Abstract
BackgroundEndothelin receptor antagonists inhibit the progression of many cancers, but research into their influence on glioma has been limited.MethodsWe treated glioma cell lines, LN-229 and SW1088, and melanoma cell lines, A375 and WM35, with two endothelin receptor type B (ETRB)-specific antagonists, A-192621 and BQ788, and quantified viable cells by the capacity of their intracellular esterases to convert non-fluorescent calcein AM into green-fluorescent calcein. We assessed cell proliferation by labeling cells with carboxyfluorescein diacetate succinimidyl ester and quantifying the fluorescence by FACS analysis. We also examined the cell cycle status using BrdU/propidium iodide double staining and FACS analysis. We evaluated changes in gene expression by microarray analysis following treatment with A-192621 in glioma cells. We examined the role of ETRB by reducing its expression level using small interfering RNA (siRNA).ResultsWe report that two ETRB-specific antagonists, A-192621 and BQ788, reduce the number of viable cells in two glioma cell lines in a dose- and time-dependent manner. We describe similar results for two melanoma cell lines. The more potent of the two antagonists, A-192621, decreases the mean number of cell divisions at least in part by inducing a G2/M arrest and apoptosis. Microarray analysis of the effects of A-192621 treatment reveals up-regulation of several DNA damage-inducible genes. These results were confirmed by real-time RT-PCR. Importantly, reducing expression of ETRB with siRNAs does not abrogate the effects of either A-192621 or BQ788 in glioma or melanoma cells. Furthermore, BQ123, an endothelin receptor type A (ETRA)-specific antagonist, has no effect on cell viability in any of these cell lines, indicating that the ETRB-independent effects on cell viability exhibited by A-192621 and BQ788 are not a result of ETRA inhibition.ConclusionWhile ETRB antagonists reduce the viability of glioma cells in vitro, it appears unlikely that this effect is mediated by ETRB inhibition or cross-reaction with ETRA. Instead, we present evidence that A-192621 affects glioma and melanoma viability by activating stress/DNA damage response pathways, which leads to cell cycle arrest and apoptosis. This is the first evidence linking ETRB antagonist treatment to enhanced expression of DNA damage-inducible genes.
Highlights
Endothelin receptor antagonists inhibit the progression of many cancers, but research into their influence on glioma has been limited
We tested the ability of endothelin receptor type B (ETRB)-specific antagonist BQ788 to reduce the viability of the glioma cells
death receptor 5 (DR5) is induced by DNA damaging compounds in malignant gliomas, including LN-229 [40]. This evidence suggests that A-192621 affects glioma viability by activating stress/DNA damage response pathways, which leads to cell cycle arrest and apoptosis
Summary
Endothelin receptor antagonists inhibit the progression of many cancers, but research into their influence on glioma has been limited. Components of the ET system have been found in many glioma tumor specimens and cell lines, and ET expression positively correlates with the degree of malignancy [1417]. Two studies demonstrated ETRA expression in the neovasculature of glioblastoma tumors, while ETRB was localized to the tumor cells [18,19]. ET-1 induces proliferation in glioblastoma through various pathways including the mitogen-activated protein kinase (MAPK) pathway, and BQ788, an ETRB-specific receptor antagonist, blocks the phosphorylation of extracellular signal-related kinase, a key step in MAPK signaling [21]. This led us to consider whether potential therapeutic candidates, the ETRB antagonists, negatively impact glioma growth
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