Manganese Superoxide Dismutase ( MnSOD Val-9 Ala ) Gene Polymorphism and Susceptibility to Gastric Cancer

Gastric cancer (GC) is one of the most important diseases that affected health in all over the world (Zare et al., 2013; Lin et al., 2014). Gastric cancer is the most general deadly cancer with approximately 738,000 deaths per year that over 70% of them occur in the developing countries (Jemal et al., 2011). Northern and northwestern regions of Iran are high risk areas for gastric cancer (Fallah, 2007; Moradi et al., 2013). Different frequency of gastric cancer in worldwide can be due to variation in the genetic background, dietary habits, living situation and also the prevalence of Helicobacter pylori (H. pylori) infection which has been classified as Group I carcinogens (Humans, 1994). H. pylori infections can induce the host inflammatory responses and oxidative stresses. Interleukin-8 is induced by the gastric epithelium cells that infected by H. pylori, contributes to the production of too much amounts of toxic reactive oxygen species (ROS) and can causes induction of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and some other interleukins (Augusto et al., 2007a). Oxidative stress that caused by ROS is involved in human carcinogenesis (Cerutti., 1985). ROS, as candidate agents in the growth of cancer, generated in normal respiration of cells and during xenobiotics metabolism, causes damage to cell membranes, mitochondria and DNA molecule. (Egan et


Introduction
Gastric cancer (GC) is one of the most important diseases that affected health in all over the world (Zare et al., 2013;Lin et al., 2014).Gastric cancer is the most general deadly cancer with approximately 738,000 deaths per year that over 70% of them occur in the developing countries (Jemal et al., 2011).Northern and northwestern regions of Iran are high risk areas for gastric cancer (Fallah, 2007;Moradi et al., 2013).Different frequency of gastric cancer in worldwide can be due to variation in the genetic background, dietary habits, living situation and also the prevalence of Helicobacter pylori (H.pylori) infection which has been classified as Group I carcinogens (Humans, 1994).H. pylori infections can induce the host inflammatory responses and oxidative stresses.
Interleukin-8 is induced by the gastric epithelium cells that infected by H. pylori, contributes to the production of too much amounts of toxic reactive oxygen species (ROS) and can causes induction of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and some other interleukins (Augusto et al., 2007a).Oxidative stress that caused by ROS is involved in human carcinogenesis (Cerutti., 1985).ROS, as candidate agents in the growth of cancer, generated in normal respiration of cells and during xenobiotics metabolism, causes damage to cell membranes, mitochondria and DNA molecule.(Egan et  ROS can induce or enable permeabilization in mitochondria, whereas glutathione and antioxidant enzymes reduce it.Some of antioxidant systems are involved in the ROS scavenging, such as the superoxide dismutase or SOD family that catalyzed the dismutation of the superoxide anion to form hydrogen peroxide (H 2 O 2 ) (Ambrosone et al., 2005).Among SOD family, only manganese superoxide dismutase (MnSOD) is essential for life that as the major antioxidant has a function against ROS or free radicals in the human mitochondria (Sun et al., 1937;Attatippaholkun and Wikainapakul., 2013).The MnSOD protein precursor, with a cleavable mitochondrial targeting sequence or MTS at N-terminal, is synthesized in the cell cytoplasm (Macmillan-Crow and Cruthirds., 2001).A polymorphism in MnSOD (rs4880) gene at the codon 16, that is located at position _9 in the complete protein and results in the substitution of either alanine (C allele) or valine (T allele) in the mitochondrial targeting sequence.This gene polymorphism of MnSOD is predicted to change the MnSOD secondary structure and may influence the efficiency for MnSOD mitochondrial transport.(Shimoda-Matsubayashi et al., 1996) Recent studies manifested that the Ala form of MnSOD is targeted into the mitochondria, whereas the Val-containing protein is partially arrested in the inner-mitochondrial membrane.(Sutton et al., 2003) However, the association between polymorphisms of the related candidate genes with gastric cancer in different ethnicities has been studied; according to our knowledge, there has been no reported data on the possible association between the polymorphism of MnSOD and gastric cancer among Iranian populations.Thus, the aim of current study was to investigate the MnSOD Val-9Ala genotype distribution and its association with gastric cancer among a sample of the Iranian population for the first time.

Enzyme and reagents
The GPP DNA isolation kit was purchased from Gen Pajohan, Iran.BSaW I as a restriction enzyme, agarose and polymerase chain reaction (PCR) materials were purchased from Fermentas.Specific primers were synthesized from Cinnaclon, Iran.The other chemicals with analytical grade were from Merck.

Participants
Gastric biopsies were taken from 52 gastric cancer patients with the mean age of 67.8±8.8 years and 100 cancer-free with the mean age of 64.2±5.1 years as controls.Control groups and patients were age and sex matched (Table 1).All gastric cancer patients were from the north and northwest provinces of Iran who admitted to the medical sciences clinics between May 2011 and June 2012.All of the patient's samples got before therapeutic actions.The gastric cancer diagnosis was according to standard clinical, radiological and histological parameters and results were analyzed by two experienced endoscopists.All procedures of recent research were accepted and approved by the ethics committee in Kermanshah University of Medical Sciences (Iran).Individuals who contributed to this study signed an informed consent in accordance with the Helsinki II declarations.All patients were informed about the aim and procedures of the research.

DNA isolation
Genomic DNA was isolated from endoscopic fresh biopsy samples.Tissue specimens were suspended in 550µl of DNA extraction buffer (2mM EDTA, 10 mM Tris-HCl (pH 8.0 and 400mM NaCl).The suspension was incubated at 20-30 ºC for 20 min, following the addition of Proteinase K and SDS (400ng and 0.6%, respectively).Then the solution was incubated at 55ºC for 15h and DNA was extracted with the GPP isolation kit (Gen Pajoohan, Iran).Isolated DNA was stored at -20 °C until use.

Quantification of extracted DNA
Isolated DNA was observed and confirmed by gel electrophoresis on 1% agarose containing DNA Safe Stain (Invitrogen).The concentration and purity of extracted DNA were analyzed using Nanodrop (Thermo) at wavelength of 260 and 280 nm and measurement of 260/280 ratio, respectively (Yari et al., 2010;Moradi, 2014).

Analysis of MnSOD Val-9Ala genotypes
The MnSOD Val-9Ala polymorphism was assessed by PCR amplification and then RFLP analysis.The specific oligonucleotide primers used to amplify as follows: 5'-CGGGCTGTGCTTTCTCGTC-3' (forward) and 5'-TCAGCCTGGAACCTACCCTT-3'(reverse).PCR amplification was carried out in a total volume of 25µl containing 500 ng of genomic DNA as template, 1x PCR reaction buffer, MgCl2 with 1.5 mM, 0.2 mM of dNTPs, 0.5 µM of each forward and reverse primers and 2 units of Taq DNA polymerase (Fermentase, USA).
Thermocycler parameters comprised of first denaturation step at 95°C for 7 min, 35 cycles of denaturation at 95°C for 45 s, annealing at 58°C for 45 s , extension at 72°C for 45 s, and then a final extension at 72°C for 5 min.
After amplification, 12µl of PCR products were subjected to overnight digestion with 10 units of BSaW I (New England Biolabs, Beverly, MA, USA) at 60°C.Digested PCR products were shown on a 2% agarose gel stained with Syber safe DNA under ultraviolet light.The patterns of the bands were as follows: 243 bp for Ala/Ala homozygotes; 243, 196, and 47 bp for Ala/Val heterozygotes; 196, and 47 bp for Val/Val homozygotes.5% of the samples were randomly selected and regenotyped for quality control purposes.

Statistical analysis
All of statistical studies were carried out with statistical software of SPSS (Version 16).Diversity in genetic distributions between control and patient groups was analyzed by Pearson's chi-square (χ 2 ) assay.95% of confidence intervals (95% CI) and Odds ratios (OR) were calculated with an unconditional logistic regression model.Results of this study were considered with statistically significant when p<0.05.

Results
Some of the personalities of the gastric cancer patients are shown in Table 1.52 gastric cancer patients and 100 healthy individuals as a control group participated in this study.The control group, 62% men and 38% women with the mean age of 64.2±5.1 years), were free from cancer signs.Further, the gastric cancer group consisted of 42.3% women and 57.7% men with the mean age of 67.8±8.8 years.There was no significant difference between patients and controls in terms of age and sex.In all participants, DNA was isolated by the GPP DNA extraction kit as previously mentioned, and quantity and quality of  Distribution of MnSOD genotypes and alleles in gastric cancer patients and controls is shown in Table 2.The frequency of MnSOD Ala/Ala, Ala/Val and Val/Val genotypes in healthy individuals were 24.3, 66.7 and 9%, respectively.However, in Gastric cancer patients, Ala/Ala, Ala/Val and Val/Val with 24, 48 and 28% were observed (p=0.01).In patients the frequency of MnSOD Val allele was higher (52%) compared to that in controls (42%).In both of patient and control groups, the frequencies of MnSOD genotypes were in Hardy-Weinberg equilibrium.

Discussion
The current research in a homogenous population of gastric cancer patients from north and northwest of Iran reports a significantly higher frequency of Val allele compared to that in healthy individuals that increased the risk of gastric cancer.Gastric cancer is the most frequent malignancy diagnosed in worldwide and it is the most common deadly cancer in Iran.Northern and northwestern regions in Iran are high risk areas for gastric cancer.Epidemiologic studies have reported numerous risk factors for gastric cancer, including environmental, genetic factors, adverse living conditions, dietary habits and the prevalence of Helicobacter pylori (H.pylori) infection (Moges et al., 2006).
Reactive oxygen species (ROS), generated during metabolism of xenobiotics and in normal cellular respiration, causes damage to membranes, mitochondria, and macromolecules including DNA, and thus are candidate agents in the development of human cancer (Margaret et al., 2011).MnSOD is the important antioxidant agent in the mitochonderia that catalyzes the dismutation of superoxide radicals to hydrogen peroxide form and detoxification of mitochondrial ROS (Sun et al., 2012).The MnSOD gene as tumor suppressor gene is located on chromosome 6 at q25.3.A number of polymorphisms have been reported in the MnSOD gene that some of them are associated with the increased risk of human cancers.Recent studies confirmed functional polymorphism of the Val-9Ala MnSOD that Val allelecontaining precursor protein with beta-sheet conformation exhibited impaired transportation, but Ala-containing precursor with alpha helical conformation showed normal transportation.Therefore, the variant allele (Ala) with higher activity suppresses carcinogenesis.MnSOD polymorphic alleles are widely variable with ethnicity, the frequency of Ala allele is 12% among Japanese (Shimoda-Matsubayashi et al., 1996) and 14% in Chinese (Cai et al., 2004), while it is more general (41-55%) in the population of Caucasian (Ambrosone et al., 1999) and 41% in Jordanian breast cancer population.
There are several available data to investigate the role of Val-9Ala MnSOD variants in susceptibility to gastric cancer.Similar to our study, Atoum et al. (2012) observed that the Val/Val allele of MnSOD Val-9Ala was correlated to a significantly decreased risk for breast cancer disease in Jordanian population.Also, Augusto et al. (2007b) indicated that gastritis was characteristic by an oxidative stress with significant absence of MnSOD and GPX expression.In meta-analysis that reported by Wang suggest that the MnSOD Val-9Ala polymorphism may contribute to cancer development through a disturbed antioxidant balance (Wang et al., 2009).
In conclusion, in a high-risk gastric cancer area, we examined the relationship between MnSOD Val/Ala polymorphism with the risk of gastric cancer.Our results of this research showed that there was a significant positive association between the distribution of the MnSOD gene polymorphism and gastric cancer disease.In addition, the results show that the polymorphism of MnSOD Val-9Ala is an important risk factor that is associated with gastric cancer in sample of the Iranian population.However, further research with a larger sample size is required to confirm these findings.The present study that reported here is the first published research on the correlation between the MnSOD genotypes and the susceptibility to gastric cancer disease in the Iranian population.This study can be used as a basis for studying polymorphisms of other important genes in correlation to gastric cancer.
extracted DNA were evaluated by spectrophotometric assay and gel electrophoresis (result not shown).To investigate the gene polymorphism, PCR-RFLP was carried out.

Figure 1 .
Figure 1. 2% Agarose Gel Electrophoresis of Digested PCR Product by BSaW I Restriction Enzyme.Lane 1 and 3 Show Mutant Genotype, Lane 2 and 5 Demonstrate the Wild Genotype.The 50 bp DNA Molecular Weight Marker is Indicated in lane 4