Endothelin-induced apoptosis of A375 human melanoma cells

Endothelin-l (ET-1) inhibited serum-dependent growth of asynchronized A375 human melanoma cells, and the growth inhibitory effect was markedly enhanced when ET-1 was applied to the cells synchronized at G(1)/S boundary by double thymidine blocks. Flow cytometric analysis revealed that ET-1 did not inhibit the cell cycle progression after the release of the block but caused a significant increase of the hypodiploid cell population that is characteristic of apoptotic cell death. ET-l-induced apoptosis was confirmed by the appearance of chromatin condensation on nuclear staining and DNA fragmentation on gel electrophoresis. The increase in the hypodiploid cell peak was manifest within 16 h of exposure to 5 nM ET-1. Within the same time range, ET-1 caused actin reorganization and drastic morphological changes of the surviving cells from epithelioid to an elongated bipolar shape. These phenotypical changes were preceded by ET-l-induced increase and nuclear accumulation of the tumor suppressor protein p53. All of these effects of ET-1 were mediated by ETB via a pertussis toxin-sensitive G protein. Flow cytometric analysis with fluorescent dye-labeled ET-1 revealed up-regulation of ETB expressed by the cells in G(1)/early S phases, and overexpression of the receptor protein by cDNA microinjection conferred the responsiveness (both apoptosis and morphological changes) to ET-1 irrespective of the position of the cell in the cell cycle. These results indicated the presence of ETB-mediated signaling pathways to apoptotic cell machinery and cytoskeletal organization. Furthermore, the densities of ET, expressed by individual A375 melanoma cells appeared to be regulated by a cell cycle-dependent mechanism, and the receptor density can be a limiting factor to control the apoptotic and cytoskeletal responses of the cells to ET-1. Although the molecular mechanisms remain to be elucidated, these findings added a new dimension to the diverse biological activities of ETs and also indicated a novel mechanism to control the responsiveness of the cell to the peptides.