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)

Excitotoxic studies using isolated chick embryo retina indicated that such an in vitro model provides a valid tool to characterize the effect of different agonists for subtypes of glutamate ionotropic receptors. In retinas maintained for 24 h in a Krebs medium, after a brief exposure (30 min) to glutamate agonists, we compared the effects produced by NMDA and non-NMDA-agonists, such as kainic acid (KA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Delayed retinal damage was assessed by measuring lactate dehydrogenase (LDH) present in the medium after exposure to the previously named agonists. Although at high concentrations, both KA and AMPA produced more relevant release than NMDA, 7-8% of total retinal LDH was released after exposure to a 50 microM concentration of non-NMDA agonists. These values were similar to those obtained after 100 microM NMDA. In this regard, retinal tissue appeared to be less sensitive to excitotoxicity based on the activation of NMDA receptor subtype. All three agents produced histopathological lesions typical for excitotoxic damage. A delayed form of excitotoxicity observed in retina segments was predominated by necrotic features. However, the activation of apoptotic machinery early during the incubation period subsequent to brief exposure to NMDA (100 microM) was also present. The activation of caspase enzymes was studied by a fluorometric protease activity assay as well as by western blot analysis. Caspase-3-like activity reached the highest value within 3 h of incubation after exposure to excitotoxin, then the level of enzyme activity declined to lower values. As confirmed by a time-related appearance of TUNEL-positive nuclei, apoptotic features appeared to be specific for retina response to NMDA. In contrast, the exposure to a 50 microM concentration of KA or AMPA induced necrotic cell damage which was evident through the incubation, leading to a delayed mechanism of excitotoxicity. These observations provide evidence that in the retinal model, with regard to agonist concentrations and subtype of glutamate receptors, the cascade of events leading to excitotoxicity may result in either apoptotic or necrotic neuronal cell damage.
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PMID:Apoptosis and necrosis occurring in excitotoxic cell death in isolated chick embryo retina. 1159 59

Prolonged liver ischemia followed by reperfusion (I/R) causes functional and structural damage to liver cells, resulting in necrosis and apoptosis. c-jun N-terminal kinase 1/stress-activated protein kinase 1 (JNK(1)/SAPK(1)) is activated during I/R and participates in the onset of the apoptosis program. Excessive blood loss during surgery can hinder postoperative recovery. Intermittent portal triad clamping (PTC) is better tolerated than prolonged continuous ischemia. This study was designed to demonstrate that intermittent ischemia could improve postischemic survival rates by a decrease of JNK(1)/SAPK(1) and caspase 3 activation, which were involved in the apoptosis process. Rats were subjected to intermittent 1-hour ischemia (15-minute ischemia/5-minute reperfusion, 4 times), followed by 220-minute reperfusion, or to continuous ischemia (1 hour), followed by 240-minute reperfusion. Mortality rates were assessed on day 7. Serum aspartate transaminase (AST), alanine transaminase (ALT), and lactate dehydrogenase levels (LDH) were measured 6 hours after ischemia. This study was completed in primary cultured isolated rat hepatocytes, subjected to the same continuous or intermittent hypoxic conditions. The activation status of JNK(1)/SAPK(1) was evaluated by immunoprecipitation or Western blotting experiments. Apoptosis was assessed by measuring caspase activation and by terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL) reaction. Eighty percent of the intermittent-ischemia group was alive 7 days after surgery and serum enzyme levels were significantly decreased. Intermittent hypoxia or ischemia did not lead to JNK(1)/SAPK(1) activation, but at least 3 hypoxia-reoxygenation (H/R) sets were necessary to inhibit kinase activation. Consequently, caspase 3 activation and apoptosis were dramatically reduced. Intermittent ischemia is a powerful, protective way to reduce I/R damage of the liver, by reduction of JNK(1)/SAPK(1) activation associated with a down-regulation of caspase 3 activity, which leads to inhibition of hepatocyte apoptosis.
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PMID:Intermittent ischemia reduces warm hypoxia-reoxygenation-induced JNK(1)/SAPK(1) activation and apoptosis in rat hepatocytes. 1167 68

The contributions of calpain and caspase-3 to apoptosis and necrosis after central nervous system (CNS) trauma are relatively unexplored. No study has examined concurrent activation of calpain and caspase-3 in necrotic or apoptotic cell death after any CNS insult. Experiments used a model of oxygen-glucose deprivation (OGD) in primary septo-hippocampal cultures and assessed cell viability, occurrence of apoptotic and necrotic cell death phenotypes, and protease activation. Immunoblots using an antibody detecting calpain and caspase-3 proteolysis of alpha-spectrin showed greater accumulation of calpain-mediated breakdown products (BDPs) compared with caspase-3-mediated BDPs. Administration of calpain and caspase-3 inhibitors confirmed that activation of these proteases contributed to cell death, as inferred by lactate dehydrogenase release. Oxygen-glucose deprivation resulted in expression of apoptotic and necrotic cell death phenotypes, especially in neurons. Immunocytochemical studies of calpain and caspase-3 activation in apoptotic cells indicated that these proteases are almost always concurrently activated during apoptosis. These data demonstrate that calpain and caspase-3 activation is associated with expression of apoptotic cell death phenotypes after OGD, and that calpain activation, in combination with caspase-3 activation, could contribute to the expression of apoptotic cell death by assisting in the degradation of important cellular proteins.
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PMID:Concurrent assessment of calpain and caspase-3 activation after oxygen-glucose deprivation in primary septo-hippocampal cultures. 1170 43

Lactic acid produced from the cells is a potential cause of extra- and intracellular acidification. Due to scarce technical tools, lactic acid that leads to acidification could not be reduced and direct evidence of the relationship between metabolic lactate and apoptosis has not yet been elucidated. In this study, we designed a cellular pH regulation system in CHO cells by a reduction of lactate dehydrogenase (LDH) activity through LDH antisense mRNA expression. This inhibited lactate production and, therefore, acidification of the cytosol. Under HCO3(-)-buffered growth conditions, both the parent CHO cells and the engineered CHO cells maintained their extracellular pH and intracellular pH fairly well. However, upon acidification of the cytosol, only the parent CHO cells underwent apoptosis under HCO3(-)-free conditions. In fact, we observed a number of apoptosis-related events only in control cells, including mitochondrial dysfunction, cytochrome c release, and an increase in caspase-3 enzymatic activity.
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PMID:Blocking of acidosis-mediated apoptosis by a reduction of lactate dehydrogenase activity through antisense mRNA expression. 1174 11

Apoptosis may be a major event in chemical-induced injury, and therefore the detection of apoptotic effects when developing new drugs is highly relevant in screening for pharmacotoxicological risk assessment. However, as apoptosis in vitro normally degenerates to secondary necrosis, it is possible that it is underestimated, unless sensitive and specific parameters are used. In this present study we have evaluated the usefulness of a set of markers associated with the pivotal steps in the execution phase of apoptosis, in order to detect apoptotic compounds in hepatocytes before significant necrosis takes place. The markers selected include several biochemical parameters (downregulation of the antiapoptotic bclX(L) gene, caspase-3 activation, and cytochrome C release from mitochondria), and flow cytometry determinations (analysis of the size of the nuclei, chromatin complexity, and DNA integrity). The effects of several well-known model apoptotic toxicants (galactosamine, tertiary-butyl-hydroperoxide, etoposide, campothecine, and curcumin) were analyzed in hepatocytes. The aim was to identify early markers of apoptosis using known inducers of apoptosis in hepatocytes, as this battery of markers is designed to identify compounds triggering apoptosis in hepatocytes prior to necrosis. Concentrations of the compounds, as low as possible in order to keep 90% of hepatocyte viability, were selected according to their intracellular lactate dehydrogenase (LDH) leakage, which is well known as an indicator of cell membrane integrity and cell viability. The results demonstrated that (1) the apoptotic effect of 4 out of 5 compounds could be detected in low concentrations of the drugs long before cell necrosis (tertiary-butyl-hydroperoxide-induced apoptosis was only detected at concentrations causing concomitant necrosis) and (2) among the markers evaluated, caspase 3 activation and nucleus and DNA analysis by flow cytometry were used to fulfil the compromise between reliability, sensitivity, and ease of performance, which are critical issues when screening for an apoptotic effect of newly developed drugs.
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PMID:Sensitive markers used to identify compounds that trigger apoptosis in cultured hepatocytes. 1181 34

1. Caspases and calpains are mediators of apoptotic cell death. The objective of this study was to determine the role of caspases and calpains in primary cerebrocortical neuronal (CCN) death in response to a range of stimuli which reportedly induce neuronal apoptosis. 2. Cell death of primary cultures of rat CCN was induced by staurosporine (STS), C2-ceramide (CER), camptothecin (CMT), hydrogen peroxide (H(2)O(2)) or N-methyl-D-aspartate (NMDA). Caspase and calpain activity were assessed by cleavage of alpha-fodrin or fluorogenic substrates. 3. Cell death was analysed by lactate dehydrogenase (LDH) assay in the absence or presence of the pan-caspase inhibitor Boc-Asp-(OMe)-Fluoromethylketone (Baf) and/or the calpain inhibitor calpeptin (CP). Cell death induced by STS, CER or CMT was accompanied by chromatin condensation and activation of multiple caspases, particularly caspase-3-type proteases. Hydrogen peroxide (H(2)O(2)) treatment was accompanied by activation of caspases -1, -6 and -8, but not -3, whereas none of the caspases tested were activated in response to NMDA. 4. With the exception of H(2)O(2), when cell death was accompanied by caspase activation, it was significantly suppressed by Baf. 5. All stimuli also induced calpain activation, but calpeptin only suppressed cell death induced by H(2)O(2). Furthermore, co-treatment with Baf and calpeptin did not alter the cell death relative to either inhibitor alone. 6. These findings suggest the existence of stimulus-dependent routes for the activation of caspases and calpains during death of cortical neurones and imply that although caspases and calpains are activated, their involvement in the execution of cell death varies with the stimulus.
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PMID:Involvement of caspases and calpains in cerebrocortical neuronal cell death is stimulus-dependent. 1186 36

The inhibitor of caspase-3-activated DNase (ICAD) is a caspase-3 substrate that controls nuclear apoptosis. ICAD has two isoforms: a functional isoform of M(r) 45,000, ICAD-L/DNA fragmentation factor (DFF) 45; and a M(r) 35,000 isoform, ICAD-S/DFF35. ICAD-deficient murine cells display resistance to apoptotic stimuli and absence of typical nuclear changes of apoptosis. Our aim was to: (a) characterize the ICAD expression in several human colonic cancer cell lines compared with human normal colonocytes; and (b) correlate the phenotypic features of apoptosis to the level of ICAD expression. ICAD expression was assessed by immunoblot analysis. Early markers of apoptosis of cultured cells included lactate dehydrogenase retention in dying cells, cytokeratin 18 cleavage, and caspase-3 activation. Nuclear markers of apoptosis were assessed by Hoechst staining of nuclei, electron microscopy, and DNA electrophoresis. Inhibition of caspases was performed using a broad-spectrum caspase inhibitor, z-Val-Ala-Asp-fluoromethyl ketone. ICAD expression was restricted to the functional ICAD-L/DFF45 isoform in colonic cancer cells as well as in human normal colonocytes. In a clonal derivative of HT29 cells (HT29-Cl.16E cells), ICAD expression was found to be down-regulated during the exponential phase of growth, and the cell death triggered by IFN-gamma, anti-Fas antibody plus Adriamycin was characterized by the expression of early markers of apoptosis, whereas the key nuclear features of apoptosis were absent. In contrast, exposure of confluent cells to this treatment led to a typical apoptotic nuclear fragmentation. Both forms of apoptosis, in exponentially growing and confluent cells, were sensitive to the broad spectrum inhibitor of caspases, z-Val-Ala-Asp-fluoromethyl ketone. Our findings support the concept that the expression of ICAD is essential to the execution of full-blown apoptosis in colonic cancer cells. Altogether, our results point to ICAD as a potential target for restoring a normal apoptotic signal transduction pathway in colonic cancer cells.
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PMID:Growth phase-dependent expression of ICAD-L/DFF45 modulates the pattern of apoptosis in human colonic cancer cells. 1192 40

Livers can be preserved only for a short period without jeopardizing the transplantation outcome. Heat shock proteins (HSPs) protect against ischemia and reperfusion injury. We studied whether their induction and, in particular, the induction of heme oxygenase 1 (HO-1), improves transplantation survival after an extended time of cold storage. Rats were subjected to heat preconditioning (42 degrees C for 20 minutes). Livers were harvested 24 hours later, preserved in cold University of Wisconsin solution for 44 hours, and transplanted in isogeneic rats (arterialized transplantation). HO-1 was specifically induced and inhibited by cobalt protoporphyrin and tin protoporphyrin, respectively. All animals receiving a graft without preconditioning and subjected to 44 hours of cold preservation died within 3 days, whereas 89% of rats who received a graft exposed to heat survived for 3 weeks (P =.0004). Preconditioning reduced serum aspartate transaminase (AST) and lactate dehydrogenase activities after reperfusion, improved bile flow, and decreased the histologic lesions of reperfusion injury. These significant effects of heat preconditioning were prevented by administration of tin protoporphyrin and could be reproduced by administration of cobalt protoporphyrin. In grafts without preconditioning, only a small fraction (<5%) of hepatocytes were positive with the terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay, and even less expressed activated caspase 3. Preconditioning tended to reduce the number of positive cells and to stimulate the expression of antiapoptotic Bcl-X(L). In conclusion, heat preconditioning and, specifically, overexpression of HO-1 improve posttransplantation survival and graft function after prolonged cold ischemia preservation. The mechanism underlying these beneficial effects does not appear to be prevention of apoptosis.
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PMID:Extended preservation of rat liver graft by induction of heme oxygenase-1. 1198 58

The hepatotoxins and neurotoxins produced by bloom-forming cyanobacteria have been the cause of human and animal health hazards and even death. The most common cyanobacterial neurotoxin is anatoxin-a, and intoxications by these toxins can be fatal through muscular paralysis causing respiratory arrest. We report here anatoxin-a-induced apoptosis in two non-neuronal cells, viz. cultured rat thymocytes and African green monkey kidney cells (Vero). Anatoxin-containing cell-free extracts (ACE) from Anabena flosaquae and purified anatoxin-a were used in the study. The toxin-induced cytotoxicity was characterized by loss of viability, lactate dehydrogenase leakage, loss of mitochondrial function, and DNA fragmentation. The toxin-induced apoptosis was characterized by plasma membrane blebbing, condensed chromatin, nuclear fragmentation and formation of apoptotic bodies. Toxin-treated thymocytes showed typical internucleosomal DNA fragmentation in agarose gel electrophoresis. Ultrastructure studies confirmed the apoptotic morphology in thymocytes. ACE and anatoxin-a showed caspase-3 activation, and pretreatment with the caspase-3-specific tetrapeptide inhibitor, acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) abolished the DNA fragmentation and reduced the incidence of apoptotic cells. The thymocytes also showed dose- and time-dependent toxin-induced generation of reactive oxygen species. The study demonstrates that anatoxin-induced apoptosis is possibly mediated by generation of reactive oxygen species and caspase activation.
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PMID:Involvement of caspase and reactive oxygen species in cyanobacterial toxin anatoxin-a-induced cytotoxicity and apoptosis in rat thymocytes and Vero cells. 1202 86

C8-ceramide, a synthetic cell-permeable analog of endogenous ceramides, interfered with cell proliferation, and was cytotoxic to papilloma virus-containing human cervix carcinoma cells, CALO, INBL, and HeLa, that match two clinical stages of tumor progression. C8-ceramide (3 microM) markedly reduced the tumor cell number after 48 h of treatment, an effect that endured even after the removal of C8-ceramide. The carcinoma cells showed morphologic changes, characteristic of necrosis and released lactate dehydrogenase (LDH). A biologically inactive analog C8-dihydro-ceramide had no effect on cell viability in any of the cell lines tested. Seventy-two hours after C8-ceramide treatment none of the biochemical and morphological markers characteristic of apoptosis: (a) nuclear chromatin condensation, (b) DNA fragmentation, (c) proteolysis of the caspase-3 substrate poly-(ADP-ribose)-polymerase (PARP), and (d) appearance of phosphatidylserine on the external cell membrane, were observed. C8-ceramide had no effect on human cervix fibroblasts and induced a mild reduction (30%) in the proliferation of normal human cervix epithelia and HeLa cells (IV-B metastatic stage). The cytotoxicity of C8-ceramide was restricted to CALO (early II-B) and INBL (IV-A non-metastatic) carcinoma cells. The possible application of ceramide in the treatment of early stages of cervical cancer is discussed.
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PMID:Ceramide promotes the death of human cervical tumor cells in the absence of biochemical and morphological markers of apoptosis. 1205 63


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