Interaction between cyclooxygenase-2 gene polymorphism and dietary n-6 polyunsaturated fatty acids on colon cancer risk: The Singapore Chinese Health Study

This case–control study of 310 colorectal cancer cases and 1177 controls in a nested prospective, population-based cohort of Singapore Chinese subjects found a statistically significant association between the cyclooxygenase (COX)-2 −765G>C gene polymorphism and colon cancer risk among high consumers of dietary n-6 polyunsaturated fatty acids (odds ratio=2.38, 95% confidence interval=1.23–4.59).

Diet is believed to be the single most important contributor to colonic carcinogenesis (Tomatis et al, 1990). Experimental data have shown that saturated fatty acids (SFAs) and n-6 polyunsaturated fatty acids (PUFAs) have tumour-enhancing properties in the colon (Reddy and Maeura, 1984;Zhao et al, 1991, Woutersen et al, 1999. Epidemiological data suggest that increased consumption of all meat or red meat, which contains high levels of SFAs, is strongly associated with colorectal cancer (Giovannucci and Goldin, 1997;Sandhu et al, 2001), but there is only limited evidence on the role of dietary n-6 PUFAs (Zock and Katan, 1998;Flood et al, 2003).
The putative mechanism through which dietary n-6 PUFAs may enhance colonic carcinogenesis is the increased formation of prostaglandins, with the rate-limiting and committal step being mediated by the cyclooxygenase (COX)-2 enzyme (Dubois et al, 1998). Prostaglandins possess a wide spectrum of procarcinogenic properties (Handler et al, 1990;Cowlen and Eling, 1993;Coffey et al, 1997;Dermott et al, 1999). We therefore hypothesised that functional COX-2 gene polymorphisms may impact on the conversion of n-6 PUFAs into prostaglandins, with consequent change in level of cancer risk. A single nucleotide polymorphism (À765G4C) in the promoter region of the COX-2 gene was recently described (Papafili et al, 2002). We therefore investigated whether this COX-2 gene polymorphism was related to colorectal cancer risk within a population-based, prospective cohort of middle-aged and older Chinese men and women in Singapore.

Study subjects
The study design and subject recruitment of the Singapore Chinese Health Study have been described (Hankin et al, 2001). Briefly, 63 257 Chinese women and men aged 45 -74 years belonging to the Hokkien or Cantonese dialect group were enrolled in the study between April 1993 and December 1998. At recruitment, information on lifestyle factors and usual diet over the last year was obtained through in-person interviews. The dietary component of the questionnaire was validated through a series of 24-h food recalls (Hankin et al, 2001). Respondents were asked to choose from predefined frequency and portion size categories for each of the 165 listed food/beverage items that he/she consumed during the past 12 months. We used the Singapore Food Composition Table to estimate average daily intake of 96 nutrient and nonnutrient compounds for each study subject (Hankin et al, 2001). The Institutional Review Boards at the University of Southern California and the National University of Singapore had approved this study.
We identified incident colorectal cancer cases through the population-based cancer registry in Singapore (Chia et al, 2000). As of 30 April 2002, 592 colorectal cancer cases had occurred among cohort participants. All cases (including one carcinoid tumour and two in situ cancers) were histologically confirmed except three (ascertained by death records and clinical evidence). Details of the biospecimen collection, processing and storage procedures have been described (Koh et al, 2003). Briefly, we attempted to collect blood and single-void urine specimens from a random 3% sample of cohort enrollees. If the subject refused to donate blood, he/she was asked to donate buccal cells. We collected blood/buccal cell samples from 1194 subjects during April 1994 -July 1999. Of these subjects, 13 developed colorectal cancer by 30 April 2002, and the remaining 1181 subjects constituted the referent group for the present study. We also attempted to collect blood/buccal cell and urine samples from all incident colorectal cancer cases. Of the 592 colorectal cancer cases, 312 (53%) donated blood/buccal cell samples.

COX-2 genotyping
Genomic DNA was extracted from buffy coats (228 cases and 895 controls) and buccal cell samples (84 cases and 286 controls) using a QIAamp 96 DNA Blood Kit (Qiagen, Valencia, CA, USA). A TaqMan assay for the À765G4C COX-2 polymorphism was developed using a TaqMan PCR Core Reagent kit (Applied Biosystems Inc., Foster City, CA, USA). The oligonucleotide primers for amplification of the polymorphic region of COX-2 were GC093 for (5 0 -CATTAACTATTTACAGGGTAACTGCTTAGG-3 0 ) and GC093rev (5 0 -CCCCCTCCTTGTTTCTTGGA-3 0 ). In addition, the fluorogenic oligonucleotide probes (TaqMan MGB Probes; ABI) used to detect each of the alleles were GC093F (5 0 -CTTTCCCGCCTCTCT-3 0 ) labelled with 6-FAM to detect the G allele and GC093V (5 0 -CTTTCCCCCCTCTCT-3 0 ) labelled with VIC to detect the C allele. Experimental samples were compared to 12 controls to identify the three genotypes at each locus (GG, GC, CC). All samples were processed without knowledge of their case/control status. Any samples that were outside the parameters defined by the controls were identified as noninformative and were retested. Four controls and two cases had noninformative COX-2 genotypes and were excluded from the present analysis.

Statistical analysis
Data were analysed by standard methods for unmatched casecontrol studies (Breslow and Day, 1980). Unconditional logistic regression models were used to examine the associations between COX-2 genotypes and risk of colorectal cancer, and their possible modification by n-6 PUFA intake. The associations were measured by odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) and P-values (two-sided). Limited by the very low frequency of the CC genotype (0.003), the GC and CC genotypes were combined when compared with the GG genotype. All ORs were adjusted for age (year) at recruitment, year of recruitment, gender, dialect group (Cantonese, Hokkien), level of education (no formal schooling, primary school, secondary school and higher), body mass index (o20, 20 to o24, 24 to o28, 28 þ kg m À2 ), smoking status (never, exsmoker, current smoker), frequency of alcohol consumption (nondrinker, monthly drinker, weekly drinker, daily drinker), and familial history of colorectal cancer (yes, no).

RESULTS
Of the 592 incident colorectal cancer cases, 282 were excluded from the present analysis due to unavailable blood/buccal cell samples (n ¼ 280) or noninformative COX-2 genotype (n ¼ 2). Cases included in the present study (n ¼ 310) were comparable to those excluded in terms of age (mean: 65.4 vs 66.1 years), but slightly different in gender (57 vs 49% male), dialect group (45 vs 37% Cantonese) and level of education (69 vs 60% attaining primary school education or higher).
In total, 180 (58%) cases had colon cancer, and the remaining cases had either rectal or rectosigmoid cancers. Table 1 shows the distributions of selected characteristics of colorectal cases and controls. Cases were older, less educated, more obese and more likely to smoke cigarettes than controls. Intakes of total calories, total fat, SFAs, monounsaturated fatty acids (MUFAs), PUFAs, n-3 PUFAs, n-6 PUFAs, fibre, calcium or folate were comparable between cases and controls.
Among control subjects, the G and C allele frequencies of the COX-2 genotype were 0.952 and 0.048, respectively, and the GG, GC and CC genotype frequencies were 0.907, 0.090 and 0.003, respectively. These genotypic distributions were in Hardy -Weinberg equilibrium (P ¼ 0.43). Overall, there was no association between colorectal cancer risk and COX-2 À765G4C genotype or n-6 PUFA intake (Table 2). When subjects were stratified into high (above median) vs low (below median) intake levels of n-6 PUFAs, a borderline statistically significant association between genotype and risk was observed among high consumers of n-6 PUFAs (OR ¼ 1.65, 95% CI ¼ 0.95 -2.87), which was mainly confined to colon cancer (OR ¼ 2.38, 95% CI ¼ 1.23 -4.59) ( Table 3). There was no association between genotype and rectal cancer risk regardless of dietary n-6 PUFA intake levels. There was indication of an interaction effect between COX-2 genotype and dietary n-6 PUFAs in colon cancer (P ¼ 0.07), which was absent in rectal cancer (P ¼ 0.51). The corresponding P-value for the gene -diet interaction effect in colorectal cancer combined was 0.10.

DISCUSSION
In this cohort of Singapore Chinese, we reported a statistically significant effect of the COX-2 À765G4C gene polymorphism on colon cancer risk among subjects with high intake of dietary n-6 PUFAs. Our data support the hypothesis that COX-2 exerts its effects on colon carcinogenesis through its influence on prostaglandin synthesis from n-6 PUFAs.
The current study has several strengths.
(1) Our prospective study design precludes the possibility of recall bias. Furthermore, reliable dietary nutrient estimates including n-6 PUFAs were assessed using a validated food frequency questionnaire. (2) The nationwide cancer registry has been in place in Singapore since 1968 (Parkin et al, 2002), and migration out of Singapore has been negligible since inception of the study. This relatively complete ascertainment of cancer and death outcomes eliminates a concern for potential selection bias. (3) Study subjects originated from two contiguous regions in Southern China, resulting in a genetically homogeneous study population that facilitated the investigation of gene -disease associations. (4) Exposure information on other known/suspected risk factors for colorectal cancer was collected and accounted for in all analyses of gene -diet -cancer risk associations.
The chief limitation of our study is the lack of information on use of COX-2 inhibitors, which may bias the effect of COX-2 genotype on risk. However, if use of COX-2 inhibitors were to exert a confounding effect on the observed COX-2 genotype/colon cancer association, our inability to control for such confounding is likely to lead to an underestimation, rather than an overestimation, of the risk associated with the putative high-activity genotype. This is because use of COX-2 inhibitors is likely to be more common among subjects with more severe symptoms of inflammation, possibly due to the possession of the high activity COX-2 genotype. Another weakness of the present study is our relatively small number of cancer cases, which may result in less precise estimation of risk factor -disease associations.
The major n-6 PUFA in most diet is linoleic acid, the precursor of arachidonic acid. The latter is consequently converted to various prostaglandins, and COX is the crucial and rate-limiting enzyme for this conversion. There is compelling evidence that prostaglandins play important roles in colorectal carcinogenesis by enhancing cell proliferation and growth, promoting angiogenesis and inhibiting apoptosis (Cao and Prescott, 2002;Stoehlmacher and Lenz, 2003). COX-2 gene expression and its mRNA and protein levels were markedly elevated in most human colorectal cancers relative to adjacent normal mucosa (Kargman et al, 1995;Sano et al, 1995). It is hypothesised that the COX-2-associated effect on colorectal carcinogenesis is due to the increased production of prostaglandins from dietary n-6 PUFAs (Eberhart and Dubois, 1995). In support of this hypothesis, high dietary n-6 PUFAs has been shown to promote colon tumorigenesis by upregulating COX-2 expression in animal studies (Singh et al, 1997).
The human COX-2 gene is mapped to chromosome 1q25.2 -q25.3 and spans about 8.3 kb pairs with 10 exons (Kosaka et al, 1994). Previous studies on the 5 0 flanking region of the human COX-2 gene show that this region contains a canonical TATA box as well as several putative factor elements that are critical in inducing COX-2 gene transcription, such as Sp1, NF-kB, GRE (glucocorticoid) and IRE (insulin) elements (Tazawa et al, 1994;Yang et al, 1997). The region from nucleotide À827 to À454 has been described as a negative region since deletion of this region led to increased luciferase activity in reporter expression studies. The À765G4C mutation lies within this region, and is also within one of the five putative Sp1 elements (Yang et al, 1997). At present, data on the functionality of the À765G4C polymorphism and the direction/magnitude of change in protein expression/activity between the C and G alleles are limited and mixed (Papafili et al, 2002;Schneider et al, 2003).
In summary, the present study provides the first epidemiological evidence for a possible cause-and-effect connection between the production of prostaglandins from n-6 PUFAs through the enzymatic activity of COX-2, and increased risk of tumour development in the colon. Our novel findings require confirmation in larger studies with varying levels of substrate intake and genotype frequency.  ORs were adjusted for age at recruitment, year of recruitment, gender, dialect group, level of education, body mass index, smoking status, frequency of alcohol consumption, and familial history of colorectal cancer; CI ¼ confidence interval. b In addition to all variables listed above, OR were adjusted for total energy intake. ORs were adjusted for age at recruitment, year of recruitment, gender, dialect group, level of education, body mass index, smoking status, frequency of alcohol consumption, and familial history of colorectal cancer; CI ¼ confidence interval. b Defined as less than or equal to the median ('low') and greater than the median ('high') intake level (6.96 g day À1 ) of dietary n-6 PUFAs among all cohort members.