In a number of experimental systems, inhibition of apoptosis by antioxidants has led to the production of radical oxygen species (ROS) in certain apoptotic forms of cell death. Since antioxidant therapies can reduce vascular dysfunctions in hypercholesterolemic patients who frequently have increased plasma levels of oxysterols constituting potent inducers of apoptosis, we speculate that oxysterol-induced apoptosis could involve oxidative stress. Here, we tested the protective effects of the aminothiols glutathione (GSH) and N-acetylcysteine (NAC), which are two potent antioxidants, on apoptosis induced by 7-ketocholesterol in U937 cells, and we present evidence indicating that oxidative processes are involved in 7-ketocholesterol-induced cell death. Thus, GSH and NAC prevented phenomenona linked to apoptosis such as reduction of cell growth, increase cellular permeability to propidium iodide, and occurrence of nuclear condensation and/or fragmentation, and they delayed internucleosomal DNA fragmentation. In addition, cell treatment with GSH impaired cytochrome c release into the cytosol and degradation of caspase-8 occurring during cell death. During 7-ketocholesterol-induced apoptosis, we also observed a rapid decrease in cellular GSH content, oxidation of polyunsaturated fatty acids, and a production of ROS by flow cytometry with the use of the dye 2', 7'-dichlorofluorescin-diacetate; both phenomena were inhibited by GSH. Prevention of cell death by GSH and NAC does not seem to be a general rule since these antioxidants impaired etoposide (but not cycloheximide) -induced apoptosis. Taken together, our data demonstrate that GSH is implied in the control of 7-ketocholesterol-induced apoptosis associated with the production of ROS.

Among oxysterols oxidized at C7 (7K K-, 7L L-hydroxycholesterol, and 7-ketocholesterol), 7L L-hydroxycholesterol and 7-ketocholesterol involved in the cytotoxicity of oxidized low density lipoproteins (LDL) are potent inducers of apoptosis. Here, we asked whether all oxysterols oxidized at C7 were able to trigger apoptosis, to stimulate interleukin (IL)-1L L and/or tumor necrosis factor (TNF)-K K secretion, and to enhance adhesion molecule expression (intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and Eselectin) on human umbilical venous endothelial cells (HUVECs). Only 7L L-hydroxycholesterol and 7-ketocholesterol were potent inducers of apoptosis and of IL-1L L secretion. TNF-K K secretion was never detected. Depending on the oxysterol considered, various levels of ICAM-1, VCAM-1 and E-selectin expression were observed. So, oxysterols oxidized at C7 differently injure and activate HUVECs, and the K K-or L L-hydroxyl radical position plays a key role in apoptosis and IL-1L L secretion.z 1998 Federation of European Biochemical Societies.

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IntroductionChemotherapeutic drugs can kill cultured leukemic cell lines by inducing apoptosis. 1 This mode of cell death may be clinically relevant because apoptotic blast cells have been detected in the peripheral blood of patients with acute leukemia who received chemotherapeutic drugs. 2 Hallmarks of apoptosis in leukemic cells include characteristic morphologic changes such as cell shrinkage, membrane blebbing, and cell fragmentation into apoptotic bodies, internucleosomal DNA fragmentation, and limited proteolytic cleavage of selective intracellular proteins such as the nuclear enzyme poly(ADP-ribose)polymerase (PARP). This cell death phenotype is induced by activation of a constitutively expressed apoptotic machinery that involves a family of cysteine proteases known as caspases, organized in a branched proteolytic cascade. These enzymes are synthesized as inactive proenzymes (procaspases) whose proteolysis at internal aspartate residues generates large and small subunits. 3 Heterodimerization of these subunits is required to form the active enzyme (caspase) that cleaves intracellular substrates, either a downstream procaspase or other cellular proteins. 4 Activation of the caspase cascade in response to specific damage induced by chemotherapeutic drugs is the consequence of a disruption of the mitochondrial membrane barrier function. 5 Although the exact mechanism remains controversial, mitochondrial changes lead to the release of soluble apoptogenic proteins such as cytochrome c, 6 apoptosis-inducing factor (AIF), 7 and Smac/Diablo 8,9 from the mitochondrial intermembrane space to the cytosol. Once in the cytosol, cytochrome c, in the presence of adenosine triphosphate (ATP), induces conformational changes of an adaptor molecule designated Apaf-1 that recruits and activates procaspase-9 molecules. 10,11 In turn, caspase-9 activates the downstream caspase cascade that involves caspase-3 and other effector enzymes. 12 Smac/Diablo was recently shown to suppress the inhibition of caspases by proteins known as inhibitor of apoptosis proteins (IAPs), thereby increasing their sensitivity to the cytochrome c/ATP pathway of activation. 8,9 Once activated, effector caspases cleave a limited set of essential cellular proteins, leading to cell dismantling. [13][14][15] Mitochondrial function during apoptosis is controlled by the Bcl-2 family of proteins that could prevent the opening of the permeability transition pore or stabilize the barrier function of the outer mitochondrial membrane and prevent the release of apoptogenic molecules. 5,16 How the specific damage induced by chemotherapeutic drugs leads to mitochondrial changes and activation of the caspase cascade under the control of Bcl-2-related proteins remains poorly understood. We have shown previously that differentiation of human myeloid leukemia cell lines along a macrophagic pathway induced by exposure to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) correlated with a resistance phenotype to apoptosis induced by a series of anticancer dru...

Biological activities of oxysterols seem tightly regulated. Therefore, the ability to induce cell death of structurally related oxysterols, such as those oxidized at C7(7a-, 7b-hydroxycholesterol, and 7-ketocholesterol), was investigated on U937 cells at different times of treatment in a concentration range of 5 ± 80 mg/ml. Whereas all oxysterols accumulate inside the cells, strong inhibition of cell growth and increased permeability to propidium iodide were observed only with 7b-hydroxycholesterol and 7-ketocholesterol, which trigger an apoptotic process characterized by the occurrence of cells with fragmented and/or condensed nuclei, and by various cellular dysfunctions: loss of mitochondrial transmembrane potential, cytosolic release of cytochrome c, activation of caspase-9 and -3 with subsequent enhanced activity of caspase-3, degradation of poly(ADP-ribose) polymerase, and increased accumulation of cellular C16 : 0 and C24 : 1 ceramide species. This ceramide generation is not attributed to caspase activation since inhibition of 7b-hydroxycholesterol-and 7-ketocholesterol-induced apoptosis by Z-VAD-fmk (100 mM), a broad spectrum caspase inhibitor, did not reduce C16 : 0 and C24 : 1 ceramide species accumulation. Conversely, when U937 cells were treated with 7b-hydroxycholesterol and 7-ketocholesterol in the presence of fumonisin B1 (100 mM), a specific inhibitor of ceramide synthase, C16 : 0 and C24 : 1 ceramide species production was completely abrogated whereas apoptosis was not prevented. Noteworthy, 7a-hydroxycholesterol induced only a slight inhibition of cell growth. Collectively, these results are consistent with the notion that the a or b hydroxyl radical position of oxysterols oxidized at C7 plays a key role in the induction of the apoptotic process. In addition, our findings demonstrate that 7b-hydroxycholesterol-and 7-ketocholesterol-induced apoptosis involve the mitochondrial signal transduction pathway and they suggest that C16 : 0 and C24 : 1 ceramide species generated through ceramide synthase play a minor role in the commitment of 7b-hydroxycholesterol-and 7-ketocholesterol-induced cell death. Cell Death and Differentiation (2001) 8, 83 ± 99.