Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.22.61 (caspase-8)
 6,833 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Neural stem cells (NSC) undergo apoptotic cell death as an essential component of neural development. Here, we present the results of our studies on the mechanisms by which NSC undergo cell death in response to neurotoxic insults. As experimental models we used primary culture of adult NSC from the subventricular zone of the rat brain, and the neural stem cell line C17.2 initially derived from developing mouse cerebellum. NSC undergo apoptosis in response to staurosporine (0.25 microM) as well as agents inducing oxidative stress such as 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). Exposed cells demonstrate an apoptotic morphology, positive TUNEL staining and phosphatidyl serine exposure as labeled with Annexin V. Using an antibody specific for cytochrome c, we found that cells exposed to staurosporine or DMNQ exhibited diffuse fluorescence throughout the cytosol, implying a release of cytochrome c from the mitochondria. In addition to positive immunoreactivity against the active fragment (p17) of caspase-3, the administration of the pan-caspase inhibitor, zVAD-fmk (40 microM), prevents apoptosis. Both NSC and C17.2 express the Fas receptor, and procaspase-8, but exposure to agonistic Fas mAb (250 ng/ml) fails to induce apoptosis. Pretreatment with cycloheximide or actinomycin D does not influence the cell response to Fas mAb, suggesting that the endogenous inhibitor of caspase-8 FLICE-inhibitory protein (FLIP) is not responsible for the inhibition of the Fas pathway. Thus, it appears that the Fas dependent cell death pathway is not operative in these cells, while the mitochondrial pathway is active and caspase-3 serves as an executioner caspase in the apoptotic machinery. It is known that Fas not only induces apoptosis, but can also deliver growth stimulatory signals through activation of the extracellular-signal regulated kinase (ERK) pathway. The Fas-induced ERK phosphorylation that we detect in C17.2 cells suggests that in NSC Fas may function as a mediator of growth rather than death.
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PMID:Neural stem cells and cell death. 1509 49

Hepatotoxicity is the major complaint during therapy with lipid-lowering agents such as statins, although the cellular mechanisms underlying the statin-induced liver injury are not fully understood. Using cultured human hepatocytes, we investigated the effects of lipophilic as well as hydrophilic statins on the cell viability. Lipophilic statins, including simvastatin, lovastatin, cerivastatin, fluvastatin and atorvastatin, reduced the viability of hepatocytes as assessed by the mitochondrial enzyme activity to reduce WST-8, however, a hydrophilic pravastatin did not cause cell injury. The simvastatin-induced loss of cell viability was attenuated by mevalonate or geranylgeranyl pyrophosphate. Simvastatin-induced DNA fragmentation and increased the number of cells stained with annexin V and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, both of which were reversed by caspase inhibitors such as zDEVD-fmk, zLEHD-fmk and zIETD-fmk. Consistent with these data, the activities of caspase-3, caspase-9 and caspase-8 were elevated by simvastatin. Simvastatin reduced the protein content and mRNA expression for bcl-2 without affecting bax mRNA expression. On the other hand, both lipophilic and hydrophilic statins significantly reduced the content of endogenous cholesterol. These findings suggest that lipophilic statins cause an apoptotic injury in human hepatocytes by stimulating caspase-3 subsequent to the activation of caspase-9 and caspase-8, in which the inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase may be involved.
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PMID:Apoptotic injury in cultured human hepatocytes induced by HMG-CoA reductase inhibitors. 1516 49

Apoptotic cell death is characterized by the early exposure of phosphatidylserine (PS) at the outer surface of the plasma membrane. The aim of the present study was to examine whether PS exposure also occurs during oncosis (early primary necrosis) and to localize PS at the subcellular level, applying a pre-embedding immunogold labeling technique with biotin conjugated annexin V. The issue was addressed by using caspase-8 deficient, Bcl-2 overexpressing JB6 cells, which die by oncosis when stimulated with synthetic dsRNA. We observed by fluorescence microscopy that oncotic cells with preserved plasma membrane integrity showed PS exposure (annexin+/propidium iodide-). The data was confirmed on the ultrastructural level and PS was localized in oncosis at the outer leaflet of the continuous plasma membrane with preserved trilamellar structure. In postoncotic necrotic cells the immunogold labels were found on the plasma membrane and on the intracellular membranes of the cells, which underwent plasma membrane disruption. In conclusion, this study reveals that PS externalization occurs not only in apoptosis but also in oncosis at least in our cell model system.
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PMID:Phosphatidylserine exposure during early primary necrosis (oncosis) in JB6 cells as evidenced by immunogold labeling technique. 1519 32

Apoptosis plays an important role in the regulation of bone turnover. Previously, we showed that 1,25(OH)2D3, the active form of vitamin D, may increase osteoblast survival by inhibiting apoptosis induced by serum deprivation. Human osteoblasts express the Fas receptor on their surface and its interaction with Fas ligand has been closely associated with human osteoblast apoptosis. To investigate the mechanism of 1,25(OH)2D3 inhibition of apoptosis in osteoblasts isolated from human calvaria, cells were exposed to Fas antibody. Visualization of apoptotic cells using annexin V revealed a significant decrease in apoptosis at 48 h in the presence of 1,25(OH)2D3 (14 +/- 4%, P < 0.04) compared with non-treated cells (52 +/- 4%). Furthermore, flow cytometric analysis of TUNEL-labeled osteoblasts showed a significant decrease in apoptotic cells in 1,25(OH)2D3-treated cultures (12 +/- 2%) at 48 h compared with non-treated cultures (44 +/- 3%, P < 0.04). Additionally, cells treated with 1,25(OH)2D3 survived longer as found by MTS analysis. To further explore the mechanism of 1,25(OH)2D3-mediated inhibition of apoptosis, we examined the changes in activation of death domain proteins, cleavage of caspases and mitochondrial regulators of apoptosis by Western blot analysis. A significant inhibition of caspase-8 cleavage and activity in 1,25(OH)2D3-treated cells was observed in conjunction with a decrease in the expression of the proapoptotic protein Bax with a significant increase in the expression of antiapoptotic protein Bcl-2. Furthermore, the levels of p21Cip1/WAF1, which inhibits the cleavage of caspase-8, was found to be highly induced in 1,25(OH)2D3-treated cells. In summary, these results demonstrate that the anti-apoptotic effect of 1,25(OH)2D3 in human osteoblasts after the activation of Fas-ligand is mediated by the regulation of components of both the mitochondrial and Fas-related pathways.
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PMID:Vitamin D inhibits Fas ligand-induced apoptosis in human osteoblasts by regulating components of both the mitochondrial and Fas-related pathways. 1520 41

Lats2, also known as Kpm, is the second mammalian member of the novel Lats tumor suppressor gene family. Recent studies have demonstrated that Lats2 negatively regulates the cell cycle by controlling G1/S and/or G2/M transition. To further understand the role of Lats2 in the control of human cancer development, we have expressed the protein in human lung cancer cells by transduction of a replication-deficient adenovirus expressing human Lats2 (Ad-Lats2). Using a variety of techniques, including Annexin V uptake, cleavage of PARP, and DNA laddering, we have demonstrated that the ectopic expression of human Lats2 induced apoptosis in two lung cancer cell lines, A549 and H1299. Caspases-3, 7, 8, and 9 were processed in the Ad-Lats2-transduced cells; however, it was active caspase-9, not caspase-8, that initiated the caspase cascade. Inhibitors specific to caspase-3 and 9 delayed the onset of Lats2-mediated apoptosis. Western blot analysis revealed that anti-apoptotic proteins, BCL-2 and BCL-x(L), but not the pro-apoptotic protein, BAX, were downregulated in Ad-Lats2-transduced human lung cancer cells. Overexpression of either Bcl-2 or Bcl-x(L) in these cells lead to the suppression of Lats2-mediated caspase cleavage and apoptosis. These results show that Lats2 induces apoptosis through downregulating anti-apoptotic proteins, BCL-2 and BCL-x(L), in human lung cancer cells.
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PMID:Putative tumor suppressor Lats2 induces apoptosis through downregulation of Bcl-2 and Bcl-x(L). 1526 83

Apoptosis appears to be the death mechanism of pericyte loss observed in diabetic retinopathy. We have previously shown that advanced glycation end-products (AGE-MGX) induce apoptosis of retinal pericytes in culture associated with diacylglycerol (DAG)/ceramide production. In the present study, we investigated possible caspase involvement in this process. Bovine retinal pericytes (BRP) were cultured with AGE-MGX and apoptosis examined after annexin V staining. Effects of peptidic inhibitors of caspases were determined on DAG/ceramide production and apoptosis. Pan-caspase inhibitor z-VAD-fmk (50 microM) was able to inhibit both DAG/ceramide production and apoptosis, whereas caspase-3-like inhibitor z-DEVD-fmk (50 microM) or caspase-9 inhibitor z-LEHD-fmk (50 microM) was only active on apoptosis. This differential effect strongly suggests involvement of initiator caspase(s) upstream and effector caspase(s) downstream DAG/ceramide production in AGE-mediated apoptosis. Pericyte treatment with caspase-8 inhibitor z-IETD-fmk (50 microM) did not protect cells against AGE-induced apoptosis and we failed to detect caspase-8 in pericytes by immunoblotting assay. Interestingly, one inhibitor of caspase-10 and related caspases z-AEVD-fmk (50 microM) inhibited both AGE-MGX-induced apoptosis and DAG/ceramide formation in pericytes. Cleavage of caspase-10 precursor into its active subunits was demonstrated by immunoblotting assay in pericytes incubated with AGE-MGX. These results strongly suggest that caspase-10, but not caspase-8, might be involved in the early phase of AGE-induced pericyte apoptosis, in contrast to caspase-9 and -3-like enzymes involved after DAG/ceramide production. This finding may provide new therapeutic perspectives for early treatment in diabetic retinopathy.
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PMID:Involvement of caspase-10 in advanced glycation end-product-induced apoptosis of bovine retinal pericytes in culture. 1527 46

Cisplatin (CDDP) is among the most widely used and most effective chemotherapeutic agent for many types of human cancer. Because killing cancer cells by chemotherapy is principally executed by apoptosis, a defective apoptotic program might acquire drug resistance. Flow cytometric Annexin V assay demonstrated that HEp-2 cells (human laryngeal cancer) were persistently resistant to CDDP as compared to HeLa cells (human uterine cervical cancer), despite the same histological type and wild-type p53 status. CDDP treatment caused steady induction of p53 protein in both cancer cell types, although it was more dramatic in CDDP-resistant HEp-2 cells, which was correlated well with p53 Ser15 phosphorylation, but not with the expression level of HPV type 18 E6 oncoprotein in these cells. Importantly, CDDP differently activated caspase cascades between HEp-2 and HeLa cells. CDDP activated the caspase-8 pathway through TNFR superfamily receptors such as Fas, but not caspase-9 in HeLa cells. On the other hand, the caspase-9 pathway was significantly activated in HEp-2 cells, although the activation of caspase-8 by CDDP was deficient. This different response to CDDP in caspase-8 activation was not related with the expression level of either Fas or FasL in these cells. We concluded from these results that loss of the caspase-8 activation pathway in HEp-2 cells was a possible mechanism for its resistance to CDDP-induced apoptosis. The caspase-8 pathway might play an important role in CDDP-induced apoptosis in HPV-positive human squamous cell carcinomas.
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PMID:Loss of caspase-8 activation pathway is a possible mechanism for CDDP resistance in human laryngeal squamous cell carcinoma, HEp-2 cells. 1528 75

Bilirubin is the principal end product of heme degradation. Prompted by epidemiologic analyses demonstrating an inverse correlation between serum bilirubin levels and cancer mortality, we examined the effect(s) of bilirubin on the growth and survival of colon adenocarcinoma cells. Adenocarcinoma cell monolayers were treated with bilirubin over a range of bilirubin:BSA molar ratios (0-0.6), and viability was assessed colorimetrically. Apoptosis was characterized by TUNEL assay, annexin V staining and caspase-3 activation. The mechanism(s) by which bilirubin induces apoptosis was investigated by Western blotting for cytochrome c release, assaying for caspase-8 and caspase-9 activation and for mitochondrial depolarization by JC-1 staining. The direct effect of bilirubin on the membrane potential of isolated mitochondria was evaluated using light-scattering and fluorescence techniques. Bilirubin decreased the viability of all colon cancer cell lines tested in a dose-dependent manner. Cells exhibited substantial apoptosis when exposed to bilirubin concentrations ranging 0-50 microM, as demonstrated by an 8- to 10-fold increase in TUNEL and annexin V staining and in caspase-3 activity. Bilirubin treatment evokes specific activation of caspase-9, enhances cytochrome c release into the cytoplasm and triggers the mitochondrial permeability transition in colon cancer monolayers. Additionally, bilirubin directly induces the depolarization of isolated rat liver mitochondria, an effect that is not inhibited by cyclosporin A. Bilirubin stimulates apoptosis of colon adenocarcinoma cells in vitro through activation of the mitochondrial pathway, apparently by directly dissipating mitochondrial membrane potential. As this effect is triggered at concentrations normally present in the intestinal lumen, we postulate a physiologic role for bilirubin in modulating colon tumorigenesis.
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PMID:Unconjugated bilirubin induces apoptosis in colon cancer cells by triggering mitochondrial depolarization. 1538 69

alpha-Santalol, an active component of sandalwood oil, has been studied in detail in recent years for its skin cancer preventive efficacy in murine models of skin carcinogenesis; however, the mechanism of its efficacy is not defined. Two major biological events responsible for the clonal expansion of transformed/initiated cells into tumors are uncontrolled growth and loss of apoptotic death. Accordingly, in the present study, employing human epidermoid carcinoma A431 cells, we assessed whether alpha-santalol causes cell growth inhibition and/or cell death by apoptosis. Treatment of cells with alpha-santalol at concentrations of 25-75 microM resulted in a concentration- and a time-dependent decrease in cell number, which was largely due to cell death. Fluorescence-activated cell sorting analysis of Annexin V/propidium iodide (PI) stained cells revealed that alpha-santalol induces a strong apoptosis as early as 3 h post-treatment, which increases further in a concentration- and a time-dependent manner up to 12 h. Mechanistic studies showed an involvement of caspase-3 activation and poly(ADP-ribose) polymerase cleavage through activation of upstream caspase-8 and -9. Further, the treatment of cells with alpha-santalol also led to disruption of the mitochondrial membrane potential and cytochrome c release into the cytosol, thereby implicating the involvement of the mitochondrial pathway. Pre-treatment of cells with caspase-8 or -9 inhibitor, pan caspase inhibitor or cycloheximide totally blocked alpha-santalol-caused caspase-3 activity and cleavage, but only partially reversed apoptotic cell death. This suggests involvement of both caspase-dependent and -independent pathways, at least under caspase inhibiting conditions, in alpha-santalol-caused apoptosis. Together, this study for the first time identifies the apoptotic effect of alpha-santalol, and defines the mechanism of apoptotic cascade activated by this agent in A431 cells, which might be contributing to its overall cancer preventive efficacy in mouse skin cancer models.
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PMID:Skin cancer chemopreventive agent, {alpha}-santalol, induces apoptotic death of human epidermoid carcinoma A431 cells via caspase activation together with dissipation of mitochondrial membrane potential and cytochrome c release. 1552 19

Apoptosis occurs during the isolation and even short-term storage and culture of hepatocytes, and in the pathogenesis of liver diseases, such as hepatic failure and hepatitis. Therapeutic hypothermia has beneficial effects in experimental models of fulminant hepatic failure. The mechanisms underlying the potential benefits of mild hypothermia on the liver have not been well investigated. We examined the effects of temperature on soluble Fas ligand-induced apoptosis in freshly isolated mouse hepatocytes. Decreasing the culture temperature from 37 degrees C to 32 degrees C produced significant suppression of Fas-mediated apoptosis in cultured hepatocytes over a 12-h period. This observation was supported by cell morphology, flow cytometry analysis of cellular DNA content, and Annexin V-FITC staining of membrane phosphatidylserine translocation. In hypothermic conditions, Fas-mediated cytochrome c release from mitochondria of hepatocytes and the proximate downstream activation of caspase-9 were suppressed under mild hypothermic conditions. Effector caspase-7 activity was also inhibited at 32 degrees C. In contrast, the activation of initiator caspase-8 and cleavage of Bid were not affected after Fas-ligand stimulation. These findings suggest that mild hypothermia suppresses Fas-mediated apoptosis of liver cells by the partial inhibition of signaling events including mitochondrial damage, cytochrome c release, and subsequent apoptosome formation and effector caspase activation.
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PMID:Hypothermia inhibits Fas-mediated apoptosis of primary mouse hepatocytes in culture. 1564 37


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