Low Virulence of Helicobacter Pylori and Gastric Cancer: the Contribution of Polymorphisms of iNOS and DNA Repair Enzymes in this Process

Special Article- Stomach cancer

Austin J Gastroenterol. 2014;1(4): 1019.

Low Virulence of Helicobacter Pylori and Gastric Cancer: the Contribution of Polymorphisms of iNOS and DNA Repair Enzymes in this Process

Isabelle Joyce de Lima Silva-Fernandes, Ana Paula Santos do Carmo, Débora Menezes da Costa, Emanuele Silva de Oliveira, Eliane dos Santos Pereira and Silvia Helena Barem Rabenhorst*

Department of Pathology and Forensic Medicine, Federal University of Ceará, Brazil

*Corresponding author: : Rabenhorst SHB, Department of Pathology and Forensic Medicine, School of Medicine, Federal University of Ceará, Coronel Nunes de Melo, 1315, Rodolfo Teófilo, Fortaleza, CE CEP 60430-270, Brazil

Received: August 06, 2014; Accepted: September 08, 2014; Published: September 10, 2014


This study aimed to investigate the interaction between the iNOS C >T polymorphism, the genotype of H. pylori strains and polymorphisms in DNA repair genes (OGG-1, APE-1 and PARP-1) in a set of gastric cancer patients. In our methods, polymorphism was assessed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and H. pylori detection/ genotyping by PCR. A significant result shows this iNOS polymorphism more frequent among young gastric cancer patients than older patients (p= 0.020). Host genetic features, such as DNA repair enzymes (OGG-1, APE-1, PARP- 1), are also important in preventing the malignancy process. Polymorphisms present in these enzymes associated with iNOS activity could lead to gastric cancer even in the presence of low virulent H. pylori strains. Within our results, iNOS homozygous wild-type (CC) genotype and APE-1 polymorphic allele (TG+GG) group were more infected by H. pylori low-virulent strains (p=0.021). In Conclusion, our study indicates the importance of H. pylori and host DNA repair enzymes genotypes in gastric carcinogenesis in interection with this specific iNOS polymorphism.

Keywords: Gastric cancer; Helicobacter pylori; PARP-1; APE-1; OGG-1


Gastric cancer is the fifth most common cancer and the second leading cause of cancer-related mortality in the world [1]. In Brazil, it is an important cause of cancer-related death in patients, with a high prevalence in the Northeast region [2]. According to Lauren’s classification, the histological subtypes, intestinal and diffuse, show distinct histological and epidemiological features, as well as a different prognosis [3,4]. Furthermore, this tumor can be located in the proximal stomach (cardia), or distal (antrum, non-cardia). Differences between tumors located in the cardia or non-cardia region, as well as intestinal or diffuse subtypes, suggests that they represent distinct diseases with different etiologies. Gastric carcinogenesis is a multifactorial process, and Helicobacter pylorus (H. pylori) is the main initiator of inflammation and atrophic changes in the gastric mucosa [5]. The association between chronic H. pylori infection and the development of gastric cancer is well established [6]. It is known that both bacterial virulence and host genetic susceptibility are associated with cancer risk [7].

H. pylori has a great genetic diversity, and virulence factors play important roles in mucosal injury, especially the genes cagA (cytotoxin associated gene A) and vacA, (vacuolating cytotoxin A), more specifically vacAs1m1. cagA is involved in many host cell alterations and tightly associated with gastric cancer risk [8,9]. The vacA gene is present in essentially all H. pylori strains. VacA is a potent toxin, where it induces the formation of vacuoles in host cells. Additionally, the cagE and virB11 genes have been found at a relevant frequency in gastric cancer patients [10]. The chronic inflammatory process in the presence of H. pylori increases the expression of iNOS which could lead to cell injury, with large amounts of NO leading to DNA lesions [11,12]. The DNA damage caused by ROS can lead to gene alterations and, therefore, requires continuous DNA repair.

Polymorphism in the iNOS gene that leads to increased expression or altered function of the enzyme could affect the level of the DNA lesions and therefore increase DNA damage. In this context, Daff et al. [13], using a bacterial culture, found a deletion located six amino acids from the currently studied C>T polymorphism in exon 16. The proximity between this deletion and C>T polymorphism has been suggested by Jing Shen et al. [14] and Jesper Johannesen et al. [15] to account for the increase in iNOS activity. The increased activity is associated with several types of diseases, such as bladder cancer [16,17], and diabetes [18], besides gastric cancer [14].

Recently, host genetic susceptibility to cancer related to polymorphisms in DNA repair enzymes has been investigated [19,20]. In gastric cancer, our team observed and reported before [21] that polymorphisms in some enzymes of the base excision repair (BER) system, responsible for recognizing and removing the damaged base, should be investigated such as the following: OGG-1 Ser326Cys [22,23], associated with reduced DNA repair capacity [24]; APE-1 Asn148Glu [25,26], associated with increased sensitivity to ionizing radiation in homozygosis [26]; and PARP-1 Val762Ala, associated with gastric cancer risk besides cag(+) H. pylori infection [27].

Therefore, this study aimed to investigate the interaction between the iNOS C >T polymorphism, the genotype of H. pylori strains and polymorphisms in DNA repair genes (OGG-1, APE-1 and PARP-1) in a set of gastric cancer patients.

Materials and Methods

Patients and specimens

This study was approved by the ethics committee of the Federal University of Ceará. A total of 109 adenocarcinoma specimens, surgically resected, were obtained from three public hospitals in Fortaleza, Ceará State, Brazil: Walter Cantideo Hospital at Federal University of Ceará, Santa Casa de Misericórdia Hospital and Cesar Cals General Hospital. Fragments of tumor were collected during gastrectomy and frozen at -80°C. Histological diagnosis and tumor classification was based on Lauren’s criteria.

DNA extraction, H. pylori detection and genotyping

Genomic DNA was extracted from frozen tumor tissue samples consisting mainly of tumor cells (>80%), using the cetyltrimethyl ammonium bromide (CTAB) method adapted from Foster and Twell [28]. H. pylori infection was detected by amplification of the urease C gene, and virulence genes were identified using specific primers and conditions, as previously described by Lage et al. [29] and Domingo et al. [30], Atherton et al. [31] and Sozzi et al. [32]. Negative (water) and positive controls were assayed in each run. PCR products were separated on 6% polyacrylamide electrophoretic gels, which were then silver stained.

DNA repair polymorphism

Single nucleotide polymorphisms (SNPs) for DNA repair genes were determined by a PCR-RFLP based method as described by Vodicka et al. [33] and Shen et al. [14]. Negative (water) and positive (DNA containing known DNA repair genes) controls were assayed in each run. The amplified fragments were visualized in 2% agarose gels containing ethidium bromide under UV light and were digested with appropriate restriction endonucleases. The fragments were resolved by 8% polyacrylamide gel electrophoresis under non-denaturing conditions and silver staining. Randomly selected samples were re-genotyped (10% of samples).

Statistical analysis

All statistical analyses were conducted with the SPSS® 15.0 version statistical software program (SPSS, Chicago, IL, USA), using the χ2 and Fisher exact tests, and p<0.05 was considered statistically significant.


In this study, the intestinal type was slightly more frequent than the diffuse (60/109 or 55.0% versus 49/109 or 45.0%), and most of the tumors were located in the non-cardia region of stomach (75.2%; 82/109). The genotype distributions were as follows: iNOS 78.0% CC (85/109), 21.1% CT (23/109) and 0.9% TT (1/109); APE-1 38.5% TT (42/109), 47.7% TG (52/109) and 13.8% GG (15/109); PARP-1 69.7% AA (76/109), 26.6% AG (29/109) and 3.7% GG (4/109); OGG-1 56% CC (61/109), 39.4% CG (43/109) and 4.6% GG (5/109).

With regard to the iNOS genotype distribution, no statistical difference was observed between histological characteristics or tumor localization (Table 1). However, considering 55 years old as the patients’ age cutoff, the iNOS wild-type (CC) was significantly (p= 0.020) more frequent in patients aged ≥ 55 years than those <55 years old (83.1% versus 16.8%, respectively), as shown in Table 1.

Citation: de Lima Silva-Fernandes IJ, do Carmo APS, da Costa DM, de Oliveira ES, dos Santos Pereira E and Rabenhorst SHB. Low Virulence of Helicobacter Pylori and Gastric Cancer: the Contribution of Polymorphisms of iNOS and DNA Repair Enzymes in this Process. Austin J Gastroenterol. 2014;1(4): 1019. ISSN:2381-9219