Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P42574 (caspase-3)
 45,978 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Physiological levels of shear stress reduce endothelial cell turnover and exert a potent antiatherosclerotic effect. Here we demonstrate that oxidative stress-induced apoptosis of human endothelial cells was inhibited by shear stress exposure (15 dynes/cm2). Incubation with H2O2 (200 mumol/L) for 18 hours induced apoptosis of human umbilical venous endothelial cells as demonstrated by an enzyme-linked immunosorbent assay specific for histone-associated DNA fragments and visual analysis of fluorescence-stained nuclei. Shear stress-mediated inhibition of apoptosis was partially prevented by pharmacological inhibition of glutathione (GSH) biosynthesis with buthionine sulfoximine (BSO) or nitric oxide (NO) synthase with NG-monomethyl-L-arginine (LNMA), whereas inhibition of catalase by aminotriazol did not affect the inhibitory action of shear stress. Combined inhibition of NO synthase and GSH biosynthesis completely reversed the protective effect of shear stress, suggesting that both NO synthase and the GSH redox cycle system are involved in the apoptosis-suppressing effect of shear stress. Similar results were obtained when apoptosis was stimulated by tumor necrosis factor alpha (TNF alpha). To gain further insights into the interference of shear stress with apoptosis signal transduction, we measured caspase-3-like activity, a cysteine protease that has been shown to play a predominant role in the cell death effector pathway. Indeed, shear stress prevented the activation of caspase-3-like activity induced by H202 or TNF alpha. The inhibitory effect of shear stress was prevented by LNMA and BSO, suggesting that the reduction of oxidative flux by shear stress prevents the activation of caspase-like proteases and thereby inhibits apoptotic cell death in human endothelial cells.
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PMID:Shear stress inhibits H2O2-induced apoptosis of human endothelial cells by modulation of the glutathione redox cycle and nitric oxide synthase. 943 9

We have recently shown that dithiocarbamate (DC) disulfides inhibit proteolytic processing of the caspase-3 proenzyme in Jurkat T lymphocytes treated with anti-CD95 (Fas/APO-1) antibody. Because the processing can be accomplished by caspase activity, we investigated the effect of DC disulfides, such as disulfiram (DSF), on active caspases. DSF showed a dose-dependent inhibition was prevented by including dithiothreitol (DTT) in the reaction buffer, thiol-disulfide exchange between inhibitor and target is suggested. Direct interaction of DSF with caspases was confirmed by its inhibition of the purified Ac-DEVD-AMC cleaving protease, caspase-3 (CPP32/apopain). An apparent rate constant (K(app)) for this inhibition was estimated to be 0.45 x 10(3)M(-1)s(-1). DSF was also observed to inhibit the purified Ac-YVAD-AMC cleaving enzyme, caspase-1 (interleukin-1 beta-converting enzyme, ICE), with a K(app) of 2.2 x 10(3) M(-1)s(-1). In this case protein mixed disulfide formation between DSF and caspase-1 was directly demonstrated using 35S-labeled DSF. The physiological disulfide GSSG was also observed to influence the activity of caspases. A glutathione buffer (5 mM) with a GSH:GSSG ratio of 9:1 decreased the Ac-DEVD-AMC cleaving activity in S100 cytosolic extracts by 50% as compared to GSH controls without GSSG. In conclusion, our study shows that caspases are quite sensitive to thiol oxidation and that DSF is a very potent oxidant of caspase protein thiol(s), being 700-fold more potent than glutathione disulfide.
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PMID:Disulfiram is a potent inhibitor of proteases of the caspase family. 943 20

Dysregulated apoptosis may underlie the etiology of T cell depletion by human immunodeficiency virus type 1 (HIV-1). We show that HIV-induced apoptosis is preceded by an exponential increase in reactive oxygen intermediates (ROIs) produced in mitochondria. This leads to caspase-3 activation, phosphatidylserine (PS) externalization, and GSH depletion. Since mitochondrial ROI levels are regulated by the supply of NADPH from the pentose phosphate pathway (PPP), the effect of transaldolase (TAL), a key enzyme of PPP, was investigated. Jurkat and H9 human CD4+ T cells were transfected with TAL expression vectors oriented in the sense or antisense direction. TAL overexpression down-regulated glucose-6-phosphate dehydrogenase activities and GSH levels. Alternatively, decreased TAL expression up-regulated glucose-6-phosphate dehydrogenase activities and GSH levels. HIV-induced 1) mitochondrial ROI production, 2) caspase-3 activation, 3) proteolysis of poly(ADP-ribose) polymerase, and 4) PS externalization were accelerated in cells overexpressing TAL. In contrast, suppression of TAL abrogated these four activities. Thus, susceptibility to HIV-induced apoptosis can be regulated by TAL through controlling the balance between mitochondrial ROI production and the metabolic supply of reducing equivalents by the PPP. The dominant effect of TAL expression on oxidative stress, caspase activation, PS externalization, and cell death suggests that this balance plays a pivotal role in HIV-induced apoptosis.
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PMID:Molecular ordering in HIV-induced apoptosis. Oxidative stress, activation of caspases, and cell survival are regulated by transaldolase. 956 23

Spermine NONOate (SpNO, a nitric oxide donor) induced apoptosis and caspase-3 activity in the macrophage cell line RAW 267.4. RES cells that have been derived from RAW 267.4 cells by repeated exposure to lipopolysaccharide and interferon-gamma (LPS/INF-gamma), followed by outgrowth of viable cells, are resistant to apoptosis and caspase-3 activation upon exposure to SpNO. In this study we have determined that RES cells have lower levels of glutathione (GSH) and a higher oxidative state than RAW cells. Subsequently, RAW and RES cells were depleted of GSH by using l-buthionine-[S,R]-sulfoximine (BSO), a specific inhibitor of GSH synthesis. GSH depleted cells did not undergo apoptosis nor demonstrate caspase-3 activity when they were exposed to SpNO. These results suggest that the redox status of the cell is one of the key factors mediating the apoptotic pathway in which glutathione plays a critical role in mediating apoptosis via NO* and reactive oxygen species (ROS).
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PMID:Glutathione levels determine apoptosis in macrophages. 964 8

Studies on the mechanism of apoptosis in this laboratory support a model in which signal transduction involving caspase 3 leads to activation of a serine protease called Mr 24,000 apoptotic protease (AP24), which then induces internucleosomal DNA fragmentation in the nucleus. This study examined the effect of Bcl-2 overexpression on activation of AP24 and the induction of DNA fragmentation by AP24 in isolated nuclei. It was demonstrated that overexpression of Bcl-2 in either HL-60 or PW leukemia cell lines suppressed activation of AP24 induced by either tumor necrosis factor or UV light and protected cells from apoptosis. Furthermore, nuclei isolated from Bcl-2-overexpressing cells were relatively resistant to internucleosomal DNA fragmentation induced by AP24 isolated from apoptotic cells. Bcl-2-overexpressing cells that were nutritionally depleted of glutathione (GSH) became sensitive to tumor necrosis factor- or UV light-induced activation of AP24 and underwent apoptotic cell death. Moreover, nuclei isolated from Bcl-2-overexpressing cells that were depleted of GSH became sensitive to AP24-induced DNA fragmentation. The addition of exogenous GSH blocked the proteolytic activity of AP24, as well as its ability to induce DNA fragmentation in normal isolated nuclei. These results indicate that Bcl-2 can attenuate at least two events in the AP24 apoptotic pathway: activation of AP24 and induction of DNA fragmentation by activated AP24. Furthermore, agents that deplete intracellular levels of GSH may have therapeutic use in the sensitization of Bcl-2-overexpressing cancer cells to apoptotic cell death.
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PMID:Bcl-2-mediated resistance to apoptosis is associated with glutathione-induced inhibition of AP24 activation of nuclear DNA fragmentation. 985 96

Glutathione (GSH) depletion caused by l-buthionine-(S,R)-sulfoximine (BSO) induced apoptosis that was recognized by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick endo-labeling (TUNEL), nuclear DNA staining with fluorescence dye, and internucleosomal DNA fragmentation in C6 rat glioma cells. The BSO-induced cell death was associated with caspase-3 activation. Lipid peroxidation and protein kinase C (PK-C) activation were observed during the apoptosis of C6 cells, and these events were inhibited by antioxidants and iron chelators without affecting BSO-induced GSH depletion. Furthermore, approximately 2 Mbp giant DNA fragments were observed in the BSO-treated cells. The giant DNA fragmentation were followed by approximately 30-700 kbp and then less than 100 kbp, including internucleosomal DNA fragmentations. Such serial DNA degradation was prevented by the antioxidants, the iron chelators, and the PK-C inhibitors. These results suggest that during apoptosis induced by GSH-depletion caused by BSO, reactive oxygen species endogenously produced cause lipid peroxidation and that the lipid peroxidation induced PK-C activation, processes which are thought to be involved in the giant DNA, high-molecular-weight DNA, and the internucleosomal DNA fragmentations.
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PMID:Glutathione depletion induces giant DNA and high-molecular-weight DNA fragmentation associated with apoptosis through lipid peroxidation and protein kinase C activation in C6 glioma cells. 1004 97

The purpose of this study was to determine if exacerbation of apoptosis precedes liver injury during chronic exposure of rats to alcohol. After 7 weeks of feeding an alcohol- or dextrin-containing liquid diet, the animals were treated with gram-negative bacterial lipopolysaccharide (1 mg x kg(-1) body weight, intravenously) or sterile saline and sacrificed 3 hr after the treatment. Alanine:2-oxoglutarate aminotransferase (ALT) and lactate:NAD oxidoreductase [lactate dehydrogenase (LDH)] were measured in plasma. The caudate lobe of the liver was resected for histology, while the rest of the organ was perfused with collagenase to isolate hepatocytes, Kupffer cells (KCs), and sinusoidal endothelial cells (SECs) by centrifugal elutriation. Hepatocyte mitochondria were isolated by differential centrifugation of the cell homogenate. Reduced and oxidized glutathione (GSH and GSSG) in isolated hepatocytes and hepatocyte mitochondria, and malondialdehyde in hepatocytes were assayed. Caspase-3 activity and Fas ligand mRNA expression were determined in hepatocytes, KCs, and SECs. Plasma ALT and LDH activity, liver histology, GSH, GSSG and their ratio, and malondialdehyde content were not affected by alcohol treatment Caspase-3 activity was significantly increased in alcohol-treated rats in all three cell types, with the lowest response observed in hepatocytes and the highest in KCs. Fas ligand mRNA expression, which had the highest level in SECs, followed by KCs and hepatocytes, was not affected by alcohol administration. Lipopolysaccharide had the following effects: an increase in ALT in both pair- and alcohol-fed rats, and LDH only in alcohol-fed rats, a decrease in GSH + GSSG levels in both mitochondria and hepatocytes, an elevation of malondialdehyde content in hepatocytes, a raise in caspase-3 activity in all groups and cell types, and an augmentation of Fas ligand expression in hepatocytes and KCs, but not in SECs. These data suggest that, during chronic alcohol consumption, an exacerbated apoptosis precedes alcohol-induced liver injury.
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PMID:Modulation of caspase-3 activity and Fas ligand mRNA expression in rat liver cells in vivo by alcohol and lipopolysaccharide. 1006 67

The mechanism of cell death caused by cytokine deprivation remains largely unknown. FL5.12 cells (a murine prolymphocytic cell line), following interleukin-3 (IL-3) withdrawal, undergo a decrease in intracellular glutathione (GSH) that precedes the onset of apoptosis. In the present study, the induction of apoptosis following IL-3 withdrawal or GSH depletion with DL-buthionine-[S,R,]-sulfoximine (BSO) was examined. Both conditions caused time-dependent increases in phosphatidylserine externalization, acridine orange and ethidium bromide staining, decreases in mitochondrial membrane potential, processing and activation of caspase-3 and proteolysis of the endogenous caspase substrate poly(adenosine diphosphate ribose)polymerase (PARP). Apoptosis induced by IL-3 deprivation but not BSO also caused lamin B1 cleavage, suggesting activation of caspase-6. Despite a more profound depletion of GSH after BSO than withdrawal of IL-3, the extent of apoptosis was somewhat lower. Benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone (z-VAD.fmk) blocked this caspase activity and prevented cell death after BSO exposure but not after IL-3 deprivation. Following IL-3 withdrawal, the caspase inhibitors z-VAD.fmk and boc-asp(OMe)fluoromethylketone (boc-asp.fmk) prevented the cleavage and activation of caspase-3, the breakdown of lamin B1 and partially mitigated PARP degradation. However, the externalization of phosphatidylserine, the fall in mitochondrial membrane potential and subsequent apoptotic cell death still occurred. These results suggest that IL-3 withdrawal may mediate cell death by a mechanism independent of both caspase activation and the accompanying loss of GSH.
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PMID:Apoptosis in hematopoietic cells (FL5.12) caused by interleukin-3 withdrawal: relationship to caspase activity and the loss of glutathione. 1020 May 49

Glutathione-doxorubicin (GSH-DXR) effectively induced apoptosis in rat hepatoma cells (AH66) at a lower concentration than DXR. After 24 h of drug treatment, DNA fragmentation of the cells was observed at the concentration of 1.0 microM DXR or 0.01 microM GSH-DXR. Increase in caspase-3 activity and DNA fragmentation were observed within 12 h and 15 h after treatment with either drug. Intracellular caspase-3 activity was increased in a dose-dependent manner after treatment with DXR or GSH-DXR, and caspase-3 activity correlated well with the ability to induce DNA fragmentation. When the cells were treated with either DXR or GSH-DXR for only 6 h, apoptotic DNA degradation and caspase-3 activation occurred 24 h after treatment. DNA fragmentation caused by these drugs was prevented completely by simultaneous treatment with the caspase-3 inhibitor, acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO), at 10 microM. By contrast, DNA fragmentation was not prevented by the caspase-1 inhibitor, acetyl-Tyr-Val-Ala-Asp-aldehyde (YVAD-CHO), at the same concentration as DEVD-CHO, and caspase-1 was not activated at all by the treatment of AH66 cells with both DXR and GSH-DXR. These results demonstrate that DXR and GSH-DXR induce apoptotic DNA fragmentation via caspase-3 activation, but not via caspase-1 activation, and that GSH-DXR enhances the activation of caspase-3 approximately 100-fold more than DXR. Moreover, the findings suggested that an upstream apoptotic signal that can activate caspase-3 is induced within 6 h by treating AH66 cells with the drug.
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PMID:Caspase-3 activation during apoptosis caused by glutathione-doxorubicin conjugate. 1036 Jun 48

Agonistic engagement of the cytokine receptor CD95 in mice leads to activation of hepatic caspases, followed by massive hepatocyte apoptosis, acute liver failure, and death. This mechanism of cell death is thought to be associated with several human liver disorders. Because hepatic glutathione represents the major defense against toxic liver injury, we investigated its role in CD95-mediated liver failure, which represents a model for hyperinflammatory organ destruction. As a tool for modulating the liver glutathione status of mice in vivo, we used the GSH transferase substrate, phorone, which rapidly depleted hepatic glutathione in a dose-dependent manner. When GSH was depleted, CD95-initiated hepatic caspase-3-like activity and DNA fragmentation were completely blocked, and animals were protected from liver injury dose-dependently as assessed by histological examination and determination of liver enzymes in plasma. Conversely, repletion of hepatic glutathione by treatment with the permeable glutathione monoethylester restored susceptibility of GSH-depleted mice toward CD95-mediated liver injury. In contrast, the antioxidants, GSH, N-acetyl cysteine, alpha-tocopherol, butyl-hydroxytoluene, and catalase failed to do so. Animals treated once with phorone survived for more than 3 months after an otherwise lethal injection of the activating anti-CD95 antibody. We investigated the thiol sensitivity of recombinant caspase-3 in vitro and observed that its activity was dependent on the presence of a reducing agent such as GSH, while GSSG attenuated proteolytic activity. Based on our finding that CD95-mediated hepatocyte apoptosis requires an intact intracellular glutathione status, we propose that the activation of apoptosis-executing caspases is controlled by reduced glutathione.
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PMID:CD95-Mediated murine hepatic apoptosis requires an intact glutathione status. 1038 54


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