Introduction

Oral cancer is malignant and usually causes extensive damage to the organs involved [1]. In Taiwan, the incidence rate of oral cancer is 20.22/100,000, and it is the 7^th prevalent malignancy [2] and the 5^th leading cause of cancer deaths among Taiwanese [3]. Interleukin-18 (IL-18), an 18-kDa cytokine, belongs to the interleukin-1 (IL-1) superfamily and is produced by various immune and non-immune cells [4-7]. It has been demonstrated that the expression and secretion of IL-18 is a crucial event against oncogenesis of oral carcinoma cells because of its modulation of cell cycle progression or its triggering of an apoptotic pathway [4,6-8]. Nilkaeo et al. found that the IL-18 suppressed KB cell line, a carcinoma cell line derived from oral cavity, proliferates in a dose-dependent manner through the modulation of cell-cycle arrest in the S phase [6]. Liu et al. demonstrated that the over-expression of IL-18 reduced cell viability and induced apoptosis of the human tongue squamous cell carcinoma cell line could be attributed to the down-regulation of cyclin D1 expression and a caspase-dependent pathway, respectively [7]. We suggested that IL-18 is a key regulator for the development of oral cancer.

The IL-18 gene is located on chromosome 11q22. Two functional gene polymorphisms -607A/C and -137G/C are found in its promoter region [9]. Giedraitis et al. analyzed IL-18 gene promoter sequence and found a change from C allele to A allele at position -607 and a change from G to C at position -137 of the IL-18 promoter region [9]. They estimated transcription activity of IL-18 gene promoter fragments and found that C allele of -607A/C or G allele of -137G/C showed higher activity of IL-18. The participants with CC homozygote of -607A/C or GG homozygote of -137G/C polymorphism exhibited somewhat higher levels of IL-18 mRNA compared with other genotypes [9].

It is demonstrated that individuals exposure to environmental risk factors such as areca, alcohol, and tobacco consumption increase their susceptibility to oral cancer [10-13]. Also, genetic polymorphisms are reported to be the main risk factors of oral cancer susceptibility [14-16]. Our study suggested that IL-18 polymorphisms -607A/C and -137G/C could regulate the protein levels of IL-18 and considerably affect the individual sensitivity to oral cancer [9]. However, to the best of our knowledge, only Vairaktaris et al. [17] and Asefi et al. [18] have estimated the impact of interleukin-18 polymorphisms -607A/C and -137G/C on oral cancer occurrence and clinical parameters. Among 238 Greek and German patients, Vairaktaris et al. recruited 149 with oral cancer and 89 who were healthy to examine the effect of -607A/C gene polymorphism of IL-18 on oral cancer risk. They found that IL-18 -607A/C polymorphism is not associated with the susceptibility to oral cancer [17]. In Iran, Asefi et al. recruited 111 patients with head and neck squamous cell carcinoma and 212 who were healthy to investigate the association of IL-18 gene polymorphisms -607A/C and -137G/C on the occurrence and clinical parameters of head and neck squamous cell carcinoma. Again, no meaningful association was found [18]. Unfortunately, their sample size limited the prediction of IL-18 -607A/C and -137G/C gene polymorphisms on the risk of oral cancer [17,18]. On the other hand, in a recently meta-analysis by Liang TJ et al. they found the -137G > C polymorphism significantly increased cancer risk in Asian population but not in Caucasian population after the stratification analyses of ethnicities [19]. We considered that the impact of genetic polymorphisms IL-18 -607A/C and -137G/C differences related to ethnicity, and their interaction with oral cancer related risk factor, including areca, tobacco, and alcohol consumption could increase oral cancer risk among Taiwanese. Therefore, for this study, we recruited 1,126 participants, including 567 patients with oral cancer and 559 healthy people to determine whether genetic variations at positions -607A/C and -137G/C of IL-18 and their interaction with oral cancer-related risk factor are associated with the susceptibility to and clinicopathological development of oral cancer among Taiwanese people.

Materials and Methods

Results

In our recruited control group, the frequencies of genetic polymorphisms such as IL-18 -607 A/C (p>0.05, χ² value: 0.08) and IL-18 -137 G/C (p>0.05, χ² value: 0.80) were in the Hardy-Weinberg equilibrium.

The study estimated differences of demographical characteristics, such as gender, age, alcohol, tobacco, and areca consumption and genetic polymorphisms between oral cancer patients and controls. A significantly different distribution of IL-18 -137 G/C gene polymorphism based on gender, age, alcohol, tobacco, and areca consumption between oral cancer patients and controls was found (Table 2). To diminish the possible interference of environmental factors, adjusted ORs (AORs) with 95% CIs were estimated by multiple logistic regression models after controlling for other covariates in each comparison.

People with G/C alleles of IL-18 -137G/C polymorphism had a 1.64-fold (95% CI=1.08-2.48; p=0.02) increased risk of developing oral cancer compared with those with G/G homozygotes. This determination was made after adjusting for gender, age, alcohol, tobacco, and areca consumption. However, there was not a significant association between IL-18 -607A/C genetic polymorphism and oral cancer. In addition, we found no gene-to-gene interaction effect on the increased susceptibility to oral cancer (Table 3).

The study also determined whether there was an interaction effect of gene-to-related-environmental-risk-factors on oral cancer susceptibility. The adjusted odd ratios and 95% confidence intervals of genotypic frequencies and oral cancer susceptibility were estimated among persons with exposure and non-exposure to oral cancer-related environmental risk factors, respectively. There was no significant association between genetic polymorphisms of IL-18 -607A/C and -137G/C and oral cancer susceptibility among participants who had no exposure to related environmental risk factors (Table 4). However, among participants who were exposed to related environmental risk factors, including areca, alcohol, and tobacco consumption, the adjusted odd ratios and 95% confidence intervals were increased to a 2.02-fold (95% CI=1.01-4.04; p=0.04), 4.04-fold (95% CI=1.65-9.87; p=0.002), and 1.66-fold (95% CI=1.00-2.84; p=0.05) risk of developing oral cancer. For -607A/C polymorphism of IL-18, among alcohol consumers, those with A/A homozygotes of IL-18 -607 A/C polymorphism had a 2.38-fold (95% CI=1.17-4.86; p=0.01) increased risk of developing oral cancer compared with those with C/C homozygotes (Table 5). For gene-to-gene interaction effect, among alcohol consumers, those with group 3 polymorphism had a 5.81 (95% CI=2.22-15.24; p=0.0003) increased risk of developing oral cancer compared with those with group 1 (Table 5). This was determined after adjusting for confounders.

Both genetic polymorphisms were analyzed with regard to the clinical status of each of our recruited 567 oral cancer patients, including the tumor stage, tumor size, lymph node metastasis, distant metastasis, and cancer cell differentiation. Patients with G/C alleles IL-18 -137G/C polymorphism showed a decreased risk of developing Stages III-IV (AOR=0.59; 95% CI=0.39-0.89; p=0.01), a tumor size > T2 (AOR=0.56; 95% CI=0.35-0.87; p=0.01), and lymph node metastasis (AOR=0.51; 95% CI=0.32-0.80; p=0.003). There was not a significant association between clinical status and IL-18 -607 A/C gene polymorphism in these patients (Table 6).

Discussion

Our study offered information that IL-18 gene promoter polymorphism -137G/C was significantly associated with oral cancer susceptibility and clinicopathological development.

IL-18 has been shown to act as a regulator of oral cancer development [5,8]. Hayes et al. suggested that oral talactoferrin, a recombinant human lactoferrin, produced a dose-dependent inhibition of oral tumors through an increased expression of IL-18 [8]. Jablonska et al. observed a considerably lower concentration of IL-18 released by polymorphonuclear leukocytes (PMN) derived from oral cavity cancer patients when compared with those of healthy people. However, the production of IL-18 by PMN was enhanced among oral carcinoma patients after cancer treatment. In our study, participants with G/C alleles of IL-18 -137 G/C polymorphism had a 1.64-fold (95% CI=1.08-2.48; p=0.02) increased risk of developing oral cancer compared with participants with G/G homozygotes, a determination made after adjusting for gender; age; and alcohol, tobacco, and areca consumption, our result was inconsistent with those of Vairaktaris et al [17] and Asefi et al. [18]., However, for -607A/C polymorphism of IL-18, our results were similar to those of Vairaktaris et al and Asefi et al., which indicated there was not a significant relationship between IL-18 -607A/C polymorphism and oral cancer risk [17,18]. We suggest that C allele of IL-18 -137G/C polymorphisms lead to a lower level of IL-18 protein synthesis [9]. Such an occurrence impedes the modulation of cell cycle arrest and the triggering of cell apoptosis, which protects the host from oral cancer development [4,6-8]. Moreover, the inconsistent results between ours and those of Vairaktaris et al and Asefi et al., indicating the impaction of genetic polymorphism -137G/C on oral cancer susceptibility may be difference related to ethnicity [19].

The exposure of patients to oral cancer-related environmental risk factors such as areca, alcohol, and tobacco consumption demonstrate an increased risk to cause mucosal fibroblast proliferation and oral epithelial hyperplasia and dysplasia, in which cancer-related tissue chronic inflammation is suggested involved [10-13,23-26]. In our study finds that the interaction of gene to oral cancer-related environmental risk factors has a synergetic effect that can further enhance oral cancer development. Among participants exposed to oral cancer-related environmental risk factors, including areca, alcohol, and tobacco consumption. the adjusted odd ratios and 95% confidence intervals increased to a 2.02-fold (95% CI=1.01-4.04; p=0.04), 4.04-fold (95% CI=1.65-9.87; p=0.002), and 1.66-fold (95% CI=1.00-2.84; p=0.05) risk of developing oral cancer for participants with G/C alleles of IL-18 -137 G/C polymorphism compared to participants with G/G homozygotes. Also, among alcohol consumers, participants with A/A homozygotes of IL-18 -607 A/C polymorphism had a 2.38-fold (95% CI=1.17-4.86; p=0.01) increased risk of developing oral cancer compared with participants with C/C homozygotes, determined after adjusting for confounders. It was known that treatment of ovalbumin-sensitized mice with areca nut extract significantly augmented inflammatory response and promoted the development of CD 11b⁺ Gr-1⁺ cells with the characteristics of myeloid-derived suppressor cells, which could skew the host immunity toward tumor-promotion and deteriorate anti-tumor immunity by down-regulating T-cell reactivity to cancer cells [25,26]. Tobacco is a heterogeneous which contains different substances classified as carcinogenic to human [27,28]. Tobacco consumption has been linked to the development of cancer-related inflammation in cancer patients [29] and to induction of oral epithelial hyperplasia and dysplasia among patients with oral cancer [23,24]. Recently, cancer-related inflammation is mentioned as one of the cancer hallmarks, because it involved in the initiation of genetic instability by inflammatory mediators [25,26,30]. We suggested that both areca nut and tobacco consumption could amplify inflammatory response and long term consumption of either areca nut or tobacco smoke could induce chronic inflammation of oral tissue, which subsequently lead to accumulation of random genetic alteration and initiate the development of oral cancer [25,26,30]. IL-18 exerts its anti-tumor activity by inducing cell cycle arrest for DNA repair, promoting cytotoxic cells activity, and triggering mutated cells apoptosis [4,6-8]. However, among individuals with C allele of IL-18 -137G/C polymorphisms result in a lower level of IL-18 protein production [9], consequently, damaged DNA does not repaired due to the fail of inducing cell cycle arrest and the defeat of triggering mutated cell apoptosis by IL-18, genetically damaged cells proliferate, giving rise eventually increase the risk to malignant neoplasm among subjects with C allele of IL-18 -137G/C polymorphisms [4,6-8]. Also, alcohol consumption is shown to modulate adaptive immune responses and inflammatory process [31,32]. Joosten et al. estimated gene expression profiles of leucocytes and circulating proteins related to immune response after moderate alcohol consumption among twenty-four healthy men. They found plasma levels of pro-inflammatory IL-1 receptor antagonist and IL-18 significantly decreased after alcohol consumption [32]. We suggested that alcohol consumption induce decreased expression of IL-18 and result in abating the function of IL-18 on the modulation of cell-cycle arrest and induction of apoptosis, particularly to the subjects with A allele of IL-18 -607 or C allele of IL-18 -137 polymorphisms because both genetic polymorphisms are suggested with lower activity of IL-18, consequently enhance the risk to have oral cancer among alcohol consumers with A allele of IL-18 -607 or C allele of IL-18 -137G/C gene polymorphisms.

However, we also found that G/C genotype IL-18 -137 polymorphism represented a protective factor for oral cancer progression. Patients with G/C alleles of IL-18 -137 correlated with a lower clinical stage, tumor size, and non-lymph node metastasis compared with patients with G/G alleles. Our results were similar to those of Jaiswal et al. [33] and Saenz-Lopez et al. [34]. Jaiswal et al. [33] recruited 200 patients with bladder cancer and 200 healthy controls to examine the impact of IL-18 gene polymorphism on bladder cancer susceptibility, they found a significant relationship of IL-18 -137 G/C heterozygous genotype (G/C) with 1.96 folds risk to bladder cancer, however, this heterozygous genotype showed a significantly reduced risk for tumor progression among patients with bladder cancer. Also, Saenz-Lopez et al. [34] found that IL-18 -137 GG genotype was significantly associated with a higher tumor size, grade, and stage among patients with renal cell carcinoma. It is found that significantly higher levels of IL-18 in serum and culture supernatants of PMN from patients with oral cancer in Stages III and IV as compared with patients in Stages I and II [5]. Our study suggests that the over-expression of IL-18 plays a vital role in protecting people from oral cancer. However, the higher concentrations of IL-18 in serum and culture supernatants of PMN from patients with oral cancer in Stages III and IV as compared with patients in Stages I and II could be the host’s response against the growth and progression of oral cancer. Furthermore, it is probable that IL-18 acts as both a suppressor and promoter in the regulation of oral cancer development [5,35-37]. One of the possible explanations for the controversial effect of IL-18 -137 G/C polymorphism in oral cancer susceptibility and clinical progression is that IL-18 has dual effects on cancer development and progression [5,37-39]. Studies have suggested that IL-18 plays a major role in angiogenesis. Specifically, it has been reported that malignant cancer cells increase their adherence to microvascular wall and even promote production of angiogenic and tumor growth-stimulating factor through the IL-18-dependendent pathway [38,39]. Over-expression of IL-18 was found among cancer patients with malignant prognosis, including oral cancer, lung cancer, gastric cancer, pancreatic cancer, and hepatocellular carcinoma [5,37,40-42]. We suggest that IL-18 can act against the occurrence of oral cancer and induce angiogenesis and metastasis, which, in part, play a role in the advanced progression of oral cancer. Our study also demonstrates that patients with G/C alleles of IL-18 -137 G/C polymorphism can express lower levels of IL-18 compared with patients with G/G homozygotes, which benefits the inhibition of angiogenesis and tumor growth and consequently protects patients from the progression of oral cancer [5,9,37].

In conclusion, our results suggest that IL-18 -137 G/C gene polymorphism may be a factor that increases the susceptibility to oral cancer and can be a protective factor against oral cancer progression. The interactions of gene to oral cancer-related environmental risk factors have a synergetic effect that can further enhance oral cancer development.