The endothelial cell-derived endothelin-1 (ET-1) is a potent mitogen for endothelial cells, vascular smooth muscle cells, and tumor cells. In this study, we analyzed the role of ET-1 on human umbilical vein endothelial cell (HUVEC) phenotype related to different stages of angiogenesis. ET-1 promoted HUVEC proliferation, migration, and invasion in a dose-dependent manner. The ET(B) receptor (ET(B)R) antagonist, BQ 788, blocked the angiogenic effects induced by ET-1, whereas the ET(A)R antagonist was less effective. ET-1 stimulated matrix metalloproteinase-2 mRNA expression and metalloproteinase-2 production, as determined by reverse transcriptase-polymerase chain reaction and gelatin zymography. Furthermore ET-1 was able to enhance HUVEC differentiation into cord vascular-like structures on Matrigel. When tested in combination with vascular endothelial growth factor (VEGF), ET-1 enhanced VEGF-induced angiogenic-related effects on endothelial cells in vitro. Finally, using the Matrigel plug neovascularization assay in vivo, ET-1 in combination with VEGF stimulated an angiogenic response comparable to that elicited by basic fibroblast growth factor. These findings demonstrated that ET-1 induces angiogenic responses in cultured endothelial cells through ET(B)R and that stimulates neovascularization in vivo in concert with VEGF. ET-1 and its receptors acting as angiogenic regulators might represent new targets for anti-angiogenic therapy.
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.
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 paclitaxelmediated 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.
Human papillomaviruses (HPV) are associated with cervical cancer and interact with growth factors that may enhance malignant transformation of cervical carcinoma cells. Endothelin-1 (ET-1) is released from HPV transfected keratinocytes and induces increased growth response in these cell lines in comparison with normal cells. In the present study several cervical carcinoma cell lines have been analyzed to investigate the expression of ET-1 and its receptors as well as their involvement in tumor growth. All HPV-positive cancer cells secreted ET-1 and expressed mRNA for ET-1 and its receptors, whereas a HPV-negative carcinoma cell line expressed only the ETBR mRNA and didn't secrete ET-1. Binding studies showed that HPV-associated cells expressed an increased number of functional ETAR. ET-1 stimulated a marked dose-dependent increase in [3H]-thymidine incorporation with respect to the normal cells whereas ET-3 and ETBR agonists had no effect. In HPV-positive cancer cells, a specific antagonist of ETAR inhibited the proliferation induced by ET-1 and substantially reduced the basal growth rate of unstimulated cervical tumor cells, whereas the ETBR antagonist had no effect. These results demonstrate that ET-1 participates in the progression of neoplastic growth in HPV-associated carcinoma, in which ETAR are increased and could be targeted for antitumor therapy.
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.
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