Ceramide generation occurring 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 to induce cell death of structurally related oxysterols, such as those oxidized at C7(7a-, 7b-hydroxycholesterol, and 7-ketocholesterol), was investigated on U937 cells at different times of treatment in a concentration range of 5 ± 80 mg/ml. Whereas all oxysterols accumulate inside the cells, strong inhibition of cell growth and increased permeability to propidium iodide were observed only with 7b-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 7b-hydroxycholesterol-and 7-ketocholesterol-induced apoptosis by Z-VAD-fmk (100 mM), a broad spectrum caspase inhibitor, did not reduce C16 : 0 and C24 : 1 ceramide species accumulation. Conversely, when U937 cells were treated with 7b-hydroxycholesterol and 7-ketocholesterol in the presence of fumonisin B1 (100 mM), a specific inhibitor of ceramide synthase, C16 : 0 and C24 : 1 ceramide species production was completely abrogated whereas apoptosis was not prevented. Noteworthy, 7a-hydroxycholesterol induced only a slight inhibition of cell growth. Collectively, these results are consistent with the notion that the a or b 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 7b-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 7b-hydroxycholesterol-and 7-ketocholesterol-induced cell death. Cell Death and Differentiation (2001) 8, 83 ± 99.

Different apoptotic mechanisms are involved in the antiproliferative effects of 7β-hydroxysitosterol and 7β-hydroxycholesterol in human colon cancer cells

“…However, DNA fragmentation was similarly enhanced in cells treated with both hydroxysterols. By using the broad-spectrum caspase inhibitor Z-VAD.fmk, 31,46,47 we demonstrated that caspases activation plays an important role in the antiproliferative effect initiated by 7b-OHsito. This was not the case for cells treated with 7b-OHchol, which were not affected by the caspase inhibitor.…”

“…23 In the presence of the pancaspase inhibitor, Z-VAD.fmk, apoptotic events were not completely suppressed. 31 Hence, 7b-OHsito may trigger apoptosis through postmitochondrial caspase cascades and DNA fragmentation. 48,49 In contrast, 7b-OHchol showed no effect on cell cycle and induced cell death mechanisms leading to DNA fragmentation that were not delayed by the inhibitor Z-VAD.fmk.…”

“…24 Furthermore, numerous studies reported atherogenic 25,26 and cytotoxic properties [27][28][29][30] of cholesterol oxides, leading to apoptosis or necrosis in various cell types. 24,[31][32][33] They may also exert their effects by lowering cholesterol availability for cell membrane formation by inhibiting 3-hydroxy-methylglutaryl coenzyme A reductase (HMG-CoA reductase), a key enzyme in cholesterol synthesis. 34 Cholesterol oxides bind to specific membrane receptors or cytosolic binding proteins, leading to the perturbation of cholesterol synthesis.…”

Fenretinide stimulates redox-sensitive ceramide production in breast cancer cells: potential role in drug-induced cytotoxicity

“…However, in other instances where induction of apoptosis is accompanied by an elevation of ceramide, FB 1 failed to prevent the apoptotic response while blocking ceramide production (Nagy et al, 2000;Miguet et al, 2001).…”

Effects of caspase inhibitors (z‐VAD‐fmk, z‐VDVAD‐fmk) on Nile Red fluorescence pattern in 7‐ketocholesterol‐treated cells: Investigation by flow cytometry and spectral imaging microscopy

“…The lipid contents of cholesterol (esterified and unesterified), phosphatidylcholine, and sphingomyelin were determined. To this end, lipids were extracted and analyzed by capillary gas chromatography and mass spectrometry (CGC/MS) as previously described (33).…”