Indian Journal of Cancer Medknow Publications on behalf of Indian Cancer Society ISSN: 0019-509X EISSN: 1998-4774 Vol. 47, Num. 3, 2010, pp. 317-321

Indian Journal of Cancer, Vol. 47, No. 3, July-September, 2010, pp. 317-321

Original Article

Association of gastric cancer incidence with MDR1 gene polymorphism in an ethnic Iranian population

Sabahi Z, Salek R¹, Heravi RE, Mosaffa F, Avanaki ZJ, Behravan J

Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad university of Medical Sciences, ¹ Omid and Imam Reza hospital, Cancer Research Center, Mashhad, Iran

Correspondence Address: Dr. Javad Behravan, Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad university of Medical Sciences, Mashhad, Iran
behravanj@mums.ac.ir

Code Number: cn10076

PMID: 20587910

DOI: 10.4103/0019-509X.64723

Abstract

Background: The allele frequency of the multidrug resistance 1 (MDR1) gene C3435T polymorphism differs with different ethnic populations, such as Asian, African, and Caucasian. This polymorphism has been reported to be associated with the increase of gastric cancer.

Objectives: The objective of this study was to find out the association of gastric cancer incidence with MDR1 gene polymorphism in an ethnic Iranian population.

Materials and Methods: In this study, 48 gastric cancer patients were diagnosed. Genomic DNA was extracted by a salting-out method. The MDR1 polymorphism was studied by a polymerase chain reaction (PCR)-restriction fragment length polymorphism method, using a standard method.

Results: The polymorphic homozygote (T/T) genotype showed significantly an association with the incidence of gastric cancer compared with controls (P < 0.05).

Conclusions: This study suggests that C3435T polymorphism of the MDR1 gene may be associated with gastric cancer in an ethnic Iranian population.

Keywords: Gastric cancer, gene polymorphism, MDR1

Introduction

P-glycoprotein (P-gp), the product of multidrug resistance gene (MDR1), is an important ATP-dependent membrane transporter, which is involved in the absorption, distribution, and elimination of numerous drugs and acts as an energy-dependent efflux pump that exports its substrates out of the cell. ^[1],[2] The MDR1 gene encodes P-gp, a 170-kDa member of adenosine triphosphate-binding cassette (ABC) superfamily of membrane transporters. The most important physiological role of P-gp is the protection of an organism against toxic xenobiotics. More than 50 single nucleotide polymorphisms (SNPs) have been reported in the MDR1 gene. ^[3] Interestingly, a C3435T polymorphism in exon 26 of chromosome 7 results in more than 2-fold lower P-gp expression in duodenum and higher plasma concentration of P-gp substrate digoxin in subjects with TT genotype compared with CC carriers. ^[4] The effects of these polymorphisms on the P-gp function or their clinical impact in most cases are unknown, but some of the SNPs are known to be of functional relevance and can also alter the pharmacokinetics of substrate drugs. Interestingly, the polymorphism at position 2677 in exon 21 of the MDR1 gene was found to co-segregate with C3435T in some studies. ^{[4],[5],[6],[7]} Therefore, in this study we aimed to answer the following question: whether C3435T polymorphism of MDR1 influences the risk of development of gastric cancer in an ethnic Iranian population.

Materials and Methods

Forty-eight patients (38 men and 10 women) with gastric cancer were enrolled in our study. The total number of observed gastric cancers (48) was not close to the expected number (96), giving a relative risk (RR) of 5.40 and P = 0.03 (95% confidence interval [CI] for RR = 1.24-24.27) because we have so limited time and the sample availability was not enough. The control group comprised 131 healthy subjects (58 men and 73 women). The gastric cancer patients (25-86 years old, 38 males (79.2%), 10 females (20.8%), with a mean age of 58.12 ± 6.55, SD = 9.8) and the control group (23-72 years old, 58 males (44.3%), 73 females (55.7%), with a mean age of 45.50 ± 7.5, SD = 9.2) were involved in our study. Diagnosis of gastric cancer was accomplished by a gastroenterologist according to established clinical guidelines and criteria based on endoscopic, radiologic, and histopathologic examinations. Demographic data (such as, sex, age, tumor stage, histology, and smoking behavior) and disease information were recorded by a registry form and interview at the time of enrolment. A written informed consent was obtained from all participants. The control group consisted of volunteers who had attended the hospital for blood sampling for biochemistry and/or hematologic analyses and who were willing to participate in the study. Subjects with any hematologic or other malignancy were excluded.

Genomic DNA was isolated from peripheral blood leukocytes, using a standard salting-out protocol. ^[8] All the polymorphism studies of the MDR1 gene were conducted by polymerase chain reaction (PCR). The C3435T variant of the MDR1 gene was identified with primers: 5´-ACTCTTGTTTTCAGCTGCTTG-3´ as the forward and 5´-AGAGACTTACATTAGGCAGTGACT-3´ as the reverse primer, yielding a 206-base pair (bp) product under the conditions described elsewhere. ^[9] For PCR reactions, 200 ng of genomic DNA was amplified in 50 μL of reaction mixture containing 250 μM each of dNTPs (dATP, dCTP, dGTP, and dTTP), 250 ng of each primer, 1.5 mM MgCl ₂ , and 1 U Taq polymerase (Cinnagen Co., Tehran, Iran). PCR amplification consisted of an initial 5-min denaturation at 94°C, followed by 35 cycles of denaturation at 94°C for 90 s, annealing at 60 °C for 30 s, and extension at 72°C for 30 s. The terminal extension was performed at 72°C for 10 min. The PCR product was then digested without further purification by MboI restriction enzyme for 24 h in 37°C. The PCR product was identified in a 1% (w/v) agarose gel stained with ethidium bromide and visualized directly under UV light. Undigested 206-bp fragment indicated the presence of the T allele and, appearance of 2 bands at 130 and 76 bp represented the C allele.

Analyses were performed by SPSS 13.0 (SPSS. Chicago, Illinois, USA) software and Chi-square and Fisher′s exact tests. For genotype and allele frequencies comparison, a level of P < 0.05 was considered statistically significant with 95% CI.

Results

In vitro DNA amplification of the MDR1 gene using the specific primers resulted in a 206-bp DNA product. Digestion of the amplified fragment (amplicon) with MboI restriction endonuclease resulted in DNA fragments of 130-bp (CC), 206-bp (TT), or 130- and 206-bp (TC). Thus, each sample revealed 1 of 3 different electrophoresis patterns [Figure - 1]. Among our population controls, the TT, TC, and CC genotypes were in Hardy-Weinberg equilibrium. The frequency of the homozygous genotypes for TT and TC were 25.2% and 57.3%, respectively. The frequency of the CC genotype was 17.6%. The mutant homozygous TT and heterozygous CT genotypes were found to be significantly associated with the occurrence of gastric cancer (P = 0.015; odds ratio [OR], 95% CI: 1.73 for TT genotype and P = 0.015; OR, 95% CI: 0.655 for TC genotype, [Table - 1]). Also, higher T allele frequency in gastric cancer patients was observed when compared with healthy controls, although this difference was not significant (P = 0.089, [Table - 2]). As we know, Helicobacter pylori is a bacterium that infects the human stomach and it has been classified as a carcinogen, and H. pylori infection status was determined by histology, culture, and the rapid urease test. Infection was diagnosed when at least one of these tests was positive. Using Chi-square test for comparison of H. pylori positive or negative frequencies between the gastric cancer patients and control groups, the results show that H. pylori infection rate was significantly higher in the gastric cancer patients compared with that of the controls (gastric cancer patients: 40 (83.3%) H. pylori positive and 8 (16.7%) H. pylori negative, controls: 62 (47.3%) H. pylori positive and 69 (52.7%) H. pylori negative, P < 0.0001). Next, we investigated the effect of MDR1 polymorphism on the risk of gastric cancer in H. pylori-positive and -negative subjects by Fisher′s exact test. It was revealed that the CC genotype held a lower risk of gastric cancer in both H. pylori-positive and -negative subjects. There were no significant differences in CT and CC genotype frequencies between the gastric cancer patients and controls (P = 0.948, [Table - 3]). Moreover, risk factors and MDR1 genotypes in patients with gastric cancer are shown in [Table - 3]. No association between the risk factors and MDR1 genotypes was observed (P > 0.05).

Discussion

Gastric adenocarcinoma (most common type of gastric cancer) is the most common gastrointestinal malignancy in Iran. ^[10] The characterization of MDR1 gene and the utilization of pharmacogenetic testing for identification of different MDR1 alleles in patients may provide a useful tool for optimizing therapy with drugs that are substrates of P-gp. So far the SNP C3435T at a wobble position in exon 26 of chromosome 7 has been correlated with intestinal P-gp expression levels and has been shown to influence the absorption of orally taken drugs that are P-gp substrates. ^[5] Information gathered on the distribution of this C3435T polymorphism in populations of different ethnic origins may be essential in explaining the interindividual and interethnic differences in drug response and their side effects. ^[11]

In this study, the association between MDR1 gene polymorphism (C3435T) and gastric cancer in Mashhad city, Iran, was investigated, and the polymorphic homozygote (T/T) genotype showed an association with the incidence of gastric cancer compared with controls (P = 0.015; OR, 95% CI: 1.73). One study reports that there were no significant differences of the CT and CC genotype frequencies between gastric cancer patients and controls and they also found that the 3435TT genotype of MDR1 was associated with a lower risk of gastric cancer. ^[12] Schwab et al, reported strong association between this polymorphism and ulcerative colitis, in a German population (P = 0.045; OR, 95% CI: 2.03). ^[13] Farnood et al, also reported the frequency of C3435T MDR1 gene polymorphism in Iranian patients with ulcerative colitis. In their study, the frequency of TT, TC, and CC genotypes were 22.1%, 53.2%, and 23.4%, respectively, showing their results are close to those of ours. ^[14]

The homozygous T allele was associated with more than 2-fold lower MDR1 expression levels compared with homozygous C/C samples. According to the protective role of P-gp against toxic substrates, the association of C3435T and P-gp expression with gastric cancer may seem to be a high possibility.

The C/T genotype was observed to be significantly more frequent in our gastric cancer patients compared with controls (P = 0.015; OR, 95% CI: 0.65). In Schwab et al.′s study, the C/T genotype was the most frequent (51.4%), but no significantly higher expression of this genotype was reported in their patients compared with controls (51.7% in patients vs. 51.0% in controls).^[13]

Our results suggest that the C/C genotype may have a protective factor against gastric cancer. In some studies on healthy populations, the C/C genotype frequency was reported from 0% in Greece to 42% in Poland. ^[15] In some other studies reported from Asia, a range of frequencies from 25% in China and Malaysia to 18% in India were observed. ^[11] In our healthy population, the C/C genotype frequency was 17.6%, which is much lower than that in Africans.

According to the studies on MDR1 gene polymorphisms worldwide, allele and genotype frequencies of C3435T polymorphism depend strongly on the ethnicity of the investigated population. A marked lower C allele frequency was observed in our healthy controls (17.6%), in comparison with other Asian, European, and American studies. ^[16] In our study, because MDR1-3435 T/T genotype is the risk factor for gastric cancer, it is expected that the prevalence of C/C genotype in gastric cancer patients (18.8%) is significantly lower than that in control (17.6%) but this paradox may be because of the small sample size of the gastric cancer patients. Within the European white population, C allele frequency ranges from 37% in Greeks to 62% in Polish population and in Asians; this allele frequency varies from 34% in southwest Asians ^[11] to 61% in Japanese. ^[17] In this study, no significance of T and C allele frequencies were found in patients compared with healthy controls (P = 0.089; OR, 95% CI: 1.5). The SNP C3435T is located at a noncoding no promoter position in the MDR1 gene and hence it is unlikely to regulate the expression of MDR1. The results of the present study provide evidence that C3435T of MDR1 gene polymorphism may be associated with susceptibility to gastric cancer. Carriers of TT genotype are more at risk for developing gastric cancer than other individuals.

Our results support the previously described role of MDR1 C3435T polymorphism and intestinal P-gp expression. Our data add to the growing literature that suggests that the relationship between genetic variation in MDR1 gene and the function of the P-gp is complex and incompletely understood, and will require larger and more detailed studies.

References

Copyright 2010 - Indian Journal of Cancer

The following images related to this document are available:

Photo images