Original Article Oncol Res Treat 2014;37:164–169 DOI: 10.1159/000361083
Received: January 20, 2014 Accepted: March 4, 2014 Published online: March 21, 2014
Glutathione S-Transferase T1 and M1 Polymorphisms Are Associated with Lung Cancer Risk in a Gender-Specific Manner Chunguo Pana Genhua Zhub Zhihong Yanb Yi Zhoua Zhihua Liua a
Radiotherapy Department of Jiangxi Cancer Hospital, Key Laboratory of Modern Preparation of TCM, Ministry of Education, Jiangxi University of Traditional Chinese Medicine, Nanchang City, Jiangxi Province, China b
Summary Background: Glutathione S-transferase (GST) T1 and M1 are detoxification enzymes which neutralize various carcinogenic compounds. Polymorphisms of the GSTT1 and GSTM1 genes which encode the enzymes could be associated with cancer risk. Patients and Methods: We investigated the association of GSTT1 and GSTM1 null polymorphisms with lung cancer risk in a tightly matched, considerably large sample in China. Genotyping was performed utilizing polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) analysis and DNA sequencing, and associations were measured by using logistic regression analysis. Results: We found that the null genotypes of both polymorphisms resulted in an increased lung cancer risk, with an odds ratio (OR) of 1.57 (95% confidence interval (CI) 1.23–2.00) for GSTT1 and OR 1.87 (95% CI 1.46–2.39) for GSTM1 (p < 0.01). Significant associations remained after stratification by histopathology (p < 0.01) and smoking status (p < 0.05). When gender-stratified association was performed, we found that the GSTT1 null genotype resulted in an increased risk among males (adjusted OR 2.95, 95% CI 2.07–4.20; p < 0.01) but not females (p > 0.05), while the GSTM1 null genotype resulted in an increased risk among females (adjusted OR 2.18, 95% CI 1.54–3.10; p < 0.01) but not males (p < 0.05). Conclusion: Polymorphisms in GSTT1 and GSTM1 are associated with the risk of lung cancer in a gender-specific manner.
C.P., G.Z., and Z.L. contributed equally to this research
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Introduction Lung cancer is a leading malignancy worldwide. The incidence of lung cancer has been steadily increasing over the past decade, and has contributed to a significant burden of cancer-related deaths [1]. Lung cancer is typically asymptomatic in its early stages. Therefore, in most patients, the disease has progressed to an advanced stage at the time of diagnosis. The lack of effective treatment strategies for these late-stage patients leads to a low average 5-year survival rate of about 16% [2]. As such, there is a need to screen for biomarkers which could help to identify individuals at risk of lung cancer so that appropriate strategies could be implemented to achieve cancer prevention in these individuals. Exposure to environmental carcinogens, such as those contained in tobacco smoke, represents a major risk factor for the occurrence of lung cancer. These environmental carcinogens may interact with genetic factors, and since the genetic makeup of each individual is unique, such a gene-environment interaction results in different effects in different individuals [3]. Genetic variations therefore play an equally important role in contributing to individual differences in lung cancer risk. Since lung cancer is primarily caused by environmental carcinogens, potential candidate genes whose variations could influence lung cancer risk include those involved in the detoxification of xenobiotics. Glutathione S-transferases (GSTs) are a group of phase II detoxification enzymes which act by conjugating harmful compounds to glutathione to neutralize the former [4]. Substrates of GSTs include the various carcinogenic chemical compounds present in the diet and tobacco smoke [5]. Inability of these enzymes to function well could therefore lead to a higher chance of developing cancers, especially those which are associated with exposure to environmental carcinogens.
Zhihua Liu, MD No. 519, Beijing Road Nanchang City, Jiangxi Province, 330029 China [email protected]
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Keywords Lung cancer · Glutathione · Risk factors · Polymorphism
2 GST polymorphisms, namely GSTT1 and GSTM1 null, have been shown to result in the elimination of the activity of GSTT1 and GSTM1 enzymes, respectively [6, 7]. These polymorphisms could therefore be associated with the risk of lung cancer, although several previous studies investigating this hypothesis have yielded contradictory results [8–12]. It is also possible that genetic variations confer their effects in a genderspecific manner [13], and hence, there is a need to stratify the analysis by sex. The above issues call for further studies involving reasonably large sample sizes and properly matched controls, with gender-based stratification of the results, to add valuable information to the currently available data on this subject. In this study, we investigated the association of GSTM1 and GSTT1 null polymorphisms (both singly and in combination) with the risk of lung cancer among Chinese subjects, employing a well-matched case-control study population with a considerably large sample size, and stratified our findings by sex.
Materials and Methods Study Subjects and Samples A total of 1,046 study subjects were included in this population-based case-control study, comprising an equal number of histopathologically confirmed lung cancer patients and tightly matched cancer-free controls (523 subjects per group). The subjects were recruited from Jiangxi Cancer Hospital from July 2008 to June 2013. All subjects were of Chinese Han ethnicity. Controls were healthy volunteers recruited from the general population, who were ascertained to be free from cancer and any other
Table 1. Characteristics of the study subjects Age, years Range Mean ± SD Median Sex, n (%) Male Female Smoking, n (%) Smoker Never smoker Histopathology, n (%) ADC SCC
Cases
Controls
42–73 57.4 ± 9.2 57
42–73 57.4 ± 9.2 57
265 (50.7) 258 (49.3)
265 (50.7) 258 (49.3)
239 (45.7) 284 (54.3)
239 (45.7) 284 (54.3)
243 (46.5) 380 (53.5)
N/A N/A
1.00
1.00 1.00 N/A
Table 2. Association of GSTT1 and GSTM1 polymorphisms with lung cancer risk
GSTT1 Non-null Null GSTM1 Non-null Null
n (%)
OR (95% CI)
p
cases
controls
253 (48.4) 270 (51.6)
311 (59.5) 212 (40.5)
1 1.57 (1.23–2.00)
< 0.01
218 (41.7) 305 (58.3)
299 (57.2) 224 (42.8)
1 1.87 (1.46–2.39)
< 0.01
OR = Odds ratio; CI = confidence interval.
GSTT1 and GSTM1 Polymorphisms and Lung Cancer Risk
Statistical Analysis SPSS version 19 (IBM Corp., Armonk, NY, USA) was used in all analyses. Chi-square test (for categorical variables) and t-test (for continuous variables) were used to calculate differences in age, gender, and smoking habits between cases and controls. Binary logistic regression analysis was used to obtain the crude and adjusted odds ratios (ORs) for measuring the association between the GSTT1 and GSTM1 null polymorphisms and lung cancer risk. For stratification analysis, the subjects were grouped according to sex and smoking status, and analyzed using the binary logistic regression model. p values of less than 0.05 were significant.
p
SD = Standard deviation; ADC = adenocarcinoma; SCC = squamous cell carcinoma; N/A = not applicable.
Genotype
Genotyping of GSTT1 and GSTM1 Polymorphisms Multiplex polymerase chain reaction (PCR) was used to genotype the GSTT1 and GSTM1 null polymorphisms as previously described [14]. The following primers were used: 5’-TTCCTTACTGGTCCTCACATCTC-3’, 5’-TCACCGGATCATGGCCAGCA-3’, 5’-GAACTCCCTGAAAAGCTAAAGC-3’, and 5’-GTTGGGCTCAAATATACGGTGG-3’. The first 2 primers amplified a 459-bp region containing a GSTT1 poly morphism, and the last 2 amplified a 219-bp region containing GSTM1 polymorphisms. Amplification of the beta-globin gene (268 bp) was used as internal control, with the following primers: 5’-CAACTTCATCCACGTTCACC-3’ and 5’-GAAGAGCCAAGGACAGTTAC-3’. Multiplex PCR was performed at an annealing temperature of 57.5 °C. The null and non-null genotypes of the genes studied were determined based on the absence or presence of the bands of interest on agarose gel, and confirmed by sequencing.
Results Characteristics of Patients and Controls Cases and controls were tightly matched in terms of age and sex, and therefore shared exactly identical characteristics (table 1). In each group, 265 (50.7%) subjects were males and 258 (49.3%) were females. The age of the subjects ranged from 42 to 73 years old, with a mean of 57.4 ± 9.2 years and a median of 57 years. 239 (45.7%) subjects were smokers, and the remaining 284 (54.3%) were never smokers. No difference was observed between the 2 groups of study subjects in terms of age, sex, and smoking status (p = 1.00). For cases, 243 (46.5%) were adenocarcinoma (ADC) by histopathology, and the remaining 380 (53.5%) were squamous cell carcinoma (SCC). Association between GSTT1 and GSTM1 null Polymorphisms and Lung Cancer Risk Table 2 shows the frequency and associated risk of GSTT1 and GSTM1 null polymorphisms with lung cancer risk in the population studied. The frequency of the non-null genotype of the GSTT1 polymorphism was 48.4% in cases and 59.5% in
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Characteristic
disease through general medical screening. 1 healthy, cancer-free control was selected to be individually matched to each case by age, sex, and smoking status. Therefore, cases and controls were exactly identical in gender distribution, age, and smoking behavior. Cases who had a previous history of other cancers, and controls who had a history of any cancer, were excluded from the study. Blood samples were obtained from each subject with informed consent, and DNA was isolated using a commercial kit. The study protocol was approved by the institutional review board of Jiangxi Cancer Hospital.
Association of GSTT1 and GSTM1 Polymorphisms with ADC and SCC of the Lung The association between GSTT1 and GSTM1 polymorphisms and ADC and SCC of the lung is shown in table 3. Among ADC patients, 48.6% had the non-null genotype and 51.4% had the null genotype for the GSTT1 polymorphism; for the GSTM1 polymorphism, 42.0% had the non-null genotype and 58.0% had the null genotype. Among SCC patients, the frequency of subjects with the non-null and the null genotype of the GSTT1 polymorphism was 48.2 and 51.8%, respectively; for the GSTM1 polymorphism, the frequency was 41.4 and 58.6%, respectively. In the control group, 59.5 and 40.5% of the subjects had the non-null and the null genotype of the GSTT1 polymorphism, respectively, whereas for the GSTM1 polymorphism, the frequency of non-null and null genotypes
Table 3. Stratification of GSTT1 and GSTM1 risk association by histopathology Genotype
n (%)
GSTT1 (ADC) Non-null Null GSTT1 (SCC) Non-null Null GSTM1 (ADC) Non-null Null GSTM1 (SCC) Non-null Null
p
cases
controls
Crude OR (95% CI)
118 (48.6) 125 (51.4)
311 (59.5) 212 (40.5)
1 1.55 (1.14–2.11)
< 0.01
135 (48.2) 145 (51.8)
311 (59.5) 212 (40.5)
1 1.58 (1.18–2.11)
< 0.01
102 (42.0) 141 (58.0)
299 (57.2) 224 (42.8)
1 1.85 (1.36–2.51)
< 0.01
116 (41.4) 164 (58.6)
299 (57.2) 224 (42.8)
1 1.89 (1.41–2.53)
< 0.01
OR = Odds ratio; CI = confidence interval; ADC = adenocarcinoma; SCC = squamous cell carcinoma.
Table 4. Association of combinations of GSTT1 and GSTM1 polymorphisms with lung cancer risk Genotype combination
n (%)
OR (95% CI)
GSTT1
GSTM1
cases
controls
Non-null Null Non-null Null
non-null non-null null null
105 (20.1) 113 (21.6) 148 (28.3) 157 (30.0)
178 (34.0) 121 (23.1) 133 (25.4) 91 (17.4)
1 1.58 (1.11–2.25) 0.01 1.88 (1.35–2.64) < 0.01 2.92 (2.05–4.16) < 0.01
OR = Odds ratio; CI = confidence interval.
166
p
Oncol Res Treat 2014;37:164–169
was 57.2 and 42.8%, respectively. Significant risk association was found for both ADC and SCC of the lung. The GSTT1 null genotype was found to increase the risk of ADC by 1.55fold (95% CI 1.14–2.11; p < 0.01), and that of SCC by 1.58-fold (95% CI 1.18–2.11; p < 0.01). For the GSTM1 polymorphism, the null genotype resulted in an OR of 1.85 (95% CI 1.36–2.51; p < 0.01) for ADC, and 1.89 (95% CI 1.41–2.53; p < 0.01) for SCC. Association of GSTT1 and GSTM1 Combination Genotypes with Lung Cancer Risk We tested the association of various combinations of GSTT1 and GSTM1 genotypes with the risk of lung cancer. The results are summarized in table 4. Significant associations were present for all the possible combinations. A total of 21.6% of cases and 23.1% of controls had GSTT1 null and GSTM1 non-null genotypes, resulting in an OR of 1.58 (95% CI 1.11–2.25; p = 0.01). On the other hand, 28.3% of cases and 25.4% of controls had GSTT1 non-null and GSTM1 null genotypes, resulting in an OR of 1.88 (95% CI 1.35–2.64; p < 0.01). 30% of cases and 17.4% of controls had the null genotype of both the GSTT1 and the GSTM1 polymorphism, and this gave an OR value of 2.92 (95% CI 2.05–4.16; p < 0.01). Stratification of the Association between GSTT1 and GSTM1 Polymorphisms and Lung Cancer Risk by Smoking Status We also stratified the association between the 2 polymorphisms and lung cancer risk according to smoking status (table 5). For the GSTT1 polymorphism, among smokers, 49.0 and 51.0% of cases had non-null and null, respectively, while the frequencies were 58.6 and 41.4% for controls. Among never smokers, 47.9 and 52.1% of cases and 60.2 and 39.8% of controls had the non-null and null genotype of the GSTT1 polymorphism, respectively. For the GSTM1 polymorphism, among smokers, 43.5 and 56.5% of cases had the non-null and null genotype, respectively, while the frequencies were 57.3 and 42.7% for controls. For non-smokers, 40.1 and 59.9% of cases and 57.0 and 43.0% of controls had the non-null and null genotype of the GSTM1 polymorphism. Significant association was observed between the polymorphisms and lung cancer risk, both among smokers and never smokers. For smokers, the null genotype of the GSTT1 polymorphism had a crude OR of 1.47 (95% CI 1.03–2.12; p = 0.04), whereas for never smokers, the crude OR was 1.65 (95% CI 1.18–2.30; p < 0.01). Adjustment for age, sex, and smoking status revealed a similar observation. For smokers, the adjusted OR of the null genotype was 1.45 (95% CI 1.01–2.09; p = 0.05), while for never smokers, the OR was 1.55 (95% CI 1.11–2.16; p = 0.01). For the GSTM1 polymorphism, the null genotype resulted in a crude OR of 1.74 (95% CI 1.21–2.50; p < 0.01) among smokers (adjusted OR 1.80, 95% CI 1.26–2.59; p < 0.01), and 1.98 (95% CI 1.42–2.77; p < 0.01) among never smokers (adjusted OR 1.93, 95% CI 1.38–2.70; p < 0.01).
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controls. For the variant null genotype, the frequency was 51.6% in cases and 40.5% in controls. The null genotype was significantly associated with the risk of lung cancer, with OR 1.57 (95% confidence interval (CI) 1.23–2.00; p < 0.01). In the case of the GSTM1 polymorphism, the frequency of the nonnull genotype was 41.7% in cases and 57.2% in controls, whereas the frequency of the null genotype was 58.3% in cases and 42.8% in controls. The null genotype of the polymorphism resulted in an increased lung cancer risk, with OR 1.87 (95% CI 1.46–2.39; p < 0.01).
Table 5. Smoking status-stratified association of GSTT1 and GSTM1 polymorphisms with lung cancer risk Genotype GSTT1 (smokers) Non-null Null GSTT1 (never smokers) Non-null Null GSTM1 (smokers) Non-null Null GSTM1 (never smokers) Non-null Null
n (%) cases
controls
Crude OR (95% CI)
p
Adjusted OR (95% CI)
p
117 (49.0) 122 (51.0)
140 (58.6) 99 (41.4)
1 1.47 (1.03–2.12)
0.04
1.45 (1.01–2.09)
0.05
136 (47.9) 148 (52.1)
171 (60.2) 113 (39.8)
1 1.65 (1.18–2.30)
< 0.01
1.55 (1.11–2.16)
0.01
104 (43.5) 135 (56.5)
137 (57.3) 102 (42.7)
1 1.74 (1.21–2.50)
< 0.01
1.80 (1.26–2.59)
< 0.01
114 (40.1) 170 (59.9)
162 (57.0) 122 (43.0)
1 1.98 (1.42–2.77)
< 0.01
1.93 (1.38–2.70)
< 0.01
OR = Odds ratio; CI = confidence interval.
Table 6. Gender-stratified association of GSTT1 and GSTM1 polymorphisms with lung cancer risk Genotype GSTT1 (male) Non-null Null GSTT1 (female) Non-null Null GSTM1 (male) Non-null Null GSTM1 (female) Non-null Null
n (%) cases
controls
Crude OR (95% CI)
p
Adjusted OR (95% CI)
p
93 (35.1) 172 (64.9)
167 (63.0) 98 (37.0)
1 3.15 (2.21–4.50)
< 0.01
1 2.95 (2.07–4.20)
< 0.01
160 (62.0) 98 (38.0)
144 (55.8) 114 (44.2)
1 0.77 (0.54–1.10)
0.15
1 0.75 (0.46–1.23)
0.26
122 (46.0) 143 (54.0)
144 (54.3) 121 (45.7)
1 1.39 (0.99–1.96)
0.06
1 1.48 (0.92–2.37)
0.11
96 (37.2) 162 (62.8)
155 (60.1) 103 (39.9)
1 2.54 (1.78–3.62)
< 0.01
1 2.18 (1.54–3.10)
< 0.01
OR = Odds ratio; CI = confidence interval.
GSTT1 and GSTM1 Polymorphisms and Lung Cancer Risk
type in females, with a crude OR of 2.54 (95% CI 1.78–3.62; p < 0.01) and an adjusted OR of 2.18 (95% CI 1.54–3.10; p < 0.01).
Discussion GSTs are a family of cytosolic enzymes which neutralize environmental carcinogens, such as polycyclic aromatic hydrocarbons. GSTM1 and GSTT1 null polymorphisms have been shown to cause elimination of GSTM1 and GSTT1 activity, respectively. Interindividual variability in enzyme activity due to these polymorphisms has been thought to confer different risks of various cancers to their carriers. The same risk modification effect has also been hypothesized for lung cancer, although the results obtained in different studies have been inconsistent. These conflicting findings could be attributed to the different characteristics of the study subjects included in the analysis, and to improper case-control matching. In this study, we sought to investigate the associations of the polymorphisms with the risk of lung cancer among Chinese subjects, and conducted a 1:1 case-control match in terms of age, gender, and smoking status. Based on the genotypes, subjects were categorized into non-null and null groups,
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Gender-Specific Association of GSTT1 and GSTM1 Polymorphisms with Lung Cancer Risk We further tested the presence of a gender-specific association between GSTT1 and GSTM1 polymorphisms and lung cancer risk (table 6). For the GSTT1 polymorphism, 35.1 and 64.9% of male cases had non-null and null, respectively. The frequencies of the genotypes in male controls were 63.0 and 37.0%. For females, the frequencies of non-null and null genotypes were 62.0 and 38.0% in cases, and 55.8 and 44.2% in controls. The null genotype was found to result in a significantly higher risk of lung cancer in males, with a crude OR of 3.15 (95% CI 2.21–4.50; p < 0.01) and an adjusted OR of 2.95 (95% CI 2.07–4.20; p < 0.01). However, no significant association was found in females (p > 0.05). For the GSTM1 polymorphism, the frequency of non-null and null genotypes in male cases was 46.0 and 54.0%, respectively. The frequency of non-null and null genotypes in male controls was 54.3 and 45.7%, respectively. No significant lung cancer risk association was found (p < 0.05). However, the null genotype was overrepresented in female cases. For the non-null genotype, the frequencies in female cases and controls were 37.2 and 60.1%, but for the null genotype, the frequency was 62.8% in female cases and only 39.9% in female controls. This resulted in a significant lung cancer risk association of the null GSTM1 geno-
where the former included both individuals who were homozygous wild type and those who were heterozygous for the polymorphisms. We found that the GSTT1 and GSTM1 null polymorphisms were associated with lung cancer risk in the Chinese population. Our observation for GSTT1 was in agreement with Cao et al. [9], Liang et al. [15], Liu et al. [16], and Yuan et al. [17], who studied the association of the polymorphisms with lung cancer risk in the same population. However, Chan-Yeung et al. [10] reported the contradictory finding of the GSTT1 polymorphism actually reducing the risk of lung cancer, while several other authors, such as Bai et al. [11] and Wang et al. [18], reported no association between the GSTT1 polymorphism and lung cancer risk. For the GSTM1 polymorphism, our results concur with those of Li et al. [12], Qian et al. [19], Wang et al. [20], Wang et al. [21], and Liu et al. [22], but are in discordance with those reported by Chan-Yeung et al. [10], Liang et al. [23], Yang et al. [24], Zeng et al. [25], and Ye et al. [26]. In the current study, we also found that the associations between the polymorphisms and lung cancer risk were present when the polymorphisms were analyzed both individually and in combination with one another. After stratification by cancer histology of cases and smoking status of all the subjects, the associations remained significant in all groups. Interestingly, when stratified by smoking status, we observed that the null genotype resulted in a slightly higher risk of lung cancer in never smokers than in smokers. This phenomenon could be explained by the hypothesis that the xenobiotic-metabolizing effect of GST enzymes is less apparent when the carcinogen is present in high doses, as is the case in smokers [27]. However,
after stratification by gender, differential risk association was observed. This suggests that there is a gender-specific association between the polymorphisms and lung cancer risk, which may be explained by higher CYP P450 enzyme activity in females, leading to the formation of high levels of DNA adduct intermediates which are the substrate of the GSTM1 enzyme. The null genotype in females jeopardizes the ability of the enzyme to detoxify the harmful DNA adducts, resulting in a higher risk of cancer [28]. In addition, we propose that hormonal differences between males and females could be an underlying reason for the presence of a gender-specific association. This is the first study reporting a gender-specific association between GSTT1 and GSTM1 polymorphisms and lung cancer risk in the Chinese population. The strengths of the current study include the relatively large sample size employed and the tight matching between cases and controls. In conclusion, GSTT1 and GSTM1 null polymorphisms were associated with lung cancer risk in the Chinese population in a gender-specific manner.
Acknowledgement We thank all doctors and subjects who participated in this study.
Disclosure Statement The authors declare no conflict of interest.
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smoking and susceptibility to lung cancer. Chin J Clin Oncol 2006;33:500–502. 20 Wang N, Zhuang DG, Wu YM: Research on relationship between GSTM1 gene deletion and lung cancer genetic susceptibility. Acta Henan Univ Sci Technol (Med Vol) 2005;23:7–8. 21 Wang DQ, Chen SD, Wang BG, Cai XL, Xian XZ: A case control study on relationship between lung cancer and genetic polymorphism of CYP1A1, CYP2E1 and GSTM1 in Han nationality, in Guangzhou area. Bulletin Chin Cancer 2006;15: 579–582. 22 Liu D, Wang F, Wang Q, Guo X, Xu H, Wang W, Zhang L: Association of glutathione S-transferase M1 polymorphisms and lung cancer risk in a Chinese population. Clin Chim Acta 2012;24:188– 190. 23 Liang GY, Pu YP, Yin LH: Studies of the genes related to lung cancer susceptibility in Nanjing
Received: January 20, 2014 Accepted: March 4, 2014 Published online: March 21, 2014
Glutathione S-Transferase T1 and M1 Polymorphisms Are Associated with Lung Cancer Risk in a Gender-Specific Manner Chunguo Pana Genhua Zhub Zhihong Yanb Yi Zhoua Zhihua Liua a
Radiotherapy Department of Jiangxi Cancer Hospital, Key Laboratory of Modern Preparation of TCM, Ministry of Education, Jiangxi University of Traditional Chinese Medicine, Nanchang City, Jiangxi Province, China b
Summary Background: Glutathione S-transferase (GST) T1 and M1 are detoxification enzymes which neutralize various carcinogenic compounds. Polymorphisms of the GSTT1 and GSTM1 genes which encode the enzymes could be associated with cancer risk. Patients and Methods: We investigated the association of GSTT1 and GSTM1 null polymorphisms with lung cancer risk in a tightly matched, considerably large sample in China. Genotyping was performed utilizing polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) analysis and DNA sequencing, and associations were measured by using logistic regression analysis. Results: We found that the null genotypes of both polymorphisms resulted in an increased lung cancer risk, with an odds ratio (OR) of 1.57 (95% confidence interval (CI) 1.23–2.00) for GSTT1 and OR 1.87 (95% CI 1.46–2.39) for GSTM1 (p < 0.01). Significant associations remained after stratification by histopathology (p < 0.01) and smoking status (p < 0.05). When gender-stratified association was performed, we found that the GSTT1 null genotype resulted in an increased risk among males (adjusted OR 2.95, 95% CI 2.07–4.20; p < 0.01) but not females (p > 0.05), while the GSTM1 null genotype resulted in an increased risk among females (adjusted OR 2.18, 95% CI 1.54–3.10; p < 0.01) but not males (p < 0.05). Conclusion: Polymorphisms in GSTT1 and GSTM1 are associated with the risk of lung cancer in a gender-specific manner.
C.P., G.Z., and Z.L. contributed equally to this research
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Introduction Lung cancer is a leading malignancy worldwide. The incidence of lung cancer has been steadily increasing over the past decade, and has contributed to a significant burden of cancer-related deaths [1]. Lung cancer is typically asymptomatic in its early stages. Therefore, in most patients, the disease has progressed to an advanced stage at the time of diagnosis. The lack of effective treatment strategies for these late-stage patients leads to a low average 5-year survival rate of about 16% [2]. As such, there is a need to screen for biomarkers which could help to identify individuals at risk of lung cancer so that appropriate strategies could be implemented to achieve cancer prevention in these individuals. Exposure to environmental carcinogens, such as those contained in tobacco smoke, represents a major risk factor for the occurrence of lung cancer. These environmental carcinogens may interact with genetic factors, and since the genetic makeup of each individual is unique, such a gene-environment interaction results in different effects in different individuals [3]. Genetic variations therefore play an equally important role in contributing to individual differences in lung cancer risk. Since lung cancer is primarily caused by environmental carcinogens, potential candidate genes whose variations could influence lung cancer risk include those involved in the detoxification of xenobiotics. Glutathione S-transferases (GSTs) are a group of phase II detoxification enzymes which act by conjugating harmful compounds to glutathione to neutralize the former [4]. Substrates of GSTs include the various carcinogenic chemical compounds present in the diet and tobacco smoke [5]. Inability of these enzymes to function well could therefore lead to a higher chance of developing cancers, especially those which are associated with exposure to environmental carcinogens.
Zhihua Liu, MD No. 519, Beijing Road Nanchang City, Jiangxi Province, 330029 China [email protected]
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Keywords Lung cancer · Glutathione · Risk factors · Polymorphism
2 GST polymorphisms, namely GSTT1 and GSTM1 null, have been shown to result in the elimination of the activity of GSTT1 and GSTM1 enzymes, respectively [6, 7]. These polymorphisms could therefore be associated with the risk of lung cancer, although several previous studies investigating this hypothesis have yielded contradictory results [8–12]. It is also possible that genetic variations confer their effects in a genderspecific manner [13], and hence, there is a need to stratify the analysis by sex. The above issues call for further studies involving reasonably large sample sizes and properly matched controls, with gender-based stratification of the results, to add valuable information to the currently available data on this subject. In this study, we investigated the association of GSTM1 and GSTT1 null polymorphisms (both singly and in combination) with the risk of lung cancer among Chinese subjects, employing a well-matched case-control study population with a considerably large sample size, and stratified our findings by sex.
Materials and Methods Study Subjects and Samples A total of 1,046 study subjects were included in this population-based case-control study, comprising an equal number of histopathologically confirmed lung cancer patients and tightly matched cancer-free controls (523 subjects per group). The subjects were recruited from Jiangxi Cancer Hospital from July 2008 to June 2013. All subjects were of Chinese Han ethnicity. Controls were healthy volunteers recruited from the general population, who were ascertained to be free from cancer and any other
Table 1. Characteristics of the study subjects Age, years Range Mean ± SD Median Sex, n (%) Male Female Smoking, n (%) Smoker Never smoker Histopathology, n (%) ADC SCC
Cases
Controls
42–73 57.4 ± 9.2 57
42–73 57.4 ± 9.2 57
265 (50.7) 258 (49.3)
265 (50.7) 258 (49.3)
239 (45.7) 284 (54.3)
239 (45.7) 284 (54.3)
243 (46.5) 380 (53.5)
N/A N/A
1.00
1.00 1.00 N/A
Table 2. Association of GSTT1 and GSTM1 polymorphisms with lung cancer risk
GSTT1 Non-null Null GSTM1 Non-null Null
n (%)
OR (95% CI)
p
cases
controls
253 (48.4) 270 (51.6)
311 (59.5) 212 (40.5)
1 1.57 (1.23–2.00)
< 0.01
218 (41.7) 305 (58.3)
299 (57.2) 224 (42.8)
1 1.87 (1.46–2.39)
< 0.01
OR = Odds ratio; CI = confidence interval.
GSTT1 and GSTM1 Polymorphisms and Lung Cancer Risk
Statistical Analysis SPSS version 19 (IBM Corp., Armonk, NY, USA) was used in all analyses. Chi-square test (for categorical variables) and t-test (for continuous variables) were used to calculate differences in age, gender, and smoking habits between cases and controls. Binary logistic regression analysis was used to obtain the crude and adjusted odds ratios (ORs) for measuring the association between the GSTT1 and GSTM1 null polymorphisms and lung cancer risk. For stratification analysis, the subjects were grouped according to sex and smoking status, and analyzed using the binary logistic regression model. p values of less than 0.05 were significant.
p
SD = Standard deviation; ADC = adenocarcinoma; SCC = squamous cell carcinoma; N/A = not applicable.
Genotype
Genotyping of GSTT1 and GSTM1 Polymorphisms Multiplex polymerase chain reaction (PCR) was used to genotype the GSTT1 and GSTM1 null polymorphisms as previously described [14]. The following primers were used: 5’-TTCCTTACTGGTCCTCACATCTC-3’, 5’-TCACCGGATCATGGCCAGCA-3’, 5’-GAACTCCCTGAAAAGCTAAAGC-3’, and 5’-GTTGGGCTCAAATATACGGTGG-3’. The first 2 primers amplified a 459-bp region containing a GSTT1 poly morphism, and the last 2 amplified a 219-bp region containing GSTM1 polymorphisms. Amplification of the beta-globin gene (268 bp) was used as internal control, with the following primers: 5’-CAACTTCATCCACGTTCACC-3’ and 5’-GAAGAGCCAAGGACAGTTAC-3’. Multiplex PCR was performed at an annealing temperature of 57.5 °C. The null and non-null genotypes of the genes studied were determined based on the absence or presence of the bands of interest on agarose gel, and confirmed by sequencing.
Results Characteristics of Patients and Controls Cases and controls were tightly matched in terms of age and sex, and therefore shared exactly identical characteristics (table 1). In each group, 265 (50.7%) subjects were males and 258 (49.3%) were females. The age of the subjects ranged from 42 to 73 years old, with a mean of 57.4 ± 9.2 years and a median of 57 years. 239 (45.7%) subjects were smokers, and the remaining 284 (54.3%) were never smokers. No difference was observed between the 2 groups of study subjects in terms of age, sex, and smoking status (p = 1.00). For cases, 243 (46.5%) were adenocarcinoma (ADC) by histopathology, and the remaining 380 (53.5%) were squamous cell carcinoma (SCC). Association between GSTT1 and GSTM1 null Polymorphisms and Lung Cancer Risk Table 2 shows the frequency and associated risk of GSTT1 and GSTM1 null polymorphisms with lung cancer risk in the population studied. The frequency of the non-null genotype of the GSTT1 polymorphism was 48.4% in cases and 59.5% in
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Characteristic
disease through general medical screening. 1 healthy, cancer-free control was selected to be individually matched to each case by age, sex, and smoking status. Therefore, cases and controls were exactly identical in gender distribution, age, and smoking behavior. Cases who had a previous history of other cancers, and controls who had a history of any cancer, were excluded from the study. Blood samples were obtained from each subject with informed consent, and DNA was isolated using a commercial kit. The study protocol was approved by the institutional review board of Jiangxi Cancer Hospital.
Association of GSTT1 and GSTM1 Polymorphisms with ADC and SCC of the Lung The association between GSTT1 and GSTM1 polymorphisms and ADC and SCC of the lung is shown in table 3. Among ADC patients, 48.6% had the non-null genotype and 51.4% had the null genotype for the GSTT1 polymorphism; for the GSTM1 polymorphism, 42.0% had the non-null genotype and 58.0% had the null genotype. Among SCC patients, the frequency of subjects with the non-null and the null genotype of the GSTT1 polymorphism was 48.2 and 51.8%, respectively; for the GSTM1 polymorphism, the frequency was 41.4 and 58.6%, respectively. In the control group, 59.5 and 40.5% of the subjects had the non-null and the null genotype of the GSTT1 polymorphism, respectively, whereas for the GSTM1 polymorphism, the frequency of non-null and null genotypes
Table 3. Stratification of GSTT1 and GSTM1 risk association by histopathology Genotype
n (%)
GSTT1 (ADC) Non-null Null GSTT1 (SCC) Non-null Null GSTM1 (ADC) Non-null Null GSTM1 (SCC) Non-null Null
p
cases
controls
Crude OR (95% CI)
118 (48.6) 125 (51.4)
311 (59.5) 212 (40.5)
1 1.55 (1.14–2.11)
< 0.01
135 (48.2) 145 (51.8)
311 (59.5) 212 (40.5)
1 1.58 (1.18–2.11)
< 0.01
102 (42.0) 141 (58.0)
299 (57.2) 224 (42.8)
1 1.85 (1.36–2.51)
< 0.01
116 (41.4) 164 (58.6)
299 (57.2) 224 (42.8)
1 1.89 (1.41–2.53)
< 0.01
OR = Odds ratio; CI = confidence interval; ADC = adenocarcinoma; SCC = squamous cell carcinoma.
Table 4. Association of combinations of GSTT1 and GSTM1 polymorphisms with lung cancer risk Genotype combination
n (%)
OR (95% CI)
GSTT1
GSTM1
cases
controls
Non-null Null Non-null Null
non-null non-null null null
105 (20.1) 113 (21.6) 148 (28.3) 157 (30.0)
178 (34.0) 121 (23.1) 133 (25.4) 91 (17.4)
1 1.58 (1.11–2.25) 0.01 1.88 (1.35–2.64) < 0.01 2.92 (2.05–4.16) < 0.01
OR = Odds ratio; CI = confidence interval.
166
p
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was 57.2 and 42.8%, respectively. Significant risk association was found for both ADC and SCC of the lung. The GSTT1 null genotype was found to increase the risk of ADC by 1.55fold (95% CI 1.14–2.11; p < 0.01), and that of SCC by 1.58-fold (95% CI 1.18–2.11; p < 0.01). For the GSTM1 polymorphism, the null genotype resulted in an OR of 1.85 (95% CI 1.36–2.51; p < 0.01) for ADC, and 1.89 (95% CI 1.41–2.53; p < 0.01) for SCC. Association of GSTT1 and GSTM1 Combination Genotypes with Lung Cancer Risk We tested the association of various combinations of GSTT1 and GSTM1 genotypes with the risk of lung cancer. The results are summarized in table 4. Significant associations were present for all the possible combinations. A total of 21.6% of cases and 23.1% of controls had GSTT1 null and GSTM1 non-null genotypes, resulting in an OR of 1.58 (95% CI 1.11–2.25; p = 0.01). On the other hand, 28.3% of cases and 25.4% of controls had GSTT1 non-null and GSTM1 null genotypes, resulting in an OR of 1.88 (95% CI 1.35–2.64; p < 0.01). 30% of cases and 17.4% of controls had the null genotype of both the GSTT1 and the GSTM1 polymorphism, and this gave an OR value of 2.92 (95% CI 2.05–4.16; p < 0.01). Stratification of the Association between GSTT1 and GSTM1 Polymorphisms and Lung Cancer Risk by Smoking Status We also stratified the association between the 2 polymorphisms and lung cancer risk according to smoking status (table 5). For the GSTT1 polymorphism, among smokers, 49.0 and 51.0% of cases had non-null and null, respectively, while the frequencies were 58.6 and 41.4% for controls. Among never smokers, 47.9 and 52.1% of cases and 60.2 and 39.8% of controls had the non-null and null genotype of the GSTT1 polymorphism, respectively. For the GSTM1 polymorphism, among smokers, 43.5 and 56.5% of cases had the non-null and null genotype, respectively, while the frequencies were 57.3 and 42.7% for controls. For non-smokers, 40.1 and 59.9% of cases and 57.0 and 43.0% of controls had the non-null and null genotype of the GSTM1 polymorphism. Significant association was observed between the polymorphisms and lung cancer risk, both among smokers and never smokers. For smokers, the null genotype of the GSTT1 polymorphism had a crude OR of 1.47 (95% CI 1.03–2.12; p = 0.04), whereas for never smokers, the crude OR was 1.65 (95% CI 1.18–2.30; p < 0.01). Adjustment for age, sex, and smoking status revealed a similar observation. For smokers, the adjusted OR of the null genotype was 1.45 (95% CI 1.01–2.09; p = 0.05), while for never smokers, the OR was 1.55 (95% CI 1.11–2.16; p = 0.01). For the GSTM1 polymorphism, the null genotype resulted in a crude OR of 1.74 (95% CI 1.21–2.50; p < 0.01) among smokers (adjusted OR 1.80, 95% CI 1.26–2.59; p < 0.01), and 1.98 (95% CI 1.42–2.77; p < 0.01) among never smokers (adjusted OR 1.93, 95% CI 1.38–2.70; p < 0.01).
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controls. For the variant null genotype, the frequency was 51.6% in cases and 40.5% in controls. The null genotype was significantly associated with the risk of lung cancer, with OR 1.57 (95% confidence interval (CI) 1.23–2.00; p < 0.01). In the case of the GSTM1 polymorphism, the frequency of the nonnull genotype was 41.7% in cases and 57.2% in controls, whereas the frequency of the null genotype was 58.3% in cases and 42.8% in controls. The null genotype of the polymorphism resulted in an increased lung cancer risk, with OR 1.87 (95% CI 1.46–2.39; p < 0.01).
Table 5. Smoking status-stratified association of GSTT1 and GSTM1 polymorphisms with lung cancer risk Genotype GSTT1 (smokers) Non-null Null GSTT1 (never smokers) Non-null Null GSTM1 (smokers) Non-null Null GSTM1 (never smokers) Non-null Null
n (%) cases
controls
Crude OR (95% CI)
p
Adjusted OR (95% CI)
p
117 (49.0) 122 (51.0)
140 (58.6) 99 (41.4)
1 1.47 (1.03–2.12)
0.04
1.45 (1.01–2.09)
0.05
136 (47.9) 148 (52.1)
171 (60.2) 113 (39.8)
1 1.65 (1.18–2.30)
< 0.01
1.55 (1.11–2.16)
0.01
104 (43.5) 135 (56.5)
137 (57.3) 102 (42.7)
1 1.74 (1.21–2.50)
< 0.01
1.80 (1.26–2.59)
< 0.01
114 (40.1) 170 (59.9)
162 (57.0) 122 (43.0)
1 1.98 (1.42–2.77)
< 0.01
1.93 (1.38–2.70)
< 0.01
OR = Odds ratio; CI = confidence interval.
Table 6. Gender-stratified association of GSTT1 and GSTM1 polymorphisms with lung cancer risk Genotype GSTT1 (male) Non-null Null GSTT1 (female) Non-null Null GSTM1 (male) Non-null Null GSTM1 (female) Non-null Null
n (%) cases
controls
Crude OR (95% CI)
p
Adjusted OR (95% CI)
p
93 (35.1) 172 (64.9)
167 (63.0) 98 (37.0)
1 3.15 (2.21–4.50)
< 0.01
1 2.95 (2.07–4.20)
< 0.01
160 (62.0) 98 (38.0)
144 (55.8) 114 (44.2)
1 0.77 (0.54–1.10)
0.15
1 0.75 (0.46–1.23)
0.26
122 (46.0) 143 (54.0)
144 (54.3) 121 (45.7)
1 1.39 (0.99–1.96)
0.06
1 1.48 (0.92–2.37)
0.11
96 (37.2) 162 (62.8)
155 (60.1) 103 (39.9)
1 2.54 (1.78–3.62)
< 0.01
1 2.18 (1.54–3.10)
< 0.01
OR = Odds ratio; CI = confidence interval.
GSTT1 and GSTM1 Polymorphisms and Lung Cancer Risk
type in females, with a crude OR of 2.54 (95% CI 1.78–3.62; p < 0.01) and an adjusted OR of 2.18 (95% CI 1.54–3.10; p < 0.01).
Discussion GSTs are a family of cytosolic enzymes which neutralize environmental carcinogens, such as polycyclic aromatic hydrocarbons. GSTM1 and GSTT1 null polymorphisms have been shown to cause elimination of GSTM1 and GSTT1 activity, respectively. Interindividual variability in enzyme activity due to these polymorphisms has been thought to confer different risks of various cancers to their carriers. The same risk modification effect has also been hypothesized for lung cancer, although the results obtained in different studies have been inconsistent. These conflicting findings could be attributed to the different characteristics of the study subjects included in the analysis, and to improper case-control matching. In this study, we sought to investigate the associations of the polymorphisms with the risk of lung cancer among Chinese subjects, and conducted a 1:1 case-control match in terms of age, gender, and smoking status. Based on the genotypes, subjects were categorized into non-null and null groups,
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Gender-Specific Association of GSTT1 and GSTM1 Polymorphisms with Lung Cancer Risk We further tested the presence of a gender-specific association between GSTT1 and GSTM1 polymorphisms and lung cancer risk (table 6). For the GSTT1 polymorphism, 35.1 and 64.9% of male cases had non-null and null, respectively. The frequencies of the genotypes in male controls were 63.0 and 37.0%. For females, the frequencies of non-null and null genotypes were 62.0 and 38.0% in cases, and 55.8 and 44.2% in controls. The null genotype was found to result in a significantly higher risk of lung cancer in males, with a crude OR of 3.15 (95% CI 2.21–4.50; p < 0.01) and an adjusted OR of 2.95 (95% CI 2.07–4.20; p < 0.01). However, no significant association was found in females (p > 0.05). For the GSTM1 polymorphism, the frequency of non-null and null genotypes in male cases was 46.0 and 54.0%, respectively. The frequency of non-null and null genotypes in male controls was 54.3 and 45.7%, respectively. No significant lung cancer risk association was found (p < 0.05). However, the null genotype was overrepresented in female cases. For the non-null genotype, the frequencies in female cases and controls were 37.2 and 60.1%, but for the null genotype, the frequency was 62.8% in female cases and only 39.9% in female controls. This resulted in a significant lung cancer risk association of the null GSTM1 geno-
where the former included both individuals who were homozygous wild type and those who were heterozygous for the polymorphisms. We found that the GSTT1 and GSTM1 null polymorphisms were associated with lung cancer risk in the Chinese population. Our observation for GSTT1 was in agreement with Cao et al. [9], Liang et al. [15], Liu et al. [16], and Yuan et al. [17], who studied the association of the polymorphisms with lung cancer risk in the same population. However, Chan-Yeung et al. [10] reported the contradictory finding of the GSTT1 polymorphism actually reducing the risk of lung cancer, while several other authors, such as Bai et al. [11] and Wang et al. [18], reported no association between the GSTT1 polymorphism and lung cancer risk. For the GSTM1 polymorphism, our results concur with those of Li et al. [12], Qian et al. [19], Wang et al. [20], Wang et al. [21], and Liu et al. [22], but are in discordance with those reported by Chan-Yeung et al. [10], Liang et al. [23], Yang et al. [24], Zeng et al. [25], and Ye et al. [26]. In the current study, we also found that the associations between the polymorphisms and lung cancer risk were present when the polymorphisms were analyzed both individually and in combination with one another. After stratification by cancer histology of cases and smoking status of all the subjects, the associations remained significant in all groups. Interestingly, when stratified by smoking status, we observed that the null genotype resulted in a slightly higher risk of lung cancer in never smokers than in smokers. This phenomenon could be explained by the hypothesis that the xenobiotic-metabolizing effect of GST enzymes is less apparent when the carcinogen is present in high doses, as is the case in smokers [27]. However,
after stratification by gender, differential risk association was observed. This suggests that there is a gender-specific association between the polymorphisms and lung cancer risk, which may be explained by higher CYP P450 enzyme activity in females, leading to the formation of high levels of DNA adduct intermediates which are the substrate of the GSTM1 enzyme. The null genotype in females jeopardizes the ability of the enzyme to detoxify the harmful DNA adducts, resulting in a higher risk of cancer [28]. In addition, we propose that hormonal differences between males and females could be an underlying reason for the presence of a gender-specific association. This is the first study reporting a gender-specific association between GSTT1 and GSTM1 polymorphisms and lung cancer risk in the Chinese population. The strengths of the current study include the relatively large sample size employed and the tight matching between cases and controls. In conclusion, GSTT1 and GSTM1 null polymorphisms were associated with lung cancer risk in the Chinese population in a gender-specific manner.
Acknowledgement We thank all doctors and subjects who participated in this study.
Disclosure Statement The authors declare no conflict of interest.
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