cytochrome-c-t and 3-aminobenzamide

Oxidative stress, continuously exerted during chronic inflammation, has been implicated as a major causative agent of cellular dysfunction and cell death. In the present study, we investigated the impact of oxidative stress on the mode of cell death in HUVECs using H2O2 as a model reagent. We found that the predominant form of cell death was necrosis. Necrosis induction was accompanied by a distinct mode of caspase-3 cleavage, yielding a 29-kDa fragment. While inhibition of caspases could not prevent the generation of the 29-kDa fragment, general protease inhibitors, such as leupeptin and LLNL, proved to be effective in inhibiting the distinct processing pattern of caspase-3. These results suggest that caspases can act as substrates for non-caspase proteases in cells primed for necrosis induction. Thus, the pattern of caspase-3 cleavage might reflect the proteolytic system engaged in the cell death machinery in HUVECs.

Topics: Amino Acid Chloromethyl Ketones; Benzamides; Caspase 3; Caspase 7; Caspase Inhibitors; Caspases; Cell Line; Collagen Type XI; Cytochromes c; Dose-Response Relationship, Drug; Endothelial Cells; Enzyme Activation; Enzyme Inhibitors; Humans; Hydrogen Peroxide; Membrane Potentials; Mitochondria; Necrosis; Oxidants; Oxidative Stress; Time Factors; Umbilical Veins

Treatment with 0.2 mM hydrogen peroxide (H(2)O(2)) or with 0.5 mM cisplatin caused caspase-9 and caspase-3 activation and death by apoptosis in U-937 human promonocytic cells. However, treatment with 2 mM H(2)O(2), or incubation with the glutathione suppressor DL-buthionine-(S,R)-sulfoximine (BSO) prior to treatment with cisplatin, suppressed caspase activation and changed the mode of death to necrosis. Treatment with 2 mM H(2)O(2) caused a great decrease in the intracellular ATP level, which was partially prevented by 3-aminobenzamide (3-ABA). Correspondingly, 3-ABA restored the activation of caspases and the execution of apoptosis. By contrast, BSO plus cisplatin did not decrease the ATP levels, and the generation of necrosis by this treatment was not affected by 3-ABA. On the other hand, while all apoptosis-inducing treatments and treatment with 2 mM H(2)O(2) caused Bax translocation from the cytosol to mitochondria as well as cytochrome c release from mitochondria to the cytosol, treatment with BSO plus cisplatin did not. Treatment with cisplatin alone caused Bid cleavage, while BSO plus cisplatin as well as 0.2 and 2 mM H(2)O(2) did not. Bcl-2 overexpression reduced the generation of necrosis by H(2)O(2), but not by BSO plus cisplatin. These results indicate the existence of different apoptosis/necrosis regulatory mechanisms in promonocytic cells subjected to different forms of oxidative stress.

Topics: Adenosine Triphosphate; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Benzamides; Blotting, Western; Buthionine Sulfoximine; Caspase 3; Caspase 9; Caspases; Cisplatin; Cytochromes c; Electron Transport Complex III; Flow Cytometry; Glutathione; Humans; Hydrogen Peroxide; Microscopy, Fluorescence; Monocytes; Necrosis; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Transfection; U937 Cells