Risk modification of colorectal cancer susceptibility by interleukin-8 -251T>A polymorphism in Malaysians

Abstract

AIM: To investigate the allele and genotype frequencies and associated risk of interleukin (IL)-8 -251T>A polymorphism on colorectal cancer (CRC) susceptibility risk.

METHODS: Peripheral blood samples of 255 normal controls and 255 clinically and histopathologically confirmed CRC patients were genotyped for IL-8 -251T>A polymorphism employing allele-specific polymerase chain reaction. The relative association of variant allele and genotypes with CRC susceptibility risk was determined by calculating the odds ratios (ORs). Corresponding χ² tests on the CRC patients and controls were carried out and 95% confidence intervals (CIs) were determined using Fisher’s exact test. The allele frequencies and its risk association were calculated using FAMHAP, haplotype association analysis software.

RESULTS: On comparing the frequencies of genotypes of patients and controls, the homozygous variant AA was significantly higher in CRC patients (P = 0.002) compared to controls. Investigation on the association of the polymorphic genotypes with CRC susceptibility risk, showed that the homozygous variant IL-8 -251AA had a significantly increased risk with OR 3.600 (95% CI: 1.550-8.481, P = 0.001). In the case of allele frequencies, variant allele A of IL-8 -251 showed a significantly increased risk of CRC predisposition with OR 1.32 (95% CI: 1.03-1.69, P = 0.003).

CONCLUSION: Variant allele and genotype of IL-8 (-251T>A) was significantly associated with CRC susceptibility risk and could be considered as a high-risk variant for CRC predisposition.

Key Words: Interleukin-8 -251T>A, Polymorphism, Colorectal cancer, Malaysians

INTRODUCTION

Colorectal cancer (CRC), the incidence of which has been increasing worldwide for the past few years, represents a significant cause of morbidity and mortality. CRC develops as a result of progressive accumulation of genetic and epigenetic alterations that lead to a series of histopathological changes, initiated by transition from normal mucosa to adenoma to carcinoma. Excluding inherited types of CRC, the susceptibility of a certain individual to development of sporadic CRC remains largely undetermined. Sporadic CRC is a multifactorial disease, therefore, environmental factors, host genotype and immunological factors all could significantly contribute to initiation and even progression of this malignancy.

Recently, chronic inflammation has been linked to increased risk of various types of cancer[1,2]. Epidemiological observations, animal models and clinical studies have established an association between continuous inflammatory conditions and CRC[1,3,4]. Patients with inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis, are at increased risk of developing CRC[5]. The associations between inflammatory response genes and IBD make them attractive candidate susceptibility genes for CRC because approximately 1:6 individuals with IBD are estimated to develop colorectal malignancy[6]. Despite this evidence strongly implicating chronic inflammation as a culprit in colorectal carcinogenesis, surprisingly little research has directly addressed the genetic predisposing factors that mediate inflammatory response and favor CRC development.

Genetic polymorphisms have emerged in recent years as important determinants of disease susceptibility and severity. Polymorphic variants of several genes are thought to play a key role in determining how individuals respond at the cellular level to various environment conditions including inflammation. If inflammation constitutes one of the molecular networks underlying susceptibility to CRC, genes that mediate inflammatory responses might be a group of candidate genes for CRC predisposition. Few genes are known to be important for inflammation of the colorectum, and their allelic variants have been shown to have biological effects[7,8]. Interleukin (IL)-8 is a chemokine and one of the major mediators of inflammatory responses, and is believed to play a role in the pathogenesis of cancer. Several polymorphisms have been detected in the IL-8 gene, and a common polymorphism at the -251 position (251T>A) of the promoter region has been associated with transcriptional activity of the gene. A case-control study was designed to investigate the IL-8 -251T>A polymorphic allele and genotype frequencies in healthy controls and sporadic CRC patients in the Malaysian population, and to determine the influence of the polymorphic genotype of IL-8 -251T>A on sporadic CRC susceptibility risk.

MATERIALS AND METHODS

Recruitment of subjects

The study was approved by the Research Review Board and Ethics Committee of Universiti Sains Malaysia and Ministry of Health (MOH) Malaysia. In this case-control study, cases comprised 255 CRC patients (139 male and 116 female), recruited from Hospital Universiti Sains Malaysia, and from a few hospitals under the MOH, Malaysia like Hospital Sultanah Bahiyah, Alor Setar, Kedah and Hospital Raja Perempuan Zainab II, Kota Bharu, Kelantan, Malaysia. An equal number of sex- and age-matched (± 5-10 years) normal healthy individuals (115 male and 140 female) were also recruited as controls. The ages of the patients ranged from 27 to 77 years with a mean age of 57.26 years. For the controls, the age ranged from 33 to 78 years with a mean age of 48.91 years. The sex and age distribution of the study subjects are shown in Table 1.

Table 1 Distribution of sex and age in cases and controls.

	Patients	Controls
Sex, n (%)
Female	116 (45.5)	140 (54.9)
Male	139 (54.5)	115 (45.1)
Age (mean ± SD)	57.26 ± 7.074	48.91 ± 12.020

DNA extraction

Peripheral blood samples of 255 normal controls and 255 clinically diagnosed and histopathologically confirmed CRC patients were collected in EDTA tubes, after obtaining written informed consent. The collected samples were stored at -20 °C till use. Genomic DNA was extracted using commercial DNA extraction kit (QIAGEN, Hilden, Germany) and the gene of interest was amplified using appropriate primers. Single nucleotide polymorphism -251 T>A in the IL-8 gene was determined using allele-specific polymerase chain reaction (PCR).

Genotyping

For genotyping, the allele-specific primers used were 5’-CCACAATTTGGTGAATTATCAAT-3’ and (251A) or 5’-TGCCCCTTCACTCTGTTAAC-3’ (251T). The consensus primer used was 5’-TGCCCCTTCACTCTGTTAAC-3’ (giving a PCR product of 366 bp). The IL-8 -251T>A polymorphic sequence was amplified using PCR with composition master mix using 100 ng DNA template, primer (0.2 μmol/L), 2.0 mmol/L MgCl₂, 10× buffer, 10 mmol dNTP (0.2 μL) and 5 U Taq DNA polymerase (Applied Biosystems, Foster City, CA, United States) with a total volume of 25 μL PCR mixture. The annealing temperature was 56.7 °C, and 35 PCR cycles were carried out. The PCR products were isolated on 2% agarose gels and visualized with SYBR Green. The IL-8 -251T>A polymorphic genotypes were categorized into homozygous wild, heterozygous and homozygous variant.

Statistical analysis

The difference in various genotype frequencies of IL-8 among the cases and controls was calculated. The relative associations of various genotypes with CRC susceptibility risk was determined by calculating the odds ratios (ORs). Corresponding χ² tests on the CRC patients and controls were carried out and 95% confidence intervals (CIs) were determined using Fisher’s exact test. Statistical analysis was carried out using SPSS version 18. The allele frequencies and risk associations were calculated using FAMHAP, haplotype association analysis software to derive ORs.

RESULTS

Visualization of PCR product of IL-8 -251T>A

Various IL-8 -251T>A genotype patterns in CRC patients in the Malaysian population are showed in Figure 1. A sample was considered as homozygous wild type (IL-8 -251TT) when only one band appeared at the T allele. When two bands appeared at both the T allele and A allele, it was considered as heterozygous variant (IL-8 -251TA). For the homozygous variant genotype (IL-8 -251AA), only one band appeared at the A allele.

Figure 1 Various interleukin-8 -251T>A genotype patterns in sporadic colorectal cancer patients observed after genotyping using allele-specific polymerase chain reaction. The genotype was considered as homozygous wild type [interleukin (IL)-8 -251TT] when only one band appeared at the T allele. When two bands appeared, each at the A allele and T allele, it was considered as heterozygous variant (IL-8 -251TA). The homozygous variant genotype was characterized by the appearance of only one band at the A allele. M: DNA ladder; 1: Homozygous wild type (IL-8 -251TT); 2: Heterozygous (IL-8 -251TA); 3 and 4: Homozygous variant (IL-8 -251AA).

Frequencies of IL-8 -251T>A genotypes in CRC cases and controls

The frequencies of IL-8 -251T>A genotypes in cases and controls are shown in Table 2. Among the 255 controls, the homozygous wild TT genotype was observed in 54 (21.18%), the heterozygous TA genotype was observed in 189 (74.12%) and the homozygous variant genotype AA was detected in 12 (4.7%) individuals. In the case of 255 CRC patients, 40 (15.69%) showed homozygous wild-type TT genotype (15.69% vs 21.18%, P = 0.11), 183 (71.76%) showed heterozygous variant TA genotype (71.76% vs 74.12%, P = 0.55), and 32 (12.55%) showed homozygous variant AA genotype. On comparing the frequencies of the polymorphic genotypes among the cases and controls, the homozygous variant genotype frequency was significantly higher among CRC cases (12.55% vs 4.7%, P = 0.002).

Table 2 Association risk and frequencies of T and A alleles of interleukin-8 -251T>A genotypes with colorectal cancer susceptibility.

	Patients	Controls	OR (95% CI)	P value
IL-8 genotype
Wild type -251TT	40 (15.69)	54 (21.18)	[1] (Ref.)	-
Hetero -251TA	183 (71.76)	189 (74.12)	1.307 (0.808-2.117)	0.250
Variant -251AA	32 (12.55)	12 (4.7)	3.600 (1.550-8.481)	0.001
Allele
Wild type	263 (51.6)	297 (58.2)	0.76 (0.59-0.97)	-
Variant	247 (48.4)	213 (41.8)	1.32 (1.03-1.69)	0.003

IL-8: Interleukin-8; OR: Odd ratio; CI: Confidence intervals.

Association risk of IL-8 -251T>A genotypes with CRC susceptibility

Table 2 shows the associated risk of IL-8 -251T>A genotypes with CRC susceptibility in this population. When the association of the polymorphic genotypes with CRC susceptibility risk was investigated, the IL-8 homozygous variant genotype (AA) showed significantly increased risk with OR 3.600 (95% CI: 1.550-8.481, P = 0.001).

Allele frequencies and risk association of inflammation response genes with CRC susceptibility

The allele frequencies and risk association of T and A alleles in cases and controls in the Malaysian population are showed in Table 2. The frequency of wild-type allele T was 51.6% and the frequency of variant allele A was 48.4% among the CRC cases. In the case of controls, the frequency of the wild-type allele T was 58.2% and 41.8% showed variant allele A. When the risk association of variant allele was examined, variant allele A of IL-8 showed a significantly higher risk with OR 1.32 (95% CI: 1.03-1.69, P = 0.003).

The patient group included patients from different parts of Malaysia and so the clinicopathological features of many of these patients could not be collected. Therefore, these details could not be specified. For the same reason, the association of genotype frequencies with patient prognostic subgroups could not be evaluated.

DISCUSSION

Inflammation, which is part of the immune response, may also induce or exaggerate some diseases through production of proinflammatory cytokines. Inflammatory cytokines are major inducers of chemokines that play a central role in leukocyte recruitment to sites of inflammation. Chemokines have pleiotropic biological effects that can play several roles in cancer progression, including angiogenesis, inflammation, cell recruitment and migration. IL-8 or chemokine CXC ligand 8 is the prototype member of the CXC chemokine family. Evidence has shown that the individual level of cytokine production is affected by single nucleotide polymorphisms in cytokine genes, and the observed differences in cytokine production among individuals can be at least partially explained by gene polymorphisms. Genetic polymorphisms might directly influence inter-individually in the magnitude of inflammatory response, and this might contribute to an individual’s ultimate clinical outcome. Genetic polymorphisms of cytokine genes have been identified to play a role in susceptibility to various diseases including cancer[9]. A common polymorphism in the -251 position (251T>A) of the promoter region of IL-8 has been identified.

We investigated the frequencies and potential risk modification of IL-8 -251T>A polymorphic genotype and allele on CRC susceptibility in the Malaysian population. Compared to controls, the prevalence of homozygous variant AA genotype was significantly higher in CRC patients (4.70% vs 12.55%, P = 0.002), whereas for the homozygous wild-type genotypes (TT) and heterozygous variant genotypes (TA), there was no significant difference in frequencies between the two groups. In a study by Yang et al[10], on the association of IL-8 -251T>A polymorphism with prostate cancer, there was no significant difference in the distribution of IL-8 polymorphic genotypes between prostate cancer cases and controls.

It has been suggested that IL-8 and its receptors are crucial to the development and progression of many malignancies[11]. Genetic polymorphisms of the IL-8 gene have been implicated in the susceptibility to a range of cancers including oral cancer[12], breast carcinoma[13] and gastric cancer[14]. Our interest was to investigate whether the genetic variants are related to the CRC risk in the Malaysian population. Our results showed that the polymorphism in the IL-8 gene was significantly associated with the risk of CRC. The -251AA genotype was associated with a significantly increased risk of CRC as compared with the -251TT genotype (OR: 3.600, 95% CI: 1.550-8.481, P = 0.001). Similarly, for allele frequencies, such an association was observed. When compared with wild-type allele T of SNP IL-8 -251, the variant allele A IL-8 -251 showed significantly increased risk for CRC predisposition with OR 1.32 (95% CI: 1.03-1.69, P = 0.003). The strong association that we observed in CRC patients prompts us to suggest that IL-8 gene -251AA polymorphism could contribute significantly to CRC susceptibility.

A few other molecular genetic epidemiological studies in diverse ethnic populations have found consistent as well as inconsistent results with ours. The IL-8 -251T>A polymorphism has been associated with the risk of gastric cancer and gastric ulcer in Japanese patients with Helicobacter pylori infection[15]. Taguchi et al[14] also have reported that IL-8 -251T>A polymorphism is associated with higher expression of IL-8 protein, more severe neutrophil infiltration, and increased risk of atrophic gastritis and gastric cancer in the Japanese population. In the study of Gunter et al[16], homozygous variant genotype of the IL-8 -251T>A had a 2.7-fold increased risk of colorectal adenoma compared to the homozygous wild type in the American population. In the French population, Küry et al[17] have reported that the heterozygous and homozygous variants of IL-8 c -352 T>A are associated with an elevated risk of CRC compared to the homozygous major genotype. Li et al[18] have found that the risk of gastric cancer in the Chinese population is significantly elevated in patients with the IL-8 -251AA genotype with OR 2.02 (95% CI: 1.27-3.21). In contrast with our study, Landi et al[19] have reported that the IL-8 -251T>A genotypes have a protective role against CRC predisposition in the Spanish population, and Theoropoulus has reported that this SNP has no effect on CRC susceptibility risk in the Greek population[8]. A few other studies have suggested that the IL-8 -251AA genotype is associated with an increased risk of prostate cancer in the Caucasian population[20] and Kaposi’s sarcoma in the Dutch population[21], but a decreased risk of CRC[4].

The 251T>A polymorphism at the promoter region of IL-8 gene is associated with the transcriptional activity of the gene[22], and to influence production and expression of IL-8[14,15]. Disrupted gene expression or altered protein formation of the IL-8 gene may contribute positively or negatively to the establishment or progression of CRC. According to Wei et al[22], higher promoter activity of IL-8 -251AA polymorphism might increase production and expression of IL-8, inducing a Th1-predominant immune response and leading to more susceptibility to CRC.

Free radicals generated as a result of oxidative stress produced by inflamed tissues may cause alteration in many metabolic reactions such as regulation of DNA, RNA and lipids, and thus can lead to cancer development[23,24]. Meira et al[25] have demonstrated that in inflamed colon cancer tissues, reactive oxygen species (ROS) and reactive nitrogen species are produced from activated inflammatory cells, and that these two species enhance DNA damage and this can cause mutation in areas of inflammation. Moreover, in CRC-associated colitis, chronic inflammation has been reported to cause oxidative damage to DNA, influence mutations in the p53 gene in the inflamed tissues, and drive the cells to malignant transformation[26]. Oxidative stress produced by inflammatory cells in inflamed tissues in the intestinal tract has been reported to influence the development of CRC in patients with IBD[27]. These studies have clearly highlighted the importance of IL-8 in modulating inflammation of the colorectum. According to Okada et al[28], ROS produced by inflammatory cells cause not only direct DNA damage but also indirect effects such as dysregulation of cell proliferation and apoptosis, stimulation of angiogenesis, and modification of gene/protein expression and protein activities that will cause cancer. Therefore, the relevance of IL-8 -251T>A polymorphism in CRC susceptibility could be explained by the enhanced transcriptional activity of the gene, resulting in functional alteration of the gene product.

To the best of our knowledge, this is the first study on the association of the IL-8 gene -251 T>A polymorphism and CRC risk in the Malaysian population. Our results show that the genetic variation of IL-8 gene influences susceptibility to CRC in the Malaysian population, and suggest inflammation-mediated pathways in the process of colorectal carcinogenesis.

ACKNOWLEDGMENTS

We express our sincere thanks to Dr. Abdullah Bayanuddin NAB (Penang Health Center, Penang) for her help in normal control sample collection, and Dr. Biswal BM, Dr. Bhavaraju VMK (Hospital Universiti Sains Malaysia), Dr. Radzi M (Hospital Sultanah Bahyah, Alor Setar, Kedah, Malaysia) and Dr. Shanwani A and Dr. Zaidi (Hospital Raja Perempuan Zainab II, Kota Bharu, Kelantan) for their help in CRC patients recruitment and sample collection.

COMMENTS

Background

Colorectal cancer (CRC), the incidence of which has been increasing worldwide for the past few years, represents a significant cause of morbidity and mortality. Genetics has a key role in predisposition to CRC and in its initiation and progression. Identifying predisposing genetic variations is important for our understanding of the carcinogenic process. Recently, chronic inflammation has been linked to increased risk to various types of cancer including CRC. Despite evidence strongly implicating chronic inflammation as a culprit in colorectal carcinogenesis, surprisingly little research has directly addressed the genetic predisposing factors that mediate inflammatory response and favor CRC development. Thus, it was of interest to explore the contribution of single nucleotide polymorphisms (SNPs) in inflammation genes as predisposing factors for CRC susceptibility.

Research frontiers

If inflammation constitutes one of the molecular networks underlying susceptibility to CRC, genes that mediate inflammatory response might be a group of candidate genes for CRC predisposition. Interleukin (IL)-8 is a chemokine and one of the major mediators of inflammatory responses, and is believed to play a role in the pathogenesis of cancer. IL-8 is a proangiogenic cytokine that is overexpressed in many human cancers and its expression promotes tumor growth, angiogenesis and metastasis. A polymorphism at -251 position (251T>A) of the promoter region of IL-8 has been associated with transcriptional activity of the gene. This study was designed to determine the frequencies and influence of the IL-8 -251T>A polymorphic genotype and alleles on sporadic CRC susceptibility risk in the Malaysian population.

Innovations and breakthroughs

There are not many data available on the contribution of SNPs in inflammation response genes in mediating CRC predisposition risk, especially from the Asian population, and none from the Malaysian population. This is believed to be the first report of an association of genetic variation of IL-8 with CRC susceptibility risk in the Malaysian population. We observed that the genetic diversity of the IL-8 gene influences patient susceptibility to CRC and implies the importance of inflammation-mediated pathways in the process of colorectal carcinogenesis. From our results, it is reasonable to pronounce that IL-8 gene may be an important candidate in the modulation of colorectal inflammation and the IL-8 251AA (homozygous variant genotype) could be considered as an important high-risk variant for CRC predisposition in the Malaysian population.

Applications

Early diagnosis is important for successful management of CRC patients and is facilitated by both invasive and noninvasive means of surveillance. Identification of genetic predisposition factors of CRC will help with better understanding of the colorectal carcinogenic process and in the design of diagnostic, therapeutic and preventive strategies. Understanding the genes and pathways that control the earliest steps of the disease and individual susceptibility can contribute to clinical management in the near future. In the future, study can be extended to a population level and individuals with high-risk predisposition genotypes can be identified. Once identified, they can be enrolled in cancer surveillance programs that will help in CRC prevention strategies.

Peer review

In this case-control study to determine the influence of the polymorphic genotype on sporadic CRC susceptibility risk in the Malaysian population, the IL-8 -251T>A polymorphic allele and genotype frequencies were evaluated in 255 patients with sporadic CRC and 255 normal healthy controls. The frequency of the homozygous variant AA was significantly higher in CRC patients compared to controls. Furthermore, the homozygous variant IL-8 -251AA was significantly associated with increased risk of CRC predisposition. This study is of interest because previous studies on the relationships between IL-8 -251T>A polymorphism and CRC have yielded contradictory results.