Introduction
Head and neck squamous cell carcinoma constitutes more than 5 per cent of all malignant neoplasms, and more than 500 000 new cases are reported each year worldwide. Laryngeal cancer is the most common neoplasm of the head and neck region and occurs most often in middle-aged men.Reference Hiyama, Yoshihara, Tanaka and Chayama1, Reference Sturgis, Wei and Spitz2 It is a multifactorial pathology that arises from complex interactions between genetic and environmental factors. The major environmental risk factor for laryngeal cancer is smoking.Reference Sapkota, Hsu, Zaridze, Shangina, Szeszenia-Dabrowska and Mates3, Reference Freedman, Abnet, Leitzmann, Hollenbeck and Schatzkin4
Although the development of laryngeal cancer is widely attributed to smoking, not all smokers develop this cancer. This situation suggests that genetic differences such as mutations or polymorphisms may play a role.
Recently, it has been reported that some single nucleotide polymorphisms are associated with increased risk of laryngeal cancer.Reference Werbrouck, De Ruyck, Duprez, Van Eijkeren, Rietzschel and Bekaert5–Reference Azarpira, Ashraf, Khademi, Darai, Hakimzadeh and Abedi7 Functional single nucleotide polymorphisms in endogenous antioxidant defence genes including superoxide dismutase, catalase and glutathione peroxidase have been linked to an increased risk of numerous organ cancers.Reference Cox, Tamimi and Hunter8–Reference Woodson, Tangrea, Lehman, Modali, Taylor and Snyder12 However, to the best of our knowledge, the role of these polymorphisms in laryngeal cancer has not previously been investigated.
We hypothesised that the three genotypes MnSOD Val16Ala, CAT-262 C < T and GPx1 Pro198Leu may be associated with an increased risk of laryngeal cancer. We studied these genotypes in heavy smokers with and without laryngeal cancer. To the best of our knowledge, this is the first genotyping study of antioxidant defence enzymes and laryngeal cancer within a smoker population.
Materials and methods
Study group
Study subjects comprised 25 patients with laryngeal cancer (age range, 38–84 years) and 23 healthy controls (age range, 40–77 years). Study participants were recruited from amongst patients admitted to the Süleyman Demirel University Hospital, Isparta, Turkey, between May 2010 and May 2012. Cancer cases consisted of consecutively treated or untreated laryngeal cancer patients admitted to the otorhinolaryngology department with histologically confirmed squamous cell carcinoma. All subjects (i.e. both laryngeal cancer patients and healthy controls) were heavy smokers (i.e. they had smoked for more than 20 years, and smoked at least 10 cigarettes per day) and were older than 35 years (Table I).
Table I Group characteristics: age and smoking
*n = 23; †n = 25. Med = median; yr = years; cig = cigarettes
The exclusion criteria were non-smokers, an age of 35 years or younger, and additional malignancy.
This study was performed according to the Declaration of Helsinki and was approved by the local ethics committee. Written, informed consent was obtained from all study subjects.
Each patient provided a venous blood sample, which was collected into ethylene diamine triacetic acid coated tubes. Blood samples were stored at −80°C until DNA isolation was conducted.
DNA extraction and genotyping
The polymorphic sites in the MnSOD Val16Ala, CAT-262 C < T and GPx1 Pro198Leu genes were analysed using polymerase chain reaction combined with restriction fragment-length polymorphism.
Isolation of DNA was carried out using the Norgen Biotek Blood Genomic DNA Isolation Kit (Norgen Biotek, Thorold, Ontario, Canada). The polymerase chain reactions contained 100 µM deoxyribonucleoside triphosphates, 20 mM Tris-HCl (pH 8.6), 50 mM KCl, 1.5 mM MgCl2 (MBI Fermentas, Burlington, Ontario, Canada), 20 pMol forward and reverse primer (MBI Fermentas), 0.2 U Taq DNA polymerase (MBI Fermentas), and 200 ng DNA in a 25 µl reaction volume.
For MnSOD Val16Ala, the forward primer was 5′-CTG TGC TTT CTC GCT TTC AG-3′ and the reverse primer was 5′-GGT GAC GTT CAG GTT GTT CA-3′; for CAT-262 C < T the forward primer was 5′-AAC TGG CAG GCC ACC AAC GG-3′ and the reverse primer was 5′-CTG ATT GGC TGC TCG GCG CT-3′; and for GPx1 Pro198Leu the forward primer was 5′-CGC CAA GAA CGA AGA GAT TC-3′ and the reverse primer was 5′-CCC ACC AGG AAC TTC TCA AA-3′ (C, cytosine; T, thymine; G, guanine; and A, adenine).
The polymerase chain reaction conditions for the polymorphisms consisted of: an initial denaturation step at 94°C for 5 minutes; 35 cycles of denaturation at 95°C for 1 minute; annealing of the primer at the appropriate annealing temperature for 1 minute; elongation at 52°C for MnSOD Val16Ala, 62°C for CAT-262 C < T and 56°C for GPx1 Pro198Leu, for 1 minute; and a final extension step at 72°C for 7 minutes. The polymerase chain reaction fragments of the polymorphisms were subsequently digested with their specific restriction enzyme (New England Biolabs, Ipswich, Massachusetts, USA). Genotypes were determined with ultraviolet light using a gel imaging system (Kodak Image Station 2000 MM; Rochester, New York, USA).
Statistical analysis
Continuous variables (i.e. age, smoking duration and smoking intensity) showed abnormal distributions and were therefore compared using the Mann–Whitney U test. Categorical variables were compared using the chi-square test. A p value of less than 0.05 was considered significant.
Results
There were no significant differences between the groups regarding age, smoking duration or smoking intensity (Table I).
There was a significant difference between the groups regarding MnSOD Val16Ala polymorphism. The homozygous AA genotype of MnSOD Val16Ala was significantly more prevalent in the cancer group (92 per cent) than the control group (13 per cent), while the heterozygous AV genotype of MnSOD Val16Ala was more significantly prevalent in the control group (87 per cent) than the cancer group (8 per cent) (p < 0.001) (Table II).
Table II Association of gene polymorphisms with laryngeal cancer
Data presented as n (%). A = alanine; V = valine; T = threonine; C = cysteine; – = not found
However, there were no significant differences between the cancer group and the control group with respect to CAT-262 C < T or GPx1 Pro198Leu polymorphisms (Table II).
Discussion
The risk of laryngeal cancer increases due to complex interactions between genetic and environmental factors such as age, heavy smoking and heavy alcohol consumption.Reference Pawlowska, Janik-Papis, Rydzanicz, Zuk, Kaczmarczyk and Olszewski13 The major environmental risk factor is smoking, although genetic differences due to mutations and polymorphisms may also play a role.Reference Hiyama, Yoshihara, Tanaka and Chayama1 Laryngeal cancer has been reported to be associated with single nucleotide polymorphisms within carcinogen metabolic enzyme and vitamin metabolism genes (i.e. glutathione S-transferase M1, cytochrome P-450 and methylenetetrahydrofolate reductase), DNA repair genes (i.e. the X-ray repair cross-complementing gene, the xeroderma pigmentosum group D gene and the xeroderma pigmentosum group G gene) and cell-cycle control genes (i.e. the p53 and cyclin D1 genes).Reference Hiyama, Yoshihara, Tanaka and Chayama1, Reference Chatzimichalis, Xenellis, Tzagaroulakis, Sarof, Banis and Gazouli14, Reference Capaccio, Ottaviani, Cuccarini, Cenzuales, Cesana and Pignataro15 To our best knowledge, there has been no previous research on the association between laryngeal cancer and functional single nucleotide polymorphism within the endogenous antioxidant defence genes superoxide dismutase, catalase and glutathione peroxidase. In the present study, we investigated the MnSOD Val16Ala, CAT-262 C < T and GPx1 Pro198Leu genotypes in heavy smokers with or without laryngeal cancer.
Previous studies have investigated single nucleotide polymorphisms in antioxidant defence genes, within the setting of various organ cancers. Tsai et al. Reference Tsai, Wu, Hou, Chen, Ma and Tsai11 showed that genetic variants of oxidative stress-related enzymes (i.e. GPx1Pro198Leu (especially GPx1Pro198Leu CT), MnSOD1183T > C, MPO-463G > A and CAT-262C > T) were associated with a reduced risk of breast cancer in non-smoking and non-alcohol-consuming women. Similarly, Cox et al. Reference Cox, Tamimi and Hunter8 reported that the GPx1 Pro198Leu CC and MnSOD Val16Ala AA genotypes were associated with an increased risk of breast cancer. Other authorsReference Ambrosone, Freudenheim, Thompson, Bowman, Vena and Marshall16, Reference Mitrunen, Sillanpää, Kataja, Eskelinen, Kosma and Benhamou17 have found the latter genotype to be strongly associated with an increased risk of breast cancer. Hansen et al. Reference Hansen, Krath, Frederiksen, Tjonneland, Overvad and Roswall10 have found the GPx1 Pro198Leu genotype to be associated with colorectal cancer in smokers. Woodson et al. Reference Woodson, Tangrea, Lehman, Modali, Taylor and Snyder12 and Li et al. Reference Li, Kantoff, Giovannucci, Leitzmann, Gaziano and Stampfer18 observed a strong association between the MnSOD Val16Ala AA genotype and prostate cancer development. Paz-y-Miño et al. Reference Paz-y-Miño, Muñoz, López-Cortés, Cabrera, Palacios and Castro9 reported an association between glutathione peroxidase-1 gene polymorphism and bladder cancer risk in an Ecuadorian population. Ezzikouri et al. Reference Ezzikouri, El Feydi, Afifi, Benazzouz, Hassar and Pineau19 found an association between variants of the manganese superoxide dismutase, catalase and glutathione peroxidase 1 genes and the development of hepatoma.
In agreement with previous studies, our study found that the MnSOD Val16Ala AA genotype was significantly more prevalent in our laryngeal cancer group than our control group. We also found that the MnSOD Val16Ala AV genotype was less prevalent in the cancer group.
• Single nucleotide polymorphisms of antioxidant defence genes have been linked with various organ cancers
• Polymorphisms of the MnSOD Val16Ala, CAT-262 C < T and GPx1 Pro198Leu genotypes may be associated with laryngeal cancer
• This was investigated in heavy smokers with or without laryngeal cancer
• The MnSOD Val16Ala AA genotype was significantly associated with increased laryngeal cancer
Limitations
This was a small study. The study contained only five women (one in the cancer group and four in the control group). This may be mainly because Turkish women are less likely to smoke than Turkish men. There were no heavy alcohol drinkers in our study because alcohol consumption is not common in the Turkish population.
Conclusion
The MnSOD Val16Ala AA genotype may be associated with an increased risk of laryngeal cancer in smokers. However, the MnSOD Val16Ala AV genotype may have a protective effect. In this study, the prevalence of GPx1 Pro198Leu and CAT-262 C < T polymorphisms was similar between the cancer and control groups. Larger studies are required to clarify these results.
