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Mutation Research 663 (2009) 52–59

Contents lists available at ScienceDirect

Mutation Research/Fundamental and MolecularMechanisms of Mutagenesis

journa l homepage: www.e lsev ier .com/ locate /molmutCommuni ty address : www.e lsev ier .com/ locate /mutres

unctional polymorphisms of cyclooxygenase-2 (COX-2) gene and risk forsophageal squmaous cell carcinoma

ohit Upadhyaya, Meenu Jaina, Shaleen Kumarb, Uday Chand Ghoshalc, Balraj Mittal a,∗

Department of Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareilly Road, Lucknow 226014, IndiaDepartment of Radiotherapy, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareilly Road, Lucknow 226014, IndiaDepartment of Gastroenterology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareilly Road, Lucknow 226014, India

r t i c l e i n f o

rticle history:eceived 10 September 2008eceived in revised form 9 January 2009ccepted 26 January 2009vailable online 4 February 2009

eywords:OX-2 gene polymorphismssophageal squamous cell carcinomanflammatory pathwayancer susceptibilityOX-2 haplotypes

a b s t r a c t

Cyclooxygenase-2 (COX-2) influences carcinogenesis through regulation of angiogenesis, apoptosis andcytokine expression. We aimed to evaluate association of COX-2 polymorphisms with predisposition toesophageal squamous cell carcinoma (ESCC), its phenotype variability and modulation of environmentalrisk in northern Indian population. We genotyped 174 patients with ESCC and 216 controls for COX-2 genepolymorphisms (−765G>C; −1195G>A; −1290A>G; 3′UTR 8473T>C) using PCR-RFLP. Data were statisti-cally analyzed using chi-square test and logistic regression model. COX-2 −765C allele carriers were atincreased risk for ESCC (OR = 1.66; 95% CI = 1.08–2.54; P = 0.004). However, −1195G>A; −1290A>G; 3′UTR8473T>C polymorphisms of COX-2 gene were not significantly associated with ESCC. We observed sig-nificantly enhanced risk for ESCC due to interaction between COX-2 −1195GA × −765GC + CC genotypes(OR = 4.60; 95% CI = 1.63–13.01; P = 0.004). High risk to ESCC was also observed with respect to COX-2haplotypes, A−1290G−1195C−765T8473 and A−1290A−1195C−765T8473 [OR = 3.35; 95% CI = 0.83–13.44; P = 0.089;OR = 4.28; 95% CI = 0.43–42.40; P = 0.246] however, it was not statistically significant. Stratification of sub-

jects based on gender showed that females were at higher risk for ESCC due to COX-2 −765C carriergenotypes (OR = 2.97; 95% CI = 1.23–7.18; P = 0.016). In association of genotypes with clinical charac-teristics, −765C carrier genotype conferred risk of ESCC in middle third of esophagus (OR = 1.78; 95%CI = 1.08–2.93; P = 0.023). In case-only analysis, interaction of environmental risk factors and COX-2 geno-types did not further modulate the risk for ESCC. In summary, COX-2 −765G>C polymorphism confersESCC susceptibility particularly in females and patients with middle third anatomical location of the

-2 −1

tumor. Interaction of COX

. Introduction

Cyclooxygenase (COX) is involved in several inflammatory path-ays [1]. One of its isoform, COX-2 or prostaglandin endoperoxide

ynthase-2 (PTGS-2) is an inducible enzyme, which catalyzeshe conversion of arachidonic acid to prostaglandins (PGs) inesponse to various pro-inflammatory and mitogenic stimuli suchs cytokines and growth factors [2]. Over-expression of COX-

influences cancer development, including hyper-proliferation,ransformation, tumor growth, invasion, metastasis and there-

ore, may increase the risk of esophageal cancer (EC). Due toncreased COX-2 expression in gastric and esophageal cancers,OX-2 inhibitors have been advocated as promising drugs forastrointestinal as well as aerodigestive tract malignancies [3,4].

∗ Corresponding author. Tel.: +91 522 2668973/004–8x2322;ax: +91 522 2668017/2668074.

E-mail addresses: balraj@sgpgi.ac.in, bml pgi@yahoo.com (B. Mittal).

027-5107/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.mrfmmm.2009.01.007

195GA and −765C carrier genotypes also modulates ESCC risk.© 2009 Elsevier B.V. All rights reserved.

However, COX-2 genetic profile should be taken in consideration todefine better treatment regimen for patients afflicted with cancer.

Transcriptional regulation has been shown to be a majormechanism in regulating gene expression. In addition, post-transcriptional mechanisms influencing stability of COX-2 mRNAare also important. Thus, it is possible that naturally occurringsequence variations in the promoter and 3′ untranslated regions ofCOX-2 gene might contribute towards differential COX-2 expressionand a substantial degree of inter-individual variability in suscepti-bility to cancer as well as response to the treatment. The humanCOX-2 gene, mapped to chromosome 1q25.2–q25.3, is about 8.3 kbpairs in size and contains 10 exons [5]. The COX-2 promoter regionconsists of several key cis acting regulatory elements includingstimulatory protein-1 (SP1) suggesting involvement of complex

array of factors in its regulation [6]. Genetic polymorphisms in theCOX-2 gene have been described to influence its expression throughtranscriptional and post-transcriptional mechanisms. Papafili et al.[7] identified −765G>C (rs20417) polymorphism; located withina putative SP1 binding site and demonstrated approximately 30%

R. Upadhyay et al. / Mutation Research 663 (2009) 52–59 53

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ig. 1. Gel pictures of COX-2 genotypes: (a) COX-2 −1290A>G polymorphism Lane 1:1195G>A polymorphism Lane 1: 50 bp ladder; Lane 2: AA genotype; Lane 3: GG gane 2: GG genotype; Lane 3: CC genotype; Lane 4: GC genotype; (d) COX-2 8473 T>C genotype.

ifference of gene expression in cells transfected with plasmids car-ying promoter variants. In contrast, Szczeklik et al. [8] showed10-fold increased biosynthesis of PGs in monocyte cultures ofatients with COX-2 −765CC genotype. Another COX-2 polymor-hism −1195G>A (rs689466) creates a c-MYB binding site, whichesults in higher transcriptional activity of the COX-2 gene whileolymorphism −1290 A>G also appears to have functional signifi-ance [9]. There are other variations like COX-2 + 8473 T>C (rs5275)ocated at 3′ untranslated region (UTR) which also alters genexpression through both messenger stability and translational effi-iency [10].

In recent years, studies have reported association of COX-2 poly-orphisms with risk of esophageal cancer from Chinese and Cau-

asian populations [9,11,12]. COX-2 haplotype A−1290A−1195C−765nd CA carriership was reported to be associated with risk ofsophageal squamous cell carcinoma (ESCC) and adenocarcinoma9,12]. Furthermore, multiplicative interaction was noted betweenhe 12-LOX Gln/Gln and COX-2 −1195AA or −765GC genotypes [11].

Incidence of esophageal cancer exhibits wide geographicalariation and COX-2 genetic variations also display remarkable eth-ic differences in esophageal cancer susceptibility [9]. Therefore,resent study was designed to evaluate the association of COX-polymorphisms with risk of ESCC, disease phenotypes, as well

s influence of environmental interactions in modulating risk ofancer in a northern Indian population.

. Materials and methods

.1. Study subjects

The study subjects for the present study were same as described in our previ-us studies [13,14] except that additional cases were added. Briefly, during a 4-yeareriod from August 2003 to September 2007, a total of 174 histopathology confirmed

atients with ESCC were enrolled from the outpatient clinics of Radiotherapy andastroenterology of Sanjay Gandhi Post-graduate Institute of Medical Sciences, Luc-now. Patients were excluded if they had non-malignant conditions like corrosivesophageal injury, Achalasia injury, Barrett’s esophagus or gastro-esophageal refluxisease (GERD). Clinical and demographic data were recorded only for the patients instandard proforma. Clinical parameters included histology, tumor location, envi-

notype; Lane 2: AG genotype; Lane 3: GG genotype; Lane 4: 50 bp ladder; (b) COX-2e; Lane 4: AG genotype; (c) COX-2 −765 G>C polymorphism Lane 1: 50 bp ladder;

morphism Lane 1: CC genotype; Lane 2: 50 bp ladder; Lane 3: TT genotype; Lane 4:

ronmental risk factors such as tobacco (smoking and non-smoking), alcohol andany work-related exposure to toxic chemicals as described previously [15]. A totalof 216 age and sex matched, healthy controls were also enrolled at the same timefrom those visiting our hospital for routine check-up and residing in adjoining areasof northern India. Selection criteria for controls included no evidence of any per-sonal or family history of cancer or other malignant conditions. Informed consentwas obtained from all recruited individuals and the study was approved by ethicalcommittee of the Institute.

2.2. Sample collection

Venous blood sample (5 ml) was collected form each subject and was kept frozentill DNA extraction. The genomic DNA was extracted from peripheral blood leuko-cytes using salting out method as described previously [15]. The quantification ofDNA was done using Nanodrop Analyzer (ND-1000) spectrophotometer (Nano DropTechnologies, Inc., Wilmington, DE, USA).

2.3. Genotyping of COX-2 (−1290A>G; −1195G>A; −765G>C) polymorphisms

Each PCR was performed in a total reaction volume of 15 �l with 20 pmol ofeach primer, genomic DNA (150 ng) and 1× PCR master mix (MBI Fermentas, USA).The primers and the reaction conditions used were as described previously [9]. PCRproducts were digested at 37 ◦C overnight with 5 U of respective restriction enzymes;RsaI, PvuII and Hha I (MBI Fermentas, USA). The digestion products were then sep-arated on a 15% polyacrylamide gel. Genotyping band patterns and band sizes havebeen depicted in Fig. 1a–c.

2.4. Genotyping of COX-2 (+8473T>C) polymorphism

PCR was conducted in a final reaction volume of 25 �l with 25 pmol of eachprimer; as reported by Hu et al. [16]. Five microliters of the PCR products weredigested at 50 ◦C overnight with 5 U of BclI (MBI Fermentas, USA). The digestionproducts were then separated on a 15% polyacrylamide gel. PCR product with 8473Callele produced two fragments of 124 and 23 bp while 8473T allele yielded a single147 bp fragment (Fig. 1d).

2.5. Quality control

The cases and controls were randomized before RFLP analysis. Twenty percentof samples from patients and controls including samples of each genotype were re-genotyped by other laboratory personnel and results showed 100% similarity in bothof the conditions. No discrepancy was found after sequencing randomly selected10% samples. Gel documentation was done by AlphaimagerTM 1220 (Alpha InnotechCorporation, San Leandro, CA, USA).

5 ion Research 663 (2009) 52–59

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Table 1Clinical characteristics of esophageal squamous cell cancer patients.

Patients (mean age ± SD = 56.79 years ± 12.07) N = 174Controls (mean age ± SD = 55.04 years ± 11.05) N = 216

Clinical characteristicsa N (%)Location (n = 174)Upper 23 (13.2)Middle 102 (58.6)Lower 49 (28.2)

Lymph nodea (n = 129)Present 83 (64.3)

Environmental factorsa (n = 138) N (%)Tobacco habitSmokers 32 (20.5)Tobacco chewers 51 (32.7)Smokers + tobacco chewers 42 (26.9)Non-tobacco users 31 (19.9)

Alcoholic habitDrinker 51 (37.0)

Occupational exposure

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.6. Statistical analysis

Data were analyzed using SPSS 15.0 (Chicago, IL, USA). �2 test was usedo determine differences in genotype/allele frequencies and deviation fromardy–Weinberg equilibrium. Logistic regression was used to calculate odds ratio

or various predictors and adjusting the confounding effect of age and gender.he homozygous genotype for the common allele of each SNP in the controlroup was used as the reference in calculating odds ratios (OR) and 95% confi-ence intervals (95% CI). Statistical analysis of the COX-2 haplotypes estimationnd linkage disequilibrium was conducted using the Arlequin software ver. 2.00y expectation–maximization algorithm [17] and SNPAnalyzer Version 1.0 (ISTECHnc.) was used for calculating D′ value which is a measure indices of linkage disequi-ibrium (LD). Since the response rate was low in controls so a case only analysis waserformed for gene–environment interaction as described earlier [18]. Age variableas expressed as mean (years) ± SD. The significance level for all the statistical testsas set at P < 0.05. P-values were corrected (Pcorr) for multiple corrections (Bonfer-

oni correction) in case of further sub grouping or stratification. The 0.05/N thresholdas set up according to the Bonferroni correction to account for the multiple testing

ssues. N is the number of tested markers (genotypes) for each gene polymorphism.herefore, for applying Bonferroni correction we have multiplied P value by theumber of comparisons (e.g.: in case of a polymorphism having three genotypes weultiplied P value by three; for carrier analysis the P value multiplied by two; and

or interactions between polymorphisms P value was multiplied by four). To avoidype one error in subgroup analyses having statistically low powered sample size,isher’s exact test was performed.

. Results

Patients were comparable to controls in age (56.8 years ± 12.1 vs.5.0 years ± 11.1, P = ns) and gender (males: 129/74.4% vs. 163/75.5%,= ns). Most patients had tumor in the middle third (102/174,8.6%) location of esophagus. Majority of patients (125/156, 80.1%)sed tobacco in some form (smoking, chewing or both). Regularsage of alcohol was noticed in 37.0% (51/138) patients and about

2% (44/138) of patients had occupational exposure, mainly fromousehold combustible fuels (Table 1). The details regarding envi-onmental risk factors for controls were not recorded and casenly analysis was performed for further possible gene environmentnteractions.

able 2ssociation of COX-2 polymorphisms with risk of esophageal squamous cell cancer.

enotype/allelea Controls (n = 216)

N (%)

1290A>GAA 156 (72.2)AG 53 (24.5)GG 7 (3.2)A 365 (84.5)G 67 (15.5)

1195G>AGG 168 (77.8)GA 45 (20.8)AA 3 (1.4)G 381 (88.2)A 51 (11.8)

765G>CGG 148 (68.5)GC 57 (26.4)CC 11 (5.1)GC + CC 68 (31.5)G 353 (81.7)C 79 (18.3)

473T>CTT 81 (37.5)TC 102 (47.2)CC 33 (15.3)T 265 (61.3)C 167 (38.7)

ignificant values shown in bold.a Total number of chromosomes.b Age and gender adjusted odds ratio.

Yes 44 (31.9)

a Information regarding lymph node status and environmental factor were notfound in some of cases.

3.1. Association of COX-2 polymorphisms with ESCC

The distribution of the four polymorphisms of COX-2 was con-sistent with Hardy–Weinberg’s equilibrium in healthy controls.Individuals with COX-2 −765GC genotype were at increased riskof ESCC (OR = 1.92; 95% CI = 1.23–2.98; P = 0.004) while individu-als having COX-2 −765CC homozygous variant genotype or C allele

were not associated with increased risk of ESCC. We clubbed GC andCC genotypes to perform carrier analysis. Using dominant model, Callele carriers were found to be at increased risk of ESCC (OR = 1.66;95% CI = 1.08–2.54; P = 0.021) (Table 2). After stratification based on

Patients (n = 174) ORb (95% CI) P

N (%)

132 (75.9) 1 (Reference)39 (22.4) 0.92 (0.57–1.49) 0.731

3 (1.7) 0.55 (0.14–2.19) 0.395303 (87.1) 1 (Reference)

45 (12.9) 0.85 (0.57–1.29) 0.450

126 72 .4) 1 (Reference)46 (26.4) 1.35 (0.84–2.17) 0.220

2 (1.1) 0.79 (0.13–4.83) 0.795298 (85.6) 1 (Reference)

50 (14.4) 1.23 (0.80–1.87) 0.343

101 (58.0) 1 (Reference)69 (39.7) 1.92 (1.23–2.98) 0.004

4 (2.3) 0.53 (0.16–1.71) 0.28673 (42.0) 1.66 (1.08–2.54) 0.021

271 (77.9) 1 (Reference)77 (22.1) 1.32 (0.92–1.88) 0.130

63 (36.2) 1 (Reference)89 (51.1) 1.17 (0.76–1.82) 0.47922 (12.6) 0.86 (0.45–1.62) 0.638

215 (61.8) 1 (Reference)133 (38.2) 0.99 (0.74–1.33) 0.967

R. Upadhyay et al. / Mutation Research 663 (2009) 52–59 55

Table 3Association of COX-2 polymorphisms with gender and risk of esophageal squamous cell cancer.

Genotype Males Females

Control (n = 163) Patients (n = 129) Control (n = 53) Patients (n = 45)

N (%) N (%) ORa (95% CI) P N (%) N (%) ORa (95%CI) P

−1290A>GAA 115 (70.6) 110 (77.5) 1 (Reference) 41 (77.4) 32 (71.1) 1 (Reference)AG 43 (26.4) 26 (20.2) 0.72 (0.41–1.26) 0.246 10 (18.9) 13 (28.9) 1.93 (0.71–5.22) 0.198GG 5 (3.1) 3 (2.3) 0.74 (0.17–3.21) 0.688 2 (3.8) 0 NC

−1195G>AGG 129 (79.1) 96 (74.4) 1 (Reference) 39 (73.6) 30 (66.7) 1 (Reference)GA 33 (20.2) 31 (24.0) 1.25 (0.72–2.19) 0.432 12 (22.6) 15 (33.3) 1.69 (0.67–4.29) 0.269AA 1 (0.6) 2 (1.6) 2.43 (0.22–27.50) 0.473 2 (3.8) 0 NC

−765G>CGG 112 (68.7) 80 (62.0) 1 (Reference) 36 (67.9) 21 (46.7) 1 (Reference)GC 43 (26.4) 47 (36.4) 1.60 (0.96–2.65) 0.072 14 (26.4) 22 (48.9) 3.52 (1.38–8.98) 0.008†1, ‡1

CC 8 (4.9) 2 (1.6) 0.36 (0.07–1.72) 0.198 3 (5.7) 2 (4.4) 1.00 (0.14–7.24) 0.998GC + CC 51 (31.9) 49 (38.0) 1.395 (0.85–2.28) 0.184 17 (32.1) 24 (53.3) 2.97 (1.23–7.17) 0.016†2, ‡2

8473T>CTT 58 (35.6) 50 (38.8) 1 (Reference) 23 (43.4) 13 (28.9) 1 (Reference)TC 79 (48.5) 64 (49.6) 0.96 (0.58–1.59) 0.877 23 (43.4) 25 (55.6) 2.25 (0.89–5.69) 0.088CC 26 (16.0) 15 (11.6) 0.68 (0.32–1.42) 0.305 7 (14.9) 7 (15.6) 1.58 (0.43–5.84) 0.494

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ignificant values shown in bold.a Age and gender adjusted odds ratio; NC: not calculated.† Fisher exact test two-sided P-value1 = 0.035; P-value2 = 0.041.‡ After Bonferroni correction Pcorr

1 = 0.024; Pcorr2 = 0.032.

ender, among all genotypes of four polymorphisms, only COX-2765C carrier genotype was found associated with risk of ESCC

or females (OR = 2.97; 95% CI = 1.23–7.17; P = 0.016) even after Bon-erroni correction (Pcorr = 0.032) and Fisher’s exact test (P = 0.041).one of the polymorphism imparted risk of ESCC in male subjects

Table 3).

.2. Haplotype analysis of COX-2 polymorphisms

There was significant linkage disequilibrium between allairs of COX-2 loci (Yate’s corrected P value < 0.02 and |D′| /= 0)

n controls as well as patients (supplementary data). A totalf 13 haplotypes were observed in the subjects. The fre-uency of haplotype combination A−1290G−1195C−765T8473 and−1290A−1195C−765T8473 was higher in patients (4.0% and 1.4%)s compared to controls (1.7% and 0.5%) [OR = 3.35, 95%

I = 0.83–13.48, P = 0.089; OR = 4.28, 95% CI = 0.43–42.40, P = 0.214]Table 4).

In addition, we performed all possible interactions among COX-2olymorphisms and found that only interaction between−1195G>And −765G>C polymorphism (COX-2 −1195GA × −765GC + CC)

able 4istribution of COX-2 haplotypes and association with risk of esophageal squamous cell c

aplotype Patients (Na = 348) Cont

−1290G−1195G−765T8473 152 (43.7%) 200 (−1290A−1195G−765T8473 38 (10.9%) 46 (−1290G−1195C−765T8473 14 (4.0%) 6 (−1290A−1195C−765T8473 6 (1.7%) 2 (−1290G−1195G−765T8473 2 (0.6%) 0−1290A−1195G−765T8473 2 (0.6%) 0−1290G−1195C−765T8473 4 (1.1%) 10 (−1290G−1195G−765C8473 74 (21.3%) 96 (−1290A−1195G−765C8473 4 (1.1%) 4 (−1290G−1195C−765C8473 14 (4.0%) 10 (−1290A−1195C−765C8473 2 (0.6%) 0−1290G−1195G−765C8473 4 (1.1%) 6 (−1290G−1195C−765C8473 32 (9.2%) 52 (

a Total number of chromosomes.b Age and gender adjusted odds ratio; NC: not calculated.

conferred significantly enhanced risk of ESCC (OR = 4.60; 95%CI = 1.63–13.01; P = 0.004; Fisher’s exact P value = 0.018). Afterapplying Bonferroni test for multiple corrections, P value was stillstatistically significant (Pcorr = 0.016) (Table 5).

3.3. Association of COX-2 polymorphisms with tumor locations

Individuals with −765C carrier genotypes were at increasedrisk for developing tumor at middle third location of esophagus(OR = 1.78; 95% CI = 1.08–2.93; P = 0.023). After Bonferroni cor-rection, association remained significant (Pcorr = 0.046). However,−1195G>A; −1290A>G; 3′UTR 8473T>C polymorphisms of COX-2were not significantly associated with development of site-specifictumors (Table 6).

3.4. Interaction of COX-2 genotype with environmental risk

factors

A case only study was performed to reveal any possiblegene–environment interactions. Environmental risk factors likealcohol intake, use of tobacco in any form, and occupational expo-

ancer.

rols (Na = 432) P-value ORb (95% CI)

46.3%) – 1 (Reference)10.6%) 0.732 1.13 (0.57– 2.23)1.4%) 0.089 3.35 (0.83– 13.44)0.5%) 0.214 4.28 (0.43– 42.40)

– NC– NC

2.3%) 0.441 0.52 (0.10– 2.78)22.2%) 1.898 1.04 (0.61– 1.75)0.9%) 0.753 1.38 (0.19– 10.04)2.3%) 0.326 1.82 (0.55– 6.04)

– NC1.4%) 0.999 1.00 (0.16– 6.26)12.0%) 0.676 0.86 (0.43– 1.73)

56 R. Upadhyay et al. / Mutation Research 663 (2009) 52–59

Table 5Interactions between COX-2 polymorphisms and their association with esophageal squamous cell cancer.

Interaction Controls (N = 216) Patients (N = 174) ORa (95% CI) P

−1195GG × −765GG 105 (48.6%) 69 (41.3%) 1 (Reference)−1195GA × −765GC 4 (1.8%) 15 (8.6%) 5.39 (1.73–16.82) 0.004†1, ‡1

−1195GA × −765CC 1 (0.5%) 1 (0.6%) 1.52 (0.09–24.60) 0.769−1195GA × −765(GC + CC) 5 (2.3%) 16 (9.2%) 4.60 (1.63–13.01) 0.004†2, ‡2

Significant values shown in bold.a Age and gender adjusted odds ratio.† Fisher exact test two-sided P-value1 = 0.025; P-value2 = 0.018.‡ After Bonferroni correction Pcorr

1 = 0.016; Pcorr2 = 0.016.

Table 6Association of COX-2 polymorphisms with tumor location and risk of esophageal squamous cell cancer.

Genotype Controls (n = 216) Upper (n = 23) Middle (n = 102) Lower (n = 49)

N (%) N (%) ORa (95% CI) P N (%) ORa (95% CI) P N (%) ORa (95% CI) P

−1290A>GAA 156 (72.2) 15 (65.2) 1 (Reference) 79 (77.5) 1 (Reference) 38 (77.6) 1(Reference)AG 53 (24.5) 8 (34.8) 1.71 (0.68–4.34) 0.257 21 (20.6) 0.85 (0.48–1.53) 0.589 10 (20.4) 0.81 (0.37–1.74) 0.587GG 7 (3.2) 0 NC 2 (2.0) 0.59 (0.12–2.93) 0.515 1 (2.0) 0.61 (0.07–5.16) 0.651

−1195G>AGG 168 (77.8) 16 (69.6) 1 (Reference) 73 (71.6) 1 (Reference) 37 (75.5) 1(Reference)GA 45 (20.8) 7 (30.4) 1.64 (0.63–4.27) 0.307 27(26.5) 1.35 (0.77–2.35) 0.294 12 (24.5) 1.18 (0.56–2.45) 0.664AA 3 (1.4) 0 NC 2 (2.0) 1.32 (0.21–8.17) 0.765 0 NC

−765G>CGG 148 (68.5) 13 (56.5) 1 (Reference) 58 (58.9) 1 (Reference) 30 (61.2) 1(Reference)GC 57 (26.4) 9 (39.1) 2.03 (0.81–5.12) 0.133 41 (40.2) 2.03 (1.21–3.42) 0.007‡1 19 (38.8) 1.80 (0.93–3.51) 0.082CC 11 (5.1) 1 (4.3) 1.06 (0.13–8.95) 0.957 3 (2.9) 0.66 (0.18–2.49) 0.541 0 NCGC + CC 68 (31.5) 10 (43.5) 1.86 (0.76–4.52) 0.173 44 (43.1) 1.78 (1.08–2.93) 0.023‡2 19 (38.8) 1.48 (0.77–2.83) 0.242

8473T>CTT 81 (37.5) 10 (43.5) 1 (Reference) 34 (33.3) 1 (Reference) 19 (38.8) 1(Reference)TC 102 (47.2) 9 (39.1) 0.71 (0.27–1.83) 0.472 55 (53.9) 1.34 (0.80–2.26) 0.271 25 (51.0) 1.08 (0.55–2.11) 0.820CC 33 (15.3) 4 (17.4) 0.94 (0.27–3.24) 0.923 13 (12.7) 0.95 (0.44–2.03) 0.886 5 (10.2) 0.65 (0.22–1.89) 0.427

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ignificant values shown in bold.a Age and gender adjusted odds ratio; NC = not calculated.‡ After Bonferroni correction Pcorr

1 = 0.021; Pcorr2 = 0.046.

ure were analyzed for interaction with COX-2 genotypes. Although

obacco use was prominent in patients (125/156, 80.1%,) but nonef genotype–environmental risk factor interactions significantlyodulated risk for ESCC (insignificant data not shown regarding

ene–environment interactions).

Fig. 2. Diagrammatic representation of pathway influenced by COX-2 polym

4. Discussion

We investigated the role of COX-2 −765G>C, −1195G>A,−1290G>A and 8473T>C 3′UTR polymorphisms in a case–controlstudy. These polymorphisms are known to modulate the transcrip-

orphisms in conferring risk of esophageal squamous cell carcinoma.

R. Upadhyay et al. / Mutation Research 663 (2009) 52–59 57

Table 7Association of COX-2 polymorphisms in different cancers from various populations.

Polymorphism MAFa incontrols (%)

Population Disease Result/association Reference

−1290A>G 15.5 Northern Indian Esophageal squamous cellcarcinoma (ESCC)

−765C allele carriers were associated with riskof ESCC, specifically for females and middlethird location of tumor

Present study−1195G>A 11.8−765G>C 18.38473T>C 38.7−1290A>G 4.5 Han Chinese ESCC Increased risk of ESCC with −1195A and −765C

allele/1195GA/AA and −765GC genotypes[9,11]

−1195G>A 50.8−765G>C 2.2−1195G>A– Dutch Esophageal adenocarcinoma

(EAC)Increased risk of EAC with CA haplotype [12]

−765G>C8473 T>C 32.4 Northern Ireland

(Caucasians)EAC C carriers were associated with EAC [21]

−765G>C 16.2 India (northern) Gastric adenocarcinoma −765C carriers were associated with gastricadenocarcinoma with or without H pylori infection

[22]

−765G>C 38 Portuguese Gastric adenocarcinoma −765C carriers were associated with risk of gastricadenocarcinoma

[23]

−765G>C 17.0 American Colorectal adenocarcinoma CC genotype were associated with low risk of colorectalcarcinoma

[24]

−765G>C 7.8 Northeast China Colorectal cancer No association; GG genotype were associated in presenceof smoking and higher BMI

[25]

−1290A>G 4.7 Han Chinese Colorectal cancer −1195GA/AA; −765GC genotypes; A−1195-C−765

haplotype were associated with Colorectalcancer

[26]−1195G>A 50.3−765G>C 2.48473T>C 46.5 Norwegian

(Caucasian)Lung cancer 8473TC/CC genotypes were associated with risk of lung

cancer[27]

8473T>C 24.4 Korea Lung cancer C carriers were associated with reduced risk of Lungadenocarcinoma

[28]

8473T>C 18.7 Han Chinese Lung cancer 8473TC/CC were associated with reduced risk of lungcancer

[16]

8473T>C 34.1 American Breast cancer Reduces risk for hormone receptor positive breast cancer [29]8 848 No8 No

tfwawt(

pe

Irgpga−tguweost

ofe(no

473T>C 29.9 Australia Breast cancer473T>C 30.9 American Breast cancer473T>C 35.9 American Breast cancer

a MAF: minor allele frequency.

ional activity and stability of mRNA. Using dominant model, weound that only −765C allele carriers were significantly associatedith risk of ESCC. The cancer risk increased specifically in females

nd also for middle third anatomical location of tumor. In addition,e also observed significant modulation of risk due to interac-

ion between −1195GA and −765GC + CC combination of genotypesFig. 2).

Recently, studies have examined the association of COX-2olymorphisms with susceptibility to both squamous and adenosophageal carcinoma in a high-risk population of China [9,11,12].

Our study was conducted in a low risk population of northernndia and we found that −765C allele carriers were at increasedisk of ESCC. The significant risk observed due to −765C carrierenotypes is apparent as previous in vitro study showed that theroduction of PGs is much higher (>10-fold) in −765CC homozy-otes than −765GG homozygotes and −765GC genotype is associ-ted with intermediate level of prostaglandin production [19]. Also,765G>C change creates a binding element for the E2F transcrip-

ion factor, a cyclin-dependent regulator of expression of severalenes. Moreover, previous studies have shown COX-2 protein wasp-regulated from normal esophagus to high grade dysplasia andas over-expressed in ESCC [4]. These results suggest that pres-

nce of COX-2 −765C carrier genotype may lead to over-expressionf COX-2 and enhance PGs production as stated previously and mayubsequently facilitate tumor progression by acting on differentia-ion and growth factors, or as immunosuppressors [20].

Three other COX-2 polymorphisms did not show modulationf risk for ESCC. We reviewed the distribution of minor allele

requencies of COX-2 polymorphisms in controls among differ-nt populations as well as various malignancies [9,11,12,16,21–32]Table 7). Minor allele frequencies of COX-2 polymorphisms inorthern Indian population were comparable to American andther Indian reports but different from Han Chinese population

73CC genotype with risk of breast cancer [30]association [31]association [32]

[9,11,16,26]. However, association of COX-2 polymorphisms in dif-ferent cancers was inconsistent, suggesting disease and populationspecific risk (Table 7).

The COX-2 polymorphisms undertaken in the present study werelocated in close proximity. Therefore, we constructed haplotype andestimated the linkage disequilibrium. Our results showed signifi-cant linkage disequilibrium between all pairs of COX-2 loci. Previousstudies have reported the association of A−1290A−1195C−765 hap-lotype with risk of ESCC [9] whereas Moons et al. [12] showedthat homozygous COX-2 CA haplotype was associated with a signif-icantly increased risk of developing esophageal adenocarcinomafrom Barrett’s esophagus and reflux esophagitis. In the presentstudy, combination of haplotype A−1290A−1195C−765T8473 showedhigh risk for ESCC but the association could not achieve statisticalsignificance probably due to limited sample size of each haplotype.However, in our study, we found that combination of −1195G>Aand −765G>C polymorphisms showed −1195GA × −765GC + CCinteraction had multiplicative effect on risk of ESCC. Recently, amultiplicative interaction was also observed between the 12-LOXGln/Gln and COX-2 −1195AA or −765GC genotype in intensifyingrisk of esophageal adenocarcinoma [11].

Sanak et al. [19] have found that instead of genotypes;haplotypes of COX-2 could better correlate with prostaglandinsbiosynthetic capacity. They observed very strong linear corre-lation between the number of C–C haplotype copies and PGslevels in monocyte cultures [C–C/C–C genotypes (locus order COX2G−765C – COX2 T8473C) revealed a 19.8-fold increase of PGE2(P < 0.001) with most contrasting wild type (G–T/G–T) genotype].

The observed high risk due to joint effect of −1195A and −765Callele may result from higher expression of COX-2 which enhancesPGs synthesis. Therefore, it may affect process of carcinogenesisthrough escape from immune surveillance mechanisms via modu-lating expression of specific cytokines.

5 ion Re

fteacitTrr−n[f8ee

rfigssfdicpu[crsfbpwm−wFf

uargosfig[usrt

oeeamTb

morphisms, Pharmacogenet. Genomics 17 (2007) 197–205.[12] L.M. Moons, E.J. Kuipers, A.M. Rygiel, A.Z. Groothuismink, H. Geldof, W.A. Bode,

K.K. Krishnadath, J.J. Bergman, A.H. van Vliet, P.D. Siersema, J.G. Kusters, COX-2

8 R. Upadhyay et al. / Mutat

In association of genotypes with clinical characteristics, weound that −765C allele carriers were at higher risk of middlehird tumor location. It has been reported that COX-2 is over-xpressed significantly in the middle and lower third of esophaguss compared to upper esophagus [33]. Findings suggest that car-inogenesis in middle and lower third ESCC may be due tonflammation caused by gastric reflux. In the presence of inflamma-ion, COX-2 may be induced by cytokines and growth factors (EGFR,GFB, HIFA and VEGF) which lead to tumor growth and heighten theisk of cancer [34–36]. Earlier, we also observed same site-specificisk with some low penetrance candidate genes [13,14]. However,1195G>A, −1290G>A and +8473T>C 3′UTR polymorphisms wereot associated with clinical characteristics of ESCC. Ferguson et al.21] have shown COX-2 8473C allele as a potential genetic markeror susceptibility to esophageal adenocarcinoma. Therefore, COX-2473T>C polymorphism may have histology-specific association insophageal cancer as squamous and adenocarcinomas are consid-red to have different etiologies.

When the subjects were stratified according to gender, the ESCCisk due to −765C carrier genotype was enhanced up to 3-fold inemales. It is known that carcinogens like benzo[a]pyrene (BaP)nduce COX-2 expression, and an interaction between BaP and estro-en in relation to COX-2 expression is suspected. Recently, it washown that estradiol activated estrogen receptor increases PGE2ecretion, which directly increases COX-2 expression via nuclearactor-kappaB (NF-kB) activation [37]. Also, animal studies haveemonstrated that females are more susceptible than males to BaP-

nduced toxicities. Therefore, it is possible that females with −765Carrier genotype are exposed to higher PG levels which promote cellroliferation by the activation of EGFR, which inhibits apoptosis byp-regulation of bcl-2, and suppression of host immune response37]. Second mechanism describing gender specific risk in C allelearriers of −765G>C polymorphism may be that, the promoteregion of COX-2 has two distinct regulatory motifs responsible forex-dependent expression of the gene. A regulatory motif upstreamrom the transcription start is located at 709–727 nucleotides andinds with progesterone receptor (which is closer to −765G>Colymorphic site) while other is located at 1206–1222 nucleotideshich binds with SRY (testis-determining factor) responsive ele-ent. Therefore, it is possible that observed female specific risk in765C allele carriers is due to linkage of −765G>C polymorphismith progesterone receptor binding regulatory motif (Transcription

actor Database, http://www.gene-regulation.com) [38]. However,urther studies are required to confirm the present finding in future.

Previous studies have pointed a strong link between tobaccose, drinking and risk of EC [39]. Guo et al. [11] showed inter-ction between the COX-2 −765GC genotype and smoking asisk for ESCC but they have employed case–control analysis forene–environment interactions. However, we have performed casenly analysis; which is considered to be more appropriate foruch interactions [15]. In our previous studies also, we did notnd association of environmental risk factors with inflammatoryenes like interleukin-1 beta, interleukin-1 RN and interleukin-613,14]. It shows that other genes may be involved in risk mod-lation for ESCC and a recent study by Leeuwen et al. [40] hashown up-regulation of certain specific genes in response to envi-onmental exposures. There may be other mechanisms underlyinghis gene–environment interaction which should also be explored.

The present study has some limitations. We have analyzednly four functional polymorphisms and in future, exploring otherxonic genetic variations in the COX-2 gene would also be inter-

sting. In addition, other gene polymorphisms (e.g. 12-LOX) maylso be explored as aberrant polyunsaturated fatty acid metabolismight have interactive role in determining susceptibility to ESCC.

he sample size is low in some of subgroup and haplotypes. It maye added that, due to low sample size in these subgroups, finding

search 663 (2009) 52–59

should be interpreted with caution and may require reconfirmationin larger samples.

In conclusion, our study observed the association of −765Callele carrier genotype with risk of ESCC particularly in femalesand patients with middle third tumor location. At the haplotypelevel, individuals with haplotype combination of −1195A−765Cwere at greater risk for ESCC. Furthermore, we observed multiplica-tive interaction between the −1195GA and −765C allele carriers. Infuture, prospective multi-centric studies are required with long-term follow-up of a cohort of patients to confirm these findings.The results obtained from such studies can be potentially impor-tant from a therapeutic point of view as ESCC cells over-expressingCOX-2 are more sensitive to COX-2 inhibitors.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgement

The study was supported by research grants from Indian Councilof Medical Research and Department of Science and Technology,Govt. of India.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.mrfmmm.2009.01.007.

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