Abstract
Background: Cytokine-mediated immune and inflammatory responses are considered to play an important role in the pathogenesis of prostate cancer. The present study investigated certain interleukin-1β (IL1β) polymorphisms and their association with prostate cancer. Materials and Methods: Genotyping of the IL1B-31(rs 1143627 G>A) and IL1B-511(rs 16944 A<G) polymorphisms were performed using real-time polymerase chain reaction for blood samples from a group of patients with prostate cancer (n=71) and controls (n=76). Results: The frequency of the IL1B-31(rs1143627) AG heterozygote genotype was found to be significantly lower in patients compared to controls (odds ratio=0.648, 95% confidence interval=0.463-0.908; p=0.036). The genotypic frequencies of IL1B-511(rs16944) between prostate cancer patients and controls were statistically significantly different (p=0.001). The frequency of AG genotype for IL1B-511(rs16944) was 0.5-fold lower in patients with prostate cancer than in the controls (odds ratio=0.546; 95% confidence interval=0.377-0.791; p=0.001). Conclusion: Our data show that individuals carrying the IL1B-31(rs1143627) and IL1B-511(rs16944) AG genotypes had a decreased risk for developing prostate cancer. Out of all the possible combinations analyzed, IL1B-31(rs1143627) G with IL1B-511(rs16944) G combination had a protective association with prostate cancer.
Prostate cancer is the most common malignancy and the second leading cause of cancer-related death among men in the world (1). Twin studies showed that up to 50% of the risk of prostate cancer may be explained by genetic factors (2). However, common genetic polymorphims are likely to play an important role in sporadic cases of prostate cancer (3).
Early identification of patients at high risk of progression could be useful to anticipate therapy. The most important prognostic markers of prostate cancer are Gleason score, preoperative serum prostate-specific antigen (PSA) and pathological stage (4). Some predictors for biochemical recurrence exist. However, the accurarcy of prediction could be improved by incorporating new prognostic markers into clinical trials. Evidence from epidemiological, histopathological, genetic and molecular research support the existence of a relation between inflammation and prostate cancer (5).
Cytokine-mediated immune and inflammatory responses have been consired to play an important role in the pathogenesis of prostate cancer (6). Interleukin-1β (IL1B) is a 17-kDa cytokine member of the interleukin-1 family, which is involved in inflammation and immunity. It is produced by B-lymphocytes, macrophages, endothelial cells, fibroblast, dendritic cells. The IL1B gene is located on chrosome 2q14 and its expression has been found to be associated with various cancer types (7). IL1B polymorphisms are associated with an increase in the production of IL1B when compared to the wild-type gene (8). IL1B polymorphisms have also been linked to several kinds of malignant tumors, such as gastric cancer (9), hepatocellular cancer (10) and lung cancer (11).
Prostate cancer cases have decreased in the past decade because of practical screening methods for PSA. PSA is a 33-kDa glycoprotein containing a polypeptide of 240 amino acids that is mainly synthesized by the epithelial cells of the prostate gland (12). Serum PSA levels can be increased in benign prostatic cases because of structural disorders of the prostate; it has been shown that higher levels of PSA are found in malignancy (13). PSA is a useful diagnostic marker for patients with prostate cancer. Although PSA has been used as tumor marker for a long time, its role in pathologies has not been precisely determinated. Recent evidence suggests that genetic variations may contribute to individual variation in serum PSA levels. However, genetic variations associated with PSA levels have not been identified (14).
To the best of our knowledge, there is a lack of studies reporting a relation between prostate cancer and IL1B -31(rs 1143627) and IL1B -511(rs 16944) polymorphism. In this study, we investigated the associations between IL1B -31 and IL1B -511 polymorphisms and prostate cancer in a Turkish population.
Materials and Methods
Patient selection. Our study included 71 patients with prostate cance (mean age=67.61±7.34 years) who were treated at the Department of Urology, Yeditepe University Hospital and Göztepe Research and Education Hospital. The diagnosis of prostate cancer was confirmed by clinical, laboratory and pathological examinations. The tumor differentiation status was evaluated using Gleason score criteria. The clinical T-stage was classified as early-stage (T1 and T2) and late-stage (T3 and T4) by clinical examinations. Pathological T-stage was classified as T2a, T2b, T2c, T3a and T3b. A total of 76 age-matched controls (mean age=67.53±8.77 years) were selected from prostate cancer-negative patients of Urology Clinics of the same hospitals. Clinical parameters (body mass index, serum PSA level, smoking habit) for each participant were collected from the hospital records. Serum samples were taken after obtaining informed consent and the study was conducted prospectively. Local Ethical Committee approval was obtained for the study (protocol no: 63/505). The protocol followed was consistent with the World Medical Association Declaration of Helsinki (Ethical Principles for Medical Research Involving Human Subjects).
DNA extraction. Blood samples from all participants were collected in tubes containing ethylene diamine tetra acetic acid (EDTA). Genomic DNA was extracted from 350 μl peripheral whole blood using Invitrogen iPrep PureLink gDNA blood isolation kit with a iPrep Purification Instrument (Invitrogen, Life Technologies, Carlsbad, CA, USA). The isolation procudure was performed in a closed system and took 45 min; 100 μl of DNA was obtained at the end of the procedure. Consequently, sample DNA concentrations (mean=80±9.98 ng/μl) and optical density ratios (at 260/280 nm) (mean=1.9±0.2) were measured by Nanodrop 2000 (Thermoscientific, Waltham, MA, USA). İsolated DNA samples were preserved at 4°C until genotyping assessments were conducted.
Genotyping. Analysis of IL1B-31 (rs 1143627) and IL1B-511 (rs 16944) were performed by Taqman Allelic Discrimination Assay (Applied Biosystems, Foster City, CA, USA). The TaqMan probes were designed using the Primer Express software (Applied Biosystems). Allelic discrimination analysis with real-time PCR was performed using fluorescein amidite (FAM) for wild-type allele and VIC fluorescent probes for mutant allele. The labeling and sequences of the probes and primers were: IL1B -31: 5-(6FAM)-TTTGAAAGCCATAAAAA-3 (probe 1), 5-VIC-TTTGAAAGCTA TAAAAACAG-3 (probe 2), forward primer: 5-CCCCTTTCCTT TAACTTGATTGTG-3 and reverse primer: 5-AGGTTTGGTATCTG CCAGTTTCTC-3; L1B-511: 5-(FAM)-AGAGCTCCCGAGGCA-3 (probe1), 5-(VIC)-AGAGCTCCTGAGGCAG-3 (probe 2), forward primer: 5-CAGCCAAGAAAGGTCAATTTTCTC-3 and reverse primer: 5-CTGTATTGAGGGTGTGGGTCTCTAC-3. The reaction mix included 20 ng genomic DNA, 10 pmol of each primer, 2 pmol of each probe, and 10 μl of 2X master mix (Applied Biosystems) in a final volume of 20 μl. The thermocycling included 40 cycles with 30 s at 95°C followed by 60 s at 60°C. The PCR reactions were performed on a 7500 Fast Real-Time PCR System (Invitrogen, Life Technologies) (11).
Statistical analyses. Statistical analyses were performed using SPSS version 21 software (SPSS Inc, Chicago, IL, USA). Values are given as the mean±standard deviation (SD). Student's t-test was used to examine the significance of differences between the two groups and χ2 and Fisher's exact tests were used to compare demographic information to expression.
The genotypic distributions in all groups was tested for deviation from Hardy–Weinberg equilibrium (HWE) using the goodness-of-fit test. Linkage disequilibrium (LOD) between variations was assessed using D0 and r2 values obtained through the Haploview program (http://www.broad.mit.edu/mpg/haploview/(documentation.php). Multivariate logistic regression analysis was used to study the effect of each genotype on the risk of prostate cancer. The relative risk at a 95% confidence interval (CI) was calculated as the odds ratio (OR). Individuals homozygous for the common genotype were used as the reference to test for any association of genotype with prostate cancer by employing logistic regression model to calculate the OR with 95% CI, p-values lower than 0.05 denoted statistical significance.
Results
The demographic characteristic of control and patient groups are given in Table I. The mean age of patients with prostate cancer and healthy controls were 67.61±7.34 and 67.53±8.77 years, respectively. No significant differences were found between patients and controls in terms of median age (p>0.05).The patient group had a higher level of PSA (p=0.006) when compared to the control group.
The allelic and genotypic frequencies for IL1B-31 (rs1143627) and IL1B-511 (rs16944) polymorphisms in patients with prostate cancer and controls are given Table II. Firstly, we evaluated the association between IL1B-31 (rs1143627) polymorphism and study groups. The frequency of the AG genotype was found to be significantly lower in patients compared to controls (χ2=6.638, p=0.036; OR=0.648, 95% CI=0.463-0.908, p=0.008). At the same time, IL1B-511 (rs16944) genotypic frequencies in patients with prostate cancer and controls were statistically significantly different (χ2=13.368, p=0.001). The frequency of the IL1B-511 (rs16944) AG genotype was 0.5-fold lower in patients than in the controls (OR=0.546, 95% CI=0.377-0.791; p=0.001).
The genotypic distributions of IL1B-31 (rs1143627) and IL1B-511 (rs16944) polymorphisms were consistent with the Hosmer and Lemeshow test. Multivariate logistic regression analysis was performed to correct the OR for the genotype to examine whether the rs1143627 AG and rs16944 AG heterozygote genotypes are independently associated with a reduced risk of prostate cancer (Table III).
We also analyzed frequencies of haplotypes of different combinations to evaluate the synergistic effect on prostate cancer. The results are shown in Table IV. The frequency of the haplotype consisting of the combination of IL1B-31 (rs1143627) G and IL1B-511 (rs16944) G alleles was significantly lower in the patients than in the control group (Table IV). Conversely, the frequency of the combination of IL1B-31 (rs1143627) G and IL1B-511 (rs16944) A alleles was significantly higher in the patients than in the control group (Table IV).
Distributions of IL1B-31 (rs1143627) and IL1B-511 (rs16944) genotypes according to stage of prostate cancer were examined. There were no significant differences in IL1B-31 (rs1143627) and IL1B-511 (rs16944) genotypic frequencies by histological tumor stage (p=0.976 and p=0.877, respectively) (Table V).
Discussion
Epidemiological, genetic and molecular studies have shown that there is a strong relation between inflammation and prostate cancer (6). However it is reasonable to assume that sequence variants in genes coding pro-inflammatory and anti-inflammatory cytokines effect prostate cancer risk, since cytokine gene polymorphisms may lead to an altered production of cytokines (5, 8). IL1 is a major pro-inflammatory cytokine which has been proposed as a molecular link between inflammation and cancer (5). IL1B polymorphisms have been investigated for association with several cancer types. It has been shown that IL1B polymorphisms (IL1B-31 and IL1B-511) are associated with increased risk of gastric cancer (9).
In our study, we found the IL1B-511 (rs16944) G allele was statistically more frequently found in the prostate cancer group (p=0.012). Wang et al. showed that IL1B-511 mutant allele, associated with high IL1B production, in the liver is a genetic marker for the development of hepatocellular carcinoma (10). Our data confirm those of Zienolddiy et al. They found that IL1B-511 (rs16944) mutan allele and IL1B-31 (rs1143627) mutant allele were statistically more frequent in the lung cancer group (11). In spite of a negative effect of carrying mutant alleles, our study showed that the AG heterozygote genotypes of IL1B (rs1143627) and (rs16944) are independently associated with reduced risk for prostate cancer (Table II).
The functional role of IL1B-31 polymorphisms are far from being clear but Zienolddiy et al. implied that the mutant allele is a pro-inflammatory allele (11). Another study also showed that the IL1B-31 mutant allele is associated with breast cancer because of the pro-inflammatory effect (15). The DNA sequence properties of IL1B-31 polymorphisms indicate the importance of IL1B-31 mutant allele. This allelic polymorphism is a TATA-box polymorphism that has been shown to affect DNA–protein interactions in vitro, hence modulating IL1B gene expression (9). Location of mutant nucleotide at this site will impair the TATA-box, which changes the affinity of regulatory proteins in binding to this sequence. Generally, the presence of a TATA-box in promoter regions is characteristic of an inducible gene. The presence of a wild-type allele at this site may reduce characteristics of the IL1B gene, leading to decreased inflammatory reaction (8, 11). Therefore, the mutant allele might be associated with higher transcription of IL1B, and inflammation.
The genotypic distributions of IL1B-31 (rs1143627) and IL1B-511 (rs16944) were also evaluated by multivariate logistic regression analysis as shown in Table III. A meta-analysis found that IL1B-31 (rs1143627) polymorphism was weakly significant under a heterozygote model in prostate cancer (16). From our results, the frequency of the IL1B-31 (rs1143627) AG genotype was found to be significantly lower in patients compared to controls, heterozygosity seemed to be protective aganist prostate cancer for both SNPs.
Among all the possible combinations analyzed here, IL1B-31 (rs1143627) G and IL1B-511 (rs16944) G combinationhad a protective association with prostate cancer. A number of studies mostly in Caucasian populations support that IL1B-511 wild-type allele in linkage disequilibrium with IL1B-31 mutant allele carriers is a risky haplotype for the development of gastric cancer (9) and hepatocellular carcinoma (10).
In conclusion, our present data show that individuals carrying the IL1B-31 (rs1143627) and IL1B-511 (rs16944) AG genotypes were at a decreased risk for developing prostate cancer. Among all possible combinations analyzed, IL1B-31 (rs1143627) G and IL1B-511 (rs16944) G combination protected against developing prostate cancer. To the best of our knowledge, the data acquired in this study highlight, for the first time, the association of IL1B gene variants with prostate cancer in a Turkish population.
- Received July 30, 2015.
- Revision received September 15, 2015.
- Accepted September 25, 2015.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved