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Caspase Activity (caspase + activity)
Selected AbstractsProgrammed cell death of the ovarian nurse cells during oogenesis of the silkmoth Bombyx moriDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 7 2006Vicky E. Mpakou In the present study, we describe the features of programmed cell death of the ovarian nurse cells occurring during vitellogenesis of the silkmoth Bombyx mori. At developmental stage 5, the nurse cells occupy one-half of the follicular volume and obtain a rather spherical shape, while the nurse cell nuclei appear large and elongated, forming impressive projections. At the following stage, stage 6, the nurse cells decrease in size and their shape becomes elliptic. The nuclei remain elongated, being also characterized by large lobes. The lobes of the ramified nurse cell nuclei seem to retain the nucleus in the center of the cell during the dumping of the nurse cell cytoplasm into the growing oocyte. At stage 7, membrane enclosed vacuoles can be easily detected into the nurse cells cytoplasm. Ultrastructural analysis and fluorescent microscopy using mono-dansyl-cadaverine staining of these vacuoles also reveal that they represent autolysosomes. Caspase activity is detected during stage 7, as it is demonstrated by using the Red-VAD-FMK staining reagent. At developmental stages 8 and 9, the nurse cells exhibit chromatin condensation, DNA fragmentation and caspase activity. Finally, during the following stage 10, the nuclear remnants are assembled into apoptotic vesicles, which, after being phagocytosed, are observed in the cytoplasm of adjacent follicle cells. We propose that apoptosis and autophagy operate synergistically during vitellogenesis of B. mori, in order to achieve an efficient and rapid clearance of the degenerated nurse cell cluster. [source] The developing embryonic cardiac outflow tract is highly sensitive to oxidant stressDEVELOPMENTAL DYNAMICS, Issue 12 2007Steven A. Fisher Abstract This study tested the hypothesis that the remodeling of the cardiac outflow tract (OFT) may represent a developmental window of vulnerability to reactive oxygen species (ROS). Chick embryos were exposed in ovo or ex ovo to increasing concentrations of the stable oxidant hydrogen peroxide (H2O2). As assessed by trypan blue staining, H2O2 induced cell injury in the stage 25,30 OFT at concentrations as low as 1 nM. Higher concentrations were required to induce cell injury in the ventricular and atrial myocardium. Using DCFDA as an indicator of oxidant stress, H2O2 also induced a greater fluorescent signal in the OFT myocardium. H2O2 at these low concentrations also induced Caspase activity, indicative of activation of the pathway of PCD. Interestingly, the induction of Caspase-3 activity was predominately in the OFT cushion mesenchymal cells. Thus, the developing OFT is particularly sensitive to ROS-mediated injury, suggesting that ROS could play a role in the development of congenital defects of the cardiac OFT. Developmental Dynamics 236:3496,3502, 2007. © 2007 Wiley-Liss, Inc. [source] Granzyme B: a natural born killerIMMUNOLOGICAL REVIEWS, Issue 1 2003Sarah J. Lord Summary:, A main pathway used by cytotoxic T lymphocytes (CTLs) and natural killer cells to eliminate pathogenic cells is via exocytosis of granule components in the direction of the target cell, delivering a lethal hit of cytolytic molecules. Amongst these, granzyme B and perforin have been shown to induce CTL-mediated target cell DNA fragmentation and apoptosis. Once released from the CTL, granzyme B binds its receptor, the mannose-6-phosphate/insulin-like growth factor II receptor, and is endocytosed but remains arrested in endocytic vesicles until released by perforin. Once in the cytosol, granzyme B targets caspase-3 directly or indirectly through the mitochondria, initiating the caspase cascade to DNA fragmentation and apoptosis. Caspase activity is required for apoptosis to occur; however, in the absence of caspase activity, granzyme B can still initiate mitochondrial events via the cleavage of Bid. Recent work shows that granzyme B-mediated release of apoptotic factors from the mitochondria is essential for the full activation of caspase-3. Thus, granzyme B acts at multiple points to initiate the death of the offending cell. Studies of the granzyme B death receptor and internal signaling pathways may lead to critical advances in cell transplantation and cancer therapy. [source] Caspase activity in newt spermatogonial apoptosis induced by prolactin and cycloheximideMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2001Takashi Yazawa Abstract We previously showed in vivo and in vitro, that among the spermatogenic stages of the newt, prolactin (PRL) induces apoptosis specifically in the penultimate stage of secondary spermatogonia. In the current report, we demonstrate in vitro that cycloheximide (CHX), an inhibitor of protein synthesis, induces morphological apoptotic changes similar to those caused by PRL, such as chromatin condensation and apoptotic body formation. Next, we found that Z-VAD-fmk, an inhibitor of various caspases, suppressed the apoptosis induced by PRL and CHX, but ICE inhibitor Ac-YVAD-CHO or caspase-3 inhibitor Ac-DEVD-CHO did not. As high caspase activity was present in extracts of testes treated with CHX, we suggest that an unidentified caspase induces the morphological changes of apoptosis in newt spermatogonia. Mol. Reprod. Dev. 59:209,214, 2001. © 2001 Wiley-Liss, Inc. [source] The combination of the antitumoural pyridyl cyanoguanidine CHS 828 and etoposide in vitro,from cytotoxic synergy to complete inhibition of apoptosisBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2002P Martinsson The present study was aimed at elucidating the apoptosis inhibitory properties of the cyanoguanidine CHS 828. CHS 828 exhibits impressive cytotoxic activity in vitro and in vivo. Apoptosis is not its main mode of cytotoxic effect, and we have previously proposed a dual mechanism, where CHS 828 inhibits its own cell death pathways. Etoposide on the other hand, is a well-established anticancer agent with documented effect in a number of malignancies, induces apoptosis through extensively studied caspase dependent pathways. Here we studied the combined effect of the two drugs in the human lymphoma cell line U-937 GTB. Cytotoxicity was evaluated as total viability measured by the fluorometric microculture cytotoxicity assay (FMCA). Caspase activity was assessed by colorimetric detection of specific cleavage products for caspases 3, 8 and 9, respectively. Morphology was evaluated in May-Grünwald/Giemsa stained preparations. Interaction analysis based on FMCA results of simple combination exposure revealed impressive synergistic effect on cell kill. Detailed investigations of the kinetics involved showed that short pre-exposure (0,12 h) to CHS 828 enhanced caspase activation by etoposide, while longer pre-exposure (18,48 h) inhibited both caspase activation and apoptotic morphology otherwise induced by etoposide. The present results support the theory that CHS 828 block specific cell death pathways. The synergistic results are promising for future combination trials in animals, however, different dosing schedules should be considered, in order to investigate whether the above findings translate into the in vivo setting. British Journal of Pharmacology (2002) 137, 568,573. doi:10.1038/sj.bjp.0704888 [source] Thyroid hormone induces the expression of 4-1BB and activation of caspases in a thyroid hormone receptor-dependent mannerFEBS JOURNAL, Issue 14 2003Toshiko Yamada-Okabe Thyroid hormone has various effects on cell proliferation, growth and apoptosis. To gain more insight into the molecular dynamics caused by thyroid hormone, gene expression in HeLaTR cells that constitutively overexpressed the thyroid hormone receptor (TR) was analyzed. Gene expression profiling of the HeLaTR cells with an oligonucleotide microarray yielded 229 genes whose expression was significantly altered by T3. Among these genes, the expression of 4-1BB, which is known to initiate a signal cascade activating NF-,B, was significantly up-regulated by T3. Although treatment of the HeLaTR cells with T3 did not induce expression of NF-,B reporter luciferase, even in the presence of the 4-1BB-Ligand, it increased the caspase activities. An increase in the caspase activities was also observed in the HeLaTR cells transfected with 4-1BB cDNA, and the 4-1BB-Ligand further increased the caspase activities of the HeLaTR cells overexpressing the 4-1BB. Furthermore, up-regulation of 4-1BB and an increase in caspase activities also occurred in the rat FRTL cells that expressed only authentic TR. These results demonstrate that the expression of 4-1BB serves as the mediator of signals from T3 to activate caspases. [source] Reduced glutathione depletion causes necrosis and sensitization to tumor necrosis factor-,,induced apoptosis in cultured mouse hepatocytesHEPATOLOGY, Issue 1 2002Hidenari Nagai The effect of reduced glutathione (GSH) depletion by acetaminophen (APAP), diethylmaleate (DEM), or phorone on the mode of cell death and susceptibility to tumor necrosis factor (TNF)-induced cell death was studied in cultured mouse hepatocytes. Dose-dependent necrosis was the exclusive mode of cell death with APAP alone, but the addition of TNF-, induced a switch to about half apoptosis without changing total loss of viability. This effect was seen at 1 and 5 mmol/L but was inhibited at 10 and 20 mmol/L APAP. The switch to apoptosis was associated with increased caspase activities, release of cytochrome c, and DNA laddering and was inhibited by caspase inhibitors. DEM and phorone also induced dose-dependent necrosis. Treatment with TNF-, under these conditions lead to incremental cell death in the form of apoptosis at 0.25 and 0.5 mmol/L DEM and 0.1 and 0.2 mmol/L phorone. At 1.0 and 2.0 mmol/L DEM and 0.5 mmol/L phorone, 90% to 100% necrosis was observed with resistance to TNF-, effects. The apoptosis with TNF-, plus DEM was confirmed by DNA laddering and inhibition by caspase inhibitors. However, in the presence of caspase inhibitors, the increment in cell death induced by TNF-, persisted as an increase in necrosis. A combination of antioxidants, vitamin E, and butylated hydroxytoluene (BHT) markedly inhibited necrosis induced by APAP or DEM alone, but the sensitization to TNF-,,induced apoptosis was unaffected. GSH monoethylester (GSH-EE) protected against necrosis and apoptosis. In conclusion, depletion of GSH by APAP, DEM, or phorone causes oxidative stress-induced necrosis and sensitizes to an oxidative stress independent TNF-,,induced apoptosis. [source] Postinjury estrogen treatment of chronic spinal cord injury improves locomotor function in ratsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 8 2010Eric A. Sribnick Abstract Spinal cord injury (SCI) causes loss of neurological function and, depending on serverity, may cause paralysis. The only recommended pharmacotherapy for the treatment of SCI is high-dose methylprednisolone, and its use is controversial. We have previously shown that estrogen treatment attenuated cell death, axonal and myelin damage, calpain and caspase activities, and inflammation in acute SCI. The aim of this study was to examine whether posttreatment of SCI with estrogen would improve locomotor function by protecting cells and axons and reducing inflammation during the chronic phase following injury. Moderately severe injury (40 g · cm force) was induced in male Sprague-Dawley rats following laminectomy at T10. Three groups of animals were used: sham (laminectomy only), vehicle (dimethyl sulfoxide; DMSO)-treated injury group, and estrogen-treated injury group. Animals were treated with 4 mg/kg estrogen at 15 min and 24 hr postnjury, followed by 2 mg/kg estrogen daily for the next 5 days. After treatment, animals were sacrificed at the end of 6 weeks following injury, and 1-cm segments of spinal cord (lesion, rostral to lesion, and caudal to lesion) were removed for biochemical analyses. Estrogen treatment reduced COX-2 activity, blocked nuclear factor-,B translocation, prevented glial reactivity, attenuated neuron death, inhibited activation and activity of calpain and caspase-3, decreased axonal damage, reduced myelin loss in the lesion and penumbra, and improved locomotor function compared with vehicle-treated animals. These findings suggest that estrogen may be useful as a promising therapeutic agent for prevention of damage and improvement of locomotor function in chronic SCI. © 2010 Wiley-Liss, Inc. [source] A caspase inhibitor, IDN-6556, ameliorates early hepatic injury in an ex vivo rat model of warm and cold ischemia,LIVER TRANSPLANTATION, Issue 3 2007Niel C. Hoglen This study examined the efficacy of the caspase inhibitor, IDN-6556, in a rat model of liver ischemia-reperfusion injury. Livers from male Sprague-Dawley rats were reperfused for 120 minutes after 24 hours of 4°C cold storage in University of Wisconsin solution. Portal blood flow measurements estimated sinusoidal resistance, and bile production, alanine aminotransferase activities, and Suzuki scores were evaluated as parameters of hepatocyte/liver injury. Treated livers were exposed to 25 or 50 ,M of IDN-6556 in University of Wisconsin storage solution and/or the perfusate. All treatment regimens with IDN-6556 significantly improved portal blood flow measured at 120 minutes, and significant improvements were seen as early as 30 minutes when inhibitor was also present in the perfusate (P < 0.01). All treatment groups with IDN-6556 significantly increased bile production by 3-4-fold compared with controls (P < 0.01), and reductions in alanine aminotransferase activities were seen within 90 minutes of reperfusion (P < 0.05). These data were confirmed by improved Suzuki scores (less sinusoidal congestion, necrosis, and vacuolization) in all treated groups. Livers from the IDN-6556,treated groups had markedly reduced caspase activities and TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling)-positive cells, suggesting reductions in apoptosis. IDN-6556 present in cold storage media ameliorated liver injury due to cold ischemia and reperfusion injury and may be a rational therapeutic approach to reduce the risk of liver ischemia in the clinical setting. Liver Transpl 13: 361,366, 2007. © 2007 AASLD. [source] Caspase-1/interleukin-1beta signaling in diabetic retinopathyACTA OPHTHALMOLOGICA, Issue 2008S MOHR Purpose The pro-inflammatory cytokine, interleukin-1, (IL-1,), is known to induce vascular dysfunction and cell death. Previously, we have shown that caspase-1 activity is increased in retinas of diabetic and galactosemic mice, and diabetic patients. Therefore, we investigated the role of IL-1, and caspase-1 (the enzyme that produces it) in diabetes-induced degeneration of retinal capillaries. Methods First, we determined the effect of agents known to inhibit caspase-1 (minocycline and tetracycline) on IL-1, production and retinal capillary degeneration in diabetic and galactose-fed mice. Diabetic and galactose-fed mice were injected intraperitoneally with minocycline or tetracycline (5mg/kg). Second, we examined the effect of genetic deletion of the IL-1, receptor on diabetes-induced caspase activities and retinal capillary degeneration using IL-1 receptor knock-out mice. Results At 2 months of diabetes, minocycline inhibited hyperglycemia-induced caspase-1 activity and IL-1, production in the retina. Long-term administration of minocycline prevented retinal capillary degeneration in diabetic (6 months) and galactose-fed (13 months) mice. Tetracycline inhibited hyperglycemia-induced caspase-1 activity in vitro, but not in vivo. Mice deficient in the IL-1, receptor were protected from diabetes-induced caspase activation and retinal pathology at 7 months of diabetes. Conclusion These results indicate that the caspase-1/IL-1, signaling pathway plays an important role in diabetes-induced retinal pathology and its inhibition might represent a new strategy to inhibit capillary degeneration in diabetic retinopathy. [source] Remodeling of the actin cytoskeleton of target hepatocytes and NK cells during induction of apoptosisCYTOSKELETON, Issue 2 2001W. Marty Blom Abstract Natural Killer cells are immune cells that recognize and eliminate altered and non-self cells from the circulation. To study the interaction between NK cells and target cells, we set up an experimental system consisting of rat Interleukin-2 activated Natural Killer cells (A-NK cells) and rat hepatocytes with a masked Major Histocompatibility Complex (MHC). The masking of the MHC induces recognition of the hepatocytes by the NK cells as non-self. We showed that in vitro apoptosis is rapidly induced in the hepatocytes [Blom et al., 1999] after co-incubation with A-NK cells. Now we describe the morphological changes that occur during and after interaction of A-NK cells with hepatocytes. Confocal laser scanning microscopy showed that the actin cytoskeleton of the NK cells was remodeled during attack of hepatocytes. Some NK cells were in close contact with the hepatocytes while others had formed actin-containing dendrites of varying length that made contact with the hepatocytes. However, dendrite formation is not obligatory for induction of apoptosis because cells that were unable to form these did induce FAS-dependent apoptosis in hepatocytes. Apparently both direct as well as distant contact resulted in apoptosis. Formation of the dendrites was calcium-dependent as EGTA largely prevented it. Importantly, chelation of the calcium also suppressed killing of the hepatocytes. Within 1 h after addition of the A-NK cells, morphological changes in hepatocytes that are characteristic of apoptosis, such as the formation of apoptotic bodies and fragmented nuclei, became apparent. Specifically, the actin cytoskeleton of the hepatocytes was remodeled resulting in the formation of the apoptotic bodies. Inhibition of caspase activity by z-Val-Ala-DL-Asp-fluoromethylketone (100 ,M) partly protected against the rearrangement of the actin filaments in the hepatocytes. Cell Motil. Cytoskeleton 49:78,92, 2001. © 2001 Wiley-Liss, Inc. [source] Programmed cell death of the ovarian nurse cells during oogenesis of the silkmoth Bombyx moriDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 7 2006Vicky E. Mpakou In the present study, we describe the features of programmed cell death of the ovarian nurse cells occurring during vitellogenesis of the silkmoth Bombyx mori. At developmental stage 5, the nurse cells occupy one-half of the follicular volume and obtain a rather spherical shape, while the nurse cell nuclei appear large and elongated, forming impressive projections. At the following stage, stage 6, the nurse cells decrease in size and their shape becomes elliptic. The nuclei remain elongated, being also characterized by large lobes. The lobes of the ramified nurse cell nuclei seem to retain the nucleus in the center of the cell during the dumping of the nurse cell cytoplasm into the growing oocyte. At stage 7, membrane enclosed vacuoles can be easily detected into the nurse cells cytoplasm. Ultrastructural analysis and fluorescent microscopy using mono-dansyl-cadaverine staining of these vacuoles also reveal that they represent autolysosomes. Caspase activity is detected during stage 7, as it is demonstrated by using the Red-VAD-FMK staining reagent. At developmental stages 8 and 9, the nurse cells exhibit chromatin condensation, DNA fragmentation and caspase activity. Finally, during the following stage 10, the nuclear remnants are assembled into apoptotic vesicles, which, after being phagocytosed, are observed in the cytoplasm of adjacent follicle cells. We propose that apoptosis and autophagy operate synergistically during vitellogenesis of B. mori, in order to achieve an efficient and rapid clearance of the degenerated nurse cell cluster. [source] Activation of JNK and PAK2 is essential for citrinin-induced apoptosis in a human osteoblast cell lineENVIRONMENTAL TOXICOLOGY, Issue 4 2009Yu-Ting Huang Abstract The mycotoxin citrinin (CTN), a natural contaminant in foodstuffs and animal feeds, exerts cytotoxic and genotoxic effects on various mammalian cells. CTN causes cell injury, including apoptosis. Previous studies by our group showed that CTN triggers apoptosis in mouse embryonic stem cells, as well as embryonic developmental injury. Here, we investigated the precise mechanisms governing this apoptotic effect in osteoblasts. CTN induced apoptotic biochemical changes in a human osteoblast cell line, including activation of c-Jun N-terminal kinase (JNK), loss of mitochondrial membrane potential, and caspase-3 and p21-activated protein kinase 2 (PAK2) activation. Experiments using a JNK-specific inhibitor, SP600125, and antisense oligonucleotides against JNK reduced CTN-induced activation of both JNK and caspase-3 in osteoblasts, indicating that JNK is required for caspase activation in this apoptotic pathway. Experiments using caspase-3 inhibitors and antisense oligonucleotides against PAK2 revealed that active caspase-3 is essential for PAK2 activation. Moreover, both caspase-3 and PAK2 require activation for CTN-induced apoptosis of osteoblasts. Interestingly, CTN stimulates two-stage activation of JNK in human osteoblasts. Early-stage JNK activation is solely ROS-dependent, whereas late-stage activation is dependent on ROS-mediated caspase activity, and regulated by caspase-induced activation of PAK2. On the basis of these results, we propose a signaling cascade model for CTN-induced apoptosis in human osteoblasts involving ROS, JNK, caspases, and PAK2. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2009. [source] Is the Cell Death in Mesial Temporal Sclerosis Apoptotic?EPILEPSIA, Issue 6 2003Hilmi Uysal Summary: Purpose: Mesial temporal sclerosis (MTS) is characterized by neuronal loss in the hippocampus. Studies on experimental models and patients with intractable epilepsy suggest that apoptosis may be involved in neuronal death induced by recurrent seizures. Methods: We searched evidence for apoptotic cell death in temporal lobes resected from drug-resistant epilepsy patients with MTS by using the terminal deoxynucleotidyl transferase (TdT) and digoxigenin-11-dUTP (TUNEL) method and immunohistochemistry for Bcl-2, Bax, and caspase-cleaved actin fragment, fractin. The temporal lobe specimens were obtained from 15 patients (six women and nine men; mean age, 29 ± 8 years). Results: Unlike that in normal adult brain, we observed Bcl-2 immunoreactivity in some of the remaining neurons dispersed throughout the hippocampus proper as well as in most of the reactive astroglia. Bax immunopositivity was increased in almost all neurons. Fractin immunostaining, an indicator of caspase activity, was detected in ,10% of these neurons. Des pite increased Bax expression and activation of caspases, we could not find evidence for DNA fragmentation by TUNEL staining. We also could not detect typical apoptotic changes in nuclear morphology by Hoechst-33258 or hematoxylin counterstaining. Conclusions: These data suggest that either apoptosis is not involved in cell loss in MTS, or a very slow rate of cell demise may have precluded detecting TUNEL-positive neurons dying through apoptosis. Increased Bax expression and activation of caspases support the latter possibility. [source] Resveratrol modulates apoptosis and oxidation in human blood mononuclear cellsEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 9 2003G. A. Losa Abstract Background, We examined the effect of resveratrol (RS), a nonflavonoid polyphenolic phytoalexin found in grapes and red wine, and RS coincubated with the oxidant 2-deoxy-D-ribose (dR), on apoptosis and on the oxidative metabolic status of normal human peripheral blood mononuclear cells (PBMNCs) isolated ex vivo from healthy donors. Material and methods, Apoptosis was measured by changes of membrane permeability to propidium iodide (PI), plasma membrane exposure of phosphatidylserine (PS) and intracellular caspase activity. Oxidative status was assessed by recording the intracellular glutathione concentration (GSH), the activities of the enzymes y -glutamyltransferase (y- GT) and glutathione-S-transferase (GST), and intracellular lipid peroxidation (MDA). Results, Neither apoptotic nor oxidative parameters were affected by culturing PBMNCs in medium containing RS up to 20 µM for 5 days, while the frequency of cells with intermediate permeability to PI (17% ± 5) increased at 50 µM of RS. Thus resveratrol was slightly toxic, but there was little apoptosis in these cells. Peripheral blood mononuclear cells were also grown first in medium plus RS for 24 h and then for 96 h in medium containing RS plus 10 mM of dR, an oxidant sugar that is apoptogenic for human lymphocytes. The apoptotic changes triggered by dR were counteracted by the phytoalexin in a dose-dependent manner, but RS activity was absent at the lowest concentration (5 µM) and significantly reduced at the highest concentration used (50 µM). In PBMNCs coincubated with 20 µM of RS and 10 mM of dR the antioxidant effect of RS manifested with a significant reduction of caspases-3, -8, y- GT, GST activities and MDA content. Conclusions, Peripheral blood mononuclear cells acquire antioxidant capacity when treated with RS. Grape resveratrol may make a useful dietary supplement for minimizing oxidative injury in immune-perturbed states and human chronic degenerative diseases. [source] Increased hepatotoxicity of tumor necrosis factor,related apoptosis-inducing ligand in diseased human liver,HEPATOLOGY, Issue 5 2007Xandra Volkmann Tumor necrosis factor,related apoptosis-inducing ligand (TRAIL) induces apoptosis in tumor cells but not in most normal cells and has therefore been proposed as a promising antitumor agent. Recent experiments suggested that isolated primary human hepatocytes but not monkey liver cells are susceptible to certain TRAIL agonists, raising concerns about the use of TRAIL in cancer treatment. Whether TRAIL indeed exerts hepatotoxicity in vivo and how this is influenced by chemotherapeutic drugs or liver disease are completely unknown. Employing different forms of recombinant TRAIL, we found that the cytokine can induce proapoptotic caspase activity in isolated human hepatocytes. However in marked contrast, these different TRAIL preparations induced little or no cytotoxicity when incubated with tissue explants of fresh healthy liver, an experimental model that may more faithfully mimic the in vivo situation. In healthy liver, TRAIL induced apoptosis only when combined with histone deacetylase inhibitors. Strikingly, however, TRAIL alone triggered massive apoptosis accompanied by caspase activation in tissue explants from patients with liver steatosis or hepatitis C viral infection. This enhanced sensitivity of diseased liver was associated with an increased expression of TRAIL receptors and up-regulation of proapoptotic Bcl-2 proteins. Conclusion: These results suggest that clinical trials should be performed with great caution when TRAIL is combined with chemotherapy or administered to patients with inflammatory liver diseases. (HEPATOLOGY 2007.) [source] Detection of apoptotic caspase activation in sera from patients with chronic HCV infection is associated with fibrotic liver injuryHEPATOLOGY, Issue 5 2004Heike Bantel Chronic hepatitis C virus (HCV) infection is characterized by inflammatory liver damage and is associated with a high risk of development of cirrhosis and hepatocellular carcinoma. Although histological examination of liver biopsies is currently the gold standard for the detection of early liver damage, there is a strong need for better noninvasive methods. We recently demonstrated that the proapoptotic activation of caspases is considerably enhanced in histological sections from HCV-infected liver tissue, suggesting an important role of apoptosis in liver damage. Here, we investigated whether caspase activation is detectable also in sera from patients with chronic HCV infection. Using a novel enzyme-linked immunosorbent assay that selectively recognizes a proteolytic neoepitope of the caspase substrate cytokeratin-18, we demonstrate that caspase activity is markedly increased in the sera of HCV patients. Interestingly, while 27% of patients with chronic HCV infection showed normal aminotransferase levels despite inflammatory and fibrotic liver damage, more than 50% of those patients exhibited already elevated serum caspase activity. Moreover, 30% of patients with normal aminotransferase but elevated caspase activity revealed higher stages of fibrosis. In conclusion, compared with conventional surrogate markers such as aminotransferases, detection of caspase activity in serum might be a more sensitive method of detecting early liver injury. Thus, measurement of caspase activity might provide a novel diagnostic tool, especially for patients with normal aminotransferases but otherwise undiagnosed histologically active hepatitis and progressive fibrosis. (HEPATOLOGY 2004;40:1078,1087.) [source] Caspase activation correlates with the degree of inflammatory liver injury in chronic hepatitis C virus infectionHEPATOLOGY, Issue 4 2001Heike Bantel Hepatitis C virus (HCV) infection is a major cause of liver disease characterized by inflammation, cell damage, and fibrotic reactions of hepatocytes. Apoptosis has been implicated in the pathogenesis, although it is unclear whether proteases of the caspase family as the central executioners of apoptosis are involved and how caspase activation contributes to liver injury. In the present study, we measured the activation of effector caspases in liver biopsy specimens of patients with chronic HCV infection. The activation of caspase-3, caspase-7, and cleavage of poly(ADP-ribose)polymerase (PARP), a specific caspase substrate, were measured by immunohistochemistry and Western blot analysis by using antibodies that selectively detect the active truncated, but not the inactive precursor forms of the caspases and PARP. We found that caspase activation was considerably elevated in liver lobules of HCV patients in comparison to normal controls. Interestingly, the immunoreactive cells did yet not reveal an overt apoptotic morphology. The extent of caspase activation correlated significantly with the disease grade, i.e., necroinflammatory activity. In contrast, no correlation was observed with other surrogate markers such as serum transaminases and viral load. In biopsy specimens with low activity (grade 0) 7.7% of the hepatocytes revealed caspase-3 activation, whereas 20.9% of the cells stained positively in grade 3. Thus, our results suggest that caspase activation is involved in HCV-associated liver injury. Moreover, measurement of caspase activity may represent a reliable marker for the early detection of liver damage, which may open up new diagnostic and therapeutic strategies in HCV infection. [source] Granzyme B: a natural born killerIMMUNOLOGICAL REVIEWS, Issue 1 2003Sarah J. Lord Summary:, A main pathway used by cytotoxic T lymphocytes (CTLs) and natural killer cells to eliminate pathogenic cells is via exocytosis of granule components in the direction of the target cell, delivering a lethal hit of cytolytic molecules. Amongst these, granzyme B and perforin have been shown to induce CTL-mediated target cell DNA fragmentation and apoptosis. Once released from the CTL, granzyme B binds its receptor, the mannose-6-phosphate/insulin-like growth factor II receptor, and is endocytosed but remains arrested in endocytic vesicles until released by perforin. Once in the cytosol, granzyme B targets caspase-3 directly or indirectly through the mitochondria, initiating the caspase cascade to DNA fragmentation and apoptosis. Caspase activity is required for apoptosis to occur; however, in the absence of caspase activity, granzyme B can still initiate mitochondrial events via the cleavage of Bid. Recent work shows that granzyme B-mediated release of apoptotic factors from the mitochondria is essential for the full activation of caspase-3. Thus, granzyme B acts at multiple points to initiate the death of the offending cell. Studies of the granzyme B death receptor and internal signaling pathways may lead to critical advances in cell transplantation and cancer therapy. [source] Chemosensitization in non-small cell lung cancer cells by IKK inhibitor occurs via NF-,B and mitochondrial cytochrome c cascadeJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 11-12 2009Xianqing Jin Abstract In this study, we demonstrated with mechanistic evidence that parthenolide, a sesquiterpene lactone, could antagonize paclitaxel-mediated NF-,B nuclear translocation and activation by selectively targeting I-,B kinase (IKK) activity. We also found that parthenolide could target IKK activity and then inhibit NF-,B; this promoted cytochrome c release and activation of caspases 3 and 9. Inhibition of caspase activity blocked the activation of caspase cascade, implying that the observed synergy was related to caspases 3 and 9 activation of parthenolide. In contrast, paclitaxel individually induced apoptosis via a pathway independent of the mitochondrial cytochrome c cascade. Finally, exposure to parthenolide resulted in the inhibition of several NF-,B transcript anti-apoptotic proteins such as c-IAP1 and Bcl-xl. These data strengthen the rationale for using parthenolide to decrease the apoptotic threshold via caspase-dependent processes for treatment of non-small cell lung cancer with paclitaxel chemoresistance. [source] Induction of oxidative stress by homocyst(e)ine impairs endothelial function,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2001Vibhas S. Mujumdar Abstract Previous studies have demonstrated a relationship between hyperhomocysteinemia and endothelial dysfunction, reduced bioavailability of nitric oxide, elastinolysis and, vascular muscle cell proliferation. In vivo decreased nitric oxide production is associated with increased matrix metalloproteinase (MMP) activity and formation of nitrotyrosine. To test the hypothesis that homocysteine neutralizes vascular endothelial nitric oxide, activates metalloproteinase, causes elastinolysis and vascular hypertrophy, we isolated aortas from normotensive Wistar rats and cultured them in medium containing homocysteine, and calf serum for 14 days. Homocysteine-mediated impairment of endothelial-dependent vasodilatation was reversed by co-incubation of homocysteine with nicotinamide (an inhibitor of peroxinitrite and nitrotyrosine), suggesting a role of homocysteine in redox-mediating endothelial dysfunction and nitrotyrosine formation. The Western blot analysis, using anti-nitrotyrosine antibody, on aortic tissue homogeneates demonstrated decreased nitrotyrosine in hyperhomocysteinemic vessels treated with nicotinamide. Zymographic analysis revealed increased elastinolytic gelatinase A and B (MMP-2, -9) in homocysteine treated vessels and the treatment with nicotinamide decreases the homocysteine-induced MMP activation. Morphometric analyses revealed significant medial hypertrophic thickening (1.4,±,0.2-fold of control, P,=,0.03) and elastin disruption in homocysteine-treated vessels as compared to control. To determine whether homocysteine causes endothelial cell injury, cross-sections of aortas were analyzed for caspase activity by incubating with Ac-YVAD-AMC (substrate for apoptotic enzyme, caspase). The endothelium of homocysteine treated vessels, and endothelial cells treated with homocysteine, showed marked labeling for caspase. The length-tension relationship of homocysteine treated aortas was shifted to the left as compared to untreated aortas, indicating reduced vascular elastic compliance in homocysteine-treated vessels. Co-incubation of homocysteine and inhibitors of MMP, tissue inhibitor of metalloproteinase-4 (TIMP-4), and caspase, YVAD-CHO, improved vascular function. The results suggest that alteration in vascular elastin/collagen ratio and activation of MMP-2 are associated with decreased NO production in hyperhomocysteinemia. J. Cell. Biochem. 82:491,500, 2001. © 2001 Wiley-Liss, Inc. [source] Early nuclear exclusion of the transcription factor max is associated with retinal ganglion cell death independent of caspase activityJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2004Hilda Petrs-Silva We examined the behavior of the transcription factor Max during retrograde neuronal degeneration of retinal ganglion cells. Using immunohistochemistry, we found a progressive redistribution of full-length Max from the nucleus to the cytoplasm and dendrites of the ganglion cells following axon damage. Then, the axotomized cells lose all their content of Max, while undergoing nuclear pyknosis and apoptotic cell death. After treatment of retinal explants with either anisomycin or thapsigargin, the rate of nuclear exclusion of Max accompanied the rate of cell death as modulated by either drug. Treatment with a pan-caspase inhibitor abolished both TUNEL staining and immunoreactivity for activated caspase-3, but did not affect the subcellular redistribution of Max immunoreactivity after axotomy. The data show that nuclear exclusion of the transcription factor Max is an early event, which precedes and is independent of the activation of caspases, during apoptotic cell death in the central nervous system. J. Cell. Physiol. 198: 179,187, 2004© 2003 Wiley-Liss, Inc. [source] 17,-estradiol prevents cytotoxicity from hydrophobic bile acids in HepG2 and WRL-68 cell culturesJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 5 2006Matteo Ricchi Abstract Background:, Epidemiological and clinical studies suggest the possibility that estrogens might have a cytoprotective effect on the liver. The aim of the present study was to test the hypothesis that 17,-estradiol (E2) prevents hepatocellular damage induced by deoxycholic acid (DCA), a hydrophobic bile acid. Methods:, HepG2 cells were exposed for 24 h to DCA (350 µmol/L). Cell viability, aspartate aminotransferase and lactate dehydrogenase activity and apoptosis were measured as indices of cell toxicity. The effect of DCA was compared to that observed using either a hydrophilic bile acid, ursodeoxycholic acid (UDCA; 100 µmol/L), or E2 at different concentrations (1 nmol/L, 10 nmol/L, 50 nmol/L and 50 µmol/L) or mixtures of E2/DCA or UDCA/DCA. The same experiments were performed using WRL-68 cells that, at variance with HepG2, express a higher level of nuclear estrogen receptor. Results:, High concentrations of E2 and UDCA prevented DCA-induced decrease in cell viability, increase in enzyme activity and apoptosis evaluated both by 4,,6-diamidino-2-phenylindole dihydrochloride (DAPI) and TdT-mediated dUTP nick-end labeling (TUNEL) assays. In addition, DCA-related apoptosis, assessed by caspase activity, was also prevented by E2 (P < 0.01) in physiological (1,10 nmol/L) doses. The cytoprotective effects of E2 and UDCA was also observed in the WRL-68 cell line. Conclusions:, 17,-Estradiol prevents DCA-induced cell damage in HepG2 and WRL-68 cell lines to an extent comparable to UDCA. The hypothesis that the protective effect of E2 may be mediated by a mechanism that is nuclear estrogen receptor independent, deserves further verification. [source] Antibacterial, antiviral, antiproliferative and apoptosis-inducing properties of Brackenridgea zanguebarica (Ochnaceae)JOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 8 2006Maren Möller Brackenridgea zanguebarica is a small tree that is used in traditional African medicine as a type of cure-all for many diseases, including the treatment of wounds. The yellow bark of B. zanguebarica was used for the preparation of an ethanolic extract, which was tested in various concentrations against eleven bacteria, Herpes simplex virus type 1 (HSV-1) and different human tumour cell lines. The extract that contains different polyphenolic substances like calodenin B. Cell growth inhibition, assessed via MTT-assay, was found in all tested human cell lines with IC50 values (concentration of extract that reduced cell viability by 50%) between 33 ,g dry extract/mL for HL-60 human myeloid leukaemia cells and 93 ,g dry extract/mL for HaCaT human keratinocytes. Staining with Annexin-V-FLUOS and JC-1 followed by subsequent analysis via flow cytometry revealed significant apoptosis-inducing properties. Analysis of caspase activity using a fluorogenic caspase-3 substrate showed a significant caspase activity in Jurkat T-cells after incubation with the extract. The bark extract had a pronounced activity against free HSV-1 and a strong antibacterial activity against Gram-positive strains (MICs: 6,24 ,g dry extract/mL), which are often involved in skin infections. Additionally, no irritating properties of the extract could be observed in hen-egg test chorioallantoic membrane (HET-CAM) assay. These findings give a rationale for the traditional use of B. zanguebarica and are a basis for further analysis of the plant's components, their biological activity, and its use in modern phytotherapy. [source] Inhibition of nuclear factor ,B and phosphatidylinositol 3-kinase/Akt is essential for massive hepatocyte apoptosis induced by tumor necrosis factor , in miceLIVER INTERNATIONAL, Issue 5 2003Motoaki Imose Abstract: Background/aims: Tumor necrosis factor (TNF)-, itself does not induce liver injury in normal mice or hepatocytes. Rather, this event, especially in vitro, is explained by the fact that the TNF-,/TNF receptor system not only triggers downstream signals leading to apoptosis but also induces an antiapoptotic pathway through the activation of nuclear factor (NF)-,B. The aim of this study was to determine whether inhibition of antiapoptotic pathways influences the susceptibility of mice to TNF-,. Here, we focused on the roles of NF-,B and phosphatidylinositol 3-kinase (PI3K)-regulated serine/threonine kinase Akt. Methods: TNF-, was administered to BALB/c mice after treatment with an adenovirus expressing a mutant form I,B, (Ad5I,B), the PI3K inhibitor wortmannin, or both. Liver injury was assessed biochemically and histologically. The expression of Bcl-2 family members and caspase activity were examined. Results: In the mice livers, treatment with Ad5I,B or the wortmannin suppressed the activation of NF-,B or Akt, respectively. Suppression of either NF-,B or Akt showed a slight increase in transaminase levels and focal liver cell death after TNF-, administration. However, in mice treated with both Ad5I,B and wortmannin, TNF-, administration resulted in massive hepatocyte apoptosis and hemorrhagic liver destruction in mice. The combination of Ad5I,B, wortmannin, and TNF-, markedly increased the activation of caspase-3 and -9, and activated caspase-8 to a lesser degree, suggesting that TNF-,-induced hepatocyte apoptosis is dependent on type II cell death signaling pathway, probably through the mitochondria. Inhibition of the NF-,B and PI3K/Akt pathways had no effect on expression of Bcl-2 families. Conclusion: The inducible activation of NF-,B and constitutive activation of Akt regulate hepatocyte survival against TNF-,, which occurs independent of Bcl-2 families. [source] Relevance of caspase activity during apoptosis in pubertal rat spermatogenesisMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 5 2008Veronica A. Codelia Abstract Caspases are a family of cysteine-proteases, activated upon several different stimuli, which execute apoptosis in many cell death models. Previous work of our group has shown rats have the highest rate of apoptosis during the first wave of spermatogenesis (between 20 and 25 days after birth), as evaluated by TUNEL and caspase activity. However, the hierarchical order of caspase activation and the relevance of each caspase during germ cell apoptosis are not clear. Thus, the goal of this work is to take a pharmacological approach to dissect the apoptosis pathway of caspase activation. Results showed that intratesticular injection of a caspase-8 inhibitor (z-IETD-fmk), or a pan-caspase inhibitor (z-VAD- fmk), significantly decreased the cleavage of p115 and PARP, two endogenous substrates of caspases, in 22-day-old rats. Additionally, these inhibitors promoted a significant reduction in the number of apoptotic germ cells. On the other hand, intratesticular injection of two different inhibitors of the intrinsic pathway (z-LEHD-fmk and minocycline) did not have any effect upon caspase substrates cleavage (p115 and PARP) or the number of apoptotic germ cells. Therefore, we conclude that the extrinsic pathway of apoptosis plays an important role in physiological germ cell apoptosis during the first round of spermatogenesis in the rat. Mol. Reprod. Dev. 75: 881,889, 2008. © 2007 Wiley-Liss, Inc. [source] Caspase activity in newt spermatogonial apoptosis induced by prolactin and cycloheximideMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2001Takashi Yazawa Abstract We previously showed in vivo and in vitro, that among the spermatogenic stages of the newt, prolactin (PRL) induces apoptosis specifically in the penultimate stage of secondary spermatogonia. In the current report, we demonstrate in vitro that cycloheximide (CHX), an inhibitor of protein synthesis, induces morphological apoptotic changes similar to those caused by PRL, such as chromatin condensation and apoptotic body formation. Next, we found that Z-VAD-fmk, an inhibitor of various caspases, suppressed the apoptosis induced by PRL and CHX, but ICE inhibitor Ac-YVAD-CHO or caspase-3 inhibitor Ac-DEVD-CHO did not. As high caspase activity was present in extracts of testes treated with CHX, we suggest that an unidentified caspase induces the morphological changes of apoptosis in newt spermatogonia. Mol. Reprod. Dev. 59:209,214, 2001. © 2001 Wiley-Liss, Inc. [source] Involvement of apoptosis and cholinergic dysfunction in Alzheimer's diseasePSYCHOGERIATRICS, Issue 2006Shinji TAGAMI Abstract As Alzheimer's disease (AD) progresses, brain atrophy becomes conspicuous, and histologically there is neuronal loss, primarily with a deficit of cholinergic neurons observed. Hitherto, the view has been that cell death, apoptosis, plays a role in this neuronal loss. Apoptosis is characterized by the morphological changes of nuclear fragmentation, chromatin condensation and cell shrinkage, with activation of caspases, members of the cysteine protease family, resulting in considerable substrate cleavage. TUNEL positive neurons have in fact been detected in AD brain, indicating increased caspase activity and resulting substrate cleavage. In AD brain, amyloid beta peptides (A,), the main constituent of senile plaque, are a specific pathological hallmark observed in extracellular spaces. In contrast, the main constituent of intracellularly observed neurofibrillary tangles (NFT) is hyperphosphorylated tau, which is observed in various neurodegenerative disorders other than AD. The viewpoint of many studies is that the A, and NFT that cause these neuropathological changes probably participate in neuronal death. However, up until now it has been thought that there was no hypothesis offering a comprehensive explanation of how the accumulation of extracellular A, and intracellular NFT leads to neuronal death. This report first covers the mechanism of apoptosis as clarified by molecular biological methods, and provides an explanation of how apoptosis could be involved in AD pathology. The subject of autophagic cell death, a type of cell death morphology that has recently been the focus of attention, is also addressed. [source] Viral ssRNA Induces First Trimester Trophoblast Apoptosis through an Inflammatory MechanismAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 1 2010Paulomi B. Aldo Citation Aldo PB, Mulla MJ, Romero R, Mor G, Abrahams VM. Viral ssRNA induces first-trimester trophoblast apoptosis through an inflammatory mechanism. Am J Reprod Immunol 2010; 64: 27,37 Problem, Infection during pregnancy represents a significant cause of mobility and mortality. While viruses pose a major threat, little is known about their effect on early pregnancy, or the mechanisms involved. The objective of this study was to characterize the trophoblast response following exposure to viral ssRNA. Method of study, First trimester trophoblast cells were treated with or without viral ssRNA. Cytokine production was measured using multiplex analysis and ELISA. Apoptosis was determined using Hoechst staining, cell viability, and caspase activity assays. Results, Treatment of trophoblasts with viral ssRNA increased their secretion of IL-8, IL-6, and IFN,. However, the ssRNA also induced trophoblast apoptosis. To test whether the viral ssRNA-induced inflammatory response was responsible for this induction of apoptosis, conditioned media (CM) from trophoblasts were added to a fresh culture of cells. The CM from viral ssRNA-treated induced higher levels of trophoblast apoptosis than the control CM. Moreover, recombinant IFN, induced trophoblast apoptosis. Conclusion, We demonstrate that viral ssRNA induces a pro-inflammatory and type I interferon response in the trophoblast and this inflammatory process may indirectly induce trophoblast apoptosis. These results provide a novel mechanism by which certain viral infections might compromise placental integrity and function, and therefore, pregnancy outcome. [source] Mahanine inhibits growth and induces apoptosis in prostate cancer cells through the deactivation of Akt and activation of caspases,THE PROSTATE, Issue 12 2006Swati Sinha Abstract BACKGROUND The present study was undertaken to evaluate anti-proliferative and -apoptotic activities of mahanine, a plant derived carbazole alkaloid, in prostate cancer cells and to determine its molecular mechanism by which it induces apoptotic cell death. METHODS The growth inhibitory and apoptotic inductive effect of mahanine on prostate cancer cells were examined by measuring cell proliferation and BrdU labeling, caspase activity, DNA fragmentation, and Western blot analyses. RESULTS Mahanine inhibited growth of PC3 and LNCaP prostate cancer cells in a dose and time-dependent manner. Mechanistically, mahanine inhibited cell-survival pathway by dephosphorylation of PIP3 dependent kinase 1 (PDK1) thereby deactivation of Akt and downregulation of Bcl-xL. In addition, mahanine activated caspase pathway (caspases 9 and 3) and eventually cleavage of DNA repair enzyme, PARP resulting DNA fragmentation and apoptosis. CONCLUSIONS Mahanine inhibits growth and induces apoptosis in both androgen-responsive, LNCaP and androgen-independent, PC3 cells by targeting cell survival pathway. Prostate © 2006 Wiley-Liss, Inc. [source] | |||||||||||||||||||||||||||||||