| Abstract|| |
Background and Aim: Oral cancer is a major health problem in South East Asia. The immunohistochemical (IHC) overexpression of COX-2 in squamous cell carcinoma is well documented. This IHC study was undertaken to understand the COX-2 expression in different grades of oral squamous cell carcinoma (OSCC) and to compare the COX-2 expression in OSCC and normal mucosa. Material and Methods: A total of 30 cases of OSCC and 10 cases of normal mucosa and positive control colon cancer were studied for IHC expression of COX-2. Of the 30 cases studied 10 cases each of well, moderately and poorly differentiated carcinoma were studied. COX-2 staining was evaluated on the basis of presence or absence of the positive tumor cells and percentage of positive tumor cells. Statistical Analysis: The various statistical tests used in this study were t-test and Chi-square test which was carried out using SPSS for Windows 22.0.0 and Minitab version 17.1.0 software package. Results: There was significant increase in COX-2 staining intensity from well to poorly differentiated OSCC. Significant difference was observed in staining intensity between moderately and poorly differentiated SCC. The percentage of positive tumor cells were high in poorly differentiated SCC compared to well and moderately differentiated OSCC. No significant expression of COX-2 was noted in normal mucosa. Interpretation and Conclusion: Our results revealed that the COX-2 enzymes were expressed, suggesting that they play complementary roles during oral carcinogenesis. In near future researches on administration of chemoradiation therapy combined with COX-2 should be evaluated to improve therapy response.
Keywords: Cyclooxygenase-2, immunohistochemistry, oral squamous cell carcinoma
|How to cite this article:|
Thomas N, Krishnapillai R, Bindhu P R, Thomas P. Immunohistochemical expression of cyclooxygenase-2 in oral squamous cell carcinoma. Indian J Dent Res 2019;30:102-6
|How to cite this URL:|
Thomas N, Krishnapillai R, Bindhu P R, Thomas P. Immunohistochemical expression of cyclooxygenase-2 in oral squamous cell carcinoma. Indian J Dent Res [serial online] 2019 [cited 2020 Jan 20];30:102-6. Available from: http://www.ijdr.in/text.asp?2019/30/1/102/254519
| Introduction|| |
The development of oral squamous cell carcinoma (OSCC) is a molecular and histological multistep process, the progression of which includes sequential histopathological alterations ranging from hyperplasia through dysplasia to carcinoma in situ and invasive carcinoma. These multiple genetic alterations in OSCC are influenced by a patient's genetic predisposition as well as by environmental influences, including tobacco, alcohol, chronic inflammation, and viral infection. In 1863, Virchow hypothesized that the origin of cancer was at sites of chronic inflammation.
Chronic inflammation has been known to induce neoplasia through increased production of reactive oxygen and nitrogen species, which results in elevated DNA damage. Interleukin (IL) 1, 6, and IL-8, granulocyte-macrophage colony-stimulating factor as well as vascular endothelial growth factor are secreted by head and neck squamous cell carcinoma (HNSCC) cell lines and found in HNSCC patient samples. The key transcription factors downstream of inflammatory processes such as NF-κB and Stat3 have also been shown to play an important role in HNSCC. The expression of Prostaglandin E2 (PGE2) and the Cyclooxygenase-2 (COX-2) which are involved in inflammatory reaction was shown to be increased in HNSCC.
COX-2 an induced pro-inflammatory enzyme involved in the metabolism of arachidonic acid and in the synthesis of prostaglandins (PGs) play a role in various steps of carcinogenesis. PGs, especially of the E series, induce cell proliferation, angiogenesis, invasion, and metastasis. Overexpression of COX-2 gene alters cell adhesion, inhibits apoptosis, and alters the response to growth regulatory signals. Upregulation of COX-2 prolongs the survival of abnormal cells thereby favors the accumulation of sequential genetic changes which increases the risk of tumorigenesis. Furthermore, the expression of COX-2 might shed some light over the pathophysiology and clinical behavior of tumors of the head and neck.
COX-2 has been shown to be upregulated in various types of cancers including those arising from colon, stomach, breast, lung, esophagus, pancreas, bladder, prostate, skin, and in OSCC. Use of nonsteroidal anti-inflammatory drugs, which are prototypic inhibitors of COX, is associated with a reduced risk of several malignancies, including OSCC.
Although overexpression of COX-2 is considered to contribute to oral carcinogenesis, literature supporting this hypothesis is relatively sparse. Therefore, the present study was designed to investigate the qualitative and quantitative immunohistochemical expression of COX-2 in normal mucosa and histopathologically diagnosed OSCC and thereby elucidate their involvement in oral carcinogenesis.
| Methodology|| |
A total of 30 cases of OSCC, 10 cases of normal mucosa and positive control, colon cancer were studied for the immunohistochemistry expression of COX-2. Of the 30 cases, ten cases each of well, moderate, and poorly differentiated carcinoma were studied.
Following approval of our institutional review board, formalin-fixed paraffin-embedded tissue blocks of histopathologically proven cases of OSCC (30) were retrieved. The OSCC cases were graded as well (10 cases), moderate (10 cases), and poorly differentiated (10 cases).
The immunohistochemical analysis for COX2 was performed using super sensitive one-step polymer-HRP technique (Biogenex Life Sciences, USA). Paraffin-embedded tissue blocks were cut into 4 μm thick sections and taken onto 2%, poly-L-Lysine (biogenex) adhesive coated slides. The sections were then deparaffinized and rehydrated through xylene and descending grades of alcohol. Antigen retrieval was done using commercial microwave antigen retrieval system where the sections were placed in a container containing EDTA (pH-9.0) at 95°C for 3 cycles of 5 min each (EZ-Retriever System, Biogenex life sciences, San Ramon, CA, USA). After rinsing in phosphate-buffered saline (PBS), the sections were treated with peroxidase block consisting of 3% H2O2 in water for 15 min to block the endogenous peroxidase activity, followed by a 20 min power block to obstruct any nonspecific antigenic sites.
The sections were then incubated for 1 h at room temperature with optimally prediluted antibody against COX2 (primary rabbit monoclonal antibody clone SP21 at 10–15 mg/ml). After washing with PBS, the sections were then incubated with one-step polymer-HRP reagent for 30 min. Visualization was performed using freshly prepared DAB (diaminobenzidine tetrahydrochloride). The slides were counterstained with Harris hematoxylin, subsequent to which sections were cleared and mounted with DPX. For each batch of staining, positive controls (colon carcinoma) were run simultaneously with the study specimens.
Evaluation of COX-2 expression was based on the extent of positivity. The presence of brown-colored end product in OSCC sections was indicative of positive reactivity. The staining was confined to the cytoplasm and perinuclear area. Positively stained cells were counted in five randomly selected fields at magnification of ×400. The total number of positive cells/100 cells per case was calculated. The positive results were assessed further for the intensity of staining as mild (1), moderate (2), and intense (3) in different grades of OSCC.
The results were tabulated and subjected to statistical analysis (SPSS for Windows 22.0.0 and Minitab version 17.1.0 software package. Minitab Company website:http://www.minitab.com and SPSS company website:http//www.spss.com). The various statistical tests used were Fisher's exact test, one-way ANOVA, Tukey's post hoc test, independent sample t-test, and Chi-square test. The staining intensity and the number of positive tumor cells were recorded.
| Results|| |
Colon carcinoma served as a positive control exhibited strong cytoplasmic COX-2 positivity in the epithelial islands (100%) [Figure 1]. OSCC demonstrated immunostaining of the tumor cells at varying levels for COX-2 [Table 1] and [Figure 2]a, [Figure 2]b, [Figure 2]c. All normal mucosa however did not show COX-2 expression.
|Table 1: Intensity of cyclooxygenase-2 expression in positive cases among different grades of oral squamous cell carcinoma|
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|Figure 2: (a) Well-differentiated squamous cell carcinoma. (b) Moderately differentiated squamous cell carcinoma (c) Poorly differentiated SCC (H and E ,X400)|
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The staining intensity and the number of positive tumor cells were recorded. Staining intensity was maximum in poorly differentiated squamous cell carcinoma (8/10 cases – intense staining, and remaining 2 cases with moderate staining) [Table 2] and [Graph 1].
|Table 2: Comparison of cyclooxygenase-2 staining intensity between different grades of oral squamous cell carcinoma|
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The percentage of COX-2 positive tumor cells were high in poorly differentiated compared to well-differentiated and moderately differentiated SCC. Poorly differentiated OSCC has the highest mean percentage of positive tumor cells followed by moderately differentiated and well-differentiated OSCC [Table 3] and [Graph 2].
|Table 3: Percentage of cyclooxygenase-2 positive tumor cells in different grades of oral squamous cell carcinoma|
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Statistical analysis for the positive tumor cells among the study groups (t = 16.1, P < 0.001) was highly significant.
| Discussion|| |
It is argued that chronic inflammation provides the ideal environment for the development of cell changes that may lead to cancer. This includes upregulation of mediators in the inflammation, such as COX-2, leading to the production of inflammatory cytokines and PGs. COX-2 is induced by stimuli such as mitogens, cytokines, growth factors, and tumor promoters. There are also various environmental stimuli (tobacco, alcohol, infectious agents, irradiation, and dietary agents) that activate these inflammatory pathways.
Studies indicate that COX-2, an induced proinflammatory enzyme involved in the metabolism of arachidonic acid and in the synthesis of PGs, plays a role in various steps of carcinogenesis. Upregulation of COX-2, at both mRNA and protein levels, may result in the enhanced synthesis of PGs, increase in cell proliferation, reduction in apoptosis rates, and promotion of tumor angiogenesis, especially during the early stages of this process thereby inhibiting immune surveillance and tumor invasiveness. These changes are mostly mediated by PGE2 and its receptor EP1-4. An elevated expression of COX-2 has been demonstrated in several human cancers, including those arising from colon, stomach, breast, lung, esophagus, pancreas, bladder, prostate, skin, and in OSCC.,,
Deregulated signaling through the EGFR pathway is recognized to be an early event in the development of head and neck tumors. Activation of the EGFR/Ras pathway thus contributes to the upregulation of COX-2 in HNSCC. Forced overexpression of COX-2 in epithelial cells leads to decreased apoptosis, which has been attributed, to some extent, to increased levels of antiapoptotic protein Bcl-2.
In the present study, COX-2 expression was recognized as brown granular/diffuse cytoplasmic staining predominantly having perinuclear location with a wide variation in staining intensity depending on the degree of differentiation of the malignant squamous cells. This finding was similar to the study by Atula et al. and Chan et al., A correlation between increased staining intensity of COX-2 expression and advanced stage of the disease was observed in OSCC of the tongue.
In the present study, all the cases of normal oral mucosa lacked COX-2 expression which is in accordance with the studies done by Nathan et al. The normal mucosa may also account for the positive staining of COX-2 as genetic alterations may be present in histologically normal tissue which has already been exposed to tobacco, alcohol, and inflammation. These results suggest that alterations of COX-2 expression can occur in the very early stages of head and neck tumorigenesis, particularly in carcinogen-exposed epithelium. This observation also supports the concept of field cancerization.
Researchers have done study to define the mechanism underlying smoke-induced elevation of COX-2 levels. One of the first steps in the process of carcinogenesis is the binding of reactive tobacco smoke and alcohol metabolites to the DNA of mucosal cells, which can lead to mutations and malignant transformation. Tobacco smoke extracts have been shown to increase the COX-2 expression in tumor cells, with concomitant increase of cell proliferation and decrease of apoptosis.
The immunoreactivity for COX-2 was expressed in cancer cells as well as in inflammatory cells including neutrophils and lymphocytes in the present study. This is in accordance with the study done by Chan et al. on SCC where a moderate to strong granular staining pattern of COX-2 expression localized to the cytoplasm.
Correlation between COX-2 expression and tumor grade has been reported by many authors. The intensity pattern of COX-2 expression varied with different grades of squamous cell carcinoma, with predominantly mild (7/10) COX-2 staining in well-differentiated squamous cell carcinoma, moderate (5/10) in moderately differentiated SCC, and intense (8/10) in poorly differentiated SCC in our study. This is comparable to the findings of Nagatsuka et al. Statistical significance was seen between COX-2 expression in well-differentiated and poorly differentiated SCC and in moderate and poorly differentiated SCC. This finding was in accordance with Sappayatosok et al. The correlation between COX-2 expression and poor histological differentiation of oral SCCs suggests that COX-2 may be a useful indicator of the malignant phenotype of squamous cells. These findings proved beyond doubt that there is a constant increase in the staining intensity as the tumor grade increased from well-differentiated to poorly differentiated.
In the present study, the mean value of positive tumor cells in the total of 30 OSCC was 32.83%. Similar mean value was found by Itoh et al. in 2003 (mean value-34.6%) but could not find a correlation between the COX-2 overexpression and histological grade. Goulart Filho et al. also found no significant differences in the expression of COX-2 protein between high- and low-grade tumors.
All the cells in the positive cases did not express COX-2 because these cells might have already crossed their G1 phase of cell cycle or have stopped proliferation. Another observation in our case was that all the cells which expressed COX-2 did not show uniform staining intensity but showed cell to cell variation. The variation in intensity reflects that the COX-2 shows characteristic cell cycle-dependent variation. Strong expression of these enzymes in tumor cells at the advancing front was noticed suggesting their role in local tumor spread.
According to some researchers, selective COX-2 inhibitors found to suppress the growth of established tumors including head and neck, colorectal, stomach, lung, breast, and prostate tumors.,
Upregulation of COX-2 in various types of human carcinomas, chemopreventive effects on cancer development by potent inhibitors of COXs, and upregulation of COX-2 linked with increased angiogenesis and metastasis support their role in carcinogenesis.
| Conclusion|| |
Our study showed a positively graded response from well-differentiated to poorly differentiated squamous cell carcinoma. This finding suggests that these proteins may play some role in carcinogenesis. The correlation seen between COX-2 expression with various grades of squamous cell carcinoma in the present study suggests that the protein level may be a very useful indicator of the malignant phenotype of squamous cells.
Immunohistochemistry is a frequently used method for analyzing COX-2 expression. This is a subjective technique that has its limitations, and reproducibility has always been an issue. We minimized methodological errors by using previously used standardized protocols for our COX-2 antibody. In conclusion, although the immunohistochemical evaluation of COX-2 expression in OSCC in our study has revealed significant results, further studies are required using a larger sample size, with the help of genetic studies to determine their role in disease progression of OSCC.
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Conflicts of interest
There are no conflicts of interest.
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Dr. Nirupa Thomas
Department of Oral Pathology and Microbiology, Annoor Dental College and Hospital, Puthuppady, Perumattom, Muvattupuzha - 686 673, Kerala
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]