Journal of Cancer Research and Therapeutics Medknow Publications on behalf of the Association of Radiation Oncologists of India (AROI) ISSN: 0973-1482 EISSN: 1998-4138 Vol. 5, Num. 4, 2009, pp. 247-253

Journal of Cancer Research and Therapeutics, Vol. 5, No. 4, October-December, 2009, pp. 247-253

Original Article

Role of nitric oxide and antioxidant enzymes in the pathogenesis of oral cancer

Patel JayendrakumarB, Shah FrankyD, Shukla ShilinN, Shah PankajM, Patel PrabhudasS

Biochemistry Research Division, The Gujarat cancer and Research Institute, Ahmadabad - 380 016

Correspondence Address:Biochemistry Research Division, The Gujarat Cancer and Research Institute, Asarwa, Ahmedabad - 380 016
prabhudas_p@hotmail.com

Code Number: cr09061

PMID: 20160357

DOI: 10.4103/0973-1482.59898

Abstract

Keywords: Catalase, nitric oxide, oral cancer, oral precancers, superoxide dismutase

Introduction

Oral cancer has been the bane of human cancers globally with its towering incidence in India. ^[1] It is a multi-disease progressing through hyperplasia, dysplasia, oral precancers (OPC), carcinoma in situ, and invasive carcinomas leading to metastasis. ^[2] The role of various genetic, environmental and life style factors in the etiology of oral cancer has been documented. ^[3] The high incidence of oral cancer and OPC in India has been linked with habits of tobacco chewing and smoking. ^[4] It is also reported that the difference in the incidence of oral cancer between western countries and India may be due to the differences in the habits of tobacco usage. ^[4]

Earlier studies have shown that pan masala and gutkha have clastogenic and carcinogenic effects. ^[5] It was first demonstrated that aqueous extracts of areca-nut and catechu were capable of generating free radicals at specific pH that are produced during auto oxidation of polyphenols in saliva of the tobacco users. ^[6] Thus, tobacco habits play an important role in etiology of oral cancer through increased generation of free radicals and reactive oxygen species (ROS). ^[7],[8] The ROS and other reactive oxygen metabolites such as superoxide anions (O ₂ ^- ), hydrogen peroxide (H ₂ O ₂ ), hydroxyl radicals (OH), malondialdehyde and nitric oxide (NO ^- ) are involved in multistep process of carcinogenesis. They cause DNA damage leading to mutations in oncogenes and tumor suppressor genes, which are implicated in oral carcinogenesis. Thus, free radicals act as initiators and/or promoters of oral carcinogenesis. ^[9],[10]

NO ^- is an uncharged molecule with an unpaired electron. It plays multiple roles in both intracellular and extracellular signaling mechanisms. ^[11] It is highly reactive, yet a simple molecule is produced in the body by the isoenzyme nitric oxide synathase (NOS) using L-arginine as a substrate. Three iso-forms of NOS have been characterized. Two of them are constitutive NOS (cNOS) and the third is inducible (iNOS) by endotoxins and cytokines. ^[12]

Reaction of NO ^- with oxygen or the free radicals generates reactive nitrogen species, which cause multiple biological effects. ^[13] NO ^- is either cytostatic or cytotoxic, interacting with a number of molecular targets within cells. Cells within different tissues display varying response to NO ^- which may relate to the presence of cellular antioxidants enzymes such as catalase and super oxide dismutase (SOD). It has been reported that NO ^- is estimated in terms its end product of nitrite+ nitrate (NO ₂ +NO ₃ ) due to short half-life. ^[11]

Previous studies have demonstrated reduced antioxidant enzymes as well as up regulation of iNOS in oral cancer patients. ^[14],[15] Most of these studies have focused on the immunohistochemically detectable expression levels of antioxidant enzymes and iNOS in oral tissues; however, there is paucity of showing involvement of NO in oral cancer and OPC. Therefore, the study evaluated NO ₂ +NO ₃ and levels of antioxidant enzymes including SOD and catalase in healthy individuals without habits of tobacco (NHT), healthy individuals with habit of tobacco (WHT), patient with OPC and oral cancer patients. The correlation between NO ₂ +NO ₃ levels and antioxidant enzyme activities with tobacco habits was also assessed.

Materials and Methods

One hundred and twenty six newly diagnosis patients with biopsy proven oral squamous cell carcinoma and 15 patients with OPC diagnosed by clinical and radiological examination were included for the study. Due consent was obtained from the subjects to participate in the study. None of the subjects included in the study had any major disease in the recent past and were not on any medication including supplementation of antioxidants. The age range for oral cancer patients was 22-75 years with a median of 45 years. For patients with OPC, the age range was 18-58 years with a median of 25 years. The study included 90 age and sex matched healthy volunteers with no major illness in recent past. Healthy individuals were grouped into WHT (n=60) and NHT (n=30). Age range of healthy individuals was 19-62 years with a median of 32 years.

The TNM staging of tumors were classified according to AJCC criteria. ^[16] Distribution of oral cancer patients with regard to various clinicopathological characteristics including age, stage and nuclear grade is provided in [Table - 1].

Blood samples were collected from the subjects prior to initiation of any anticancer therapy. Plasma were separated and stored at -20 ^o C until analyzed. Erythrocytes were washed twice with saline and centrifuged at 10,000 R.P.M. Washed erythrocytes were stored at -20 ^o C until analyzed.

NO ₂ +NO ₃ levels were measured by the Griess reagent method. ^[17],[18] In the two-step assay procedure, the first step was the conversion of nitrate using cadmium metal powder and the second step was the addition of sulphanilamide and N-(naphthyl) ethylenediamine (Griess reagent). The deep purple azo compound of nitrite was measured spectrophotometrically at 550 nm. NO ₂ +NO ₃ were expressed as µM. Erythrocyte SOD enzyme was estimated by spectrophotometeric method. ^[19] The SOD enzyme activity was expressed as unit/gm Hb. Erythrocyte catalase was estimated by spectrophotometric method. ^[20] The expression of catalase enzyme activity was expressed as unit/gm Hb.

The data were statistically analyzed using SPSS statistical software (Version 10). Student′s ′t′ test was performed to compare levels between controls and patients. Pearson′s correlation was studied to assess association between the biomarkers. Receivers operating characteristic (ROC) curves were constructed to evaluate discriminatory efficacy of the parameters between patients and controls. Survival curves were plotted and median survival duration was estimated by the Kaplan Meier method. The survival duration and the strength of association between categories with variables were compared with the log-rank test. The difference was considered statistically significant when ′p′ values were 0.05 or less.

Results

Mean plasma NO ₂ +NO ₃ levels in controls, patients with OPC and oral cancer patients are depicted in [Figure - 1]. Plasma NO ₂ +NO ₃ levels were comparable between NHT and WHT. The patients with OPC and oral cancer patients showed significantly elevated plasma NO ₂ +NO ₃ levels as compared to NHT (p=0.0001) as well as WHT (p=0.0001).

As illustrated in [Figure - 2], mean erythrocyte SOD activity was higher in WHT and patients with OPC as compared to NHT. Oral cancer patients showed significantly decreased erythrocyte SOD activity as compared to NHT, WHT and patients with OPC (p=0.041, p=0.0001 and p=0.008, respectively). Mean erythrocyte catalase activity in WHT and patients with OPC was significantly elevated as compared to NHT (p=0.036 and p=0.0001, respectively). Patients with OPC also showed significantly higher erythrocyte catalase activity than WHT (p=0.0001). Erythrocyte catalase activity was comparable between NHT and oral cancer patients. Mean erythrocyte catalase activity was lower in oral cancer patients than WHT. A significant decrease in erythrocyte catalase activity was observed in oral cancer patients as compared to the patients with OPC (p=0.0001).

[Table - 2] demonstrates erythrocyte levels of the parameters in NHT, WHT and oral cancer patients. Mean erythrocyte SOD and catalase activities were higher in WHT (both tobacco smokers and chewers) than NHT. Oral cancer patients having tobacco habits showed significant increase in mean plasma NO ₂ +NO ₃ levels as compared to NHT (p=0.0001). The erythrocyte SOD levels were significantly lower in oral cancer patients with tobacco smoking and chewing habits than NHT (p=0.033 and p=0.050, respectively).

Mean plasma NO ₂ +NO ₃ and SOD levels were comparable between poor and moderate differentiation tumors of oral cancer patients Erythrocyte SOD activity was higher in advanced disease as compared to early disease. Mean plasma NO ₂ +NO ₃ and catalase levels were comparable between early and advanced stages of oral cancer patients. Oral cancer patients were divided into well, moderate and poor differentiation according to their pathological tumor differentiation. Erythrocyte catalase activities were significantly lower in poorly and moderately differentiated tumors than well-differentiated tumors (p=0.011 and p=0.046, respectively). Mean erythrocyte catalase activities were also decreased in poorly differentiated tumors as compared to moderately differentiated tumors. Mean plasma NO ₂ +NO ₃ and SOD levels were comparable between early and advanced stages of oral cancer patients [Table - 3].

ROC curve analysis is a more meaningful way of discriminating two groups under the study as it simultaneously considers sensitivity and specificity of the parameters. ROC curve analysis revealed that plasma NO ₂ +NO ₃ and erythrocyte catalase levels could significantly discriminate between controls and patients with OPC (p=0.0001 and p=0.0001, respectively). Erythrocyte SOD levels also could also differentiate between controls and patients with OPC. The area under curve for the plasma NO ₂ +NO ₃ , erythrocyte catalase and erythrocyte SOD were 0.981, 0.890 and 0.571, respectively [Figure - 3]a. As depicted in [Figure - 3]b, plasma NO ₂ +NO ₃ and erythrocyte SOD could significantly differentiate between controls and oral cancer patients (p=0.0001 and p=0.0001, respectively). Erythrocyte catalase activity also showed higher accuracy in discriminating between controls and oral cancer patients. The area under curve for the plasma NO ₂ +NO ₃ , erythrocyte SOD and erythrocyte catalase were 0.939, 0.693 and 0.491, respectively. [Figure - 3]c displays erythrocyte SOD and catalase activities which could discriminate between patients with OPC and oral cancer patients (p=0.024 and p=0.0001, respectively). The areas under curve for erythrocyte SOD and catalase were 0.709 and 0.939, respectively.

Pearson′s correlation analysis was performed to study correlation between (i) antioxidant enzymes (erythrocyte SOD and catalase) and plasma NO ₂ +NO ₃ and (ii) SOD levels and catalase activity. [Table - 4] shows Pearson′s correlation analysis of plasma NO ₂ +NO ₃ with the changes in antioxidant enzymes. The changes in erythrocyte SOD revealed significant negative correlation (r=-0.581, p=0.0001) with plasma NO ₂ +NO ₃ . The alterations in erythrocyte SOD activities were significantly associated with changes in erythrocyte catalase activities (r=-0.178, p=0.026).

Kaplan Meier survival curves were plotted to evaluate prognostic significance of the markers in 79 oral cancer patients [Figure - 4]. The mean + SD of healthy individuals was considered as cut off value. Survival of patients with levels of biomarkers above cut-off values were compared with their counterparts. Survival curve of erythrocyte SOD and catalase enzymes indicated significantly better overall survival in patients with erythrocyte SOD and catalase levels above cut-off values as compared with their counterparts (p=0.0191 and p= 0.0270, respectively).

Discussion

The role of NO ^- is multidimensional because of it functions as an intracellular messenger and is also implicated as a deleterious agent in various pathological conditions including cancer. Chronic inflammation can lead to the production of NO ^- , which in turn has the potentials to mediate DNA damage directly or indirectly through the generation of more persistent reactive nitrogen species. ^[21],[22] The present study measured plasma NO ₂ +NO ₃ levels to estimate the levels of NO ^- formation, since NO ^- is highly unstable and has a very short half-life. Evidence of the role of NO ^- in carcinogenesis has been provided by the fact that iNOS expression was found in various human cancers. ^[23],[24] A higher expression of iNOS in oral cancer patients has also been reported. ^{[25],[26],[27]} It was documented as expression of iNOS in a high-grade tumor in biopsy samples of human cancer. ^[12] Earlier studies have reported significantly elevated NO ₂ +NO ₃ levels in various malignancies. ^{[28],[29],[30]} Previous study has reported significantly elevated NO _2, NO ₃ and NO ₂ +NO ₃ levels in oral cancer patients as compared to normal healthy subjects, ^[8] However, earlier studies have not included patients with OPC for study of plasma NO ₂ +NO ₃ levels along with oral cancer. In addition to the patients with OPC, the present study compared plasma NO ₂ +NO ₃ levels between NHT and WHT. The patients with OPC and oral cancer patients showed significantly elevated plasma NO ₂ +NO3 levels as compared with NHT and WHT. As evidence by other investigators, this could result from a generalized increase in NOS through NO degradation promoted by oxidative stress. ^[8]

Antioxidant enzymes such as SOD and catalase can directly counter balance the oxidant attack and may protect cells against DNA damage. SOD inhibits OH ^- production; therefore it acts as inhibitor at the initiation and promotion stages during carcinogenesis. In the present study, erythrocyte SOD activities were found to be higher in WHT as compared to NHT. Erythrocyte SOD levels were higher in patients with OPC than WHT and NHT. It has been reported that erythrocyte SOD levels were significantly elevated in patients with OPC as compared to healthy individuals. ^[31] This might be due to tobacco associated changes in response to free radical generation in WHT and patients with OPC. It has been reported that SOD and catalase play an important role as first line of defense in response to ROS mediated changes. ^[32],[33] Moreover, contradictory reports for SOD activity in cancer patients are documented. Several authors have reported significantly elevated erythrocytes SOD activity in various malignancies. ^[34] On the other hand, decreased erythrocytes SOD activity in various malignancies has also been documented. ^{[35],[36],[37]} The present study showed that erythrocyte SOD activity was decreased in oral cancer patients than healthy individuals (NHT and WHT) and patients with OPC. ^[37] The low activity of erythrocyte SOD in the present study might be due to the depletion of antioxidant defense system, which could occur as the consequence of overwhelming free radicals. A previous report has also suggested that lower antioxidant enzymes activities in oral cancer patients might be due to the depletion of the antioxidant defense system. ^[8] This could occur as a consequence of overwhelming free radicals by the elevated levels of lipid peroxides in the circulation of oral cancer patients.

Results on erythrocytes catalase activity in cancer patients are contradictory. Previous studies have reported both increased and decreased erythrocytes catalase activity in oral cancer patients. ^[38],[39] In the present study, erythrocyte catalase activity was significantly elevated in WHT and patients with OPC than NHT. However, erythrocyte catalase activity was decreased in oral cancer patients than OPC. The decrease in catalase activity may be due to higher OH ^- production, which may adversely affect the activity of catalase. It has reported that low activity of erythrocyte catalase in oral cancer patients might be due to increased endogenous production of the superoxide anion by increased NO ^- end products or decreased activity of antioxidant enzymes or all of these factors. ^[8] The present study documented that the erythrocyte SOD activity was higher in advanced malignant disease than early disease. Erythrocyte catalase activity was lower in tumor with poor differentiation than well and moderately differentiated tumors. These results indicated that the alterations in antioxidant enzymes might be useful in the molecular diagnosis of oral cancer. ^[14]

In conclusion, the results from the present study illustrate a potential involvement of nitric oxide and antioxidant enzymes in the pathogenesis of oral cancer as evident from enhanced nitric oxide products with deranged antioxidant defense system. Thus, the present study suggests that alterations in NO2 +NO3 and antioxidant enzymes might have a significant role in oral carcinogenesis.

Acknowledgment

The project was funded by Department of Atomic Energy (DAE), Government of India (Grant No: 2004/37/4/BRNS with BSC, BRNS)

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