Introduction

The relationship between inflammation and cancer is well established, mainly in the gastrointestinal tract [1]. Many of the factors involved in chronic inflammation and immune response play a role in cell proliferation, stimulation of angiogenesis and inhibition of apoptosis [2]. Gastric cancer is a model of multifactorial disease resulting from environmental and genetic factors, following a chronic inflammation of the gastric mucosa induced by Helicobacter pylori infection [3].

H. pylori is the most important risk factor for gastric cancer, triggering chronic inflammation of the stomach and leading to stepwise development of the malignancy [4]. It is widely accepted that chronic H. pylori infection induces gastric atrophy and hypochlorhydria, which are precursors of all pathophysiological changes of gastric carcinogenesis [5].

Although H. pylori infection is widespread throughout the world’s human population, only about 1–2% of exposed individuals progress to gastric cancer [6]. Thus, besides virulence factors of the bacterium, host genetic patterns, mainly those that regulate the immunological and inflammatory response, may also contribute to a neoplastic progression [7]. Genetic polymorphisms in genes that codify pro- and anti-inflammatory cytokines have been associated with an increase in the synthesis of those interleukins and have emerged as a hypochlorhydria-determining factor for cancer susceptibility [8, 9].

The interleukin-1 (IL-1) gene cluster on chromosome 2q contains three related genes, IL-1A, IL-1B and IL-1RN, which encode, respectively, the pro-inflammatory cytokines IL-1α and IL-1β, as well as their endogenous receptor antagonist (IL-1ra) [10]. IL-1ra, encoded by the IL-1RN gene, is an anti-inflammatory cytokine that competitively binds to IL-1 receptors but does not elicit a response, thereby modulating the potentially damaging effects of IL-1 [11, 12]. The IL-1RN gene has penta-allelic 86 bp variable number of tandem repeats (VTNR) in the second intron, resulting in a short allele (IL-1RN*2, with two repeats) or long alleles (IL-1RN*L, with three to six repeats) [13].

The short allele is associated with chronic inflammation, autoimmune conditions and increased secretion of IL-1β [9]. In 2000, El-Omar et al. [10] reported an increased risk for developing gastric cancer in individuals having the IL-1RN*2/*2 genotypes. This finding was subsequently confirmed in other population studies [7, 8, 11, 14, 15], but the data are still controversial [16, 17].

In addition, genetic polymorphisms in pro-inflammatory cytokines such as TNF-β are known to be related to several autoimmune, infectious and neoplastic diseases [18–21]. Tumor necrosis factor β (TNF-β) or lymphotoxin-α (LT-α) is encoded by the same gene cluster for TNFA, located in tandem within the MHC complex on chromosome 6p21.3 [22]. TNF-β is a lymphokine that also plays an important role in the regulation of the immune response as part of the cytokine network. It is mainly produced by stimulated T-cells, B and natural killer-cell lymphocytes [23].

An increased level of TNF-β expression in vitro has been shown for the Single Nucleotide Polymorphism (SNP) TNF-B+252A/G [24]. Also, there are studies showing that the TNF-B+252G-carriage is associated with a high serum concentration of C-reactive protein, a biomarker of inflammation [25, 26]. Therefore, this polymorphism has been investigated as a potential factor of susceptibility to gastric cancer [27].

The aim of this study was to determine if there is an association between anti- and pro-inflammatory cytokines polymorphisms, such as the IL-1RN VTNR and SNP of TNF-B+252A/G and the risk of chronic gastritis and gastric cancer in a population from Southeast Brazil, and whether gastric lesions are associated with risk factors such as gender, age, smoking, alcohol intake and H. pylori infection.

Methods

Population

The Brazilian National Research Ethics Committee approved this work, and written informed consent was obtained from all individuals participating.

This was a case–control study on chronic gastritis and gastric cancer, in which a total of 675 DNA samples extracted from peripheral blood leukocytes were genotyped. It was used DNA samples stored in our laboratory collected between the periods 2002 and 2004 and used in previous studies [28, 29] and the current period (2007–2010).

All subjects of case groups were recruited from the Service of Endoscopy or Surgery Center, Hospital de Base, Medical School, Sao Jose do Rio Preto-SP, Brazil. In cases groups were included, incident cases recruited from the moment that started the sample collection in those years, with participation rate about 90% of patients interviewed. Recurrent malignancies were excluded.

The chronic gastritis groups (CG) comprised 229 individuals (113 men and 116 women) with a mean age of 52.3 ± 14.5 years (range 19 –86 years). All had a histopathologically confirmed diagnosis of chronic gastritis, mainly nonatrophic, but also was included gastritis atrophic and with metaplasia area (Sydney System) [30], but individuals who presented specific gastritis types such as autoimmune and ischemic were excluded.

The gastric cancer group (GC) comprised 200 individuals (156 men and 44 women) with a mean age of 61.7 ± 12.5 years (range 28–93 years), and with a histopathologically confirmed diagnosis of intestinal or diffuse gastric cancer (Lauren’s classification) [31]. Information on H. pylori infection was obtained when available from the patients’ medical records (95 with gastric cancer and 177 with chronic gastritis). The diagnosis was established using the Giemsa staining technique or the urease test, performed at the respective Pathology Service.

The cancer-free control group (C) was composed of 246 healthy individuals (126 men and 120 women), with a mean age of 55.5 ± 17.7 years (range 20–93 years), blood donors recruited from the Blood Bank of Base Hospital, Medical School, Sao Jose do Rio Preto-SP, Brazil. Individuals were selected without diagnoses of cancer according to government guidelines for donated blood that tests for 20 related diseases (http://www.hemonline.com.br/portarias/rdc153/index frame.htm), and no previous history of gastric disease. The participation rate was about 100% of subjects interviewed.

Epidemiological data on the study population were collected using a standard interviewer-administered questionnaire, with questions about current and past occupation, smoking habits, alcohol intake, and family history of cancer. Considering the ethnic admixture of the evaluated Brazilian population, the study subjects were visually classified for ethnicity, based on their appearance, as Caucasians (90%) or Negroids (10%). Smokers were defined as individuals who consumed at least 100 cigarettes during their lifetime, and alcohol consumers were those who drank more than four drinks per week [32].

IL-1RN and TNFB+252A/G genotyping

About 5 mL of whole blood were collected from all study participants in sterile EDTA-coated vacutainer tubes. DNA was extracted as described in a previous report [33] and stored at −20°C until use.

IL-1RN polymorphisms were investigated using the allele-specific polymerase chain reaction (PCR) method [10], and the PCR–RFLP (restriction fragment length polymorphism) technique was employed to identify the TNF-β genotype [21], in both cases and control groups. In brief, the assay was carried out in a total reaction volume of 25 μL, containing 2.5 μL 10× PCR buffer, 2 μL dNTPs (1.25 μmol/L, Invitrogen, Carlsbad, CA, USA), 0.5 μL MgCl2 (25 mmol/L), 1.25 μL of each primer (25 mmol/L, Sigma–Aldrich, St. Louis, MO, USA), 15.3 μL dH2O, 2 μL DNA (100 ng/μL), and 0.2 μL Taq DNA polymerase (5 U/μL, Invitrogen, Carlsbad, CA, USA). The PCR procedure for IL-1RN was as follows: an initial denaturation step at 94°C for 3 min, 35 amplification cycles at 94°C for 45 s, 61°C for 45 s, and 72°C for 1.5 min, followed by a final elongation cycle at 72°C for 7 min. The size of the amplified fragments is shown in Table 1. Alleles 1, 3, 4 and 5 were grouped together and named long alleles (L) and analyzed separately from the short allele 2 (two repeats). So, the genotypes were further classified as L/L, L/2, and 2/2. PCR for the TNF-β polymorphism was performed as follows: an initial denaturation step at 94°C for 5 min, 35 amplification cycles at 94°C for 1 min, 65°C for 1 min, and 72°C for 1 min, followed by a final elongation cycle at 72°C for 7 min. Then, 10 μL of PCR products were digested with 0.5 μL of the specific enzyme NcoI (5 U/μL) in a 10 μL volume containing 2.5 μL 10× buffer 1 (New England Biolabs, MA, USA) and 7.0 μL dH2O (Table 1). The products were then electrophoresed on a 1.5% agarose gel (Invitrogen, Carlsbad, CA, USA) and visualized by ethidium bromide staining. Random samples were selected and performed their duplicates by others researchers of the laboratory for quality control, but there were not discrepancies, and the concordance rate was 98%.

Table 1 Primer sequences, restriction enzyme and fragment sizes for IL1RN and TNFB gene polymorphisms
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Statistical analysis

Hardy–Weinberg equilibrium for TNF-β polymorphism was assessed using Chi-square test. Multiple logistic regression analysis was conducted using the following models: log-additive (major allele homozygotes vs heterozygotes vs minor allele homozygotes), dominant (major allele homozygotes vs heterozygotes + minor allele homozygotes), and recessive (major allele homozygotes + heterozygotes vs minor allele homozygotes). The age, gender, smoking, drinking and H. pylori infection as covariates were adjusted to obtain statistical significance in both the gastric cancer and the chronic gastritis groups for two polymorphisms. Statistical analyses were performed using the GraphPad Instat, SPSS (11.5 version) and SNPStats (http://bioinfo.iconcologia.net/index.php) computer software programs [34]. A probability level (p) of less than 0.05 was adopted as significance criterion.

Results

The samples obtained from the 675 subjects in the GC, CG and C groups were genotyped for the IL-1RN VNTR and TNF-β+252A/G polymorphisms. The genotype and allele frequency distributions of the TNF-β+252A/G polymorphism were in agreement with the Hardy–Weinberg equilibrium, in both the case and the control groups (data not shown).

Genotype frequencies and logistic regression analysis adjusted for age, gender, smoking and drinking in all three analysis models (log-additive, dominant, and recessive models for rare alleles) of both polymorphisms in the gastric cancer, chronic gastritis and control group (GC vs C and CG vs C) are presented in Table 2.

Table 2 Genotype frequencies and logistic regression analysis of IL1RN-VNTR and TNFβ polymorphisms in the gastric cancer (GC), chronic gastritis (CG) and control (C) groups (GC vs C, CG vs C)
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In the GC group, the genotype frequencies of IL1RN VNTR were 40.5% for the L/L genotype, 44.5% for L/2 and 15.0% for 2/2. In the CG group, the genotype frequencies were 52.0, 37.5 and 10.5%, respectively, while in the C group, these frequencies were respectively 64.2, 31.3 and 4.5%. As showed in the Table 2, the 2/2 genotype was associated with risk of gastric cancer and chronic gastritis in all 3 models evaluated, evidencing higher values of OR in the recessive model (OR = 3.04, 95% CI = 1.41–6.56, p < 0.01 and OR = 2.32, 95% CI = 1.10–4.90, p = 0.02, respectively). However, when compared both case groups there was a statistically significant difference in genotype frequencies only in the log-additive and dominant models (OR = 1.42, 95% CI = 1.04–1.95, p = 0.02 and OR = 1.70, 95% CI = 1.11–2.62, p = 0.01, respectively) (data not showed in Table 2). In contrast, for TNF-β+252A/G, no significant difference was found among the three groups evaluated (Table 2).

We evaluated the combined effect of the two polymorphisms studied and the combination at least one variant allele IL1-RN*2 with TNF-β+252A/G+G/G remained the risk for GC (OR = 3.69; 95% CI = 2.13–6.41; p < 0.01) and CG (OR = 2.34; 95% CI = 1.42–3.86; p < 0.01) in relation to control group (Table 3).

Table 3 Combined effect of IL-1RN (L/L, L/2+2/2 genotypes) and TNFB+252A/G (A/A, A/G+G/G genotypes) polymorphisms on risk of gastric cancer (GC) and chronic gastritis (CG)
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The multiple logistic regression for risk factors showed that in the GC group male gender (OR = 2.27, 95% CI = 1.42–3.62, p < 0.01), age above 60 years (OR = 1.70, 95% CI = 1.10–2.61, p < 0.01) and alcohol intake (OR = 3.09, 95% CI = 1.91–5.02, p < 0.01) were associated with greater susceptibility to the development of this neoplasm (Table 4). While the comparisons between gastritis and control groups showed that only alcohol intake was associated with increased risk of chronic gastritis (OR = 1.81, 95% CI = 1.16–2.83, p < 0.01), but age above 53 years (OR = 0.66, 95% CI = 0.45–0.96, p = 0.03) and smoking (OR = 0.58, 95% CI = 0.38–0.87, p < 0.01) were negatively associated with the development of gastritis.

Table 4 Distribution of risk factors, genotypes of IL-1RN and TNFB+252A/G polymorphisms, OR and 95% confidence intervals (CI) between gastric cancer (GC) and chronic gastritis (CG) with control (C) group
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Because the reduced number of cases tested for H. pylori infection (95 with gastric cancer and 177 with gastritis), the Table 5 shows another multiple logistic regression analysis between both case groups also adjusted for the other risk factors. There was no statistically significant association for H. pylori (OR = 1.39, 95% CI = 0.78–2.48, p = 0.25, Table 5). There was also no association of both polymorphisms when addressed separately in H. pylori positive and H. pylori negative subjects within a group of gastric cancer and gastritis (data not shown).

Table 5 Distribution of risk factors, genotypes of IL-1RN and TNFB+252A/G polymorphisms, OR and 95% confidence intervals (CI) between gastric cancer (GC) and chronic gastritis (CG) groups tested for H. pylori
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Discussion

Studies on cytokine gene polymorphisms have generated further evidence to support the role of inflammation in gastrointestinal carcinogenesis, mediated by an array of pro- and anti-inflammatory cytokines. Polymorphisms of these genes are associated with differences in gastric mucosal cytokine mRNA level, which result in an individual final clinical outcome [35]. Considering this scenario, we investigated the effect of polymorphisms of two cytokine genes, one anti-inflammatory (IL-1RN VNTR) and the other pro-inflammatory (TNF-β+252A/G), on the risk of developing GC and CG in a population from Southeast Brazil.

Our results demonstrated that in this population there was an association of the IL-1RN*2 allele variant and of the combined effect of polymorphisms IL1-RN*2 and TNF-β+252*G with susceptibility to GC and CG. The presence of polymorphism TNF-β+252A/G alone was not associated with risk of any of the gastric lesions studied.

The results of our study add to the current controversy about the role of IL-1RN VNTR polymorphism in the gastric carcinogenesis risk, recently addressed in three meta-analysis studies [12, 36, 37]. The first one, by Kamangar et al. [36] observed that the overall associations between IL-1RN pro-inflammatory polymorphisms and gastric cancer were null, even in studies conducted in Western countries. The second meta-analysis, reported by Peleteiro et al. [37] showed that allele IL-1RN*2 was associated with increase of the risk of gastric precancerous lesions, supporting a role for this polymorphism in the early stages of gastric carcinogenesis. The last meta-analysis mentioned, by Xue et al. [12] observed that allele IL-1RN*2 was significantly associated with an increased risk of developing gastric carcinoma and even more significantly with noncardia gastric carcinoma or with intestinal-type gastric carcinoma. However, this association was evident among Caucasians, but not among Asians or Hispanics. These discrepancies may be due to the low frequency of the risk allele in some populations, such as the Asians.

Rocha et al. [38] conducted an assessment study on cytokine polymorphisms in an admixed population of Southeast Brazil (Minas Gerais state) with a background of approximately 33% Portuguese, 33% Amerindian and 33% African ancestry. They demonstrated an association between the polymorphic allele IL-1RN*2 and the development of gastric cancer, and also an association with the cagA-positive H. pylori status. However, they found a low 2/2 homozygous frequency (3.6%) compared to our study (15.0%) and to studies conducted in European Caucasian (28%) and Asian populations (0.4%) [39]. More recently, another study [7] performed in a population of northern Brazil, with a strong genetic contribution of Portuguese (50%) and Amerindians (40%) but low African ancestry (10%), also showed a positive association between allele IL-1RN*2 and gastric ulcer and gastric adenocarcinoma and CagA + H. pylori infection. In this study, the frequency of homozygous 2/2 was about 6% [7]. However, the 2/2 homozygous frequency in the control group of the present study (4.5%) is more similar than those reported by Rocha et al. (3.9%) [38], but discrepant of Asian population (0.4%) [39].Those results indicate that the genotype frequencies of IL-1RN are related to the ethnic composition of the population studied.

In the present study, focusing on another population of Southeast Brazil (São Paulo state), the Caucasian ethnicity was around 90% for the control and cases groups. These data when compared with other epidemiological studies in the Southeast (84.1%), Central-Western (87.2%), Northern (60%) and Southern (94.0%) in Brazilian population demonstrated a variation of the frequency of Caucasians in healthy individuals in different Brazilian regions [40–42]. Because of the broad geographic extension of Brazil, there is variation in population composition among different regions that dates back to its colonization by Europeans and Africans, which eventually got admixed with the native Amerindians [43], and consequently there are differences in genotype distribution.

The polymorphic allele IL1-RN*2 has been linked to an increased production of the pro-inflammatory cytokine IL1-β, which causes a chronic inflammatory process and reduced production of gastric acid (hypochlorhydria) [44]. The reactive oxygen species and nitric oxide, products derivatives of the inflammatory process, are known as mutagens, whereas hypochlorhydria can favor infections by bacteria, thus increasing the production of carcinogens. The DNA damage caused by this cascade of factors may be amplified by increased cell division inherent to the inflammatory process. Thus, genotypes that enhance the production of pro-inflammatory cytokines may be related to the process of gastric carcinogenesis [45].

In addition, about the importance of pro-inflammatory cytokines, that can also modify the immune/inflammatory responses upon H. pylori infection, and influence gastric carcinogenesis, we assessed the TNF-β+252A/G polymorphism. Although we found a higher frequency of genotypes A/G and G/G in groups GC and CG compared to controls, the difference was not statistically significant. Similarly, no association was found by Lee et al. [46] in the Korean population, and Guo et al. [21] also found no relationship between the polymorphic allele and the groups of gastric cardiac adenocarcinoma and squamous cell carcinoma of the esophagus. However, a recent study in the Japanese population showed association of genotypes G/G and A/G with an increased risk of noncardia gastric cancer of the diffuse type and attributed this association to the ability of the 252G allele to increase the production of pro-inflammatory cytokines such as TNF-β [25]. These discrepant results may be linked to ethnicity, among others factors.

When analyzing the influence of H. pylori infection on the risk for the gastric lesions evaluated, no association was observed. However, several studies have found this association between IL-1RN polymorphisms and gastric lesions risk in the presence of the bacterium H. pylori [11, 47, 48]. Furthermore, Rocha et al. and Melo-Barbosa et al. [7, 38] found a strong association of this polymorphism and infection by H. pylori CagA+ strains with the development of gastric lesions and adenocarcinoma. This influence of H. pylori infection is well defined in the progression of gastric cancer in some populations. Individuals with pro-inflammatory genotypes overexpress gastric IL-1β in response to H. pylori infection, leading to increased inflammation, gastric atrophy, hypochlorhydria and the development of gastric carcinoma [8]. However, in the present study, we did not find this association. This discrepancy might be due to the small number of cases with the available information in their medical records for H. pylori infection (95 with gastric cancer and 177 with chronic gastritis). Despite the relevance of the study, also should be considered some limitations that did not enable the analysis of association by cancer histological type and anatomical site.

Other risk factors such as male gender, old age and alcohol intake were found to be significantly associated with the risk of gastric cancer. According to the data of the Brazilian National Cancer Institute (INCA), the highest incidence of GC occurs in men around age 70, and about 65% of patients diagnosed with this type of cancer are over 50 years old. In Brazil, GC is the third in incidence among men and fifth in women, so our results are consistent with the epidemiological data reported in the literature [49]. Another risk factor well established in the literature in relation to gastric carcinogenesis is the excessive consumption of alcohol. The oxidation of ethanol generates acetaldehyde, which primarily has direct carcinogenic and mutagenic effects. This metabolite interferes with many sites of synthesis and DNA repair, which can lead to tumor development [32].

Conclusions

In conclusion, our findings do not evidence any association of the TNF-β+252A/G polymorphism alone with susceptibility to gastric lesions, but indicate a significant role of the IL-1RN*2 allele for both GC and CG in the Southeast Brazilian population evaluated. Thus, they show the importance of polymorphisms in genes for anti-inflammatory factors in gastric carcinogenesis, reinforcing the contribution of the host genetic factors in the development of gastric cancer.