Endothelin-induced Apoptosis of A375 Human Melanoma Cells

Abstract

Endothelin-1 (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-1-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-1-induced increase and nuclear accumulation of the tumor suppressor protein p53. All of these effects of ET-1 were mediated by ET B via a pertussis toxin-sensitive G protein. Flow cytometric analysis with fluorescent dye-labeled ET-1 revealed up-regulation of ET B 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 ET B -mediated signaling pathways to apoptotic cell machinery and cytoskeletal organization. Furthermore, the densities of ET B 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.

Specific inhibition of the endothelin A receptor with ZD4054: clinical and pre-clinical evidence

“…In most cells, activation of ET A promotes cell growth , whereas activation of ET B induces cell death via apoptosis (Okazawa et al, 1998). In addition, binding of ET-1 to ET B results in clearance of ET-1 from the circulation.…”

Silencing of HTR1B and reduced expression of EDN1 in human lung cancers, revealed by methylation-sensitive representational difference analysis

“…A recent study showed that endothelin-1 induces apoptosis of human melanoma cells and inhibits their growth (Okazawa et al, 1998). This signal is mediated by endothelin (Okazawa et al, 1998). Hypermethylation of the 5' CGI of the ETRB gene and its decreased expression have been reported in human prostate cancers (Nelson et al, 1996(Nelson et al, , 1997.…”

“…Endothelin-1 was originally identi®ed as a potent vasoconstrictive peptide (Yanagisawa et al, 1988), and subsequent studies revealed its diverse biological roles (Masaki et al, 1992). A recent study showed that endothelin-1 induces apoptosis of human melanoma cells and inhibits their growth (Okazawa et al, 1998). This signal is mediated by endothelin (Okazawa et al, 1998).…”

Endothelin-1 in smooth muscle cells and mast cells of mouse uterus after parturition

“…As the ET-1 mRNA levels gradually decreased over 14 days postpartum, we also observed a gradual decrease in the thickness of the uterine wall, which was enlarged and thickened for pregnancy, particularly the myometrium (data not shown). It is well known that ET-1 has not only mitogenic activity but also an ability to induce apoptosis via the ETB receptor (Okazawa et al 1998). In vascular smooth muscle, it has been proposed that ET-1 caused apoptosis via the ETB receptor induced by stretch stimuli (Cattaruzza et al 2000).…”