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Cancer is the second leading cause of death after heart disease, accounting for 23% of all deaths in developed countries . With the emergence of increasingly sensitive and widely used diagnostic testing, cancer diagnosis has increased in the last decade. Known as a multifactorial disease, prostate cancer is considered one of the most frequently diagnosed tumours and second leading cause of cancer-related death amongst men . Racial and geographic differences, as well as age-associated changes, such as chronic inflammation and hormonal status may contribute to the aetiology . Notwithstanding, little is known about the aetiological factors in carcinogenesis and malignant progression.
Inflammation unalterably leads to oxidative stress producing reactive oxygen species (ROS). In addition, oxidative stress is an inevitable consequence of aerobic life, and there is growing understanding that the endogenous generation of toxic oxidative stress products may play a major role in age-related diseases, such as prostate cancer . The arrest or induction of transcription, the induction of signal transduction pathways, replication errors or genomic instability are the results of oxidative damage to nuclear DNA, which can result in carcinogenesis . ROS, such as superoxide radical (O2−), hydrogen peroxide (H2O2) and hydroxyl radical (OH) are capable of causing lipid peroxidation, altering the activity of antioxidant enzymes and damaging DNA . There is a dynamic equilibrium between the production of ROS and the antioxidant capacity of the cell . Oxidative stress occurs when ROS levels exceed the antioxidant capacity of a cell . To control the effects of ROS, aerobic cells have antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT) to fortify against oxidative stress.
MnSOD, encoded by the nuclear chromosome 6q25.3, has been identified as a tumour suppressor gene . It docks in the mitochondrial matrix in a dimeric or tetrameric form, catalysing the superoxide anion (O2−) into H2O2 and oxygen (O2) and is considered as one of the key enzymes involved in the antioxidant system in clinical disorders . The most frequently studied polymorphisms of MnSOD are Val16Ala and Ile58Thr. The Ile58Thr polymorphic locus is located in exon 3 and is reported to have an association with the activity of the SOD enzyme . Polymorphism at codon +58 has been reported to have association with breast cancer . To our knowledge, no data on prostate cancer and MnSOD Ile58Thr gene polymorphism has been reported to date.
CAT is an enzyme involved in ROS neutralising pathways participating in defence mechanisms against oxidative stress. CAT, encoded by the nuclear chromosome 11p13, controls intracellular concentration of H2O2 by converting it into H2O and O2 . Battisti et al.  reported a significant reduction of CAT activity in prostate cancer, implicating oxidative damage in prostate carcinogenesis. The variant T allele of the CAT C-262T gene polymorphism has been associated with lower enzyme activity compared with the C allele and thus, increased levels of ROS . Several studies have reported a relationship between CAT C-262T gene polymorphism and various types of cancers including prostate cancer  and breast cancer .
Myeloperoxidase (MPO), encoded by the nuclear chromosome 17q23.1, is a lysosomal enzyme located in neutrophils and monocytes and it produces a strong oxidant, hypochlorous acid (HOCl), for microbicidal activity [16, 17]. Through the release of ROS, MPO causes oxidative damage in vivo to biomolecules, e.g. DNA, protein and lipid, and causes cellular disintegrity that may lead to carcinogenesis . A MPO -463G to A substitution located in the consensus binding site of a SP1 transcription factor in the 5′-untranslated region, confers lower transcriptional activity (≈25 times) than the -463G common allele in vitro, due to disruption of the binding site and hence, lower inflammatory potential . There have been several epidemiological studies concerning MPO G-463A polymorphism and lung , breast , bladder  and oropharyngeal cancer  risk; however, only one study on prostate cancer risk has been reported to date . To our knowledge, there is no study investigating the association between the MnSOD Ile58Thr, CAT C-262T and MPO G-463A gene polymorphisms and prostate cancer progression and metastasis in Turkish men.
The objective of the present study was to investigate the influence of the MnSOD Ile58Thr, CAT C-262T and MPO G-463A gene polymorphisms on the susceptibility and clinicopathological characteristics; e.g. Gleason score, T stage, and metastasis of prostate cancer in a Turkish male population.
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The sample consisted of 155 patients with histologically diagnosed prostate cancer, at the Department of Urology, Istanbul Faculty of Medicine, Istanbul University, between 2008 and 2011. In all, 195 men with normal DRE and serum PSA levels of <4 ng/mL were included as controls. Informed consent was obtained from each participant and ethical approval was obtained from the Ethics Committee of Istanbul Faculty of Medicine.
All patients and controls were evaluated with a detailed medical history and physical examination. Additionally, body mass index (BMI) was calculated and smoking history was recorded. The serum PSA level was measured using an auto analyser Elecsys E170 (Roche Diagnostics, Basel, Switzerland). Serum PSA levels of ≥4 ng/mL or <4 ng/mL and with suspicious DRE were biopsied.
Furthermore, for the purposes of analysis, patients with prostate cancer were divided into different groups for each of the following parameters: pathological grade was recorded as the Gleason score and classified into two groups. Gleason score ≥7 was accepted as high and <7 as low grade. The tumour stage was defined according to TNM 2002 classification system and grouped into low (T1 and T2) and high (T3 and T4, M0 or M1) T-stage disease. The presence or absence of bone metastasis was determined by bone scanning of patients with prostate cancer with serum PSA levels of >10 ng/mL. There were 93 (60%) patients with low-grade (Gleason score <7) and 62 (40%) with high-grade (Gleason score ≥7) prostate cancer. The number of patients with prostate cancer showing low T stage (T1 and T2) and high T stage (T3 and T4, M0 or M1) was 115 (74.2%) and 40 (25.8%), respectively. Of these, 27 (17.4%) patients with prostate cancer had bone metastasis.
Blood samples from all study participants were collected in EDTA-containing tubes. Genomic DNA was extracted from peripheral whole blood with the use of commercially available PCR template preparation kit (Roche Diagnostics, Mannheim, Germany). The extracted DNA was stored at 4 °C until analysis. For the MnSOD Ile58Thr polymorphism, sense primer 5′-AGC TGG TCC CAT TAT CTA ATA G-3′ and antisense primer 5′-TCA GTG CAG GCT GAA GAG AT-3′ were used ; for the CAT C-262T polymorphism, sense primer 5′-ATT CCG TCT GCA AAA CTG GC-3′ and antisense primer 5′-GAG CCT CGC CCC GCC GGC CCG-3′ were used , for the MPO G-463A polymorphism, sense primer 5′-CGG TAT AGG CAC ACA ATG GTG AG-3′ and antisense primer 5′-GCA ATG GTT CAA GCG ATT CTT C-3′ were used . The PCR mixture contained 100 ng DNA, 10 x PCR buffer (pH 8.8), 20 pmol of each primer, 2 mm MgCl2, 200 μm of each dNTP and 1.25 U Taq DNA polymerase (MBI Fermentas). For the MnSOD Ile58Thr polymorphism, the reaction mixture was subject to 35 cycles of denaturating for 30 s at 94 °C, 30-s annealing at 60 °C and 30-s extension at 72 °C. The PCR product (140 base pairs [bp]) was digested with EcoRV restriction endonuclease. Digested products were separated by electrophoresis on 3% agarose gel and observed using ethidium bromide. Fragment patterns for MnSOD Ile58Thr genotypes were CC (140 bp), CT (140 bp, 117 bp), TT (117 bp). For the CATC-262T polymorphism the reaction mixture was subject to 35 cycles of denaturating for 30 s at 94 °C, 45-s annealing at 60 °C and 30-s extension at 72 °C. The 129 bp product was digested with Sma I restriction endonuclease. Fragment patterns for CATC-262T genotypes were TT (129 bp); CT (129 bp, 99 bp); CC (99 bp). For MPO G-463A polymorphism, the reaction mixture was subject to 34 cycles of denaturating for 30 s at 95 °C, 30-s annealing at 61 °C and 40-s extension at 72 °C. The 350 bp product was digested with Aci I restriction endonuclease. Fragment patterns for MPO G-463A genotypes were GG (169 bp, 120 bp, 61 bp), GA (289 bp, 169 bp, 120 bp, 61 bp), AA (289 bp, 61 bp).
Clinical laboratory data are expressed as the mean (sd). Clinical variables, e.g. age, BMI and PSA levels in patients and controls were compared by Student's t-test (for equal variances) or Mann–Whitney U-test (for unequal variances). Differences in the distribution of MnSOD Ile58Thr, CAT C-262T and MPO G-463A genotypes or alleles between patients and controls were tested using chi-squared analysis. The association of genotypes with tumour grade (low vs high) and pathological T stage (low vs high) and metastasis (metastasis – vs metastasis +) were also calculated by chi-squared test. Logistic regression was used to estimate age, BMI and smoking status adjusted odds ratio (aOR) with the corresponding 95% CI. Based on our sample size, the NCSS 2000 Statistical package (NCSS Inc; Kayswille, UT) was used to determine an effect size (w) of 0.20 using 2 degrees of freedom (2; 0.005) and the power of the study was calculated as 92%. A P < 0.05 was considered to indicate statistical significance.
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There were no statistically significance differences in demographic data and smoking status between the groups. As expected, total PSA levels were significantly elevated in patients with prostate cancer compared with the controls (Table 1).
Table 1. Demographic and clinical data of controls and patients with prostate cancer
|Variable||Controls||Patients with prostate cancer||P*|
|Mean (sd):|| || || |
|Age, years||62.8 (7.58)||64.3 (7.47)||0.069|
|BMI, kg/m2||26.3 (2.96)||26.6 (2.98)||0.438|
|PSA level, ng/mL||1.44 (1.55)||13.64 (16.8)5||<0.001|
|N (%):|| || || |
|Smoking status:|| || || |
|Yes||85 (43.6)||83 (53.5)|| |
|No||110 (56.4)||72 (46.5)||0.064|
|Gleason score:|| || || |
|<7|| ||93 (60.0)|| |
|≥7|| ||62 (40.0)|| |
|T stage:|| || || |
|T1+T2|| ||115 (74.2)|| |
|T3+T4|| ||40 (25.8)|| |
|Bone metastasis:|| || || |
|–|| ||128 (82.6)|| |
|+|| ||27 (17.4)|| |
The genotype distributions of MnSOD Ile58Thr, CAT C-262T and MPO G-463A gene polymorphisms were consistent with Hardy–Weinberg equilibrium in controls and patients with prostate cancer (P > 0.05). The TT genotype and T allele in the MnSOD Ile58Thr gene polymorphism, the CC genotype and C allele in the CAT C-262T gene polymorphism and the GG genotype and G allele in the MPO G-463A gene polymorphism were the predominant genotypes and alleles in the present population of Turkish men. There was no relationship between MnSOD Ile58Thr gene polymorphism and prostate cancer risk. However, there was a significant difference in the genotype distribution of CAT C-262T gene polymorphism between patients with prostate cancer and controls. Individuals carrying the TT genotype had a 1.57-fold increased prostate cancer risk (aOR 1.57, 95%CI 1.11–2.21; P = 0.010) compared with those carrying the CC genotype. Moreover, individuals with at least one T allele (CT and TT) of the CAT C-262T gene polymorphism had a 1.36-fold increased prostate cancer risk (aOR 1.36, 95%CI 1.09–1.71; P = 0.007) compared with those with the CC genotype. The frequency of the T allele in CAT C-262T polymorphism was also higher in patients with prostate cancer than in controls (aOR 1.88, 95%CI 1.37–2.58; P < 0.001). There was a significant difference in the genotype distribution of MPO G-463A gene polymorphism between patients with prostate cancer and controls. Individuals carrying the GG genotype had a 1.78-fold increased risk of prostate cancer (aOR 1.78, 95%CI 1.15–2.76; P = 0.009) compared with those carrying the AA genotype. Moreover, individuals with at least one G allele (GA and AA) of the MPO G-463A gene polymorphism had a 1.65-fold increased risk of prostate cancer (aOR 1.65; 95%CI 1.08–2.56; P = 0.020) compared with those with the AA genotype. The frequency of the G allele in the MPO G-463A gene polymorphism was also higher in patients with prostate cancer than in controls (aOR 1.65; 95%CI 1.18–2.30; P = 0.003) (Table 2).
Table 2. Genotype distribution and allelic frequency of MnSOD Ile58Thr, CAT C-262T and MPO G-463A polymorphisms between controls and patients with prostate cancer
| ||Controls, n (%)||Patients, n (%)||aOR (95% CI)*||P|
|MnSOD Ile58Thr|| || || || |
|TT||127 (65.1)||100 (64.5)||Reference|| |
|TC||64 (32.8)||50 (32.3)||0.98 (0.77–1.25)||0.895|
|CC||4 (2.1)||5 (3.2)||1.29 (0.65–2.57)||0.456|
|TC+CC||68 (34.9)||55 (35.5)||1.03 (0.82–1.30)||0.766|
|Allele|| || || || |
|T||318 (81.5)||250 (80.6)||Reference|| |
|C||72 (18.5)||60 (19.4)||1.06 (0.72–1.55)||0.764|
|CAT C-262T|| || || || |
|CC||107 (54.9)||58 (37.4)||Reference|| |
|CT||68 (34.9)||64 (41.3)||1.28 (1.00–1.64)||0.048|
|TT||20 (10.2)||33 (21.3)||1.57 (1.11–2.21)||0.010|
|CT+TT||88 (45.1)||97 (62.6)||1.36 (1.09–1.71)||0.007|
|Allele|| || || || |
|C||282 (72.3)||180 (58.0)||Reference|| |
|T||108 (27.7)||130 (42.0)||1.88 (1.37–2.58)||<0.001|
|MPO G-463A|| || || || |
|AA||28 (14.4)||12 (7.7)||Reference|| |
|GA||77 (39.5)||50 (32.3)||0.83 (0.65–1.06)||0.138|
|GG||90 (46.1)||93 (60.0)||1.78 (1.15–2.76)||0.009|
|GA+GG||167 (85.6)||143 (92.3)||1.65 (1.08–2.52)||0.020|
|Allele|| || || || |
|A||133 (34.1)||74 (23.9)||Reference|| |
|G||257 (65.9)||236 (76.1)||1.65 (1.18–2.30)||0.003|
MnSOD Ile58Thr and MPO G-463A gene polymorphisms were not associated with clinicopathological characteristics including pathological grade, T stage and metastasis among patients with prostate cancer (Data not shown). However, the CAT C-262T gene polymorphism was associated with clinicopathological characteristics including T stage and metastasis among patients with prostate cancer. The patients with prostate cancer carrying the TT genotype had a significantly higher risk of high pathological T stage disease than patients carrying the CC genotype (aOR 1.94, 95%CI 1.14–3.23; P = 0.014). The T allele was significantly associated with high pathological T stage when compared with the C allele (aOR 2.04, 95%CI 1.22–3.42; P = 0.005). Patients with prostate cancer carrying the TT genotype in the CAT C-262T polymorphism were more susceptible to developing metastasis than those carrying the CC genotype (aOR 3.83, 95%CI 1.75–6.59; P < 0.001). Moreover, patients with prostate cancer with at least one T allele (CT and TT) of CAT C-262T had a higher risk of developing metastasis as compared with the CC genotype (aOR 1.81, 95%CI 1.00–3.26; P = 0.047). The T allele was significantly associated with metastasis (aOR 4.23, 95%CI 2.23–8.09; P < 0.001) compared with the C allele (Table 3).
Table 3. Genotype distribution and allelic frequency of the CAT C-262T gene polymorphism according to the tumour grade, T stage and metastasis of the disease.
|CAT C-262T||Grade, n (%)||Stage, n (%)||Metastasis, n (%)|
|Low*||High†||aOR (95% CI)¶||P||Low‡||High§||aOR (95% CI)¶||P||–||+||aOR (95% CI)¶||P|
|CC||37 (39.8)||21 (33.9)||Reference|| ||46 (40.0)||12 (30.0)||Reference|| ||53 (41.4)||5 (18.5)||Reference|| |
|CT||42 (45.2)||22 (35.5)||0.86 (0.56–1.31)||0.488||52 (45.2)||12 (30.0)||0.82 (0.48–1.42)||0.491||58 (45.3)||6 (22.2)||0.90 (0.42–1.92)||0.791|
|TT||14 (15.0)||19 (30.6)||1.54 (0.94–2.52)||0.081||17 (14.8)||16 (40.0)||1.94 (1.14–3.23)||0.014||17 (13.3)||16 (59.3)||3.83 (1.75–6.59)||<0.001|
|CT + TT||56 (60.2)||41 (66.1)||1.06 (0.73–1.55)||0.743||69 (60.0)||28 (70.0)||1.23 (0.79–1.93)||0.348||75 (58.6)||22 (81.5)||1.81 (1.00–3.26)||0.047|
|Allelle|| || || || || || || || || || || || |
|C||116 (62.4)||64 (51.6)||Reference|| ||144 (62.6)||36 (45.0)||Reference|| ||164 (64.1)||16 (26.6)||Reference|| |
|T||70 (37.6)||60 (48.4)||1.55 (0.98–1.46)||0.060||86 (37.4)||44 (55.0)||2.04 (1.22–3.42)||0.005||92 (35.9)||38 (70.4)||4.23 (2.23–8.09)||<0.001|
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Progressive changes in cellular metabolism occurring over years may play a considerable role in the initiation and development of cancer . Inadequacy in the antioxidant defence system or excessive ROS production may cause oxidative stress leading to development of many cancers including prostate cancer . Information about the activities of antioxidant enzymes are conflicting in patients with cancer .
MnSOD acts as an antioxidant by scavenging oxygen free radicals, and its activity is induced by the amount of ROS . The known polymorphic sites in MnSOD include the ones in exon 2 (Val–9Ala) and exon 3 (Ile+58Thr) . In a meta-analysis, Mao et al.  indicated that the Ala/Ala or Val/Ala genotypes of the MnSOD gene polymorphism were associated with an increased risk of prostate cancer development. However, in another meta-analysis, Liwei et al.  found no significant association between the Val-9Ala gene polymorphism in the MnSOD gene and prostate cancer susceptibility. The MnSOD Ile58Thr polymorphism has been investigated in other cancer types but not prostate cancer. The MnSOD protein synthesised by the CC genotype has been reported to have half (in vitro) or one-third (in vivo) of the activity of the protein synthesised by the TT genotype, and the possible disadvantage of lower MnSOD protection against long-term oxidative damage. In the present study, there was no relationship between MnSOD Ile58Thr polymorphism and prostate cancer. Neither the TT nor CC genotype of MnSOD was associated with increased prostate cancer risk. Tombyln et al.  reported <1% of the C allele in a Caucasian study population, furthermore, Galecki et al.  demonstrated that the C allele was very rare in Caucasians. Consistent with previous studies, in the present study, the CC and TC genotypes of MnSOD polymorphism were found in 2.1% and 32.8% of the studied Turkish population, respectively. The T allele was reported to have higher tumour suppressive effects on human breast cancer than the C allele . Meanwhile, Kucukgergin et al.  reported a high risk of bladder cancer among Turkish patients carrying the Ala/Ala genotype of MnSOD. To our knowledge, the present study is the first investigating the relationship between the MnSOD Ile58Thr gene polymorphism and prostate cancer and no relationship was found. Similarly, Eeles et al.  identified single nucleotide polymorphisms (SNPs) in a genome-wide association study accounting for some portion of the familial predisposition to prostate cancer but have not reported risk of SNPs in antioxidant genes. Nevertheless, evaluation of this polymorphism in larger series would be important considering its behaviour in the biology of MnSOD.
CAT activity has been reported to be lower in patients with prostate cancer compared with controls . Botswick et al.  reported low levels of CAT in prostate cancer, leading to lower H2O2 detoxification, increased oxidative stress and implicating oxidative DNA damage in prostate carcinogenesis. The CAT CC genotype was reported to have higher enzyme activity compared with the CAT CT and TT genotypes and was associated with reduced risk of breast cancer in higher fruit and vegetable consumers . Ahn et al.  also showed lower enzyme activity of the TT and CT genotypes in the CAT C-262T gene polymorphism. The T allele of the CAT C-262T gene polymorphism was shown to protect against the development of asthma in non-smokers , diabetic neuropathy  and to have an association with hypertension . In the present study, we found a CC genotype predominance in Turkish men similar to Choi et al.  where 90% of the study group consisted of Caucasians. To our knowledge, Choi et al.  were the first to evaluate the association between the risk of prostate cancer and the CAT C-262T gene polymorphism. They did not find a notable effect of CAT genotypes overall in relation to prostate cancer risk but showed that the TT genotype of the CAT gene polymorphism was significantly associated with increased risk of prostate cancer among men diagnosed aged <65 years. However, in the present study, we found a higher risk of prostate cancer in patients with the TT genotype of the CAT gene polymorphism irrespective of age. In addition, patients with the TT genotype had significantly higher stage disease compared with those with the CC genotype. Furthermore, the present study showed a higher risk of bone metastasis of the TT genotype compared with the CC genotype of the CAT gene polymorphism. Therefore, the TT genotype may have a major role in the progression of prostate cancer. It is rational to conclude that the lower activity of CAT in carriers with the TT genotype may explain its risk and susceptibility to advanced and metastatic prostate cancer.
CAT is the key enzyme reducing H2O2 to H2O, thus the lower the CAT activity, the higher the H2O2 concentration. It has been suggested that in the presence of high H2O2 concentrations, the step of nicotinamide adenine dinucleotide phosphate (NADP) reduction becomes rate limiting as the decomposition rate decreases by 100 times . If not catalyzed, the H2O2 participates in the Fenton reaction to form OH radicals, which can damage DNA and may promote carcinogenesis . Moreover, OH. radicals may activate some oncogenes, further contributing to cancer development . Additionally, high H2O2 concentrations upregulate matrix metalloproteinases, proteins responsible for extracellular matrix degradation, increasing the metastatic potential . The TT genotype of CAT confers lower activity and thus increases the H2O2 concentration, which may promote aggressiveness and metastatic potential. Furthermore, the TT genotype of the CAT gene polymorphism may be considered as a molecular marker of more aggressive disease.
The MPO enzyme is found in primary granules of neutrophils and monocytes and functions as an oxidative antimicrobial agent by catalyzing the generation of genotoxic HOCl and other ROS . MPO gene polymorphism has been studied most commonly in relation to lung cancer [20, 45]. Feyler et al.  reported a reduction in the risk of lung cancer for carriers of the homozygous AA variant. However, Ambrosone et al.  reported better survival in patients with the GG genotype after treatment of breast cancer. In a study investigating the risk of pancreatic adenocarcinoma, the A allele was associated with a lower risk of pancreatic cancer . Similarly, Zhu et al.  reported a protective effect of the A allele in gastric cancer relative to the GG genotype. Furthermore, Ahn et al.  found more pronounced radiotoxicity of the GG genotype of MPO among women receiving radiation therapy after lumpectomy for breast cancer. In addition, Hung et al.  reported a protective effect of the AA genotype of the MPO gene polymorphism on bladder cancer risk. There were similar findings in the present study, with a 1.78 fold increased prostate cancer risk of the GG genotype. Whereas, Choi et al.  reported no association of prostate cancer risk overall and a 2-fold reduced risk of the AA genotype among men with aggressive prostate cancer compared with the GG genotype of the MPO gene polymorphism. Notwithstanding, in the present study, there was no association regarding high stage and metastatic prostate cancer risk after adjustment for age, BMI and cigarette smoking. This inconsistency between the two studies may be attributed to environmental and genetic differences among the cohorts. A meta-analysis of MPO G-463A polymorphism and cancer risk has suggested that there may be no significant association of MPO G-463A polymorphism when stratified by cancer type . MPO produces HOCl from H2O2 and chloride anions . HOCl reacts with other biological molecules to generate secondary oxidation products, thus increases oxidative stress . Additionally, HOCl may cause DNA damage and lead to the mutation of oncogenes and tumour suppressor genes . It has been suggested that the A allele confers lower transcriptional MPO activity and thus lower HOCl production as compared with the G allele . Therefore, the G allele producing more HOCl may increase metabolic activation of procarcinogens and thus lead to the development of prostate cancer. The present study showed an increased risk of the GG genotype of the MPO gene polymorphism but no association with aggressiveness and metastatic potential in patients with prostate cancer.
In conclusion, the GG genotype in the MPO G-463A polymorphism and the TT genotype in the CAT C-262T polymorphism may be considered as risk factors for the susceptibility to prostate cancer. Additionally, the TT genotype in the CAT C-262T gene polymorphism may be associated with prostate cancer progression and metastasis among Turkish men. Further studies are needed to confirm the results of the present report.