Endothelin receptor B antagonists decrease glioma cell viability independently of their cognate receptor

BMC Cancer. 2008 Nov 28;8:354. doi: 10.1186/1471-2407-8-354.

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.

Conclusion: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis / drug effects
  • Cell Cycle / drug effects
  • Cell Line, Tumor
  • Cell Proliferation / drug effects
  • Cell Survival / drug effects
  • Ciprofloxacin / analogs & derivatives
  • Ciprofloxacin / pharmacology
  • DNA Damage / drug effects
  • DNA Damage / genetics
  • Endothelin B Receptor Antagonists*
  • Gene Expression Profiling
  • Gene Expression Regulation, Neoplastic
  • Glioma / genetics
  • Glioma / metabolism*
  • Glioma / pathology
  • Humans
  • Isoquinolines
  • Melanoma / genetics
  • Melanoma / metabolism*
  • Melanoma / pathology
  • Oligonucleotide Array Sequence Analysis
  • Pyrroles
  • Pyrrolidines / pharmacology*
  • RNA, Small Interfering
  • Receptor, Endothelin A / metabolism
  • Receptor, Endothelin B / genetics
  • Receptor, Endothelin B / metabolism

Substances

  • A 192621
  • Endothelin B Receptor Antagonists
  • Isoquinolines
  • Pyrroles
  • Pyrrolidines
  • RNA, Small Interfering
  • Receptor, Endothelin A
  • Receptor, Endothelin B
  • WM5 compound
  • Ciprofloxacin