[ Main Page | Editorial Board | About | Instructions ]
[ Table of Contents | Archive | Archive Search | Online Submission | Sponsor | E-mail ]

Turkish Journal of Cancer
2006, Volume 36, Number 1, Page(s) 005-010
[ Summary ] [ PDF ] [ Similar Articles ] [ Mail to Editor ]
The relationship between the risk of lung cancer and the exon 5 (ILE105VAL) polymorphism of glutathione S-transferase P1 (GSTP1) gene
FATMA SÖYLEMEZ1, ETEM AKBAŞ1, ERTUĞRUL SEYREK2, HİCRAN MUTLUHAN1, HANDAN ÇAMDEVİREN3
1Mersin University, Faculty of Medicine, Medical Biology and Genetics, Departments of Mersin-Turkey
2Mersin University, Faculty of Medicine, Oncology and Departments of Mersin-Turkey
3Mersin University, Faculty of Medicine, Biological Statistics, Departments of Mersin-Turkey
Keywords: Lung cancer, genetic polymorphisms, GSTP1 gene
Summary
Lung cancer is one of the cancers which are seen all around the world and whose mortality rate is high. Many environmental factors and genotypes of individuals play an important role in the process of lung cancer. Previous epidemiologic studies have suggested that cancer risks are modified by some genetic polymorphisms. Glutathione S-transferase P1 (GSTP1) gene polymorphism is the most explored in all of these polymorphisms. GSTP1, the most abundant GST isoform in the lung, metabolizes numerous carcinogenic compounds including polycyclic aromatic hydrocarbons (PAH), a tobacco carcinogen. Utilizing a hospital-based case-control study, we investigated the association between GSTP1 polymorphisms at exon 5 with lung cancer risk in the Turkish population living in Mersin. The study population consisted of 50 lung cancer cases that were diagnosed at the Clinical Oncology Department of the Mersin University Hospital and 50 healthy controls matched for sex and age. Genomic DNA from patients and healthy controls was extracted from peripheral blood leukocytes and PCR-RFLP assay were used to genotype the GSTP1 polymorphisms. A statistically significant difference was observed only in the frequencies of the GSTP1 exon 5 allele and genotype between the control group and the case group (p=0.037). There was an association between the exon 5 mutant genotype (GG) and overall lung cancer risk (OR 7.55, 95% CI 1.127-50.596). [Turk J Cancer 2006;36(1):5-10].
  • Top
  • Summary
  • Introduction
  • Methods
  • Results
  • Disscussion
  • References
  • Introduction
    Lung cancer is the most widespread and the most fatal type of cancer worldwide. Polymorphisms of glutathione S-transferase enzymes show differences according to the races in the world. The frequency of lung cancer in men is higher than women [1]. The survival is rather low, only 15% of the individuals with lung cancer can manage to survive for 5 years. According to the WHO guidelines, lung cancer is histologically divided into 5 subtypes; squamous cell carcinoma, adenocarcinoma, small cell carcinoma, nonsmall cell carcinoma and adenosquamous carcinoma [2]. Lung cancer generally appears after the age of 45 and its incidence increases by age. Together with smoking which is the most important risk factor (92% in men, 78% in women) conditions of labour, nourishment, age, gender, socioeconomic condition and genetic susceptibility have a big role on the etiology of lung cancer [35]. Genetic factors consist of the interactions of tumor suppressor genes, oncogenes, the genes coding the enzymes of xenobiotic metabolism and gene amplification [6]. In the etiology of lung cancer, the role of genetic susceptibility is important [7]. The incidence of lung cancer and polymorphisms of glutathione S-transferase enzymes show differences according to race.

    Among the constituents of tobacco smoke, the polycyclic aromatic hydrocarbons (PAHs), such as benzo (a) pyrene, play a major role in the chemical lung carcinogenesis [8]. Glutathione S-transferases (GSTs) consist of a super family of phase II metabolic enzymes, such as glutathione Stransferase P1 (GSTP1), that catalyze the conjugation of reduced glutathione (GSH- L-Y-glutamyl-L-cysteinylglycine) with electrophilic groups of a wide variety of compounds including PAH, tobacco carcinogen [9]. The human cytosolic GST isoenzymes are comprised of the four gene families and are classified according to their biochemical characteristics: alpha (GSTA), mu (GSTM), theta (GSTT) and pi (GSTP). A new form of GSTs named zeta (GSTZ1) has been discovered [10]. Also, although chromosomal localization has not been determined exactly yet, there are studies on a mitochondrial glutathione Stransferase named GST kappa. GSTM1, GSTM3, GSTT2 and GSTP1 are polymorphic in the human populations. GSTP1 has been mapped to chromosome 11q13 and contains seven exons [11]. The GSTP1 gene has a polymorphism at exon 5 (ile105val). This GSTP1 polymorphism is connected with an A→G substitution in the nucleotides of 313 causing amino acid change of the GST pi protein. It has been suggested that this genetic polymorphism of GSTP1 exon 5 has functional effects on the GST gene product resulting in reduced enzyme activity [12]. GSTP1 is widely expressed in different human tissues and is the most abundant GST isoform in the lung. Thus, alterations in the structure, function or expression levels of GSTP1 due to genetic polymorphisms could alter the ability to detoxify carcinogens and modulate lung cancer risk. In the epidemiological studies about this matter, controversial results are reported. For instance; though there are studies reporting that the activity of GSTP1 gene in the individuals having valine variant is rather low against diol epoxides and especially benzo(a)pyrene diol epoxide (BPDE), in a contrary study it has been shown that GSTP1/val1054 has higher catalytic function against the carcinogenic epoxides than GSTP1/ile105 [81315].

    A lot of studies have been carried out in order to determine the relationship between lung cancer and the polymorphism of exon 5 in GSTP1 gene. Some studies suggested that exon 5 polymorphisms were associated with lung cancer development [81522]. On the other hand, other studies reported that polymorphisms at exon 5 were not related with lung cancer [23-29]. These contradictory results necessitate more studies about this issue. The purpose of this study was to examine the association of the GSTP1 polymorphisms of exon 5 with lung cancer.

  • Top
  • Introduction
  • Methods
  • Results
  • Disscussion
  • References
  • Material and Methods
    The study population consisted of 50 lung cancer cases (43 male, 7 female; mean age 54.4±8.6) who were diagnosed at the Oncology Clinic of the Mersin University Hospital and 50 healthy controls (43 male, 7 female; mean age 51.8±8.5) matched for ethnicity, sex and age (±5 years). All individuals gave informed consent before participating in the research. All the study subjects were interviewed with a standard questionnaire for their demographic characteristics, previous family history of cancer, cigarette consumption and smoking habits. A venous blood sample was drawn from each individual. The samples were collected into tubes containing ethylenediaminetetraacetic acid (EDTA). DNA was extracted from whole blood by a salting out procedure [30].

    Polymerase Chain Reaction - Restriction Fragment Length Polymorphisms (PCR-RFLP) assay was used to genotype GSTP1 exon 5 polymorphism. The specific primers for the GSTP1 exon 5 polymorphism were F 5’- GTA GTT TGC CCA AGG TCA AG-3’ and R 5’- AGC CAC CTG AGG GGT AAG-3’. Amplification was performed in an automated thermal cycler (Techne Progene, Cambridge, UK). The PCR conditions were 5 min for initial denaturation at 94° C, 30 cycles at 94° C for 1 min for denaturation, 90 sec at 59° C for annealing and 90 sec at 72° C for extension, followed by 7 min at 72° C for final extension. The amplified product of 431 bp was digested with restriction endonuclease Alw26I (MBI Fermentas) for 2 hours at 37° C. The gel visualizing system was used (Vilber Lourmat, France). The fragments were separated on a 3% agarose gel stained with ethidium bromide. The wild type (AA), heterozygous genotype (AG) and mutant genotype (GG) yielded 2 bands (329 and 107 bp), 3 bands (329, 222 and 107 bp) and 2 bands (222 and 107 bp), respectively (Figure 1).

    Fig 1: PCR-RFLP patterns of the exon 5 polymorphism. In the wild type sequence (AA), bands of 329 bp and 107 bp (lane 4) were generated, whereas in the homozygous mutant (GG), bands at 222 and 107 bp (lanes 2, 8-10) were produced. In the heterozygous AG, all three bands were present (lanes 1,3,5-7)

    Statistical Analysis
    All statistical analyses were performed using SPSS for Windows software. Allele and genotype frequencies were compared in lung cancer cases and healthy controls by using χ2 test. The Student’s t test was used to compare quantitative demographic data in study objects. The strength of association was tested by multivariate logistic regression analysis. A p value of <0.05 was regarded as significant.

  • Top
  • Introduction
  • Methods
  • Results
  • Disscussion
  • References
  • Results
    When we studied the relationship between age and the risk of lung cancer, the risk of lung cancer was found to increase with age (p=0.001). The average age of those with lung cancer is 54.4±8.6. Lung cancer risk increases 1.102 times with every 1 year increase above average. As for the relationship between smoking and lung cancer; 15 controls were non-smokers, 10 of were smoking one packet a day, and 25 were smoking more than one packet a day. On the other hand, 8 of those with lung cancer were nonsmokers, 14 of them were smoking one packet a day and 28 were smoking more than one packet a day. There was a relationship between smoking and the risk of lung cancer. This difference was more apparent for those who smoke more than a packet a day (p=0.001). As for the relationship between gender and lung cancer, 7 of our patients with lung cancer were female and 43 were male. The lung cancer risk was higher in men (p<0.001).

    When the relationship between tumor types and genotype rates of exon 5 polymorphisms was studied, it was found that AA genotype was higher in squamous cell carcinoma and small cell carcinoma than adenocarcinoma. The AG genotype was found to be lowest in squamous cell carcinoma (p=0.035). The GG genotype was determined to be at similar rates in all tumor types. In this study, the proportion of women with adenocarcinoma and men with squamous cell carcinoma were found to be high in the total population of patients.

    The allele and genotype distribution of GSTP1 exon 5 polymorphism is shown in table 1. There was a connection between lung cancer and exon 5 gene polymorphism genotype (p=0.047). Allele A of exon 5 gene polymorphism was found to be higher in the control group whereas allele G was found to be higher in the group with lung cancer (p=0.005). Lung cancer risk was 7.55 times higher in homozygote (GG) individuals than those with the AA genotype (p=0.037).

    Table 1: Distribution of GSTP1 exon 5 genotypes in cases and controls

  • Top
  • Introduction
  • Methods
  • Results
  • Disscussion
  • References
  • Discussion
    In this study that investigated the role of polymorphic variants of glutathione S-transferase P1 (GSTP1) gene, it was found that there is a relationship between lung cancer and the exon 5 gene polymorphism genotype (p=0.037). Of exon 5 gene polymorphism frequencies, the frequency of allele A was found to be higher in the control group and allele G was found to be higher in the patients with lung cancer. Lung cancer risk was found to be 7.6 times higher in homozygote (GG) individuals than in those with AA genotype. When groups were evaluated in terms of exon 6 polymorphisms of the GSTP1 gene, no difference was observed between the groups.

    Studies on tumor types reveal that the most common lung tumor is adenocarcinoma in women and squamous cell carcinoma in men [31,32]. In this study also, the rates of women with adenocarcinoma and men with squamous cell carcinoma were found to be high in the total population of patients. There are a lot of studies about GSTP1 exon 5 polymorphism in different populations. Nazar-Steward et al. [13], in their study on patients with lung cancer, found a connection between lung cancer risk and 105 val allele (G) and especially homozygote genotype (GG). Ryberg [15] found that patients with lung cancer had a higher homozygote allele G frequency compared to the control group. In this study which measured DNA adduct levels as well, they also found that adduct level in individuals with allele G (val) was higher compared to individuals with allele A (ile). In addition, they also determined that lung cancer risk was two-fold in homozygote individuals for allele G. To-Figueras et al. [19] determined no important difference between the patient and control groups in terms of allele frequencies in exon 5 polymorphism. Lin et al. [20] reported that lung cancer risk was high in those with squamous cell carcinoma who had mutant valine allele (G) in exon 5 of the gene GSTP1. Stucker et al. [22], in their study on 251 patients with lung cancer, determined a two-fold lung cancer risk increase in individuals with homozygote 105val (GG) genotype. Katoh et al. [23] studied this A→G polymorphism on the 313th nucleotide of GSTP1 in terms of its connection with various cancer types in the Japanese population but could not find any relationship with lung cancer. To-Figueras et al. [26], in their study on 164 North-West Mediterranean Caucasian patients with lung cancer, determined no difference between the patient and control groups in terms of allele frequencies in exon 5 polymorphism. Lewis et al. [33], in their study analyzing the relationship between lung cancer risk and GSTM1, GSTT1 and GSTP1 polymorphisms, determined that there wasn’t any connection between lung cancer risk and P1’s exon 5 genotypes. Kihara et al. [34] reported that exon 5 polymorphism of the gene GSTP1 did not change lung cancer risk by itself but the interaction of GSTP1 exon 5 genotypes with GSTM1 null genotype increased the risk.

    Findings in some studies indicate that these polymorphisms on the gene GSTP1 do not affect the risk of lung cancer by themselves individually, but they can change the risk only if seen together with other genetic polymorphisms. Miller et al. [16] reported that in case of the combination of GSTP1 GG (105ile) + GSTM1 null or GSTP1 GG (105ile) + p53 Arg/Pro, Pro/Pro genotypes, this variant of GSTP1 influences the increase in lung cancer risk. Jourenkova et al. [27] determined that GSTP1 105 val allele has no connection with the increase in lung cancer risk by itself, however in combination with GSTM1 null and GSTM3 AA polymorphisms it contributes to increased risk.

    The fact that the greatest factor in lung cancer is smoking shows the importance of various enzymes that play a role in the excretion through xenobiotic metabolism of toxic materials, such as PAH contained in the cigarette smoke. GSTP enzyme is an important one that catalyzes phase II reactions of this mechanism. The importance of GSTP enzyme in lung cancer, in addition to being the most expressed enzyme to the lungs, is being the most important enzyme that metabolizes activated products of benzo(a)pyren, a carcinogenic material contained in the cigarette smoke, such as BPDE. As a result of single nucleotide changes in this polymorphism in the GSTP1 gene, isoleucine aminoacid is synthesized at the 105th codon of GSTP1 gene’s exon 5 instead of alanine and this aminoacid change causes differences in the substrate binding active region of the enzyme. There are contradictions in most of the studies about the way this difference affects enzyme activity. Watson et al. [35] reported that individuals with the variant allele of exon 5 polymorphism have significantly lower GSTP enzyme activity. Sundberg et al. [8] argue that valine aminoacid synthesis in related regions in GSTP polypeptide due to the changes in GSTP1 gene’s exon 5 increases enzyme activity against BPDE, which is one of the major substrates of the enzyme and related to lung cancer because of its existence in the cigarette smoke, and thus aminoacid changes do not affect the risk for lung cancer. Contrary to this, Harries et al. [14] and Ryberg et al. [15] argue that valine variant has a low activity against polycyclic aromatic hydrocarbon diol epoxides, particularly against BPDE, and for this reason cancer risk will be higher in individuals with valine variant as detoxification potential will be lower. They have supported this argument with the finding that the frequency of valine variant is higher in patients with lung cancer compared to controls.

    Our findings regarding the relationship between lung cancer and smoking, age and gender are consistent with the literature. It was determined in our study that GSTP1 exon 5 (ile105val) polymorphism is associated with the increase in lung cancer risk and the risk for lung cancer is about 7.5 times higher in individuals who have homozygote allele G. As a result of our evaluations taking into account the age, smoking habit, and tumor types of individuals; it is evident that the risk for lung cancer especially for adenocarcinoma is high in individuals who carry the mutant allele G of exon 5 of GSTP1 gene as homozygote. While adenocarcinoma is more prevalent in women, squamous cell carcinoma is more prevalent in men. The findings of our study parallels those of a major portion of literature about exon 5 polymorphism and its association with lung cancer. However, there is some literature arguing otherwise, stressing the effect of race in the relationship between these two polymorphisms and lung cancer. The findings regarding the effects of the GSTP1 polymorphisms on enzyme function and their relationship with lung cancer risk are contradicting. Therefore, more comprehensive studies evaluating the interaction of this polymorphism with genes related to lung cancer might prove useful in the future.

    ACKNOWLEDGEMENT
    This study has been supported by Turkish Association for Cancer Research and Control, Terry Fox Research Grant.

  • Top
  • Introduction
  • Methods
  • Results
  • Discussion
  • References
  • References

    1) Magrath I, Litvak J. Cancer in developing countries: opportunity and challenge. J Natl Cancer Inst 1993;85:862-74.

    2) Topuz E. Biology, Diagnosis and Treatment of Lung Cancer. İstanbul University, Oncology Inst. Press, 2001.

    3) Radzikowska E, Roszkowski K, Giaz P. Lung cancer in patients under 50 years old. Lung Cancer 2001;33:203-11.

    4) Skarin AT, Herbst RS, Leong TL, et al. Lung cancer in patients under age 40. Lung Cancer 2000;32:255-64.

    5) Garfinkel L, Stellman SD. Smoking and lung cancer in women: findings in a prospective study. Cancer Res 1988;48:6951-5.

    6) Haugen A, Ryberg D, Mollerup S, et al. Gene-environment interactions in human lung cancer. Toxicol Lett 2000;112:233-7.

    7) Wu AH, Fontham ET, Reynolds P. Family history of cancer and risk of lung cancer among lifetime nonsmoking women in the United States. Am J Epidemiol 1996;143:535-42.

    8) Sundberg K, Johansson AS, Stenberg G, et al. Differences in the catalytic efficiencies of allelic variants of glutathione S transferase P1-1 towards carcinogenic diol epoxides of polycyclic aromatic hydrocarbons. Carcinogenesis 1998;19:433-6.

    9) Rahman I, MacNee W. Lung glutathione and oxidative stress: implications in cigarette smoke-induced airway disease. Am J Physiol 1999;277:1067-88.

    10) Eaton DL, Bammler TK. Concise review of the glutathione S-transferases and their significance to toxicology. Toxicol Sci 1999;49:156-64.

    11) Ali-Osman F, Akande O, Antoun G, et al. Molecular cloning, characterization and expression in Escherichia coli of fulllenght cDNAs of three human glutathione S-transferase pigene variants. J Biol Chem 1997;272:10004-12.

    12) Ishii T, Matsuse T, Teramoto S, et al. Glutathione S-transferase P1 (GSTP1) polymorphism in patients with chronic obstructive pulmonary disease. Thorax 1999;4:693-6.

    13) Nazar-Steward V, Vaugan TL, Stapleton P, et al. A populationbased study of glutathione S-transferase M1, T1 and P1 genotypes and risk for lung cancer. Lung Cancer 2003;40:247-58.

    14) Harries LW, Stubbins MJ, Forman D, et al. Identification of genetic polymorphisms at the glutathione S-transferase Pi locus and association with susceptibility to bladder, testicular and prostate cancer. Carcinogenesis 1997;18:641-4.

    15) Ryberg D, Skaug V, Hewer A, et al. Genotypes of glutathione transferase M1 and P1 and their significance for lung DNA adduct levels and cancer risk. Carcinogenesis 1997;18:1285-9.

    16) Miller DP, Liu G, De Vivo I, et al. Combinations of the variant genotypes of GSTP1, GSTM1 and p53 are associated with an increased lung cancer risk. Cancer Res 2002;62:2819-23.

    17) Risch A, Wikman H, Thiel S, et al. Glutathione S-transferase M1, M3, T1 and P1 polymorphisms and susceptibility to non-small-cell lung cancer subtypes and hamartomas. Pharmacogenetics 2001;11:757-64.

    18) Miller DP, De Vivo I, Neuberg D, et al. Association between self-reported environmental tobacco smoke exposure and lung cancer: modification by GSTP1 polymorphism. Int J Cancer 2003;104:758-63.

    19) To-Figueras J, Gene M, Gomez-Catalan J, et al. Lung cancer susceptibility in relation to combined polymorphisms of microsomal epoxide hydrolase and glutathione S-transferase P1. Cancer Lett 2001;173:155-62.

    20) Lin P, Hsueh YM, Ko JL, et al. Analysis of NQO1, GSTP1 and MnSOD genetic polymorphisms on lung cancer risk in Taiwan. Lung Cancer 2003;40:123-9.

    21) Reszka E, Wasowicz W. Significance of genetic polymorphisms in glutathione S-transferase multigene family and lung cancer risk. Int J Occup Med Environ Health 2001;14:99-113.

    22) Stucker I, Hirvonen A, de Waziers I, et al. Genetic polymorphisms of glutathione S-transferases as modulators of lung cancer susceptibility. Carcinogenesis 2002;23:1475-81.

    23) Katoh T, Kaneko S, Takasawa S, et al. Human glutathione S-transferase P1 polymorphism and susceptibility to smoking related epithelial cancer; oral, lung, gastric, colorectal and urothelial cancer. Pharmacogenetics 1999;9:165-9.

    24) Frias FR, Gonzales C, Costa X, et al. Screening for polymorphisms in exon 5 of the glutathione S-transferase P1 gene. Thorax 2000;55:535-6.

    25) Lewis SJ, Cherry NM, Niven RM, et al. GSTM1, GSTT1 and GSTP1 polymorphisms and lung cancer risk. Cancer Lett 2002;180:165-71.

    26) To-Figueras J, Gene M, Catalan JG, et al. Genetic polymorphism of glutathione S-transferase P1 gene and lung cancer risk. Cancer Causes Control 1999;10:65-70.

    27) Jourenkova N, Wikman H, Bouchardy C, et al. Role of glutathione s-transferase GSTM1, GSTM3, GSTP1 and GSTT1 genotypes in modulating susceptibility to smokingrelated lung cancer. Pharmacogenetics 1998;8:495-502.

    28) Yang M, Coles BF, Delongchamp R, et al. Effects of the ADH3, CYP2E1 and the GSTP1 polymorphisms on their expressions in Caucasian lung tissue. Lung Cancer 2002;38:15-20.

    29) Wang Y, Spitz MR, Schhabath MB, et al. Association between glutathione S-transferase P1 polymorphisms and lung cancer risk in Caucasians: a case-control study. Lung Cancer 2003;40:25-32.

    30) Miller SA, Dykes DD, Polesky HF, et al. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16:1215.

    31) Dresler CM, Fratelli C, Babb J, et al. Gender differences in genetic susceptibility for lung cancer. Lung Cancer 2000;30:153-60.

    32) Koyi H, Hillerdal G, Branden E, et al. A prospective study of a total material of lung cancer from a county in Sweden 1997-1999: gender, symptoms, type, stage and smoking habits. Lung Cancer 2002;36:9-14.

    33) Lewis SJ, Niven RM, Cherry NM, et al. A molecular epidemiological study of genetic susceptibility to lung cancer. Mutat Res 1997;379:161-9.

    34) Kihara M, Kihara M, Noda K. Lung cancer risk of the GSTM1 null genotype is enhanced in the presence of the GSTP1 mutated genotype in male Japanese smokers. Cancer Lett 1999;137:53-60.

    35) Watson MA, Steward RK, Smith BJ, et al. Human glutathione S-transferase P1 polymorphisms: relationship to lung tissue enzyme activity and population frequency distribution. Carcinogenesis 1998;19,275-80.

  • Top
  • Introduction
  • Methods
  • Results
  • Discussion
  • References
  • [ Top ] [ Summary ] [ PDF ] [ Similar Articles ] [ Mail to Editor ]
    Turkish Journal of Cancer web sitesi Novartis Onkoloji'nin karşılıksız eğitim katkılarıyla hazırlanmıştır.
    [ Main Page | Editorial Board | About | Instructions ]
    [ Table of Contents | Archive | Archive Search | Online Submission | E-mail ]