ABSTRACT
Chemotherapy-induced apoptosis by DNA-damaging drugs is thought to be generally dependent on the release of cytochrome c and the subsequent activation of caspase-9 and -3. However, the molecular mechanism of how damaged DNA triggers the apoptotic process is not clear. To better understand the mechanisms underlying this process, we examined drug-induced apoptosis in cultured H-460 cells. Using cell fractionation, western blotting, and immunofluorescence assays, we show that the activation of nuclear caspases-7 and -3, and poly(ADP-ribose) polymerase (PARP) cleavage, are early events in camptothecin-induced apoptosis. Moreover, we demonstrate that these events precede the release of cytochrome c and apoptotic inducing factor, and the activation of caspases 2, 8, 9 and 12. Together our results suggest that drugs acting at the DNA level can initiate apoptosis via nuclear caspase activation.
Subject(s)
Apoptosis/drug effects , Apoptosis/physiology , Camptothecin/pharmacology , Caspase 3/metabolism , Caspase 7/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Amino Acid Chloromethyl Ketones/pharmacology , Caspase Inhibitors , Cell Line, Tumor , Cell Nucleus/drug effects , Cell Nucleus/enzymology , Cysteine Proteinase Inhibitors/pharmacology , DNA Damage , Enzyme Activation/drug effects , Humans , Oligopeptides/pharmacology , Poly (ADP-Ribose) Polymerase-1ABSTRACT
Arachidonic acid metabolism is modulated during differentiation induced by 1alpha,25(OH)(2)D(3) in HL-60 cells. Antioxidants that affect arachidonic acid metabolism enhance this differentiation program. Ascorbate also enhances differentiation in 1alpha,25(OH)(2)D(3)-induced cells depending on the induction of cAMP. The aim of this work was to study if this cAMP rise depends on modulation of arachidonic acid metabolism by ascorbate. Cyclooxygenase inhibitors, indomethacin and aspirin, increased cAMP levels and also enhanced 1alpha,25(OH)(2)D(3)-induced differentiation in HL-60 cells. Ascorbate did not affect the release of arachidonic acid-derived metabolites but decreased the levels of TXB(2) and PGE(2), suggesting the inhibition of cyclooxygenase. On the other hand, free arachidonic acid increased both cAMP levels and differentiation in the absence or presence of 1alpha,25(OH)(2)D(3). Neither cyclooxygenase inhibitors nor ascorbate modified AA effect. Then, inhibition of cyclooxygenase activity by ascorbate could accumulate free arachidonic acid or other metabolites that increase cAMP levels and enhance differentiation in 1alpha,25(OH)(2)D(3)-induced HL-60 cells.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Arachidonic Acid/metabolism , Ascorbic Acid/metabolism , Cell Differentiation/physiology , Cyclic AMP/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Vitamin D/analogs & derivatives , Vitamin D/pharmacology , Cell Division , Cells, Cultured , Cyclooxygenase Inhibitors/pharmacology , HL-60 Cells , Humans , Oxidation-Reduction , Vitamin D/metabolismABSTRACT
Coenzyme Q10 (CoQ10) is a component of the antioxidant machinery that protects cell membranes from oxidative damage and decreases apoptosis in leukemic cells cultured in serum-depleted media. Serum deprivation induced apoptosis in CEM-C7H2 (CEM) and to a lesser extent in CEM-9F3, a subline overexpressing Bcl-2. Addition of CoQ10 to serum-free media decreased apoptosis in both cell lines. Serum withdrawal induced an early increase of neutral-sphingomyelinase activity, release of ceramide, and activation of caspase-3 in both cell lines, but this effect was more pronounced in CEM cells. CoQ10 prevented activation of this cascade of events. Lipids extracted from serum-depleted cultures activated caspase-3 independently of the presence of mitochondria in cell-free in vitro assays. Activation of caspase-3 by lipid extracts or ceramide was prevented by okadaic acid, indicating the implication of a phosphatase in this process. Our results support the hypothesis that plasma membrane CoQ10 regulate the initiation phase of serum withdrawal-induced apoptosis by preventing oxidative damage and thus avoiding activation of downstream effectors as neutral-sphingomyelinase and subsequent ceramide release and caspase activation pathways.
Subject(s)
Apoptosis , Caspase Inhibitors , Ceramides/antagonists & inhibitors , Ubiquinone/metabolism , Caspase 3 , Cell Membrane/metabolism , Cell-Free System , Coenzymes , Culture Media, Serum-Free , Electron Transport Complex IV/metabolism , Enzyme Activation/drug effects , Humans , Leukemia/metabolism , Lipid Metabolism , Mitochondria/metabolism , Okadaic Acid/metabolism , Oxidative Stress , Sphingomyelin Phosphodiesterase/metabolism , Time Factors , Tumor Cells, Cultured , Ubiquinone/analogs & derivativesABSTRACT
Serum withdrawal is a model to study the mechanisms involved in the induction of apoptosis caused by mild oxidative stress. Apoptosis induced by growth factors removal was prevented by the external addition of antioxidants such as ascorbate, alpha-tocopherol and coenzyme Q (CoQ). CoQ is a lipophilic antioxidant which prevents oxidative stress and participates in the regeneration of alpha-tocopherol and ascorbate in the plasma membrane. We have found an inverse relationship between CoQ content in plasma membrane and lipid peroxidation rates in leukaemic cells. CoQ10 addition to serum-free culture media prevented both lipid peroxidation and cell death. Also, CoQ10 addition decreased ceramide release after serum withdrawal by inhibition of magnesium-dependent plasma membrane neutral-sphingomyelinase. Moreover, CoQ10 addition partially blocked activation of CPP32/caspase-3. These results suggest CoQ of the plasma membrane as a regulator of initiation phase of oxidative stress-mediated serum withdrawal-induced apoptosis.
