Ceramide generation occuring during 7β-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis is caspase independent and is not required to trigger cell death

Abstract

Biological activities of oxysterols seem tightly regulated. Therefore, the ability of induce cell death of structurally related oxysterols, such as those oxidized at C7(7α-, 7β-hydroxycholesterol, and 7-ketocholesterol), was investigated on U937 cells at different times of treatment in a concentration range of 5-80 μg/ml. Whereas all oxysterols accumulate inside the cells, strong inhibition of cell growth and increased permeability to propidium iodide were observed only with 7 β-hydroxycholesterol and 7-ketocholesterol, which trigger an apoptotic process characterized by the occurrence of cells with fragmented and/or condensed nuclei, and by various cellular dysfunctions: loss of mitochondrial transmembrane potential, cytosolic release of cytochrome c, activation of caspase-9 and -3 with subsequent enhanced activity of caspase-3, degradation of poly(ADP-ribose) polymerase, and increased accumulation of cellular C16:0 and C24:1 ceramide species. This ceramide generation is not attributed to caspase activation since inhibition of 7 β-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis by Z-VAD-fmk (100 μM), a broad spectrum caspase inhibitor, did not reduce C16:0 and C24:1 ceramide species accumulation. Conversely, when U937 cells were treated with 7β-hydroxycholesterol and 7-ketocholesterol in the presence of fumonisin B1 (100 μM), a specific inhibitor of ceramide synthase, C16:0 and C24:1 ceramide species production was completely abrogated whereas apoptosis was not prevented. Noteworthy, 7α-hydroxycholesterol induced only a slight inhibition of cell growth. Collectively, these results are consistent with the notion that the α or β hydroxyl radical position of oxysterols oxidized at C7 plays a key role in the induction of the apoptotic process. In addition, our findings demonstrate that 7β-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis involve the mitochondrial signal transduction pathway and they suggest that C16:0 and C24:1 ceramide species generated through ceramide synthase play a minor role in the commitment of 7β-hydroxycholesterol- and 7-ketocholesterol-induced cell death.