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A Functional Promoter Polymorphism (-174 G/C) of the Interleukin-6 Gene and Risk for Incident, Sporadic Colorectal Adenomas
Zonglin Deng2, Zhihong Gong1,2, Roberd M. Bostick3, Franklin G.
Berger2,4, James R. Hebert1,2,5,
and Dawen Xie1,2
Interleukin-6 (IL-6) is an important pro-inflammatory cytokine that has
multiple effects on inflammation and cell growth. A genetic polymorphism
within the IL-6 promoter region (-174G/C) modulates IL-6 expression in
vitro and in vivo. Researching the association between IL-6 -174G/C
polymorphism and risk of colorectal neoplasia, whether the association
differs according to adenoma characteristics, and whether IL-6 genotype
interacts with environmental factors to modify risk for incident, sporadic
colorectal adenomas, findings suggests that the IL-6 -174G/C polymorphism,
in conjunction with other risk factors, may reduce risk for colorectal
adenomas.
ABSTRACT
Interleukin-6 (IL-6) is an
important pro-inflammatory cytokine that has multiple effects on
inflammation and cell growth. A genetic polymorphism within the IL-6
promoter region (-174G/C) modulates IL-6 expression in vitro and in
vivo. The C allele has been associated with a higher incidence of
colorectal cancer in some patients. To investigate whether this IL6
-174G/C polymorphism influences risk for incident, sporadic
colorectal adenoma, we used PCR to genotype the -174G/C polymorphism
in 157 subjects with incident sporadic adenoma and 205 disease-free
controls aged 30-74 years in a colonoscopy-based case-control
study. At least one C allele was found in 36% of cases and 39% of
controls. After multivariable adjustment for potential confounders
there was no evidence of an overall association between IL-6
genotypes and adenoma; however, there was a pattern of reduced risk
among women who had at least one C allele (odds ratio [OR] = 0.50,
95% confidence interval [CI] 0.26 – 0.95), non-smokers (OR = 0.32,
95% CI 0.15 – 0.69), or nonsteroidal anti-inflammatory drug users
(OR = 0.43, 95% CI 0.20 – 0.92). These data suggest that the IL-6
-174G/C polymorphism, in conjunction with other risk factors, may
reduce risk for colorectal adenomas. Further studies with larger
patient groups are needed to confirm these findings and to elucidate
the biological mechanisms underlying the possible effect of IL-6
-174G/C polymorphism.
INTRODUCTION
In the United States, colorectal cancer is currently the third most
common cancer, with an estimated 147,500 new cases diagnosed in 2005
[1]. Ecological and migrant studies indicate that environmental
factors, including various aspects of diet and other lifestyle
factors, may strongly influence risk for colorectal cancer and
account for the marked difference in incidence rates between
developed and undeveloped countries [2;3]. Data from numerous
analytical observational studies and laboratory animal experiments
indicate that risk for colorectal neoplasms is increased with
lifestyle-related factors such as red meat intake, physical
inactivity, large body mass, and smoking [4], and decreased with
nonsteroidal anti-inflammatory drug (NSAID) use and higher calcium
intakes [5-7]. While various environmental factors clearly play a
role in colorectal cancer etiology, they do not appear to be the
sole determinants because large proportions of the “exposed” never
get the disease.
Interleukin-6 (IL-6) is a multifunctional, pro-inflammatory cytokine
that has been implicated in the pathogenesis of several chronic
diseases associated with aging, including colorectal cancer [8].
Because of its multidimensional and complex actions, dysregulation
of IL-6 is linked to numerous disorders, including a variety of
neoplastic processes [8]. Elevated plasma levels of IL-6 are
associated with an increased risk of, and worse outcomes from,
colorectal cancer [9-12]. Belluco demonstrated that carcinoembryonic-secreting
tumors (such as colon cancers) induce IL-6 production and that IL-6
stimulates tumor cell growth at metastatic sites [13].
Although a common G/C polymorphism at nucleotide -174 in the
promoter region of the IL-6 gene was identified and found to affect
IL-6 expression [14-16], there have been few studies on the
association of -174 G/C polymorphism with risk for colorectal cancer
in humans [17-19]. One case-control study found that -174G/C
polymorphism influences circulating levels of IL-6 in patients with
colorectal cancer [20]. Theodoropoulos et al. reported that G allele
is associated with
higher rates of CRC [21]. Another study reported significant
positive associations between -174G/C polymorphism and risk for
colorectal cancer and that the -174 C allele was associated with
increased risk of colorectal cancer among those who did not
habitually take NSAIDs but did consume alcohol [22]. However, to
date, there are no published epidemiologic studies that have
evaluated IL-6 174G/C polymorphism and risk for incident, sporadic
colorectal adenomatous polyps. In addition, no studies have
investigated potential interactions between dietary and other
environmental risk factors, IL-6 genotypes, and colorectal adenomas.
Herein we report a molecular epidemiological study of the
association between IL-6 -174G/C polymorphism and risk of colorectal
neoplasia, whether the association differs according to adenoma
characteristics, and whether IL-6 genotype interacts with
environmental factors to modify risk for incident, sporadic
colorectal adenomas.
MATERIALS AND METHODS
Study Design and Study Population
The design and population characteristics of this community-,
colonoscopy-based case-control study were described previously
[23-26]. Briefly, participants were recruited through community
gastroenterology practices in Winston-Salem and Charlotte, North
Carolina. All patients scheduled for a colonoscopy between 1994 and
1997 were screened for specific study eligibility criteria and then
recruited before colonoscopy. Eligibility criteria included English
speaking, 30-74 years of age, no previous adenoma, no individual
history of cancer (except non-melanoma skin cancer), no known
genetic syndromes associated with predisposition to colonic neoplasia, no history of ulcerative colitis or Crohn’s disease, and
resident of either of the two North Carolina metro areas. The study
was approved by the Institutional Review Board of Wake Forest
University. Informed consent was obtained from each participant
prior to data collection.
Cases were defined as patients with at least one adenomatous polyp,
and controls were identified as patients without adenomatous polyps.
Polyps were removed at colonoscopy and examined by a study index
pathologist using criteria from the National Polyp Study [27].
Information on polyp location, number, size, shape, histological
type, and degree of dysplasia were collected.
Study participants were sent self-administered questionnaires to
complete before their colonoscopy visit. Information on diet,
smoking habits, alcohol intake, NSAID use, physical activity,
medical history, anthropometrics (height, weight, and waist and hip
circumferences), family history of colorectal cancer, hormonal and
reproductive history, and reasons for colonoscopy were collected
using the mailed questionnaires which were collected from each
subject at the colonoscopy visit. Dietary information was assessed
using an adaptation of the Willett semi-quantitative food frequency
questionnaire (FFQ) (153 items), which was expanded to include
additional vegetables, fruit, and low-fat foods [28]. Physical
activity information was obtained using a modified Paffenbarger
questionnaire [29]. Alcohol intake data were collected using alcohol
consumption questions on both the FFQ and the personal history
portion of the questionnaire. Blood was drawn and stored at -70˚C
for possible later measurement of various genotypes.
Among all four clinical sites, 669 participants were found to be
initially eligible for the study. Of these, 617 were contacted, and
417 participants (62.3% of total eligible participants) signed the
consent form and participated in the study. Of the 417 participants,
259 had some type of polyp, and of these, 179 had adenomatous
polyps. Nine of the 417 total participants were subsequently
determined ineligible for the study, and an additional 8
participants had incident colon cancer and were not eligible for the
primary case-control analyses; thus, 400 possible participants were
available for genotypic analysis. Of these 400 patients, viable DNA
was isolated from 362 (157 cases and 205 controls) for genotyping.
Laboratory Methods
Genomic DNA was obtained from stored white blood cells (WBCs)
digested in 500 μl of lysis buffer (50 mM Tris/HCl, pH 8.5, 1 mM
EDTA, 0.2% SDS, 200 g/ml proteinase K) overnight at 55°C with
shaking. The digestion was precipitated directly with isopropanol
and the pellets were washed with 70% ethanol. The genomic DNA
pellets (50 - 100 μg) were dissolved in 300 - 800 μl of TE buffer,
of which about 1 μl was used for each PCR reaction. DNA was
amplified by primers designed for the promoter region of the IL-6
gene. The primers used were: 5’-CCTAAGCTGCACTTTTCCCCCTAGTTGTGT-3’
(forward) and 5’-GGTTGAGACTCTAATATTGAG-3’ (reverse). The PCR
reactions were performed on a Perkin Elmer GeneAmp System 9700
according to the manufacturer’s protocol. Specifically, these
reactions were carried out in 50 μl volume of 20 mM Tris-HCl (pH
8.4), 50 mM KCI, 1.0 mM MgCl2, 0.2 mM dNTP, 1 unit Taq polymerase (Gibco-Invitrogen),
and 0.4 μM of each oligonucleotide primer. The reactions were heated
to 95°C for 2 minutes followed by 35 cycles of 95°C for 30 seconds,
62°C for 30 seconds, and 72°C for 30 seconds. At the end, the
reactions were extended for 7 minutes at 72°C. After PCR
amplification, PCR products (174bp) were digested with the Nla III
restriction enzyme overnight and electrophoresed on 3% 2:1 Nusiev/SeaKem
agarose gel. The presence of a cytosine (C allele) at nucleotide
-174 was revealed by the presence of the Nla III cutting site. The
genotypes identified at the -174 G/C locus were classified as G/G
(homozygous for the absence of the Nla III cutting site), G/C
(heterozygous for the absence/presence of the Nla III cutting site),
and C/C (homozygous for the presence of the Nla III cutting site).
The allele types were determined as follows: three fragments of
111bp, 37bp, and 26bp for the C/C; two fragments of 148bp and 26bp
for the G/G; and four fragments of 148bp, 111bp, 37bp, and 26bp for
the C/G genotypes.
Statistical Analyses
IL-6 -174G/C allelic frequencies found in this study population were
compared to those in previous study populations. IL-6 -174G/C
genotype (GG, GC, CC) distributions for cases and controls were
tested for adherence to the Hardy-Weinberg equilibrium.
All statistical inquiries were conducted using SAS Software version
8.2e from the SAS Institute in Cary, NC. Descriptive comparisons
(i.e. means, SD, frequencies as percents) of cases and controls were
conducted utilizing chi-square tests for categorical variables, and
analysis of covariance (ANCOVA) for continuous variables.
Logistic regression was utilized to calculate odds ratios (ORs) and
corresponding 95% confidence intervals (CI), adjusted only for age
and sex, to estimate the strength of an association between IL-6
-174G/C genotype and risk for incident sporadic colorectal adenomas.
The effect of IL-6 -174G/C genotype was analyzed using the most
common homozygote genotype, GG, as the referent group. A test for
trend was calculated across genotypes to assess the pattern of
association.
Several risk factors were scrutinized as possible confounders or
effect modifiers of the IL-6 -174G/C genotype-colorectal adenoma
association. Among these were age, sex, race, body mass index [BMI =
weight(kg)/height(m)2], family history of colon cancer in a first
degree relative (FHCC), smoking, alcohol consumption, NSAID use, and
intakes of total fat, energy, calcium, fiber, folate, meat,
vegetables and fruit, and various antioxidant micronutrients.
Criteria for inclusion of any covariate in the final model included:
1) biological plausibility, 2) whether it fit the model at p ≤ 0.1
for Ho: b = 0 (i.e., OR = 1), and 3) whether it altered the OR for
the primary exposure variable, IL-6 -174G/C genotype, by 10 percent
or more. Covariates for the final models for genotype effects
included only age and sex. Covariates for final models to assess
possible interactions between genotypes and various anti- and
pro-inflammatory and other key risk factors included age, sex, FHCC,
NSAID use, smoking status, and total intakes of energy, calcium, and
alcohol.
To examine the separate and combined effects of IL-6 -174G/C
genotype and certain risk factors, stratified analyses were
conducted. Continuous variables were dichotomized on median values
for controls; furthermore, continuous dietary variables were
categorized as sex-specific. Criteria for assessing effect modifiers
were based on previous literature, biological plausibility, and
whether or not risk estimates differed substantially across strata.
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