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Year : 2014  |  Volume : 10  |  Issue : 2  |  Page : 244-250

Do tobacco stimulate the production of nitric oxide by up regulation of inducible nitric oxide synthesis in cancer: Immunohistochemical determination of inducible nitric oxide synthesis in oral squamous cell carcinoma - A comparative study in tobacco habituers and non-habituers

Department of Oral Pathology and Microbiology, Coorg Institute of Dental Sciences, Virajpet, India

Date of Web Publication14-Jul-2014

Correspondence Address:
B Karthik
Department of Oral Pathology and Microbiology, Coorg Institute of Dental Sciences, Virajpet
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0973-1482.136542

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 > Abstract 

Background: Oral cancer accounts for 6% of all cancers. The most prevalent form of oral cancer is oral squamous cell carcinoma (OSCC), which accounts for 90% of the oral cancer cases. The major risk factor for development of OSCC is the use of tobacco in various forms. NO has been studied widely over the years due to its role in various physiological and pathophysiological processes, including its complex role in carcinogenesis.
Materials and Methods: A total of 20 cases of OSCC in tobacco habituers and tobacco non-habituers were retrieved respectively from the archival biopsy specimens. Immunohistochemistry was done to assess the inducible nitric oxide synthase (iNOS) protein.
Results: This study was performed to assess the correlation between tobacco and nitric oxide in OSCC in order to know the association of these two in the process of carcinogenesis. The results showed the enhanced expression of iNOS in tobacco habituers in comparison with tobacco non-habituers. Though the increased expression of iNOS is found, significant difference is not obtained with scores, but significant difference was found with intensity of staining.
Conclusions: The results of the present study indicate the enhanced expression in OSCC of tobacco habituers when compared to OSCC of tobacco non-habituers indicating the effect of tobacco on nitric oxide. Carcinogenic chemical compounds in Tobacco induce nitric oxide production by iNOS, by its tumor-promoting effects which may enhance the process of carcinogenesis.

 > Abstract in Chinese 


Keywords: Carcinogenic chemical, inducible, oral squamous cell carcinoma, tobacco

How to cite this article:
Karthik B, Shruthi D K, Singh J, Tegginamani AS, Kudva S. Do tobacco stimulate the production of nitric oxide by up regulation of inducible nitric oxide synthesis in cancer: Immunohistochemical determination of inducible nitric oxide synthesis in oral squamous cell carcinoma - A comparative study in tobacco habituers and non-habituers. J Can Res Ther 2014;10:244-50

How to cite this URL:
Karthik B, Shruthi D K, Singh J, Tegginamani AS, Kudva S. Do tobacco stimulate the production of nitric oxide by up regulation of inducible nitric oxide synthesis in cancer: Immunohistochemical determination of inducible nitric oxide synthesis in oral squamous cell carcinoma - A comparative study in tobacco habituers and non-habituers. J Can Res Ther [serial online] 2014 [cited 2022 Nov 29];10:244-50. Available from: https://www.cancerjournal.net/text.asp?2014/10/2/244/136542

 > Introduction Top

Oral squamous cell carcinoma (OSCC) accounts for more than 90% of all oral cancers, the overall 5-year survival rate in OSCC has not significantly increased in the last few years. The overall disease-free survival rates are 56% and 58% respectively. The most important task is to establish an early diagnosis at initial stages of the disease. [1] Oral cancer is one of the most common cancers representing 6% of all cancers in populations. [2],[3] In India, it represents the commonest among males and third most common among females. [4]

The known classic risk factor of oral cancer is tobacco use and other etiological factors include alcohol, infections, dietary factors, and chemical irritants. [5],[6] Tobacco products contain a large array of carcinogens, such as benzopyrene and other polycyclic aromatic carcinogens which are the most important carcinogenic agents in cigarette smoke, but in chewable tobacco, nitrosamines are the strongest carcinogens. The metabolites of the above-mentioned compounds are found in saliva of the oral cavity as well as in other body fluids. These agents are known to cause toxic effects, particularly cancer and other cellular and DeoxyriboNucleic Acid(DNA) changes. Oral carcinogenesis is a multistep process in which multiple genetic events occur that alter the normal functions of oncogenes and tumor-suppressor genes. This can result in increased production of growth factors or number of cell-surface receptors, enhanced intracellular messenger signaling, and/or increased production of transcription factors. In combination with the loss of tumor-suppressor activity, this leads to a cell phenotype capable of increased cell proliferation with loss of cell cohesion and the ability to infiltrate local tissue and spread to distant sites. Despite treatment advances, patients with advanced disease have a poor prognosis. More than two-thirds of the individuals with Head & neck squamous cell carcinoma (HNSCC) at diagnosis will present with stage III and stage IV disease. Despite aggressive therapy based on combination of surgery and radiotherapy, loco-regional recurrence will develop in 50-60% of patients and distant metastasis in 10-20%. [7],[8],[9]

Nitric oxide (NO) is a highly reactive oxygen radical found in normal and malignant tissues. However, its levels are much higher in malignant tissue, which is thought to be generated by a family of enzymes called nitric oxide synthase (NOS). NOS is available in three isoforms NOS1 or type 1 or nNOS (neuronal), NOS2 or type 2 or iNOS (inducible), NOS3 or type 3 or eNOS (endothelial). Out of these, iNOS produces continuous NO and is shown to be expressed in many malignant tumors. NO which is produced by iNOS has shown to have a dual role in cancer process. NO has been implicated in both tumor-promoting and tumor-inhibiting actions in tumor biology. Inhibiting actions like inducing apoptosis and promoting actions being induction of angiogenesis, degrading extracellular matrix, inactivation of p53, and inducing metastasis. The actions are shown to be concentration-dependent. [10],[11]

Tobacco increases production of free radicals such as NO, superoxide anions etc., By increased production of these radicals, they yield by-products which are capable of inducing various type of stress and biological effects and has been concerned in the process of carcinogenesis. [12]

Considering the potential role of NO in carcinogenesis, and the causative relation between tobacco and development of oral cancers, analysis of any correlation between tobacco use and NO expression and to assess how tobacco use is linked to NO is deemed an important aspect in development of oral cancer.

 > Materials and methods Top

The sample size of the present study consisted of 20 cases each of OSCC in tobacco habituers and tobacco non-habituers, respectively, from the archival biopsy specimens. Two sections were cut for each of the formalin-fixed paraffin-embedded specimens from the rotary microtome; one section with thickness of four microns, stained with Harris haemotoxylin and other section of four microns thickness were taken in poly-L-lysine-coated slides for immunohistochemistry.


Immunostaining was performed by a standard avidin-biotin-peroxidase complex method. The previously fixed tissues were embedded in paraffin. Five-micrometer specimens were made which were mounted on poly-L-lysine-coated glass slides and dried overnight at room temperature. After the sections had been deparaffinized in xylene and rehydrated using graded ethanol, they were immersed in 3% hydrogen peroxide in methanol (V/V) for 15 min in order to quench the endogenous peroxidase activity. They were then washed in tris(hydroxymethyl)aminometh (TRIS) buffer and incubated with normal 1% bovine serum albumin in TRIS buffer for 1 h to reduce the nonspecific binding of the primary antibody (NOS2 [N-20] - {SC-651}, rabbit polyclonal Immunoglobulin G (IgG) [Santa Cruz Biotechnology Secondary antibody: Polymer/HRP sensitive kit - Biogenix life sciences]). After washing in TRIS buffer, the tissues were stained for detecting the presence of iNOS protein using a primary rabbit polyclonal antibody at 1:200 dilutions overnight in a humidified chamber. They were then incubated for 30 min at room temperature with biotin-conjugated and then for 30 min with ABC. To detect the immunoreactivity, the sections were treated with diaminobenzidine and counterstained with hematoxylin. Negative controls for the specificity of anti-iNOS antisera were included by omitting the primary antibody.


Tumor markers - brown in color and nucleus - blue in color.

Counting criteria

Neoplastic cells and inflammatory cells were counted in all the samples in four different microscopic fields under 100 × magnifications. Mean of three observations was taken into considerations.


0 - No cell stained

1 - Less than 25% of cells stained

2 - Between 25-50% of cells stained

3 - Between 50-75% cells stained

4 - More than 75% cells stained

The scores ranged from 0-16 after counting all the four fields in each section.

Statistical analysis

Statistical significance was analyzed by using the Student's t-test for two groups and one-way analysis of variance for multi-group comparison.

  1. OSCC in tobacco habituers (Both neoplastic and inflammatory cells)
  2. OSCC in tobacco non-habituers (Both neoplastic and inflammatory cells)

 > Results Top

The scores are given as 0, 1+, 2+, 3+, 4 + according to the percentage of the positive cells, counted within group, neoplastic and inflammatory, and compared between groups.

When the tobacco habituers group was analyzed for neoplastic cells, no field showed 0 scores, 1.3% showed 1+, 8.8% showed 2+, 32.5% showed 3+, and 57.5% showed 4+, and inflammatory cells, 1.3% showed 0, 5.0% showed 1+, 32.5 showed 2+, 52.5 showed 4+.

Within the group when compared between neoplastic and inflammatory cells in tobacco habituers, neoplastic cells showed 57.5% of score 4+, but which was only 8.8% in inflammatory cells. The majority of the neoplastic cells was 3 + and 4 + which is 32.5% and 57.5% respectively. Whereas in inflammatory cells, majority is found to be in 2 + and 3 + which is 32.5% and 52.5% respectively [Graph 1] [Additional file 1] and [Graph 2]. [Additional file 2]

The group tobacco non-habituers assessed for neoplastic cells showed 1.3% of 0 score, 3.8% showed 1+, 15% showed 2+, 35% showed 3+, and 45% showed 4+. Inflammatory cells showed 0% of 0+, 3.8% of 1+, 52.5% of 2+, 32.5% of 3 + and 11.3% of 4+.

The overall score were assessed in tobacco habituers and tobacco non-habituers. In tobacco habituers 6% showed 0, 3.1% showed 1+, 20.6% showed 2+, 42.5% showed 3+, 33.1% showed 4+. Whereas in tobacco non-habituers, 6% showed 0, 3.8% showed 1+, 33.8% showed 2+, 33.8% showed 3+, 28.1% showed 4+ [Graph 3]. [Additional file 3]

Even though there was an increase in the expression of iNOS in the habituers group, the scoring comparison between group does not show statistical significance [Figure 1] and [Figure 2].
Figure 1: Enhanced expression of inducible (iNOS) in protein by tumor cells in oral squamous cell carcinoma of tobacco habituers (iNOS, ×200)

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Figure 2: Expression of iNOS protein by tumor cells in OSCC of tobacco habituers (iNOS, ×200)

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Intensity of tobacco habituers and tobacco non-habituers was scored as 0, 1+, 2+. 3+. The tobacco habituers group showed 0% of 1+, 50% of 2+, and 50% of 3+. Whereas in the tobacco non-habituers group, 15% showed 1+, 75% showed 2+, 10% showed 3+. Tobacco habituers group had majority of its cases showing 2 + and 3 + that is 50% in both and without tobacco showing majority 2 + comprising 75% of cases [Graph 4]. [Additional file 4]

With respect to positive cells in comparison between groups, in tobacco habituers, neoplastic and inflammatory cells shows higher mean of 36.13 and 26.35, respectively, when compared to tobacco non-habituers 30.40 and 25.95 respectively [Figure 3].
Figure 3: Expression of iNOS protein by inflammatory cells in OSCC of tobacco habituers (iNOS, ×200)

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Even though an enhanced expression of iNOS is seen with tobacco habituers group when compared to tobacco non-habituers, the statistically significant difference was not found with scoring but the significant difference was found in intensity, showing more intense staining in tobacco habituers when compared to non-habituers.

 > Discussion Top

The major risk factor for the development of OSCC is the use of tobacco in various forms and other etiological causes being alcohol, infections, dietary factors, and chemical irritants. Oral carcinogenesis is a multistep process in which genetic events lead to disruption of normal regulatory pathways that control the basic cellular functions including cell division, differentiation, and cell death. The crucial event in transformation of cell to a malignant cell involves the complex step of activation and inactivation of certain regulatory genes. [1],[7],[8],[9]

In the developed countries, oral and pharyngeal cancers rarely occur in people who neither smoke nor drink alcohol. However, many epidemiological studies conducted over the last three decades in the Americas, Europe, and Asia have provided strong evidence of an association between alcohol and tobacco use (both separately and in combination) and an increased risk of oral and pharyngeal tumors. The risk of both oral and pharyngeal cancer rises steeply with the level of alcohol consumption. An analysis that pooled data (i.e. a meta-analysis) from 26 studies of oral and pharyngeal cancers found that consumption of 25, 50, or 100 g pure alcohol/day was associated with a pooled relative risk of 1.75, 2.85, and 6.01, respectively, of oral and pharyngeal cancer. Thus, there appears to be sufficient evidence in laboratory animal studies to support a link between alcohol and the development of oral cancer. Oral streptococci may contribute significantly to the normal individual variation of salivary acetaldehyde levels after alcohol drinking and thereby, also to the risk of oral cancer. This may in fact be the mechanism that explains the observed phenomena that individuals with poor oral hygiene have an increased risk of developing oral cancer. People who drank heavily and smoked had a 300 times higher risk of oral and pharyngeal cancer than people who neither drank nor smoked. It has been difficult to distinguish the separate effects of these agents, however, since drinkers of alcoholic beverages tend to be users of tobacco, and vice versa. Moreover, studies have shown that heavy intake of alcoholic beverages is associated with nutrient deficiency, which appears to contribute independently to oral carcinogenesis. Although there is considerable controversy and debate about how to define oral leukoplakia, there is little doubt that both tobacco, in any form, and areca nut use are major risk factors for developing this condition. This condition is well known for its potential for malignant change, and transformation rates between 0.1% and 17.5% have been quoted in the literature. The figures for malignant transformation based on population studies reported from India are much lower than those reported from Europe and the United States based on hospital series, but this may be due to a selection bias. There is historical evidence dating back to nearly a century that suggests that the areca nut may be involved in the development of OSCC, although it is widely accepted that the presence of tobacco in betel quid plays an important role in the pathogenesis of OSCC the carcinogenic potential of areca in the absence of other ingredients is less clear. In addition to human data, there are also a large number of experimental studies, which have attempted to evaluate the carcinogenic potential of areca and its derivatives both in vivo and in vitro in a recent review that examined the mutagenicity and genotoxicity of areca alkaloids to target cells in the oral mucosa. Nitric oxide has been studied widely over the years due to its role in various physiological, pathophysiological processes, including its complex role in carcinogenesis. The functions being vascular functions, neurological functions, and at a relatively high concentration, cytotoxic function. Various diseases and conditions are seen to be associated with altered nitric oxide production giving a clue of possibility of participation of NO in the pathogenesis of disease. The production of NO and expression of NOS can be seen in association with infectious diseases, autoimmune and chronic inflammatory diseases, chronic neuro-degeneration, ischemic events, noxious insults, transplantations, and various tumors. NO has a dual role in tumor biology by promoting and inhibiting tumor progression and metastasis. The effects of NO in tumor seem to depend on the activity and localization of NOS isoforms concentration and duration of NO exposure and cellular sensitivity to NO. NO production may promote cancer progression by providing a selective growth advantage to tumor cells with mutant p53. Induction of angiogenesis by modulating angiogenic factors, thus promoting tumorigenic activity or by activating p53 leading to apoptosis exhibiting tumoricidal activity. [10],[13],[14],[15],[16],[17],[18],[19] NO is one of the 10 smallest molecules. The arrangement of one atom of nitrogen and one atom of oxygen leaves an unpaired electron which makes the molecule a highly reactive free radical. [20] Its reaction with oxygen or oxygen-related reactive intermediates yield numerous reactive nitrogen as well as oxygen species. These account for most of so-called indirect effects attributed to NO through oxidation, nitrosation, and nitration reactions leading to formation of certain substances which cluster carcinogenic substances, which may lead to damage of DNA. NO is thought to be formed by NOS. NOS catalyzes NO biosynthesis via a reaction involving the conversion of L-arginine to L-citruline. There are three distinct isoforms of NOS which differ both in their structure and function. eNOS NOS (eNOS, NOS III) and nNOS NOS (nNOS, NOS I) are generally referred to as constitutively expressed, Ca + 2 dependent enzyme, while iNOS NOS (iNOS, NOS II) is expressed in high levels and produces continuous NO and is formed by induction by cytokines or other inflammatory agents and its activity is independent of Ca + and is expressed in a wide variety of human cells. NO production by tumor cells may influence tumor cell growth, differentiation, metastatic capability, chemosensitivity, radiosensitivity, and tumor-induced immunosuppression. [10],[11],[14],[21]

High expression of iNOS has been shown in the carcinomas of different organs and parts of the body and has been implicated in the role of development of those malignancies. Contrasting to this, studies which have shown expression of NO to be associated with tumoricidal activities also exist. [22],[23],[24]

Studies on OSCC and oral dysplasias has shown that, there is an increased expression of iNOS on OSCCs when compared to oral dysplasia and is absent in normal epithelium indicating the role of NO in neoplastic transformation and carcinogenesis. [25]

The expression of iNOS in dysplasia correlated with p53 expression, the iNOS level increased the severity of the dysplasia and correlated with p53 expressions, hence it was concluded that there was a possibility of this relationship being a major contributing factor in development of cancer from premalignant and in-situ lesions. Suggestions had also been made that high levels of NO are required in the first stages of HNSCC tumor growth, probably as a result of iNOS activity to activate angiogenesis and enhance adhesibility and permeability. The high iNOS expression seen in severe dysplasia might contribute to these processes before the transformation into invasive cancer. [26] It has also been demonstrated that enhanced iNOS expression in the premalignant lesions of the oral cavity was more in severe dysplasia indicating the vital participation of iNOS in the process of carcinogenesis. [27] It further showed the correlation between the iNOS and Vascular Endothelial Growth Factor (VEGF) in oral dysplasia. The enhanced iNOS, VEGF expression, and correlation with severity of dysplasia may provide an information on complex transformation of oral epithelial dysplasia to invasive carcinoma and the role of angiogenesis. [28] A number of studies have shown the enhanced expression of iNOS in OSCC indicating the role of NO in carcinogenesis. [25],[29],[30] Through these correlations and enhanced expression, the iNOS can be implicated in the development of cancer.

Tobacco includes specific chemicals like polycyclic aromatic hydrocarbons, N-Nitrosamines aromatic amines, ethylene oxide, 1,3-butadine, and other tobacco-specific nitrosamines. Polycyclic aromatic hydrocarbons are the most effective carcinogens in humans. [30] Tobacco causes increased generation of free radicals and reactive oxygen species, such as NO, superoxide anions, hydroxyl radicals, etc., Tobacco-. Nicotine from cigarette smoke has shown to stimulate NO production neurologically and also shown to stimulate angiogenesis and promotes tumor growth thought to be mediated by the production of NO and other factors. [31],[32],[33]

The present study included 20 cases of OSCC in tobacco habituers and 20 cases of tobacco non-habituers. By immunohistochemistry, the expression of iNOS was assessed in 40 cases of OSCC.

Expression of iNOS was assessed in the tumor cells and inflammatory cells present in the stroma. The expression of iNOS was compared between groups based on scores and intensity of staining. The scoring was given as 1+, 2+,3+, 4+ according to the percentage of the cells stained, and enhanced expression was seen in the tobacco habituers, as more percentage showed 4+ score indicating relatively more positive cells. Even though there was enhanced iNOS expression in tobacco habituers, no statistically significant difference was found. In tobacco habituers, with a mean of 48.7 neoplastic cells counted, it showed 36.1 mean positive cells and inflammatory cells showed mean 46.2 counted cells showing cells showing 26.3.In tobacco non-habituers, with a mean of 44.0 neoplastic cells counted, it showed mean positive cells of 30.4 and inflammatory cells showed a mean of 53.1 and mean positive cells were 25.The staining intensity was assessed and compared between groups. The tobacco group showed majority of its cases showing 2+ and 3+, that is, 50% in both and without tobacco showing majority 2 + comprising of 75% of cases and least being 3+ comprising of 10% and 1+ in 15% of the cases. With intensity assessment, the high-intensity expression was seen within the tobacco habituers group, while moderate expression was seen in the non-habituers group and showed a statistically significant difference between tobacco habituers and non-habituers. By comparing the score and intensity, the enhanced expression of iNOS is seen in tobacco habituers compared to non-habituers, inferencing the possible influence of tobacco on iNOS expression and NO production.

The findings of the present study are consistent with the previous study on lung carcinoma by Chen et al., who demonstrated the increased levels of iNOS expression in lung carcinoma of smokers when compared to lung carcinoma of non-smokers and also provided the evidence for causative relation between smoking and lung cancer by NNK, the most potent tobacco-specific carcinogen and its correlation to iNOS and proliferation of cells. [34] It has also been demonstrated that inhalation of cigarette smoke causes increased iNOS and increased eNOS in pulmonary vasculatures gives support for the association between tobacco and NO. [35] A study in hamster cheek pouch to assess the tobacco-related compound-induced nitrosative stress showed that the NO radicals released from tobacco-related compounds were shown to cause nitrosative stress and DNA strand break in immortalized hamster cheek pouch cells. Dose-dependent DNA strand break was seen. Nitrosative stress injury is caused by the formation of peroxynitrite, so the NO-mediated pathogenesis may play a vital role in tobacco-associated conditions. [31]

Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis in mouse models and both process are thought to be mediated via production of NO, prostaglandin, and VEGF. This may be correlated with the number cases showing lymph-node metastasis in OSCC of tobacco habituers. [32]

In the present study, twelve cases of OSCC had lymph-node metastasis in tobacco habituers and four cases of non-habituers had lymph-node metastasis, so a proposition can be made that tobacco habituers show enhanced growth and metastasis via NO-mediated pathogenesis as NO causes induction of angiogenesis and enhanced tumor growth and expression has been shown to be associated with extra-capsular spread.

The increase in nitric oxide metabolites in exhaled breath condensate after exposure to tobacco smoke and the deleterious effect of oxidant radicals induced by smoking may contribute to the epithelial damage seen in smokers. [36] In contrast, it is showed decreased exhaled NO by cigarette smoking and hypothesized that oxides of nitrogen in cigarette smoke solution may exert negative feedback mechanism upon NO release from epithelial and basophilic cells. [37] Smokeless tobacco extract causes dose-dependent increase in the levels of NO and the high levels of NO-induced apoptosis. [38]

Contrasting results also have been reported that smoking damages the production of nitric oxide or the expression of NOS is decreased in airway compartments and lung epithelial cells. Studies have also shown that smoking has no effect of NO production or NOS expression. [39],[40],[41],[42]

In conclusion, we found an enhanced expression of iNOS in tobacco habituers when compared to non-habituers. Still, the statistically significant difference between the scores for iNOS expression in tobacco habituers and non-habituers was not found, but a significant difference was found with respect to intensity showing intense staining in OSCC of tobacco habituers. The findings of the present study are in agreement with previous studies in the literature. Within the confines of the present study, the enhanced expression of iNOS in OSCC of tobacco habituers when compared to OSCC of tobacco non-habituers indicates the role of tobacco on production and action of nitric oxide, in the complex process of carcinogenesis. An increased iNOS expression was found in association with tobacco in the present study, though statistically non-significant. Tobacco may stimulate the production of NO, probably via the up-regulation of iNOS in cancer cells. Carcinogenic chemical compounds of tobacco may contribute to promotion of NO in cancer and the increased iNOS may enhance the ability of cancer cells to proliferate and grow. Further prospective studies with larger samples will provide greater insights into the prognostic implication of iNOS.

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  [Figure 1], [Figure 2], [Figure 3]

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