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

Cell Death Differ. 2001 Jan;8(1):83-99. doi: 10.1038/sj.cdd.4400792.


Biological activities of oxysterols seem tightly regulated. Therefore, the ability to induce cell death of structurally related oxysterols, such as those oxidized at C7(7alpha-, 7beta-hydroxycholesterol, and 7-ketocholesterol), was investigated on U937 cells at different times of treatment in a concentration range of 5-80 microg/ml. Whereas all oxysterols accumulate inside the cells, strong inhibition of cell growth and increased permeability to propidium iodide were observed only with 7beta-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 7beta-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis by Z-VAD-fmk (100 microM), a broad spectrum caspase inhibitor, did not reduce C16 : 0 and C24 : 1 ceramide species accumulation. Conversely, when U937 cells were treated with 7beta-hydroxycholesterol and 7-ketocholesterol in the presence of fumonisin B1 (100 microM), a specific inhibitor of ceramide synthase, C16 : 0 and C24 : 1 ceramide species production was completely abrogated whereas apoptosis was not prevented. Noteworthy, 7alpha-hydroxycholesterol induced only a slight inhibition of cell growth. Collectively, these results 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 7beta-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 7beta-hydroxycholesterol- and 7-ketocholesterol-induced cell death.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Chloromethyl Ketones / pharmacology
  • Apoptosis* / drug effects
  • Carboxylic Acids / pharmacology
  • Caspase 3
  • Caspase 9
  • Caspase Inhibitors
  • Caspases / metabolism*
  • Cell Death / drug effects
  • Cell Division / drug effects
  • Cell Membrane Permeability / drug effects
  • Ceramides / biosynthesis*
  • Cytochrome c Group / metabolism
  • Cytosol / metabolism
  • Dose-Response Relationship, Drug
  • Enzyme Inhibitors / pharmacology
  • Fumonisins*
  • Humans
  • Hydroxycholesterols / pharmacokinetics
  • Hydroxycholesterols / pharmacology*
  • Ketocholesterols / pharmacokinetics
  • Ketocholesterols / pharmacology*
  • Membrane Potentials / drug effects
  • Mitochondria / drug effects
  • Mitochondria / metabolism
  • Poly(ADP-ribose) Polymerases / metabolism
  • Propidium / pharmacokinetics
  • U937 Cells / cytology
  • U937 Cells / drug effects*
  • U937 Cells / metabolism


  • Amino Acid Chloromethyl Ketones
  • Carboxylic Acids
  • Caspase Inhibitors
  • Ceramides
  • Cytochrome c Group
  • Enzyme Inhibitors
  • Fumonisins
  • Hydroxycholesterols
  • Ketocholesterols
  • benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone
  • Propidium
  • fumonisin B1
  • cholest-5-en-3 beta,7 alpha-diol
  • Poly(ADP-ribose) Polymerases
  • CASP3 protein, human
  • CASP9 protein, human
  • Caspase 3
  • Caspase 9
  • Caspases
  • 7-ketocholesterol