Ceramide generation occurring during 7 beta-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis is caspase independent and is not required totrigger cell death

Biological activities of oxysterols seem tightly regulated, Therefore, the ability to induce cell death of structurally related oxysterols, such as th ose oxidized at C7(7 alpha-, 7 beta -hydroxycholesterol, and 7-ketocholeste rol), was investigated on U937 cells at different ti mes of treatment in a concentration range of 5-80 mug/ml. Whereas ail oxysterols accumulate insid e the cells, strong inhibition of cell growth and increased permeability to propidium iodide were observed only with 7 beta -hydroxycholesterol and 7- ketocholesterol, which trigger an apoptotic process characterized by the oc currence of cells with fragmented and/or condensed nuclei, and by Various c ellular dysfunctions: loss of mitochondrial transmembrane potential, cytoso lic 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 inhi bition of 7 beta -hydroxycholesterol- and 7-ketocholesterol induced apoptos is by Z-VAD-fmk (100 muM), a broad spectrum caspase inhibitor, did not redu ce C16:0 and C24:1 ceramide species accumulation. Conversely, when U937 cel ls were treated with 7 beta -hydroxycholesterol and 7-ketochotesterol in th e presence of fumonisin B1 (100 muM), a specific inhibitor of ceramide synt hase, C16:0 and C24:1 ceramide species production was completely abrogated whereas apoptosis was not prevented, Noteworthy, 7 alpha -hydroxycholestero l induced only a slight inhibition of cell growth. Collectively, these resu lts are consistent with the notion that the alpha or beta 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 7 beta -h ydroxycholesterol- and 7-ketochotesterol-induced apoptosis involve the mito chondrial 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 7 beta -hydroxycholesterol- and 7-ketocholesterol-induced cell death.