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INTRODUCTION
Cervical cancer is one of the most common types of cancer observed in women. The largest contributing factor in its development is the progression of untreated, high-risk types of human papillomavirus (HPV) in cervical tissue. Oncoproteins E6 and E7 have been shown to be activated by HPV, disrupting the normal cell cycle and DNA structures and leading to the development of cervical cancer.1-3 Epidemiological studies have shown that certain etiological factors other than HPV may also play a role in the development of cervical cancer. These may include marrying at a very early age, early childbirth, multiple births, low socio-economic status and heavy cigarette consumption.4-7 Smoking cigarettes may also play a role in the development of high-risk human papillomaviruses (HR-HPVs), which appear to interact with active cigarette smoking to increase the risk of high-grade cervical squamous intraepithelial lesions (HSIL).8,9 The nicotine in cigarettes has been found in cervical mucus, which may have a mutagenic impact.4,10,11
GST enzymes, which are encoded by GST genes, are responsible for the detoxification of chemicals found in the environment and naturally synthesized metabolites, and they play an important role in protecting tissue from oxidative damage. An increase or decrease in the tendency of certain types of cancer observed in a group of individuals is often linked to the genetic polymorphism observed in enzymes that play a role in the detoxification of xenobiotics. A significant relationship is observed between the risk of developing cancer and xenobiotic metabolism enzyme gene polymorphism. This relationship has highlighted the role of genetics in cancer etiology.12-14 The relation between GST gene polymorphism and cervical cancer has been investigated in various studies, which demonstrated that the risk of cervical cancer increases in women with GST gene polymorphism.15,16
In this study, we aimed to investigate the relationship between the development of cancer and general polymorphisms that play a role in detoxification reactions. Specifically, glutathione S-transferase M1 (GSTM1), glutathione S-transferase T1 (GSTT1), and glutathione S-transferase P1 (GSTP1) polymorphisms were studied in Turkish patients with cervical cancer.
MATERIALS AND METHODS
1. Study subjects
This study was conducted as a prospective study in the Department of Obstetrics and Gynecology at the Medical School of Uludag University between 2008 and 2009. In this study, the patient group consisted of 46 cases diagnosed with cervical cancer, and the age-matched control group consisted of 52 cases with no cancer history. Both groups visited our clinic during same period. Members of both the patient group and the control group were asked to sign an informed consent form.
2. DNA extraction and GST genotyping
Blood samples from both the patient and the control groups were taken in EDTA tubes. DNA isolation was performed according to the procedures of the Dr. Zeydanlı (DZ) DNA isolation kit, and samples were stored at -20℃ until PCR. Multiplex PCR method was used to determine GSTM1 and GSTT1 polymorphisms in the isolated DNAs. For the GSTT1 polymorphism, forward 5'-TTCCTTACTGGTCCTCACATCTC-3' and reverse 5'-TCACCGGATCATGGCCAGCA-3' primers were used. For the GSTM1 polymorphism, forward 5'-GAACTCCCTGAAAAGCTAAAGC-3' and reverse 5'-GTTGGGCTCAAATATACGGTGG-3' primers were used. Albumin forward 5'-GCCCTCTGCTAACAAGTCCTAC-3' and reverse 5'-GCCCTAAAAAGAAAATCCCCAATC-3' primers were used as internal controls.17 Albumin 350 bp, GSTM1 219 bp and GSTT1 459 bp PCR products were formed. PCR conditions required denaturation for 5 minutes at 94℃ and then 35 cycles as follows: 1 minute at 94℃ (denaturation), 1 minute at 58℃ (annealing), one minute at 72℃ (elongation) and finally 10 minutes at 72℃ (final elongation). Genotypes were determined by migration of the products in agarose gel with added 2% ethidium bromide (Fig. 1).
GSTP1 (Ile105Val) gene polymorphism was determined using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. For the GSTP1 polymorphism, forward 5'-ACCCCAGGGCTCTATGGGAA-3' and reverse 5'-TGAGGGCACAAGAAGCCCCT-3' primers were used.17 To identify the GSTP1 (Ile105Val) gene polymorphism among the products, the Alw 26 I (Genemark, Russia) enzyme was used. In the analysis conducted in 4% agarose gel after cutting the enzyme, genotypes were determined as follows: if the 176 bp PCR product from the GSTP1 gene was cut into two distinct products of 85 bp and 91 bp, then the genotype was identified as Val/Val; if three distinct products formed as 176 bp, 91 bp and 85 bp, then the genotype was identified as Ile/Val; and if the product was 176 bp then the genotype was identified as Ile/Ile (Fig. 2).
RESULTS
The characteristics of the study population are shown in Table 1. The age distribution was no different between patients and controls, the mean age being 53.73±10.35 and 51.32±8.86 years for patients and controls, respectively (p=0.11). No significant difference was seen between the two groups in terms of year of menopause and abortus. However, gravida, parity and number of living children were significantly different in the cervical cancer group and control group, respectively (p=0.03, p=0.01, and p=0.01).
Table 2 shows the distribution of GSTM1, GSTT1, and GSTP1 (Ile105Val) genotype prevalence in patients and controls. We found a prevalence of GSTM1 null genotype in 54.3% of patients compared with 57.7% in controls. The homozygous null genotype GSTT1 was found in 32.6% of patients and in 30.8% of controls. When the groups were compared in terms of GSTT1 and GSTM1 genotype, no statistical significance was determined (p=0.84 and p=0.73, respectively). Using subject with the Val/Val homozygote as a reference group, we found no association between the Ile/Ile and Ile/Val genotypes and the risk of cervical cancer after statistical analysis (p=0.730 and p=0.053, respectively). The prevalence of Ile allele was 71.8% and 70.1% in the cervical cancer group and control group, respectively.
DISCUSSION
Genetic variation in susceptibility to chemical carcinogens among individuals is one of the main factors leading to cancer development among human beings. Genetic variations among the genes forming the enzymes that play a role in metabolism, such as CYP and GST, have been shown to lead to susceptibility to the development of various cancers.18,19 The mu and theta classes of GST isozymes (GSTM1 and GSTT1, respectively) have a common and broad range of substrate specificities, and they detoxify the reactive metabolites of benzo-a-pyrene and other polycyclic aromatic hydrocarbons.12,20 The null genotype developing from homozygote deletion of GSTM1 or GSTT1 genes is frequently observed in lung and bladder cancers.21-26
When patients with cervical cancer were reviewed, 54.3% of them showed the GSTM1 null genotype. The GSTM1 null genotype among the control group was 57.7%. While the GSTM1 null genotype rate among the patients was lower than that of the control group, this difference is not statistically significant (p=0.73). On the other hand, while the GSTT1 null genotype rate among patients with cervical cancer was determined to be 32.6%, it was determined to be 30.8% in the control group. While the GSTT1 null genotype rate among patients was higher than that of the control group, this difference is not statistically significant (p=0.84).
Our study seems to be more in compliance with the studies of Warwick et al.,27 de Carvalho et al.,28 Settheetham-Ishida et al.,29 and Chen et al.,30 which did not demonstrate any relationship between the GSTM1 and GSTT1 null genotype and cervical cancer (p>0.05). Warwick et al.27 reported that no difference was found in the frequency of GSTM1 and GSTT1 null genotypes between controls and cervical carcinoma cases, including cervical intraepithelial neoplasia. Chen et al.30 did not observe any increase in incidences of cervical cancer in patients with the GSTM1 null genotype. In the study conducted by Sharma et al.31 on Indian patients with cervical cancer, the combined analysis of both GSTM1 null and GSTT1 null genotypes did not appear to influence the susceptibility. Contrary to these studies, Kim et al.12 reported that, among patients who carried HPV, the risk of developing cervical cancer before 40 is high (p<0.05) (Table 3).
The presence of the GSTP1 (Ile105Val) polymorphism produced no difference between the patient and control groups. Jee et al.32 reported that the polymorphism of GSTP1 in women who smoke cigarettes was associated with a higher risk of developing cervical cancer. In a study by Sobti et al.,4 it was reported that, in women with GSTM1 (null), GSTP1 (null) and GSTP1 (Ile105Val) genotype, the cervical cancer development rate was elevated in passive smokers. In our study, no relation could be found between the GSTP1 polymorphism and the development of cervical cancer. Since the number of cases in our study was limited, our findings would need to be supported with studies conducted on a larger number of cases (Table 4).
To conclude, demonstrating a relation between cancer types and genes that code the enzymes that act in detoxification metabolism is of great importance for determining risk groups for various cancer types. While the GST polymorphism is related to the development of cervical cancer in certain studies, no relation is seen in other studies. Many more studies should be conducted with larger patient and control groups to resolve this conflict.
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