Journal of Surgical Oncology 2012;105:643–649 Expression Patterns of Endothelin-1 and Its Receptors in Colorectal Cancer PARASKEVI LIAKOU, MD, 1 KOSTANTINOS TEPETES, MD, PhD, 1 ANASTASIOS GERMENIS, MD, PhD, 2 VASILIKI LEVENTAKI, MD, 3 VASSILIS ATSAVES, MSc, 4 EFSTRATIOS PATSOURIS, MD, PhD, 4 NIKOLAOS ROIDIS, MD, PhD, DSc, 5 KONSTANTINOS HATZITHEOPHILOU, MD, PhD, 1 AND GEORGE Z. RASSIDAKIS, MD, PhD 4 * 1 Department of Surgery, University of Thessaly, Larissa, Greece 2 Department of Immunology & Histocompatibility, University of Thessaly, Larissa, Greece 3 Department of Pathology, University of Utah, Salt Lake, Utah 4 First Department of Pathology, National and Kapodistrian University of Athens, Athens, Greece 5 3rd Department of Orthopaedics & Traumatology, KAT Hospital NHS, Athens, Greece Background and Objectives: Endothelin-1 (ET-1), a potent vasoconstricting peptide, plays an important role in carcinogenesis. Previous in vitro studies have shown that colorectal cancer cells produce ET-1. Methods: ET-1 and its receptors ET-A (ET A R) and ET-B (ET B R) were analyzed in colorectal cancer cell lines and tumors by Western blot and immunohistochemistry. Also, ET-1 levels were measured by ELISA in blood samples collected before and after tumor resection. Results: ET-1 was immunohistochemically expressed by tumor cells at a variable level in 39 cases tested. The adjacent normal mucosa was negative for ET-1 expression. Strong ET A R expression observed in the deeper infiltrating areas at the periphery of neoplastic tissue correlated significantly with tumor stage. ET B R levels were very low or undetectable. Western blot analysis in paired (normal, tumor) fresh-frozen samples of colorectal cancers and in four colon carcinoma cell lines confirmed these findings. In addition, lower levels of ET-1 in the peripheral circulation after the tumor resection were found by ELISA as compared to those observed before surgery. Conclusions: ET-1 and ET A R, but not ET B R, are expressed at a higher level in primary and cultured colon carcinoma cells as compared to normal colon mucosa cells. Further functional studies are needed to explore the role of ET-1/ET A R axis in colon carcinogenesis. J. Surg. Oncol. 2012;105:643–649. ß 2011 Wiley Periodicals, Inc. KEY WORDS: endothelin-1; endothelin receptors; colon carcinoma INTRODUCTION Endothelin-1 (ET-1) is a potent vasoconstricting peptide—100 times more powerful than norepinephrine in a molar basis. It was first iso- lated in 1985 from aortic endothelial cell lines and was considered responsible for coronary vessels spasm. Three forms of the molecule have been identified so far: endothelin 1, 2, and 3, produced from different genes. The gene for ET-1, located on chromosome 6, gives rise to a 212-amino acid protein, the pre-proendothelin. This is cleaved into a 39 amino acid peptide, the proendothelin or big endo- thelin which is catalyzed by a metal peptidase (ECE, endothelin con- verting enzyme—2 types, 4 isoforms each) into the final molecule of 21 amino acids [1,2]. ET-1 is produced mainly by the endothelial and smooth muscle cells of the vessels under hypoxia, ischemia and shear stress, among other stimuli such as IL-1b, TNF-a, TGF-b, PDGF, vasopressin. It is not however stored into the cells but secreted directly to the adjacent tissues acting in an autocrine and paracrine way. Seventy-five percent of the produced ET-1 is conjugated to its receptors on the neighbor- ing smooth muscle cells and regulates immediately the vascular tone. The half life of ET-1 mRNA is 15–20 min and of the molecule itself 4–7 min; 80–90% of the circulating ET-1 is cleared by the lungs during the first passage. ET-1 is also produced by neurons and astro- cytes, hepatocytes, kidney mesangial cells, endometrial cells, breast epithelial cells, Sertoli cells. Endothelin-2 is expressed basically in the kidney and gastrointestinal tract, while endothelin-3 is found in the brain, GI tract, lungs, and kidney [3,4]. ET-1 acts by binding to two distinct receptors: ET-A (ET A R) and ET-B (ET B R), which belong to the G-protein-coupled receptor subtype (GPCRs). Their activation produces multiple effects, as the coupling with G proteins leads to induction of several intracellular pathways. Additionally, it seems that ET B R counteracts the ET A R signaling [5]. Though ET-1 had been associated with cardiovascular conditions at first, there is increasing evidence of its role in carcinogenesis as well, exerted through a network of important mechanisms. First, a mitogenic activity is found, mainly through activation of ET A R, by stimulating DNA synthesis and cell proliferation and interacting with known growth factors in epithelial tumors, such as prostate, cervical, and ovarian [6–8]. Also, an angiogenic effect is established, through mitogenesis of endothelial cells (ET B R), vascular smooth muscle cells, fibroblasts and pericytes (ET A R) and increase of VEGF expres- sion in tumor cells (via hypoxia-inducible factor HIF-1a and cyclo- oxygenase COX-1 and -2). By endothelial cell proliferation, migration, invasion, protease production and tubule formation, ET-1 appears to modulate neovascularization, especially in aggressive ovarian cancer cells, as well as in those of melanoma, prostate, and breast carcinomas [9–14]. Besides these, anti-apoptotic function, mostly through ET A R mediated modulation of cell survival pathways has been reported [15]. Also, metastatic function (i.e., invasiveness and progression) through several mechanisms, is noticed. These mechanisms include induction of metastasis-related proteinases (MMP and uPA), reduction of intercellular communications and *Correspondence to: George Z. Rassidakis, MD, PhD, First Department of Pathology, National and Kapodistrian University of Athens, 75 Mikras Asias Str. Goudi, Athens, Greece. Fax: þ30-210-746-2179. E-mail: grassidakis@hotmail.com Received 24 September 2011; Accepted 1 December 2011 DOI 10.1002/jso.23017 Published online 27 December 2011 in Wiley Online Library (wileyonlinelibrary.com). ß 2011 Wiley Periodicals, Inc.