No association between ACE polymorphism and risk of nasopharyngeal carcinoma

Transcription

1 6168JRA / Li et al.journal of the Renin-Angiotensin-Aldosterone System Article No association between ACE polymorphism and risk of nasopharyngeal carcinoma Journal of the Renin-Angiotensin- Aldosterone System 13(1) The Author(s) 2011 Reprints and permission: sagepub.co.uk/journalspermissions.nav DOI: / jra.sagepub.com Zhao-Hui Li 1,*, Xin-Min Pan 2,*, Bao-Wei Han 1, Hong-bin Han 1, Zhen Zhang 2 and Lin-Bo Gao 3,* Abstract Emerging evidence has shown that angiotensin I-converting enzyme (ACE) plays pivotal roles not only in the regulation of cardiovascular homeostasis but also in the process of tumorigenesis. A common ACE I/D polymorphism has been found to be functional, with the D allele displaying a higher plasma ACE level and ACE activity. The purpose of this study was to investigate whether the ACE I/D polymorphism was related to the risk of nasopharyngeal carcinoma (NPC). The study included 175 patients with NPC and 279 age- and sex-matched control subjects. The ACE I/D polymorphism was identified by a polymerase chain reaction analysis. No association was found between the ACE I/D polymorphism and risk of NPC (ID vs. II: odds ratio [OR] = 0.77, 95% confidence interval [CI] ; DD vs. II: OR = 0.98, 95%CI , respectively). This finding indicates that the ACE I/D polymorphism may not play a role in susceptibility to NPC. Further studies are warranted to confirm this finding, especially in ethnically disparate populations. Keywords ACE, nasopharyngeal carcinoma, polymorphism Introduction Nasopharyngeal carcinoma (NPC) is a malignancy originating in the nasopharynx with a distinct geographical distribution. Although it is rare in most parts of the world, it is extremely common in southern China, where the annual incidence is about 25 times higher than in the rest of the world. 1,2 In past years, many risk factors for the development of NPC have been widely accepted, including Epstein Barr virus infection, intake of salted fish, and tobacco smoking. 3 5 However, only a small fraction of individuals suffer from the disease in the endemic regions, even though all the people are exposed to the same risk factors, suggesting that a genetic factor, such as a single nucleotide polymorphism, may influence individual susceptibility to NPC. Our previous studies have shown that IL-16 and NFKB1 gene polymorphisms may contribute to the development of NPC. 6,7 Angiotensin I-converting enzyme (ACE) is a key component of the renin angiotensin system (RAS) and catalyzes the conversion of angiotensin I to angiotensin II. Angiotensin II has been reported to be involved in tumorigenesis, including tumor cell proliferation, angiogenesis, migration, and metastatic behavior. 8 Growing evidence has shown that ACE levels are differentially expressed in several cancers and that administration of ACE inhibitors could attenuate tumor growth in experimental studies. 9,10 These observations imply that ACE may be a candidate cancer gene. The gene encoding ACE is located on the long arm of chromosome 17 (17q23) in humans and contains 26 exons and 25 introns. A common insertion/deletion (I/D) polymorphism in the non-coding region of the ACE gene was identified in The I allele results from the presence of a 287bp DNA fragment in intron 16, and the D allele results from the absence of this DNA sequence. 11 It has 1 Secondary Department of General Surgery, Luo Yang Central Hospital, PR China 2 Department of Forensic Pathology, College of Forensic Medicine, Henan University of Science and Technology, PR China 3 Laboratory of Molecular and Translational Medicine, West China Institute of Women and Children s Health, West China Second University Hospital, Sichuan University, PR China * These authors contributed equally to this work. Corresponding author: Lin-Bo Gao, Laboratory of Molecular and Translational Medicine, West China Institute of Women and Children s Health, West China Second University Hospital, Sichuan University, Chengdu, Sichuan , PR China cdchglb@gmail.com

2 Li et al. 211 been reported that individuals with the D allele have higher plasma ACE activity Recently, numerous studies have been carried out to examine the association between the ACE I/D polymorphism and susceptibility to a range of cancers, including breast cancer, gastric cancer, colorectal cancer, prostate cancer, lung cancer, 35,36 oral cancer, 37,38 and renal cancer. 39 No studies to date have evaluated the relationship between the ACE I/D polymorphism and NPC risk. In this case control study, we aimed to explore whether the ACE I/D polymorphism is associated with susceptibility to NPC in a Chinese population. Materials and methods Study population The study protocol was approved by the hospital ethics committee and all subjects provided informed consent. One hundred and seventy-five NPC patients (mean age: 45.6 ± 11.6 years) and 279 control subjects (mean age: 43.3 ± 12.7 years) were enrolled in this study between July 2006 and September 2010 (Table 1). All subjects were genetically unrelated Chinese individuals residing in Luo Yang Central Hospital. The diagnosis of NPC was confirmed by histopathological examination. Patients with recurring NPC or NPC occurring in combination with other cancers were excluded. Healthy individuals without any evidence of personal or family history of cancer were defined as controls. The controls were frequency-matched to the cases based on age, sex, and residential area. Genotyping Genomic DNA was extracted from peripheral blood by using a DNA isolation kit (Bioteke Corporation, Beijing, China). The polymerase chain reaction (PCR) polyacrylamide gel electrophoresis (PAGE) method was used to genotype the ACE I/D polymorphism. The PCR primers were based on Ruggenenti et al. 40 PCR reaction was performed in a total volume of 10 µl containing 1.0 µl 10 PCR buffer, 0.15 mmol/l deoxy-ribonucleoside triphosphates (dntps), 1.5 mmol/l MgCl 2, 0.1 µl each primer, 50 ng genomic DNA and 0.3 U of Taq DNA polymerase. The reactions were run under the following conditions: initial denaturation at 94 C for 4 min, followed by 35 cycles of 30 s at 94 C, 90 s at 64 C and 90 s at 72 C, and a final elongation at 72 C for 10 min. There is a possibility of misclassification of the ID heterozygote as DD homozygote due to preferentially amplifying the D allele. An additional PCR, therefore, was conducted to avoid the misclassification. The PCR procedure was described previously. 41 All PCR products were visualized after electrophoresis on 6% polyacrylamide gel with silver nitrate staining. Table 1. Characteristics of the study population. Variable NPC patients (n = 175) Controls (n = 279) Age (years ± SD) 45.6 ± ± 12.7 Sex Male 129 (73.7%) 207 (74.2%) Female 46 (26.3%) 72 (25.8%) Clinical stages I II 27 (15.4%) - III IV 148 (84.6%) - NPC: nasopharyngeal carcinoma, SD, standard deviation. Statistical analysis All analyses were done by the SPSS statistical software package version 11.5 (SPSS Inc., Chicago, IL, USA). Hardy Weinberg equilibrium was assessed by the χ 2 test. Continuous variables were compared using the unpaired Student s t-test. The distribution of genotype and allele frequencies of ACE I/D polymorphism between NPC cases and controls was compared using the χ 2 test. The strength of the gene cancer association was estimated by computing odds ratio (OR) and 95% confidence intervals (CI). All p values were two-sided, and statistical significance was set at p < Results The genotype and allele frequencies of the ACE I/D polymorphism in NPC patients and controls are summarized in Table 2. Genotype distributions did not deviate from Hardy Weinberg equilibrium in either cases or controls. The frequencies of the II, ID, and DD genotypes were 33.7, 50.9, and 15.4% in controls and 38.3, 44.6, and 17.1% in NPC patients, respectively. No significant association was observed between the ACE I/D polymorphism and risk of NPC (ID vs. II: OR = 0.77, 95%CI ; DD vs. II: OR = 0.98, 95%CI , respectively). Discussion Angiotensin II, a key effector of the RAS, plays important roles not only in the regulation of cardiovascular homeostasis but also in the process of tumor cell proliferation, angiogenesis, migration, and metastatic behavior. 8 The generation of Ang II depends on the activity of ACE. Emerging evidence has shown that ACE is implicated in tumorigenesis. Alterations of plasma ACE that may influence cancer biology occur in several cancers. 29,42 The administration of ACE inhibitors had a significantly protective effect against tumor growth and angiogenesis in animal models of cancer. 9,10 A common ACE I/D

3 212 Journal of the Renin-Angiotensin-Aldosterone System 13(1) Table 2. The association between the ACE I/D polymorphism and NPC risk. Control, n = 279 (%) NPC, n = 175 (%) OR (95% CI) p value Genotype II 94 (33.7) 67 (38.3) 1.0 (Ref) ID 142 (50.9) 78 (44.6) 0.77 ( ) 0.22 DD 43 (15.4) 30 (17.1) 0.98 ( ) 0.94 Allele I 330 (59.1) 212 (60.6) 1.0 (Ref) D 228 (40.9) 138 (39.4) 0.94 ( ) 0.67 ACE: angiotensin I-converting enzyme, CI: confidence interval, NPC: nasopharyngeal carcinoma, OR: odds ratio polymorphism has been found to be functional, with the D allele displaying a higher plasma ACE level and ACE activity Recently, the polymorphism has been reported to be associated with the etiology of a variety of cancers, including breast cancer, 18,19 gastric cancer, 25 prostate cancer, 32 and oral cancer. 37 Based on these backgrounds, we hypothesized that the ACE I/D polymorphism might modulate susceptibility to NPC. Nevertheless, no significant association was observed between the ACE I/D polymorphism and NPC risk, indicating that the polymorphism may not contribute to susceptibility to NPC in the Chinese population. Quanto software (Gauderman WJ, Morrison JM. QUANTO 1.2: A computer program for power and sample size calculations for genetic-epidemiology studies) was used to calculate the power of the case control study according to the current sample size. Our study had 84% power to detect an effect of the ACE I/D polymorphism on NPC susceptibility with an estimated relative risk of 1.8 under a dominant genetic model. In concordance with our findings, some authors reported that the ACE I/D polymorphism was not significantly associated with the risk of colorectal cancer, 27,28,30,31 breast cancer, 17 gastric cancer, 22 lung cancer, 35 or renal cancer. 39 In contrast to our findings, Röcken et al. reported that the ACE I/D polymorphism correlated with the number of lymph node metastases and tumor stage in gastric cancer. Patients with the II genotype were found to have a smaller number of lymph node metastases and lower clinical stage than patients with the DD genotype. 24 Ebert et al. reported that the II genotype was associated with a decreased risk of gastric cancer compared with the DD genotype (OR = 0.20, 95%CI ) in a German population. 25 Goto et al. reported that the I/D genotype conferred a 1.59-fold greater risk of developing gastric cancer on Helicobacter pylori positive Japanese patients with atrophic gastritis. 26 Sierra Diaz et al. reported that the D allele conferred a significantly higher risk of developing prostate cancer. 32 Moreover, some researchers reported that the II genotype was associated with a significantly increased risk of breast cancer 18,19 and oral cancer. 37 Although the reason for these discrepancies remains unknown, it is likely that the ACE I/D polymorphism may have different roles in different cancer types. This phenomenon was confirmed by a recently published metaanalysis study that reported a significantly decreased incidence of the ACE I/D polymorphism in patients with prostate cancer or breast cancer, but not in patients with colorectal cancer. 43 Another important issue may be environmental factors that influence the risk for tumor development. Cancer is a multifactorial disease, and individuals with different environmental exposure may be responsible for the conflicting results. In addition, the genetic trait differences in diverse ethnicities may be a potential explanation for the inconclusive results. After comparing the distribution of the ACE I/D polymorphism in diverse populations, we found that the genetic polymorphism varied significantly between different ethnic groups. The genotype and allele frequencies of the ACE I/D polymorphism in healthy controls from various populations are summarized in Table 3. The detection rate of the I allele in Asians ( %) was remarkably higher than in Caucasians ( %). 14,19,24,30,38,39,49,50 The heterogeneity of allelic frequency among the ethnicities may affect individual susceptibility to cancer in different ethnic groups. Replicated studies are of great value to confirm these findings. Some limitations should be discussed. All the participants in our study were Chinese, and thus the results of this study may not be automatically extrapolated to other ethnic groups. Moreover, gene gene or gene environment interactions were not considered in this study, which may restrict our comprehensive understanding of the effect of the ACE I/D polymorphism on NPC risk. In summary, to our knowledge, this the first study to investigate the relationship between the ACE I/D polymorphism and the occurrence of NPC. We failed to find any association between the ACE I/D polymorphism and risk of NPC. However, additional studies are required to explore the gene gene and gene environment interactions in susceptibility to NPC, especially in ethnically disparate populations.

4 Li et al. 213 Table 3. Distribution of the ACE I/D polymorphism in different populations. Population n Genotype Allele II, n (%) ID, n (%) DD, n (%) I, n (%) D, n (%) Present study (Chinese Han) (33.7) 142 (50.9) 43 (15.4) 330 (59.1) 228 (40.9) Chinese Han (41.3) 78 (41.3) 33 (17.5) 234 (61.9) 144 (38.1) Chinese Han (42.7) 130 (43.1) 43 (14.2) 388 (64.2) 216 (35.8) Chinese Han (22.0) 107 (45.3) 77 (32.6) 211 (44.7) 261 (55.3) Japanese (42.0) 355 (47.3) 80 (10.7) 985 (65.7) 515 (34.3) Korean (36.5) 3549 (49.1) 1041 (14.4) 8827 (61.0) 5631 (39.0) Turkish (13.0) 12 (26.1) 28 (60.9) 24 (26.1) 68 (73.9) Greek and German (5.9) 66 (43.1) 78 (51.0) 84 (27.5) 222 (72.5) Greek (5.9) 44 (43.1) 52 (51.0) 56 (27.5) 148 (72.5) Brazilian (17.3) 113 (36.8) 141 (45.9) 219 (35.7) 395 (64.3) Italian (13.5) 118 (46.8) 100 (39.7) 186 (36.9) 318 (63.1) French (18.1) 288 (51.4) 171 (30.5) 490 (43.8) 630 (56.2) German (21.7) 95 (50.3) 53 (28.0) 177 (46.8) 201 (53.2) American (18.3) 295 (53.3) 157 (28.4) 492 (44.5) a 614 (55.5) a a Evaluated from published data. ACE: angiotensin I-converting enzyme. Conflict of interest The authors declare that they have no conflict of interest. Funding This study was supported by the PhD scientific research foundation of Henan University of Science and Technology (No ). References 1. McDermott AL, Dutt SN and Watkinson JC. The aetiology of nasopharyngeal carcinoma. Clin Otolaryngol Allied Sci 2001; 26: Chang ET and Adami HO. The enigmatic epidemiology of nasopharyngeal carcinoma. Cancer Epidemiol Biomarkers Prev 2006; 15: Chen DL and Huang TB. A case-control study of risk factors of nasopharyngeal carcinoma. Cancer Lett 1997; 117: Yu MC and Yuan JM. Epidemiology of nasopharyngeal carcinoma. Semin Cancer Biol 2002; 12: Zheng X, Yan L, Nilsson B, Eklund G and Drettner B. Epstein-Barr virus infection, salted fish and nasopharyngeal carcinoma. A case-control study in southern China. Acta Oncol 1994; 33: Gao LB, Liang WB, Xue H, et al. Genetic polymorphism of interleukin-16 and risk of nasopharyngeal carcinoma. Clin Chim Acta 2009; 409: Zhou B, Rao L, Li Y, et al. A functional insertion/deletion polymorphism in the promoter region of NFKB1 gene increases susceptibility for nasopharyngeal carcinoma. Cancer Lett 2009; 275: Suzuki Y, Ruiz-Ortega M, Lorenzo O, Ruperez M, Esteban V and Egido J. Inflammation and angiotensin II. Int J Biochem Cell Biol 2003; 35: Volpert OV, Ward WF, Lingen MW, et al. Captopril inhibits angiogenesis and slows the growth of experimental tumors in rats. J Clin Invest 1996; 98: Yoshiji H, Kuriyama S, Kawata M, et al. The angiotensin-i-converting enzyme inhibitor perindopril suppresses tumor growth and angiogenesis: possible role of the vascular endothelial growth factor. Clin Cancer Res 2001; 7: Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P and Soubrier F. An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest 1990; 86: Nakai K, Itoh C, Miura Y, et al. Deletion polymorphism of the angiotensin I-converting enzyme gene is associated with serum ACE concentration and increased risk for CAD in the Japanese. Circulation 1994; 90: Winkelmann BR, Nauck M, Klein B, et al. Deletion polymorphism of the angiotensin I-converting enzyme gene is associated with increased plasma angiotensin-converting enzyme activity but not with increased risk for myocardial infarction and coronary artery disease. Ann Intern Med 1996; 125: Yaren A, Turgut S, Kursunluoglu R, et al. Association between the polymorphism of the angiotensin-converting enzyme gene and tumor size of breast cancer in premenopausal patients. Tohoku J Exp Med 2006; 210: Yaren A, Turgut S, Kursunluoglu R, et al. Insertion/deletion polymorphism of the angiotensin I-converting enzyme gene in patients with breast cancer and effects on prognostic factors. J Investig Med 2007; 55: Yuan JM, Koh WP, Sun CL, Lee HP and Yu MC. Green tea intake, ACE gene polymorphism and breast cancer risk among Chinese women in Singapore. Carcinogenesis 2005; 26: Koh WP, Yuan JM, Sun CL, et al. Angiotensin I-converting enzyme (ACE) gene polymorphism and breast cancer risk

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