Download Chapter 5 Genetic polymorphisms and Expression of

Chapter 5
GENETIC POLYMORPHISMS AND
EXPRESSION OF INTERLEUKIN-8 AND
RISK OF GASTRIC CANCER
Introduction
Cancer is one of the leading causes of adult deaths worldwide. In India, the International
Agency for Research on Cancer estimated indirectly that about 635 000 people died from cancer
in 2008, representing about 8% of all estimated global cancer deaths and about 6% of all deaths
in India (Ferlay et al., 2010). Gastric cancer is the second leading cause of cancer deaths
worldwide after lung cancer, resulting in more than 800,000 deaths worldwide every year
(N°297, 2009). The Kashmir valley (India) which borders the southern part of the high incidence
belt represents a moderately high incidence area where incidence rates for gastric cancer were:
men 36.70/lack per annum, women 9.9/ lack per annum (Khuroo et al., 1992). Alterations in
various genetic factors are important in increasing gastric cancer risk (Jung et al., 2011). IL-8 a
chemoattractant of neutrophils and lymphocytes, a wide variety of normal and tumour cells could
express IL-8, and role of IL-8 is to initiate and amplify acute inflammatory reactions. Growing
evidence has shown that the important roles IL-8 may play in the pathogenesis of cancer,
including angiogenesis, tumour growth, and metastasis (Lin et al., 2010). Numbers of molecular
epidemiological studies have been done to evaluate the association between IL-8 –251 A/T
polymorphism and tumor risk in diverse populations. The tumour types included gastric
cancer,(Taguchi A 2005) breast cancer (Kamali et al., 2007) colorectal cancer, (Cacev et al.,
2008) and lung cancer, (Campaet al., 2005). Taguchi and colleagues reported that the IL-8–
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251AA genotype of IL-8 was associated with a significantly increased risk of gastric cancer in a
Japanese population (Taguchi et al., 2005); nevertheless, Savage and colleagues did not find any
significant association between –251A/T polymorphism of IL-8 and gastric cancer in a case–
control study based on a Polish population (Savage et al., 2006). Many studies have
demonstrated the relationship between IL-8 and the risk of GC. IL-8 expression is also strongly
correlated with neovascularization in the tissues from GC patients. The -251 A/T polymorphism
in the IL-8 promoter region has been associated with increased expression of IL-8. Many
researchers reported that the -251 A genotype is associated with the risk of GC as well as antral
atrophy and metaplasia compared with the T genotype. Furthermore, the association between the
-251 A genotype and the risk of GC varied according to histological type and tumor location.
Meta analysis done by Liu et al showed IL-8 -251 A/T polymorphism was not associated with
the risk of GC and the association may be varied when stratified for histological type, tumor
location and ethnicity/country (Liu et al., 2010).
Here we describe a population-based case–control study of 150 incident gastric cancer
cases and 250 cancer-free controls frequency-matched to the cases by age and sex from Kashmir
(India), an area of high risk of gastric cancer, to test the hypothesis that these two promoter
variants of IL-8 and its expression contribute to host susceptibility to gastric cancer.
Subject characteristics
This study included 150 primary gastric cancer patients. All blood and tissue with
adjacent normal samples for this study were taken from gastric cancer patients registerd in the
Department of Surgical Gastroenterology, Sher-I-Kashmir Institute of Medical Sciences, from
March 2009 to March 2012. Tumor types and stages were determined by two experienced
pathologists. Blood samples of 250 age, gender, dwelling and smoking matched cases with no
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signs of any malignancy or any other disease were collected for controls (Table 1). Data on all
gastric cancer patients were obtained from personal interviews with patients and or guardians,
medical records and pathology reports. The data collected included gender, age, dwelling, tumor
location, lymph node status, site of growth, EGD biopsy and salt tea consumption. All patients
and or guardians were informed about the study, and their consent to participate in this study was
obtained on a predesigned questionnaire (available on request). The collection and use of tumor
and blood samples for this study was approved by the appropriate Institutional Ethics
Committee.
DNA extraction, PCR-RFLP and Sequencing
Genomic DNA samples were obtained from blood lymphocytes using a genomic DNA
extraction kit (Bioserve Biotechnologies Pvt. Ltd., India) or by phenol/chloroform method.
Primers and PCR is described in methodology section
For RFLP, the PCR products of IL-8-251 and IL-8-845 SNPs were digested with MunI
and VspI (15 U at 37°C for 16 h) (Fermentas). In the case of IL8-251A/T polymorphism, The A
allele sequence was cut into two fragments by Mun I (500 and 298 bp band), while the T allele
sequence remained intact (798 bp), and the A/T heterozygote was expected to have three bands
(798, 500 and 298 bp) (Fig.1), In the case of IL-8-845T/C polymorphism, only twoband were
seen (337 and 196) (Fig.3),. DNA fragments were electrophoresed through a 3% agarose gel for
resolution. The genotypes of >20% of the samples were double blindly reassessed to confirm the
results by two independent researchers. Also the Purified PCR products showing digestion by
RFLP analysis as well as randomly chosen samples were used for direct DNA sequencing using
the ABI prism 310 automated DNA sequencer (Fig.2 and Fig.4). To minimize the sequencing
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artifacts by PCR, products from at least two different PCRs were sequenced using both forward
and reverse primers.
Immunostaining methods
Immunostaining was done by the method of (Ahmad et al., 2011).
Quantitative evaluation of IL-8
Semi-Quantitative intensity scale ranging from 0 for no staining and +3 for maximum
staining is used for calculating Immunohistochemical score. According to the diffuseness of the
DAB staining, sections were graded as 0 (no staining), 1 (staining, 25%), 2 (staining between
25% and 50%), 3 (staining between 50% and 75%), or 4 (staining >75%). According to staining
intensity, sections were graded as follows: 0 (no staining), 1 (weak but detectable staining), 2
(distinct staining) or 3 (intense staining) (Fig. 5). Immunohistochemical staining scores were
obtained by adding the diffuseness and intensity scores. All slides were examined by two
independent observers who were unaware of the experimental protocol. The slides with
discrepant evaluations were reevaluated, and a consensus was reached. Measurements were
carried out using an Olympus BX51 (Hamburg, Germany) microscope using objectives with 10×
and 40× magnifications.
Semiquantitative Reverse Transcription-PCR (RT-PCR)
Total RNA was extracted after homogenisation with 1 ml/100 mg of gastric tissues by
TRIzol (Invitrogen Life Technologies, USA). RNA extracted’s quantity and quality was
analyzed by UV spectrophotometer and agarose gel electrophoresis. One microgram total RNA
was reverse transcribed using RevertAidTM First Strand cDNA Synthesis Kit (Fermentas Life
Sciences, USA). By use of first-strand cDNA as a template, the specific primers for IL-8 sense
5- AAGGAACCATCTCACTG-3 and antisense 5-GATTCTTGGATACCACAGAG-3 primers
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yield a 352-bp product and GAPDH sense 5-CAAGGTCATCCATGACAACTTTG-3 and
antisense 5-GTCCACCACCCTGTTGCTGTAG-3 primers yeild 496 bp (as reference gene) were
subjected to 35 cycles of PCR amplification (30 s denaturation at 94 ◦C, 30 s annealing
temperature (IL-8 700c and GAPDH 580c, 2 min extension at 72 ◦C) in a thermal cycler (Fig.
6). The amplified products were resolved by gel electrophoresis on 1.5% agarose and visualized
by ethidium bromide (0.5µg/ml). Images of the RT-PCR ethidium bromide stained agarose gel
were acquired using AlphaImagerTM Gel Documentation, USA. Band intensity of the band of
tumours and corresponding normal’s were compared and measured by software Gelquant.NET
Biochemicalsolutions.com Version 1.8.2. with gel scale of 1.The intensities of bands of above
mentioned genes were normalized relative to that of GAPDH bands by dividing the former by
the GAPDH specific PCR product densities. GAPDH acted as control for sample to sample
variations in reverse transcription and PCR conditions and the extent of degradation and
recovery of RNA.
Results
In the present study, we analyzed two SNPs in IL-8 i.e. IL-8-251A/T and IL-8-845 gene in
gastric cancer patients and matched controls in order to evaluate their association with the risk of
gastric cancer in Kashmiri population, and expression of 90 samples by immnohistochemistry
and RT PCR in order to evaluate the correlation of polymorphism versus expression and risk of
gastric cancer in Kashmiri population. Observed frequencies of genotypes in gastric cancer
patients were compared to controls using chi-square or Fisher exact tests when expected
frequencies were small. The chi-square test was used to verify whether genotype distributions
were in Hardy-Weinberg equilibrium. Statistical significance was set at P < 0.05. Odds ratio at
95% confidence intervals (95% CI) and P values were computed by binary logistic regression,
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and all results were adjusted for age, gender, Hot salt tea consumption, smoking, and dwelling.
The independent sample Student’s t-test was applied to check association between cases and
controls. The P values < 0.05 were considered to indicate statistical significance in these tests.
All analyses were performed using the statistical package SPSS ver. 16 (SPSS Inc., Chicago,
IL).Haplotype analysis was performed by Shesis online version.
The correlation of IL-8-251 A/T polymorphic status with the clinicopathological
characteristics was carefully analyzed. It was found that the IL-8-251 A/T was associated with
clinical age, smoking status, lymph node involvement and stage with gastric cancer (Table 2),
but after analyzing by multivariate regression analysis (Table 3) lack of association of
hetrozygous form of IL-8 251 A/T with gastric cancer with odds ratio of 0.493 and P value of
0.007 and association of AA genotype of IL-8-251 A/T with odds ratio of 1.013 and P value of
0.944 was found. Further statistics for mutant of IL-8-251 AA Vs AT/TT shows association with
odds ratio of 1.413 and P value of 0.398 with gastric cancer.
Expression study of 90 samples by immunohistochemistry and RT PCR does not show
any association with gastric cancer (Table 4) as genotype of IL-8-251 A/T showed no association
with gastric cancer (Table 5). Semiquantitative analysis by RT-PCR showed expression of all
samples to almost equal extent thus correlating with IHC and no association was found with the
gastric carcinoma. It was found after correlating the expression with polymorphism no
association was found (Fig.7).
IL-8-845 SNP is not found in our population. Hence only two bands were found in both
cases and controls in our study population.
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Table 1: Clinicopathological characteristics of gastric cancer patients and
healthy controls used for Polymorphic and expression studies
Variables
Cases
N=150(%)
Controls
N=250(%)
P Value
<45
>45
110(74.7)
40(25.3)
170(68.0)
80(32.0)
P>0.05
Male
Female
112(73.3)
38(26.5)
162(64.8)
88(35.2)
P>0.05
Rural
Urban
90(60.0)
60(40.0)
170(68.0)
80(32.0)
P> 0.05
94(62.7)
56(37.3)
190(76.0)
60(24.0)
P< 0.01
98(65.3)
52(34.7)
130(52.0)
120(48.0)
P<0.05
120(80)
30(20)
-
-
102(68)
48 (32)
-
-
80(53.3)
70(46.7)
-
-
-
-
-
-
Age
Sex
Dwelling
Salt tea consumption
<4 cups/day
>4 cups /day
Smoking
Ever
Never
Site of growth
GE junction
Others
EGD biopsy
Intestinal
Diffuse
Lymph node involvement
Yes
No
Clinical tumor stage
I
II
III
Grade
I
II+III
67(44.7)
40(26.7)
43(28.7)
123(82)
27 (18)
EGD=Esophagogastroduodenoscopy, GE Junction=Gastroespophageal junction
The P values < 0.05 were considered to indicate statistical significance in these tests.
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Table 2: Association between IL-8 251A/T genotypes and clinicopathological
characteristics
Clinical Parameters
IL 8 251 genotype
AT AA
Total
TT
P value
Age
<45
>45
30
21
70
16
10
3
110
40
Male
Female
44
7
59
27
9
4
112
38
0.06
Rural
Urban
36
15
45
41
9
4
90
60
0.08
38
13
48
38
12
1
98
52
0.009
33
18
51
35
10
3
94
56
0.44
38
13
71
15
11
2
120
30
0.47
29
22
65
21
8
5
102
48
0.07
0.014
Sex
Dwelling
Smoking
Ever
Never
Salt tea consumption
<4 cups/day
>4 cups /day
Site of growth
GE junction
Others
EGD biopsy
intestinal
Diffuse
Lymph node involvement
Yes
No
Clinical tumor stage
I
II
III
Grade
I
II+III
40
11
30
56
10
3
80
70
<0.001
19
17
15
46
2
18
8
22
3
67
43
40
>1
123
27
0.007
40
11
76
10
7
6
EGD=Esophagogastroduodenoscopy, GE Junction=Gastroespophageal junction
The P values < 0.05 were considered to indicate statistical significance in these tests.
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Table 3: Multivariate logistic regression model for association of IL 8 251 A/T polymorphism and gastric
cancer risk
Genotype
Controls (%)
IL 8 251
TT
AT
AA
T
A
AA/AT Vs TT(Dominant)
AA Vs AT/TT(Recessive)
152(60.8)
68(27.2)
30 (12)
372(74.4)
128 (25.6)
98/152
30/210
Casesl (%)
51 (34)
86(57.3)
13(8.7)
188(62.7)
112(37.3)
99/51
13/137
ORa
Ref.1
0.2653
0.7743
Ref. 1
0.5776
0.3321
1.55
CI (95%)a
0.1693-0.4157
0.3754-1.5971
0.4243-0.7862
0.2177-0.5068
0.7585-2.988
P valuea
<0.001
0.488
0.0004
<0.001
0.240
ORB
0.493
1.013
0.573
1.413
CI(95%)b
0.295-0.822
0.446-2.383
P b value
0.007
0.944
0.354-0.930 0.024
0.633-3.153 0.398
ORb _ crude odds ratio calculated at 95% confidence interval (95% CI) for adjusted model for sex (categorical), dwelling
(continuous), smoking(categorical), and hot salt tea consumption (categorical). The P values < 0.05 were considered to indicate
statistical significance in these tests. OR ratio less than 1 is associated in decreasing risk.
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Table 4: Immunohistochemical scores of IL-8 expression in Gastric cancers
Parameters
Immunohistochemical score of IL-8 expression (N=90)
0
1
2
3
P Value
Grade
I
II
III
5
7
8
8
6
9
6
8
6
5
9
13
0.8163
I
II
III+IV
6
5
5
9
10
12
8
7
6
8
9
5
0.9295
Stage
0 is absent staining, 1is weak staning, 2 is moderate staining, and 3 is strong staining.
The P values < 0.05 were considered to indicate statistical significance in these tests.
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Table 5: Relationship of IL-8 polymorphisms and IL-8 expression in gastric
cancer
Genotypes
Number
underexpression
overexpression
(0+1)
(1+2)
13
13
17
30
11
17
19
36
P value
IL-8
TT
AT
AA
AT+AA
24
30
36
66
Ref 1
0.60
0.79
0.62
a=one tailed and b=pearsons
The P values < 0.05 were considered to indicate statistical significance in these tests.
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Figure 1: Showing IL-8-251A/T amplicons and RFLPs
A.
800bp
500bp
300bp
798bp
200bp
100bp
B.
800bp
798bp
500bp
500bp
100bp
298bp
Fig . 1:
A: IL-8-251
A/T (798bp) gel picture. Lane M: 100bp DNA marker, Lanes 1-5: cases and Lanes6-9: controls
B: RFLP picture of IL-8-251 A/T after restriction digestion with MunI enzyme
Lane M: 100bp DNA marker, Lane1: undigested sample, Lanes 5 wild (TT) genotype, Lanes 1,2,3 and 6:
heterozygous (AT)
(798, 500 and 298 bp) genotype and Lane 4:(AA) (500 and 298 bp) Variant genotype.
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Fig. 2: Partial electropherogram sequences of IL-8-251A/T.
Normal
Heterozygous
Variant
Fig. 2: Partial electropherogram sequences (forward) of normal, heterozygous and variant of
IL-8-251A/T.
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Figure 3: Showing IL-8-845 amplicons and RFLPs
A.
500bp
530bp
100bp
B.
500bp
337bp
300bp
196bp
100bp
Fig. 3:
A: IL-845 (530bp) gel picture. Lane M: 100bp DNA marker, Lanes 1-5: cases and Lanes6-9:
controls
B: RFLP picture of IL-8-845 after restriction digestion with VspI enzyme
Lane M: 100bp DNA marker, Lane1-9: digested sample only two bands 337bp and 196bp.
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Fig.4: Partial electropherogram of IL-8-845.
Normal
Fig. 4: Partial electropherogram sequences (forward) of normal of IL-8-845.
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Fig.5: IL-8 expression by IHC
Fig. 5: IHC representative photomicrographs (400×) of IL-8 of Gastric cancer tissue. a. absent,
b. weak, c. moderate and d. strong expression
.
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Fig. 6: Intensities of IL-8
400bp
352
bpIL-8
IL-8
352bp
300bp
200bp
50bp
400bp
496bp GAPDH
300bp
200bp
50bp
Fig. 6: Intensities of IL-8 and GAPDH cDNA bands (agarose gel) in gastric tissues M=marker
and 1-6 represents band intensities. Gastric cancer tissues from ninety patients were removed for
the preparation of total cellular RNA. IL-8 mRNA expression. A 352bp sequence, corresponding
to the total open reading frame, was amplified and separated on 1.5% agarose gel
electrophoresis. As an internal control GAPDH mRNA (496 bp) was assayed in parallel. The
amplified sequence was visualized using ethidium bromide staining under UV light. M, 50bp
Molecular size marker.
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Fig.7 Graphs showing IL-8 mRNA expression of tumor in comparison with
their respective normal’s by RT-PCR.
1
0.8
0.6
0.4
0.2
0
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
T14
T15
T16
T17
T18
T19
T20
T21
T22
T23
T24
T25
T26
T27
T28
T29
T30
Band intensity
Gel scale(1)
IL-8 mRNA expression by RT-PCR
N1 N2 N3 N4 N5 N6 N7 N8 N9N10N11N12N13N14N15N16N17N18N19N20N21N22N23N24N25N26N27N28N29N30
Cancerous Vs Normal tissues 1-30
1
0.8
0.6
0.4
0.2
0
T31
T32
T33
T34
T35
T36
T37
T38
T39
T40
T41
T42
T43
T44
T45
T46
T47
T48
T49
T50
T51
T52
T53
T54
T55
T56
T57
T58
T59
T60
Band intensity
Gel scale(1)
IL-8 mRNA expression by RT-PCR
N31N32N33N34N35N36N37N38N39N40N41N42N43N44N45N46N47N48N49N50N51N52N53N54N55N56N57N58N59N60
Cancerous Vs Normal tissues 31-60
1
0.8
0.6
0.4
0.2
0
T61
T62
T63
T64
T65
T66
T67
T68
T69
T70
T71
T72
T73
T74
T75
T76
T77
T78
T79
T80
T81
T82
T83
T84
T85
T86
T87
T88
T89
T90
Band intensity
Gel scale(1)
IL-8 mRNA expression by RT-PCR
N61N62N63N64N65N66N67N68N69N70N71N72N73N74N75N76N77N78N79N80N81N82N83N84N85N86N87N88N89N90
Cancerous Vs Normal tissues 60-90
Fig.7: IL-8 mRNA expression in gastric cancer tissues. Tumoral tissues (T1- T50) and non
Tumoral mucosa (N1-N50) are indicated, with the numbers shown corresponding to the patient
number. Semi-Quantitative analysis of IL-1B mRNA expression by Reverse transcriptase PCR.
Band intensity of the band of tumours and corresponding normal’s were compared and measured
by software Gelquant.NET Biochemicalsolutions.com Version 1.8.2. with gel scale of 1.
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Discussion
In this population-based case–control study, we found significantly increased risk of
gastric cancer associated with the IL-8-251 A/T variant genotypes and the risk was significantly
more evident among individuals with smoking status and upper age, suggesting that the promoter
variant of IL-8-251A/T may play an important role in increasing gastric cancer in Kashmiri
population and may modulate the risk of gastric cancer. IL-8, a member of the chemokine family,
is produced by a wide range of normal cells including monocytes, neutrophils, fibroblasts, and
endothelials cells, as well as by several types of tumor cells (Hongyan et al., 2002). It was
originally described as a chemoattractant for neutrophils and lymphocytes (K et al., 2001) and
recently linked to cancer progression through its functions as mitogenic, motogenic, and
angiogenic factor (Taguchi et al., 2005). Recent studies revealed that IL-8 is overexpressed in a
range of human cancers including nasopharyngeal (Beck et al.,2001), breast (Yokoe et al.,
2000), and gastric cancers (Kitadai et al., 1998) and may, thus, constitute a risk factor in the
development of solid tumors. Studies have shown that epithelial injury and repeated regeneration
caused by inflammation increases the risk for several cancers including: ulcerative colitis,
chronic gastritis, chronic pancreatitis, Barrett esophagus and chronic hepatitis (Liwei et al.,
2010).
In our study it was found that polymorphism of IL-8-251A/T is not associated with
increased expression of IL-8. Many studies have demonstrated the relationship between IL-8 and
the risk of gastric cancer. IL-8 expression is also strongly correlated with neovascularization in
the tissues from gastric cancer patients (Kitadai et al., 1998). The IL-8-251 A/T polymorphism
in the IL-8 promoter region has been associated with increased expression of IL-8 (Hull et al.,
2000), IL-8 may stimulate the expression of Reg protein in stomach cells, which intensifies the
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proliferation of gastric mucosal cells and may indirectly promote GC initiation (Yoshino et al.,
2005). Moreover, IL-8 was reported to be involved in lung cancer etiopathogenesis as well as in
the initiation and progression of multiple myeloma (Xie et al., 2010; Vetvicka et al., 2011).
In our study it was found that A genotype is associated with gastric cancer risk as is
consistent with many researchers that reported IL-8-251 A genotype is associated with the risk
of gastric cancer as well as antral atrophy and metaplasia compared with the T genotype
Furthermore, the association between the IL-8-251 A genotype and the risk of gastric cancer
varied according to histological type and tumor location, as was found in our study that genotype
associated with tumour stage (Liu et al., 2010 ). Many studies have been performed to evaluate
the relationship between genetic polymorphisms of various cytokines and gastric cancer (ElOmar et al., 2000). It has been reported that the IL-8-251 A allele is associated with progression
of gastric atrophy in patients, and increases the risk of gastric cancer in Korean and Japanese
patients (Ohyauchi et al., 2005; Taguchi et al., 2005; Ye et al., 2009).
In our study it was found that AA genotype is associated with increased risk of gastric
cancer. Which is same as that of Chinese population, subjects who carried the IL-8-251 AA
genotype have an increased risk of gastric cancer (Lu et al., 2005), whereas the IL-8- 251 T
allele is significantly associated with increased risk of diffuse- type or mixed-type gastric cancer
(Lee et al., 2005).
It was found in our study that the IL-8-251 A/T was associated with clinical age, smoking
status, lymph node involvement and stage with gastric cancer as several studies performed in
Western countries did not find any significant relationship between IL-8-251 A/T polymorphism
and risk of gastric cancer (Savage et al., 2006; Canedo et al., 2008). These conflicting results
suggest that IL-8-251 A/T polymorphism may be ethnic-specific and the relevant host-susceptible
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factor for gastric cancer (Ji et al., 2010 ). The analysis of Sugimoto et al. (Sugimoto et al., 2010)
indicated an absence of association between A allele carriers and noncardiac GC risk (OR 0.99), whereas peptic ulcer risk was significantly higher among Western compared with Asian
non-GC patients (OR-1.49). Most studies in the meta-analysis have reported positive
associations for Asians and negative associations for Caucasians. The sample size was
approximately equal to that in the study of Lu et al. (Lu et al., 2007).Gastric cancer, like many
malignancies, is a result of interaction between genetic factors of the host together with dietary
and other factors in the environment.
Our study finds that salt Tea consumption is a risk factor of gastric cancer in case of
Kashmiri population. Epidemiological studies on Northern Chinese and American Japanese in
Hawaii lent strong support to the effects of lack of fresh fruit and vegetable, smoking, and
consumption of salty food in the development of gastric cancer (You et al., 1988; Nomura et
al., 1990).
Some studies have suggested that the IL-8-251AA genotype was associated with an
increased risk of prostate cancer and Kaposi’s sarcoma, and a decreased risk of colorectal cancer.
IL-8 has potent activity in the activation and migration of neutrophils and, therefore, may play an
important role in H.pylori-associated gastric inflammation. It is possible that increased IL-8
might amplify the inflammatory response to H.pylori by recruiting neutrophils and monocytes,
and thus results in an advanced degree of gastritis, ultimately predisposing to the development of
gastric cancer. Moreover, the expression of IL-8 directly correlates with gastric peritumoral
cellular infiltrates, hypostatic migration of tumor cells and angiogenesis of gastric cancer.
Studies in vitro have shown that the IL-8-251 A allele tended to be associated with increased IL8 production by lipopolysacharide- stimulated whole blood. Wanli Lu observed an elevated
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riskfor IL-8-251 AA genotype and gastric cancer in subjects without H.pylori infection, but the
CIs included the null value. The data suggested that the IL-8 polymorphism might be an
independent risk factor for the pathogenesis of gastric cancer. Cigarette smoking is an
established risk factor of gastric cancer. Although alcohol drinking is an established risk factor
for cancers of the upper aero digestive tract, the evidence for an association with gastric cancer is
weak. However, studies have demonstrated that smoking and drinking may influence the
production of cytokines. Therefore, smoking and drinking may interact with cytokine genotypes
to enhance gastric inflammation. Presence of both smoking or drinking significantly elevated the
risk of the cancer (Wanli et al., 2005).
Conclusion
This study provides evidence to support a relationship of increased susceptibility to
gastric cancer in individuals of the Kashmir valley with IL-8 251 AA genotype, especially for
those individuals who do smoke and have older age.
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