some cancers (9–15), including endometrial cancer (12–15).
Recent studies have found that tea polyphenols, and isoflavones moderately inhibit the activity of aromatase (CYP19A1) (16–17), the crucial enzyme converting androstenedione and testosterone to estrone and estradiol, respectively. Genetic variations in the CYP19A1 gene have been shown to alter aromatase activity and affect hormone levels (18, 19), possibly influencing the pathogenesis of endometrial cancer (20–23). We hypothesized that isoflavones and tea polyphenols may interact with polymorphisms in the CYP19A1 gene in the development of endometrial cancer and tested this hypothesis in the Shanghai Endometrial Cancer Study (SECS).
The SECS is a population-based case-control study of 1,204 incident cases diagnosed between the ages of 30 and 69 years from 1997 to 2003 and 1,212 age-frequency matched controls. Cancer cases were identified through the population-based Shanghai Cancer Registry. A total of 1,454 eligible endometrial cancer cases were identified during the study period, and 1,204 (82.8 percent) completed an in-person interview. Cases were confirmed by medical chart review. The median interval between diagnosis and interview for cases was 5.6 months. Controls were randomly selected from the general population of Shanghai using the Shanghai Resident Registry and matched to cases according to the age distribution (in 5-year intervals) of endometrial cancer cases in 1996. Women with a history of cancer or hysterectomy were not eligible. Of the 1,629 eligible women identified, 1,212 (74.4 percent) participated in the study. The study protocols were approved by the Institutional Review Boards of all institutes involved in the study, and written informed consent was obtained from all subjects.
Study participants were interviewed in person by trained interviewers. Weight, height and circumferences of the waist and hips were measured according to a standardized protocol at the time of interview. A structured questionnaire was used to elicit detailed information on demographic factors, menstrual and reproductive history, hormone use, prior disease history, physical activity, tobacco and alcohol use, weight history, and family history of cancer. Regular smokers were defined as women who had ever smoked at least 1 cigarette per day for 6 months or more, while alcohol drinkers were defined as women who had ever drunk alcoholic beverages at least 3 times a week for at least 6 months. Similarly, tea drinkers were defined as women drinking tea at least three times per week for 6 months or longer. Tea drinkers were also asked to provide the age at which they started to drink tea regularly, the total number of years they drank tea, and the usual frequency, amount and major types of tea consumed. Participants were asked if they had engaged in regular exercise/sports (at least once a week for at least three months) during the preceding five years. Usual dietary habits over the preceding 5 years were assessed using a validated, quantitative food-frequency questionnaire (FFQ), which included 71 food items and covered more than 85 percent of the commonly consumed foods in Shanghai (24). During the interview, each participant was first asked how frequently she consumed a specific food or group of foods (per day, week, month, year, or never), followed by a question on how many liang (=50 grams) were consumed per unit of time (day, week, month, or year) during the preceding 5 years, ignoring any recent dietary changes. For seasonal foods, each participant was asked to describe her consumption during the month(s) when the food was available. Average daily intake of each seasonal item was estimated by calculating the percentage of months that the food was on the market over a 1-year period. Total soy food intake was measured by soy protein intake and was estimated by multiplying the amount of soy food consumed with the amount of protein in that food according to the Chinese Food Composition Tables (25).
Of the study participants, 857 cases and 837 controls donated a blood sample and 282 cases and 286 controls provided a buccal cell sample. Among those who provided a buccal cell sample, 189 cases and 198 controls provided samples using a mouthwash method; and 93 cases and 88 controls provided samples using a buccal swab method. Due to the very low DNA yield of the buccal swab method, we did not include buccal swab DNA samples in the genotyping. In addition, DNA from blood samples donated by 19 control subjects were not available because of their use in previous studies. Thus, DNA samples from 1,046 (86.9 percent, 857 blood and 189 buccal cell) cases and 1,035 (85.4 percent, 837 blood and 198 buccal cell) controls were included in the genotyping study. Genotyping success rates were between 98.1 percent and 99.6 percent.
The samples were processed on the same day as collection, typically within 6 hours, at the Shanghai Cancer Institute. The buffy coat (WBC) and the buccal cell pellet samples were stored at −70 ºC. Genomic DNA was extracted from buffy coat fractions or buccal cells by using a QIAmp DNA mini Kit (Qiagen, Valencia, CA) following the manufacturer’s protocol. The allelic discrimination of the CYP19A1 polymorphisms at rs28566535 (originally named hcv1664178), rs1065779, rs752760 and rs1870050 were assessed with the ABI PRISM 7900 Sequence Detection System (Applied Biosystems, Inc. (ABI), Foster City, CA) using TaqMan Assay-on-Demand obtained from ABI. The CYP19 rs700519 polymorphism was genotyped using MGB Eclipse (3’ hybridization triggered fluorescence reaction) assay (Epoch Biosciences, Bothell, WA) (A detailed lab protocol has been described elsewhere (23)).
The laboratory staff was blind to the identity of the subjects. Quality control (QC) samples were included in the genotyping assays. Each 384-well plate contained four water, eight CEPH 1347–02 DNA, eight blinded QC DNA, and eight unblinded QC DNA samples. The concordance rates for the quality control samples were 97.4 percent for rs752760 and rs1870050, 98.7 percent for rs700519, 100 percent for rs1065779 and rs28566535. In addition, the DNA of Chinese samples that were used in the HapMap (N=45) and Perlegen (N=24) projects were purchased from Coriell Cell Repositories (http://locusumdnj.edu/ccr/) and genotyped for SNPs rs752760, rs1870050 and rs700519. The consistence rate was 100.0 percent for all 3 SNPs when compared with the data from HapMap (http://www.hapmap.org) and/or Perlegen (http://genome.perlegen.com). The other two SNPs, rs1065779 and rs28566535, were not genotyped in either of these two databases.
Chi-square tests were used to evaluate case-control differences in the distribution of genotypes. Multivariate analyses were performed to adjust for potential confounding variables, including age (continuous variable), education (no formal education, elementary, middle school, high school, college), menopausal status, years of menstruation (<25, <30, <35, ≥35 yrs), number of pregnancies (0, 1, 2, 3, 4, ≥5), body mass index (by quintile), alcohol consumption (never/ever), diagnosis of diabetes (never/ever), physical activity in metabolic equivalent tasks (METs) (by quintile), total energy intake (by quintile), and total fruit and vegetable intake (by quintile). Logistic regression models were used to estimate odds ratios (ORs) and their 95 percent confidence intervals (95 percent CIs). Haplotypes for all five SNPs were constructed based on their chromosome position (rs1065779- rs700519- rs28566535- rs752760-rs1870050) via a Bayesian approach using PHASE software (26, 27). Due to the significant interaction of SNPs rs1065779, rs752760 and rs1870050 with tea consumption in the risk of endometrial cancer, haplotypes were reconstructed based on these three SNPs (rs1065779-rs752760-rs1870050). The differences of haplotype frequencies between cases and controls were tested with 100 times of permutation (28). The haplotype data were analyzed using weighted haplotype probability. Logistic regression was employed to assess the association of endometrial cancer risk with each haplotype under dominant, recessive, and additive genetic models. Stratified and joint association analyses were performed to evaluate whether intake of soy food and tea modified the associations of CYP19A1 genotypes with endometrial cancer risk. The likelihood ratio test was conducted to formally test multiplicative interactions. P-values of less than 0.05 (two-sided probability) were interpreted as statistically significant.
Selected demographic and risk factors were compared between cases and controls as shown in Table 1. Cases and controls were similar in age, marital status, and education. There were no significant differences between cases and controls with respect to use of hormone replacement therapy, cigarette smoking, or total fruit and vegetable intake. Compared with controls, cases were significantly more likely to have a younger age at menarche, an older age at menopause, a greater number of years of menstruation, to have a first degree relative with any cancer or a hormone-related female cancer, a higher body mass index, to be pre-menopausal, nulliparous, to be diagnosed with diabetes, and to consume more total energy. Cases were less likely to be physically active, to drink alcohol, or to use oral contraceptives. There were no appreciable differences between subjects included in the present study and those included in the parent study (data not shown).
The distribution of all five SNPs was consistent with Hardy-Weinberg equilibrium among controls (data not shown in the table). The frequencies of the genotypes were significantly different between cases and controls for SNPs rs1065779 and rs1870050 (Table 1).
High intake of soy protein was associated with a decreased risk of endometrial cancer, with an OR of 0.8 (95 percent CI: 0.6,1.0) for the highest versus the lowest tertile of intake (Table 2). Compared to non tea drinkers, tea drinkers had a 20 percent (OR: 0.8, 95 percent CI: 0.6,0.9) reduced risk of endometrial cancer. Of 737 tea drinkers, 665 (90.2 percent) drank primarily green tea and 46 (6.2 percent) drank mainly black tea. Excluding the 72 women who primarily drank non-green tea did not change the results substantially. Among tea drinkers, we did not observe a significant dose-response association when frequency, amount or duration of tea consumption was considered. Soy intake and tea consumption was not significantly correlated in this population (data not shown).
We evaluated the joint effect of diet and CYP19A1 genetic polymorphisms on endometrial cancer risk (Table 3). Soy protein intake did not significantly interact with any of the five CYP19A1 SNPs. We estimated the amount of soy isoflavone intake by using the Chinese Food Composition Tables (25) and assessed their association with endometrial cancer risk and their potential modifying effect on the gene-cancer associations.
We found that intake of isoflavones was inversely related to endometrial cancer risk but did not modify the gene-disease association (data not shown). We found that tea consumption significantly modified the association of endometrial cancer risk with three SNPs: rs1065779 (P for interaction=0.01), rs752760 (P for interaction=0.04), and rs1870050 (P for interaction=0.01). The most notable finding was that SNPs rs1065779 and rs1870050 were related to a reduced risk of endometrial cancer only among women who drank tea regularly. Additionally adjusting for soy protein intake did not materially change the results (data not shown).
We present in Table 4 the results of association analyses between the common (frequency>5 percent) CYP19A1 haplotypes (for all five SNPs and only those three SNPs that significantly interacted with tea consumption, rs1065779-rs752760-rs1870050). A global significant difference in haplotype frequencies was found for both the 5-SNP and 3-SNP haplotypes (P<0.05). Haplotypes TCATC and TTC were related to a reduced risk of endometrial cancer and the associations were more pronounced among tea drinkers. For haplotypes GCCTA and GTA, conversely, a positive association with endometrial cancer was observed (Table 4).
We conducted further exploratory analyses by grouping women together by the number of minor alleles of these SNPs that they carried. Compared to women with no or one minor allele in the diplotype derived from all five SNPs or three SNPs (rs1065779, rs752760 and rs1870050), the risk of endometrial cancer decreased with an increasing number of minor alleles (Table 5). This reduction in risk was stronger among tea drinkers than among non tea drinkers, although the interaction tests were only significant for the three-SNP-based diplotype (P for interaction = 0.02).
In this population-based case-control study, we found that dietary consumption of soy foods and tea was inversely associated with endometrial cancer risk. Three polymorphisms (rs1065779, rs752760, and rs1870050), in both single marker and haplotype analyses, were found to be related to the risk of endometrial cancer and their effects were modified by tea consumption.
Our findings of an inverse association of endometrial cancer with intake of soy food and tea are consistent with previous studies (12–15, 28), including our earlier reports from subsets of the SECS (12–15). Soy foods are rich in isoflavones, which have estrogenic or anti-estrogenic properties due to the similarity of their structure to estradiol (7,8). It has been suggested that these phytoestrogens, as well as tea polyphenols, decrease estrogen biosynthesis and produce anti-estrogenic effects through lowering the activity of aromatase (16–17, 29, 30). In addition to this anti-estrogenic effect, polyphenols also have other anti-carcinogenic properties, such as an antioxidant effect (31).
Aromatase is a crucial enzyme catalyzing three consecutive hydroxylation reactions converting C19 androgens to aromatic C18 estrogenic steroids, namely, androstenedione and testosterone to estrone and estradiol, respectively. The CYP19A1 gene codes aromatase and its transcription is regulated by tissue-specific promoters (32). The SNPs rs1870050 and rs752760 are located in the first exon, close to promoter I.1, the major promoter for the placenta. The SNP rs28566535 is located in the first exon close to promoter I.4, which is the promoter for adipose tissue, bone, and skin. The SNP rs1065779 is located in intron 9, 53 bp upstream of exon 10, which may affect transcription or expression of aromatase. The SNP rs700519 (Arg264Cys) is located in exon 7. By causing an amino acid change from Arg to Cys, this SNP may result in a change of enzyme activity. Previous studies have found that variants in the CYP19A1 gene lead to altered aromatase activity and estrogen levels (18, 19, 33), which are related to risk of hormone-related cancers (34, 35), including endometrial cancer (21, 22). We also found that several polymorphisms, SNPs rs1065779, rs752760 and rs1870050, were associated with risk of endometrial cancer (23), suggesting that these polymorphisms may play a critical role in the development of endometrial cancer.
In this study, we observed significant interactions between three CYP19A1 genetic polymorphisms and tea consumption. Although these findings are new, they are biologically possible. It has been shown that green tea catechins and black tea polyphenol theaflavins decrease aromatase enzyme activity (17, 31). Our finding that the associations of rs1065779, rs752760 and rs1870050 with endometrial cancer are more pronounced in tea drinkers is in agreement with these results. It is possible that SNP rs1065779 and rs752760, two polymorphisms close to the promoter for the placenta, may play an important role in the pathogenesis of endometrial cancer. SNP rs1065779, through its effect on transcription or the expression of aromatase, may also be involved in the carcinogenesis of endometrial cancer. The tea and single SNP interactions were also confirmed in the haplotype analyses. Of note, these interactions were most evident under the recessive model. Given that the functionality of SNPs and associated haplotypes are not well understood and that the genetic regulation of aromatase has not been extensively studied in endometrial carcinoma, further studies with a more comprehensive SNP coverage of the CYP19A1 gene are needed to evaluate our findings.
We did not observe a significant interaction between intake of soy foods and any of the five polymorphisms in the CYP19A1 gene. These results are not surprising because previous studies have relatively consistently found that the inhibitory activity of isoflavones on aromatase, if any (30, 36–38), was much weaker than that of other polyphenols (30, 37–40). On the other hand, in vitro studies have found that soy isoflavones can inhibit the activity of 17β-hydroxysteroid dehydrogenase type I (17β-HSD1), a key enzyme in catalyzing estrone (E1) to the biologically more active estradiol (E2) (2, 41, 42). We have previously reported from the same study that soy consumption may interact with polymorphisms in the 17β- HSD1 gene in relation to endometrial cancer risk (43). Taken together, these findings suggest that isoflavones and tea polyphenols may modify the associations of genetic polymorphisms in different estrogen-related genes with endometrial cancer risk.
As with all case-control studies, the possibility for recall bias could not be completely eliminated. We tried to minimize recall bias by shortening the interval between diagnosis and interview for cases and by asking participants to ignore any dietary change over the preceding year. Furthermore, it is unlikely that recall bias would depend on an individual’s genotype. In this study, we collected information on tea consumption according to the amount of tea leaves consumed because Chinese people drink tea by putting loose tea leaves in a cup and repeatedly adding water using the same tea leaves. Variability in the amount of water added to the tea may have introduced measurement errors with regard to tea consumption. Likewise, soy intake was also subject to measurement errors. Misclassification of tea and soy consumption, which is most likely to be non-differential, may bias the results towards the null. Although we adjusted for many potential confounding factors, we still cannot exclude the possibility that residual confounding or related dietary patterns may partially explain our results. Finally, we only included five polymorphisms in this study. These SNPs were chosen based on literature review of published data and the potential functionality of the SNPs. Thus, our study is not as comprehensive as other studies which have applied the haplotype tagging SNP approach (44, 45). Therefore, we cannot exclude the possibility that there may be other polymorphisms that interact with dietary polyphenols and that these unstudied polymorphisms may be responsible for the gene-tea interaction observed in this study.
Strengths of this study included the population-based study design, high response rate, high DNA sample donation rate, and the low frequency of hysterectomy in the population (3.6 percent), which minimized the selection bias. The homogeneous ethnic background (>98 percent Han Chinese) of the study participants avoided potential confounding from ethnicity. The large sample size enabled us to explore potential gene-environment interactions.
In summary, this population-based study suggests that consumption of soy food and tea was related to a reduced risk of endometrial cancer. Tea consumption may interact with genotypes of the CYP19A1 gene in the etiology of endometrial cancer.
We thank Dr. Fan Jin for her contributions to implementing the study in Shanghai, Drs. Qiuyin Cai, Hongmei Wu, and Regina Courtney for their contributions to the genotyping and Ms. Bethanie Hull for her assistance in the preparation of this manuscript. This study would not have been possible without the support of all of the study participants and research staff of the Shanghai Endometrial Cancer Study.
1This work was supported by USPHS grant R01CA92585 from the National Cancer Institute.