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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 28  |  Issue : 2  |  Page : 134-139

Expression of cyclo-oxygenase-2 enzyme in the tissue samples of patients with various clinicopathological stages of oral leukoplakia and oral squamous cell carcinoma


1 Department of Oral Medicine and Radiology, Rajas Dental College, Tirunelveli, Tamil Nadu, India
2 Department of Oral Medicine and Radiology, Rajah Muthaiah Dental College, Annamalai University, Chidambaram, Tamil Nadu, India
3 Department of Oral, Basic and Clinical Sciences, Division of Oral Diagnosis, Faculty of Dentistry, Qassim Private Colleges, Buraydah, Qassim Province, Kingdom of Saudi Arabia

Date of Submission30-Sep-2015
Date of Acceptance27-Aug-2016
Date of Web Publication02-Dec-2016

Correspondence Address:
Nelson Aruldoss
3, Sowdambigai Nagar, Thirupapuliyur, Cuddalore - 607 002, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-1363.195096

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   Abstract 

Aim: The purpose of this study was to evaluate the expression of cyclo-oxygenase-2 (COX-2) enzyme in the tissue samples of patients with various clinicopathological stages of oral leukoplakia and oral squamous cell carcinoma (OSCC). Materials and Methods: The samples for the study were divided into 4 groups. Group A comprised 20 healthy individuals with no habits. Twenty healthy individuals with habitual tobacco usage and no oral lesions were included in Group B. Twenty cases of leukoplakia diagnosed clinically and histopathologically were included in Group C. Staging was done using the modified classification and staging system of oral leukoplakia. Twenty cases of OSCC diagnosed clinically and histopathologically were included in Group D. Immunohistochemical staining was done on these 80 samples (paraffin blocks) for COX-2 expression by indirect method using polymer based Horseradish peroxidase system. Statistical analysis was performed using Kruskal-Wallis test and Spearman's rank correlation test. Results: Significant and proportional increase of COX-2 staining was noted with the increase in the severity of dysplasia. Eighty percent of OSCC expressed COX-2, increasing in its intensity of staining with the decrease in differentiation. Seventy five percent of leukoplakia showed positive COX-2 expression. Only 15% of positive controls were COX-2 positive. No normal mucosa showed positive expression of COX-2. Conclusion: High expression of COX-2 is seen in advanced stages of leukoplakia and OSCC. Hence, COX-2 enzyme increases cell proliferation, promotes angiogenesis and inhibits immune surveillance in carcinogenesis; it can be an early detection marker in oral leukoplakia and a prognostic marker of OSCC.

Keywords: Cyclo-oxygenase-2, immunohistochemistry, leukoplaia, oral squamous cell carcinoma, prostaglandin


How to cite this article:
Aruldoss N, Sarvathikari R, Srivastav CK, Austin RD. Expression of cyclo-oxygenase-2 enzyme in the tissue samples of patients with various clinicopathological stages of oral leukoplakia and oral squamous cell carcinoma. J Indian Acad Oral Med Radiol 2016;28:134-9

How to cite this URL:
Aruldoss N, Sarvathikari R, Srivastav CK, Austin RD. Expression of cyclo-oxygenase-2 enzyme in the tissue samples of patients with various clinicopathological stages of oral leukoplakia and oral squamous cell carcinoma. J Indian Acad Oral Med Radiol [serial online] 2016 [cited 2019 Mar 24];28:134-9. Available from: http://www.jiaomr.in/text.asp?2016/28/2/134/195096


   Introduction Top


With approximately 500,000 new cases and 250,000 deaths annually, squamous cell carcinoma of the head and neck (HNSCC) represents the sixth most common cancer in world. [1] Despite recent advances in radiotherapy and chemotherapy, the survival of patients with HNSCC has not improved significantly. Hence, new molecular targets are needed for the prevention and treatment of HNSCC and related cancers. Nonsteroidal anti-inflammatory drugs, which are potent inhibitors of cyclooxygenase (COX), have been shown to exert chemopreventive effects on cancer development. [2] COX, which are prostaglandin endoperoxide synthase, are rate-limiting enzymes for the conversion of arachidonic acid to prostaglandins. There are two isoforms of COX; COX-1 is constitutively expressed and COX-2 is inducible. COX-2 is expressed in inflammatory cells, such as macrophages, fibroblasts and vessel endothelial cells after being stimulated by antigens or cytokine. [3] Nevertheless, how COX-2 overexpression results in tumorigenesis remains poorly understood. [4] The importance of COX-2 in tumorigenesis was first demonstrated in rodent models of familial adenomatous polyposis, a genetic disease leading to gastrointestinal cancer, in which loss of COX-2 activity by either genetic deletion or selective enzymatic inhibition suppressed intestinal polyp formation. [5] Recent studies showed increased levels of COX-2 in premalignant and malignant lesions and genetic evidence of COX-2 implication in tumorigenesis. [6] In the present study, we investigated whether there was overexpression of COX-2 in premalignant lesion (leukoplakia) and HNSCC compared with normal mucosa of healthy volunteers. We also estimated and compared COX-2 levels in various clinicopathological stages of oral leukoplakia and oral squamous cell carcinoma (OSCC) and evaluated the possibility to propose COX-2 as a potential early detection marker and/or as a prognostic tool.


   Materials and Methods Top


The participants for the study were selected among the outpatients who visited the Department of Oral Medicine and Radiology between November 2012 and August 2013. The design of the study was approved by the Institute of Human Ethical committee, Annamalai University and informed consent was obtained from all the patients. The study group comprised a sample size of 80 patients divided into 4 groups: Group A, 20 healthy individuals with no potential oral habits and no oral lesions. Group B, 20 participants with potential oral habits but no oral lesions. Group C, 20 participants with clinically and histopathologically proven cases of oral leukoplakia. Group D, 20 participants with clinically and histopathologically proven OSCC.

A detailed case history of the patients with emphasis on their habits (Betel quid, gutkha, smoking, alcohol, etc.) was obtained, and a thorough clinical examination was done and recorded on a standard proforma. The participants for these groups were selected from patients who required extraction and other minor surgical procedures. Patients who underwent previous treatment for oral leukoplakia and OSCC, pregnant women, and patients suffering from systemic diseases were excluded. In patients with leukoplakia and OSCC, appearance of lesion and size was measured. Staging for the patients with leukoplakia was done using the method proposed by van der Waal et al. [7] After establishing the clinical diagnosis of leukoplakia and oral cancer, the patients were subjected to routine blood investigations (hemoglobin%, total count, differential count, clotting and bleeding time, random blood sugar) to rule out any systemic ailments. Incisional biopsy was performed in patients with leukoplakia and OSCC, which was divided into two specimens. One specimen was stained with routine hematoxylin-eosin stain to analyze histopathological criteria followed to diagnose leukoplakia (graded as mild/moderate/severe dysplasia) and SCC (well/moderate/poorly differentiated) [8] and another for immunohistochemical staining. Primary reagent for COX-2 used was Rabbit Monoclonal antibody to COX-2 Receptor (Thermo Scientific, UK); secondary antibody was poly Horseradish peroxidase (HRP) enzyme (SCYTEK, USA) and the mountant was distrene dibutylpthalate xylene (DPX).

The indirect method using polymer-based HRP system was performed. All the 80 formalin-fixed paraffin embedded samples were covered with 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity followed by treatment with protein block for 10 min to avoid cross reaction. The sections were then covered completely with primary antibody Rabbit Monoclonal antibody against COX-2 receptor. The slides were washed and treated with secondary antibody poly HRP enzyme for 30 min. The slides were then washed with phosphate buffer solution. Immunostaining was carried out with 3, 3-diaminobenzidine tetrahydrocholoride (DAB) for 5 min. The slides were counterstained by immersion in Mayer's hematoxylin for 7 min. The sections were dehydrated and mounted. In positive and negative control groups, only one specimen was taken from each patient for immunohistochemical staining. [9]

Cytosolic staining for COX-2 was scored as follows: 0, no staining of cells in any microscopic field; 1+, less than 25% of tissue stained positive; 2+, between 25 and 50% staining positive; 3+, between 50 and 75% staining positive; 4+, greater than 75% staining positive. The intensity of staining (viewed at a magnification of ×100) was on the following scale: 0, no staining seen; 1+, mild staining; 2+, moderate staining; 3+, intense staining. The scores of area and intensity of staining were combined to give a value between 0 and 7.


   Results Top


A total of 80 participants included 20 cases of OSCC (15 male patients and 5 female patients with a mean age of 55.75 ± 13.21 years); 20 cases of Leukoplakia (16 male patients and 4 female patients with a mean age of 40.55 ± 11.83 years). Twenty cases of positive control (12 male patients and 8 female patients with a mean age of 34.15 ± 7.19 years) and 20 cases of normal oral mucosa (12 male patients and 8 female patients with a mean age of 23.20 ± 4.24 years) were included in this study.

All our patients in Group D had a habit of chewing tobacco with or without additives. Group D patients had an average duration of habit of 22 ± 11.13 years with an average frequency of 5.25 ± 3.38 times/day, ranging from 3-10 times/day. Thus, our data confirms the gravity of these risk factors. The site distribution of the leukoplakia and OSCC seen in our samples is shown in [Table 1]. Among specimens of dysplasia in this study, 7 cases (35%) showed mild dysplasia, 5 cases (25%) moderate dysplasia, and 8 cases (40%) severe dysplasia; among samples of SCC, 12 samples (60%) were well differentiated, 7 cases (35%) were moderately differentiated, and 1 case (5%) was poorly differentiated. The study sample showed maximum participants with well-differentiated grade of OSCC.
Table 1: Distribution of location of lesions in samples of oral squamous cell carcinoma and leukoplakia


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The comparison of COX-2 expression in 4 groups, i.e., negative control, positive control, leukoplakia, and oral cancer is depicted in [Table 2]. Kruskal-Wallis test (nonparametric test of significance; two tailed) was applied at 99% confidence interval (CI). The mean value of COX-2 expression was 0, 29.72, 48.10, and 59.18 in groups A-D, respectively. A very high significant increase in expression was seen from control through leukoplakia to oral cancer. The comparison of COX-2 expression among three grades of leukoplakia is shown in [Table 3]. Kruskal-Wallis test (nonparametric test of significance; two tailed) showed a significant increase in expression along increasing dysplasia and statistically significant value (P < 0.000). A high significant correlation was found between COX-2 expression and grades of dysplasia [Figure 1] and [Figure 2]. The comparison of COX-2 expression among three grades of OSCC is presented in [Table 4]. Kruskal-Wallis test showed an increase in expression along increasing loss of differentiation and statistically significant value (P < 0.001). A high significant correlation was found between COX-2 expression and grades of differentiation [Figure 3] [Figure 4] [Figure 5].
Figure 1: Mild dysplasia

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Figure 2: Moderate dysplasia

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Figure 3: Well differentiated carcinoma

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Figure 4: Moderately differentiated carcinoma

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Figure 5: Poorly differentiated carcinoma

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Table 2: Group comparison showing positive for COX-2


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Table 3: Correlation between grades of dysplasia for group C using Spearman's rank correlation test


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Table 4: Comparison of grades of differentiation within group D using Spearman's rank correlation test


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   Discussion Top


Squamous cell carcinoma of the head and neck is one of the most common human cancers and remains a major cause of mortality and morbidity throughout the world. [10] Age, gender, site-wise distribution of leukoplakia and OSCC participants of our study is in accordance with the previous studies done by Napier et al. and Sankaranarayanan et al. [11],[12],[13] In our study, COX-2 staining was not demonstrated in normal oral mucosa. In group B, COX-2 expression was seen in 15% of the cases. In group C, it was 60% of the cases. In group D, COX-2 expression was seen in 80% of the cases. Comparison of COX-2 expression in 4 groups, i.e., negative control, positive control, leukoplakia and oral cancer revealed a significant and proportional increase in expression from normal to cancer. Similar to oral cancers, leukoplakia also involves all intraoral sites. The location distribution of leukoplakia varies in different geographic areas according to the habitual usage. Our study samples showed higher prevalence in buccal mucosa which correlated with the literature. [12],[13] The shortcoming of this study was the inability to collect age and sex matched samples for the control groups because of ethical clearance and patient unavailability. The primary concern of this study was to select the samples devoid of inflammation, not correlating on the age and gender in COX-2 expression.

In HNSCC, diffuse faint COX-2 expression was also observed in cancer cells without mitosis. This result was similar to the study done by Kutchera et al. who investigated esophageal squamous cell carcinoma. [14] In agreement with our study, Segawa et al. and Chan et al. also found the expression and upregulation of COX-2 in human oral epithelial dysplasia and HNSCC. [15],[16] In our study, the comparison of COX-2 expression among three grades of oral cancer showed a significant increase in expression along with the decrease in differentiation. Previous studies by various researchers correlates with our results. [15],[16] A study by Mittal et al. showed similar results and also indicates that functional genetic variants of 50 and 30 UTR of COX-2 gene may have potential risk-modifying effects in tobacco-related oral squamous cell carcinoma in Asian Indians. [17] Abrahao had done a study which supports the emerging notion that PGE2 is produced in the tumor microenvironment by the over expression of COX-2 in tumoral and inflammatory cells which may promote the growth of HNSCC. [18] The study done by Hao et al. in 2009 showed that overexpression of COX-2 in oral mucosa has been associated with an increased risk of HNSCC. [19] Another study has shown that HNSCC development is closely associated with inflammation. [20] COX-2 is an important mediator of inflammation. According to Yang et al., PGE2 and EP1 interaction might act through the PKCd, c-Src, c-Jun, and AP-1 pathway to induce ICAM-1 activation in human oral cancer cells. [21]

On the basis of these findings, it can be inferred that the COX-2 expression is upregulated in leukoplakia and highly expressed in OSCC. Increasing scores and values suggests enhanced synthesis of prostaglandins, which is a consequence of upregulation of COX-2 which can increase cell proliferation, promote angiogenesis and inhibit immune surveillance. [22] Renkonen et al. reported that overexpression of COX-2 in tongue SCC is almost associated with histological grade. [23] The increased expression of COX-2 during oral carcinogenesis may depend on the developmental stage of the tumor, as well as etiologic factors such as the types of mutations and distinct types of injuries affecting different regions. Thus, there are wide variations in pathways and factors that lead to or affect oral carcinogenesis. [24] In addition, further studies are required to determine which of these mechanisms are more important in the development and progression of OSCC.


   Conclusion Top


Our study showed that there was an increased expression of COX-2 from leukoplakia to OSCC. Therefore, COX-2 can be used as an early detection marker in oral leukoplakia and a prognostic marker in OSCC. However, further studies are needed with a larger sample size to validate the use of COX-2 expression as a marker of oral leukoplakia and as a prognostic marker of OSCC.

Acknowledgements

The histopathological sectioning for the study was carried out in the Department of Oral and Maxillofacial Pathology, Rajah Muthaiah Dental College, Annamalai University, Chidambaram, under the supervision of Dr. Madhava Nirmal, Professor and Head of the Department of Oral and Maxillofacial Pathology, Rajah Muthaiah Dental College.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

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Mittal M, Kapoor V, Mohanti BK, Das SN. Functional variants of COX-2 and risk of tobacco-related oral squamous cell carcinoma in high-risk Asian Indians. Oral Oncol 2010;46:622-6.  Back to cited text no. 17
    
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Renkonen J, Wolff H, Paavonen T. Expression of cyclo-oxygenase-2 in human tongue carcinoma and its precursor lesions. Virchows Arch 2002;440:594-7.  Back to cited text no. 23
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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