ET-1, ET-2 and ET-3, are characterized by a single α-helix and two disulfide bridges. The three peptides are encoded by distinct genes and are regulated at the level of mRNA transcription. The primary translation product of the ET-1 gene is the 212-aa prepro-ET-1, which is cleaved by an endopeptidase to form the 38-aa big-ET-1. The biologically active ET-1 is formed by endothelin-converting-enzyme (ECE), an enzyme with intracellular and membrane-bound isoforms 7. The half-life of ET-1 in circulation is seven minutes 8. Two pathways have been described for clearance of endothelin: ET_B receptor-mediated uptake followed by lisosomal degradation 910 and catabolism by extracellular neutral endopeptidase (NEP) 1112. ET-1 production is stimulated by a variety of cytokines and growth factors, including IL-1β, TNF-α, TGF-β, PDGF, vasopressin, hypoxia, and shear stress. Inhibitory factors include nitric oxide, prostacyclin and atrial natriuretic peptide 6.

Endothelins exert their effects by binding to two distinct cell surface ET receptors, ET_A and ET_B. The ET_B receptor (ET_BR) binds the three peptide isotypes with equal affinity. In contrast, ET_AR binds ET-1 with higher affinity than the other isoforms. Both receptors belong to the G protein-coupled receptor (GPCR) system and mediates biological responses from a variety of stimuli, including growth factors, vasoactive polypeptides, neurotransmitters, hormones, and phospholipids 12.

ET-1 is produced by a variety of normal cells, including endothelial cells, vascular smooth muscle cells, and various epithelial tissues (eg, bronchial, endometrial, mammary, and prostatic) and is mitogenic for a variety of cell types including endothelial cells, vascular and bronchial smooth muscle cells, fibroblasts, keratinocytes, mesangial cells, osteoblasts, melanocytes, and endometrial stromal cells. This peptide, which is the most common circulating form of ETs, is produced also by many epithelial tumors where it acts as an autocrine or paracrine growth factor 4. Ligand binding to the endothelin receptor results in activation of a pertussis toxin-insensitive G protein that stimulates phospholipase C activity and increases intracellular Ca²⁺ levels, activation of protein kinase C, mitogen activated protein kinase (MAPK) and p125 focal adhesion kinase (FAK) phosphorylation. Among downstream events after endothelin receptor activation, ET-1 causes EGF receptor transactivation, which is partly responsible for MAPK activation 1314.

ET-1 stimulates DNA synthesis and cell proliferation in various cells, including vascular smooth muscle, osteoblasts, glomerular mesangial cells, fibroblasts and melanocytes. ET-1 is also a mitogen for different cell types including prostate, cervical, ovary cancer cells. In primary cultures and established ovarian carcinoma cell lines, spontaneous growth was significantly inhibited in the presence of ET_AR antagonist. ET_BR antagonist lacked this activity demostrating that endogenous ET-1 acts as an autocrine modulator of ovarian carcinoma cell proliferation only through ET_AR 15.

The mitogenic activity of ET-1 can be amplified by synergistic interactions with other growth factors including epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin, insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF), and interleukin-6 (IL-6) 16.

ETs, which are mitogens for endothelial cells, vascular smooth muscle, fibroblasts, and pericytes, are also angiogenic factors. Endothelial cell mitogenesis is mediated by ET_BR, while vascular smooth muscle cells and pericyte mitogenesis is mediated predominantly or solely by the ET_AR. ET-1 modulates various stages of neovascularization, including endothelial cell proliferation, migration, invasion, protease production, tube formation and stimulates neovascularization in vivo 17. Elevated expression of ET-1 and its cognate receptor is significantly associated with microvessel density (MVD) and vascular endothelial growth factor (VEGF) expression in ovarian carcinomas 18, suggesting that ET-1 and VEGF might have a complementary and coordinated role during neovascularization in this tumor. Thus, in ovarian carcinoma cell lines, ET-1 increases VEGF mRNA expression and induces VEGF levels in a time- and dose-dependent fashion, and does so to a greater extent during hypoxia. These activities were mediated through the ET_AR, because the specific antagonist, BQ 123, reversed the stimulation of VEGF production 18.

The transcriptional upregulation of VEGF has been linked to a critical mediator of hypoxia signaling, the hypoxic inducible factor-1α (HIF-1α). ET-1 promotes VEGF production through HIF-1α and this mechanism might be responsible for increasing tumor angiogenesis. Degradation of HIF-1α was infact reduced in ET-1-treated ovarian carcinoma cells under both hypoxic and normoxic conditions. After ET-1 stimulation, HIF-1α protein levels increase in the cells, the HIF-1 transcription complex is formed and binds to the HRE binding site. Therefore, ET-1-induced HIF-1 accumulation activates all the signals necessary for a complete HIF-1 response 19. The HIF-1α-mediated transcription of VEGF by ET-1 under normoxic conditions points to a general mechanism through which oncogenes and growth factors might upregulate VEGF, and could synergize with hypoxia during tumor growth. Addition of a specific ET_AR antagonist blocked the ET-1-induced upregulation of VEGF expression and secretion as well as the ET-1-induced activation of HIF-1 transcription complex. In tumor cells, ET-1 might be upregulated by hypoxia and could promote angiogenesis by increasing VEGF production through an HIF-1α-dependent mechanism. Thus, under hypoxic conditions, ET-1 potentiates hypoxia stimulus by amplifying HIF-1α stability and VEGF production 25.

Prostaglandins (PG) and their rate-limiting enzymes cyclooxygenase (COX)-1 and -2 are involved in the onset and progression of a variety of malignancies 21. Moreover high COX-1 and -2 have been reported in association with elevated levels of proangiogenic factors in ovarian cancer 22232425. In ovarian carcinoma cells, ET-1 significantly increases the expression of COX-1 and -2, at mRNA and protein level, the COX-2 promoter activity and PGE₂ production. These effects depend on the ET_AR activation and involve multiple MAPK signal pathways, including p42/44 MAPK, p38 MAPK and the transactivation of EGFR.

There is increasing evidence that PGE₂ contributes to tumor progression also by promoting angiogenesis and that this effect is mediated by VEGF. COX-2 and-1 inhibitors blocked ET-1-induced PGE₂ and VEGF release demonstrating that both enzymes, although by a different extent, participate to PGE₂ and VEGF production. These results indicate that impairing COX-1 and -2 and their downstream effect by targeting ET_AR can be therapeutically advantageous 26 also in view that elevated COX-2 levels are associated with tumor progression and chemoresistance 24.

ET-1 is an antiapoptotic factor in different cell types, indicating that the peptide may also modulate cell survival pathways. In ovarian carcinoma cells, the addition of ET-1 markedly inhibited serum-withdrawal and paclitaxel-induced apoptosis in a concentration-dependent manner. Paclitaxel-induced apoptosis resulted in the phosphorylation of Bcl-2 that was suppressed by the addition of ET-1. Further analysis of the survival pathway demonstrated that ET-1 stimulated Akt activation dependent on PI3-K. Interestingly, the addition of a specific ET_AR antagonist blocked the ET-1-induced resistance to paclitaxel-mediated apoptosis indicating that ET-1 contributes to trigger resistance to paclitaxel through ET_AR binding via activation of anti-apoptotic signaling pathways such as Akt.

Specific ET_AR antagonists may therefore provide an additional approach to the treatment of ovarian carcinoma in which ET_AR blockade could result in the tumor growth inhibition by reducing tumor growth as well as by inducing apoptosis. Furthermore when combined with the conventional chemotherapy the ET_AR antagonists would more effectively induce apoptosis by contributing to the reversal of paclitaxel resistance 27.

As mentioned, high levels of ET-1 are detected in the majority of ascitic fluids of ovarian cancer patients and are significantly correlated with VEGF ascitic concentrations, suggesting that ET-1 may enhance the secretion of extracellular matrix-degrading proteinases and metastatization 18. Thus, ET-1 acting through the ET_AR consistently induced the activity of two families of metastasis-related proteinases, the matrix metalloproteinases (MMPs) and the urokinase type plasminogen activator system at several levels: mRNA transcription, zymogen secretion and pro-enzymes activation. ET-1 in fact activates MMP-2, MMP-9, MMP-3, MMP-7 and MMP-13. In addition to soluble MMPs, ET-1 enhanced the activation of MT1-MMP and the secretion of tissue inibitor of MMP (TIMP-1 and -2) increasing the net MMP/TIMP balance and the gelatinolytic capacity that results in rapid degradation of extracellular matrix (ECM). In the ovarian carcinoma cells, co-induction of uPA system, by the concomitant stimulation of production and secretion of uPA and uPAR, and MMPs by ET-1 resulted into the highest invasive potential of tumor cells through the Matrigel. Interestingly, the addition of BQ 123 blocked the ET-1-induced proteinase activation and tumor cell migration and invasion. Furthermore in these cells, ET-1 stimulated FAK and paxillin phosphorylation through ET_AR binding 13 which directly correlated with tumor cell migration and invasion suggesting that ET_AR antagonist can inhibit cell migration and possibly other FAK-associated processes which also contributes to invasion and metastasis in this tumor 28.

Following malignant transformation, stepwise changes in intercellular communications enable tumor cells to escape microenvironmental control from the normal surrounding tissue, thus allowing local invasiveness and metastatization. Human ovarian surface epithelial cells exhibit extensive gap junction intercellular communications (GJIC) and expression of different types of connexin (Cx), predominantly Cx43. Defects in intercellular communication, including reduced or inappropriate expression of Cx43 has emerged as key factors in ovarian carcinoma progression. In ovarian carcinoma cells, ET-1 via ET_AR induces a transient and a dose-dependent reduction of GJIC (50–75%) and phosphorylates Cx43 through Src tyrosine kinase pathway indicating that ET-1 promotes cellular uncoupling at the level of connexin maturation and subsequent degradation 29. The capacity of ET-1 to disrupt gap junctions could serve as a basis to further evaluate the cell-cell metabolic uncoupling and the cell detachment that occurs during tumor progression and adds further information on the overall relevance of ET_AR in regulating the complex array of cell-cell or cell-matrix interactions promoting ovarian carcinoma growth 29.

Several observations implicate ET-1 in the osteoblastic response. Osteoblasts display a high density of ET receptors, and respond to ET-1 by increasing synthesis of both collagenous and noncollagenous proteins, including two osteoblast messengers such as osteopontin and osteocalcin 30. Osteoblasts are also sensitive to the mitogenic and comitogenic activity of ET-1. Furthermore ET-1 inhibits osteoclasts activity and motility, upsetting the balance of osteoblasts and osteoclasts to favor new bone formation 31. Alkaline phosphatase, a marker of new bone formation, is elevated in the presence of exogenous ET-1 32. ET-1 production is stimulated by IL-1β, TNF-α and TGF-β, which are normally secreted by immunocompetent cells, as well as osteoblasts and endothelial cells. This suggests that paracrine mediators secreted by osteoblasts and tumor cells are involved in a paracrine growth loop that results in the derangement of bone remodelling.

ET-1 is described as a new pain mediator implicated in the pathogenesis of pain states. Local cutaneous injection of ET-1 causes pain and excitation of nociceptors, both phenomena depending on ET_AR. The peptide can trigger pain through ET_AR of local nociceptors, and concurrently produces analgesia through ET_BR by inducing the release of β-endorphin and the activation of opioid pool 33. It is reasonable to speculate that the refractory pain of certain metastatic cancers is related to the direct nociceptive effects of ET-1. Thus antagonist of ET_AR have been shown to ameliorate pain. This knowledge may lead to improved, targeted analgesia in patients with advanced cancer 34.