Small Interfering RNA Molecules Targeting Endothelin-Converting Enzyme-1 Inhibit Endothelin-1 Synthesis and the Invasive Phenotype of Ovarian Carcinoma Cells

Abstract

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 Ecadherin, an epithelial adhesion molecule, and reduced Ncadherin 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. [Cancer Res 2008;68(22):9265-73]

HIF1A-dependent increase in endothelin 2 levels in granulosa cells: role of hypoxia, LH/cAMP, and reactive oxygen species

“…ThGCs were trypsinized and plated immediately on six-well plates and cultured for 24 h. Then cells were transfected using Lipofectamine 2000 reagent in 1% FCS, as described previously (Rayhman et al 2008, Klipper et al 2010. Cells were transfected with 50 nM siRNA constructs targeting HIF1A or with scrambled siRNA (the negative control).…”

Osteopontin-c Splicing Isoform Contributes to Ovarian Cancer Progression

“…We chose the approach to ectopically overexpress each of these splicing isoforms in OvCar-3 cells, once these cells are a well-established model for ovarian cancer functional studies (23,24).…”

Expression of endothelin receptor subtypes in bronchial tumors

“…However, to date, there is no clinical assay on the effects of endothelin receptor antagonists on lung cancer. Endothelin receptor antagonists have shown promising preclinical results in prostate and ovarian cancer treatment (31,40,41). In prostate cancer, several phase II and phase III clinical trials with atrasentan (ABT-627) or ZD4054, two highly ET A R selective antagonists, did not demonstrate beneficial effects on time to progression (40,(42)(43)(44).…”

“…In addition, elevated ET-1 concentrations in the saliva of oral cancer patients or breath condensate of lung cancer patients has been proposed as indicators to monitor patients at risk for oral or lung carcinoma (26,27). Several studies have provided evidence that ET-1 is involved in proliferation and migration of cancer cells such as prostate, melanoma, colorectal and ovarian cancer cells (18,(28)(29)(30)(31). In non-small cell lung carcinomas (NSCLC) and in breast carcinomas, a positive correlation has been found between ET-1 and the expression of vascular endothelial growth factor (VEGF) mRNA, suggesting that ET-1 might be involved in angiogenesis in these carcinomas (25,32).…”