cytochrome-c-t and 25-hydroxycholesterol

Cholesterol oxidation products are suggested to be involved in neuronal degeneration. Apocynin has demonstrated to have anti-inflammatory and anti-oxidant effects. We assessed the effect of apocynin on the cholesterol oxidation product-induced programmed cell death in neuronal cells using differentiated PC12 cells in relation to NF-κB-mediated cell death process. 7-Ketocholesterol and 25-hydroxycholesterol decreased the levels of Bid and Bcl-2, increased the levels of Bax and p53, and induced loss of the mitochondrial transmembrane potential, release of cytochrome c and activation of caspases (-8, -9 and -3). 7-Ketocholesterol caused an increase in the levels of cytosolic and nuclear NF-κB p65, cytosolic NF-κB p50 and cytosolic phospho-IκB-α, which was inhibited by the addition of 0.5 μM Bay11-7085 (an inhibitor of NF-κB activation). Apocynin attenuated the cholesterol oxidation product-induced changes in the programmed cell death-related protein levels, NF-κB activation, production of reactive oxygen species, and depletion of GSH. The results show that apocynin appears to attenuate the cholesterol oxidation product-induced programmed cell death in PC12 cells by suppressing the activation of the mitochondrial pathway and the caspase-8- and Bid-dependent pathways that are mediated by NF-κB activation. The preventive effect appears to be associated with the inhibitory effect on the production of reactive oxygen species and depletion of GSH.

Topics: Acetophenones; Animals; Antioxidants; Cell Death; Cell Differentiation; Cytochromes c; Hydroxycholesterols; NF-kappa B; Oxidation-Reduction; PC12 Cells; Rats; Reactive Oxygen Species

To characterize the possible cytotoxic effects of oxysterols (7beta-hydroxycholesterol (7beta-OH), 25-hydroxycholesterol (25-OH)) in human retinal pigment epithelial cells (ARPE-19) and to detail the relationships between some of these effects.. ARPE-19 cells were treated with 7beta-OH and 25-OH. Cell viability was measured with the MTT assay. Membrane permeability, mitochondrial potential, and lysosomal integrity were measured by flow cytometry with propidium iodide, DiOC6(3), and acridine orange, respectively. Cell death was characterized by staining with Hoechst 33342, transmission electron microscopy, and analysis of the DNA fragmentation pattern. Caspase activity was examined with fluorochrome-labeled inhibitors of caspases (FLICA) and Western blotting. Immunofluorescence staining was used to visualize the cellular distribution of cytochrome c (Cyt-c) and apoptosis-inducing factor (AIF). The effect of the cathepsin inhibitor (z-FA-fmk) on oxysterol-induced cell death was evaluated.. Cell viability of ARPE-19 cells was decreased with 7beta-OH, whereas 25-OH had no cytotoxic effects. Loss of mitochondrial potential and lysosomal destabilization was associated with 7beta-OH-induced cell death, few morphologically apoptotic cells were identified, and no internucleosomal DNA fragmentation was found. Slight caspase activation was detected with FLICA, and no caspase-3 activation was revealed. A pronounced relocalization of Cyt-c and AIF was observed. Noteworthy, z-FA-fmk was able to prevent cell death.. 7beta-OH induced a caspase-3-independent mode of cell death associated with lysosomal destabilization, which could play a key role in the signaling pathways leading to cell death, as shown by the ability of z-FA-fmk to counteract the cytotoxic effects of 7beta-OH.

Topics: Apoptosis Inducing Factor; Blotting, Western; Caspase 3; Cell Death; Cell Membrane Permeability; Cell Survival; Cells, Cultured; Cysteine Proteinase Inhibitors; Cytochromes c; Dipeptides; DNA Fragmentation; Electrophoresis, Agar Gel; Enzyme Activation; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Humans; Hydroxycholesterols; Ketones; Lysosomes; Membrane Potentials; Mitochondrial Membranes; Pigment Epithelium of Eye

Cells of the vasculature, including macrophages, smooth muscle cells, and endothelial cells, exhibit apoptosis in culture upon treatment with oxidized low density lipoprotein, as do vascular cells of atherosclerotic plaque. Several lines of evidence support the hypothesis that the apoptotic component of oxidized low density lipoprotein is one or more oxysterols, which have been shown to induce apoptosis through the mitochondrial pathway. Activation of the mitochondrial pathway of apoptosis is regulated by members of the BCL family of proteins. In this study, we demonstrate that, in the murine macrophage-like cell line P388D1, oxysterols (25-hydroxycholesterol and 7-ketocholesterol) induced the degradation of the prosurvival protein kinase AKT (protein kinase B). This led, in turn, to the activation of the BCL-2 homology-3 domain-only proteins BIM and BAD and down-regulation of the anti-apoptotic multi-BCL homology domain protein BCL-xL. These responses would be expected to activate the pro-apoptotic multi-BCL homology domain proteins BAX and BAK, leading to the previously reported release of cytochrome c observed during oxysterol-induced apoptosis. Somewhat surprisingly, small interfering RNA knockdown of BAX resulted in a complete block of the induction of apoptosis by 25-hydroxycholesterol.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; bcl-X Protein; Carrier Proteins; Caspase 3; Caspases; Cell Line; Cholesterol; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; Down-Regulation; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Green Fluorescent Proteins; Hydroxycholesterols; Immunoblotting; Luminescent Proteins; Membrane Proteins; Mice; Mitochondria; Phospholipases A; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Recombinant Fusion Proteins; Sterols; Time Factors