title: Endothelin receptor B antagonists decrease glioma cell viability independently of their cognate receptor.
abstract: BACKGROUND
  Endothelin receptor antagonists inhibit the progression of many cancers, but research into their influence on glioma has been limited.
  
  
  METHODS
  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).
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: California Institute of Technology, 1200 E, California Blvd, MC 216-76, Pasadena, CA 91125, USA. jpmontgom@gmail.com
authors.forename: Jennifer P
authors.initials: JP
authors.lastname: Montgomery
authors.forename: Paul H
authors.initials: PH
authors.lastname: Patterson
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002939:Ciprofloxacin / Q000031:analogs & derivatives / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics; D065131:Endothelin B Receptor Antagonists; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D005910:Glioma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007546:Isoquinolines; D008545:Melanoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis; D011758:Pyrroles; D011759:Pyrrolidines / Q000494:pharmacology*; D034741:RNA, Small Interfering; D044022:Receptor, Endothelin A / Q000378:metabolism; D044023:Receptor, Endothelin B / Q000235:genetics / Q000378:metabolism
chemical_list: C120508:A 192621; D065131:Endothelin B Receptor Antagonists; D007546:Isoquinolines; D011758:Pyrroles; D011759:Pyrrolidines; D034741:RNA, Small Interfering; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; C486449:WM5 compound; D002939:Ciprofloxacin