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Year : 2013  |  Volume : 56  |  Issue : 2  |  Page : 151-154
Immunohistochemical expression of endothelin protein in oral squamous cell carcinoma

1 Department of Oral Pathology, Faculty of Oral and Dental Medicine, Cairo University, Giza, Egypt; Department of Oral Pathology, Faculty of Oral and Dental Medicine, Sana'a University, Yemen
2 Department of Oral Pathology, Faculty of Oral and Dental Medicine, Cairo University, Giza, Egypt

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Date of Web Publication23-Sep-2013


Context: Endothelin-1 (ET-1) plays a significant role in cancer biology and is considered as a potential target for molecular therapy. Alteration of ET-1 was detected in oral squamous cell carcinoma (OSCC). Aim: To evaluate the immunohistochemical expression and distribution of ET-1 in OSCC and to correlate its expression with the degree of histopathologic differentiation as well as the regional metastatic status of OSCC. Materials and Methods: The immunohistochemical expression and distribution of ET-1 was evaluated in total number of 30 cases of OSCC. The specimens were retrieved as formalin-fixed, paraffin-embedded blocks from the archival files. Detection of ET-1 expression in different grades of metastatic and non-metastatic OSCC was quantified using an image analyzer computer system. Statistical Analysis Used: Statistical software package SPSS v. 19. Results: ET-1 immunoreactivity was observed in all studied specimens. On comparing optical density values for ET-1 expression in different grades of OSCC, poorly differentiated OSCC showed a significantly greater expression than moderately differentiated OSCC, which was also significantly greater than well-differentiated OSCC ( P = 0.001). Moreover, ET-1 expression was higher in metastatic OSCC than that in non-metastatic OSCC ( P = 0.018). Conclusions: Increased ET-1 expression could enhance the aggressive behavior of poorly differentiated OSCC, especially metastasis. Accordingly, ET-1 could be a therapeutic target in OSCC.

Keywords: Endothelin-1, immunohistochemical, oral squamous cell carcinoma

How to cite this article:
Alaizari NA, Abdelbary SN, Amin NR. Immunohistochemical expression of endothelin protein in oral squamous cell carcinoma. Indian J Pathol Microbiol 2013;56:151-4

How to cite this URL:
Alaizari NA, Abdelbary SN, Amin NR. Immunohistochemical expression of endothelin protein in oral squamous cell carcinoma. Indian J Pathol Microbiol [serial online] 2013 [cited 2022 Aug 14];56:151-4. Available from: https://www.ijpmonline.org/text.asp?2013/56/2/151/118677

   Introduction Top

Cancer is an important public health problem in many parts of the world, and oral cancer is among the 10 most common cancers worldwide. Squamous cell carcinoma (SCC) is the most prevalent malignant neoplasm in the oral cavity. [1] It accounts for more than 90% of oral cancers. [2] It is the most frequently observed form of head and neck cancer in Southeast Asia and is the sixth most common cancer worldwide. [3]

The endothelin (ET) axis which includes ET-1, ET-2, ET-3, and 2 G protein-coupled receptor subtypes, ETAR and ETBR, promotes growth and progression of a variety of tumors such as prostatic, ovarian, renal, pulmonary, colorectal, cervical, breast, lung, bladder, endometrial carcinoma, Kaposi's sarcoma, brain tumors, and melanoma. [4]

The components of the ET systems are altered in cancer and appear to aid tumor growth and progression in a number of epithelial cancer types via direct and indirect mechanisms. [5]

Direct effects of ET-1 in neoplastic cells appear to primarily involve proliferation, migration, invasion, and resistance to apoptosis. The mechanism of indirect effects of ET-1 may be related to its ability to regulate various kinases which in turn may have direct effects on cell proliferation, survival, angiogenesis, epithelial to mesenchymal transition, cell motility and invasion, and even cell adherence and metastasis. Further, ET-1 may have indirect effects on tumor metastasis; this appears to occur by stimulation of angiogenesis resulting from ET-1 stimulation of proangiogenic factors such as vascular endothelial growth factors. Additional indirect effects of ET-1 in cancers include an extracellular matrix deposition and remodeling. [6]

The combination of ET-1 direct cellular effects and indirect effects in the cancer milieu emphasizes the important cell biologic effects of this peptide in carcinogenesis. [7]

The significant role of ET-1 in tumor biology has gained a lot of interest and to our knowledge, little data concerning its expression and distribution in oral SCC (OSCC) is available. Therefore, the aim of this study was to evaluate the immunohistochemical expression and distribution of ET-1 in OSCC and to correlate its expression with the degree of histopathologic differentiation as well as the regional metastatic status of OSCC.

   Materials and Methods Top

A total of 30 formalin-fixed, paraffin-embedded archival tissue blocks of OSCC (10 cases well differentiated, 10 moderately differentiated, and 10 poorly differentiated) were retrieved from the Department of Pathology, National Cancer Institute. The specimens were further subclassified into regional lymph node metastatic and non-metastatic OSCC. Hematoxylin-eosin-stained sections were reviewed to ensure conformity with accepted diagnostic criteria.

Two 4-μm-thick sections were cut from each block and mounted on positively charged slides. One was used for the primary antibody (positive test slide) and the other one served as a negative control (same immunostaining procedures, but omitting the primary antibody and replacing it by IgG of the respective species).

A positive control slide (a tissue known to contain the antigen under study) was also included in which a tissue section of breast cancer; as ascribed in the datasheet of the used marker, was cut and immunostained by ET-1 antibody.

The sections were stained immunohistochemically using the goat polyclonal antibody to ET-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, Product No. ET-1 (N-8): sc-21625) that was applied at a dilution of 1:50 for 30 min.

For ET-1 immunostaining, before inactivation of endogenous peroxidase activity, sections were immersed in 10 mM citrate buffer (in distilled water, pH 6.0) and heated in a microwave oven at 95°C for 5 min. The binding of primary antibodies was detected by using streptavidin biotin immunoperoxidase method. Diaminobenzidine was used as chromogen. To assure specificity, the primary antibody was omitted (negative control in all cases). The immunoreactivity of breast ductal carcinoma was used as a positive ET-1 stain.

Evaluation of Immunostaining

Immunostaining was examined by the image analyzer computer system with the software Leica Quin 500 (LEICA Imaging Systems Ltd, Cambridge, England). The ET-1 immunoreactivity was measured in the form of optical density and area percentage.

Regarding the intensity of reaction within the malignant cells, the optical density was measured after transforming the image into gray mode. Areas with maximum gray were masked by binary blue color. The gray levels ranging from pure black to pure white were automatically converted to optical density values and stored [Figure 1]a.
Figure 1: : A copy of display as seen on the monitor's screen of the image analyser showing the blue color for optical density values of ET-1 immunoexpression inside a small measuring frame (10 micro meter) in well-differentiated OSCC (a); (b) and the red color for area percent values of ET-1 immunoexpression inside a small measuring frame (10 micro meter) in moderately-differentiated OSCC

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The area percentage was measured in a standard measuring frame of 10 μm per 10 fields using a magnification ×400 by light microscopy transferred to the monitor's screen. The most intense immunostained areas were masked by red binary color using the computer system [Figure 1]b.

Statistical Analysis

Data obtained were collected, expressed as mean values ± standard deviation, and statistically evaluated by using the statistical software package SPSS v.19 in order to determine if there is a significant difference in the staining intensity or the area percentage for ET-1 immunoexpression in different grades of metastatic and non-metastatic OSCC. Differences were considered statistically significant when P values were <0.05 and highly significant if the values were <0.01.

   Results Top

Positivity of ET-1 immunoreaction was brown homogenous staining in almost all malignant epithelial cells. A granular pattern of immunostaining was also noticed in some cases. The distribution of ET-1 immunoreactivity was mainly cytoplasmic with some cases showing a membranous expression [Figure 2].
Figure 2: Representative examples of staining for ET-1 in malignant epithelial cells of OSCCs (a) with granular pattern (b) immunoreaction in the cell membrane (c) and cytoplasm (d) [(a) IHC, ×200; (b) IHC, ×400; (c) IHC, ×200; and (d) IHC, ×200]

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ET-1 expression in adjacent stromal fibroblasts, endothelial cells and nerve fibers.

On comparing optical density, ET-1 expression significantly correlated with the histopathologic grade (P = 0.001) and with the lymph node metastatic status (P = 0.018) [Table 1]. Moreover, a direct relationship existed between the area percentage of ET-1 expression with the high histological grade OSCC and the metastatic OSCC. However, these relationships did not reach statistical significance [Table 2].
Table 1: Comparing the numerical values for means±standard deviations of optical density and area percent for different histopathologic grades of OSCC

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Table 2: Numerical values for means±standard deviations of optical density and area percent for metastatic and non-metastatic OSCC

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   Discussion Top

OSCC largely compromises the patient's life quality. Therefore, the identification of biomarkers for this kind of cancer is essential to provide a better diagnosis and prognosis for the patient. [8]

In this study, ET-1 expression was detected in OSCC. The distribution of ET-1 immunoreactivity was mainly cytoplasmic and this observation is consistent with Pickering et al. who detected a strong homogenous cytoplasmic staining for ET-1 in OSCC. [9] The ET-1 reaction in the cytoplasm may denote its role in the important cellular functions encountered in the cytoplasm. ET-1 exerts a lot of biological effects utilizing multiple intracellular signal transduction pathways, including activation of GTP-binding protein s, phospholipases, protein kinases, and phosphatases, and various transcription factors. [10]

Adding to the cytoplasmic positive reaction, some cases of OSCC in the current study showed weak membranous expression. This is in accordance with the findings of Li et al. who found membranous ET-1 immunoreaction in neoplastic adrenal glands. [11] This could be due to the binding of ET-1 to its receptors on the cell membrane to mediate its different functions.

The pattern of ET-1 staining in some of the studied cases, especially of the moderately and poorly differentiated carcinomas, was more or less granular, but with no significance. This is in agreement with Lu et al. who noticed brownish grains of ET immunostaining localized in the cytoplasm of tumor cells and normal breast epithelial cells. [12]

Most of the studied cases of well-differentiated OSCC in this study showed weak ET-1 immunostaining. This was in accordance with a study by Satomura et al. in which a slight expression of ET-1 was noted in some neoplastic keratinocytes of non-pigmented well-differentiated SCCs. [13] This denotes the mild effect of ET-1 in well-differentiated OSCC. It could be concluded that the weak ET-1 expression might be one of the reasons for the low proliferative activity, low metastatic potential, and less aggressive behavior of the well-differentiated grades of OSCC.

In the present study, on comparing optical density values for ET-1 expression in different grades of OSCC, poorly differentiated OSCC showed a significantly greater expression than moderately differentiated OSCC, which was also significantly greater than well-differentiated OSCC. These results are compatible with those of Yang et al. who studied ET-1 expression in lung cancer and found that lower the lung cancer differentiation, higher is the ET-1 expression. They also demonstrated that ET-1 analysis may be a monitoring index of SCC and adenocarcinoma. [14]

Moreover, this study also compared the optical density values for ET-1 expression in metastatic and non-metastatic OSCC. It showed that metastatic OSCC demonstrated a higher expression than non-metastatic OSCC. This result agrees with Ishibashi et al. who observed a significant direct relationship between ET staining in esophageal SCC and lymphatic vessel invasion, as well as regional lymph node and distant metastasis. [15] In addition, Wülfing et al. illustrated the presence of a significant correlation between increased ET-1 expression and its receptors with lymphovascular invasion and metastasis in breast cancer status. [16] This finding was explained by Bagnato et al. who demonstrated that ET played an important role in enhancing angiogenesis in ovarian carcinoma, thus causing metastasis. [17] Knowles et al. also showed that ET had a direct angiogenic effect on endothelial and peri-vascular cells and an indirect effect through increased release of vascular endothelial growth factor (VEGF), via hypoxia inducible factor-1. [18] Furthermore, Spinella et al. demonstrated that ET stimulates lymphatic endothelial cells and lymphatic vessels to grow and invade. [19] These previous findings could explain the mechanism by which ET-1 acted to enhance OSCC metastasis. ET-1 enhances cell motility and decreases cell adherence through regulation of various kinases. [6]

Stromal expression of ET-1 in fibroblasts in some cases studied was noticed. This finding is in accordant with that of Knowles et al. who found that ET-1 could activate fibroblasts within colorectal cancers, thus modulating tumor stroma development. This indicates that ET-1 plays a role in invasion and metastasis of OSCC. [20] Hinsley et al. demonstrated that ET-1 is able to stimulate the migration of Head and Neck Squamous Cell Carcinoma. This paracrine stimulation is the result of the ET-1 action in stimulating proteolytic release of bioactive ligands from fibroblasts, which subsequently bind to EGFR (Epidermal growth factor receptor) on HNSCC cells, triggering an increase in COX-2 expression which in turn is involved in tumor progression by inducing proliferation, survival, invasion, and metastasis. [21]

Based on the collectible findings, it could be identified that the increased expression of ET-1 by tumor cells of OSCC could thus facilitate their growth and invasion not only directly via tumor cell receptors, but also indirectly via its effects on vascular elements. Hoffmann et al. provided a scheme regarding the involvement of endothelins and their receptors in OSCC. They stated that ETAR activation by ET-1 largely contributes to tumor growth and progression by inducing cell proliferation, survival, angiogenesis, and metastatic spread. Moreover, ET-1 could cause modulation of angiogenesis either directly by acting on ETBR or indirectly through induction of VEGF. [8] Overall, this may reveal the importance of ET-1 as a potential marker for differentiation, metastasis, and prognosis of OSCC.

   Acknowledgments Top

It was an honor for me to be a candidate of Prof. Dr. Sawsan Abdelbary, whose encouragement, guidance, and support from the initial to the final level enabled me to achieve this work.

This thesis would not have been possible without the great efforts and help of Dr. Nermine Raouf Amin. Really, I want to express my deep thanks and appreciation for her valuable advice, close supervision, and endless help throughout this work.

I owe my deepest gratitude to Prof. Dr. Heba Farag, Prof. of Oral Pathology, Faculty of Oral and Dental Medicine, Cairo University, who kindly helped me with the image analysis interpretation.

   References Top

1.Neville BW, Day TA. Oral cancer and precancerous lesions. CA Cancer J Clin 2002;52:195-215.  Back to cited text no. 1
2.Neville B, Damm DD, Allen CM, Bouquot J. Squamous cell carcinoma. In: Dolan J, St Louis, editors. Oral and Maxillofacial Pathology. 3 rd ed. Chapter 10. Philadelphia: Saunders; 2009.  Back to cited text no. 2
3.Al-Swiahb JN, Chen CH, Chuang HC, Fang FM, Tasi HT, Chien CY. Clinical, pathological and molecular determinants in squamous cell carcinoma of the oral cavity. Future Oncol 2010;6:837-50.  Back to cited text no. 3
4.Bagnato A, Rosano L. The endothelin axis in cancer. Int J Biochem Cell Biol 2008;40:1443-51.  Back to cited text no. 4
5.Grant K, Loizidou M, Taylor I. Endothelin-1: A multifunctional molecule in cancer. Br J Cancer 2003;88:163-6.  Back to cited text no. 5
6.Khimji A, Rockey DC. Endothelin - biology and disease. Cell Signal 2010;22:1615-25.  Back to cited text no. 6
7.Bagnato A, Catt KJ. Endothelins as autocrine regulators of tumor cell growth. Trends Endocrinol Metab 1998;9:378-83.  Back to cited text no. 7
8.Hoffmann RR, Yurgel LS, Campos MM. Endothelin and their receptors as biological markers for oral cancer. Oral Oncol 2010;46:644-7.  Back to cited text no. 8
9.Pickering V, Jordan RC, Schmidt BL. Elevated salivary endothelin levels in oral cancer patients - a pilot study. Oral Oncol 2007;43:37-41.  Back to cited text no. 9
10.Sokolovsky M. Endothelin receptor subtypes and their role in transmembrane signaling mechanisms. Pharmacol Ther 1995;68:435-71.  Back to cited text no. 10
11.Li Q, Zhang X, Lar G. Ultrastructural and immunohistochemical localization of endothelin-1 in nonneoplastic, hyperplastic and neoplastic adrenal gland. Zhonghua Yi Xue Za Zhi 1996;76:128-31.  Back to cited text no. 11
12.Lu J, Liang Y, Wang X. Immunohistochemical study of endothelin-1 in breast cancer. Zhonghua Zhong Liu Za Zhi 1995;17:13-5.  Back to cited text no. 12
13.Satomura K, Tokuyama R, Yamasaki Y, Yuasa T, Tatehara S, Ishimaru N, et al. Possible involvement of stem cell factor and endothelin-1 in the emergence of pigmented squamous cell carcinoma in oral mucosa. J Oral Pathol Med 2007;36:621-4.  Back to cited text no. 13
14.Yang X, Liu L, Qi H. Study on endothelin-1 positive expression and quantitative analysis in lung cancer. Zhonghua Jie He He Hu Xi Za Zhi 1998;21:111-3.  Back to cited text no. 14
15.Ishibashi Y, Hanyu N, Nakada K, Suzuki Y, Yamamoto T, Takahashi T, et al. Endothelin protein expression as a significant prognostic factor in oesophageal squamous cell carcinoma. Eur J Cancer 2003;39:1409-15.  Back to cited text no. 15
16.Wülfing P, Diallo R, Kersting C, Wülfing C, Poremba C, Rody A, et al. Expression of endothelin-1, endothelin-A, and endothelin-B receptor in human breast cancer and correlation with long-term follow-up. Clin Cancer Res 2003;9:4125-31.  Back to cited text no. 16
17.Bagnato A, Spinella F, Rosanò L. Emerging role of the endothelin axis in ovarian tumor progression. Endocr Relat Cancer 2005;12:761-72.  Back to cited text no. 17
18.Knowles J, Loizidou M, Taylor I. Endothelin-1 and angiogenesis in cancer. Curr Vasc Pharmacol 2005;3:309-14.  Back to cited text no. 18
19.Spinella F, Caprara V, Garrafa E, Di Castro V, Rosanò L, Natali PG, et al. Endothelin axis induces metalloproteinase activation and invasiveness in human lymphatic endothelial cells. Can J Physiol Pharmacol 2010;88:782-7.  Back to cited text no. 19
20.Knowles JP, Shi-Wen X, Haque SU, Bhalla A, Dashwood MR, Yang S, et al. Endothelin-1 stimulates colon cancer adjacent fibroblasts. Int J Cancer 2012;130:1264-72.  Back to cited text no. 20
21.Hinsley EE, Hunt S, Hunter KD, Whawell SA, Lambert DW. Endothelin-1 stimulates motility of head and neck squamous carcinoma cells by promoting stromal-epithelial interactions. Int J Cancer 2012;130:40-7.  Back to cited text no. 21

Correspondence Address:
Nermine Raouf Amin
Faculty of Oral and Dental Medicine, Cairo University, Giza, Egypt

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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.118677

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