Genetic Polymorphisms of Glutathione S -Transferase P1 and Bladder Cancer Susceptibility in a Chinese Population

Abstract

Background: Glutathione S-transferase P1 (GSTP1) is an important phase II enzyme involved in the metabolism of xenobiotics and whose polymorphisms have been related to individual cancer risks. The objective of this study was to explore the association of GSTP1 A1578G (Ile105Val) polymorphism with susceptibility and clinicopathologic characteristics of bladder cancer in a Chinese population. Methods: We investigated the distribution of GSTP1 A1578G polymorphism in 200 bladder cancer patients and in 200 age- and sex-matched cancer-free controls. The polymorphisms were analyzed using polymerase chain reaction-restriction fragment length polymorphism assay. Genotype and allele frequencies and their associations with demographic factors, smoking status, stage, grade, and histological type of bladder cancer were investigated. Results: The prevalence of GSTP1 GG genotype in cases was 22.5%, compared with 13.5% in the control group (odds ratio = 1.86, 95% confidence interval = 1.10-3.14, p = 0.02). There was an association between smoking status and bladder cancer (odds ratio = 1.77, 95% confidence interval = 1.17-2.67, p = 0.007). When stratifying by the stage, grade, and histological type of bladder cancer, we found no statistical association. Conclusions: These data seem to indicate that the GSTP1 GG genotype is associated with a modest increase in the risk of bladder cancer in a Chinese population.

Introduction

An estimated 357,000 cases of bladder cancer occurred worldwide in 2002, making this the ninth most common cause of cancer for both sexes combined. Bladder cancer is the second most common genitourinary malignant disease in the United States, with an expected 69,000 newly diagnosed cases in 2008, and 14,000 deaths (Jemal et al., 2008). It has been reported in literature that tobacco is one of the main risk factors for bladder cancer; approximately half of the cases of male urinary tract cancer and one-third of the cases of female urinary tract cancer could be attributable to cigarette smoking (Zeegers et al., 2000). Risk factors for the development of bladder cancer can be classified into three subsets: genetic and molecular abnormalities, chemical or environmental exposures, and chronic irritation (Williams and Stein, 2004; Bryan et al., 2005a, 2005b; Kaufman et al., 2009). Several genetic susceptibility factors have been studied in relation to bladder cancer (Mitra et al., 2006; Wallerand et al., 2008).

Glutathione S-transferases (GSTs) are a family of phase II enzymes that catalyze the conjugation of many endogenous and exogenous electrophilic compounds to glutathione. GSTs play an important role in the protection of cells from the products of oxidative stress, as well as from several environmental carcinogens (Hayes and Pulford, 1995; Strange and Fryer, 1999). In humans, eight distinct gene families encode soluble GSTs; among them, four are mainly expressed in human tissues: GSTA, GSTM, GSTT, and GSTP (Strange et al., 2001). A recent meta-analysis of GSTT1 and bladder cancer, including 37 studies (6986 cases and 9166 controls), found that GSTT1 null status is associated with a modest increase in the risk of bladder cancer (Zeng et al., 2010). There also has been one meta-analysis that suggested that GSTM1 null status was associated with a modest increase in the risk of bladder cancer. A previous meta-analysis of 16 studies, 4273 cases and 5081 controls, suggested that GSTP1 A1578G (Ile105Val) was associated with the risk of bladder cancer (Kellen et al., 2007).

The objective of this study was to explore the association of GSTP1 A1578G polymorphism with susceptibility and clinicopathologic characteristics of bladder cancer in a Chinese population.

Materials and Methods

Study population

The subjects studied were 200 bladder cancer patients and 200 age- and sex-matched cancer-free controls who were enrolled in the Second Affiliated Hospital of Chongqing Medical University in Southwest China, from March 2008 to March 2010. The Chinese Han population was collected from the same geographic region. Informed consents were obtained according to the Declaration of Helsinki. Informed consent was obtained from both groups that participated in this study. The protocol was approved by the Ethical Committee of the Second Affiliated Hospital of Chongqing Medical University. All cases were confirmed by clinical histopathology and staged according to the TNM staging system of the Union Internationale Contre le Cancer. Tumors were graded according to the World Health Organization classification. Smoking habit was defined as nonsmoker (smoked <100 cigarettes in lifetime) and smoker.

DNA extraction and genotyping

Blood samples were obtained from peripheral veins of patients and controls. Genomic DNA isolation was performed by following a standard procedure for phenol-chloroform extraction and ethanol precipitation. Genomic DNA samples were stored at −20°C until use. The GSTP1 A1578G genotype was determined by polymerase chain reaction (PCR)-restriction fragment length polymorphism assay. The following primers were used: 5'-ACCCCAGGGCTCTATGGGAA-3' and 5'-TGAGGGCACAAGAAGCCCT-3'. PCR conditions were 95°C for 3 min, followed by 30 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, with a final extension at 72°C for 5 min. For restriction fragment length polymorphism, the PCR products were digested overnight with 5 units of restriction enzyme Alw26I (Promega, Madison, WI), which distinguishes between the Ile allele and the Val allele. The digested PCR products were analyzed on a premade 4% NuSieve 3:1 Plus agarose gel stained with ethidium bromide (BioWhittaker Molecular Applications, Rockland, ME) and photographed under UV light.

Statistical analysis

The associations between GSTP1 and risks of bladder cancer were estimated with the odds ratio (OR) and 95% confidence interval (CI) derived from logistic regression analysis in SAS (SAS, Cary, NC). Comparison of GSTP1 A1578G genotype frequencies between study and control groups was assessed by chi-square test. Statistical significance was set at p < 0.05. All statistical tests were two sided. Genotype distribution and allele frequencies were compared with the Hardy-Weinberg equilibrium model using the Pearson χ² test.

Results

Subject characteristics

The characteristics of the study population are shown in Table 1. The mean ages were 54.6 years for bladder cancer patients and 63.8 years for controls. As expected, cases and controls did not differ in age (p = 0.37) and gender (p = 0.84). Cases were higher in percentage of former or current cigarette smokers than controls but lower in percentage of never-smokers. There was an association between smoking status and bladder cancer (OR = 1.77, 95% CI = 1.17-2.67, p = 0.007). Among 200 case patients, 147 (73.5%) were invasive (T2-T4) bladder cancer, 53 (26.5%) were superficial (Tis-T1) bladder cancer, 152 (76.0%) were high-grade (G2+G3) bladder cancer patients, and 48 (24.0%) were low-grade (G1) case patients. For the histological type of these patients, 164 (82.0%) were papillary bladder cancer and 36 (18.0%) were nonpapillary bladder cancer. Genotype and allele frequencies were in Hardy-Weinberg equilibrium (p = 0.18).

Table 1.

Characteristics and Risk Factors in Patients with Bladder Cancer and Controls

Variations	Bladder cancer	Controls	p
Subjects (n)	200	200
Gender (male/female)	105/95	103/97	0.84
Age (years)	54.6 ± 8.3	53.8 ± 9.5	0.37
Smoking status (smokers/nonsmokers)	85/115	59/141	0.007
Stage (%)
Invasive (T2-T4)	147 (73.5)
Superficial (Tis-T1)	53 (26.5)
Grade (%)
High (G2+G3)	152 (76.0)
Low (G1)	48 (24.0)
Histological type (%)
Papillary	164 (82.0)
Nonpapillary	36 (18.0)

The prevalence of GSTP1 GG genotype in cases was 22.5%, compared with 13.5% in the control group (OR = 1.86, 95% CI = 1.10-3.14, p = 0.02) (Table 2). When stratifying by the stage, grade, and histological type of bladder cancer, we found no statistical association (Table 3).

Table 2.

Genotype and Allele Frequencies of Glutathione S-Transferase P1 A1578G Polymorphism Among Bladder Cancer Patients and Controls

Genotypes	Cases (%)	Controls (%)	OR (95% CI)	p
AA	83 (41.5)	92 (46.0)	0.83 (0.56, 1.24)	0.37
AG	72 (36.0)	81 (40.5)	0.83 (0.55, 1.24)	0.36
GG	45 (22.5)	27 (13.5)	1.86 (1.10, 3.14)	0.02
A allele frequency	238 (59.5)	265 (66.3)	0.75 (0.56, 1.00)	0.05
G allele frequency	162 (40.5)	135 (33.7)	1.34 (1.00, 1.78)	0.05

CI, confidence interval; OR, odds ratio.

Table 3.

Stratification Analysis of Glutathione S-Transferase P1 A1578G Polymorphism in Bladder Cancer Patients

		AA			AG			GG
Variations	Cases (n = 200)	n (%)	OR (95% CI)	p	n (%)	OR (95% CI)	p	n (%)	OR (95% CI)	p
Stage	200	83 (41.5)	1 (reference)		72 (36.0)	1 (reference)		45 (22.5)	1 (reference)
Invasive	147	60 (40.8)	0.97 (0.63, 1.50)	0.90	52 (35.4)	0.97 (0.62, 1.52)	0.90	35 (23.8)	1.08 (0.65, 1.78)	0.78
Superficial	53	23 (43.4)	1.08 (0.59, 1.99)	0.80	20 (37.7)	1.08 (0.58, 2.01)	0.82	10 (18.9)	0.80 (0.37, 1.72)	0.57
Grade	200	83 (41.5)	1 (reference)		72 (36.0)	1 (reference)		45 (22.5)	1 (reference)
High	152	60 (39.5)	0.92 (0.60, 1.41)	0.70	56 (36.8)	1.04 (0.67, 1.61)	0.87	36 (23.7)	1.07 (0.65, 1.76)	0.79
Low	48	23 (47.9)	1.30 (0.69, 2.44)	0.42	16 (33.3)	0.89 (0.46, 1.73)	0.73	9 (18.8)	0.79 (0.36, 1.76)	0.57
Histological type	200	83 (41.5)	1 (reference)		72 (36.0)	1 (reference)		45 (22.5)	1 (reference)
Papillary	164	69 (42.1)	1.02 (0.67, 1.56)	0.91	61 (37.2)	1.05 (0.69, 1.62)	0.81	34 (20.7)	0.90 (0.54, 1.49)	0.68
Nonpapillary	36	14 (38.9)	0.90 (0.43, 1.86)	0.77	11 (30.6)	0.78 (0.36, 1.68)	0.53	11 (30.6)	1.52 (0.69, 3.32)	0.30

Discussion

We attempted to explore the association of GSTP1 A1578G polymorphism with susceptibility and clinicopathologic characteristics of bladder cancer in a Chinese population. We investigated the distribution of GSTP1 A1578G polymorphism in 200 bladder cancer patients and in 200 age- and sex-matched cancer-free controls. In our study, we found that the GSTP1 GG genotype is associated with a modest increase in the risk of bladder cancer in a Chinese population.

Cigarette smoking is the predominant risk factor for bladder cancer (Cohen et al., 2000; Pashos et al., 2002; Zeegers et al., 2004). Systematic review and meta-analysis by Zeegers et al. (2000) concluded that current cigarette smokers have an approximately threefold higher risk of urinary tract cancer than nonsmokers. In Europe, approximately half of urinary tract cancer cases among males and one-third of cases among females might be attributable to cigarette smoking (Zeegers et al., 2000). Carcinogens such as aminobiphenyls found in tobacco have been implicated in bladder cancer etiology in smokers (Probst-Hensch et al., 2000). Although many people are exposed to this risk factor, bladder cancer develops in only a fraction of these individuals, which suggests variation in individual susceptibility to bladder carcinogenesis (Manolio et al., 2008; Rahim et al., 2008). A genetic mechanism that could account for this variability is the activity of polymorphically expressed xenobiotic-metabolizing enzymes that activate procarcinogens or, conversely, detoxify carcinogens. In our study, there was an association between smoking status and bladder cancer.

The mechanism of the association between GSTP1 A1578G (Ile105Val) polymorphism and bladder cancer remains relatively unclear. GSTP1 is an important phase II enzyme involved in the metabolism of xenobiotics. GSTP1 exhibits a polymorphism within its coding region, which leads to reduced enzyme activity and can be related to individual cancer risk. GSTP1 A1578G polymorphism was associated with elevated breast cancer risk in a race-specific manner (Egan et al., 2004; Lee et al., 2008; Sergentanis and Economopoulos, 2010). GSTP1 A1578G polymorphism also may be associated with gastric cancer among Caucasians (Zhou et al., 2009). The varied G allele of GSTP1 may be considered as a factor contributing to increased susceptibility of oral cancer in Chinese (Chen et al., 2010). A potential effect modification by the GSTP1 Ile/Ile genotype on smoking and the risk of prostate cancer is suggested (Mao et al., 2004). GSTP1 Val/Val genotype was a potential risk modifier in lung cancer development (Reszka et al., 2003). Although these studies supported our results, functional studies of the GSTP1 need further research.

Although numerous studies have investigated the relationship between GSTP1 A1578G polymorphism and bladder cancer, the results remain controversial and ambiguous. Some studies support the relationship between GSTP1 A1578G polymorphism and bladder cancer. In a French cohort, Fontana et al. (2009) found that GSTP1 A1578G variants showed a marginally increased risk of bladder cancer. The study by Ma et al. (2002) showed that GSTP1 AG or GG genotype had a higher frequency in the patients with benzidine-related occupational bladder cancer in a Shanghai (China) population. But other studies do not support the relationship. The case-control study by Grando et al. (2009) found no association between GSTP1 A1578G polymorphism and bladder cancer in a Brazilian population. A study by Altayli et al. (2009) found no association between GSTP1 A1578G polymorphism and bladder cancer in the Turkish population. A study in Germany by Kopps et al. (2008) indicated that on exposure to aromatic amines the GSTP1 A1578G polymorphism did not appear to play a significant role as a predisposing factor for bladder cancer incidence. The results vary widely and are often discordant likely because of ethnic and geographic differences of the enrolled subjects. Other possible reasons for such a discrepancy include the frequency of the polymorphism in the population studied or linkage disequilibrium with other, perhaps undiscovered, functional single-nucleotide polymorphisms in the GSTP1 gene.

There are shortcomings in our study. First of all, this is a hospital-based case-control study, selection bias may not be avoidable, and the subjects may not be representative of the general population. Second, only one single-nucleotide polymorphism was investigated, so we could not get more information about gene-gene or gene-environment interactions for bladder cancer. Finally, as only a Chinese population was studied, it does not permit extrapolation of the results to other ethnic groups.

In conclusion, our data seem to indicate that the GSTP1 GG genotype is associated with a modest increase in the risk of bladder cancer in a Chinese population. However, currently, we have no indication as to the meaning and biological events related to this finding; functional studies and the role of GSTP1 need further research in the field of bladder cancer.

Footnotes

Acknowledgments

The authors thank Dr. Yanjun Lin (Department of Urology, the Second Affiliated Hospital, Chongqing Medical University) for assisting in data collection and preparation of this article.

Disclosure Statement

No competing financial interests exist.

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