Involvement of mitochondria and caspase-3 in ET-18-OCH(3)-induced apoptosis of human leukemic cells.
The induction of cell death in leukemic HL-60 cells by the ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH(3); edelfosine) followed the typical apoptotic changes in ultrastructural morphology, including blebbing, chromatin condensation, nuclear membrane breakdown and extensive vacuolation. Using a cytofluorimetric approach, we found that ET-18-OCH(3) induced disruption of the mitochondrial transmembrane potential (DeltaPsi(m)) followed by production of reactive oxygen species (ROS) and DNA fragmentation in leukemic cells. ET-18-OCH(3) also induced caspase-3 activation in human leukemic cells, as assessed by cleavage of caspase-3 into the p17 active form and cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP). ET-18-OCH(3) analogues unable to induce apoptosis failed to disrupt DeltaPsi(m) and to activate caspase-3. ET-18-OCH(3)-resistant Jurkat cells generated from sensitive Jurkat cells showed no caspase-3 activation and did not undergo DeltaPsi(m) disruption upon ET-18-OCH(3) incubation. Cyclosporin A partially inhibited DeltaPsi(m) dissipation, caspase activation and apoptosis in ET-18-OCH(3)-treated leukemic cells. Overexpression of bcl-2 by gene transfer prevented DeltaPsi(m) collapse, ROS generation, caspase activation and apoptosis in ET-18-OCH(3)-treated leukemic T cells. Pretreatment with the caspase inhibitor Z-Asp-2, 6-dichlorobenzoyloxymethylketone prevented ET-18-OCH(3)-induced PARP proteolysis and DNA fragmentation, but not DeltaPsi(m) dissipation. ET-18-OCH(3) did not affect the expression of caspases and bcl-2-related genes. ET-18-OCH(3)-induced apoptosis did not require protein synthesis. Our data indicate that DeltaPsi(m) dissipation and caspase-3 activation are critical events of the apoptotic cascade triggered by the antitumor ether lipid ET-18-OCH(3), and that the sequence of events in the apoptotic action of ET-18-OCH(3) on human leukemic cells is: DeltaPsi(m) disruption, caspase-3 activation and internucleosomal DNA degradation.
Gajate
C
C
Centro de Investigación del Cáncer, Instituto de Biología Molecular y Celular del Cáncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, Spain.
Santos-Beneit
A M
AM
Macho
A
A
Lazaro
M d
Md
Hernandez-De Rojas
A
A
Modolell
M
M
Muñoz
E
E
Mollinedo
F
F
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D053938:DNA Fragmentation; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007938:Leukemia / Q000201:enzymology / Q000473:pathology* / Q000503:physiopathology; D015470:Leukemia, Myeloid, Acute / Q000473:pathology; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000473:pathology; D008564:Membrane Potentials / Q000187:drug effects; D008854:Microscopy, Electron; D008928:Mitochondria / Q000502:physiology*; D010742:Phospholipid Ethers / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D010742:Phospholipid Ethers; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; C026659:edelfosine; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases