IL-10 Promoter Polymorphism and Gastric Cancer Risk

Chapter 6

IL-10 promoter polymorphism and gastric cancer risk

Introduction

Chronic inflammation is thought to contribute to the development of cancer (Balkwill and Mantovani,2001). Progressive inflammation leads to activation of inflammatory cytokines, recruitment of inflammatory cells, generation of free radical species, and subsequent malignant transformation. Individual responses to inflammation, mediated by genetic variation, may influence the degree of inflammation and thus the risk for cancer.

Interleukin-10 (IL-10) is an anti-inflammatory cytokine, which is involved in down-regulating cell-mediated and cytotoxic inflammatory responses(Wu et al., 2002). The gene encoding IL-10 is located on chromosome 1 (1q31-1q32). IL-10 has three confirmed biallelic polymorphisms in the gene promoter region: -1082 A/G, -819 C/T, and -592 C/A. Presence of -1082A is associated with lower production of IL-10 in vitro and in vivo, and an accordingly stronger inflammatory response(Rad et al., 2004). Interleukin-10 (IL-10) is an immunoregulatory Th2 cytokine with a polymorphic promoter (-1082, rs1800896; and -592, rs1800896), with variants correlated with increased IL-10 production(De VF 2001). These IL-10 polymorphisms have been associated with noncardia gastric cancer risk(Suarez et al., 2003).

In the multistage model of gastric carcinogenesis, gastric inflammation is a prerequisite for the development of GC(Correa, 1992). Accordingly, factors involved in initiation and regulation of the inflammatory response may confer susceptibility to or protection against GC. In this respect, cytokines that play a crucial role in regulating inflammation are potential candidates for correlating with such variation. The in vitro maximal capacity to produce different cytokines varies among different individuals. Family studies indicate that much of this variability is genetically determined (Westendsop et al., 1997). Such inter individual differences can be attributed to several molecular mechanisms, including single nucleotide polymorphisms (SNPs) in the coding or promoter regions of cytokine or cytokine receptor genes. These polymorphisms may affect the overall expression and secretion of cytokines.

Subject characteristics

A total of 150 cases (gastric cancer patients) and 250 normal healthy controls were studied for polymorphic analysis. The clinical characteristics of study subjects are summarized in Table 1. The subjects were considered never smokers if they had never smoked before sample collection and ever smokers if they are smoking presently or had quit smoking since last 6 months or less before sample collection. There were no significant differences among cases and controls in terms of age, gender distribution, dwelling and blood group. Since the consumption of hot beverages, is considered as a possible risk factor for gastric cancer, we designated the cases on the basis of the daily consumption of salted tea, the cases were divided into <4 cups/day and ≥ 4 cups/day consumers. 62.7% (n=94) belonged to the latter group while as 37.3% (n=56) belonged to the former group.60.78% patients were having A+ve blood group while 39.22% of patients had the rest blood group types.65.3% of patients were ever smokers and only 34.7% were never smokers.

Genotyping

Genomic DNA was extracted from 10 ml EDTA (Ethylene di amine tetracetate) treated venous blood samples using the standard phenol-chloroform extraction protocol. DNA purity was assessed by a UV–Vis spectrophotometer estimating the A260/A280 ratio or by running samples on 1% agarose.

IL-10-1082 and -592 promoter polymorphisms were analyzed with polymerase chain reaction (PCR) amplification followed by restriction fragment length polymorphism (RFLP). Primers used were as described by (Ja YL 2005). For IL-10-1082 genotyping, PCR condition was as follows; 94oC for 2 min, then 40 cycles of 94oC for 1 min, 58oC for 1 min, 72oC for 1 min, and finally 72oC for 10 min. The PCR products were digested with MnlI (Fermentas Inc., Hanover, MD) at 37oC for 4 h and separated by electrophoresis on a 3% agarose gel. A fragment containing the MnlI polymorphic site at position -1082 of the IL-10 gene was separated as follows; The A allele was designated if 2 bands of 125 and 65 bp were obtained, and the G allele was designated if 3 bands of 93, 65, and 32 bp were obtained (Fig.1).

The PCR condition for IL-10-592 site polymorphism was as follows; 94oC for 2 min, then 40 cycles of 94oC for 1 min, 59oC for 1 min, 72oC for 1 min, and finally 72oC for 10 min. The PCR products were digested with RsaI (Fermentas Inc., Hanover, MD) at 37oC for 4 h and separated by electrophoresis on a 3% agarose gel. A fragment containing the RsaI polymorphic site at position -592 of the IL 10 gene was separated as follows; The C allele was designated if 2 bands of 302 and 97bp were obtained, and the A allele was designated if 3 bands of 240, 97bp, and 35bp were obtained(Fig.2).

For quality control, distilled water was used instead of DNA as a negative control in each PCR reaction, and more than 20% of the samples were reanalyzed blindly by another co researcher. Results were reproduced up to 98% satisfactorily.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. To minimize the sequencing artifacts by PCR, products from at least two different PCRs were sequenced using both forward and reverse primers (Fig.3and Fig.4).

Results

In the present study, we analyzed two SNPs in IL-10i.e. IL-10-1082 G/A and IL-10-59 C/A gene in gastric cancer patients and matched controls in order to evaluate their association with the 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, 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).

One hundred fifty gastric cancer patients and two hundred fifty six controls were included in the study. The demographic features of the population under study have been listed in Table 1. The risk factor profile revealed that hot salt tea consumption was the most common risk factor in patients followed by smoking and blood group in gastric cancer patients. The genotypic distribution and allelic frequencies of IL-10 gene polymorphism in patients and controls are given in Table2 and Table 3. There were statistically significant differences in the genotypic distribution and allelic frequency between the patients and healthy controls [for AA vs. GG genotype: X2 = 21.802; OR = 0.2857 (95% CI: 0.166-0.488) and p < 0.00015; for AA vs. (GG + GA): X2 = 63.268; OR = 0.172 (95% CI: 0.109–0.269) and (p =<0.0001)]. The X2 for A vs. G allele was 46.875; OR = 0.355 (95% CI: 0.263–0.480) and p =<0.0001 (Table 4). There were statistically no significant differences in the genotypic distribution and allelic frequency between the patients and healthy controls in case of IL-10-592 C/A polymorphic study.

The correlation of IL-10-1082 polymorphic status with the clinicopathological characteristics was carefully analyzed. It was found that the IL-10-1082 polymorphism was significantly related to EGD biopsy, salt tea consumption, blood group, sex, age and dwelling and not to other clinicopathological features (Table 5).

Table 1: Clinical characteristics of Gastric cancer patients and controls

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Table 2: Distribution of IL-10-1082 genotypes and allelic frequencies of the study population

Table 3: Distribution of IL-10-592 genotypes and allelic frequencies of the study population

Table 4: Odds ratio and P values

The P values < 0.05 were considered to indicate statistical significance in these tests.

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Table 5: Association between IL-10-1082 phenotypes and clinicopathological characteristics

The P values < 0.05 were considered to indicate statistical significance in these tests.

Figure 1a: Showing IL-10-1082 amplicons and RFLPs

Fig .1:

a: IL-10-1082A/G (190bp) gel picture.Lane M: 50bp DNA marker, Lanes 1-3: cases and Lanes4-5: controls

a: RFLP picture of IL-10-1082A/Gafter restriction digestion with MnlIenzyme, Lane M: 100bp DNA marker

Lane2: (GG) genotype, Lanes 1,3,6 and 7: heterozygous (GA)genotype and Lane 4: (AA) genotype .

Fig. 2: Partial electropherogram sequences of IL-10-1082A/G

Normal

Heterozygous

Variant

Fig.2:Partial electropherogram sequences (forward) of normal, heterozygous and variant of IL-10-1082G/A.

Figure 3: Showing IL-10-592 amplicons and RFLPs

Fig. 3:

A: IL-10-592 A/C (337bp) gel picture.Lane M: 100bp DNA marker, Lanes 1-3: cases and Lanes4-5: controls

B: RFLP picture of IL-10-592 A/C after restriction digestion with RsaIenzyme

Lane M: 100bp DNA marker, Lane 2, 6: C Allele(302bp, 97bp and 35bp), A allele: (240bp,97bp and 35bp).

Fig. 4: Partial electropherogram of IL-10-592 A/C.

Normal

Heterozygous

Variant

Fig. 4:Partial electropherogram sequences (forward) of normal, heterozygous and variant of IL-10-592 A/C.

Discussion

The incidences of polymorphism in genomic DNA, their susceptibility to genetic alterations, and the risk of tumor progression in patients with cancer can vary substantially between different racial groups(Perez et al., 2006; Bojesen et al., 2008; Katkoori et al., 2009). Although most polymorphisms are functionally neutral, some affect regulation of gene expression or the function of the coded protein(Costa et al., 2008). Interleukin-10 (IL-10) is an anti-inflammatory cytokine, which is involved in down-regulating cell-mediated and cytotoxic inflammatory responses. The gene encoding IL-10 is located on chromosome 1 (1q31-1q32). IL-10 has two confirmed biallelic polymorphisms in the gene promoter region: -1082A/G, and -592 C/A. Presence of -1082Ais associated with lower production of IL-10 in vitro and in vivo, and an accordingly stronger inflammatory response.

The objective of this study was to find any association between IL-10 polymorphism and gastric cancer from Kashmir valley. The meta analysis performed by Yong Zhou (Yang et al., 2000) suggests that the IL-10-1082 promoter polymorphism may be associated with gastric cancer among Asians, and that differences in genotype distribution may be associated with the location of gastric cancer(Yong et al., 2008). IL-10 is a potent anti-inflammatory cytokine which has multiple functions. It is secreted by many cell types and a number of cell types are responsive to it (Moore et al., 1993). The IL-10 gene is located on chromosome 1 at q 31–32 and is highly polymorphic(Eskdale et al., 1999). Endogenously produced IL-10 is a potent immunosuppressant and important modulator of acute and chronic inflammation(Goldman et al.,1997; Rennick et al., 1997). It limits the production of pro-inflammatory cytokines by providing a negative feedback mechanism and therefore down-regulates the deleterious action of pro-inflammatorycytokines(Spera et al., 1998; Dietrich et al., 1999; Grilli et al., 2000; Yang et al., 2000; Ooboshi et al., 2002).

In this study, we assessed the polymorphism of two SNPs of Interleukin-10, IL-10-1082 and IL-10-592 polymorphism and found a significant association of IL-10-1082 with gastric cancer patients.We also found that the IL-10-1082 polymorphism was significantly related to EGD biopsy, salt tea consumption, blood group, sex and dwelling and not to other clinicopathological features. Many recent in vitro and in vivo investigations observed an impact of IL-10 on autoimmune diseases and progression of malignancies (Asadullah et al., 2003). Throughout the past 2 decades intensive genetic association studies have demonstrated controversial results on polymorphisms in the IL-10 gene, but maintain that they promote cancer appearance and development one way or another. There are 3 functional promoter SNPs in the IL-10 locus at –1082 (A to G, rs1800896), –819 (C to T, rs1800871), and –592 (A to C, rs1800872) pairs from the transcriptional start site. Most studies on these polymorphisms have investigated haplotypes, but not distinct polymorphisms, which is important. The GCC haplotype for IL-10-1082/-819/-592 polymorphisms is associated with higher cytokine production compared with the ATA haplotype. Alteration of the IL-10 protein level may be directly linked to the fact that polymorphisms of the IL-10 gene are associated with susceptibility to prostate, cervical, bladder, and breast carcinoma(Ahirwar et al., 2009; Gerger et al., 2010; Liu et al., 2010; Matsumoto et al., 2010). Furthermore, a high serum level of IL-10 was reported to contribute to chronic hepatitis C and several autoimmune diseases(Edwards et al.,1999). Wu et al.(Wu et al., 2003) revealed that the high-producing GCC haplotype of IL-10-1082/-819/-592 genetic polymorphisms was more frequent among GC patients in comparison with healthy controls in a Thai population (OR - 2.67). The effect was more evident for cardiac GC (OR -3.21) compared with a noncardiac tumor location (OR- 1.96). A high GC risk was also reported for smokers and H pylori.-infected carriers of the GCC haplotype, whereas for both smokers and H pylori.-positive patients the risk was extremely high (OR -3.05, 2.32, and 6.00, respectively). Sugimoto et al. (Sugimoto et al., 2007) also identified the GCC combination as a risk haplotype for GC in comparison with the ATA haplotype among H pylori.-positive Japanese individuals (OR- 2.805). In contrast with previous papers, El-Omar et al. (El-Omar et al., 2003) demonstrated the hypoactive ATA haplotype to be associated with noncardiac GC (OR -2.5). Carriers of the low-producing haplotype apparently have a stronger inflammatory response to H pylori. infection, which may result in the reduction of acid production, atrophy of the stomach, and progressive growth of neoplastic cells. However, it remains unclear how the high-producing GCC haplotype correlates with increased cancer risk. Perhaps this correlation may be explained by the immunosuppressive properties of IL-10 or by the inhibitory effect on other cytokines involved in antitumor defense. Finally, separate gene polymorphism investigations, among which are the IL-10–1082A/G, IL-10–592A/C, and IL-10–819C/T polymorphisms, demonstrated the absence of significant differences in genotype distribution between patients with gastric cancer and healthy controls(Zhou et al., 2008; Chen et al., 2010; Zhuang et al., 2010). In addition, Kang et al.(Kang et al., 2009) reported that CC carriers of the IL-10–592A/C polymorphism have a decreased risk of intestinal GC (OR -0.4). These findings revealed the advantage of haplotype analysis, which allows to define plausible and consistent results. The investigations on IL-10 gene polymorphisms allow to suggest that they impact GC(Arseniy, 2011).

IL-10 has emerged as a potent immunosuppressive cytokine by down-regulating the expression of Th1 cytokines and co stimulatory molecules and this gene is highly polymorphic. The association of the A to G substitution at position -1082 of the IL-10 gene promoter with increased transcription of IL-10 has been shown in vitro. The IL-10 genotype may influence predisposition to a number of solid tumors. Some studies have described an association between the IL-10-1082 G allele and decreased susceptibility to cancers (10,15). The mechanism for this may be via the ability of IL-10 to down-regulate synthesis of IL-1β,TNFa and vascular endothelial growth factor. However, other studies drew an opposite conclusion.One suggestive explanation for these intriguing findings is that IL-10 generally protects the host against inflammation after toxin-induced injury, but physiologically inadequate responses related to IL-10 after systemic injury may render the host susceptible to malignant change(Wanli et al., 2005).

IL-10 is also an important regulatory cytokine that is involved in many aspects of the immune response, and it seems to dysregulate in human autoimmune(Llorente et al., 1995; Chai et al., 2005; Duan et al., 2005; Mekala et al., 2005), malignant(Luscher et al., 1994; Kamiya and Abe, 2003; Azar et al., 2004; Murtaand Miranda,2004; Chan et al., 2005; Guo et al., 2005) and infectious (Chung et al., 2005; Migita et al., 2005; Stoycheva et al., 2005)disease. IL-10 production has also been implicated in the tumorigenesis of various types of cancers (Pisa et al., 1992; Huang et al., 1995). IL-10 polymorphism has also been extensively studied and reported to be associated with other cancers and diseases. Stratifying for race, patients with gastric cancer had a significantly lower frequency of AA and higher frequency AG than non cancer patients among Asians (Yong et al.,2008).

Conclusion

Our results suggest that A allele of IL-10 gene is an important genetic risk factor for Gastric cancer in the North Indian population from ethnic Kashmiri population.

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