The Ca(2+) channel blocker flunarizine induces caspase-10-dependent apoptosis in Jurkat T-leukemia cells.
ABSTRACT Flunarizine is a Ca(2+) channel blocker that can be either cytoprotective or cytotoxic, depending on the cell type that is being examined. We show here that flunarizine was cytotoxic for Jurkat T-leukemia cells, as well as for other hematological maligancies, but not for breast or colon carcinoma cells. Treatment of Jurkat cells with flunarizine resulted in caspase-3 activation, poly (ADP-ribose) polymerase cleavage, and laddering of DNA fragments, all of which are hallmarks of apoptosis. Flunarizine-induced DNA fragmentation was inhibited by the caspase-3 inhibitor z-DEVD-fmk, the caspase-8/caspase-10 inhibitor z-IETD-fmk, and the caspase-10 inhibitor z-AEVD-fmk, but was not reduced in caspase-8-deficient Jurkat cells, indicating the involvement of caspase-10 upstream of caspase-3 activation. Interestingly, FADD recruitment to a death receptor was not involved since flunarizine caused DNA fragmentation in FADD-deficient Jurkat cells. Flunarizine treatment of Jurkat cells also resulted in reactive oxygen species production, dissipation of mitochondrial transmembrane potential, release of cytochrome c from mitochondria, and caspase-9 activation, although none of these events were necessary for apoptosis induction. Collectively, these findings indicate that flunarizine triggers apoptosis in Jurkat cells via FADD-independent activation of caspase-10. Flunarizine warrants further investigation as a potential anti-cancer agent for the treatment of hematological malignancies.
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ABSTRACT: Apoptosis inducing factor (AIF) is a novel apoptotic effector protein that induces chromatin condensation and large-scale ( approximately 50 kbp) DNA fragmentation when added to purified nuclei in vitro. Confocal and electron microscopy reveal that, in normal cells, AIF is strictly confined to mitochondria and thus colocalizes with heat shock protein 60 (hsp60). On induction of apoptosis by staurosporin, c-Myc, etoposide, or ceramide, AIF (but not hsp60) translocates to the nucleus. This suggests that only the outer mitochondrial membrane (which retains AIF in the intermembrane space) but not the inner membrane (which retains hsp60 in the matrix) becomes protein permeable. The mitochondrio-nuclear redistribution of AIF is prevented by a Bcl-2 protein specifically targeted to mitochondrial membranes. The pan-caspase inhibitor Z-VAD. fmk does not prevent the staurosporin-induced translocation of AIF, although it does inhibit oligonucleosomal DNA fragmentation and arrests chromatin condensation at an early stage. ATP depletion is sufficient to cause AIF translocation to the nucleus, and this phenomenon is accelerated by the apoptosis inducer staurosporin. However, in conditions in which both glycolytic and respiratory ATP generation is inhibited, cells fail to manifest any sign of chromatin condensation and advanced DNA fragmentation, thus manifesting a 'necrotic' phenotype. Both in the presence of Z-VAD. fmk and in conditions of ATP depletion, AIF translocation correlates with the appearance of large-scale DNA fragmentation. Altogether, these data are compatible with the hypothesis that AIF is a caspase-independent mitochondrial death effector responsible for partial chromatinolysis.The FASEB Journal 05/2000; 14(5):729-39. · 5.70 Impact Factor
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ABSTRACT: Prolonged ERK/MAPK activation has been implicated in neuronal cell death in vitro and in vivo. We found that HEK293 cells, recently reported to express neuronal markers, are exquisitely sensitive to long term ERK stimulation. Activation of an inducible form of Raf-1 (Raf-1:ER) in HEK293 cells induced massive apoptosis characterized by DNA degradation, loss of plasma membrane integrity and PARP cleavage. Cell death required MEK activity and protein synthesis and occurred via the death receptor pathway independently of the mitochondrial pathway. Accordingly, prolonged ERK stimulation activated caspase 8 and strongly potentiated Fas signaling. The death receptor adaptator FADD was found to be rapidly induced upon ERK activation. However using RNA interference and ectopic expression, we demonstrated that neither FADD nor Fas were necessary for caspase 8 activation and cell death. These findings reveal that prolonged ERK/MAPK stimulation results in caspase 8 activation and cell death.APOPTOSIS 04/2006; 11(3):337-46. · 3.95 Impact Factor
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ABSTRACT: The antioxidant effect of Ca2+ antagonists on sheep brain microsomal membranes and on liposomes prepared with total lipids extracted from the membranes was studied. Microsomal membranes were isolated from three brain areas: frontal cortex, hippocampus and caudate nucleus. Lipid peroxidation was induced by ascorbic acid and measured by malondialdehyde formation. Seven Ca2+ antagonists representative of the major chemical classes (dihydropyridines, benzothiazepines, phenylalkylamines, alkylamines, diphenylpiperazines) were tested for their antioxidant activity over a wide range of concentrations (0-500 microM). The order of antioxidant activity on frontal cortex membrane phospholipids, expressed as 50% inhibition of peroxidation (antioxidant IC50), was: nifedipine (IC50 = 4 microM) greater than flunarizine (IC50 = 48 microM) greater than bepridil (IC50 = 50 microM) greater than verapamil (IC50 = 74 microM). The dihydropyridines, nitrendipine and nimodipine, and the benzothiazepine, diltiazem, did not affect peroxidation even at a concentration of 500 microM. Membrane phospholipids are the substrate for free radical-induced damage since the extent of peroxidation in brain microsomal membranes was equal to that produced in liposomes prepared from membrane lipids. Although the lipophilicity of certain Ca2+ channel antagonists can enhance their antioxidant activity, our data suggest that Ca2+ antagonists inhibit peroxidation of the membrane lipid bilayer by a free radical scavenger effect that may be related to their chemical structure.European Journal of Pharmacology 12/1991; 204(3):315-22. · 2.59 Impact Factor