Protective effect of Cox-2 allelic variants on risk of colorectal adenoma development in African Americans.
ABSTRACT Recent evidence indicates that single nucleotide polymorphisms (SNPs) in the Cox-2 gene may modulate the risk of colorectal adenoma development.
We explored possible associations between Cox-2 polymorphisms and risk of adenoma development in an African American case-control study comprising 72 cases of advanced adenomas and 146 polyp-free controls. An exhaustive approach of genotyping 13 haplotype-tagging SNPs (ht SNPs) distributed over the entire COX-2 gene was used.
Statistically significant inverse associations were observed between the heterozygous genotypes at the 5229 G>T polymorphism in intron 5 [odds ratio (OR)=0.42; confidence interval (CI)=0.19-0.92; p=0.03] and at the 10935 A>G polymorphism in the 3' flanking region downstream from the poly A signals (OR=0.39; CI=0.18-0.83;p=0.01) and the risk for colorectal adenoma development.
The data from our pilot study suggest that allelic variants of the COX-2 gene significantly influence the risk of adenoma development in the African American population.
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ABSTRACT: Patients with familial adenomatous polyposis have a nearly 100 percent risk of colorectal cancer. In this disease, the chemopreventive effects of nonsteroidal antiinflammatory drugs may be related to their inhibition of cyclooxygenase-2. We studied the effect of celecoxib, a selective cyclooxygenase-2 inhibitor, on colorectal polyps in patients with familial adenomatous polyposis. In a double-blind, placebo-controlled study, we randomly assigned 77 patients to treatment with celecoxib (100 or 400 mg twice daily) or placebo for six months. Patients underwent endoscopy at the beginning and end of the study. We determined the number and size of polyps from photographs and videotapes; the response to treatment was expressed as the mean percent change from base line. At base line, the mean (+/-SD) number of polyps in focal areas where polyps were counted was 15.5+/-13.4 in the 15 patients assigned to placebo, 11.5+/-8.5 in the 32 patients assigned to 100 mg of celecoxib twice a day, and 12.3+/-8.2 in the 30 patients assigned to 400 mg of celecoxib twice a day (P=0.66 for the comparison among groups). After six months, the patients receiving 400 mg of celecoxib twice a day had a 28.0 percent reduction in the mean number of colorectal polyps (P=0.003 for the comparison with placebo) and a 30.7 percent reduction in the polyp burden (the sum of polyp diameters) (P=0.001), as compared with reductions of 4.5 and 4.9 percent, respectively, in the placebo group. The improvement in the extent of colorectal polyposis in the group receiving 400 mg twice a day was confirmed by a panel of endoscopists who reviewed the videotapes. The reductions in the group receiving 100 mg of celecoxib twice a day were 11.9 percent (P=0.33 for the comparison with placebo) and 14.6 percent (P=0.09), respectively. The incidence of adverse events was similar among the groups. In patients with familial adenomatous polyposis, six months of twice-daily treatment with 400 mg of celecoxib, a cyclooxygenase-2 inhibitor, leads to a significant reduction in the number of colorectal polyps.New England Journal of Medicine 07/2000; 342(26):1946-52. · 51.66 Impact Factor
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ABSTRACT: Cyclooxygenase-2 (COX-2) is the inducible isoform of cyclooxygenase, the enzyme that catalyzes the rate-limiting step in prostaglandin synthesis from arachidonic acid. Various prostaglandins are produced in a cell type-specific manner, and they elicit cellular functions via signaling through G-protein coupled membrane receptors, and in some cases, through the nuclear receptor PPAR. COX-2 utilization of arachidonic acid also perturbs the level of intracellular free arachidonic acid and subsequently affects cellular functions. In a number of cell and animal models, induction of COX-2 has been shown to promote cell growth, inhibit apoptosis and enhance cell motility and adhesion. The mechanisms behind these multiple actions of COX-2 are largely unknown. Compelling evidence from genetic and clinical studies indicates that COX-2 upregulation is a key step in carcinogenesis. Overexpression of COX-2 is sufficient to cause tumorigenesis in animal models and inhibition of the COX-2 pathway results in reduction in tumor incidence and progression. Therefore, the potential for application of non-steroidal anti-inflammatory drugs as well as the recently developed COX-2 specific inhibitors in cancer clinical practice has drawn tremendous attention in the past few years. Inhibition of COX-2 promises to be an effective approach in the prevention and treatment of cancer, especially colorectal cancer.Journal of Cellular Physiology 04/2002; 190(3):279-86. · 4.22 Impact Factor
- Current Problems in Cancer 01/2007; 31(3):123-33. · 1.56 Impact Factor
Abstract. Background: Recent evidence indicates that
single nucleotide polymorphisms (SNPs) in the Cox-2 gene
may modulate the risk of colorectal adenoma development.
Patients and Methods: We explored possible associations
between Cox-2 polymorphisms and risk of adenoma
development in an African American case–control study
comprising 72 cases of advanced adenomas and 146 polyp-
free controls. An exhaustive approach of genotyping 13
haplotype-tagging SNPs (ht SNPs) distributed over the entire
COX-2 gene was used. Results: Statistically significant
inverse associations were observed between the heterozygous
genotypes at the 5229 G>T polymorphism in intron 5 [odds
ratio (OR)=0.42; confidence interval (CI)=0.19-0.92;
p=0.03] and at the 10935 A>G polymorphism in the 3’
flanking region downstream from the poly A signals
(OR=0.39;CI=0.18-0.83;p=0.01) and the risk for colorectal
adenoma development. Conclusion: The data from our pilot
study suggest that allelic variants of the COX-2 gene
significantly influence the risk of adenoma development in
the African American population.
Colon cancer accounts for approximately 10% of all cancer-
related deaths and remains the third deadliest killer among
cancer types in the United States (1). Epidemiological data
show that African Americans have higher age-specific
incidence and mortality rates and lower 5-year survival rates
compared to Caucasians (2). Although reasons for this
disparity are not clear, evidence implicates genetic,
environmental and lifestyle factors as contributors to this
multi-factorial disease (3).
There is mounting evidence that chronic inflammation is
involved in the etiology of cancer. Previous studies have
reported an association between genetic variants of pro-
inflammatory genes and the risk of developing colorectal
adenoma and carcinoma (4-6). One such gene, encoding the
enzyme cyclooxygenase-2 (Cox-2) plays a significant role
in inflammation and carcinogenesis (7). Epidemiological
observations as well as randomized prevention clinical trials
have provided evidence for a significant role of Cox-2 in
colon carcinogenesis (8-11). More recently, several studies
have explored association between genetic variants of Cox-2,
alone or in interaction with environmental factors, and risk
of developing colorectal adenoma/carcinoma mostly in
Relatively few studies have addressed the influence of
genetic variants of Cox-2 on cancer risk in the African
Americans replacing the amino acid valine with alanine at
position 511 in exon 10 of Cox-2 has been described to
reduce the risk of colorectal cancer (16, 17). Another study
reported different patterns of association between the
genetic variants in the regulatory regions of Cox-2 and
prostate cancer risk in three different ethnic populations
including African Americans (18). This observation is not
surprising as patterns of genetic polymorphisms may vary
within and between populations. The haplotype block
structures of human genome containing regions of high
linkage disequilibrium are of shorter size and reduced
diversity in African Americans compared to Caucasians
(19). To better understand the significance of genetic
variations in the Cox-2 gene in influencing colorectal cancer
risk in African Americans, we used a case–control study of
advanced adenomas to exhaustively analyze a possible
population.A polymorphism in African
*Both authors contributed equally to this work.
Correspondence to: Iqbal U. Ali, Molecular Oncology Program,
Panjwani Center for Molecular Medicine and Drug Research,
International Center for Chemical and Biological Sciences,
University of Karachi, Karachi, Pakistan. e-mail: firstname.lastname@example.org
Key Words: Cox-2 polymorphisms, colorectal adenoma, African
ANTICANCER RESEARCH 28: 3119-3124 (2008)
Protective Effect of Cox-2 Allelic Variants on Risk of Colorectal
Adenoma Development in African Americans
HASSAN ASHKTORAB1*, SHIRLEY TSANG2*, BRIAN LUKE3, ZHONGHE SUN2,
LUCILE ADAM-CAMPBELL1, JOHN KWAGYAN1, RICHARD POIRIER4, SHAHINA AKTER1,
AHMAD AKHGAR1, DUANE SMOOT1, DAVID J. MUNROE2and IQBAL UNNISA ALI4
1Department of Medicine and Cancer Center, Howard University College of Medicine, Washington, D.C.;
2Laboratory of Molecular Technology, and
3Advanced Biomedical Computing Center, SAIC-Frederick, Frederick, MD;
4Division of Cancer Prevention, National Cancer Institute, Bethesda, MD, U.S.A.
association between Cox-2 polymorphisms and colorectal
nucleotide polymorphisms (htSNPs) in the Cox-2 gene.
bygenotyping 13 haplotype-taggingsingle
Materials and Methods
Patient selection. The study was approved by the Howard University
Institutional Review Board. Study participants were recruited from
patients referred for colonoscopy to the gastroenterology division at
Howard University Hospital between September 2000 and October
2003. Indications for colonoscopy included rectal bleeding, irregular
bowel habit, weight loss, family history of colon polyp/cancer,
personal history of colon polyp and routine screening. Cases were
eligible if colonoscopy resulted in a first diagnosis of colorectal
adenomatous or hyperplastic polyp as confirmed by histology.
malabsorption, any cancer, current or past chemotherapy or
interferon treatment were excluded. Patients with distal or proximal
polyps and with adenomatous or hyperplastic pathology, as
determined by independent pathologists were selected as cases.
Based on these criteria, 72 patients qualified as cases. Controls had
to be free of all polyps and self-described with no previous history
of colorectal adenomas/cancer. All patients were as African
Americans. Clinical and demographic data collected on each patient
included race, gender, past medical history, family history of
colorectal polyp/cancer and information on smoking, alcohol
consumption and medication use. DNA was extracted from samples
from 72 patients and 146 controls.
Genotyping. The htSNPs of the Cox-2 gene for the population of
African American descent, together with the respective primers and
probes used in this study are displayed in Table I. The positions of the
polymorphisms refer to the Genbank entry AY382629 and as detailed
at http://pga.gs.washington.edu/data/ptgs2/. All assays were designed
and developed using Assay-by-Design (Applied Biosystems Inc, CA,
USA). All oligo primers and probes were synthesized by Applied
Biosystems, Inc. Assays were validated and optimized using in-house
collected human DNA samples. Positive control DNAs of known
genotypes as well as a no-template control were run on each assay
plate for quality control. All SNPs were tested by the Taqman assay
using the MGB chemistry (Applied Biosystems, Inc.) and the ABI
7900HT Sequence Detector. SDS 2.1 (Applied Biosystems, Inc.) was
used to determine the genotype calls. Specific experimental details
about genotyping will be provided upon request from the authors.
Data analysis. Odds ratios (ORs) were estimated using logistic
regression models with the PROC LOGISTIC function of the SAS
software package (version 9.1; SAS Institute, Cary, NC, USA)
adjusting for gender and smoking. Departure from Hardy–Weinberg
equilibrium was assessed by comparing the expected to observed
genotype frequencies using the asymptomatic Pearson’s χ2test.
ANTICANCER RESEARCH 28: 3119-3124 (2008)
Table I. Polymorphisms, primers and probes used in this study.
Polymorphism Forward primersReverse primersReporter _VICReporter _FAM
Numbers for polymorphism refer to positions in the Genbank entry AY382629 and as detailed at http://pga.gs.washington.edu/data/
Characteristics of the study population and the association
with colorectal cancer in this group of cases and controls are
displayed in Table II. The only highly significant positive
association was observed between current smokers and the
risk of adenoma development [odds ratio (OR)=2.73, 95%
confidence interval (CI) =1.20-6.19, p=0.03). A non-
significant association was also present between former
smokers and adenoma development (OR=1.52, CI=0.81-
The data of the association analysis for the main effect of
the 13 polymorphisms distributed over the entire Cox-2 gene
are displayed in Table III. Of the 13 htSNPs, an intronic
polymorphism and another in the 3’ flanking region (FR),
when adjusted for gender and smoking, were associated with
a lower risk of adenomas. Adjusting for age and smoking
also resulted in very similar associations (data not shown).
Individuals with the heterozygous genotype at the intron 5-
5229 had a statistically significant decrease in the risk of
developing adenomas (OR=0.42, CI=0.19-0.92, p=0.03).
Similarly, a highly significant protective effect for adenoma
risk was observed in individuals with the heterozygous
genotype at position 3’FR-10935 (OR=0.39, CI=0.18-0.83,
p=0.01). The risk of adenoma in individuals with the variant
homozygous genotypes at intron 5-5229 and 3’ FR-10935,
however, was no different from that of the control group.
Ashktorab et al: Cox-2 Variants and Adenoma Risk in African Americans
Table III. COX-2 genotypes and the risk of advanced colorectal
Genotype Cases/controlsOR 95% CI
Ancestral alleles are treated as wild-type. OR, odds ratio; CI, confidence
interval. Values are adjusted for gender and smoking.
Table II. Characteristics of cases and controls.
Characteristic Cases Controls OR95% CI
Body mass index
OR, odds ratio; CI, confidence interval.
Besides the polymorphisms at intron 5- 5229 and 3’FR-
10935, two other polymorphisms in the promoter region
showed a trend for a protective effect for adenoma
development. There was a marginally significant lower risk
of adenoma development in individuals with the rare
homozygous genotype at the –861 position (OR=0.29,
CI=0.08-1.04, p=0.06) and a statistically non-significant
protective trend for the risk of adenomas (OR=0.29,
CI=0.06-1.38, p=0.12) in individuals with another rare
homozygous variant at the –663 polymorphism (Table III).
To our knowledge, our pilot study represents the first
exhaustive approach to determine the influence of genetic
variants of Cox-2 on the risk of colorectal adenoma
development in African Americans. We evaluated 13 htSNPs
with a minor allele frequency ranging between 0.13-0.43 that
were distributed over the entire Cox-2 gene and captured
most common variations in the African American population.
Two polymorphisms located in intron 5 and in the 3’ FR
showed a protective effect for adenoma development.
A reduced risk of adenoma development in African
Americans in carriers of the heterozygous genotype at intron
5-5229 in the Cox-2 gene is consistent with the previous
finding of the protective effect of this polymorphism on
development of colorectal adenoma in Caucasians (12).
Interestingly, a Swedish study reported a protective effect of
the same polymorphism (rs20432) (referred to as position
+3100) for prostate cancer (20). It is also noteworthy that,
similar to our study in African Americans, the heterozygous
but not the variant homozygous genotype at intron 5-5229 had
an inverse association with prostate cancer risk in a Swedish
population (20). Intronic sequences are believed to harbor
transcriptional regulatory elements. The intronic variants may
therefore modulate disease risk by regulating gene expression,
gene splicing, or transcript stability (21). A protective effect
of the variant G allele at intron 5-5229 of the Cox-2 gene in
colorectal and prostate cancer may indicate a transcription
regulatory role of intronic sequences. Alternatively, the intron
5-5229 polymorphism may be in linkage disequilibrium with
a nearby functional polymorphism.
Another polymorphism with a protective effect for
adenoma development was detected at position 10935 in the
3’ flanking region of the Cox-2 gene. This is located
downstream of the polyadenylation signal and AU-rich
elements. Previously, disease-associated variants have been
described in the 3’ flanking region of genes that affect
transcription factor-binding sites (22). Polymorphisms in
the Cox-2 gene located upstream of the 3’FR-10935
position have been reported to have cancer-modulating
effect. Especially, both positive and negative association of
the 3’ UTR-8494 polymorphism with various types of
cancer has been widely reported (12). In particular, the
heterozygous, but not the variant homozygous genotype at
3’ UTR-10494 was found to be protective for prostate
cancer in a Swedish population (20). Although, there was
no evidence of a risk-modulating effect of the previously
reported 8494 or 10494 variants of the Cox-2 gene in our
small study, the protective effect of the nearby 3’ FR-10935
polymorphism underscores the significance of allelic
variants in the 3’ regulatory region of the Cox-2 gene in
affecting the risk of cancer development.
In summary, our study underscores the relevance of genetic
variants in the regulatory regions of Cox-2 in modulating
cancer risk. In the absence of any information on the
functional significance of the intron 5-5229 and 3’FR-10935
polymorphisms in development of colorectal adenoma in
African Americans, future studies with larger numbers of
cases and controls will be necessary to rule out the possibility
that the protective effect of Cox-2 variants in the regulatory
regions on adenoma development is a chance finding.
This work was supported by Grant #CA102681, funded by
the National Cancer Institute and GCRC.
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Received May 14, 2008
Revised July 25, 2008
Accepted August 4, 2008
Ashktorab et al: Cox-2 Variants and Adenoma Risk in African Americans