The COX-2-1195AA Genotype Is Associated with Diffuse-Type Gastric Cancer in Korea.

BACKGROUND/AIMS: The potential role of the cyclooxygenase (COX)-2 polymorphism has been reported in relation to the risk of gastrointestinal tract malignancies. Therefore, we investigated whether COX-2 polymorphisms are associated with the risk of gastric cancer (GC) in Korea, one of the areas with a high prevalence of this condition.
METHODS: We evaluated the genotypic frequencies of COX-2-765 and -1195 in 100 peptic ulcer patients, 100 GC patients, and 100 healthy controls. The polymorphisms of the COX-2-765 and -1195 genes were analyzed by polymerase chain reaction and restriction fragment length polymorphisms.
RESULTS: The frequencies of the COX-2-1195 GG, GA, and AA genotype were 20%, 60%, and 20% in intestinal-type GC and 8%, 48%, and 44% in diffuse-type GC, respectively (p=0.021). There were no significant differences in the frequency of COX-2-765 genotypes between intestinal-type GC and diffuse-type GC (p=0.603). Age- and sex-adjusted logistic regression analysis showed that the COX-2-1195 AA genotype was the independent risk factor of diffuse-type GC compared with the COX-2-1195 GG genotype (p=0.041; odds ratio, 6.22; 95% confidence interval, 1.077 to 35.870).
CONCLUSIONS: The COX-2-1195 AA genotype may render subjects more susceptible to diffuse-type GC.

INTRODUCTION

Although Helicobacter pylori infection has been generally accepted as the main risk factor for gastric cancer (GC), carcinogenesis of GC is still unclear.1,2 H. pylori infection alone is not enough to explain the gastric carcinogenesis because GC develops in only a small portion of infected subjects. It has been suggested that GC may have a more complex mechanism involving bacterial, dietary, and host factors that are intimately interconnected. Among the host factors, single nucleotide polymorphisms (SNPs) of interleukin (IL)-1, IL-10, and tumor necrosis factor (TNF)-α have been thought to play an important role in Caucasians.3-5 The impact of polymorphisms of IL-1, IL-10, and TNF-α on the development of GC, however, is still controversial in Asians including our previous studies.6-9 Recently, several studies suggested that cyclooxygenase (COX)-2 polymorphisms are related with the high risk of various human malignancies, predominantly in gastrointestinal tracts including GC.10-14 As well known, COX is a rate-limiting enzyme that converts arachidonate to prostaglandins. As of today, three isoenzymes COX-1, COX-2, and COX-3 were found in human.15 Among them, COX-2, only expressed by various stimuli such as cytokines, growth factors, and mitogens, is responsible for inflammatory process and carcinogenesis. In particular, increased COX-2 expression is linked to progression of gastric pre-malignant lesions and gastric carcinogenesis by activating angiogenesis, inhibiting apoptosis, and accelerating invasion and metastasis.16-19 Moreover, it has been reported that SNPs in the promoter region of the COX-2 encoding gene have a direct effect on COX-2 expression and its functional activity. That is, COX-2-765 C allele increased the production of PGE2 and PGD2 in asthma,20,21 and COX-2-1195 A allele is associated with increased COX-2 expression in esophageal cancer.22 In GC, to our knowledge, few studies have been published on the SNPs of the promoter region -765 (rs689466) or -1195 (rs20417) of the COX-2 encoding gene.10-13 Moreover, it is still inconclusive although it is probable that those polymorphisms are helpful to identify the high-risk subjects for GC development.23

GC can be subdivided into two pathologic entities with intestinal and diffuse types, which have distinct epidemiological and clinical features. It is generally accepted that the carcinogenesis of intestinal type GC shows a multi-step progression of gastric mucosal lesions from atrophic gastritis to adenoma, followed by dysplasia and then eventually GC.24 To date, few data about polymorphisms of sporadic diffuse type GC has been reported.25 GC is highly prevalent in the Far East, especially in Korea, Japan, and China. According to a recent survey, in South Korea, GC incidence in men and women reaches 62.8 and 25.7 cases per one hundred thousand, respectively.26 Such a high incidence of GC in South Korea remains an etiologic and treatment challenge. Therefore, in this study, we focused whether COX-2 polymorphisms are associated with risk of GC, H. pylori infection, and pathologic types of GC in South Korea, one of the most prevalent areas of GC in the world.

MATERIALS AND METHODS

1. Patients and samples

A total of three hundred cases with peptic ulcer diseases (PUDs) (n=100), GC (n=100), and healthy controls (n=100) were enrolled and analyzed. Healthy controls were recruited voluntarily, who were conducted a routine medical check-up at Hallym University Kangdong Sacred Heart Hospital, Seoul, South Korea. All enrolled subjects underwent endoscopy, and the endoscopic findings were reviewed by two experienced endoscopists with a blind fashion. The control group has superficial gastritis or normal appearance of the gastric mucosa endoscopically. PUD and GC were diagnosed by endoscopic findings combined with histology. GC was subdivided into two distinct pathologic entities according to the Lauren's classification by an experienced pathologist blindly. The tumor, node, metastasis (TNM) stages were assigned according to the 6th American Joint Committee on Cancer TNM staging system.27 GC was grouped into cadiac and non-cadiac cancer by the locations of GC. H. pylori infection was evaluated by histologic examination, rapid urease test, and/or anti-H. pylori immunoglobulin G quantification (GCRL Co., Seoul, Korea; sensitivity, 98.10%; specificity, 90.82%). We defined H. pylori infection as being positive when at least one test of them showed positivity. All the subjects had no past history of H. pylori eradication. Buffy coat was separated from the whole blood and stored immediately at -70℃ until use. The study protocol was approved by Institutional Review Board at Hallym University Kangdong Sacred Heart Hospital.

2. Analysis of COX-2 gene polymorphism

Genomic DNA was extracted from the buffy coat using a commercialized kit (QIAamp; QIAGEN, Valencia, CA, USA). Following polymerase chain reaction (PCR) amplification using the primers as listed in Table 1, the COX-2 polymorphism was analyzed by restriction fragment length polymorphism (RFLP) method. Briefly, PCR conditions for COX-2-765 were as follows: 94℃ for 2 minutes, then 35 cycles of 94℃ for 1 minute (denaturation), 60℃ for 1 minute (annealing), 72℃ for 1 minute (extension), and finally 72℃ for 10 minutes. And then, the PCR products were digested with AciI (Promega, Madison, WI, USA) at 37℃ for 4 hours and separated by electrophoresis on a 2% agarose gel. A fragment containing the AciI polymorphic site at position -765 of COX-2 gene was separated as follows: the G allele was designated if two bands of 100 and 210 bp were obtained, and the C allele was designated if a single band of 310 bp was obtained (Fig. 1A).

Table 1

Primers Used for the Polymerase Chain reaction

COX, cyclooxygenase.

Fig. 1

Restriction patterns for cyclooxygenase (COX)-2-765 with AciI and COX-2-1195 with PvuII. (A) The genotypes of COX-2-765 were designated as follows: GG, 2 bands of 100/210 bp; GC, 3 bands of 100, 210, and 310 bp; and CC, a single band of 310 bp. A 100-bp ladder is displayed in Lane 1 (Marker). Lanes 2, 3, 4, 5, and 6 are GC, GG, GC, GG, and GG, respectively. (B) The genotypes of COX-2-1195 were designated as follows: GG, 2 bands of 50/220 bp; GA, 3 bands of 50, 220, and 270 bp; and AA, a single band of 270 bp. A 100-bp ladder is displayed in Lane 1 (Marker). Lanes 2, 3, and 4 are AA, GG, and GA, respectively.

The PCR conditions for the COX-2-1195 polymorphism were as follows: 94℃ for 2 minutes, then 35 cycles of 94℃ for 1 minute, 60℃ for 1 minute, 72℃ for 1 minute, and finally 72℃ for 10 minutes. A fragment containing the PvuII (Promega, Madison, WI, USA) polymorphic site at position -1195 of COX-2 gene was separated as follows: the G allele was designated if two bands of 220 and 50 bp were obtained, and the A allele was designated if a single band of 270 bp was obtained (Fig. 1B).

3. DNA sequencing of PCR products to validate RFLP

Ten samples were randomly selected for DNA sequences analysis from each group. Following PCR for COX-2-765 and -1195, DNA sequences were analyzed by automated DNA fluorescent sequencer using ABI prism™ Bigdye™ terminator cycle sequencing Ready reaction kit version 3.1 and ABI 3730XL capillary DNA Sequencer (Applied Biosystems, Foster City, CA, USA). The deduced sequences were compared with the sequences from the Genbank database in order to confirm the genotype of -765 and -1195 COX-2 genes (Fig. 2A and B).

Fig. 2

Sequences of the cyclooxygenase-2 promoter region. (A) The single nucleotide polymorphism (SNP) located on nucleotide -765 is represented by arrows. (B) The SNP located on nucleotide -1195 is represented by arrows.

4. Statistical analysis

The Hardy-Weinberg equilibrium of alleles at individual loci was assessed by chi-square test. Univariate analysis was performed with chi-square test and Fisher's exact test for the comparison of genotype frequencies among the groups. Age and sex adjusted odds ratios (ORs) with 95% confidence intervals (CIs) for genotyping were calculated by logistic regression analysis. The 2-sided p-value less than 0.05 was considered statistically significant. All statistical analyses were carried out using dBSTAT for Windows version 4.0 (dBSTAT Co., Seoul, Korea).

RESULTS

1. Clinical characteristics of enrolled subjects

Baseline characteristics were listed in Table 2. Mean age was significantly older in GC group than in PUD and control groups (p<0.0001). Female-to-male ratio was significantly different among the groups (p=0.03). Prevalence of H. pylori infection was 47%, 39.6%, and 35.9% in control, PUD, and GC group, respectively. There was no difference in the prevalence of H. pylori infection among the groups (p>0.05).

Table 2

Clinical Characteristics and Genotypic Frequencies of -765 and -1195 COX-2 in the Subjects Enrolled

HP, Helicobacter pylori; COX, cyclooxygenase; GC, gastric cancer.

2. COX-2-765 and -1195 genotype frequencies of study groups

Genotypic frequencies of the individual loci were in the Hardy-Weinberg equilibrium. The genotypic and allelic frequencies of COX-2-765 and -1195 were summarized in Table 2. No COX-2-765 CC genotype was found. There were no significant differences in the distribution of COX-2-765 and -1195 genotypes among the groups (p=0.302).

3. COX-2-765 and -1195 genotype frequencies according to the pathologic type of GC

We summarized the baseline characteristics of the patients with GC subdivided by Lauren's classification in Table 3. GC was confirmed as having diffuse type in 50 (50%) cases and intestinal type in 50 (50%) cases. There were no differences in the distribution of age, sex, smoking, and the prevalence of H. pylori infection between intestinal type GC and diffuse type GC. Significant difference was observed in the distribution of TNM stages between two groups (p<0.0001). Advanced GCs (AGCs) were observed more frequently in diffuse type GC (73.9% vs 43.8%, p=0.003). The location of GC was significantly different between the two groups (p=0.007). All the cardiac GC was diffuse type. There were significant differences in the frequency of COX-2-1195 genotypes between two groups (p=0.021). However, no significant differences were found in the frequency of COX-2-765 genotypes between two groups.

Table 3

The Clinicopathologic Characteristics and Genotypic Frequencies of -765 and -1195 COX-2 according to the Histological Type of Gastric Cancer

Data are presented as number (%).

HP, Helicobacter pylori; TNM, tumor, node, metastasis; COX, cyclooxygenase; GC, gastric cancer.

4. Risk of intestinal or diffuse type GC according to genotype of COX-2-1195

Comparing control group with diffuse type GC group, the age and sex adjusted logistic regression analysis showed that the subjects with COX-2-1195 AA genotype had the OR of 6.22 for diffuse type GC compared with the subjects carrying the COX-2-1195 GG genotype (p=0.041; 95% CI, 1.077 to 35.870). Comparing intestinal type GC with diffuse type GC, the age, sex, and smoking adjusted OR for COX-2-1195 AA genotype relative to GG genotype was 5.55 (p=0.001; 95% CI, 2.044 to 15.065) (Table 4).

Table 4

The Risk of Intestinal- or Diffuse-Type Gastric Cancer according to the Genotype of -1195 COX-2

Adjusted for age and sex.

COX, cyclooxygenase; GC, gastric cancer; OR, odds ratio; CI, confidence interval.

*Adjusted for age, sex, and smoking.

5. GC risks according to H. pylori infection and COX-2-1195 genotype frequencies

There were no significant differences in the distribution of COX-2-1195 genotypes and status of H. pylori infection between control and GC (Table 5).

Table 5

Gastric Cancer Risks according to Helicobacter pylori Infection and COX-2-1195 Genotype Frequencies

COX, cyclooxygenase; HP, Helicobacter pylori; C, control; I, intestinal type gastric cancer; D, diffuse type gastric cancer; GC, gastric cancer; OR, odds ratio; CI, confidence interval.

DISCUSSION

The role of genetic backgrounds for diffuse type GC remains to be clarified. In addition, it is still unknown whether diffuse type GC develops through a multi-step progression like intestinal type GC. In the present study, for the first time, we revealed that COX-2-1195 AA genotype was the independent risk factor of diffuse type GC in the prevalent area of GC.

Although the carcinogenesis of diffuse type GC in relation to COX-2 polymorphism is unknown, a few studies suggested the possible role of COX-2 polymorphism in gastric carcinogenesis. Liu et al.11 reported the significant association between COX-2 overexpression and the -1195 AA genotype in pre-malignant gastric lesions. Another study demonstrated that the -1195 A allele creates a C-MYB binding site, a transcriptional factor related in the regulation of the balance among cell division, survival, and differentiation,28 resulting in a higher transcriptional activity of the COX-2 gene.23 Additionally, COX-2 overexpression increases adhesion to extracellualr matrix proteins, and inhibits programmed cell death by reduction of transforming growth factor β2 receptor and E-cadherin levels, down-regulation of which is associated with diffuse-type GC.29-33 Taken together, COX-2 overexpression may be involved in intestinal epithelial cell differentiation, apoptosis, and local invasion of tumor cells.

However, the COX-2-1195 gene polymorphism in our study did not increase the overall risk of GC, which differs from the Chinese study.11 The relatively high frequency of COX-2-1195 AA genotype in our control group could explain the reason why the genotypic frequencies of COX-2-1195 were not different between control and GC group. In fact, the frequency of COX-2-1195 AA genotype in our control group (37%) was similar to that in GC group (35.5%) of the Chinese study. Considering that the frequency of COX-2-1195 AA genotype in our control group (37%) was higher than that of Chinese study (23.7%), it might be due to the different distribution between two populations. Although the number of our control group was relatively small, the frequencies of COX-2-1195 genotype in this study were very similar to the results from the large population based study of rheumatoid arthritis in Korea that included 1,000 subjects.34 Therefore, the present study suggests that a certain type of COX-2 polymorphism can be considered as a risk factor for diffuse-type GC in Korea.

The frequencies of COX-2-765 genotype were not different among the groups in our study. COX-2-765 C carriers have been reported to have 3-8 fold increased risk of GC in a few studies,10,12 while its polymorphism was not associated with GC in the other studies.11,13 These conflicts may come from the different genotypic frequencies among ethnic populations as well as the different environmental, dietary, and H. pylori virulent factors. For instance, only 5% of control group in our study and Chinese population had COX-2-765 C allele,11 while 22% in Portuguese10 and 16% in Northern Indian did.12

In this study, sub-analysis was carried out to investigate the interaction between H. pylori infection and COX-2 polymorphism. No significant difference, however, was observed in the distribution of genotypic frequencies of COX-2 polymorphism and status of H. pylori infection between control and GC. Liu et al.11 reported that H. pylori infected tissues could stimulate the host COX-2 expression much stronger in subjects with COX-2-1195 AA genotype than GG genotype. However, Saxena et al.12 described that COX-2-765 C carriers were susceptible to developing GC, which were out of relation to H. pylori infection. Therefore, the degree of the contribution of H. pylori infection, even though it is classified as a definite carcinogen, on the gastric carcinogenesis might vary according to ethnicity and prevalence of GC.35,36 Putting theses previous reports together, it is thought that interaction between H. pylori infection and the COX-2 polymorphism is not conclusive especially in the prevalent area of GC such as Korea. Interestingly, the prevalence of H. pylori was lower in GC than that of expected, though there was no statistical difference between control and GC group. This result can be explained by the results of some reports, which showed a high rate of false negative for detecting current H. pylori infection by rapid urease test or histology in atrophic and/or metaplastic gastric mucosa.37-39 Recent Korean study showed H. pylori-positive rates of 24% in antrum, 50% in gastric body by rapid urease test in metaplastic gastric mucosa.39

In conclusion, it suggests that COX-2-1195 AA genotype may make the subjects more susceptible to diffuse type GC in Korea. However, GC risk according to H. pylori infection and COX-2-1195 genotype frequencies was not conclusive, because there were many subgroups according to H. pylori infection status and COX-2-1195 genotypes, resulting too small number of subjects in each group to be compared with statistical significance. Considering the epidemiological, clinical, and genetic differences between intestinal and diffuse type GC, larger study including various host factors, bacterial factors, and ethnics will be warranted in the future.