Signaling pathways involved in polyamine analog-induced apoptosis in melanoma cell lines.

摘要

N1, N 11-diethylnorspermine (DENSPM) is a polyamine analog having clinical relevance as an experimental anticancer agent. We previously reported that DENSPM induced a G1 arrest in MALME-3M human melanoma cells containing wild type p53 and rapid apoptosis in SK-MEL-28 cells containing mutant p53. While the G1 arrest was found to be mediated by p53/p21Waf1/Cip1/Rb checkpoint, the mechanism involved in DENSPM-induced apoptosis was not determined. The goal of this study was to characterize effectors and signaling pathways that contribute to polyamine analog-induced apoptosis and to examine the genetic determinants for the differential cellular outcomes seen in SK-MEL-28 cells versus MALME-3M cells. Our findings indicate that DENSPM-induced apoptosis was initiated by massive increases in SSAT activity and related oxidative events followed by activation of mitochondrial apoptotic signaling as indicated by cytochrome c release and caspase activation in SK-MEL-28 cells. We also demonstrate that DENSPM induction of EGR-1 and activation of MAPK signaling were linked to induction of SSAT and related oxidative events but neither contributed to the rapid apoptosis seen in SK-MEL-28 cells. However, DENSPM induction of ERK1/2 and p38 did contribute to delayed apoptosis seen in MALME-3M cells. Since p53 status may contribute to the differential cellular response to DENSPM, we examined the possible role of the downstream gene p21 Waf1/Cip1 in delaying apoptosis in SK-MEL-28 cells. Whether pharmacologically induced or overexpressed by transfection, p21Waf1/Cip1 delayed DENSPM-induced apoptosis as indicated by caspase-3 activation and annexin V staining.; Although DENSPM accumulates to similar intracellular levels and initiates similar polyamine effects in SK-MEL-28 and MALME-3M human melanoma cells, the downstream cellular responses appear to be highly dependent on genetic context. The more fulminant apoptotic response seen in SK-MEL-28 cells appears to be due to unrestrained activation of the mitochondria-mediated caspase cascade while in MALME-3M cells delaying factors involving p53-dependent induction of p21Waf1/Cip1 and activation of the MAPK pathway serve to modulate this response. It is anticipated that further definition of such genetic determinants of outcome will have therapeutic and prognostic implications for the clinical use of DENSPM.