a-192621 and Glioma

The proliferative and antiapoptotic actions of endothelin (ET)-1 in cancer cells have been documented and ET receptor antagonists have been exploited as potential anticancer drugs. Glioblastoma cell lines express both ETA and ETB receptors and previous works have shown that ETB receptors are involved in the proliferation of different cancer cell types. In this study we have investigated the effects of two structurally unrelated ETB receptor antagonists, BQ788 and A192621, on cell survival, proliferation and apoptosis in 1321-N1, U87 and IPDDCA2 glioma cell lines. BQ788 and A192621 reduced glioma cells viability and proliferation assessed by BrdU incorporation and cell cycle analysis by flow cytometry, while in contrast the ETA receptor antagonist BQ123 had no effect on cell survival. TUNEL assay and immunocytochemical experiments showed that BQ788 and A192621 trigger apoptotic processes mainly via activation of the intrinsic mitochondrial pathway involving caspase-9 activation, AIF release and cytochrome c translocation. Furthermore, treatment with ETB antagonists downregulates ERK- and p38MAPK-dependent pathways but does not affect VEGF mRNA levels. Our findings support the hypothesis that ETB antagonists represent a new promising therapeutic strategy for the treatment of high grade gliomas.

Topics: Apoptosis; Apoptosis Inducing Factor; Caspase 9; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Down-Regulation; Drug Screening Assays, Antitumor; Endothelin B Receptor Antagonists; Glioma; Humans; Oligopeptides; Piperidines; Pyrrolidines; Signal Transduction; Vascular Endothelial Growth Factor A

Endothelin receptor antagonists inhibit the progression of many cancers, but research into their influence on glioma has been limited.. We 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).. We 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.. While 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.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Ciprofloxacin; DNA Damage; Endothelin B Receptor Antagonists; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glioma; Humans; Isoquinolines; Melanoma; Oligonucleotide Array Sequence Analysis; Pyrroles; Pyrrolidines; Receptor, Endothelin A; Receptor, Endothelin B; RNA, Small Interfering