endothelin-1 and Ovarian-Neoplasms

Ovarian cancer is a highly metastatic disease and the leading cause of death from gynecologic malignancy. Hence, and understanding of the molecular changes associated with ovarian cancer metastasis could lead to the identification of targets for novel therapeutic interventions. The conversion of an epithelial cell to a mesenchymal cell plays a key role both in the embryonic development and cancer invasion and metastasis. Cells undergoing epithelial-mesenchymal transition (EMT) lose their epithelial morphology, reorganize their cytoskeleton and acquire a motile phenotype through the up- and down-regulation of several molecules including tight and adherent junctions proteins and mesenchymal markers. EMT is believed to be governed by signals from the neoplastic microenvironment including a variety of cytokines and growth factors. In ovarian cancer EMT is induced by transforming growth factor-beta (TGF-beta), epidermal growth factor (EGF), hepatocyte growth factor (HGF) and endothelin-1 (ET-1). Alterations in these cellular pathways candidate them as useful target for ovarian cancer treatment.

Topics: Bone Morphogenetic Protein 4; Cadherins; Cell Differentiation; Endothelin-1; Epidermal Growth Factor; Epithelial Cells; Female; Hepatocyte Growth Factor; Humans; Mesoderm; Neoplasm Metastasis; Ovarian Neoplasms; Transforming Growth Factor beta

The endothelin-1 (ET-1)/ET A receptor (ET(A)R) axis is involved in the pathobiology of different tumors, including ovarian carcinoma. Acting selectively on ET(A)R, ET-1 regulates, through multiple signaling pathways, mitogenesis, cell survival, angiogenesis, lymphangiogenesis, invasion, and metastatic dissemination. Moreover, ET-1/ET(A)R axis appears to be critical in epithelial-to-mesenchymal transition (EMT), providing a mechanism of escape to a new, less adverse niche, in which resistance to apoptosis ensures cell survival in conditions of stress in the primary tumor, and acquisition of "stemness" ensures generation of the critical mass required for tumor progression. Emerging experimental and preclinical data demonstrate that interfering with ET(A)R pathways provides an opportunity for the development of new mechanism-based antitumor strategies by using ET(A)R antagonists alone and in combination with cytotoxic drugs or molecular inhibitors. A specific ET(A)R antagonist in combination with standard chemotherapy is currently evaluated in clinical and translational studies to provide us with new options to treat ovarian cancer and to predict response to therapy. Deeper understanding of molecular mechanism activated by ET(A)R in ovarian cancer will be of paramount importance in the study of ET(A)R-targeted therapy that, regulating EMT and other tumor-associated processes, represents an attractive but challenging approach to improve clinical management of ovarian cancer.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Transformation, Neoplastic; Disease Progression; Drug Resistance, Neoplasm; Endothelin A Receptor Antagonists; Endothelin-1; Female; Humans; Ovarian Neoplasms; Receptor, Endothelin A; Signal Transduction

The 21 amino acid signaling peptide endothelin-1 is commonly elevated in epithelial ovarian cancer, and it mediates or facilitates much of this cancer's aggressive behavior. Ambrisentan (Letairis; Gilead Sciences Inc.) is an antagonist of endothelin-1 at its cognate receptor that has just been approved to treat pulmonary hypertension. Ambrisentan is a well-tolerated pill taken once daily. In theory, it should retard and inhibit lodgement and establishment of disseminated peritoneal micrometastases after debulking surgery.

Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Chemotherapy, Adjuvant; Endothelin-1; Female; Gynecologic Surgical Procedures; Humans; Molecular Sequence Data; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Phenylpropionates; Pyridazines

The endothelin system comprises the three peptide hormones endothelin (ET)-1, -2, -3, their G protein-coupled receptors, endothelin-A-receptor (ET(A)R) and endothelin-B-receptor (ET(B)R), and the enzymes of endothelin biosynthesis and degradation. In the past two decades, an impressive amount of data has been accumulated investigating the role of the endothelin system in a variety of malignancies. In many cancers, ET-1/ET(A)R interaction induces proliferation, angiogenesis, antiapoptosis and resistance to chemotherapy. Data indicate a pivotal role of the endothelin system in tumorigenesis, local progression and metastasis. Subsequently, novel drugs have been designed inhibiting ET-1 biosynthesis or ET(A)R interaction. A wide range of preclinical data is available on the role of ET(A)R antagonists in gynecological, urological and breast cancers providing evidence for their antiangiogenic, proapoptotic and growth inhibitory effects. Of particular interest is the anti-invasive and antimetastatic efficacy of ET(A)R antagonists and synergism when co-administered with established cancer therapies. Data indicate a future role of ET(A)R antagonists in oncologic therapies.

Topics: Animals; Antineoplastic Agents; Aspartic Acid Endopeptidases; Breast Neoplasms; Endometrial Neoplasms; Endothelin Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Female; Humans; Kidney Neoplasms; Male; Metalloendopeptidases; Ovarian Neoplasms; Prostatic Neoplasms; Receptors, Endothelin; Urinary Bladder Neoplasms; Urogenital Neoplasms; Uterine Cervical Neoplasms

In ovarian carcinoma, acquisition of invasiveness is accompanied by the loss of the epithelial features and the gain of a mesenchymal phenotype, a process known as epithelial-mesenchymal transition (EMT). The endothelin A receptor (ET(A)R)/endothelin-1 (ET-1) axis is overexpressed in primary and metastatic ovarian carcinoma. In this tumor type, the ET-1/ET(A)R axis has a critical role in ovarian carcinoma progression by inducing proliferation, survival, neoangiogenesis, loss of intercellular communication and invasion. Recently, we demonstrated that the ET-1/ET(A)R autocrine pathway drives EMT in ovarian tumor cells by inducing an invasive phenotype through downregulation of E-cadherin, increased levels of beta-catenin, Snail and other mesenchymal markers, and suppression of E-cadherin promoter activity. Activation of ET(A)R by ET-1 triggers a phosphatidylinositol 3-kinase-dependent integrin-linked kinase (ILK)-mediated signaling pathway leading to glycogen synthase kinase-3beta (GSK-3beta) inhibition, Snail and beta-catenin stabilization and transcriptional programs that control EMT. Transfection of dominant negative ILK or exposure to an ILK inhibitor suppresses the ET-1-induced phosphorylation of GSK-3beta as well as Snail and beta-catenin protein stability, transcriptional activity and invasiveness, indicating that ET-1/ET(A)R-induced EMT depends on ILK activity. ET(A)R blockade by specific antagonists, or reduction by ET(A)R RNA interference, reverses EMT and cell invasion by inhibiting autocrine signaling pathways. In ovarian carcinoma xenografts, the specific ET(A)R antagonist ABT-627 suppresses EMT determinants and tumor growth. In human ovarian cancers, ET(A)R expression is associated with E-cadherin downregulation, N-cadherin expression and tumor grade. In conclusion, our findings demonstrate that ET(A)R activation by ET-1 is a key mechanism of the complex signaling network that promotes EMT as well as ovarian cancer cell invasion. The small molecule ET(A)R antagonist achieves concomitant suppression of tumor growth and EMT effectors, providing a new opportunity for therapeutic intervention in which targeting ILK pathway and the related Snail and beta-catenin signaling cascade via ET(A)R blockade may be advantageous in the treatment of ovarian cancer.

Topics: Animals; Disease Progression; Endothelin-1; Epithelial Cells; Epithelium; Female; Humans; Mesoderm; Models, Biological; Neoplasm Invasiveness; Ovarian Neoplasms; Receptor, Endothelin A

The endothelin axis (ET axis), comprising the three peptides endothelin (ET)-1, -2, -3 and their receptors ET(A)R and ET(B)R, is expressed in various cells and tissues. The biologically active ET-1 is formed by endothelin-converting enzyme (ECE) from inactive big-ET-1. ET-1 has emerged as an important peptide in a host of biological functions, including development, cellular proliferation, apoptosis and angiogenesis, thereby playing an important physiological and pathophysiological role. As these effects are mediated by ET(A)R, activation of ET(B)R prevents apoptosis, inhibits ECE expression and mediates the clearance of ET-1. Emerging data indicate that the ET axis is involved in tumourigenesis and tumour progression of various cancers. Expression of the ET axis has been demonstrated in a wide range of human tumours. Since most data have been reported for female malignancies, this review will focus on the role of the ET axis in cancers of the ovary, the cervix and the breast. In ovarian cancer, activation of ET(A)R by ET-1 is a key mechanism in the cellular signalling network promoting cancer growth and progression. Similar effects have been shown for cervical and endometrial cancer. In breast cancer, ET-1 via ET(A)R promotes proliferation and invasion, mediates bone metastases and predicts unfavourable response to chemotherapy. The outstanding role of ET-1 and ET(A)R in carcinogenesis and tumour progression has led to an extensive search for interfering agents, resulting in the development of selective ET(A)R antagonists on the one hand and inhibitors of the endothelin-converting enzyme (ECE) on the other. Targeting the ET axis via ET(A)R or ECE blockade seems to be a promising approach in the treatment of female malignancies.

Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Aspartic Acid Endopeptidases; Breast Neoplasms; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Endothelin-2; Endothelin-3; Endothelin-Converting Enzymes; Enzyme Inhibitors; Female; Humans; Metalloendopeptidases; Molecular Sequence Data; Neoplasms; Ovarian Neoplasms; Receptor, Endothelin A; Receptor, Endothelin B; Uterine Cervical Neoplasms

The endothelin (ET) system consists of three ET isopeptides, several converting enzyme isoforms and two G-protein-coupled receptors, ETA and ETB, which are linked to multiple signaling pathways. Less than 20 years after the initial detection of ET-1 in granulosa cells, the ovarian ET network continues to expand with the discovery of new members and functions. ETs influence a broad range of essential reproductive processes, such as ovulation, steroidogenesis and luteolysis. Therefore, a more comprehensive understanding of the ovarian ET network might provide new strategies for controlling reproduction. This review presents up-to-date findings on the ET network in the ovary.

Topics: Aspartic Acid Endopeptidases; Corpus Luteum; Endothelin-1; Endothelin-2; Endothelin-Converting Enzymes; Endothelins; Female; Humans; Metalloendopeptidases; Ovarian Neoplasms; Ovary; Receptors, Endothelin; RNA, Messenger

Endothelins are a family of peptide compounds which exert regulatory control over cellular processes important for growth, survival, invasion, and angiogenesis. In particular, endothelin-1, acting primarily through the endothelin-A receptor, is implicated in the neoplastic growth of multiple tumor types. In preclinical models, endothelin antagonism inhibits tumor cell proliferation, invasiveness, and new vessel formation, as well as attenuates osteoblastic and pain-related responses to tumor. Clinical testing of an orally bioavailable endothelin antagonist has demonstrated benefit in PSA progression, markers of bone turnover, and pain in men with prostate cancer, but has not demonstrated significant improvement in survival or time to cancer progression. Although this class of drugs is promising for targeted anti-cancer therapy, their role in treatment remains to be defined by completion of future clinical trials.

Topics: Atrasentan; Bosentan; Clinical Trials as Topic; Endothelin Receptor Antagonists; Endothelin-1; Female; Humans; Male; Neoplasms; Ovarian Neoplasms; Prostatic Neoplasms; Pyrimidines; Pyrrolidines; Receptors, Endothelin; Signal Transduction; Sulfonamides

PARP inhibitors (PARPi) have changed the treatment paradigm of high-grade serous ovarian cancer (HG-SOC). However, the impact of this class of inhibitors in HG-SOC patients with a high rate of TP53 mutations is limited, highlighting the need to develop combinatorial therapeutic strategies to improve responses to PARPi. Here, we unveil how the endothelin-1/ET-1 receptor (ET-1/ET-1R) axis, which is overexpressed in human HG-SOC and associated with poor prognosis, instructs HG-SOC/tumor microenvironment (TME) communication via key pro-malignant factors and restricts the DNA damage response induced by the PARPi olaparib. Mechanistically, the ET-1 axis promotes the p53/YAP/hypoxia inducible factor-1α (HIF-1α) transcription hub connecting HG-SOC cells, endothelial cells and activated fibroblasts, hence fueling persistent DNA damage signal escape. The ET-1R antagonist macitentan, which dismantles the ET-1R-mediated p53/YAP/HIF-1α network, interferes with HG-SOC/stroma interactions that blunt PARPi efficacy. Pharmacological ET-1R inhibition by macitentan in orthotopic HG-SOC patient-derived xenografts synergizes with olaparib to suppress metastatic progression, enhancing PARPi survival benefit. These findings reveal ET-1R as a mechanistic determinant in the regulation of HG-SOC/TME crosstalk and DNA damage response, indicating the use of macitentan in combinatorial treatments with PARPi as a promising and emerging therapy.

Topics: Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Endothelial Cells; Endothelin-1; Female; Humans; Ovarian Neoplasms; Poly(ADP-ribose) Polymerase Inhibitors; Receptor, Endothelin A; Tumor Microenvironment; Tumor Suppressor Protein p53

Topics: Acyltransferases; Animals; Apoptosis; beta-Arrestin 1; Cell Cycle Proteins; Cell Line, Tumor; Drug Resistance, Neoplasm; Endothelin-1; Female; Humans; Mice; Ovarian Neoplasms; Protein Serine-Threonine Kinases; Receptor, Endothelin A; Transcription Factors

Topics: beta-Arrestin 1; Cell Line, Tumor; Cystadenocarcinoma, Serous; Endothelin-1; Extracellular Matrix; Female; Gene Expression Regulation, Neoplastic; Humans; Ovarian Neoplasms; Podosomes; Proteolysis; ras GTPase-Activating Proteins; Receptor, Endothelin A; Signal Transduction; Vinculin

Topics: Adaptor Proteins, Signal Transducing; Animals; Antineoplastic Agents; beta-Arrestin 1; Cell Line, Tumor; Cell Survival; Cystadenocarcinoma, Serous; Disease Models, Animal; Endothelin-1; Female; Gene Expression Regulation, Neoplastic; Guanine Nucleotide Exchange Factors; Humans; Mice, Nude; Mutation; Ovarian Neoplasms; Protein Serine-Threonine Kinases; Pyrimidines; Receptor, Endothelin A; rho GTP-Binding Proteins; rhoA GTP-Binding Protein; Signal Transduction; Sulfonamides; Transcription Factors; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays; YAP-Signaling Proteins

Topics: Animals; beta-Arrestin 1; Cystadenocarcinoma, Serous; Cytoskeleton; Endothelin A Receptor Antagonists; Endothelin-1; Female; Gene Expression Regulation, Neoplastic; Humans; Mice, Nude; Microfilament Proteins; Ovarian Neoplasms; Podosomes; Pyrimidines; Receptor, Endothelin A; rhoC GTP-Binding Protein; Sulfonamides; Xenograft Model Antitumor Assays

Hypoxia-inducible factor-1α (HIF-1α) mediates the response to hypoxia or other stimuli, such as growth factors, including endothelin-1 (ET-1), to promote malignant progression in numerous tumors. The importance of cofactors that regulate HIF-1α signalling within tumor is not well understood. Here we elucidate that ET-1/ET(A) receptor (ET(A)R)-induced pathway physically and functionally couples the scaffold protein β-arrestin1 (β-arr1) to HIF-1α signalling. In epithelial ovarian cancer (EOC) cells, ET-1/ET(A)R axis induced vascular-endothelial growth factor (VEGF) expression through HIF-1α nuclear accumulation. In these cells, activation of ET(A)R by ET-1, by mimicking hypoxia, promoted the nuclear interaction between β-arr1 and HIF-1α and the recruitment of p300 acetyltransferase to hypoxia response elements on the target gene promoters, resulting in enhanced histone acetylation, and HIF-1α target gene transcription. Indeed, β-arr1-HIF-1α interaction regulated the enhanced expression and release of downstream targets, such as ET-1 and VEGF, required for tumor cell invasion and pro-angiogenic effects in endothelial cells. These effects were abrogated by β-arr1 or HIF-1α silencing or by pharmacological treatment with the dual ET-1 receptor antagonist macitentan. Interestingly, ET(A)R/β-arr1 promoted the self-amplifying HIF-1α-mediated transcription of ET-1 that sustained a regulatory circuit involved in invasive and angiogenic behaviors. In a murine orthotopic model of metastatic human EOC, treatment with macitentan, or silencing of β-arr1, inhibits intravasation and metastasis formation. Collectively, these findings reveal the interplay of β-arr1 with HIF-1α in the complexity of ET-1/ET(A)R signalling, mediating epigenetic modifications directly involved in the metastatic process, and suggest that targeting ET-1-dependent β-arr1/HIF-1α pathway by using macitentan may impair EOC progression.

Topics: Animals; beta-Arrestin 1; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Disease Progression; Endothelin-1; Female; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mice, Nude; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms

In epithelial ovarian cancer (EOC), activation of endothelin-1 (ET-1)/endothelin A receptor (ETAR) signalling is linked to many tumor promoting effects, such as proliferation, angiogenesis, invasion and metastasis. These effects are dependent by the activation of critical signalling pathways, such as MAPK, Akt, and β-catenin, through specific cytosolic and nuclear scaffolding functions of β-arrestin 1 (β-arr1). Here, we have assessed the potential role of ET-1/ETAR in promoting NF-κB signalling in EOC cells through β-arr-1 recruitment.. We used cultured HEY EOC cells cultured in the presence or absence of ET-1 and the ETAR antagonist BQ123. The phosphorylation of p65 and Iκ-Bα was evaluated by immunoblotting analysis. The interaction between p65 and β-arr1 was evaluated by immunoprecipitation experiments in nuclear extracts. NF-κB promoter activity was evaluated by transfection with NF-κB-driven luciferase reporter construct. Assessment of the function of β-arr1 was achieved by β-arr1 silencing with shRNA and expression of β-arr1-FLAG expression vector.. In EOC cells, ET-1 promotes the phosphorylation of p65 subunit and the cytoplasmic inhibitor IκB that in turn led to increased NF-κB transcriptional activity. These effects were inhibited by the use of BQ123, as well as by β-arr-1 silencing, suggesting that ET-1 through ETAR promotes the recruitment of β-arr1 to regulate NF-κB signalling. Moreover, the nuclear physical interaction between p65 and β-arr1 indicates a nuclear function of β-arr-1 in ETAR-driven NF-κB transcriptional activity.. Altogether these findings reveal a previously unrecognized pathway that depends on β-arr1 to sustain NF-κB signalling in response to ETAR activation in ovarian cancer.

Topics: Arrestins; beta-Arrestin 1; beta-Arrestins; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Endothelin-1; Female; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Models, Biological; Neoplasms, Glandular and Epithelial; NF-kappa B; Ovarian Neoplasms; Receptor, Endothelin A; Receptor, Endothelin B; RNA, Small Interfering

The endothelin-1 (ET-1)/endothelin A receptor (ETAR, a G protein-coupled receptor) axis confers pleiotropic effects on both tumor cells and the tumor microenvironment, modulating chemo-resistance and other tumor-associated processes by activating Gαq- and β-arrestin-mediated pathways. While the precise mechanisms by which these effects occur remain to be elucidated, interference with ETAR signaling has emerged as a promising antitumor strategy in many cancers including ovarian cancer (OC). However, current clinical approaches using ETAR antagonists in the absence of a detailed knowledge of downstream signaling have resulted in multiple adverse side effects and limited therapeutic efficacy. To maximize the safety and efficacy of ETAR-targeted OC therapy, we investigated the role of other G protein subunits such as Gαs in the ETAR-mediated ovarian oncogenic signaling. In HEY (human metastatic OC) cells where the ET-1/ETAR axis is well-characterized, Gαs signaling inhibits ETAR-mediated OC cell migration, wound healing, proliferation and colony formation on soft agar while inducing OC cell apoptosis. Mechanistically, ET-1/ETAR is coupled to Gαs/cAMP signaling in the same ovarian carcinoma-derived cell line. Gαs/cAMP/PKA activation inhibits ETAR-mediated β-arrestin activation of angiogenic/metastatic Calcrl and Icam2 expression. Consistent with our findings, Gαs overexpression is associated with improved survival in OC patients in the analysis of the Cancer Genome Atlas data. In conclusion, our results indicate a novel function for Gαs signaling in ET-1/ETAR-mediated OC oncogenesis and may provide a rationale for a biased signaling mechanism, which selectively activates Gαs-coupled tumor suppressive pathways while blocking Gαq-/β-arrestin-mediated oncogenic pathways, to improve the targeting of the ETAR axis in OC.

Topics: Adenylyl Cyclases; Apoptosis; Arrestins; beta-Arrestins; Carcinogenesis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Endothelin-1; Enzyme Activators; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; GTP-Binding Protein alpha Subunits, Gs; Humans; Models, Biological; Neoplasm Metastasis; Neovascularization, Pathologic; Ovarian Neoplasms; Receptor, Endothelin A; Signal Transduction; Survival Analysis

The high mortality of epithelial ovarian cancer (EOC) is mainly caused by resistance to the available therapies. In EOC, the endothelin-1 (ET-1, EDN1)-endothelin A receptor (ETAR, EDNRA) signaling axis regulates the epithelial-mesenchymal transition (EMT) and a chemoresistant phenotype. However, there is a paucity of knowledge about how ET-1 mediates drug resistance. Here, we define a novel bypass mechanism through which ETAR/β-arrestin-1 (β-arr1, ARRB1) links Wnt signaling to acquire chemoresistant and EMT phenotype. We found that ETAR/β-arr1 activity promoted nuclear complex with β-catenin and p300, resulting in histone acetylation, chromatin reorganization, and enhanced transcription of genes, such as ET-1, enhancing the network that sustains chemoresistance. Silencing of β-arr1 or pharmacologic treatment with the dual ETAR/ETBR antagonist macitentan prevented core complex formation and restored drug sensitivity, impairing the signaling pathways involved in cell survival, EMT, and invasion. In vivo macitentan treatment reduced tumor growth, vascularization, intravasation, and metastatic progression. The combination of macitentan and cisplatinum resulted in the potentiation of the cytotoxic effect, indicating that macitentan can enhance sensitivity to chemotherapy. Investigations in clinical specimens of chemoresistant EOC tissues confirmed increased recruitment of β-arr1 and β-catenin to ET-1 gene promoter. In these tissues, high expression of ETAR significantly associated with poor clinical outcome and chemoresistance. Collectively, our findings reveal the existence of a novel mechanism by which ETAR/β-arr1 signaling is integrated with the Wnt/β-catenin pathway to sustain chemoresistance in EOC, and they offer a solid rationale for clinical evaluation of macitentan in combination with chemotherapy to overcome chemoresistance in this setting.

Topics: Arrestins; beta-Arrestin 1; beta-Arrestins; Cell Line, Tumor; Drug Resistance, Neoplasm; Endothelin-1; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Ovarian Neoplasms; Receptor, Endothelin A; Wnt Signaling Pathway

Despite the fundamental pathophysiological importance of β-catenin in tumor progression, the mechanism underlying its final transcriptional output has been partially elucidated. Here, we report that β-arrestin-1 (β-arr1) is an epigenetic regulator of endothelin (ET)-1-induced β-catenin signaling in epithelial ovarian cancer (EOC). In response to ET A receptor (ETAR) activation by ET-1, β-arr1 increases its nuclear translocation and direct binding to β-catenin. This in turn enhanced β-catenin nuclear accumulation and transcriptional activity, which was prevented by expressing a mutant β-arr1 incapable of nuclear distribution. β-arr1-β-catenin interaction controls β-catenin target gene expressions, such as ET-1, Axin 2, Matrix metalloproteinase 2, and Cyclin D1, by promoting histone deacetylase 1 (HDAC1) dissociation and the recruitment of p300 acetyltransferase on these promoter genes, resulting in enhanced H3 and H4 histone acetylation, and gene transcription, required for cell migration, invasion and epithelial-to-mesenchymal transition. These effects are abrogated by β-arr1 silencing or by mutant β-arr1, as well as by β-catenin or p300 silencing, confirming that nuclear β-arr1 forms a functional complex capable of regulating epigenetic changes in β-catenin-driven invasive behavior. In a murine orthotopic model of metastatic human EOC, silencing of β-arr1 or mutant β-arr1 expression, as well as ETAR blockade, inhibits metastasis. In human EOC tissues, β-arr1-β-catenin nuclear complexes are selectively enriched at β-catenin target gene promoters, correlating with tumor grade, confirming a direct in vivo β-arr1-β-catenin association at specific set of genes involved in EOC progression. Collectively, our study provides insights into how a β-arr1-mediated epigenetic mechanism controls β-catenin activity, unraveling new components required for its nuclear function in promoting metastasis.

Topics: Animals; Arrestins; Axin Protein; beta Catenin; beta-Arrestin 1; beta-Arrestins; Carcinoma, Ovarian Epithelial; Cell Nucleus; Cyclin D1; Endothelin-1; Epigenesis, Genetic; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase 1; Histones; Humans; Matrix Metalloproteinase 2; Mice, Nude; Mutation; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Promoter Regions, Genetic; Protein Transport; Receptor, Endothelin A; Signal Transduction; Xenograft Model Antitumor Assays

The Rho-associated serine-threonine protein kinase (ROCK) is a downstream effector of Rho GTPases that is frequently activated in the epithelial to mesenchymal transition (EMT) of human ovarian cancer cells. On the other hand, endothelin-1 (ET-1) and its receptor endothelin A receptor (ET(A)R) are overexpressed in primary and metastatic ovarian carcinoma, which suggests that ET-1 promotes tumor dissemination. Hence, two human ovarian carcinoma cell lines, SK-OV-3 and CaOV3, were chosen to study the effects of ET-1/ET(A)R and ROCK in promoting EMT of ovarian cancer cells. We found that ET-1 exposure induced EMT of SK-OV-3 and CaOV3 by monitoring cells morphology, enhanced fibronectin, and reduced E-cadherin protein. At the same time, ET-1/ET(A)R enhanced the level of transcription of SLUG a transcriptional repressor of E-cadherin. More importantly, a constitutively active mutant of ROCK enhanced the transcription of SLUG by stimulating SLUG promoter activity. Furthermore, ROCK inhibitor Y27632 reversed the increase in fibronectin induced by ET-1/ET(A)R. Our data suggest that ROCK cooperated with ET-1/ET(A)R to promote EMT of human ovarian carcinoma cells through upregulation of SLUG mRNA.

Topics: Cadherins; Cell Line, Tumor; Endothelin-1; Epithelial-Mesenchymal Transition; Female; Humans; Ovarian Neoplasms; Receptor, Endothelin A; rho-Associated Kinases; RNA, Messenger; Snail Family Transcription Factors; Transcription Factors; Transcription, Genetic; Up-Regulation

Emerging evidence suggests molecular and phenotypic association between chemoresistance and epithelial-mesenchymal transition (EMT) in cancer. Endothelin-1 (ET-1)/endothelin A receptor (ET(A)R) axis is implicated in the pathobiology of epithelial ovarian cancer (EOC) by driving tumor-promoting effects, including EMT. Here, we analyzed how ET(A)R regulates chemoresistance and EMT in EOC.. The effects of ET-1 axis on cell proliferation, drug-induced apoptosis, invasiveness, and EMT were analyzed in cultured EOC cells sensitive and resistant to cisplatinum and taxol. Tumor growth in response to ET(A)R antagonist was examined in EOC xenografts. ET(A)R expression was examined in 60 human EOC tumors by immunohistochemistry and correlated with chemoresistance and EMT.. In resistant EOC cells ET-1 and ET(A)R are upregulated, paralleled by enhanced mitogen activated protein kinase (MAPK) and Akt phosphorylation and cell proliferation. Moreover, in these cells the expression of E-cadherin transcriptional repressors, including Snail, Slug, and Twist, as well as of mesenchymal markers, such as vimentin and N-cadherin, were upregulated and linked with enhanced invasive behavior. Interestingly, ET(A)R blockade with zibotentan, a specific ET(A)R antagonist, or its silencing, downregulated Snail activity, restored drug sensitivity to cytotoxic-induced apoptosis, and inhibited the invasiveness of resistant cells. In vivo, zibotentan inhibited tumor growth of sensitive and resistant EOC xenografts, and sensitized to chemotherapy. Analysis of EOC human tissues revealed that ET(A)R is overexpressed in resistant tumors and is associated with EMT phenotype.. Our data provide the first evidence that blockade of ET(A)R-driven EMT can overcome chemoresistance and inhibit tumor progression, improving the outcome of EOC patients' treatment.

Topics: Aged; Animals; Antineoplastic Combined Chemotherapy Protocols; Blotting, Western; Cell Line, Tumor; Cisplatin; Drug Resistance, Neoplasm; Endothelin A Receptor Antagonists; Endothelin-1; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Middle Aged; Ovarian Neoplasms; Paclitaxel; Pyrrolidines; Receptor, Endothelin A; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Treatment Outcome; Xenograft Model Antitumor Assays

Ovarian tumours have a low incidence in bitch. Endothelin (ET-1) and endothelin A receptor (ET-A) are overexpressed in human ovarian cancer. Twenty canine ovarian tumours and five normal samples were first evaluated by western blotting and then immunohistochemically for ET-1 and ET-A expression. Seventeen out of twenty tumours were ET-1 positive. Eight out of twenty tumours were ET-A immunohistochemically positive. At molecular level both proteins were proven to be expressed in normal as well as in tumour samples. Our results show that ET-1 and ET-A are overexpressed in canine ovarian tumours, suggesting a potential role of these two molecules in canine ovarian carcinogenesis.

Topics: Animals; Carcinoma, Papillary; Cystadenocarcinoma; Dog Diseases; Dogs; Endothelin-1; Female; Gene Expression Regulation, Neoplastic; Granulosa Cell Tumor; Ovarian Neoplasms; Receptor, Endothelin A

The contribution of the endothelin-1 (ET-1)/ET A receptor (ETAR) axis in tumor growth and progression is investigated in many tumor types, including ovarian carcinoma. In ovarian cancer cells, ET-1 acts as an autocrine growth factor selectively through the ETAR triggering the concomitant activation of multiple pathways. In these cells, the involvement of beta-arrestin-1 as signal transducer in ET-1-dependent signalling pathways has been recently highlighted. Because several G protein-coupled receptors have been shown to activate signalling pathways in a beta-arrestin-dependent manner, in this study we explored whether beta-arrestin-1 is involved in a distinct signalling mechanism linking the ETAR to phosphoinositide 3-kinase (PI3K)/integrin-linked kinase (ILK)/Akt in HEY ovarian cancer cells. The inhibitory effects of ZD4054 (zibotentan), a specific ETAR antagonist, in ET-1-dependent phosphorylation of ILK, Akt, and glycogen synthase kinase (GSK-3beta) demonstrated the involvement of the ETAR in these effects. By using a kinase assay, we demonstrate that beta-arrestin-1 silencing inhibits the ET-1-induced ILK activity in a time-dependent manner and downstream Akt and GSK-3beta phosphorylation. These results reveal that beta-arrestin-1 is implicated as an ETAR-transducer in the activation of ILK and Akt and in the inactivation of GSK-3beta in response to ET-1 and further support the role of beta-arrestin-1 as a multifunctional adaptor facilitating interprotein interactions critically involved in ETAR-mediated signalling that regulate invasive and metastatic behaviour of ovarian cancer.

Topics: Arrestins; beta-Arrestin 1; beta-Arrestins; Cell Line, Tumor; Endothelin A Receptor Antagonists; Endothelin-1; Enzyme Activation; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Models, Biological; Ovarian Neoplasms; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Pyrrolidines; RNA, Small Interfering; Signal Transduction

Assessment of endothelin-1 (ET-1) level for the differential diagnosis of ovarian tumors was performed.. Fifty-three women with ovarian tumors (12 malignant, 41 benign) treated at the Division of Gynecological Surgery, Poznan, Poland were analyzed. ET-1 serum levels were compared with CA125 and Doppler parameters (RI, PI, PSV). The differences were evaluated using the unpaired t-test with Welch's correction (p < 0.05).. There was statistically significant difference in ET-1 levels between the ovarian cancer and benign tumor groups (3.130 pg/ml and 1.819 pg/ml, respectively). The ROC area for ET-1 and CA125 were 0.727 and 0.791, respectively. A negative linear correlation between the level of ET-1 and RI and a positive linear correlation between the level of ET-1 and PSV in the ovarian cancer group were found.. ET-1 as a new biomarker for predicting ovarian cancer is not satisfying. A correlation between ET-1 levels and Doppler parameters is very interesting and its role in angiogenesis should be studied extensively.

Topics: Adult; Biomarkers, Tumor; CA-125 Antigen; Disease Progression; Endothelin-1; Female; Humans; Middle Aged; Neoplasm Staging; Ovarian Neoplasms; ROC Curve; Ultrasonography, Doppler

Endothelin-1 (ET-1) has been implicated in the progression of various cancers, including ovarian carcinoma. We found that the ovarian carcinoma cell lines ES2 and OVCAR3 and tumors from different anatomic sites expressed ET-1 system members [ET receptor A and ET-converting enzyme-1 (ECE-1)]. However, only ECE-1 was significantly higher in the solid tumors compared with effusions. We therefore investigated the effect of RNA interference-induced knockdown of ECE-1, the key enzyme in ET-1 production, on these two ovarian carcinoma cell lines. Small interfering RNA (siRNA) targeting of ECE-1 markedly reduced ECE-1 mRNA and protein levels, which subsequently led to 80% to 90% inhibition of ET-1 peptide secretion by the cells. ECE-1 silencing also profoundly affected the behavior of tumor cells compared with cells treated with scrambled siRNA. Silenced cells exhibited (a) reduced ET-1-dependent p44/42 mitogen-activated protein kinase phosphorylation; (b) decreased invasiveness and matrix metalloproteinase-2 activity; (c) improved adhesion to basal lamina proteins, laminin-1, and collagen IV; and (d) increased E-cadherin, an epithelial adhesion molecule, and reduced N-cadherin expression, a mesenchymal marker. Altered cell adherence is one of the hallmarks of the transformed phenotype, often characterized by the loss of the epithelial features and the gain of a mesenchymal phenotype. ECE-1 ablation did not, however, alter viable ovarian carcinoma cell numbers. Addition of exogenous ET-1 reversed the effects cited above. Taken together, these data indicate that siRNA is an effective tool for manipulating ECE-1 expression, ET-1 biosynthesis, and invasiveness of ovarian carcinoma. ECE-1 silencing may therefore develop into a promising novel anticancer therapy.

Topics: Aspartic Acid Endopeptidases; Cell Line, Tumor; Endothelin-1; Endothelin-Converting Enzymes; Female; Humans; Metalloendopeptidases; Neoplasm Invasiveness; Ovarian Neoplasms; Receptor, Endothelin A; RNA, Messenger; RNA, Small Interfering

The endothelin-1 (ET-1) axis represents a novel target in several malignancies, including ovarian carcinoma. Upon being activated, the endothelin A receptor (ET(A)R) mediates multiple tumor-promoting activities, including mitogenesis, escape from apoptosis, angiogenesis, metastasis-related protease activation, epithelial-mesenchymal transition, and invasion. Integrin-linked kinase (ILK) is a multidomain focal adhesion protein that conveys intracellular signaling elicited by beta1-integrin and growth factor receptors. In this study, we investigate whether the signaling triggered by ET(A)R leading to an aggressive phenotype is mediated by an ILK-dependent mechanism. In HEY and OVCA 433 ovarian carcinoma cell lines, activation of ET(A)R by ET-1 enhances the expression of alpha2beta1 and alpha3beta1 integrins. ILK activity increases as ovarian cancer cells adhere to type I collagen through beta1 integrin signaling, and do so to a greater extent on ET-1 stimulation. ET-1 increases ILK mRNA and protein expression and activity in a time- and concentration-dependent manner. An ILK small-molecule inhibitor (KP-392) or transfection with a dominant-negative ILK mutant effectively blocks the phosphorylation of downstream signals, Akt and glycogen synthase kinase-3beta. The blockade of ET-1/ET(A)R-induced ILK activity results in an inhibition of matrix metalloproteinase activation as well as of cell motility and invasiveness in a phosphoinositide 3 kinase-dependent manner. In ovarian carcinoma xenografts, ABT-627, a specific ET(A)R antagonist, suppresses ILK expression, Akt and glycogen synthase kinase-3beta phosphorylation, and tumor growth. These data show that ILK functions as a downstream mediator of the ET-1/ET(A)R axis to potentiate aggressive cellular behavior. Thus, the ILK-related signaling cascade can be efficiently targeted by pharmacologic blockade of ET(A)R.

Topics: Animals; Base Sequence; Cell Line, Tumor; Chemotaxis; DNA Primers; Endothelin-1; Female; Humans; Immunoblotting; Mice; Mice, Nude; Neoplasm Invasiveness; Ovarian Neoplasms; Protein Serine-Threonine Kinases; Receptor, Endothelin A; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Transplantation, Heterologous

The green tea polyphenol, epigallocatechin-3-gallate (EGCG), has been shown to prevent cancer; however, a precise mechanism responsible for tumor growth inhibition has not yet been clearly described. The endothelin (ET) A receptor (ET(A)R)/ET-1 autocrine pathway is overexpressed in ovarian carcinoma and triggers tumor growth, neoangiogenesis, and invasion. These latter tumor-promoting effects are mediated through the activation of cyclooxygenase (COX)-1- and COX-2-dependent pathways by ET-1. In the present study, pretreatment of HEY and OVCA 433 ovarian carcinoma cell lines with green tea and EGCG inhibited ET-1/ET(A)R expression, ET(A)R-mediated COX-1/2 mRNA expression, and COX-2 promoter activity. These effects were associated with a significant reduction in the COX-1/2-derived prostaglandin E2 (PGE2) production. These results provide a novel insight into the mechanism by which EGCG, by affecting ET(A)R-dependent COX-1/2 pathways may inhibit ovarian tumors suggesting that EGCG may be useful in preventing and treating ovarian carcinoma in which activation of ET(A)R by ET-1 plays a critical role in tumor growth and progression.

Topics: Antineoplastic Agents; Catechin; Cell Line, Tumor; Cyclooxygenase Inhibitors; Dinoprostone; Endothelin A Receptor Antagonists; Endothelin-1; Female; Humans; Ovarian Neoplasms; Promoter Regions, Genetic; RNA, Messenger; Tea

In a range of human cancers, tumorigenesis is promoted by activation of the endothelin A receptor (ET(A)R)/endothelin-1 (ET-1) axis. ET-1 and ET(A)R are overexpressed in primary and metastatic ovarian carcinomas, and high levels of ET-1 are detectable in patient ascites, suggesting that ET-1 may promote tumor dissemination. Moreover, in these tumors, engagement of ET(A) receptor by ET-1 triggers tumor growth, survival, angiogenesis, and invasiveness. Thus, ET-1 enhances the secretion of matrix metalloproteinases, disrupts intercellular communications, and stimulates cell migration and invasion. Therefore, we investigated the role of the ET-1/ET(A)R autocrine axis in promoting epithelial to mesenchymal transition (EMT) in ovarian tumor cells, a key event in cancer metastasis, in which epithelial cells depolarize, disassemble cell-cell contacts, and adopt an invasive phenotype. Here, we examine the potential role of ET-1 in regulating cell morphology and behavior and epithelial and mesenchymal proteins employing an in vitro 3-D culture system. We found that in 3-D serum-free collagen I gel cultures, HEY and OVCA 433 ovarian carcinoma cells undergo fibroblast-like morphologic changes between 3 and 5 days of ET-1 treatment. In these cells, ET-1 induces loss of adherens and tight-junction protein expression, E-cadherin, beta-catenin, and zonula occludens-1, and gain of N-cadherin and vimentin expression. These results confirm the ability of ET-1 to promote EMT, a metastable process involving sustained loss of epithelial markers and gain of mesenchymal markers. Collectively, these findings provide evidence of a critical role for the ET-1/ET(A)R axis during distinct steps of ovarian carcinoma progression, thus underlining this axis as a potential target in the treatment of ovarian cancer.

Topics: Cell Line, Tumor; Collagen Type I; Culture Media, Serum-Free; Disease Progression; Endothelin A Receptor Antagonists; Endothelin-1; Epithelial Cells; Female; Humans; Mesoderm; Neoplasm Invasiveness; Organ Culture Techniques; Ovarian Neoplasms; Receptor, Endothelin A; Time Factors

Endothelin-1 (ET-1) is present at high concentrations in ovarian cancer ascites and is overexpressed in primary and metastatic ovarian carcinomas. In these tumors, the presence of ET-1 correlates with tumor grade, enhanced neovascularization, and with vascular endothelial growth factor (VEGF) expression. ET-1 acts as an autocrine factor selectively through ET(A) receptor (ET(A)R), predominantly expressed in ovarian carcinoma cells resulting in increased VEGF production and VEGF-mediated angiogenic effects. Previous results demonstrated that in ovarian carcinoma cells, activation of the ET-1/ET(A)R axis promotes cell proliferation, neovascularization, and invasion, which are the principal hallmarks of tumor progression. The present study was designed to investigate the in vitro effects of trans, trans-2(4-methoxydhenyl)-4-(1-3-benzodiazol-5-yl)-1-(dibutylaminocarbonylmethyl)-pyrrolidine-3-carboxylic acid (ZD4054), an orally active specific ET(A)R antagonist, on the ET-1-induced mitogenic effect in OVCA 433 and HEY ovarian carcinoma cell lines secreting ET-1 and expressing ET(A)R and ET(B)R mRNA. We show that ET(A)R blockade by ZD4054 inhibits ET-1-induced mitogenic effects, while the ET(B)R antagonist, BQ 788, is ineffective. In conclusion, our data demonstrate that ZD4054 is capable in inhibiting the proliferative activity of ET-1, indicating that this specific ET(A)R antagonist may be a potential candidate in developing novel treatment of ovarian carcinoma.

Topics: Cell Line, Tumor; Cell Proliferation; Endothelin A Receptor Antagonists; Endothelin-1; Female; Humans; Ovarian Neoplasms; Pyrrolidines; RNA, Messenger

The polyphenol epigallocatechin-3-gallate (EGCG), the principal mediator of the green tea, has been known to possess antitumor effect. The endothelin A receptor (ET(A)R)/endothelin-1 (ET-1) axis is overexpressed in ovarian carcinoma representing a novel therapeutic target. In this study, we examined the green tea and EGCG effects on two ovarian carcinoma cell lines, HEY and OVCA 433. EGCG inhibited ovarian cancer cell growth and induced apoptosis that was associated with a decrease in Bcl-X(L) expression and activation of caspase-3. Treatment with green tea or EGCG inhibited ET(A)R and ET-1 expression and reduced the basal and ET-1-induced cell proliferation and invasion. The EGCG-induced inhibitory effects were associated with a decrease of ET(A)R-dependent activation of the p42/p44 and p38 mitogen-activated protein kinases and phosphatidylinositol 3-kinase pathway. Remarkably, EGCG treatment resulted in a lowering of basal and ET-1-induced angiogenesis and invasiveness mediators, such as vascular endothelial growth factor and tumor proteinase activation. Finally, in HEY ovarian carcinoma xenografts, tumor growth was significantly inhibited by oral administration of green tea. This effect was associated with a reduction in ET-1, ET(A)R, and vascular endothelial growth factor expression, microvessel density, and proliferation index. These results provide a novel insight into the mechanism by which EGCG, affecting multiple ET(A)R-dependent pathways, may inhibit ovarian carcinoma growth, suggesting that EGCG may be useful in preventing and treating ovarian carcinoma in which ET(A)R activation by ET-1 plays a critical role in tumor growth and progression.

Topics: Animals; Anticarcinogenic Agents; Apoptosis; bcl-X Protein; Caspase 3; Caspases; Catechin; Cell Proliferation; Endothelin-1; Enzyme Activation; Female; Humans; Mice; Mice, Nude; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Receptor, Endothelin A; Signal Transduction; Tea; Transplantation, Heterologous; Urokinase-Type Plasminogen Activator; Vascular Endothelial Growth Factor A

Despite considerable efforts to improve early detection and advances in chemotherapy, metastatic relapses remain a major challenge in the management of ovarian cancer. The endothelin A receptor (ET(A)R)/endothelin-1 (ET-1) axis has been shown to have a significant role in ovarian carcinoma by promoting tumorigenesis. Here we show that the ET-1/ET(A)R autocrine pathway drives epithelial-to-mesenchymal transition (EMT) in ovarian tumor cells by inducing a fibroblastoid and invasive phenotype, down-regulation of E-cadherin, increased levels of beta-catenin, Snail, and other mesenchymal markers, and suppression of E-cadherin promoter activity. Activation of ET(A)R by ET-1 triggers an integrin-linked kinase (ILK)-mediated signaling pathway leading to glycogen synthase kinase-3beta (GSK-3beta) inhibition, Snail and beta-catenin stabilization, and regulation of transcriptional programs that control EMT. Transfection of dominant negative ILK or exposure to an ILK inhibitor suppresses the ET-1-induced phosphorylation of GSK-3beta as well as Snail and beta-catenin protein stability, activity, and invasiveness, indicating that ET-1/ET(A)R-induced EMT-promoting effects depend on ILK. ET(A)R blockade by specific antagonists or reduction by ET(A)R RNA interference reverses EMT and cell invasion by inhibiting autocrine signaling pathways. In ovarian carcinoma xenografts, ABT-627, a specific ET(A)R antagonist, suppresses EMT determinants and tumor growth. In human ovarian cancers, ET(A)R expression is associated with E-cadherin down-regulation, N-cadherin expression, and tumor grade. Collectively, these findings provide evidence of a critical role for the ET-1/ET(A)R axis during distinct steps of ovarian carcinoma progression and identify novel targets of therapeutic intervention.

Topics: Animals; beta Catenin; Blotting, Northern; Blotting, Western; Cadherins; Cell Nucleus; Down-Regulation; Endothelin A Receptor Antagonists; Endothelin-1; Enzyme Inhibitors; Epithelial Cells; Female; Fibroblasts; Gene Expression Regulation, Neoplastic; Genes, Dominant; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Luciferases; Mesoderm; Mice; Mice, Nude; Neoplasm Invasiveness; Ovarian Neoplasms; Phenotype; Phosphorylation; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Protein Transport; Receptor, Endothelin A; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

New therapies against cancer are based on targeting cyclooxygenase (COX)-2. Activation of the endothelin A receptor (ET(A)R) by endothelin (ET)-1 is biologically relevant in several malignancies, including ovarian carcinoma. In this tumor, the ET-1/ET(A)R autocrine pathway promotes mitogenesis, apoptosis protection, invasion, and neoangiogenesis. Because COX-1 and COX-2 are involved in ovarian carcinoma progression, we investigated whether ET-1 induced COX-1 and COX-2 expression through the ET(A)R at the mRNA and protein level in HEY and OVCA 433 ovarian carcinoma cell lines by Northern blot, reverse transcription-PCR, Western blot, and immunohistochemistry; we also investigated the activity of the COX-2 promoter by luciferase assay and the release of prostaglandin (PG) E(2) by ELISA.. ET-1 significantly increases the expression of COX-1 and COX-2, COX-2 promoter activity, and PGE(2) production. These effects depend on ET(A)R activation and involve multiple mitogen-activated protein kinase (MAPK) signaling pathways, including p42/44 MAPK, p38 MAPK, and transactivation of the epidermal growth factor receptor. COX-2 inhibitors and, in part, COX-1 inhibitor blocked ET-1-induced PGE(2) and vascular endothelial growth factor release, indicating that both enzymes participate in PGE(2) production to a different extent. Moreover, inhibition of human ovarian tumor growth in nude mice after treatment with the potent ET(A)R-selective antagonist ABT-627 is associated with reduced COX-2 and vascular endothelial growth factor expression.. These results indicate that impairing COX-1 and COX-2 and their downstream effect by targeting ET(A)R can be therapeutically advantageous in ovarian carcinoma treatment. Pharmacological blockade of the ET(A)R is an attractive strategy to control COX-2 induction, which has been associated with ovarian carcinoma progression and chemoresistance.

Topics: Animals; Atrasentan; Blotting, Northern; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dinoprostone; Endothelin A Receptor Antagonists; Endothelin-1; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Indomethacin; Isoenzymes; Luciferases; MAP Kinase Signaling System; Membrane Proteins; Mice; Mice, Nude; Nitrobenzenes; Ovarian Neoplasms; Promoter Regions, Genetic; Prostaglandin-Endoperoxide Synthases; Pyrazoles; Pyrrolidines; Receptor, Endothelin A; Recombinant Fusion Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sulfonamides; Transfection; Up-Regulation; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Cyclooxygenase (COX)-1- and COX-2-derived prostaglandins are implicated in the development and progression of several malignancies. We have recently demonstrated that treatment of ovarian carcinoma cells with endothelin-1 (ET-1) induces expression of both COX-1 and COX-2, which contributes to vascular endothelial growth factor (VEGF) production. In this study, we show that in HEY and OVCA 433 ovarian carcinoma cells, ET-1, through the binding with ETA receptor (ETAR), induces prostaglandin E2 (PGE2) production, as the more represented PG types, and increases the expression of PGE2 receptor type 2 (EP2) and type 4 (EP4). The use of pharmacological EP agonists and antagonists indicates that ET-1 and PGE2 stimulate VEGF production principally through EP2 and EP4 receptors. At the mechanistic level, we prove that the induction of PGE2 and VEGF by ET-1 involves Src-mediated epidermal growth factor receptor transactivation. Finally, we demonstrate that ETAR-mediated activation of PGE2-dependent signaling participates in the regulation of the invasive behavior of ovarian carcinoma cells by activating tumor-associated matrix metalloproteinase. These results implicate EP2 and EP4 receptors in the induction of VEGF expression and cell invasiveness by ET-1 and provide a mechanism by which ETAR/ET-1 can promote and interact with PGE2-dependent machinery to amplify its proangiogenic and invasive phenotype in ovarian carcinoma cells. Pharmacological blockade of ETAR can therefore represent an additional strategy to control PGE2 signaling, which has been associated with ovarian carcinoma progression.

Topics: Blotting, Western; Cell Line; Cell Line, Tumor; Dinoprostone; Disease Progression; Dose-Response Relationship, Drug; Endothelin-1; Enzyme-Linked Immunosorbent Assay; Female; Humans; Neoplasm Invasiveness; Ovarian Neoplasms; Phenotype; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP2 Subtype; Receptors, Prostaglandin E, EP4 Subtype; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; src-Family Kinases; Transcriptional Activation; Up-Regulation; Vascular Endothelial Growth Factor A

Ovarian carcinoma cells release high amounts of endothelin-1 and exhibit increased expression of endothelin-A receptor. Engagement of the endothelin-A receptor triggers tumor growth, survival, neoangiogenesis and invasion. Cyclooxygenase-1 and cyclooxygenase-2 are enzymes involved in the production of prostaglandins and play a role in the regulation of tumor progression in several malignancies, including ovarian carcinomas. Endothelin-1 significantly increases the expression of cyclooxygenase-1 and cyclooxygenase-2 mRNA and protein, the activity of the cyclooxygenase- 2 promoter, and the release of prostaglandin E2 from two ovarian carcinoma cell lines, HEY and OVCA 433. The cyclooxygenase- 2 inhibitor, NS-398 drastically decreased the endothelin- 1-induced prostaglandin E2 production and vascular endothelial growth factor upregulation, indicating a role for cyclooxygenase-2 in endothelin-1-induced vascular endothelial growth factor-mediated angiogenesis. In this study we demonstrated that endothelin-1-induced cyclooxygenase-2 and related prostaglandin E2 release were dependent upon the activation of endothelin-A receptor and of multiple mitogen-activated protein kinase signal pathways, including extracellular signal-regulated kinase 1/2 kinase, p38 mitogen-activated protein kinase and the transactivation of the epidermal growth factor receptor. In human ovarian xenografts, the levels of cyclooxygenase-2 protein expression were significantly reduced following treatment with the endothelin-A receptor selective antagonist, atrasentan, compared with untreated mice. These results suggest that the pharmacological blocking of endothelin-A receptor is an attractive strategy to control the cyclooxygenase-2 protein expression in ovarian carcinoma.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cyclooxygenase 1; Cyclooxygenase 2; Dinoprostone; Endothelin A Receptor Antagonists; Endothelin-1; Enzyme Induction; ErbB Receptors; Female; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Ovarian Neoplasms; p38 Mitogen-Activated Protein Kinases; Peptides, Cyclic; Protein Kinase Inhibitors; Pyridines; Quinazolines; Receptor, Endothelin A; Signal Transduction; Transcriptional Activation; Tyrphostins

Endothelin-1 (ET-1) is overexpressed in ovarian carcinoma and acts as an autocrine factor selectively through the ETA receptor (ETAR) to promote tumor cell proliferation, survival, neovascularization, and invasiveness. Loss of gap junctional intercellular communication (GJIC) is critical for tumor progression by allowing the cells to escape growth control. Exposure of HEY and OVCA 433 ovarian carcinoma cell lines to ET-1 led to a 50-75% inhibition in intercellular communication and to a decrease in the connexin 43 (Cx43)-based gap junction plaques. To investigate the phosphorylation state of Cx43, ovarian carcinoma cell lysates were immunoprecipitated and transient tyrosine phosphorylation of Cx43 was detected in ET-1-treated cells. BQ 123, a selective ETAR antagonist, blocked the ET-1-induced Cx43 phosphorylation and cellular uncoupling. Gap junction closure was prevented by tyrphostin 25 and by the selective c-Src inhibitor, PP2. Furthermore, the increased Cx43 tyrosine phosphorylation was correlated with ET-1-induced increase of c-Src activity, and PP2 suppressed the ET-1-induced Cx43 tyrosine phosphorylation, indicating that inhibition of Cx43-based GJIC is mainly mediated by the Src tyrosine kinase pathway. In vivo, the inhibition of human ovarian tumor growth in nude mice induced by the potent ETAR antagonist, ABT-627, was associated with a reduction of Cx43 phosphorylation. These findings indicate that the signaling mechanisms involved in GJIC disruption on ovarian carcinoma cells depend on ETAR activation, which leads to the Cx43 tyrosine phosphorylation mediated by c-Src, suggesting that ETAR blockade may contribute to the control of ovarian carcinoma growth and progression also by preventing the loss of GJIC.

Topics: Animals; Cell Division; Cell Line, Tumor; Connexin 43; Disease Progression; Endothelin-1; Female; Humans; Immunoblotting; Kinetics; Mice; Mice, Nude; Neoplasm Transplantation; Ovarian Neoplasms; Phosphorylation; Precipitin Tests; Time Factors; Tyrosine

Endothelin-1 (ET-1) is a potent mitogenic and angiogenic factor for ovarian carcinoma cell lines, which acts selectively through the ET(A) receptor (ET(A)R). A previous study demonstrated that ET-1 is present at high concentrations in ovarian cancer ascites, indicating a direct role in the progression and metastasis of ovarian carcinoma. In this study, we investigated whether ET-1 could induce production and activation of tumour-associated proteinases in ovarian carcinoma cells. As demonstrated by ELISA, we found that the secretion of matrix metalloproteinase (MMP)-2 and MMP-9, urokinase-type plasminogen activator and plasminogen activator inhibitor type-1 and -2 was upregulated by ET-1 in a dose-dependent manner in the HEY cell line. In addition, the MMPs in ET-1-treated cells are consistently active, as shown by MMP gelatinase activity assay. Finally, we demonstrated that BQ-123, an antagonist of ET(A)R, inhibited the ET-1-induced tumour protease secretion and activity, suggesting that ET-1/ET(A)R may play an important role in the progression and metastasis of ovarian carcinoma, activating multiple proteinase cascades.

Topics: Dose-Response Relationship, Drug; Endopeptidases; Endothelin Receptor Antagonists; Endothelin-1; Female; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Ovarian Neoplasms; Peptides, Cyclic; Plasminogen Activator Inhibitor 1; Plasminogen Activator Inhibitor 2; Receptor, Endothelin A; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

The aim of this study was to evaluate the role of endothelin-1 (ET-1) in the sensitivity of ovarian carcinoma to paclitaxel, one of the most common drugs used for the management of this tumour histotype. ET-1 is a powerful mitogenic peptide produced by ovarian carcinomas and it acts as an autocrine growth factor, selectively through ET(A) receptor (ET(A)R), which is predominantly expressed in this tumour. OVCA 433 and HEY, two ovarian carcinoma cell lines, which produce elevated amounts of ET-1 and express abundantly high-affinity ET(A)Rs, were used. As demonstrated by sub-G(1) peak in DNA content histograms and terminal transferase deoxytidyl uridine end labelling assay, we found that paclitaxel induces cytotoxic effect through the activation of apoptosis in both cell lines. When the treatment with paclitaxel was performed in association with ET-1, paclitaxel-induced apoptosis was inhibited. In order to evaluate which ET-1 receptor mediated the effect of ET-1 on protection from paclitaxel-induced apoptosis, we performed experiments using two selective antagonists for ET(A)R (BQ-123) and for ET(B)R (BQ-788). We showed that ET(A)R blockade inhibits the ET-1-induced survival activity against paclitaxel-mediated apoptosis. However, no effect was observed on blocking ET(B)R with BQ-788. Our results establish a novel role for ET-1 in determining survival of ovarian carcinoma cells and suggest that pharmacological ET(A)R blockade using a specific ET(A)R antagonist may provide a novel approach to the treatment of ovarian carcinoma in combination therapy.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Survival; Endothelin Receptor Antagonists; Endothelin-1; Female; Humans; Oligopeptides; Ovarian Neoplasms; Paclitaxel; Peptides, Cyclic; Piperidines; Receptor, Endothelin A; Receptor, Endothelin B; Tumor Cells, Cultured

Endothelin-1 (ET-1) is present at high concentrations in ovarian cancer ascites and is overexpressed in primary and metastatic ovarian carcinomas. In these tumours the presence of ET-1 is associated with enhanced neovascularization and with vascular endothelial growth factor (VEGF) expression. In these tumour cells, ET-1 acts as an autocrine growth factor selectively through the receptor ET(A), which is predominantly expressed in tumour cells. Furthermore, ET-1 produced by ovarian tumour cells stimulates VEGF production and VEGF-mediated angiogenic effects through ET(A) binding. These results demonstrate that activation of the ET(A) in ovarian carcinoma cells promotes cell proliferation, neovascularization and invasion, which are the principal hallmarks of malignant transformation. The present study was designed to investigate the effects of the ET(A)-selective antagonist ABT-627 on the ET-1-induced mitogenic effect in both primary cultures (PMOV1 and PMOV2) and cell lines (OVCA 433 and HEY) of ovarian carcinoma. All tumour cells express the components of the ET-1 system and secrete ET-1. ET(A) blockade by ABT-627 inhibits ET-1-induced mitogenic effects. The ET(B) antagonist BQ-788 is ineffective although all cell lines express both ET(A) and ET(B) mRNAs. In conclusion, our results demonstrate that ABT-627 is capable of inhibiting the proliferative activity of ET-1, suggesting that this potent ET(A) antagonist may provide a novel approach to the multidisciplinary treatment of ovarian carcinoma.

Topics: Atrasentan; Binding, Competitive; Cell Division; Endothelin Receptor Antagonists; Endothelin-1; Enzyme-Linked Immunosorbent Assay; Female; Humans; Ovarian Neoplasms; Pyrrolidines; Receptor, Endothelin A; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured

Endothelin-1 (ET-1) is a powerful mitogenic peptide produced by different tumors. In ovarian carcinoma cells, ET-1 acts as an autocrine growth factor, selectively through ET(A) receptor (ET(A)R), which is predominantly expressed in tumor cells. The aim of this study was to examine whether ET-1 plays a role in the sensitivity of three ovarian carcinoma cell lines (OVCA 433, HEY, and SK-OV-3) to apoptosis induced by two different stimuli. Our results demonstrated that the addition of ET-1 markedly inhibited serum withdrawal and paclitaxel-induced apoptosis in a concentration-dependent manner, as demonstrated by Annexin-V assay, sub-G(1) peak in DNA content histograms, internucleosomal DNA fragmentation, and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick-end labeling method. Pretreatment of the cells with an ET(A)R antagonist, BQ 123, reversed the ET-1-induced protective effect. Paclitaxel-induced apoptosis resulted in the phosphorylation of Bcl-2 that was suppressed by the addition of ET-1. Further analysis of the signaling pathway demonstrated that ET-1 stimulated Akt activation. The phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin blocked ET-1-induced Akt phosphorylation. Inhibition of ET-1-stimulated mitogen-activated protein kinase activity did not affect ET-1 protection from paclitaxel-mediated apoptosis. Moreover, BQ 123 blocked the Akt-mediated pathway activated by ET-1, sensitizing ovarian carcinoma cells to paclitaxel treatment. These results establish a novel role for ET-1 in determining protection of ovarian carcinoma cells against paclitaxel-induced apoptosis through Bcl-2-dependent and PI3-K-mediated Akt pathways and suggest that ET-1 and ET(A)R could represent important targets for anticancer therapy.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Culture Media, Serum-Free; Drug Interactions; Endothelin-1; Female; Humans; Ovarian Neoplasms; Paclitaxel; Phosphorylation; Protective Agents; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Receptor, Endothelin A; Receptors, Endothelin; Tumor Cells, Cultured

Angiogenesis is an essential prerequisite for tumor growth, invasion, and metastasis. In ovarian carcinoma cells, endothelin-1 (ET-1) stimulates the secretion of vascular endothelial growth factor (VEGF), a major mediator of tumor angiogenesis. In OVCA 433 and HEY ovarian carcinoma cell lines, ET-1 treatment increases VEGF mRNA expression and induces VEGF protein levels in a time- and dose-dependent fashion, and do so to a greater extent under hypoxic conditions. ET-1 also increases hypoxia-inducible factor-1alpha (HIF-1alpha) accumulation and activates the HIF-1 transcription complex under both normoxic and hypoxic conditions, suggesting a role for HIF-1 in the induction of VEGF expression. These effects are inhibited by the selective ET(A) receptor (ET(A)R) antagonist, BQ123. The ET-1-induced increase in HIF-1alpha protein levels is due to the enhanced HIF-1alpha stabilization. These results implicate HIF-1alpha in the induction of VEGF expression in ET-1-stimulated ovarian carcinoma cells, and provide a mechanism whereby ET-1 acting selectively through ET(A)R can interact with the HIF-1alpha-dependent machinery of angiogenesis. Our results suggest that new therapeutic strategies using specific ET(A)R antagonists could provide an additional approach to the treatment of ovarian carcinoma by inhibiting neovascularization as well as tumor cell growth.

Topics: Cell Hypoxia; Cell Nucleus; DNA Primers; Endothelial Growth Factors; Endothelin-1; Female; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Kinetics; Lymphokines; Ovarian Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Endothelin-1 (ET-1) is present at high concentrations in ovarian cancer ascites and is overexpressed in primary and metastatic ovarian carcinoma. In these cells, ET-1 acts as an autocrine mitogenic and angiogenic factor selectively through the ET(A) receptor (ET(A)R). We investigated at mRNA and protein levels whether ET-1 could affect the expression and activation of metastasis-related proteinases and whether this process was associated with ovarian tumor cell invasion. ELISA, gelatin zymography, Western blot, and reverse transcription-PCR analyses demonstrated that in two ovarian carcinoma cell lines (HEY and OVCA 433), the expression of matrix metalloproteinase (MMP) -2, -9, -3, -7, and -13 was up-regulated and activated by ET-1. Moreover we observed that ET-1 was able to enhance the secretion and activation of membrane-type metalloproteinase-1, a critical mediator of invasiveness. The secretion of tissue inhibitor of metalloproteinase-1 and -2 was decreased by ET-1, which increased the net MMP/tissue inhibitor of metalloproteinase balance and the gelatinolytic capacity. In addition, ET-1 induced overexpression of urokinase-type plasminogen activator, its receptor, and plasminogen activator inhibitor type-1 and -2. Finally, we demonstrated that, in HEY and OVCA 433 cells, ET-1 dose-dependently increased migration and MMP-dependent invasion through Matrigel. BQ123, an antagonist of the ET(A)R, inhibited the ET-1-induced tumor protease activity and subsequent increase in cell migration and invasion. These findings demonstrate that ET-1 promotes ovarian carcinoma cell invasion, acting through the ET(A)R by up-regulating secretion and activation of multiple tumor proteinases. Therefore, ET-1 may represent a key component of more aggressive ligand-induced invasiveness of ovarian carcinoma.

Topics: Cell Movement; Endothelin-1; Enzyme Activation; Female; Humans; Matrix Metalloproteinases; Neoplasm Invasiveness; Ovarian Neoplasms; Plasminogen Inactivators; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Endothelin (ET)-1 is produced in ovarian carcinoma cells and is known to act through ET(A) receptors as an autocrine growth factor in vitro and in vivo. In OVCA 433 human ovarian carcinoma cells, ET-1 caused phosphorylation of the epidermal growth factor receptor (EGF-R) that was accompanied by phosphorylation of Shc and its recruitment complexed with Grb2. These findings suggested that an EGF-R/ras-dependent pathway may contribute to the activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (Erk) 2 and mitogenic signaling induced by ET-1 in these cells. Specific inhibition of EGF-R kinase activity by tyrphostin AG1478 prevented ET-1-induced transactivation of the EGF-R, as well as Shc phosphorylation and recruitment with Grb2. Furthermore, ET-1-induced activation of Erk 2 was partially inhibited by tyrphostin AG1478. In accord with this finding, the mitogenic action of ET-1 in OVCA 433 cells was also significantly reduced by a concentration of tyrphostin AG1478 that abolished the growth response of EGF-stimulated cells. Inhibition of protein kinase C activity, which contributes to the proliferative action of ET-1 in OVCA 433 cells, had no effect on the activation of Erk 2 by ET-1, which suggests that this effect of protein kinase C does not involve ras-independent activation of Erk 2. Inhibition by wortmannin of PI3-kinase activity, which has been implicated in ET-1 and other G protein-coupled receptor (GPCR)-mediated signaling pathways, reduced Erk 2 activation by ET-1 but had no effect on ET-1-induced EGF-R and Shc phosphorylation. These findings indicate that ET-1-induced stimulation of Erk 2 phosphorylation, and mitogenic responses in OVCA 433 ovarian cancer cells are mediated in part by signaling pathways that are initiated by transactivation of the EGF-R.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Cell Division; Endothelin Receptor Antagonists; Endothelin-1; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Female; GRB2 Adaptor Protein; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Ovarian Neoplasms; Peptides, Cyclic; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase C; Proteins; Quinazolines; Receptor, Endothelin A; Shc Signaling Adaptor Proteins; Src Homology 2 Domain-Containing, Transforming Protein 1; Transcriptional Activation; Tumor Cells, Cultured; Tyrosine; Tyrphostins

Endothelin-1 (ET-1) is overexpressed in ovarian carcinomas and acts, via ET(A) receptors (ET(A)R), as an autocrine growth factor. In this study we investigate the role of ET-1 in the neovascularization of ovarian carcinoma. Archival specimens of primary (n = 40) and metastatic (n = 8) ovarian tumors were examined by immunohistochemistry for angiogenic factor and receptor expression and for microvessel density using antibodies against CD31, ET-1, vascular endothelial growth factor (VEGF), and their receptors. ET-1 expression correlated with neovascularization and with VEGF expression. The localization of functional ET(A)R and ET(A)R mRNA expression, as detected by autoradiography and in situ hybridization, was evident in tumors and in intratumoral vessels, whereas ET(B)R were expressed mainly in endothelial cells. High levels of ET-1 were detected in the majority of ascitic fluids of patients with ovarian carcinoma and significantly correlated with VEGF ascitic concentration. Furthermore ET-1, through ET(A)R, stimulated VEGF production in an ovarian carcinoma cell line, OVCA 433, by an extent comparable to hypoxia. Finally, conditioned media from OVCA 433 as well as ascitic fluids caused an increase in endothelial cell migration and the ET-1 receptor blockade significantly inhibited this angiogenic response. These findings indicate that ET-1 could modulate tumor angiogenesis, acting directly and in part through VEGF.

Topics: Adenocarcinoma; Adult; Aged; Ascitic Fluid; Blood Vessels; Carcinoma; Cell Movement; Endothelial Growth Factors; Endothelin-1; Endothelium, Vascular; Female; Humans; Lymphokines; Middle Aged; Neovascularization, Pathologic; Ovarian Neoplasms; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

In the present study, we have investigated the expression of endothelin 1 (ET-1) and the ET(A) receptor (ET(A)R) and ET(B) receptor (ET(B)R) in primary (n = 30) and metastatic (n = 8) ovarian carcinomas and their involvement in tumor growth. By reverse transcription-PCR and Northern blot analysis, we detected ET-1 mRNA in 90% of primary and 100% of metastatic ovarian carcinomas. ET-1 mRNA expression was significantly higher in tumors than in normal ovarian tissues (n = 12; P < 0.01). ET(A)R mRNA was also detected in 84% of the carcinomas examined, whereas ET(B)R mRNA was expressed in 50% of the tumors. The in vivo presence of mature ET-1 and ET(A)R was confirmed by immunohistochemistry, demonstrating a higher expression in primary and metastatic cells. Ten primary cultures of ovarian tumors secreted ET-1 and were positive for ET-1 and ET(A)R mRNA, whereas only 40% expressed ET(B)R mRNA. Radioligand binding studies showed that ET-1-producing cells also expressed functional ET(A)R, whereas no specific ET(B)R could be demonstrated. ET-1 stimulated dose-dependent [3H]thymidine incorporation and enhanced the mitogenic effect of epidermal growth factor. The ET(A)R-selective antagonist BQ 123 strongly inhibited ET-1-stimulated growth and substantially reduced the basal growth rate of unstimulated cells, whereas the ET(B)R-selective antagonist BQ 788 had no effect. In conclusion, the present data demonstrate a novel mechanism in the growth control of ovarian carcinoma in vivo mediated by the ET-1 autocrine loop that selectively occurs via the ET(A)R.

Topics: Adult; Aged; Blotting, Northern; Cell Division; Endothelin Receptor Antagonists; Endothelin-1; Female; Humans; Middle Aged; Ovarian Neoplasms; Peptides, Cyclic; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured

Previous studies have demonstrated that endothelin (ET) isoforms (ET-1, ET-2 and ET-3) can act in an autocrine manner in ovarian cancer while in breast cancer their role has been proposed to be that of a paracrine mitogen. To explore the possibility that endothelin isoforms might function not only as autocrine regulators but also as paracrine mitogens in ovarian cancers, we investigated their effects on the growth of ovarian fibroblasts derived from ovarian carcinomas, the interaction between ovarian carcinoma and fibroblast cells and the location of the isoform expression in primary ovarian tumours. ET-1, ET-2 and ET-3 stimulated the growth of three ovarian fibroblast cell lines at concentrations ranging from 10(-12) M to 10(-7) M. Inhibition of 125I-ET binding by the ETA receptor antagonist BQ123 and the ETB receptor antagonist BQ788 suggested the presence of both types of ET receptors in fibroblast cells. In the absence of ET-1, neither BQ 123 nor BQ 788 inhibited growth. However, both antagonists inhibited ET-1 stimulated growth suggesting the involvement of both receptor types in ET-1 growth regulation. In contrast to carcinoma cells which secrete measurable levels of ET-1, fibroblast cell lines did not secrete detectable protein. Co-culture experiments (using porous membrane insert wells) of fibroblasts with carcinoma cells demonstrated that growth of both populations of cells was increased compared with either grown in isolation. In this system, growth of the fibroblast cell line was partially inhibited by both BQ123 and BQ788, whilst growth of the PE014 carcinoma cell line was inhibited by only BQ123. RT-PCR measurements detected the presence of the ETA receptor subtype in 10/10 primary ovarian cancers but the presence of ETB receptor in only 6/10 cancers. Using specific antibodies, ET-1 was found in 11/15, ET-2 in 5 of 7 and ET-3 in 5/7 primary ovarian cancers predominantly in the epithelial cells but with some stromal expression. These data indicate that the ET isoforms may stimulate growth of the fibroblast population within an ovarian cancer in addition to stimulating the epithelial cells and since the ETs are expressed in the majority of ovarian cancers, this paracrine effect may contribute to the overall growth of the tumour.

Topics: Cell Division; Endothelin Receptor Antagonists; Endothelin-1; Endothelin-2; Endothelin-3; Endothelins; Female; Fibroblasts; Humans; Immunohistochemistry; Ovarian Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured

Endothelin 1 (ET-1) is produced in ovarian cancer cell lines and has been shown to act through ET(A) receptors as an autocrine growth factor to promote tumor cell proliferation in vitro. In OVCA 433 cells, the efficacy of ET-1 as a stimulus of [3H]thymidine incorporation was equivalent to that of epidermal growth factor. ET-1 also stimulated the rapid expression of c-fos, an action mediated by ET(A) receptors. The mitogenic action of ET-1 was not mediated by a pertussis toxin-sensitive G protein. An analysis of the effects of inhibition and depletion of protein kinase C (PKC) on mitogenic responses demonstrated that PKC was necessary but not sufficient for maximal stimulation by ET-1. In quiescent OVCA 433 cells, ET-1-induced stimulation of [3H]thymidine incorporation was prevented by two structurally distinct inhibitors of tyrosine kinase, herbimycin A and genistein. These results indicate that both PKC and protein tyrosine kinase participate in ET-1-stimulated mitogenic signaling. ET-1 rapidly stimulated tyrosine phosphorylation of several cellular proteins, among which p125FAK and p42 mitogen-activated protein kinase were identified. The additivity between the potent mitogenic actions of ET-1 and epidermal growth factor is consistent with the independence of their signal transduction pathways in ovarian cancer cells. These findings also indicate that intracellular signaling between the ET(A) receptor and a yet unidentified tyrosine kinase is involved in the mitogenic response to ET-1.

Topics: Benzoquinones; Blotting, Northern; Calcium-Calmodulin-Dependent Protein Kinases; Cell Adhesion Molecules; Cell Division; DNA, Neoplasm; Endothelin-1; Enzyme Inhibitors; Epidermal Growth Factor; Female; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation; Genes, fos; Genistein; Humans; Immunoblotting; Indoles; Isoflavones; Lactams, Macrocyclic; Maleimides; Ovarian Neoplasms; Pertussis Toxin; Phosphorylation; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Rifabutin; Signal Transduction; Tumor Cells, Cultured; Virulence Factors, Bordetella

To elucidate the potential role of endothelins (ETs) as growth regulators in ovarian carcinoma cells in culture, expression of endothelins and their receptors were measured in two ovarian cancer cell lines (PEO4 and PEO14), together with the effect of the exogenous addition of endothelins on the growth of these cell lines in vitro. RT-PCR analysis of mRNA prepared from PEO4 and PEO14 indicated the presence of ET-1 and ET-3 mRNA. Immunoreactive ET-1-like peptide was found in media from cultures of both PEO4 (1.7 +/- 0.4 fmol/10(6) cells/72 h) and PEO14 (20.2 +/- 6.8 fmol/10(6) cells/72 h) cell lines. Radioligand binding studies using 125I-ET-1 and membrane fractions were consistent with PEO4 cells having two receptor sites of either high affinity (Kd = 0.065 nM, Bmax = 0.047 pmol/mg protein) or lower affinity sites (Kd = 0.49 nM, Bmax = 0.23 pmol/mg protein). Studies using membrane fractions of PEO14 cells indicated that this cell line has only a single lower affinity binding site (Kd = 0.56 nM, Bmax = 0.31 pmol/mg protein). However, RT-PCR analysis indicated the presence of mRNA from both ETA and ETB receptors in PEO4 and PEO14 cell lines. Exogenous addition of ETs to PEO4 and PEO14 cells at concentrations of 10(-10)-10(-7)M resulted in specific dose-dependent increases in cell number for ET-1 (with maximum effects at 10(-10) and 10(-9)M for PEO4 and PEO14, respectively) and ET-2 (maximum effects at 10(-8) and 10(-9)M for PEO4 and PEO14, respectively) but not for ET-3. Experiments on the growth of PEO14 cells using BQ123 (ETA-R) antagonist and "antisense" oligonucleotide against the ETA-R, in the absence of exogenous ETs, suggested that immunoreactive ET-1-like material secreted by PEO14 cells can affect their growth in an autocrine manner. These results would be consistent with ET-1 acting as a possible autocrine growth regulator in human ovarian carcinoma cells.

Topics: Cell Division; Endothelin Receptor Antagonists; Endothelin-1; Endothelin-2; Endothelin-3; Endothelins; Female; Humans; Oligopeptides; Ovarian Neoplasms; Peptides, Cyclic; Piperidines; Polymerase Chain Reaction; Protein Binding; Receptors, Endothelin; RNA, Messenger; Tumor Cells, Cultured

The production of endothelin 1 (ET-1) and its receptor-mediated actions on calcium signaling and growth responses were analyzed in human ovarian carcinoma cells. Immuno-reactive ET-1 was released from three of four ovarian tumor cell lines as a function of time in amounts ranging from 56 to 74 fmol/10(6) cells. Reverse-phase HPLC and radioimmuno-assay of conditioned media from tumor cells revealed a single peak coeluting with authentic ET-1. Radioligand binding studies showed that the ET-1-producing cell lines also expressed high-affinity ETA receptors (Kd < 0.1 nM) that ranged in abundance from 2,600 to 43,600 sites/cell. In fura-2-loaded ovarian carcinoma cells, ET-1 induced dose-dependent increases in cytoplasmic Ca2+ concentration. ET-1 also stimulated thymidine incorporation in the three cell lines that expressed ET receptors. In OVCA 433 cells, BQ 123 inhibited the stimulatory actions of ET-1 on thymidine incorporation and cell proliferation, and substantially reduced the basal growth rate of unstimulated ovarian tumor cells. These results demonstrate that ET-1 is produced in ovarian cancer cells and acts as an autocrine growth factor on ETA receptors to stimulate calcium signaling and proliferative responses. Such findings suggest that ET-1 participates in the progression of neoplastic growth in certain ovarian tumors.

Topics: Calcium; Cell Division; Endothelin-1; Female; Humans; Neoplasm Proteins; Ovarian Neoplasms; Receptor, Endothelin A; Receptors, Endothelin; Thymidine; Tumor Cells, Cultured