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Clinical Cancer Research Vol. 11, 6431-6441, September 15, 2005
© 2005 American Association for Cancer Research

Human Cancer Biology

The –251T Allele of the Interleukin-8 Promoter Is Associated with Increased Risk of Gastric Carcinoma Featuring Diffuse-Type Histopathology in Chinese Population

Wei-Ping Lee1,4, Dar-In Tai8, Keng-Hsin Lan1,5, Anna Fen-Yau Li2,6, Hou-Ching Hsu1, En-Ju Lin1, Yi-Ping Lin1, Meei-Ling Sheu7, Chung-Pin Li1,6, Full-Young Chang1,6, Yee Chao3,6, Shang-Heu Yen3,6 and Shou-Dong Lee1,6

Authors' Affilliations: 1 Division of Gastroenterology, Department of Medicine, 2 Department of Pathology, and 3 Cancer Center, Taipei Veterans General Hospital; 4 Department and Institute of Biochemistry, 5 Department and Institute of Pharmacology, and 6 School of Medicine, National Yang-Ming University; and 7 Department of Toxicology, National Taiwan University College of Medicine, Taipei, Taiwan; and 8 The Liver Research Unit, Department of Gastroenterology and Hepatology, Chang Gung Memorial Hospital, Taoyuan, Taiwan

Requests for reprints: Wei-Ping Lee, Division of Gastroenterology, Department of Medicine, Taipei Veterans General Hospital, 201 Shi-Pai Road Section 2, Taipei 112, Taiwan. Phone: 886-2-28712121-2017; Fax: 886-2-2874-9425; E-mail: wleemc{at}yahoo.com.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Persistent interleukin-8 (IL-8) production contributes to chronic inflammation of the stomach. The proinflammatory IL-1ß polymorphisms, which enhance the cytokine production, are associated with increased risk of gastric cancer. The –251A/T polymorphism of the IL-8 promoter is involved in several human diseases. Particularly, the –251A is associated with decreased risk of colorectal cancer. We aimed to determine whether the –251 allele resulting in high IL-8 expression was associated with increased risk of gastric carcinoma.

Experimental Design: The –251A/T promoters were cloned and analyzed by luciferase assay. Binding of nuclear proteins to the –251A/T promoters was analyzed by electrophoretic mobility shift assay. The –251A/T promoters were differentiated by PCR-RFLP. Comparison of gastric cancer risk between the –251A/T promoters was done by a case-control study.

Results: The –251T allele possessed transcriptional activity 2- to 5-fold stronger than the –251A counterpart. Electrophoretic mobility shift assay showed that the –251A promoter had strong ability to bind to an unknown protein or multiprotein complex. The –251T allele was associated with increased risk of noncardia (Ptrend = 0.012) and cardia (Ptrend = 0.029) carcinomas. Gastric carcinoma patients with the low-risk AA genotype had a tendency to sustain intestinal-type carcinomas ({chi}2 = 6.816; P = 0.033); however, the high-risk –251T allele was associated with >2-fold increased risk of diffuse-type (AA versus AT + TT: odds ratio, 2.52; 95% confidence interval, 1.16-5.49; P = 0.017) and mixed-type (AA versus AT + TT: odds ratio, 2.22; 95% confidence interval, 1.12-4.40; P = 0.019) carcinomas.

Conclusions: The IL-8 –251T allele is significantly associated with increased risk of gastric carcinoma, particularly the diffuse and mixed types in Chinese population.

Gastric cancer is a major cancer death in the world. Helicobacter pylori, a member of a large family of related bacteria that colonize the mammalian stomach, have been associated with peptic ulcers, non-Hodgkin's lymphoma of the stomach, gastric atrophy, and gastric carcinoma (1, 2). However, only <3% of individuals who carry H. pylori ever develop gastric neoplasias related to its presence, indicating that other factors are involved (3). Progression to gastric malignancy is likely due to the concerted effects of bacterial pathogenicity, host susceptibility, and environmental factors.

Interleukin (IL)-1ß is the first cytokine showing gene polymorphism related to gastric cancer. It is a potent proinflammatory cytokine, and its production is stimulated by H. pylori (4, 5). The IL-1B gene cluster contains IL-1B encoding IL-1ß and IL-1RN encoding its naturally occurring receptor antagonist and has several functionally relevant polymorphisms that correlate with high incidence of gastric carcinoma, including the IL-1B –31T, IL-1B –551T, and IL-1RN*2 alleles (6, 7). In addition, the –308A allele of proinflammatory TNF-A and the –1082A and –592A alleles of anti-inflammatory IL-10 are also associated with more than doubling of the risk for noncardia gastric cancer (8, 9).

IL-8, a member of the CXC chemokine family, is a chemoattractant of neutrophils and lymphocytes (10, 11). H.pylori adhere to gastric epithelial cells, activate nuclear factor-{kappa}B, and stimulate IL-8 production (12, 13). Gastric mucosal levels of IL-8 are in parallel with the histologic severity of gastritis (12). The role of IL-8-induced chronic inflammation in gastric carcinogenesis remains unclear; however, IL-8 is a potent angiogenic factor involved in tumor invasion and metastasis (14–18). IL-8-mediated vascularization in the rat cornea assay (18) has led to its implication in angiogenesis in a variety of diseases, such as psoriasis (19), rheumatoid arthritis (20), idiopathic pulmonary fibrosis (21), and some neoplasms (14, 22–25). Gastric carcinoma cells in surgical specimens overexpressed IL-8 compared with corresponding normal mucosa (24, 26), and the IL-8 mRNA level directly correlated with the vascularity of the tumors (24). Furthermore, transfection of gastric carcinoma cells with the IL-8 gene enhanced their tumorigenic and angiogenic potentials in the gastric wall of nude mice (27). Thus, IL-8 seems to play a role in invasion and metastasis of gastric carcinoma through angiogenesis.

The IL-8 promoter is estimated to be 1,500 bp. Several reports have shown relationship between IL-8 gene polymorphisms and human diseases (28–32), and all of them have focused on the A/T polymorphism at –251 upstream from the transcriptional start site. The IL-8 gene –251T has recently been reported to be a risk factor of bronchial asthma (28), whereas –251A confers increased incidence in bronchiolitis caused by respiratory syncytial virus (29, 30). AIDS patients with the –251TT genotype are protected from the development of severe visceral Kaposi's sarcoma (31). Individuals with the –251TT genotype have lower chance in developing Parkinson's disease (32). The combined genotypes of IL-8 –251TT and IL-10 –819TT are associated with a high probability of persistent H. pylori infection (33).

The association of the proinflammatory cytokines IL-1ß and tumor necrosis factor-{alpha} with risk of gastric carcinoma (6–9) raises a possibility that high level of IL-8 in the gastric mucosa may also be associated with increased risk of the malignancy. Therefore, we hypothesized that the IL-8 promoter allele, which resulted in higher IL-8 secretion, was associated with increased risk of gastric carcinoma. To test this hypothesis, we cloned the IL-8 promoter, analyzed promoter activity of –251A/T alleles, and evaluated the relationship between IL-8 –251A/T polymorphism and gastric cancer risk in a case-control study.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Study subjects. A hospital-based case-control study was conducted to evaluate the association of IL-8 –251A/T genotypes with incidence of gastric carcinoma in Taipei Veterans General Hospital (Taipei, Taiwan). The hospital was established to take care of retired servicemen and their family, and it also admits ordinary citizens now. The majority of retired servicemen ages >60 years were from mainland China when they were young. Subjects included 470 gastric carcinoma patients (92 cardia and 378 noncardia) and 308 control blood donors who were health checkup examinees in the hospital. Cases were patients with new diagnosis of gastric carcinoma attending the hospital from January 1999 to December 2003. All cases had histologic confirmation of their tumor diagnosis. Patients included were those able to endure surgical treatment or chemotherapy, and those with major systemic diseases or mental disability were excluded from the study. Individuals participating in this study signed an agreement approved by the board of human clinical study of the hospital. Each subject was scheduled for an interview after informed consent was obtained, and a structured questionnaire was administered by interviewers to collect information on demographic data. Some gastric carcinoma samples were obtained from surgical tissues embedded in parafilm blocks because of failed genomic DNA purification from blood samples.

Cell lines and cell cultures. MKN45 is a well-known gastric carcinoma cell line (34). SC-M1 is also a gastric carcinoma cell line derived from a surgical sample in Taiwan (35). Both cell lines were grown in DMEM + F-12 (1:1; Life Technologies, Gaithersburg, MD) supplemented with 10% FCS in a humidified atmosphere of 5% CO2 at 37°C.

Genomic DNA purification. Human leukocyte genomic DNA was purified with standard phenol/chloroform extraction (36). Briefly, heparin-treated whole blood was centrifuged at 1,000 x g, and buffy coat on the interface between RBC and plasma was collected. RBCs were lysed by 10-fold volume of distilled water. The resulting genomic DNA and cell debris were precipitated by centrifugation at 10,000 x g. The pellet was resuspended in TE [10 mmol/L Tris, 1 mmol/L EDTA (pH 8.0)] containing 0.5% SDS, 10 µg/mL proteinase K (Roche, Indianapolis, IN), and 1 µg/mL RNase A (Roche) and then incubated at 55°C overnight. Then, genomic DNA was purified by phenol/chloroform extraction. Genomic DNA of some samples was purified from parafilm-embedded blocks (37). The surgical tissues were thin-sliced, and parafilm was removed with sufficient amount of xylene. The tissues were cleaned twice with 95% ethanol and then air-dried. DNA from the tissues was dissolved in TE containing 0.5% SDS and then further purified as described for genomic DNA purification of blood samples.

PCR for generation of the full-length interleukin-8 promoter. The full-length IL-8 promoter (1,524 bp, from –1,479 to +45) was synthesized by PCR with 0.5 µg genomic DNA from a healthy volunteer with heterozygote AT at –251 locus of the IL-8 promoter. The forward and reverse primers for PCR were 5'-CCGCTCGAGATTCAGTAACCCAGGCATTATT and 5'-CCCAAGCTTGTGTGCTCTGCTGTC, respectively. The KpnI and HindIII sites (underlined bases) were flanked at 5' and 3' ends of the promoter, respectively. The reaction condition for PCR was 95°C for 1 minute, 55°C for 1 minute, and 72°C for 5 minutes. The PCR product was analyzed by 1% agarose gel electrophoresis.

Analysis of interleukin-8 promoter activity. The two full-length IL-8 promoters with –251A and –251T were cloned to the multiple cloning sites (KpnI and HindIII) of the pGL3-Basic plasmid (Promega, Madison, WI) in which the IL-8 promoters (–251A or –251T) drove the expression of the firefly luciferase gene. The –251A and –251T were differentiated by the restriction enzymes HindIII and MfeI and further confirmed by DNA sequencing. Plasmid DNA of the –251A and –251T IL-8 promoters in different amounts (0.05, 0.1, and 0.2 µg) was separately transfected with 0.01 µg of the pRL-TK control plasmid (carrying the gene coding for Renilla luciferase) into MKN45 (3 x 105) and SC-M1 (2 x 105) gastric carcinoma cells in 3-cm tissue culture dishes. The transfection was done by using Lipofectin (Life Technologies) mixed with plasmid DNA in serum-free medium. The medium was replaced with serum-enriched medium at 6 hours after transfection. Then, cells were cultured for 36 hours and subjected to luciferase assay. The –251A and –251T promoter activities were determined by intensity of firefly luciferase, which was carried out by the Dual-Luciferase Assay System (Promega). The firefly luciferase activity was adjusted with the internal control activity of Renilla luciferase.

Electrophoretic mobility shift assay. The assay was carried out as described previously (38). MKN45 and SC-M1 gastric carcinoma cells (1 x 106) were washed twice with PBS, scrapped into 1.5 mL cold PBS, pelleted for 10 seconds, and then resuspended in 200 µL cold buffer A [10 mmol/L HEPES-KOH (pH 7.9), 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L DTT, 0.2 mmol/L phenylmethylsulfonyl fluoride]. Cells were allowed to swell on ice for 10 minutes and then vortexed for 10 seconds. Samples were centrifuged for 10 seconds, and the supernatant was discarded. The pellet was resuspended in 100 µL cold buffer C [20 mmol/L HEPES-KOH (pH 7.9), 25% glycerol, 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.5 mmol/L DTT, 0.2 mmol/L phenylmethylsulfonyl fluoride] and incubated on ice for 20 minutes for high-salt extraction. Cell debris was removed by centrifugation for 2 minutes at 4°C, and the supernatant fraction (nuclear extract) containing DNA-binding proteins was stored at –70°C until use. The nuclear extract (5 µg protein) was incubated with 1 µg poly(deoxyinosinic-deoxycytidylic acid) (Roche) on ice for 20 minutes and then with the allele-specific 32P-labeled double-stranded oligonucleotides 5'-TAAAAAAGCATACA (A/T) TTGATAATTCACCAA designed according to the IL-8 promoter sequence. The underlined nucleotides represent the MfeI site (CAATTG) in –251A promoter. Binding of the radioactive double-stranded oligonucleotides to nuclear DNA-binding proteins was carried out at room temperature for 20 minutes. The resulting double-stranded oligonucleotide-protein complexes were resolved in 4% nondenaturing PAGE followed by X-ray film autoradiography.

Determination of the interleukin-8 promoter –251A/T genotypes. An 816-bp partial IL-8 promoter was generated by PCR. To enrich the products, PCR was done twice but with different primers. The first PCR was done with the primers for the full-length IL-8 promoter as described above. The template was 0.5 µg genomic DNA. The condition for the first PCR was 95°C for 1 minute, 55°C for 1 minute, and 72°C for 5 minutes. The PCR product was diluted 50 times with TE buffer. The second PCR was done with the primers 5'-GATTCTGCTCTTATGCCTCCA and 5'-CCCAAGCTTGTGTGCTCTGCTGTC. The template was 2 µL of the diluted PCR product with a total reaction mixture of 50 µL. The reaction condition for the second PCR was the same as that for the first PCR. The resulting PCR products were digested with MfeI, which recognized CAATTG and thereby differentiated A from T at –251 locus.

Detection of Helicobacter pylori infection. H. pylori infection was detected by gastric specimens of endoscopic biopsy, which were either processed for Giemsa staining (39) or subjected to urease test (CLO test, Delta West, Bentley, Australia). The subjects tested were those who had not received anti–H. pylori treatment. Some of the study subjects were subjected to 13C-urea breath test (Otsuka Pharmaceutical Co., Chiyoda-ku, Tokyo, Japan).

Statistical analysis. The Hardy-Weinberg equation was used to determine whether the proportion of each genotype obtained was in agreement with expected values. Differences in selected demographic variables and genotype frequencies between cases and controls were evaluated by using the {chi}2 test. The association between the IL-8 –251 genotypes and risk of gastric carcinoma was estimated by computing odds ratios (OR) and their 95% confidence intervals (95% CI) from univariate and multivariate logistic regression models. Stratification analysis was used to estimate risk for subgroups by age, sex, ethnicity, and tumor histology. These statistical analyses were done with the SPSS software program (SPSS Science, Chicago, IL). All tests were two sided, and differences were considered to be significant at P < 0.05.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Promoter activity of interleukin-8 –251A/T alleles. The IL-8 promoter contains several elements to which transcriptional factors bind (40), including nuclear factor-{kappa}B, activator protein-1, CAAT/enhancer-binding protein, Lef/TCF (for ß-catenin), etc. (Fig. 1A). There is no major polymorphism within these sites. The first polymorphism potentially involved in the promoter's activity is located at –251 upstream from the transcriptional start site (Fig. 1A). To determine whether –251A/T polymorphism could influence activity of the promoter, the full-length –251A/T promoters of 1,524 bp (Fig. 1B) were cloned into the pGL3-Basic plasmid. The two different promoters were differentiated by HindIII + MfeI digestion, which released a fragment of 290 bp in the plasmid carrying the promoter of –251A allele (Fig. 1C). The resulting IL-8 –251A/T promoters drove the expression of the firefly luciferase gene in the pGL3-Basic plasmid (Fig. 2). The –251A/T promoter plasmids were separately transfected into MKN45 and SC-M1 gastric carcinoma cells. The promoter strength was determined by luciferase activity. IL-8 –251T showed luciferase activity 2- to 5-fold stronger than IL-8 –251A with statistically significant Ps (Fig. 2). The luciferase activities between –251A and –251T were best contrasted in cells transfected with 0.05 µg plasmid (Fig. 2). A control study was done with the pGL3-Basic plasmid without carrying any gene; luciferase activity from the control study was extremely low in comparison with experiments done with the plasmid carrying the –251A/T promoters (data not shown).



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  		Fig. 1. Molecular cloning of the IL-8 promoter into the pGL3-Basic plasmid. A, schematic representation of the IL-8 promoter showing the site of IL-8 –251A/T variation. Modified from a previous report (39). GR, glucocorticoid receptor; HNF-1, hepatocyte nuclear factor-1; IRF-1, IFN-regulatory factor-1. B, PCR product of a 1,524-bp IL-8 promoter. C, cloning of the IL-8 –251A/T promoters. The IL-8 promoters were cloned into the pGL3-Basic plasmid through 5'-KpnI and 3'-HindIII sites. The –251A/T alleles of the IL-8 promoter were differentiated by HindIII and MfeI digestion. IL-8 pmt, IL-8 promoter.

 


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  		Fig. 2. Luciferase assay showing stronger promoter activity in the IL-8 –251T allele. The firefly luciferase gene was driven by the IL-8 –251A/T promoters. The promoters of 0.05, 0.1, and 0.2 µg were transfected with 0.01 µg of the pRL-TK control plasmid (carrying the gene coding for Renilla luciferase) into MKN45 and SC-M1 gastric carcinoma cell lines. Promoter strength was determined by luciferase activity (Dual-Luciferase Assay System), which was presented by values relative to the one for cells transfected with 0.05 µg of the –251A allele plasmid. The firefly luciferase activity was adjusted with the internal control activity of Renilla luciferase. The assays were done with triplicate samples. Differences between the –251A/T promoter activities were determined by the Student's t test.

 
Analysis of the interleukin-8 –251A/T allele-specific binding protein by electrophoretic mobility shift assay. As shown in Fig. 2, the IL-8 –251T allele had stronger promoter activity than its –251A counterpart. To investigate the underlying molecular mechanism, we did electrophoretic mobility shift assay to determine whether there might exist transcription factors bound to –251 locus of the IL-8 promoter with differential binding ability between A and T alleles. The 30-base allele-specific oligonucleotides were synthesized and reannealed to double-stranded DNA, with which to perform electrophoretic mobility shift assay using nuclear extracts from MKN45 and SC-M1 gastric cancer cells (Fig. 3). The –251A double-stranded oligonucleotides had stronger binding activity to a nuclear protein or multiprotein complex than did the –251T double-stranded oligonucleotides (indicated by an arrow in Fig. 3, lanes 1 and 6). Formation of the DNA-protein complex on the radiolabeled –251A double-stranded oligonucleotides was specifically blocked by competition with the unlabeled –251A (lanes 2 and 7) but not the –251T double-stranded oligonucleotides (lanes 3 and 8). This study suggests that the protein or multiprotein complex bound to the –251A may function as a negative regulator for IL-8 transcription. Lipopolysaccharide (Sigma-Aldrich, Saint Louis, MO) was added to treat cells in one set of experiment but did not cause any change in DNA-protein binding shown in Fig. 3 (data not shown).



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  		Fig. 3. Electrophoretic mobility shift assay showing binding of a protein or multiprotein complex to the –251A allele-specific double-stranded oligonucleotides. The 30-bp allele-specific double-stranded oligonucleotides for IL-8 –251A/T (top) were labeled with [{gamma}-32P]ATP, incubated with nuclear extracts from MKN45 and SC-M1 gastric carcinoma cell lines, and then subjected to electrophoretic mobility shift assay. Hot oligo, radioactive oligonucleotides; Cold oligo, oligonucleotides not labeled with [{gamma}-32P]ATP. Cold oligonucleotides were used to compete with radioactive oligonucleotides.

 
Interleukin-8 –251A/T genotyping. An 816-bp fragment of the IL-8 promoter was synthesized by PCR, which possessed a MfeI site at the 520th base. The –251A/T alleles were differentiated by MfeI digestion, resulting in two fragments of 296 and 520 bp in the –251A allele. The PCR products from the AA homozygotes were fully digested by MfeI, those from the AT heterozygotes were half digested, and those from the TT homozygotes were not digested (Fig. 4). The base differences between –251 A and T alleles were further confirmed by DNA sequencing.



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  		Fig. 4. Differentiation of the IL-8 –251A/T promoters by MfeI digestion. An 816-bp fragment of the IL-8 promoter was synthesized by PCR, digested with MfeI, and then subjected to 2% agarose gel electrophoresis. The –251A allele was digested into two fragments of 520 and 296 bp. The –251A/T alleles were further confirmed by DNA sequencing.

 
Characteristics of study subjects. Subjects in controls (n = 308) and cases (n = 470) came from north Taiwan but had different ethnic background (Table 1). There was no significant difference in the distributions of age ({chi}2 test; P = 0.970) and sex (P = 0.301) between the two groups. The median age was 64.2 years for cases (33-82 years) and 62.6 years for controls (29-84 years). The cancer group included 287 patients who either emigrated from mainland China to Taiwan around the year 1949 or were born in Taiwan but with parents from mainland China (ethnic A) and 174 patients (ethnic B) who were born in Taiwan with ancestry as Hoklo or Hakka, originating from south Fujian (Hoklo) or north Guangdong (Hakka) of China and rooting in Taiwan for several generations. Nine cancer cases had unknown ethnicity. The control group included 118 healthy individuals with ethnic A, 185 with ethnic B, and 5 with uncertain ethnicity (Table 1).


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  		Table 1. Demographic data of study subjects

 
Patients admitted to Taipei Veterans General Hospital mainly are retired servicemen and their family, and the majority of them originated from different provinces of China. Thus, more than half of our gastric cancer patients belonged to ethnic A (Table 2). However, ethnic A should normally be 12% to 15%, Hoklo 70% to 75%, and Hakka 12% to 15% in Taiwan. To determine whether ethnicity had significant effect on modulating IL-8 –251A/T genotype frequency, ethnicities and genotype frequencies of cases and controls were subjected to the {chi}2 test. As shown in Table 2, ethnicity had no significant impact on IL-8 –251A/T genotype frequency in the three ethnic groups (ethnic A, Hoklo and Hakka), including controls (P = 0.995) and cases (P = 0.997).


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  		Table 2. Frequencies of IL-8 –251A/T genotypes across ethnicity in Taiwan

 
H. pylori infection was assessed in controls and gastric cancer cases (Table 3). Controls (n = 291) and noncardia gastric cancer cases (n = 364) had ever received tests of H. pylori infection, which included CLO urease test, Giemsa staining of biopsy specimens, and 13C-urea breath test. More than 50% of the subjects tested were infected by the bacteria. Individuals infected by H. pylori had 2.27-fold increased risk in developing noncardia gastric carcinoma in comparison with those without H. pylori infection (OR, 2.27; 95% CI, 1.66-3.12). This result was consistent with previous reports (i.e., H. pylori infection is a risk factor of noncardia gastric carcinoma; refs. 1–3). However, there was no significant difference in frequencies of H. pylori infection among three IL-8 –251 genotypes (total subjects, {chi}2 = 0.089; P = 0.956; controls, {chi}2 = 0.029; P = 0.985; gastric cancer cases, {chi}2 = 0.047; P = 0.977; Table 3); that is, none of the AA, AT, or TT genotype would cause increased or decreased risk of H. pylori infection.


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  		Table 3. Frequencies of H. pylori infection in controls and noncardia gastric cancer cases of different IL-8 –251 genotypes

 
Association of the interleukin-8 –251T allele with increased risk of gastric carcinoma. In the control group, the IL-8 –251A/T genotypes showed no evidence of deviation from the Hardy-Weinberg equilibrium ({chi}2 = 0.977; P = 0.641; Table 4A). The –251T allele frequency was more common among the cases (64.8%) than among the controls (57.5%), and this difference was statistically significant ({chi}2 = 8.453; P = 0.004), suggesting that the –251T allele may be the risk or marker allele (Table 4A). Similarly, the AT heterozygotes and TT homozygotes were more common among the cases than among the controls, and these differences were also statistically significant ({chi}2 = 9.239; P = 0.010). Compared with the AA homozygotes, the AT heterozygotes had >60% increased risk of gastric carcinoma (OR, 1.62; 95% CI, 1.07-2.46; P = 0.022), and the TT homozygotes had >90% increased risk (OR, 1.93; 95% CI, 1.26-2.95; P = 0.002; Table 4A). The risk of –251T exhibited an allele-dose effect (i.e., the ORs increased as the number of the T allele increased; Ptrend = 0.004).


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  		Table 4.
 
H. pylori infection has been known to be a risk factor of noncardia gastric carcinoma. In our study, the IL-8 –251T allele was associated with increased risk of noncardia carcinoma in both H. pylori–negative and H. pylori–positive individuals (Table 4B), although statistics for this association in H. pylori–negative subjects were not significant (P > 0.05). Compared with the H. pylori–negative group, the H. pylori–positive AT heterozygotes were associated with 42% increased risk of noncardia carcinoma (OR subtraction; Table 4B), and the H. pylori–positive TT homozygotes were associated with 79% increased risk. Although none of the IL-8 –251 genotypes was susceptible to H. pylori infection (Table 3), H. pylori predisposed individuals with the –251T carrier to noncardia carcinoma.

When the cases were divided into cardia and noncardia carcinomas (Table 5), the AT heterozygotes also had >60% increased risk of noncardia carcinoma (OR, 1.63; 95% CI, 1.05-2.52; P = 0.028), and the TT homozygotes had >85% increased risk (OR, 1.87; 95% CI, 1.19-2.93; P = 0.006) compared with the AA homozygotes (Table 5A). For the cardia carcinoma, the AT genotype was associated with 59% increased risk in comparison with the AA homozygotes, but this was not statistically significant (OR, 1.59; 95% CI, 0.77-3.32; P = 0.211), and the TT genotype was associated with 2.19-fold increased risk (OR, 2.19; 95% CI, 1.05-4.57; P = 0.033; Table 5B). The risk of the –251T allele with cardia and noncardia carcinomas also showed allele-dose effects (Ptrend for cardia carcinoma = 0.029 and Ptrend for noncardia carcinoma = 0.012).


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  		Table 5.
 
To evaluate the risk of the –251T allele for subgroups, we stratified the associations between –251T allele (i.e., AA versus TT, AA versus AT + TT) and gastric cancer risk by age, sex, ethnicity (Table 6), and tumor histopathology (Table 7B). We found that the risk associated with the –251T allele was more evident in older individuals (greater double equals50 years; AA versus TT: OR, 2.00; 95% CI, 1.27-3.14; P = 0.002; AA versus AT + TT: OR, 1.80; 95% CI, 1.19-2.71; P = 0.005) than in younger individuals (<50 years; AA versus TT: OR, 1.40; 95% CI, 0.37-5.27; P = 0.616; AA versus AT + TT: OR, 1.45; 95% CI, 0.44-4.78; P = 0.540). The sample size for the younger age group was small, so it was hard to draw any conclusion from this result. It seemed that sex and ethnicity were not associated with the –251T-mediated risk. In addition, there was no evidence for an interaction of the –251A/T genotypes with age, sex, and ethnicity as evaluated in the multiple logistic regression models (data not shown).


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  		Table 6. Stratification analysis of IL-8 –251A/T genotypes, ORs, and 95% CIs by selected variables

 

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  		Table 7.
 
Among the 470 patients with gastric carcinoma, 339 received surgical treatment, whose samples were subjected to Laurén classification of histopathology (41, 42). The mixed-type carcinoma represented a histopathology containing both intestinal and diffuse entities. The patients with the AT or TT genotype did not show a predisposition to have a certain type of gastric carcinoma (AT versus TT: {chi}2 = 0.092; P = 0.955; Table 7A). However, the patients with the AA genotype tended to sustain intestinal-type (19.5%) carcinoma rather than diffuse (9.1%) and mixed (10.2%) types, and this was statistically significant (AA versus AT + TT: {chi}2 = 6.816; P = 0.033; AT and TT were grouped because the statistic for AT versus TT was not significant; Table 7A). Because the mixed-type carcinoma contained intestinal and diffuse features, the trend for the patients with the AA genotypes to sustain intestinal carcinoma was also statistically significant by a 3 x 2 cross-table {chi}2 test with the histologic sequences of "intestinal to mixed to diffuse type" x "AA versus AT + TT" (Ptrend = 0.016; Table 7A). In addition, the patients of different genotypes did not have a tendency to sustain cardia or noncardia carcinoma ({chi}2 = 0.586; P = 0.746).

When the data of genotype frequencies were stratified according to histologic types, the TT homozygotes had 2.73-fold increased risk of diffuse-type carcinoma compared with the AA homozygotes (AA versus TT: OR, 2.73; 95% CI, 1.20-6.22; P = 0.014; Table 7B). The –251T allele was associated with >2-fold increased risk of diffuse-type carcinoma (AA versus AT: OR, 2.36; 95% CI, 1.05-5.32; P = 0.035; AA versus AT + TT: OR, 2.52; 95% CI, 1.16-5.49; P = 0.017). Likewise, the TT homozygotes also had 2.30-fold increased risk of mixed-type carcinoma in comparison with the AA homozygotes (AA versus TT: OR, 2.30; 95% CI, 1.11-4.77; P = 0.023), and the –251T allele was associated with >2-fold increased risk of mixed-type carcinoma (AA versus AT: OR, 2.16; 95% CI, 1.06-4.43; P = 0.032; AA versus AT + TT: OR, 2.22; 95% CI, 1.12-4.40; P = 0.019; Table 7B). There also seemed to be an allele-dose effect in –251T allele-mediated risk of diffuse-type carcinoma (Ptrend = 0.026), which was not significant in mixed-type carcinoma (Ptrend = 0.050). Finally, it was evident that the occurrence of intestinal-type carcinomas was not associated with the –251A/T genotypes.


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
It has been well studied that the IL-1B (encoding IL-1ß) and IL-1RN (encoding IL-1RA) gene polymorphisms (IL-1B –31T, IL-1B –511T, and IL-1RN*2 alleles) are associated with increased risk of gastric cancer (6, 7). In addition to IL-1ß, polymorphisms of IL-10 (–592A and –1082A) and TNF-A (–308A) have also been associated with increased risk of the malignancy (8, 9). In the current report, we assessed the relationship between the IL-8 –251A/T polymorphism and gastric cancer risk in a population from north Taiwan and found a significant association between –251T allele (i.e., the high expresser of IL-8) and increased risk of diffuse-type and mixed-type gastric carcinomas by a hospital-based case-control study.

We are aware that there are potential limitations in any case-control study and that the use of hospital controls is not ideal. Because the controls were not selected from the same population from which the cases arose, we could not rule out the possibility of selection bias. We tried to minimize potential bias by careful study design and by use of validated questionnaires administrated by trained interviewers. By matching on age, sex, and ethnicity, potential confounding factors might be minimized. H. pylori infection was also taken into consideration and showed no significant association with IL-8 –251 genotypes (Table 3); however, H. pylori infection increased the risk of noncardia gastric carcinoma in –251T carriers compared with –251AA homozygotes (Table 4B). In addition, we restricted our analysis to Chinese subjects, so it is uncertain whether these results are applicable to other populations. Carcinogens ingested from daily diet, such as smoked and pickled food, may contribute to carcinogenesis of the stomach. However, it is very hard to formulate a suitable questionnaire in which carcinogens ingested are well quantified. In this report, we have emphasized molecular analysis of the IL-8 –251A/T promoters and confined our data analyses to the association between –251A/T polymorphism and gastric cancer risk. Although the Ps in some tests only reached borderline significance (Tables 4 and 5), all of these case-control studies showed allele-dose effects, giving additional evidence of the –251T allele as a risk marker of gastric carcinoma. Increased sample size would improve statistical significance. Therefore, to confirm the role of the IL-8 –251 polymorphism in gastric cancer risk requires additional larger studies in different populations. Multifactor analysis by addition of dietary habits is also worth doing through deliberate study design in the future.

There are two mechanisms by which the gastric mucosa progresses to carcinoma, both starting from chronic gastritis. One mechanism is via gastric atrophy, intestinal metaplasia, and adenomatous dysplasia leading to intestinal-type carcinomas characterized by glandular formation; the other is via hyperplastic or de novo changes leading to diffuse-type carcinomas characterized by isolated cancer cells with an infiltrative growth (41, 42). Approximately one-third of gastric carcinomas have both entities of histopathology. IL-1B –511T and IL-1RN*2 result in high IL-1ß secretion (43–47) and increased risk of intestinal-type gastric carcinoma (6, 7). In this report, we observed that IL-8 –251T was not associated with a risk of intestinal-type carcinoma, and conversely, this allele was associated with increased risk of diffuse-type and mixed-type carcinomas (Table 7B). The Ps for the association of –251A/T genotypes with histologic types of gastric cancer were between 0.01 and 0.03 (Table 7B), suggesting that the sample size could be enlarged to make our conclusions more convincing. It is noteworthy that the mixed type also contains diffuse-type entity. Eck et al. reported 22 gastric carcinoma cases examined by immunohistopathology and found that IL-8 expression was significantly stronger in diffuse-type tumors (n = 10) than in intestinal-type tumors (n = 12; ref. 26). IL-8 is a potent stimulator of angiogenesis and has been involved in tumor invasion and metastasis (14–19). Individuals with the –251T allele are expected to express higher local IL-8 in the gastric mucosa in response to tissue insult, which may assist early malignant cells in diffuse spread rather than adenomatous transformation into intestinal-type carcinoma.

The IL-1B alleles on increased risk of gastric carcinoma can be explained by allele-specific nucleotide variations. IL-1B –31C/T is a TATA-box polymorphism that markedly affects DNA-protein interactions in vitro, hence modulating the expression of IL-1ß(6). The IL-1B –31T allele possesses classic TATA box and results in stronger promoter activity in response to extracellular stimuli than the –31C allele (6). Individuals with the IL-1B –31T allele have increased risk of gastric carcinoma (6). In the early stage of the present study, we made an attempt to determine IL-8 expression in individuals with the –251 AA, AT, and TT genotypes from human leukocytes; however, we failed to obtain constant data because it was difficult to isolate homogenous monocyte, macrophage, or T lymphocyte for in vitro study. Thus, we cloned the IL-8 –251A/T promoters into the pGL3-Basic plasmid in which the IL-8 promoter controlled luciferase expression. We found that the –251T allele drove luciferase expression 2- to 5-fold stronger than the –251A allele (Fig. 2). We also found that a protein or multiprotein complex bound to the –251A allele and might negatively regulate the expression of IL-8 (Fig. 3). As presented in the IL-1B –31T allele, we also observed that the IL-8 –251T allele was associated with increased risk of gastric carcinoma. Identification of the protein or multiprotein complex bound to the IL-8 –251A allele is ongoing in our laboratory.

The IL-8 –251A allele has also been shown to be associated with decreased risk of colorectal cancer (48). The authors cited a report (29) describing that the –251A allele produced more IL-8 than the –251T allele following lipopolysaccharide treatment and contributed to increased incidence of infantile bronchitis in exposure to respiratory syncytial virus. The results of IL-8 expression level in this report (29) are contrary to ours. We reviewed the report (29) and found that the authors used mixed leukocytes to detect the IL-8 level by ELISA in healthy blood donors of –251 AA, AT, and TT genotypes. It could be questioned that the major WBCs that secret IL-8 are T lymphocyte, monocyte, and macrophage, and the percentage of these IL-8-secreting blood cells varies greatly in healthy individuals. We ever did the same assays with purified monocyte and macrophage but failed to obtain constant data showing that the –251A allele was the high expresser of IL-8. The IL-8 –251T allele has recently been reported to be a risk factor of bronchial asthma (28), and the disease has also been associated with increased IL-8 secretion in airway epithelia (49). It could be deduced from these reports and our study that it is the IL-8 –251T allele that results in high local IL-8 in the bronchioles and gastric mucosa. Because colorectal and gastric cancers have similar histologic origin, the –251A allele (i.e., the low expresser of IL-8) induces less chronic inflammation and hence reduces the risk of the malignancies. Our study makes clear the previously controversial results about the association of the IL-8 –251A allele with reduced risk of colorectal cancer (48) based on the –251A allele as the high expresser of IL-8 (29).

In conclusion, our study provides evidence that the IL-8 –251T allele is significantly associated with increased risk of gastric carcinoma, particularly the diffuse-type and mixed-type carcinomas in Chinese population. To further explore the role of the IL-8 gene in the etiology of gastric cancer, studies on the IL-8 polymorphisms other than the –251A/T variant may be warranted. It is a noteworthy idea from our gastric cancer and the previous colorectal cancer (48) studies that the IL-8 –251T allele may be a common risk factor of gastrointestinal carcinomas.


   Acknowledgments
 
We thank Dr. Ming-Wei Lin for her kind help in statistical analysis.


   Footnotes
 
Grant support: National Science Council, Taiwan grants NSC91-2315-B-182A003 (D-I. Tai) and NSC92-2314-B-075-053 (W-P. Lee), Taipei Veterans General Hospital grants VGH 92-A-027, VGH 93-A-052, and VGH 93-B2-183 (W-P. Lee), and Chang Gung Memorial Hospital, Taiwan grant CMRP1061 (W-P. Lee and D-I. Tai).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 4/30/05; revised 5/24/05; accepted 6/ 6/05.


   References
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 Materials and Methods
 Results
 Discussion
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