study on polymorphism and activities of gstm1 … · study on polymorphism and activities of gstm1...

2020 Biotechnol. & Biotechnol. eq. 24/2010/3

Article DOi: 10.2478/v10133-010-0069-4 mb

Biotechnol. & Biotechnol. eq. 2010, 24(3), 2020-2025Keywords: GSTM1, CYP1A1, cancer, gene polymorphism

Introductionin traditional cancer diagnostics, pathologists examine biopsies in order to make diagnostic assessments largely based on cell morphology and tissue distribution. traditionally, pathologists use histopathological images of biopsy samples removed from patients, examine them under a microscope, and make judgments based on their personal experience. While examining such images, a pathologist typically assesses the deviations in the cell structures and/or the change in the distribution of the cells across the tissue under examination. the goal is to automatically evaluate the existence of cancer in the tissue and/or determine the malignancy level of cancer by examining the histopathological properties of the tissue. the cancer process is usually a multistep phenomenon, during which successive somatic cell mutations occur. Genes involved in cell cycle control, genetic repair systems, or encoded enzymes for biotransformation of environmental carcinogens have important roles in this process (5, 10).

Variations in metabolic genes can result in alterations in the efficiency of phase I and phase II detoxification mechanisms as well as in other metabolic pathways of micronutrients, like folate. Specific variations occur in cytochrome P450 (CYPs)

glutathione S-transferases (GSts). these same polymorphisms may also modulate genetic susceptibility to cancer (6). Previous studies have implicated CYP1A1 and GSTM1 polymorphisms as risk factors for various cancers. A number of studies have been devoted to the association of CYP1A1 or GSTM1 polymorphisms with susceptibility to oral carcinoma and have yielded conflicting results (7, 12, 22). The aim of the present study was to assess the possible associations of various cancer types with CYP1A1 genetic variations and GSTM1 null genotype via systematic analyses. the data suggest that variant genotypes of CYP1A1 might not be risk factors for lung and larynx cancer, whereas GSTM1 null genotype significantly increases susceptibility to endometrial cancer, lung, tyroid cancer, etc. in turkey population.

CYP1A1 is co-expressed in many human tissues. Though constitutive extrahepatic CYP1A1 expression is generally extremely low, CYP1A1 can be found in tissues such as prostate, mammary gland, intestine, thymus, colon, adrenal, ovary, uterus, lung, and testis. Various CYP1A1 gene polymorphisms have been found to be differentially associated with increased risk of several cancers (including cancer of the lung, breast, and colon) in different specific ethnic groups (1, 4, 5, 14, 20). enzymes, glutathione S-transferase (GSts), affect and activate metabolites of carcinogens in order to be subjected to metabolic conjugation and other kinds of detoxifications. Homozygous

STUDY ON POLYMORPHISM AND ACTIVITIES OF GSTM1 AND CYP1A1 GENES IN CONNECTION WITH VARIOUS CANCER TYPES IN TURKEY POPULATION

h.c. Vural1, n. turaclar2 and S. elagoz3

1Selcuk University, Department of Biology, Molecular Biology, Selcuklu, Konya, Turkey2Selcuk University, Vocational School of Health Services, Konya, Turkey3Cumhuriyet University, Cumhuriyet Medical Faculty, Department of Pathology, Sivas, Turkeycorrespondence to: nesrin turaclare-mail: [email protected]

ABSTRACTThe metabolism of drugs and chemical carcinogens involves a variety of isoenzymes such as members of the Cytochrome P450 (CYP) and Glutathione S-transferase (GSTs) families, with polymorphisms described in these genes appearing to be responsible for differences in individual susceptibility to cancer. Furthermore, the cytochrome P450 family (CYPs) and the glutathione S-transferase (GSTs) enzymes also play an important role in the metabolism of environmental carcinogens and of estrogen and can affect various cancer types. In this study we examine the role of the genes CYP1A1 and GSTM1, in different cancer types in Turkey population. The aim of our work was to evaluate the association between CYP1A1 and GSTM1 genetic polymorphisms and activities of genes indicative for susceptibility to various cancer types in Turkey population. The polymorphic inheritance of human drug-metabolizing enzymes, such as those encoded by the GST and CYP systems, has been implicated in all the cancer risk and prognostic. The study population consisted of 80 different incident cancer cases and 100 healthy controls. Genotyping analyses were performed by PCR based methods. In conclusion, in our population GSTM1 was associated with breast, gastric, endometrium, testis, parotis, lymph node, pancreas, tyroid, and lung cancer risk in Turk population. The analysis of patients by histological types of different cancer showed no association between histopathologic types of cancer and CYP1A1 gene polymorphism (p=0.6). Genetic researches using specific biomarkers are expected to be helpful in evaluation the risks for every cancer type.

2021Biotechnol. & Biotechnol. eq. 24/2010/3

deletions or null genotypes of GSTT1 and GSTM1 (mu class) genes may be associated with an increased risk of cancer (12, 14, 20). in search for genetic markers for cancer susceptibility as well as for diagnostic prognosis, polymorphisms of known oncogenes, such as CYPA1 and GSTM1, are of particular interest. in our studies we examined patients with lung, breast, gastric, endometrial, testicular, lymph node, parotis, pancreatic and thyroid cancer to determine whether genetic variations in CYP1A1 and GSTM1 genes were associated with increased risk and higher susceptibility to cancer. Polymerase Chain Reaction (PCR), Restriction Fragment Length Polymorphism (RFLP), Agarose Gel Electrophoresis Techniques were used to determine these genotypes in the patient and control groups. The results were evaluated using SPSS 15.0 statistical software.

Materials and MethodsCasesWe enrolled 80 different incident cancer cases and 100 healthy controls in this study. tissue samples were also collected at the same time as the blood samples for control and were processed within 5 h of collection. the study was approved by the medical ethics board of cumhuriyet University, Sivas, turkey.

Histopathology studyDuring the period from 2002 to 2008 80 new cases were included in the research. Fresh tissue samples were sent to Medical Faculty of Cumhuriyet University, Department of Pathology, with the prediagnosis of malign tumor and studied. For the purpose of molecular genetic analysis about 3 mm were taken from the tumoral pieces and were put into deep freezer (at -20ºC). For histopathologic investigation, tissues were fixed in 10% formalin and were embedded in parafine; then stained with hematoxylin-eosin. the histological slides were examined under a microscope by a pathologist- a medically qualified specialist. The medical diagnosis was formulated as a pathology report describing the histological findings and the opinion of the pathologist. then each tissue sample with cancer, 5 μm thick sections of each biopsy specimen were placed in eppendorf tubes with coded labels and sent to molecular laboratory for DNA extraction and PCR assays.

the sample histopathological images of different cancer types (stained using hematoxylin-and-eosin technique) are shown in Fig. 1, Fig. 2 and Fig. 3, and Table 1. As illustrated in these figures, cell distribution is completely different for cancerous and healthy samples.

Patients and DNA isolationThe DNA used for polymorphic analysis was isolated from the biopsy samples of patients with cancers by using DNA isolation kit (Qiagen, Almanya) according to the manufacturer’s instructions (Fig. 4). Isolated DNA was stored at -20°c prior further analysis. the control group consisted of healthy unrelated volunteers without a medical history of cancer or other chronic diseases. All patients and controls were of turkish population.

Fig. 1. Normal Lung Tissue (HE; X200)

Fig. 2. Adenocarsinoma of Lung (HE; X100)

Fig. 3. Invasive Ductal Carsinom of Breast (HE; X100)


Fig. 4. Genomic DNAs were loaded in a 1% agarose gel, seperated by electrophoresis and visualised with transillumination after ethidium bromide staining. Respectively: Lane 1, 2, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and 17- genomic DNAs isolated from breast tumor tissues with Bio Robot EZ1; Lane 3, 4 and 5- genomic DNAs isolated from lung tumor tissues with Bio Robot EZ1

Genotyping assaysCYP1A1 genetic polymorphisms were determined as previously described (8, 9, 11, 14). Primer pair sets were used to detect the polymorphism within exon 7 of the CYP1A1

gene by PCR where the praimer set consisted of sense primer 1A1A: 5’-GAAGTGTATCGGTGAGACCA-3’ and antisense primer c53: 5’-GTAGACAGAGTCTAGGCCTCA-3’, located about 190 bp downstream of the polymorphism site (Fig. 5).

the other primer pair (G5: 5’-GAACTCCCTGAAAAGCTAAAGC-3’ and G6: 5’-GTTGGGCTCAAATATACGGTGG-3’), which are located about 215 and 286 bp downstream of the polymorphic sites, was used to determine the GSTM1 genetic polymorphism as previously described (4). (Fig. 6 A and B).

PCR amplifi cation conditions for GSTM1PCR were carried out in two tubes of a total volume of 25 μl containing 0.4 μg of genomic DNA, 0.5 μl of each primer, 3.0 μl of 10X PCR buffer, 2.5 μl of 25 mmol/l MgCl2, 3.0 μl of 10 mmol/l dNTPs and 2U of Taq DNA polymerase. The program was iniated on BioRad PCR amplifi er with 4 min of denaturation, followed by 25 cycles of amplifi cation with denaturation for 15 sec at 94°C, fi rst annealing for 20 sec at 52°C, and an extention at 72°C for 15 sec; followed by 10 cycles of amplifi cation with denaturation for 15 sec,

TABLE 1Patients with various cancers were analyzed with respect to the histologic diagnoses

Tissue Histopatolojik diagnosis Numbers of samples Stage TNMBreast Apocrine CA 3 IIA T2N0MXBreast Apocrine CA 3 i T1N0MXBreast Mix invasive ductal+lobular CA 1 IIIA T3N2MXBreast Invasive ductal CA 4 IIA T2N0MXBreast Invasive ductal CA 4 i T1N0MXBreast Invasive ductal CA 5 IIA T1N1MXBreast Invasive ductal CA 2 IIIA T2N2aMxBreast Invasive ductal CA 5 IIA T2N0MxBreast Invasive ductal CA 4 iiic T3N3MxBreast Infl ammatory CA 3 iiiB T4dN2MxBreast Invasive lobular CA 4 IIA T1cN1aMxlung Adenocarsinoma 3 iiB T3N0Mxlung Adenocarsinoma 5 IA T1N0Mxlung carsinoid tumor 2 IA T1N0Mxlung Differentiate adenocarsinoma 1 iiiB T4N0Mxlung Adenosquamous carsinoma 2 iiB T2N1Mxlung Large cell CA 6 iB T2N0Mx

Gastric neuroendocrine carsinoma 2 IIIA T3N1MxEndometrıum Adenocarsinoma 4 iB T1BN0Mx

Pancreas Periampuller duodenal adenocarsinoma 3 iiB T3N0Mxtestis combine germ cell tumor 3 iiB T3NxMx

thyroid Papillar CA 4 iii T3N0Mxthyroid Papillar CA 5 i T1N0MxParotis Metastasis of Malign Melanom 1 iV clark iV

lymph node Metastasis of epidermoid carsinom 1


second annealing for 20 sec at 48°C, an extention at 72°C for 15 sec, and a fi nal elongation at 72°C for 3 min. The PCR products were then subjected to electrophoresis on 2% agarose gels. the presence of intensive bands at 215 and 268 bp was indicated and recorded as +/+ genotype. If there was only a light band of 268 bp, the genotype was determined to be -/-. if there was a faint band of 215 bp and a more intense band of 268 bp, the genotype was recorded as +/-. All results were confi rmed by repeated experiments. The fi nal PCR product from co-amplifi cation of GSTM1 (215 bp) was visualized on an ethidium bromide-stained 2% agarose gel.

PCR amplifi cation conditions for CYP1A1The reaction mixture contained 200 pmol of each dNTPs, 3 µl of 10X PCR buffer (100 mM Tris-HCl pH 9.0 25°C, 500 mM KCl), 1.6 mM MgCl2 and 1U of Taq DNA polymerase. Amplifi cation consisted of melting at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 2 min, annealing at 59°C for 1 min and extension at 72°C for 10 min. Products were separated by 2% (w/v) agarose gel electrophoresis. A 312 bp band, corresponding to the CYP1A1 gene, was always present and was used as an internal control to indicate successful PCR amplifi cation. All results were confi rmed by repeated experiments.

Statistical analysisDifferences between different cancer risk types for categorical variables were analyzed by chi Square test, and odds ratios were calculated. cancer type was evaluated by Student’s test. The association between CYP1A1 and GSTM1 polymorphisms was evaluated by multiple logistic regression analysis controlling for cancer types. Adjusted odds ratios and their confi dence intervals were calculated. P values less than 0.05 were considered signifi cant. SPSS for 15.0 Windows was used for statistical analysis.

Results and DiscussionCYP1A1 and CYP1B1 are members of the cytochrome P450 superfamily of enzymes that catalyze the oxidation and sometimes reduction of a large number of xenobiotic chemicals such as drugs, toxic chemicals, carcinogens and endogenous compounds such as steroids, fatty acids, vitamins, and prostaglandins. Metabolism of these compounds generally results in detoxifi cation by forming polar metabolites that are easily excreted from the body. however, in some cases metabolism results in the bioactivation of the parent compound. CYP1A1 can be found in tissues such as prostate, mammary gland, intestine, thymus, colon, adrenal, ovary, uterus, lung, and testis (3, 8, 13, 16, 17, 18, 19, 21, 23, 24). Several of these genes (GSTM1 and CYP1A1) modulate response to cancer therapy and/or treatment associated toxicity. the two genes analyzed in this article include GSTM1 and CYP1A1. Variations within these genes have been previously demonstrated to infl uence drug effi cacy and toxicity and also to modify individual susceptibility to cancer.

Fig. 5. Agarose gel electrophoresis of PCR products from CYP1A1 gene. Lane 1, 100 bp ladder size standard; Lane 2, tissue sample with PCR product of genomic DNA from endometrial cancer; Lane 3, PCR product of blood sample genomic DNA from healty person; Lane 4, PCR product of tissue sample genomic DNA from breast cancer; Lane 5, PCR product of blood sample genomic DNA from healty person

A

BFig. 6. Agarose gel electrophoresis of PCR products from CYP1A1 gene. Lane 1, 100 bp ladder size standard; Respectively: Lane 1-10 (breast, lung, thyroid, gastric, endometrium, lymph node, pancreas, breast, testis, parotis) at A; and lane 1-5 (thyroid, parotis, pancreas, breast, testis) at B


We have presented the genotype distribution of two susceptibility genes of the GSTM1 and CYP1A1 families observed in different tissue samples with cancer from turk population in Turkey. A better understanding of polymorphic chemical metabolizing genes can contribute, in the future, to preventive actions or for risk assessment of humans exposed to environmental carcinogens. For this purpose, however, it is important to take into account the different cancer types. From the present study it is evident that distribution of CYP1A1 genotypes and GSTM1 genotypes was different from the normal controls. here we studied the combined effects of the genotypes of both CYP and GST polymorphisms on development of pathology. Metabolism of carcinogens under genetic control is an important factor in modulating individual susceptibility to cancer. information on susceptibility to cancer is valuable for identifying high-risk individuals, allowing for early diagnosis and reduction of risk exposure to carcinogens. in conclusion it can be said that the studied population is bearing a significant fraction of genotypes associated with increased risk for development of cancers. Most studies describe CYP1A1 polymorphisms in relation to lung cancer, because CYP1A1 is involved in the activation of PAHs, which are present in cigarette smoke, for example. Several studies have investigated the association between CYP1A1 polymorphisms and breast cancer risk. For the CYP1A1 polymorphism the reports are also inconclusive; one study reports a decrease in breast cancer risk in Japanese women associated with the CYP1A1 (11). Seven studies examined, individually, the frequencies of both GSTM1 and GSTT1 null genotypes in cases and controls. however, only three studies (2, 9, 15) examined the risk of combined GSTM1-GSTT1 null genotype. one study (2) also examined the effect of combined genotype of GSTM1, and CYP1A1, which only mentioned in passing that the combined genotype did not increase the risk for endometriosis. In our study, GSTM1 and CY1PA1 genes were studied in relation to breast, gastric, endometrial, testicular, parotis, pancreatic, lymph node, tyroid, and lung cancer risks. According to the results of the present study, there was a relation between the type of genetic aberration in a gene and the resulting cancer, and furthermore, there is a causal relation between the altered gene, the tissue type and the type of cancer.

Genetic polymorphismsPolymorphisms in the estrogen metabolizing enzymes may define subpopulations of women that are more susceptible to breast cancer development. A polymorphism is a common mutation in the gene that is present in at least 2% of the Turk population. the contribution of one single gene containing a polymorphism might not increase the relative risk for breast cancer, but a combination of polymorphisms might be associated with a small to moderate increased risk for breast cancer. these genes are also called low penetrance genes (3, 21). Effects of the polymorphisms are difficult to investigate since a large study population is needed to filter out subtle effects on, for instance, breast cancer risk. in addition,

comparison of the various studies are further complicated because polymorphisms are mostly ethnically related (21). in this study, significant relations were found between CYP1A1 and GSTM1 polymorphisms in tissues with solid tumours.

Histopathologic and Molecular Genetic Assaysit is critical for us to detect cancer, distinguish cancerous structures from benign and healthy ones and identify malignancy level. Breast cancer is the most common cancer and the second leading cause for cancer associated deaths among turk females (3). the current incident rates predict that 1 in 10 women in turkey will develop breast cancer. currently, long-term survival is approximately 70%. Early diagnosis of breast cancer is crucial and the diagnosis and staging for prognosis is based on histopathological examination and grading of surgically removed breast tissue and axillary lymph nodes. Prognostic analysis of breast cancer in individual patients currently depends on established clinical, and laboratory parameters such as histopatological grading and hormonal receptor status of individual tumor tissues. lung cancer accounts for 19% of all types of cancer and a mortality rate of 29% for all cancer related deaths, particularly in men, and is second only to breast cancer in women. the major cause of lung cancer is tobacco smoking, which is also associated with the risk of cancer of larynx, mouth, esophagus, urinary bladder and kidney (21). it has been found that the progression of precancerous lesions to gastric cancer is associated with cigarette smoking, alcohol drinking, and Helicobacter pylori infection in high incidence areas of china. individual susceptibility to cancer may be partly due to exposure to environmental risk factors, which can be partly explained by genetic variability in metabolic activities related to phases i and II detoxification enzyme pathways. Polymorphisms in these metabolic susceptible genes have been linked to increased risk of cancer in several case-control studies (8).

Oncogenes and proto-oncogenes are DNA sequences that encode factors that drive the cell cycle and include growth factors, their ligands, internal signaling pathway protein kinases, cyclins, cyclin associated kinases, and DNA transcription factors, many of which can be demonstrated in tumor tissues. conversely, anti-oncogene or tumor suppressor gene protein products retard or inhibit the cell cycle or activate pathways that lead to programmed cell death (apoptosis). loss of suppressor gene product or inactivation by mutation of both alleles will favor cell proliferation and malignancy. these proteins can also be detected in tissues by immunohistochemical and molecular analytic methods. the aim of our work was to evaluate the association between CYP1A1 and GSTM1 genetic polymorphisms and activities of genes indicative for susceptibility to various cancer types in turkey population. the polymorphic inheritance of human drug-metabolizing enzymes, such as those encoded by the GST and CYP systems, has been implicated in all cancer risk assessment and prognosis. the study population consisted of 80 different incident cancer cases and 100 healthy controls. Genotyping analyses were performed by PCR based methods.


ConclusionsAmong our population GSTM1 was associated with breast, gastric, endometrial, testicular, ileum, pancreatic, tyroid, prostate and lung cancer risk in turk population. the analysis of patients by histological types of different cancer showed no association between histopathologic types of cancer and CYP1A1 gene polymorphism (p=0.6). Genetic researches using specific biomarkers are expected to be helpful in monitoring the risks of all cancer types. Furthermore, in this study the use of PCR to detect specific mutations identified in the primary tumor in the histopathologically negative surgical margin could be also useful.

AcknowledgmentsWe thank Dr Sahande elagoz and her working fellowships (University of Cumhuriyet, Medical Faculty, Department of Pathology, Sivas, Turkey) for assistance in case recruitment. We thank Dr. hasibe cingilli Vural’s working team and Archeametry and Biotechnology laboratory workers, her and Dr. nesrin turaclar laboratory for molecular genetic techniques.

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study on polymorphism and activities of gstm1 … · study on polymorphism and activities of gstm1...

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