Induction of apoptosis by arachidonic acid in human retinoblastoma Y79 cells: involvement of oxidative stress.
Arachidonic acid administration caused apoptosis in Y79 cells, as shown by typical morphological changes, phosphatidylserine externalization, chromatin condensation, processing and activation of caspase-3 and cleavage of the endogenous caspase substrate poly-(ADP-ribose)-polymerase. Arachidonic acid also caused lamin B cleavage, suggesting caspase-6 activation. Arachidonic acid treatment was accompanied by increased formation of the lipid peroxidation end products malondialdehyde and 4-hydroxy-2-nonenal, lowering in reduced glutathione content and in mitochondrial membrane potential. Inhibiting glutathione synthesis sensitized Y79 cells to apoptosis-inducing stimuli, whilst replenishing reduced glutathione attenuated arachidonic acid toxicity. Similar findings were obtained using hydroperoxyeicosatetranoic acids (oxygenated metabolites of arachidonic acid which deplete the reduced glutathione pool) and nordihydroguaretic acid, a general inhibitor of lipooxygenase pathway. which may also trigger rapid depletion of reduced glutathione. Melittin, which is known to activate phospholipase A2, also potently induced apoptosis. Arachidonic acid toxicity was inversely related to cell density. This could depend on an increased production of molecules with antiapoptotic effect; insulin-like growth factors could most likely be one of these molecules. These results propose a role for oxidative stress in the cytotoxicity induced by arachidonic acid in Y79 cells and suggest that these cells could be protected from such toxicity as long as sufficient levels of reduced glutathione and survival factors are present.
Vento
R
R
Institute of Biological Chemistry, University of Palermo, Policlinico, Italy. rvento@mbox.unipa.it
D'Alessandro
N
N
Giuliano
M
M
Lauricella
M
M
Carabillò
M
M
Tesoriere
G
G
D017209:Apoptosis / Q000187:drug effects*; D016718:Arachidonic Acid / Q000627:therapeutic use*; D015153:Blotting, Western; D020169:Caspases / Q000187:drug effects; D002452:Cell Count; D002470:Cell Survival; D003124:Colorimetry; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D005978:Glutathione / Q000032:analysis; D006801:Humans; D008564:Membrane Potentials; D008928:Mitochondria / Q000502:physiology; D018384:Oxidative Stress / Q000187:drug effects*; D011065:Poly(ADP-ribose) Polymerases / Q000187:drug effects; D012175:Retinoblastoma / Q000188:drug therapy* / Q000503:physiopathology; D014343:Trypan Blue; D014407:Tumor Cells, Cultured
D016718:Arachidonic Acid; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases; D005978:Glutathione; D014343:Trypan Blue