Caffeine induces sustained apoptosis of human gastric cancer cells by activating the caspase-9/caspase-3 signalling pathway

PubMed Central

Liu, Hanyang; Zhou, Yan; Tang, Liming

2017-01-01

Caffeine is one of the most widely consumed substances found in beverages, and has demonstrated anticancer effects in several types of cancer. The present study aimed to examine the anticancer effects of caffeine on gastric cancer (GC) cells (MGC-803 and SGC-7901) in vitro, and to determine whether the apoptosis-related caspase-9/−3 pathway is associated with these effects. The sustained antiproliferative effects of caffeine on gastric cancer were also investigated. GC cell viability and proliferation were evaluated using cell counting and colony forming assays, following treatment with various concentrations of caffeine. Flow cytometry was performed to assess cell cycle dynamics and apoptosis. Western blot analysis was conducted to detect the activity of the caspase-9/−3 pathway. The results indicated that caffeine treatment significantly suppressed GC cell growth and viability and induced apoptosis by activating the caspase-9/−3 pathway. Furthermore, the anticancer effects of caffeine appeared to be sustained, as the caspase-9/−3 pathway remained active following caffeine withdrawal. In conclusion, caffeine may function as a sustained anticancer agent by activating the caspase-9/−3 pathway, which indicates that it may be useful as a therapeutic candidate in gastric cancer. PMID:28677810

An endoplasmic reticulum stress-specific caspase cascade in apoptosis. Cytochrome c-independent activation of caspase-9 by caspase-12.

PubMed

Morishima, Nobuhiro; Nakanishi, Keiko; Takenouchi, Hiromi; Shibata, Takehiko; Yasuhiko, Yukuto

2002-09-13

Activation of caspase-12 from procaspase-12 is specifically induced by insult to the endoplasmic reticulum (ER) (Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner, B. A., and Yuan, J. (2000) Nature 403, 98-103), yet the functional consequences of caspase-12 activation have been unclear. We have shown that recombinant caspase-12 specifically cleaves and activates procaspase-9 in cytosolic extracts. The activated caspase-9 catalyzes cleavage of procaspase-3, which is inhibitable by a caspase-9-specific inhibitor. Although cytochrome c released from mitochondria has been believed to be required for caspase-9 activation during apoptosis (Zou, H., Henzel, W. J., Liu, X., Lutschg, A., and Wang, X. (1997) Cell 90, 405-413, Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., and Wang, X. (1997) Cell 91, 479-489), caspase-9 as well as caspase-12 and -3 are activated in cytochrome c-free cytosols in murine myoblast cells under ER stress. These results suggest that caspase-12 can activate caspase-9 without involvement of cytochrome c. To examine the role of caspase-12 in the activation of downstream caspases, we used a caspase-12-binding protein, which we identified in a yeast two-hybrid screen, for regulation of caspase-12 activation. The binding protein protects procaspase-12 from processing in vitro. Stable expression of the binding protein renders procaspase-12 insensitive to ER stress, thereby suppressing apoptosis and the activation of caspase-9 and -3. These data suggest that procaspase-9 is a substrate of caspase-12 and that ER stress triggers a specific cascade involving caspase-12, -9, and -3 in a cytochrome c-independent manner.

Inhibition of cathepsin B by caspase-3 inhibitors blocks programmed cell death in Arabidopsis.

PubMed

Ge, Y; Cai, Y-M; Bonneau, L; Rotari, V; Danon, A; McKenzie, E A; McLellan, H; Mach, L; Gallois, P

2016-09-01

Programmed cell death (PCD) is used by plants for development and survival to biotic and abiotic stresses. The role of caspases in PCD is well established in animal cells. Over the past 15 years, the importance of caspase-3-like enzymatic activity for plant PCD completion has been widely documented despite the absence of caspase orthologues. In particular, caspase-3 inhibitors blocked nearly all plant PCD tested. Here, we affinity-purified a plant caspase-3-like activity using a biotin-labelled caspase-3 inhibitor and identified Arabidopsis thaliana cathepsin B3 (AtCathB3) by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Consistent with this, recombinant AtCathB3 was found to have caspase-3-like activity and to be inhibited by caspase-3 inhibitors. AtCathepsin B triple-mutant lines showed reduced caspase-3-like enzymatic activity and reduced labelling with activity-based caspase-3 probes. Importantly, AtCathepsin B triple mutants showed a strong reduction in the PCD induced by ultraviolet (UV), oxidative stress (H2O2, methyl viologen) or endoplasmic reticulum stress. Our observations contribute to explain why caspase-3 inhibitors inhibit plant PCD and provide new tools to further plant PCD research. The fact that cathepsin B does regulate PCD in both animal and plant cells suggests that this protease may be part of an ancestral PCD pathway pre-existing the plant/animal divergence that needs further characterisation.

FRET analysis demonstrates a rapid activating of caspase-3 during PDT-induced apoptosis

NASA Astrophysics Data System (ADS)

Wu, Yunxia; Chen, Qun

2006-09-01

Apoptosis is a very important cellular event that plays a key role in pathogeny and therapy of many diseases. In this study, a recombinant caspase-3 substrate was used as a fluorescence resonance energy transfer (FRET) probe to detect the activation of caspase-3, and to monitor apoptosis in human lung adenocarcinoma (ASTC-a- 1) cells. With laser scanning confocal microscopy, we found that Photofrin were localized primarily in mitochondria, the primary targets of Photofrin-PDT. By analyzing the dynamic changes of FRET fluorescence, the results indicate that the onset and completion of caspase-3 activation induced by PDT is more rapidly than that by tumor necrosis factor-α (TNF-α). The activation of caspase-3 by PDT started 20 minutes after treatment and completed in about 15 minutes. In comparison, the onset of caspase-3 activation by TNF-a was delayed by 3 hours and the completion of caspase-3 activation required a significantly longer time (approximately 90 minutes). These results indicated that the initiation and process of caspase-3 activation are different corresponding to different treatment methods. Our data suggest that caspase-3 activation mediated by the cell surface death receptors is slower than that of the mitochondrial pathway and the mitochondria is an efficient target to induce apoptosis.

Local pruning of dendrites and spines by caspase-3-dependent and proteasome-limited mechanisms.

PubMed

Ertürk, Ali; Wang, Yuanyuan; Sheng, Morgan

2014-01-29

Synapse loss occurs normally during development and pathologically during neurodegenerative disease. Long-term depression, a proposed physiological correlate of synapse elimination, requires caspase-3 and the mitochondrial pathway of apoptosis. Here, we show that caspase-3 activity is essential--and can act locally within neurons--for regulation of spine density and dendrite morphology. By photostimulation of Mito-KillerRed, we induced caspase-3 activity in defined dendritic regions of cultured neurons. Within the photostimulated region, local elimination of dendritic spines and dendrite retraction occurred in a caspase-3-dependent manner without inducing cell death. However, pharmacological inhibition of inhibitor of apoptosis proteins or proteasome function led to neuronal death, suggesting that caspase activation is spatially restricted by these "molecular brakes" on apoptosis. Caspase-3 knock-out mice have increased spine density and altered miniature EPSCs, confirming a physiological involvement of caspase-3 in the regulation of spines in vivo.

Hsp70 suppresses apoptosis of BRL cells by regulating the expression of Bcl-2, cytochrome C, and caspase 8/3.

PubMed

Kong, Fanzhi; Wang, Hui; Guo, Jingru; Peng, Mengling; Ji, Hong; Yang, Huanmin; Liu, Binrun; Wang, Jianfa; Zhang, Xu; Li, Shize

2016-05-01

During cold stress, liver cells undergo apoptotic injury as a result of oxidative stress. Heat shock 70 kDa protein (Hsp70) is a protein involved in modulating a variety of physiological processes, including stress responses, proliferation, and apoptosis. In addition, Hsp70 regulates apoptotic signaling pathways in different manners, promoting or suppressing apoptosis. In this study, we investigated the effects of Hsp70 overexpression on hydrogen peroxide (H2O2)-induced apoptosis of Buffalo rat liver (BRL) cells and the underlying mechanisms of these effects. Our results show that in comparison with the control group, Hsp70 overexpression displayed increased protein levels of Bcl-2, and decreased cytochrome c (Cyt c), cleaved caspase 3, and cleaved caspase 8, but no apparent differences were found in levels of Bax. Furthermore, Hsp70 overexpression significantly suppresses the amount of apoptotic cells. Such findings indicate that overexpression of Hsp70 inhibits H2O2-mediated activation of caspase 8 and caspase 3, upregulates the expression of Bcl-2 which is a known anti-apoptotic protein, and decreases the release of Cyt c from the mitochondria into the cytoplasm, collectively decreasing cell apoptosis.

Oxidized low-density lipoprotein induces calpain-dependent cell death and ubiquitination of caspase 3 in HMEC-1 endothelial cells.

PubMed Central

Pörn-Ares, M Isabella; Saido, Takaomi C; Andersson, Tommy; Ares, Mikko P S

2003-01-01

Oxidized low-density lipoprotein (oxLDL) is known to induce apoptosis in endothelial cells, and this is believed to contribute to the progression of atherosclerosis. In the present study we made the novel observation that oxLDL-induced death of HMEC-1 cells is accompanied by activation of calpain. The mu-calpain inhibitor PD 151746 decreased oxLDL-induced cytotoxicity, whereas the general caspase inhibitor BAF (t-butoxycarbonyl-Asp-methoxyfluoromethylketone) had no effect. Also, oxLDL provoked calpain-dependent proteolysis of cytoskeletal alpha-fodrin in the HMEC-1 cells. Our observation of an autoproteolytic cleavage of the 80 kDa subunit of mu-calpain provided further evidence for an oxLDL-induced stimulation of calpain activity. The Bcl-2 protein Bid was also cleaved during oxLDL-elicited cell death, and this was prevented by calpain inhibitors, but not by inhibitors of cathepsin B and caspases. Treating the HMEC-1 cells with oxLDL did not result in detectable activation of procaspase 3 or cleavage of PARP [poly(ADP-ribose) polymerase], but it did cause polyubiquitination of caspase 3, indicating inactivation and possible degradation of this protease. Despite the lack of caspase 3 activation, oxLDL treatment led to the formation of nucleosomal DNA fragments characteristic of apoptosis. These novel results show that oxLDL initiates a calpain-mediated death-signalling pathway in endothelial cells. PMID:12775216

Imaging Caspase-3 Activation as a Marker of Apoptosis-Targeted Treatment Response in Cancer

PubMed Central

Chen, Delphine L.; Engle, Jacquelyn T.; Griffin, Elizabeth A.; Miller, J. Philip; Chu, Wenhua; Zhou, Dong; Mach, Robert H.

2016-01-01

Purpose We tested whether positron emission tomography (PET) with the caspase-3 targeted isatin analog [18F]WC-4-116 could image caspase-3 activation in response to an apoptosis-inducing anticancer therapy. Procedures [18F]WC-4-116 uptake was determined in etoposide-treated EL4 cells. Biodistribution studies with [18F]WC-4-116 and [18F]ICMT-18, a non-caspase-3-targeted tracer, as well as [18F]WC-4-116 microPET imaging assessed responses in Colo205 tumor bearing mice treated with death receptor 5 (DR5) targeted agonist antibodies. Immunohistochemical staining and enzyme assays confirmed caspase-3 activation. Two-way analysis of variance or Student’s t-test assessed for treatment-related changes in tracer uptake. Results [18F]WC-4-116 increased 8 ± 2-fold in etoposide-treated cells. The [18F]WC-4-116 %ID/g also increased significantly in tumors with high caspase-3 enzyme activity (p < 0.05). [18F]ICMT-18 tumor uptake did not differ in tumors with high or low caspase-3 enzyme activity. Conclusions [18F]WC-4-116 uptake in vivo reflects increased caspase-3 activation and may be useful for detecting caspase-3 mediated apoptosis treatment responses in cancer. PMID:25344147

Nuclear Condensation during Mouse Erythropoiesis Requires Caspase-3-Mediated Nuclear Opening.

PubMed

Zhao, Baobing; Mei, Yang; Schipma, Matthew J; Roth, Eric Wayne; Bleher, Reiner; Rappoport, Joshua Z; Wickrema, Amittha; Yang, Jing; Ji, Peng

2016-03-07

Mammalian erythropoiesis involves chromatin condensation that is initiated in the early stage of terminal differentiation. The mechanisms of chromatin condensation during erythropoiesis are unclear. Here, we show that the mouse erythroblast forms large, transient, and recurrent nuclear openings that coincide with the condensation process. The opening lacks nuclear lamina, nuclear pore complexes, and nuclear membrane, but it is distinct from nuclear envelope changes that occur during apoptosis and mitosis. A fraction of the major histones are released from the nuclear opening and degraded in the cytoplasm. We demonstrate that caspase-3 is required for the nuclear opening formation throughout terminal erythropoiesis. Loss of caspase-3 or ectopic expression of a caspase-3 non-cleavable lamin B mutant blocks nuclear opening formation, histone release, chromatin condensation, and terminal erythroid differentiation. We conclude that caspase-3-mediated nuclear opening formation accompanied by histone release from the opening is a critical step toward chromatin condensation during erythropoiesis in mice. Copyright © 2016 Elsevier Inc. All rights reserved.

Nuclear condensation during mouse erythropoiesis requires caspase-3-mediated nuclear opening

PubMed Central

Zhao, Baobing; Mei, Yang; Schipma, Matthew J; Roth, Eric Wayne; Bleher, Reiner; Rappoport, Joshua Z.; Wickrema, Amittha; Yang, Jing; Ji, Peng

2016-01-01

SUMMARY Mammalian erythropoiesis involves chromatin condensation that is initiated in the early stage of terminal differentiation. The mechanisms of chromatin condensation during erythropoiesis are unclear. Here, we show that the mouse erythroblast forms large, transient, and recurrent nuclear openings that coincide with the condensation process. The opening lacks nuclear lamina, nuclear pore complexes, and nuclear membrane, but it is distinct from nuclear envelope changes that occur during apoptosis and mitosis. A fraction of the major histones are released from the nuclear opening and degraded in the cytoplasm. We demonstrate that caspase-3 is required for the nuclear opening formation throughout terminal erythropoiesis. Loss of caspase-3 or ectopic expression of a caspase-3 non-cleavable lamin B mutant blocks nuclear opening formation, histone release, chromatin condensation, and terminal erythroid differentiation. We conclude that caspase-3-mediated nuclear opening formation accompanied by histone release from the opening is a critical step towards chromatin condensation during erythropoiesis in mice. PMID:26954545

Caspase-like activity is essential for long-term synaptic plasticity in the terrestrial snail Helix.

PubMed

Bravarenko, N I; Onufriev, M V; Stepanichev, M Yu; Ierusalimsky, V N; Balaban, P M; Gulyaeva, N V

2006-01-01

Although caspase activity in the nervous system of mollusks has not been described before, we suggested that these cysteine proteases might be involved in the phenomena of neuroplasticity in mollusks. We directly measured caspase-3 (DEVDase) activity in the Helix lucorum central nervous system (CNS) using a fluorometrical approach and showed that the caspase-3-like immunoreactivity is present in the central neurons of Helix. Western blots revealed the presence of caspase-3-immunoreactive proteins with a molecular mass of 29 kDa. Staurosporin application, routinely used to induce apoptosis in mammalian neurons through the activating cleavage of caspase-3, did not result in the appearance of a smaller subunit corresponding to the active caspase in the snail. However, it did increase the enzyme activity in the snail CNS. This suggests differences in the regulation of caspase-3 activity in mammals and snails. In the snail CNS, the caspase homolog seems to possess an active center without activating cleavage typical for mammals. In electrophysiological experiments with identified snail neurons, selective blockade of the caspase-3 with the irreversible and cell-permeable inhibitor of caspase-3 N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp-(OMe)-fluoro-methylketone prevented development of the long-term stage of synaptic input sensitization, suggesting that caspase is necessary for normal synaptic plasticity in snails. The results of our study give the first direct evidence that the caspase-3-like activity is essential for long-term plasticity in the invertebrate neurons. This activity is presumably involved in removing inhibitory constraints on the storage of long-term memory.

Cadmium Induces Liver Cell Apoptosis through Caspase-3A Activation in Purse Red Common Carp (Cyprinus carpio)

PubMed Central

Qiao, Panpan; Liu, Shen; Zhang, Li; He, Penghui; Zhang, Xiaoyan; Wang, Yannan; Min, Weiping

2013-01-01

Caspase-3, the essential effector caspase, plays a pivotal role during caspase-dependent apoptosis. In this study, we isolated and characterized caspase-3A gene from common carp. The common carp caspase-3A comprising 273 amino acids showed 71.8% sequence similarity and 59.3% sequence identity to human caspase-3. It exhibited an evolutionarily conserved structure of mammalian caspase-3 genes, including a pro-domain, a large subunit, a small subunit and other motifs such as the pentapeptide active-site motif (QACRG) and the putative cleavage sites at the aspartic acids. Phylogenetic analysis demonstrated that common carp caspase-3A formed a clade with cyprinid fish caspase-3. To assess whether caspase-3A is involved in cadmium (Cd)-induced cell apoptosis in common carp, a Cd exposure experiment was performed. TUNEL analysis showed that Cd triggered liver cell apoptosis; caspase-3A activity was markedly increased; its proenzyme level was significantly decreased, and the levels of its cleaved forms were markedly increased. However, real-time quantitative PCR analysis revealed that the mRNA transcript level of caspase-3A was not significantly elevated. Immunoreactivities were observed in the cytoplasm of hepatocytes by immunohistochemical detection. The findings indicates that Cd can trigger liver cell apoptosis through the activation of caspase-3A. Caspase-3A may play an essential role in Cd-induced apoptosis. PMID:24349509

Caspase-3 short hairpin RNAs: a potential therapeutic agent in neurodegeneration of aluminum-exposed animal model.

PubMed

Zhang, Qinli; Li, Na; Jiao, Xia; Qin, Xiujun; Kaur, Ramanjit; Lu, Xiaoting; Song, Jing; Wang, Linping; Wang, Junming; Niu, Qiao

2014-01-01

There is abundant evidence supporting the role of caspases in the development of neurodegenerative disease, including Alzheimer's disease (AD). Therefore, regulating the activity of caspases has been considered as a therapeutic target. However, all the efforts on AD therapy using pan-caspase inhibitors have failed because of uncontrolled adverse effects. Alternatively, the specific knockdown of caspase-3 gene through RNA interference (RNAi) could serve as a future potential therapeutic strategy. The aim of the present study is to down-regulate the expression of caspase-3 gene using lentiviral vector-mediated caspase-3 short hairpin RNA (LV-Caspase-3 shRNA). The effect of LV-Caspase-3 shRNA on apoptosis induced by aluminum (Al) was investigated in primary cultured cortical neurons and validated in C57BL/6J mice. The results indicated an increase in apoptosis and caspase-3 expression in primary cultured neurons and the cortex ofmice exposed to Al, which could be down-regulated by LV-Caspase-3 shRNA. Furthermore, LV-Caspase-3 shRNA reduced neural cell death and improved learning and memory in C57BL/6J mice treated with Al. Our results suggest that LV-caspase-3 shRNA is a potential therapeutic agent to prevent neurodegeneration and cognitive dysfunction in aluminum- exposed animal models. The findings provide a rational gene therapy strategy for AD.

Exploiting differences in caspase-2 and -3 S₂ subsites for selectivity: structure-based design, solid-phase synthesis and in vitro activity of novel substrate-based caspase-2 inhibitors.

PubMed

Maillard, Michel C; Brookfield, Frederick A; Courtney, Stephen M; Eustache, Florence M; Gemkow, Mark J; Handel, Rebecca K; Johnson, Laura C; Johnson, Peter D; Kerry, Mark A; Krieger, Florian; Meniconi, Mirco; Muñoz-Sanjuán, Ignacio; Palfrey, Jordan J; Park, Hyunsun; Schaertl, Sabine; Taylor, Malcolm G; Weddell, Derek; Dominguez, Celia

2011-10-01

Several caspases have been implicated in the pathogenesis of Huntington's disease (HD); however, existing caspase inhibitors lack the selectivity required to investigate the specific involvement of individual caspases in the neuronal cell death associated with HD. In order to explore the potential role played by caspase-2, the potent but non-selective canonical Ac-VDVAD-CHO caspase-2 inhibitor 1 was rationally modified at the P(2) residue in an attempt to decrease its activity against caspase-3. With the aid of structural information on the caspase-2, and -3 active sites and molecular modeling, a 3-(S)-substituted-l-proline along with four additional scaffold variants were selected as P(2) elements for their predicted ability to clash sterically with a residue of the caspase-3 S(2) pocket. These elements were then incorporated by solid-phase synthesis into pentapeptide aldehydes 33a-v. Proline-based compound 33h bearing a bulky 3-(S)-substituent displayed advantageous characteristics in biochemical and cellular assays with 20- to 60-fold increased selectivity for caspase-2 and ∼200-fold decreased caspase-3 potency compared to the reference inhibitor 1. Further optimization of this prototype compound may lead to the discovery of valuable pharmacological tools for the study of caspase-2 mediated cell death, particularly as it relates to HD. Copyright © 2011 Elsevier Ltd. All rights reserved.

Progesterone production requires activation of caspase-3 in preovulatory granulosa cells in a serum starvation model.

PubMed

An, Li-Sha; Yuan, Xiao-Hua; Hu, Ying; Shi, Zi-Yun; Liu, Xiao-Qin; Qin, Li; Wu, Gui-Qing; Han, Wei; Wang, Ya-Qin; Ma, Xu

2012-11-01

Granulosa cells proliferate, differentiate, and undergo apoptosis throughout follicular development. Previous studies have demonstrated that stimulation of progesterone production is accompanied by caspase-3 activation. Moreover, we previously reported that arsenic enhanced caspase-3 activity coupled with progesterone production. Inhibition of caspase-3 activity can significantly inhibit progesterone production induced by arsenic or follicle-stimulating hormone (FSH). Here, we report that serum starvation induces caspase-3 activation coupled with augmentation of progesterone production. Serum starvation also increased the levels of cytochrome P450 cholesterol side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory (StAR) protein, both of which may contribute to progesterone synthesis in preovulatory granulosa cells. Inhibition of caspase-3 activity resulted in a decrease in progesterone production. Deactivation of caspase-3 activity by caspase-3 specific inhibitor also resulted in decreases in P450scc and StAR expression, which may partly contribute to the observed decrease in progesterone production. Our study demonstrates for the first time that progesterone production in preovulatory granulosa cells is required for caspase-3 activation in a serum starvation model. Inhibition of caspase-3 activity can result in decreased expression of the steroidogenic proteins P450scc and StAR. Our work provides further details on the relationship between caspase-3 activation and steroidogenesis and indicates that caspase-3 plays a critical role in progesterone production by granulosa cells. Copyright © 2012 Elsevier Inc. All rights reserved.

Tissue Inhibitor of Metalloproteinase-3 (TIMP3) Promotes Endothelial Apoptosis via a Caspase-Independent Mechanism

PubMed Central

Qi, Jian Hua; Anand-Apte, Bela

2015-01-01

Tissue Inhibitor of Metalloproteinases-3 (TIMP3) is a tumor suppressor and a potent inhibitor of angiogenesis. TIMP3 exerts its anti-angiogenic effect via a direct interaction with vascular endothelial growth factor (VEGF) receptor-2 (KDR) and inhibition of proliferation, migration and tube formation of endothelial cells (ECs). TIMP3 has also been shown to induce apoptosis in some cancer cells and vascular smooth muscle cells via MMP inhibition and caspase-dependent mechanisms. In this study, we examined the molecular mechanisms of TIMP3-mediated apoptosis in endothelial cells. We have previously demonstrated that mice developed smaller tumors with decreased vascularity when injected with breast carcinoma cells overexpressing TIMP3, than with control breast carcinoma cells. TIMP3 overexpression resulted in increased apoptosis in human breast carcinoma (MDA-MB435) in vivo but not in vitro. However, TIMP3 could induce apoptosis in endothelial cells (ECs) in vitro. The apoptotic activity of TIMP3 in ECs appears to be independent of MMP inhibitory activity. Furthermore, the equivalent expression of functional TIMP3 promoted apoptosis and caspase activation in endothelial cells expressing KDR (PAE/KDR), but not in endothelial cells expressing PDGF beta-receptor (PAE/β-R). Surprisingly, the apoptotic activity of TIMP3 appears to be independent of caspases. TIMP3 inhibited matrix-induced focal adhesion kinase (FAK) tyrosine phosphorylation and association with paxillin and disrupted the incorporation of β3 integrin, FAK and paxillin into focal adhesion contacts on the matrix, which were not affected by caspase inhibitors. Thus, TIMP3 may induce apoptosis in ECs by triggering a caspase-independent cell death pathway and targeting a FAK-dependent survival pathway. PMID:25558000

Tissue inhibitor of metalloproteinase-3 (TIMP3) promotes endothelial apoptosis via a caspase-independent mechanism.

PubMed

Qi, Jian Hua; Anand-Apte, Bela

2015-04-01

Tissue inhibitor of metalloproteinases-3 (TIMP3) is a tumor suppressor and a potent inhibitor of angiogenesis. TIMP3 exerts its anti-angiogenic effect via a direct interaction with vascular endothelial growth factor (VEGF) receptor-2 (KDR) and inhibition of proliferation, migration and tube formation of endothelial cells (ECs). TIMP3 has also been shown to induce apoptosis in some cancer cells and vascular smooth muscle cells via MMP inhibition and caspase-dependent mechanisms. In this study, we examined the molecular mechanisms of TIMP3-mediated apoptosis in endothelial cells. We have previously demonstrated that mice developed smaller tumors with decreased vascularity when injected with breast carcinoma cells overexpressing TIMP3, than with control breast carcinoma cells. TIMP3 overexpression resulted in increased apoptosis in human breast carcinoma (MDA-MB435) in vivo but not in vitro. However, TIMP3 could induce apoptosis in ECs in vitro. The apoptotic activity of TIMP3 in ECs appears to be independent of MMP inhibitory activity. Furthermore, the equivalent expression of functional TIMP3 promoted apoptosis and caspase activation in ECs expressing KDR (PAE/KDR), but not in ECs expressing PDGF beta-receptor (PAE/β-R). Surprisingly, the apoptotic activity of TIMP3 appears to be independent of caspases. TIMP3 inhibited matrix-induced focal adhesion kinase (FAK) tyrosine phosphorylation and association with paxillin and disrupted the incorporation of β3 integrin, FAK and paxillin into focal adhesion contacts on the matrix, which were not affected by caspase inhibitors. Thus, TIMP3 may induce apoptosis in ECs by triggering a caspase-independent cell death pathway and targeting a FAK-dependent survival pathway.

TRIF Licenses Caspase-11-Dependent NLRP3 Inflammasome Activation by Gram-Negative Bacteria

PubMed Central

Rathinam, Vijay A.K.; Vanaja, Sivapriya Kailasan; Waggoner, Lisa; Sokolovska, Anna; Becker, Christine; Stuart, Lynda M.; Leong, John M.; Fitzgerald, Katherine A.

2013-01-01

SUMMARY Systemic infections with Gram-negative bacteria are characterized by high mortality rates due to the “sepsis syndrome,” a widespread and uncontrolled inflammatory response. Though it is well recognized that the immune response during Gram-negative bacterial infection is initiated after the recognition of endotoxin by Toll-like receptor 4, the molecular mechanisms underlying the detrimental inflammatory response during Gram-negative bacteremia remain poorly defined. Here, we identify a TRIF pathway that licenses NLRP3 inflammasome activation by all Gram-negative bacteria. By engaging TRIF, Gram-negative bacteria activate caspase-11. TRIF activates caspase-11 via type I IFN signaling, an event that is both necessary and sufficient for caspase-11 induction and autoactivation. Caspase-11 subsequently synergizes with the assembled NLRP3 inflammasome to regulate caspase-1 activation and leads to caspase-1-independent cell death. These events occur specifically during infection with Gram-negative, but not Gram-positive, bacteria. The identification of TRIF as a regulator of caspase-11 underscores the importance of TLRs as master regulators of inflammasomes during Gram-negative bacterial infection. PMID:22819539

Role of Caspase-3 in acute light damage to retina of rats.

PubMed

Wang, Xiao; Hu, Shi-Xing; Li, Wei; Lin, Shao-Chun

2007-03-01

To investigate the role of Caspase-3 in retinal damage caused by light exposure in rats. Light injury to retina was induced by persistent exposure to illumination (intensity: 30 000 +/- 50 lux) of operating microscope for 30 minutes in the right eyes of Sprague-Dawley rats. The pathological changes of retina were observed under optical and electron microscopies at different time points, which were 6 hours, 1, 3, 7, and 15 days after the light exposure. Apoptosis of retinal cells was analyzed by flow cytometry. The activity of Caspase-3 was evaluated by using the Caspase-3 assay kit. At the same time, the expression of Caspase-3 protease was determined with Western blot analysis. The examination results of optical and transmission electron microscopes showed that edema of inner and outer segments of the retina, especially the chondriosome inside the inner segment, became obvious 6 hours after the light exposure. The change was deteriorated along with the increasing time. The structures of the discoidal valve dissociated in the outer segment simultaneously. Disorderly arranged nuclei, karyopycnosis, and thinning in the outer nuclear layer were observed. The retinal pigment epithelium almost disappeared during the later stage. The staining results of Annexin-V combined with PI demonstrated that the proportion of apoptotic cells increased with time. The proportion between 7th day (82.7%) and 15th day (80.4%), however, showed no significant difference. Caspase-3 became remarkably active with the lapse of time, which increased from 0.02 at 6th hour to the peak of 9.8 at 7th day before it started to descend. The Western blot detected a expression of the active form of Caspase-3 at 7th day and 15th day. Apoptosis of photoreceptor cells is markedly involved in the light damage and Caspase-3 protease may play an important role in the apoptotic process of the retina after light exposure in rats.

Targeting caspase-6 and caspase-8 to promote neuronal survival following ischemic stroke.

PubMed

Shabanzadeh, A P; D'Onofrio, P M; Monnier, P P; Koeberle, P D

2015-11-05

Previous studies show that caspase-6 and caspase-8 are involved in neuronal apoptosis and regenerative failure after trauma of the adult central nervous system (CNS). In this study, we evaluated whether caspase-6 or -8 inhibitors can reduce cerebral or retinal injury after ischemia. Cerebral infarct volume, relative to appropriate controls, was significantly reduced in groups treated with caspase-6 or -8 inhibitors. Concomitantly, these treatments also reduced neurological deficits, reduced edema, increased cell proliferation, and increased neurofilament levels in the injured cerebrum. Caspase-6 and -8 inhibitors, or siRNAs, also increased retinal ganglion cell survival at 14 days after ischemic injury. Caspase-6 or -8 inhibition also decreased caspase-3, -6, and caspase-8 cleavage when assayed by western blot and reduced caspase-3 and -6 activities in colorimetric assays. We have shown that caspase-6 or caspase-8 inhibition decreases the neuropathological consequences of cerebral or retinal infarction, thereby emphasizing their importance in ischemic neuronal degeneration. As such, caspase-6 and -8 are potential targets for future therapies aimed at attenuating the devastating functional losses that result from retinal or cerebral stroke.

Multiple Mechanisms of Zinc-Mediated Inhibition for the Apoptotic Caspases-3, -6, -7, and -8.

PubMed

Eron, Scott J; MacPherson, Derek J; Dagbay, Kevin B; Hardy, Jeanne A

2018-05-18

Zinc is emerging as a widely used and important biological regulatory signal. Cellular zinc levels are tightly regulated by a complex array of zinc importers and exporters to control processes such as apoptotic cell death. While caspase inhibition by zinc has been reported previously, the reported inhibition constants were too weak to suggest a critical biological role for zinc-mediated inhibition. In this work, we have adopted a method of assessing available zinc. This allowed assessment of accurate inhibition constants for apoptotic caspases, caspase-3, -6, -7, and -8. Each of these caspases are inhibited by zinc at intracellular levels but with widely differing inhibition constants and different zinc binding stoichiometries. Caspase-3, -6, and -8 appear to be constitutively inhibited by typical zinc levels, and this inhibition must be lifted to allow activation. The inhibition constant for caspase-7 (76 nM) is much weaker than for the other apoptotic caspases (2.6-6.9 nM) suggesting that caspase-7 is not inactivated by normal zinc concentrations but can be inhibited under conditions of zinc stress. Caspase-3, -7, and -8 were found to bind three, one, and two zincs, respectively. In each of these caspases, zinc was present in the active site, in contrast to caspase-6, which binds one zinc allosterically. The most notable new mechanism to emerge from this work is for zinc-mediated inhibition of caspase-8. Zinc binds caspase-8 directly at the active site and at a second site. Zinc binding inhibits formation of the caspase-8 dimer, the activated form of the enzyme. Together these findings suggest that zinc plays a critical role in regulation of apoptosis by direct inactivation of caspases, in a manner that is unique for each caspase.

Executioner Caspase-3 and 7 Deficiency Reduces Myocyte Number in the Developing Mouse Heart

PubMed Central

Cardona, Maria; López, Juan Antonio; Serafín, Anna; Rongvaux, Anthony; Inserte, Javier; García-Dorado, David; Flavell, Richard; Llovera, Marta; Cañas, Xavier; Vázquez, Jesús; Sanchis, Daniel

2015-01-01

Executioner caspase-3 and -7 are proteases promoting cell death but non-apoptotic roles are being discovered. The heart expresses caspases only during development, suggesting they contribute to the organ maturation process. Therefore, we aimed at identifying novel functions of caspases in heart development. We induced simultaneous deletion of executioner caspase-3 and -7 in the mouse myocardium and studied its effects. Caspase knockout hearts are hypoplastic at birth, reaching normal weight progressively through myocyte hypertrophy. To identify the molecular pathways involved in these effects, we used microarray-based transcriptomics and multiplexed quantitative proteomics to compare wild type and executioner caspase-deficient myocardium at different developmental stages. Transcriptomics showed reduced expression of genes promoting DNA replication and cell cycle progression in the neonatal caspase-deficient heart suggesting reduced myocyte proliferation, and expression of non-cardiac isoforms of structural proteins in the adult null myocardium. Proteomics showed reduced abundance of proteins involved in oxidative phosphorylation accompanied by increased abundance of glycolytic enzymes underscoring retarded metabolic maturation of the caspase-null myocardium. Correlation between mRNA expression and protein abundance of relevant genes was confirmed, but transcriptomics and proteomics indentified complementary molecular pathways influenced by caspases in the developing heart. Forced expression of wild type or proteolytically inactive caspases in cultured cardiomyocytes induced expression of genes promoting cell division. The results reveal that executioner caspases can modulate heart’s cellularity and maturation during development, contributing novel information about caspase biology and heart development. PMID:26121671

VX-induced cell death involves activation of caspase-3 in cultured rat cortical neurons.

PubMed

Tenn, Catherine C; Wang, Yushan

2007-05-01

Exposure of cell cultures to organophosphorous compounds such as VX can result in cell death. However, it is not clear whether VX-induced cell death is necrotic or involves programmed cell death mechanisms. Activation of caspases, a family of cysteine proteases, is often involved in cell death, and in particular, caspase-3 activation appears to be a key event in programmed cell death processes including apoptosis. In this study, we investigated VX-induced neuronal cell death, as well as the underlying mechanism in terms of its effect on caspase-3 activity. Primary cortical neuronal cultures were prepared from gestational days 17 to 19 Sprague Dawley rat fetuses. At maturation, the cells were treated with varying concentrations of VX and cell death was evaluated by lactate dehydrogenase (LDH) release. VX induced an increase in LDH release in a concentration-dependent manner. Morphological VX-induced cell death was also characterized by using nuclear staining with propidium iodide and Hoechst 33342. VX induced a concentration- and time-dependent increase in caspase-3 activation. Caspase-3 activation was also confirmed by the proteolytic cleavage of poly(ADP-ribose)polymerase (PARP), an endogenous caspase-3 substrate. These data suggested that in rat cortical neurons, VX-induced cell death via a programmed cell death pathway that involves changes in caspase-3 protease.

Caspase-3 activity, response to chemotherapy and clinical outcome in patients with colon cancer.

PubMed

de Oca, Javier; Azuara, Daniel; Sanchez-Santos, Raquel; Navarro, Matilde; Capella, Gabriel; Moreno, Victor; Sola, Anna; Hotter, Georgina; Biondo, Sebastiano; Osorio, Alfonso; Martí-Ragué, Joan; Rafecas, Antoni

2008-01-01

The prognostic value of the degree of apoptosis in colorectal cancer is controversial. This study evaluates the putative clinical usefulness of measuring caspase-3 activity as a prognostic factor in colonic cancer patients receiving 5-fluoracil adjuvant chemotherapy. We evaluated caspase-3-like protease activity in tumours and in normal colon tissue. Specimens were studied from 54 patients. These patients had either stage III cancer (Dukes stage C) or high-risk stage II cancer (Dukes stage B2 with invasion of adjacent organs, lymphatic or vascular infiltration or carcinoembryonic antigen [CEA] >5). Median follow-up was 73 months. Univariate analysis was performed previously to explore the relation of different variables (age, sex, preoperative CEA, tumour size, Dukes stage, vascular invasion, lymphatic invasion, caspase-3 activity in tumour and caspase-3 activity in normal mucosa) as prognostic factors of tumour recurrence after chemotherapy treatment. Subsequently, a multivariate Cox regression model was performed. Median values of caspase-3 activity in tumours were more than twice those in normal mucosa (88.1 vs 40.6 U, p=0.001), showing a statistically significant correlation (r=0.34). Significant prognostic factors of recurrence in multivariate analysis were: male sex (odds ratio, OR=3.53 [1.13-10.90], p=0.02), age (OR=1.09 [1.01-1.18], p=0.03), Dukes stage (OR=1.93 [1.01-3.70]), caspase-3 activity in normal mucosa (OR=1.02 [1.01-1.04], p=0.017) and caspase-3 activity in tumour (OR=1.02 [1.01-1.03], p=0.013). Low caspase-3 activity in the normal mucosa and tumour are independent prognostic factors of tumour recurrence in patients receiving adjuvant 5-fluoracil-based treatment in colon cancer, correlating with poor disease-free survival and higher recurrence rate.

Dynamic detection of caspase-3 activation during photosensitization by fluorescence resonance energy transfer

NASA Astrophysics Data System (ADS)

Wu, Yunxia; Xing, Da; Chen, Qun; Chen, Tongsheng; Tang, Yonghong; Wan, Qingling

2005-04-01

Apoptosis is one of the important modes in PDT-induced cell death. Activation of caspase-3 is considered to be the final step in many apoptosis pathways. In this study, we used SCAT3, a fluorescence resonance energy transfer (FRET) probe containing caspase-3 substrate, to study the dynamics of caspase-3 activation in living ASTC-a-1 cells expressing stably SCAT3. The FRET analysis results indicated that caspase-3 activation in response to tumor necrosis factor-α or PDT resulted in cleavage of the linker peptide and subsequent disruption of the FRET signal. The SCAT3 was cleaved immediately after PDT treatment, but that for TNF-a treatment was delayed two hours. Our experimental results suggested that the different apoptotic pathways induced by TNF-α or PDT caused different cleavage kinetics of SCAT3. This study shows that FRET technique based on GFPs could be used to study the mechanism of PDT-induced apoptosis in living cells.

Engineering a light-activated caspase-3 for precise ablation of neurons in vivo

PubMed Central

Smart, Ashley D.; Pache, Roland A.; Thomsen, Nathan D.; Kortemme, Tanja; Davis, Graeme W.; Wells, James A.

2017-01-01

The circuitry of the brain is characterized by cell heterogeneity, sprawling cellular anatomy, and astonishingly complex patterns of connectivity. Determining how complex neural circuits control behavior is a major challenge that is often approached using surgical, chemical, or transgenic approaches to ablate neurons. However, all these approaches suffer from a lack of precise spatial and temporal control. This drawback would be overcome if cellular ablation could be controlled with light. Cells are naturally and cleanly ablated through apoptosis due to the terminal activation of caspases. Here, we describe the engineering of a light-activated human caspase-3 (Caspase-LOV) by exploiting its natural spring-loaded activation mechanism through rational insertion of the light-sensitive LOV2 domain that expands upon illumination. We apply the light-activated caspase (Caspase-LOV) to study neurodegeneration in larval and adult Drosophila. Using the tissue-specific expression system (UAS)-GAL4, we express Caspase-LOV specifically in three neuronal cell types: retinal, sensory, and motor neurons. Illumination of whole flies or specific tissues containing Caspase-LOV–induced cell death and allowed us to follow the time course and sequence of neurodegenerative events. For example, we find that global synchronous activation of caspase-3 drives degeneration with a different time-course and extent in sensory versus motor neurons. We believe the Caspase-LOV tool we engineered will have many other uses for neurobiologists and others for specific temporal and spatial ablation of cells in complex organisms. PMID:28893998

Engineering a light-activated caspase-3 for precise ablation of neurons in vivo.

PubMed

Smart, Ashley D; Pache, Roland A; Thomsen, Nathan D; Kortemme, Tanja; Davis, Graeme W; Wells, James A

2017-09-26

The circuitry of the brain is characterized by cell heterogeneity, sprawling cellular anatomy, and astonishingly complex patterns of connectivity. Determining how complex neural circuits control behavior is a major challenge that is often approached using surgical, chemical, or transgenic approaches to ablate neurons. However, all these approaches suffer from a lack of precise spatial and temporal control. This drawback would be overcome if cellular ablation could be controlled with light. Cells are naturally and cleanly ablated through apoptosis due to the terminal activation of caspases. Here, we describe the engineering of a light-activated human caspase-3 (Caspase-LOV) by exploiting its natural spring-loaded activation mechanism through rational insertion of the light-sensitive LOV2 domain that expands upon illumination. We apply the light-activated caspase (Caspase-LOV) to study neurodegeneration in larval and adult Drosophila Using the tissue-specific expression system (UAS)-GAL4, we express Caspase-LOV specifically in three neuronal cell types: retinal, sensory, and motor neurons. Illumination of whole flies or specific tissues containing Caspase-LOV-induced cell death and allowed us to follow the time course and sequence of neurodegenerative events. For example, we find that global synchronous activation of caspase-3 drives degeneration with a different time-course and extent in sensory versus motor neurons. We believe the Caspase-LOV tool we engineered will have many other uses for neurobiologists and others for specific temporal and spatial ablation of cells in complex organisms.

Cleavage of the actin-capping protein alpha -adducin at Asp-Asp-Ser-Asp633-Ala by caspase-3 is preceded by its phosphorylation on serine 726 in cisplatin-induced apoptosis of renal epithelial cells.

PubMed

van de Water, B; Tijdens, I B; Verbrugge, A; Huigsloot, M; Dihal, A A; Stevens, J L; Jaken, S; Mulder, G J

2000-08-18

Decreased phosphorylation of focal adhesion kinase and paxillin is associated with loss of focal adhesions and stress fibers and precedes the onset of apoptosis (van de Water, B., Nagelkerke, J. F., and Stevens, J. L. (1999) J. Biol. Chem. 274, 13328-13337). The cortical actin cytoskeletal network is also lost during apoptosis, yet little is known about the temporal relationship between altered phosphorylation of proteins that are critical in the regulation of this network and their potential cleavage by caspases during apoptosis. Adducins are central in the cortical actin network organization. Cisplatin caused apoptosis of renal proximal tubular epithelial cells, which was associated with the cleavage of alpha-adducin into a 74-kDa fragment; this was blocked by a general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk). Hemagglutinin-tagged human alpha-adducin was cleaved into a similar 74-kDa fragment by caspase-3 in vitro but not by caspase-6 or -7. Asp-Arg-Val-Asp(29)-Glu, Asp-Ile-Val-Asp(208)-Arg, and Asp-Asp-Ser-Asp(633)-Ala were identified as the principal caspase-3 cleavage sites; Asp-Asp-Ser-Asp(633)-Ala was key in the formation of the 74-kDa fragment. Cisplatin also caused an increased phosphorylation of alpha-adducin and gamma-adducin in the MARCKS domain that preceded alpha-adducin cleavage and was associated with loss of adducins from adherens junctions; this was not affected by z-VAD-fmk. In conclusion, the data support a model in which increased phosphorylation of alpha-adducin due to cisplatin leads to dissociation from the cytoskeleton, a situation rendered irreversible by caspase-3-mediated cleavage of alpha-adducin at Asp-Asp-Ser-Asp(633)-Ala.

Blazeispirol A from Agaricus blazei fermentation product induces cell death in human hepatoma Hep 3B cells through caspase-dependent and caspase-independent pathways.

PubMed

Su, Zheng-Yuan; Tung, Yen-Chen; Hwang, Lucy Sun; Sheen, Lee-Yan

2011-05-11

Currently, liver cancer is a leading cause of cancer-related death in the world. Hepatocellular carcinoma is the most common type of liver cancer. Previously, it was reported that blazeispirol A (BA) is the most active antihepatoma compound in an ethanolic extract of Agaricus blazei fermentation product. The aim of this study was to understand the antihepatoma mechanism of BA in human liver cancer Hep 3B cells. The results showed that BA inhibited the growth of Hep 3B cells and increased the percentage of cells in sub-G1 phase in a concentration- and time-dependent manner. In addition, BA treatment resulted in DNA fragmentation, caspase-9 and caspase-3 activations, poly(ADP-ribose)polymerase (PARP) degradation, down-regulation of Bcl-2 and Bcl-xL expressions, up-regulation of Bax expression, and disruption of the mitochondrial membrane potential (MMP) in Hep 3B cells. Furthermore, z-VAD-fmk, a caspase inhibitor, did not enhance the viability of BA-treated Hep 3B cells, and BA induced the release of HtrA2/Omi and apoptosis-inducing factor (AIF) from mitochondria into the cytosol. These findings suggested that BA with novel chemopreventive and chemotherapeutic potentials causes both caspase-dependent and caspase-independent cell death in Hep 3B cells.

[Expression and clinical significance of Xiap and Caspase-3 protien in primary epithelia ovarian cancer].

PubMed

Chen, Wei; Peng, Ping

2010-07-01

To study the expression and clinical significance of Xiap, Caspase-3 protein in primary epithelia ovarian cancer. The Xiap and Caspase-3 were detected by immunohistochemical in 40 cases of epithelial ovarian cancer 20 cases of borderline ovarian tumor, 15 cases of benign ovarian tumor, and 15 normal ovarian tissues. There were significantly different between the expression of Xiap in epithelial ovarian cancer, borderline ovarian tumor, benign ovarian tumor and normal ovarian tissues. The expression of Caspase-3 in epithelial ovarian cancer and borderline ovarian tumor was significantly lower than that in benign ovarian tumor and normal ovarian tissue (P<0.01). The expression of Xiap in epithelial ovarian cancer was related to clinc stage, pathological grade and living. The expression of caspase-3 in epithelial ovarian cancer was related to clinc stage and living (P<0.01). The expressions of Xiap and Caspase-3 may be important roles for the formation and development of epithelia ovarian cancer. The expressions of Xiap and Caspase-3 are the poor prognostic factors in epithelial ovarian carcinomas.

Caspase-1 and IL-1β Processing in a Teleost Fish

PubMed Central

Reis, Marta I. R.; do Vale, Ana; Pereira, Pedro J. B.; Azevedo, Jorge E.; dos Santos, Nuno M. S.

2012-01-01

Interleukine-1β (IL-1β) is the most studied pro-inflammatory cytokine, playing a central role in the generation of systemic and local responses to infection, injury, and immunological challenges. In mammals, IL-1β is synthesized as an inactive 31 kDa precursor that is cleaved by caspase-1 generating a 17.5 kDa secreted active mature form. The caspase-1 cleavage site strictly conserved in all mammalian IL-1β sequences is absent in IL-1β sequences reported for non-mammalian vertebrates. Recently, fish caspase-1 orthologues have been identified in sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) but very little is known regarding their processing and activity. In this work it is shown that sea bass caspase-1 auto-processing is similar to that of the human enzyme, resulting in active p24/p10 and p20/p10 heterodimers. Moreover, the presence of alternatively spliced variants of caspase-1 in sea bass is reported. The existence of caspase-1 isoforms in fish and in mammals suggests that they have been evolutionarily maintained and therefore are likely to play a regulatory role in the inflammatory response, as shown for other caspases. Finally, it is shown that sea bass and avian IL-1β are specifically cleaved by caspase-1 at different but phylogenetically conserved aspartates, distinct from the cleavage site of mammalian IL-1β. PMID:23226286

Structural basis for alpha fetoprotein-mediated inhibition of caspase-3 activity in hepatocellular carcinoma cells.

PubMed

Lin, Bo; Zhu, Mingyue; Wang, Wenting; Li, Wei; Dong, Xu; Chen, Yi; Lu, Yan; Guo, Junli; Li, Mengsen

2017-10-01

Alpha-fetoprotein (AFP) is an early serum growth factor in the foetal liver development and hepatic carcinogenesis; However, the precise biological role of cytoplasmic AFP remains elusive. Although we recently demonstrated that cytoplasmic AFP might interact with caspase-3 and inhibit the signal transduction of apoptosis in human hepatocellular carcinoma (HCC) cells, the details of this interaction are not clear. To reveal the molecular relationship between AFP and caspase-3, we performed molecular docking, co-immunoprecipitation (Co-IP), laser confocal microscopy, site-directed mutagenesis and functional experiments to analyse the key amino acid residues in the binding site of caspase-3. The results of Co-IP, laser confocal microscopy and functional analyses were consistent with the computational model. We also used the model to explain why AFP cannot bind to caspase-8. These results provide the molecular basis for the AFP-mediated inhibition of caspase-3 activity in HCC cells. Altogether, we found that AFP interacts with caspase-3 through precise amino acids, namely loop-4 residues Glu-248, Asp-253 and His-257. The results further demonstrated that AFP plays a critical role in the inhibition of the apoptotic signal transduction that mediated by caspase-3. Thus, AFP might represent a novel biotarget for the therapy of HCC patients. © 2017 UICC.

Caspase-3 controls AML1-ETO-driven leukemogenesis via autophagy modulation in a ULK1-dependent manner.

PubMed

Man, Na; Tan, Yurong; Sun, Xiao-Jian; Liu, Fan; Cheng, Guoyan; Greenblatt, Sarah M; Martinez, Camilo; Karl, Daniel L; Ando, Koji; Sun, Ming; Hou, Dan; Chen, Bingyi; Xu, Mingjiang; Yang, Feng-Chun; Chen, Zhu; Chen, Saijuan; Nimer, Stephen D; Wang, Lan

2017-05-18

AML1-ETO (AE), a fusion oncoprotein generated by t(8;21), can trigger acute myeloid leukemia (AML) in collaboration with mutations including c-Kit, ASXL1/2, FLT3, N-RAS, and K-RAS. Caspase-3, a key executor among its family, plays multiple roles in cellular processes, including hematopoietic development and leukemia progression. Caspase-3 was revealed to directly cleave AE in vitro, suggesting that AE may accumulate in a Caspase-3-compromised background and thereby accelerate leukemogenesis. Therefore, we developed a Caspase-3 knockout genetic mouse model of AML and found that loss of Caspase-3 actually delayed AML1-ETO9a (AE9a)-driven leukemogenesis, indicating that Caspase-3 may play distinct roles in the initiation and/or progression of AML. We report here that loss of Caspase-3 triggers a conserved, adaptive mechanism, namely autophagy (or macroautophagy), which acts to limit AE9a-driven leukemia. Furthermore, we identify ULK1 as a novel substrate of Caspase-3 and show that upregulation of ULK1 drives autophagy initiation in leukemia cells and that inhibition of ULK1 can rescue the phenotype induced by Caspase-3 deletion in vitro and in vivo. Collectively, these data highlight Caspase-3 as an important regulator of autophagy in AML and demonstrate that the balance and selectivity between its substrates can dictate the pace of disease. © 2017 by The American Society of Hematology.

SfDronc, an initiator caspase involved in apoptosis in the fall armyworm Spodoptera frugiperda

PubMed Central

Huang, Ning; Civciristov, Srgjan; Hawkins, Christine J.; Clem, Rollie J.

2013-01-01

Initiator caspases are the first caspases that are activated following an apoptotic stimulus, and are responsible for cleaving and activating downstream effector caspases, which directly cause apoptosis. We have cloned a cDNA encoding an ortholog of the initiator caspase Dronc in the lepidopteran insect Spodoptera frugiperda. The SfDronc cDNA encodes a predicted protein of 447 amino acids with a molecular weight of 51 kDa. Overexpression of SfDronc induced apoptosis in Sf9 cells, while partial silencing of SfDronc expression in Sf9 cells reduced apoptosis induced by baculovirus infection or by treatment with UV or actinomycin D. Recombinant SfDronc exhibited several expected biochemical characteristics of an apoptotic initiator caspase: 1) SfDronc efficiently cleaved synthetic initiator caspase substrates, but had very little activity against effector caspase substrates; 2) mutation of a predicted cleavage site at position D340 blocked autoprocessing of recombinant SfDronc and reduced enzyme activity by approximately 10-fold; 3) SfDronc cleaved the effector caspase Sf-caspase-1 at the expected cleavage site, resulting in Sf-caspase-1 activation; and 4) SfDronc was strongly inhibited by the baculovirus caspase inhibitor SpliP49, but not by the related protein AcP35. These results indicate that SfDronc is an initiator caspase involved in caspase-dependent apoptosis in S. frugiperda, and as such is likely to be responsible for the initiator caspase activity in S. frugiperda cells known as Sf-caspase-X. PMID:23474489

Increased expression and processing of caspase-12 after traumatic brain injury in rats.

PubMed

Larner, Stephen F; Hayes, Ronald L; McKinsey, Deborah M; Pike, Brian R; Wang, Kevin K W

2004-01-01

Traumatic brain injury (TBI) disrupts tissue homeostasis resulting in pathological apoptotic activation. Recently, caspase-12 was reported to be induced and activated by the unfolded protein response following excess endoplasmic reticulum (ER) stress. This study examined rat caspase-12 expression using the controlled cortical impact TBI model. Immunoblots of fractionalized cell lysates found elevated caspase-12 proform (approximately 60 kDa) and processed form (approximately 12 kDa), with peak induction observed within 24 h post-injury in the cortex (418% and 503%, respectively). Hippocampus caspase-12 proform induction peaked at 24 h post-injury (641%), while processed form induction peaked at 6 h (620%). Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis confirmed elevated caspase-12 mRNA levels after TBI. Injury severity (1.0, 1.2 or 1.6 mm compression) was associated with increased caspase-12 mRNA expression, peaking at 5 days in the cortex (657%, 651% and 1259%, respectively) and 6 h in the hippocampus (435%, 451% and 460%, respectively). Immunohistochemical analysis revealed caspase-12 induction in neurons in both the cortex and hippocampus as well as in astrocytes at the contusion site. This is the first report of increased expression of caspase-12 following TBI. Our results suggest that the caspase-12-mediated ER apoptotic pathway may play a role in rat TBI pathology independent of the receptor- or mitochondria-mediated apoptotic pathways.

Melanoma differentiation associated gene-7/interleukin-24 induces caspase-3 denitrosylation to facilitate the activation of cancer cell apoptosis.

PubMed

Tian, Hui; Zhang, De-Fang; Zhang, Bao-Fu; Li, Hui-Zhong; Zhang, Qing; Li, Lian-Tao; Pei, Dong-Sheng; Zheng, Jun-Nian

2015-03-01

Melanoma differentiation-associated gene-7 (mda-7)/interleukin-24 (IL-24) induces caspase-3 cleavage and subsequent activation via the intrinsic or extrinsic pathway to result in cancer cell-selective apoptosis, but whether mda-7/IL-24 may directly regulate caspase-3 through the post-translational modification remains unknown. Here, we reported that tumor-selective replicating adenovirus ZD55-IL-24 led to caspase-3 denitrosylation and subsequent activation, indicating that caspase-3 denitrosylation played a crucial role in ZD55-IL-24-induced cancer cell apoptosis. To confirm the relationship between caspase-3 denitrosylation and its activation in response to ZD55-IL-24, we treated carcinoma cells with the different nitric oxide (NO) regulators to modulate caspase-3 denitrosylation level, then observed the corresponding caspase-3 cleavage. We found that NO inhibitor 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy-3-oxide (PTIO) promoted caspase-3 denitrosylation and caspase-3 cleavage, thereby exacerbating ZD55-IL-24-induced cancer cell apoptosis, whereas NO donor sodium nitroprusside (SNP) showed the opposite effect. Moreover, caspase-3 denitrosylation facilitated its downstream target poly ADP-ribose polymerase (PARP) degradation that further increased the apoptotic susceptibility. Although caspase-3 activation controlled by denitrosylation modification has emerged as an important regulator of programmed cell death, the detailed molecular mechanism by which caspase-3 exerts its denitrosylation modification in response to ZD55-IL-24 still needs to be elucidated. Thus, our results demonstrated that ZD55-IL-24 increased Fas expression to enhance thioredoxin reductase 2 (TrxR2), which was responsible for caspase-3 denitrosylation. Collectively, these findings elucidate that ZD55-IL-24 induces caspase-3 denitrosylation through Fas-mediated TrxR2 enhancement, thereby facilitating caspase-3 cleavage and the downstream caspase signaling pathway activation, which

3-Monochloro-1,2-propanediol (3-MCPD) induces apoptosis via mitochondrial oxidative phosphorylation system impairment and the caspase cascade pathway.

PubMed

Peng, Xiaoli; Gan, Jing; Wang, Qian; Shi, Zhenqiang; Xia, Xiaodong

2016-11-30

3-Monochloro-1,2-propanediol (3-MCPD) is the most toxic chloropropanols compounds in foodstuff which mainly generated during thermal processing. Kidney is one of the primary target organs for 3-MCPD. Using human embryonic kidney cell (HEK293FT) as an in vitro model, we found that 3-MCPD caused concentration-dependent increase in cytoxicity as assessed by dye uptake, lactatedehydrogenase (LDH) leakage and MTT assays. HEK293FT cell treated with 3-MCPD suffered the decrease of mitochondrial membrane potential and the impairment of mitochondrial oxidative phosphorylation system, especially the reduced amount of mRNA expression and protein synthesis of electron transport chain complex II, complex IV, and complex III. More importantly, energy release (ATP synthesis) was significantly inhibited by 3-MCPD resulting from the down regulation expressions of ATP synthase (ATP6 and ATP8), as well as the loss of transmembrane potential required for synthesis of ATP. The decreased ratio of mitochondrial apoptogenic factors Bax/Bcl-2 and the cytochrome-c release from mitochondria to cytosol followed by the activation of apoptotic initiators caspase 9 and apoptotic executioners (caspase 3, caspase 6 and caspase 7) leading to apoptosis. The activation of caspase 8 and caspase 2 implied that there were probably other factors to induce the caspase-dependent apoptosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Procaspase-activating compound 1 induces a caspase-3-dependent cell death in cerebellar granule neurons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aziz, Gulzeb; Akselsen, Oyvind W.; Hansen, Trond V.

2010-09-15

Procaspase-activating compound 1, PAC-1, has been introduced as a direct activator of procaspase-3 and has been suggested as a therapeutic agent against cancer. Its activation of procaspase-3 is dependent on the chelation of zinc. We have tested PAC-1 and an analogue of PAC-1 as zinc chelators in vitro as well as their ability to activate caspase-3 and induce cell death in chicken cerebellar granule neuron cultures. These neurons are non-dividing, primary cells with normal caspase-3. The results reported herein show that PAC-1 chelates zinc, activates procaspase-3, and leads to caspase-3-dependent cell death in neurons, as the specific caspase-3-inhibitor Ac-DEVD-cmk inhibitedmore » both the caspase-3 activity and cell death. Thus, chicken cerebellar granule neurons is a suitable model to study mechanisms of interference with apoptosis of PAC-1 and similar compounds. Furthermore, the present study also raises concern about potential neurotoxicity of PAC-1 if used in cancer therapy.« less

Antiapoptotic property of human alpha-synuclein in neuronal cell lines is associated with the inhibition of caspase-3 but not caspase-9 activity.

PubMed

Li, Wenxue; Lee, Michael K

2005-06-01

Abnormalities of alpha-synuclein (alpha-Syn) are mechanistically linked to Parkinson's disease (PD) and other alpha-synucleinopathies. To gain additional insights into the relationships between alpha-Syn expression and cell death, we examined the effects of expressing human alpha-Syn (Hualpha-Syn) variants on the cellular vulnerability to apoptotic stimuli. We show that the expression of wild-type (WT) and A30P mutant, but not A53T mutant, Hualpha-Syn leads to the protection of neuronal cell lines from apoptosis but not necrosis. Significantly, Hualpha-Syn did not protect non-neuronal cell lines from apoptosis. We also show that A53T mutant is a loss of function in regards to the antiapoptotic property since the expression of WT Hualpha-Syn with an excess of A53T mutant Hualpha-Syn leads to protection of the cells from apoptosis. The antiapoptotic property is specific to human alpha-Syn as neither beta-Syn nor mouse alpha-Syn protected cells from apoptosis, and the carboxy-terminal 20 amino acids are required for the antiapoptotic property. Analyses of capase-3 and caspase-9 activation reveal that the antiapoptotic property of Hualpha-Syn in neuronal cell lines is associated with the attenuation of caspase-3 activity without affecting the caspase-9 activity or the levels of cleaved, active caspase-3. We conclude that Hualpha-Syn modulates the activity of cleaved caspase-3 product in neuronal cell lines.

SfDronc, an initiator caspase involved in apoptosis in the fall armyworm Spodoptera frugiperda.

PubMed

Huang, Ning; Civciristov, Srgjan; Hawkins, Christine J; Clem, Rollie J

2013-05-01

Initiator caspases are the first caspases that are activated following an apoptotic stimulus, and are responsible for cleaving and activating downstream effector caspases, which directly cause apoptosis. We have cloned a cDNA encoding an ortholog of the initiator caspase Dronc in the lepidopteran insect Spodoptera frugiperda. The SfDronc cDNA encodes a predicted protein of 447 amino acids with a molecular weight of 51 kDa. Overexpression of SfDronc induced apoptosis in Sf9 cells, while partial silencing of SfDronc expression in Sf9 cells reduced apoptosis induced by baculovirus infection or by treatment with UV or actinomycin D. Recombinant SfDronc exhibited several expected biochemical characteristics of an apoptotic initiator caspase: 1) SfDronc efficiently cleaved synthetic initiator caspase substrates, but had very little activity against effector caspase substrates; 2) mutation of a predicted cleavage site at position D340 blocked autoprocessing of recombinant SfDronc and reduced enzyme activity by approximately 10-fold; 3) SfDronc cleaved the effector caspase Sf-caspase-1 at the expected cleavage site, resulting in Sf-caspase-1 activation; and 4) SfDronc was strongly inhibited by the baculovirus caspase inhibitor SpliP49, but not by the related protein AcP35. These results indicate that SfDronc is an initiator caspase involved in caspase-dependent apoptosis in S. frugiperda, and as such is likely to be responsible for the initiator caspase activity in S. frugiperda cells known as Sf-caspase-X. Copyright © 2013 Elsevier Ltd. All rights reserved.

Feasibility study for clinical application of caspase-3 inhibitors in Pemphigus vulgaris.

PubMed

Hariton, William V J; Galichet, Arnaud; Vanden Berghe, Tom; Overmiller, Andrew M; Mahoney, My G; Declercq, Wim; Müller, Eliane J

2017-12-01

The potentially severe side effects of systemic corticosteroids and immunosuppressants used in Pemphigus vulgaris (PV) call for novel therapeutic approaches. In this context, pharmacological inhibition of major pathogenic signalling effectors represents a promising alternative. However, we have also shown that overinhibition of effectors required for epidermal homeostasis can exacerbate PV pathophysiology implicating transepidermal keratinocyte fragility. A feedforward target validation therefore preferentially includes studies on knockout mouse models. We previously reported on successful amelioration of PV blisters following inhibition of non-apoptotic, low-level caspase-3. Here, we use conditional, keratinocyte-specific caspase-3-deficient mice (casp3 EKO ) to demonstrate (i) absence of keratinocyte fragility upon injection of the potent Dsg3-specific antibody AK23 and (ii) amelioration of blistering on the background of known signalling effectors. Our results provide the experimental proof of concept justifying translation of the caspase-3 inhibitor approach into PV clinical trials. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Molecular cloning and expression of caspase-3 in the protandrous cinnamon clownfish, Amphiprion melanopus, during sex change.

PubMed

Kim, Na Na; Lee, Jehee; Habibi, Hamid R; Choi, Cheol Young

2013-06-01

The caspase-3 appears to be a key protease in the apoptotic pathway. We identified caspase-3 complementary DNAs from the ovaries of the protandrous cinnamon clownfish (Amphiprion melanopus), and investigated its mRNA and proteins, and activity levels during the sex change (I, mature male; II, male at 90 days after removing of the female; and III, mature female). The nucleotide sequence of the caspase-3 cDNA was 969 base pairs in length with open reading frames encoding peptides of 282 amino acids. The caspase-3 mRNA and protein, and activity levels in stages of the mature gonad are higher than those of the development gonad stage. To understand the effect of gonadotropin-releasing hormone (GnRH) on gonad apoptosis, we examined expression of genes caspase-3 mRNA and activity level in immature cinnamon clownfish gonads after GnRH analogue (GnRHa). The findings support the hypothesis that caspase-3 expression is associated with both testicular and ovarian development, and suggests that it may play a role in the control of ovarian development in cinnamon clownfish. Also, we demonstrate that GnRH agonists stimulate caspase-3 production which can in turn stimulate apoptosis. The present study provides a framework for better understanding of the role of caspase-3 during sex change processes in fish.

Cooperation of bisphenol A and leptin in inhibition of caspase-3 expression and activity in OVCAR-3 ovarian cancer cells.

PubMed

Ptak, Anna; Rak-Mardyła, Agnieszka; Gregoraszczuk, Ewa L

2013-09-01

This study was designed to investigate the effect of bisphenol A and leptin on caspase-3 expression and activity in OVCAR-3 ovarian cancer cells. Caspase-3 and survivin expression was measured at the transcript level by real-time PCR and at the protein level by Western blotting. In addition, caspase-3 activity was measured, using a fluorometric assay, upon exposure to bisphenol A (40 nM) alone, leptin (2.5 nM) alone, and the combination of both agents. 17β-estradiol (40 nM) was used as a positive control for estrogenic properties of bisphenol A. Results showed that the interaction between bisphenol A and leptin, which was similar to that observed between 17β-estradiol and leptin, led to the inhibition of caspase-3 expression and activity in OVCAR-3 cells. Surprisingly, survivin was found to not be involved in the anti-apoptotic activity of either agent. Also, results showed that leptin inhibits caspase-3 activity by acting on the signal transducers and activators of transcription 3 (STAT3) pathway, but bisphenol A and 17β-estradiol by the extracellular-signal-regulated kinases 1/2 (ERK1/2) pathway. In conclusion, the study reveals that bisphenol A and leptin interact to inhibit caspase-3 expression and activity by modulating STAT3 and ERK1/2 signaling pathways in OVCAR-3 cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

Inhibition of apoptosis by knockdown of caspase-3 with siRNA in rat bone marrow mesenchymal stem cells.

PubMed

Hua, Ping; Liu, Li-Bao; Liu, Jia-Liang; Wang, Meng; Jiang, Hui-Qi; Zeng, Kuan; Yang, Yan-Qi; Yang, Song-Ran

2013-09-01

Transplantation of bone marrow mesenchymal stem cells is a promising new strategy for the repair of infarcted cardiac tissue. However, the majority of transplanted bone marrow mesenchymal stem cells (BMSCs) die soon after transplantation, due in part to oxidative stress in the ischemic region. Oxidative stress is known to induce apoptosis through the activation of caspase-3. The aim of this study is to determine whether small interfering RNA targeting caspase-3 can inhibit the apoptosis of rat BMSCs in vitro. Caspase-3 siRNA expression vectors were prepared and transfected into rat BMSCs in the presence of liposomes. Western blot assay and real-time polymerase chain reaction (RT-PCR) were performed to detect caspase-3 expression. A retrovirus packaging system was employed to package 293FT cells producing caspase-3 siRNA virus, which were transfected into rat BMSCs. Those stably expressing caspase-3 siRNA were screened by Western blot assay and RT-PCR to determine caspase-3 expression levels. Stable transfection of caspase-3 siRNA significantly decreased caspase-3 protein (0.26 ± 0.001 vs. 0.42 ± 0.004, P < 0.05) and mRNA expression (0.19 ± 0.002 vs. 1, P < 0.05) in BMSCs compared to non-transfected BMSCs. Cells were incubated in H2O2 to induce apoptosis, which was detected by TUNEL staining, and BMSC morphology was not altered by either transient or stable transfection of caspase-3 siRNA. H2O2-induced apoptosis of BMSCs stably transfected with caspase-3 siRNA was dramatically reduced compared to that of normal BMSCs (11.0 ± 3.2 vs. 25.8 ± 4.2, P < 0.05). Caspase-3 knockdown BMSCs are thus more resistant to apoptosis than normal BMSCs, potentially increasing their survival rates under conditions that cause oxidative stress.

A Biotin Switch-Based Proteomics Approach Identifies 14-3-3ζ as a Target of Sirt1 in the Metabolic Regulation of Caspase-2

PubMed Central

Andersen, Joshua L.; Thompson, J. Will; Lindblom, Kelly R.; Johnson, Erika S.; Yang, Chih-Sheng; Lilley, Lauren R.; Freel, Christopher D.; Moseley, M. Arthur; Kornbluth, Sally

2011-01-01

While lysine acetylation in the nucleus is well characterized, comparatively little is known about its significance in cytoplasmic signaling. Here we show that inhibition of the Sirt1 deacetylase, which is primarily cytoplasmic in cancer cell lines, sensitizes these cells to caspase-2-dependent death. To identify relevant Sirt1 substrates, we developed a novel proteomics strategy, enabling the identification of a range of putative substrates, including 14-3-3ζ, a known direct regulator of caspase-2. We show here that inhibition of Sirtuin activity accelerates caspase activation and overrides caspase-2 suppression by nutrient abundance. Furthermore, 14-3-3ζ is acetylated prior to caspase activation, and supplementation of Xenopus egg extract with glucose-6-phosphate, which promotes caspase-2/14-3-3ζ binding, enhances 14-3-3ζ-directed Sirtuin activity. Conversely, inhibiting Sirtuin activity promotes 14-3-3ζ dissociation from caspase-2 in both egg extract and human cell lines. These data reveal a role for Sirt1 in modulating apoptotic sensitivity, in response to metabolic changes, by antagonizing 14-3-3ζ acetylation. PMID:21884983

A pro-apoptotic 15-kDa protein from Bacopa monnieri activates caspase-3 and downregulates Bcl-2 gene expression in mouse mammary carcinoma cells.

PubMed

Kalyani, Manjula Ishwara; Lingaraju, Sheela Mysore; Salimath, Bharathi P

2013-01-01

In diseases such as cancer, induction of apoptosis has been a new target for mechanism-based drug discovery. The central component of the process of apoptosis is a proteolytic system involving a family of proteases called caspases. Apoptosis involves characteristic morphological and biochemical events ultimately leading to cell demise. Apoptotic induction is evidently central to the mechanism of action of plant-derived anticancer drugs. Extract of the medicinal plant, Bacopa monnieri, inhibits tumor cell proliferation and accumulation of malignant ascites fluid. The crude sample when subjected to Soxhlet extraction yielded different solvent extracts of which the aqueous extract showed biological activity of apoptosis in Ehrlich ascites tumor cell lines (EAT). Bacopa monnieri water extract (BMWE) treatment of EAT cells produced apoptotic morphological characteristics and in-vivo DNA fragmentation, which is due to the activity of an endogenous endonuclease. The endonuclease responsible for DNA fragmentation acts downstream of caspase-3 activity and is also referred to as caspase-activated DNase (CAD). The CAD constitutively expressed in the cell cytoplasm is translocated into the nucleus upon BMWE treatment, as verified by Western blotting, leading to DNA fragmentation and to programmed cell death. The expression of the pro-apoptotic gene Bax was increased and the expression of the anti-apoptotic gene Bcl-2 was decreased by BMWE treatment. Considering the above results, BMWE was able induce apoptosis in EAT cells via Bax-related caspase-3 activation. This may provide experimental data for the further clinical use of BMWE in cancer.

Artemisinin induces ROS-mediated caspase3 activation in ASTC-a-1 cells

NASA Astrophysics Data System (ADS)

Xiao, Feng-Lian; Chen, Tong-Sheng; Qu, Jun-Le; Liu, Cheng-Yi

2010-02-01

Artemisinin (ART), an antimalarial phytochemical from the sweet wormwood plant or a naturally occurring component of Artemisia annua, has been shown a potential anticancer activity by apoptotic pathways. In our report, cell counting kit (CCK-8) assay showed that treatment of human lung adenocarcinoma (ASTC-a-1) cells with ART effectively increase cell death by inducing apoptosis in a time- and dose-dependent fashion. Hoechst 33258 staining was used to detect apoptosis as well. Reactive oxygen species (ROS) generation was observed in cells exposed to ART at concentrations of 400 μM for 48 h. N-acetyl-L-cysteine (NAC), an oxygen radical scavenger, suppressed the rate of ROS generation and inhibited the ART-induced apoptosis. Moreover, AFC assay (Fluorometric assay for Caspase3 activity) showed that ROS was involved in ART-induced caspase3 acitvation. Taken together, our data indicate that ART induces ROS-mediated caspase3 activation in a time-and dose-dependent way in ASCT-a-1 cells.

A novel Death Defying Domain in Met entraps the active site of Caspase-3 and blocks apoptosis in hepatocytes

PubMed Central

Ma, Jihong; Zou, Chunbin; Guo, Lida; Seneviratne, Danushka S.; Tan, Xinping; Kwon, Yong-Kook; An, Jiyan; Bowser, Robert; DeFrances, Marie C.; Zarnegar, Reza

2013-01-01

Met, the transmembrane tyrosine kinase receptor for hepatocyte growth factor (HGF) is known to function as a potent anti-apoptotic mediator in normal and neoplastic cells. Herein we report that intracellular cytoplasmic tail of Met has evolved to harbor a tandem pair of Caspase-3 cleavage sites, which bait, trap and disable the active site of Caspase-3, thereby blocking the execution of apoptosis. We call this Caspase-3 cleavage motif the ‘Death Defying Domain’ (DDD). This site consists of the following sequence: DNAD-DEVD-T (where the hyphens denote caspase cleavage sites). Through functional and mechanistic studies, we show that upon DDD cleavage by Caspase-3, the resulting DEVD-T peptide acts as a competitive inhibitor and entraps the active site of Caspase-3 akin to DEVD-CHO, which is a potent, synthetic inhibitor of Caspase-3 activity. By gain and loss-of-function studies using restoration of DDD expression in DDD deficient hepatocytic cells, we found that both Caspase-3 sites in DDD are necessary for inhibition of Caspase-3 and promotion of cell survival. Employing mutagenesis studies, we show that DDD could operate independently of Met’s enzymatic activity as determined by using kinase-dead human Met mutant constructs. Studies of both human liver cancer tissues and cell lines uncovered that DDD cleavage and entrapment of Caspase-3 by DDD occur in vivo, further proving that this site has physiological and pathophysiological relevance. Conclusion Our findings show that Met can directly inhibit Caspase-3 via a novel mechanism and promote hepato-cyte survival. Results presented here will further our understanding of the mechanisms that control not only normal tissue homeostasis but also abnormal tissue growth such as cancer and degenerative diseases in which apoptotic caspases are at play. PMID:24122846

Caspase-3 activation in the guinea pig cochlea exposed to salicylate.

PubMed

Feng, Hao; Yin, Shi-Hua; Tang, An-Zhou; Cai, Hong-Wu; Chen, Ping; Tan, Song-Hua; Xie, Li-Hong

2010-07-19

In the current study, we explored whether chronic salicylate exposure could induce apoptosis in outer hair cells (OHCs) and spiral ganglion neurons (SGNs) of the cochlea. Guinea pig received sodium salicylate (400 mg/kg/d) or saline vehicle for 10 consecutive days. Programmed cell death (PCD) executioner was evaluated with immunohistochemistry detection of activated caspase-3. Apoptosis was examined with a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. Repeated salicylate administration activated caspase-3 and caused apoptosis in OHCs and SGNs (p<0.01 vs. saline control for both measures and in both cell types). Cell counting showed a significant loss in OHCs (p<0.01 vs. saline control), but not in inner hair cells (IHCs). Transmission electron microscopy (TEM) revealed chromatin condensation and nucleus margination in salicylate-treated cochlea. Scanning electron microscopy (SEM) demonstrated stereociliary bundles breakdown and fusion at the apical of OHCs, villous matter was discovered to attach on the surface of SGNs. These findings suggest that long-term administration of high-dose salicylate can activate caspase-3 pathway to induce OHC and SGN apoptosis. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Neural Cell Apoptosis Induced by Microwave Exposure Through Mitochondria-dependent Caspase-3 Pathway

PubMed Central

Zuo, Hongyan; Lin, Tao; Wang, Dewen; Peng, Ruiyun; Wang, Shuiming; Gao, Yabing; Xu, Xinping; Li, Yang; Wang, Shaoxia; Zhao, Li; Wang, Lifeng; Zhou, Hongmei

2014-01-01

To determine whether microwave (MW) radiation induces neural cell apoptosis, differentiated PC12 cells and Wistar rats were exposed to 2.856GHz for 5min and 15min, respectively, at an average power density of 30 mW/cm2. JC-1 and TUNEL staining detected significant apoptotic events, such as the loss of mitochondria membrane potential and DNA fragmentation, respectively. Transmission electron microscopy and Hoechst staining were used to observe chromatin ultrastructure and apoptotic body formation. Annexin V-FITC/PI double staining was used to quantify the level of apoptosis. The expressions of Bax, Bcl-2, cytochrome c, cleaved caspase-3 and PARP were examined by immunoblotting or immunocytochemistry. Caspase-3 activity was measured using an enzyme-linked immunosorbent assay. The results showed chromatin condensation and apoptotic body formation in neural cells 6h after microwave exposure. Moreover, the mitochondria membrane potential decreased, DNA fragmentation increased, leading to an increase in the apoptotic cell percentage. Furthermore, the ratio of Bax/Bcl-2, expression of cytochrome c, cleaved caspase-3 and PARP all increased. In conclusion, microwave radiation induced neural cell apoptosis via the classical mitochondria-dependent caspase-3 pathway. This study may provide the experimental basis for further investigation of the mechanism of the neurological effects induced by microwave radiation. PMID:24688304

Caspase-1 from the silkworm, Bombyx mori, is involved in Bombyx mori nucleopolyhedrovirus infection.

PubMed

Wang, Qiang; Ju, Xiaoli; Chen, Liang; Chen, Keping

2017-03-01

Caspase-1 is one of the effector caspases in mammals that plays a central role in apoptosis. However, the lepidopteran caspase-1, especially the Bombyx mori caspase-1 (Bm-caspase-1), has not been investigated in detail. In this study, Bm-caspase-1 was identified from an expressed sequence tag database in B. mori by BLAST search. The open reading frame of Bm-caspase-1 contained 879 nucleotides and encoded 293 amino acids with a predicted molecular mass of 33 kDa. Bm-caspase-1 contained two consensus amino acid motifs of caspase cleavage sites, DEGDA and TETDG. Caspase activity assays revealed significant proteolytic activity of the Ac-DEVD-pNA substrate. Bm-caspase-1 can be detected in all tissues and developmental stages by a semi quantitative polymerase chain reaction assay. More importantly, the expression level of Bm-caspase-1 is increased upon baculovirus infection and up-regulated in BmNPV-resistant silkworms. Taken together, these results indicate that Bm-caspase-1 plays an important role during baculovirus infection.

Immunoexpression of cleaved caspase-3 shows lower apoptotic area indices in lip carcinomas than in intraoral cancer.

PubMed

Leite, Ana Flávia Schueler de Assumpção; Bernardo, Vagner Gonçalves; Buexm, Luisa Aguirre; Fonseca, Eliene Carvalho da; Silva, Licínio Esmeraldo da; Barroso, Danielle Resende Camisasca; Lourenço, Simone de Queiroz Chaves

2016-01-01

This study aimed to evaluate apoptosis by assessing cleaved caspase-3 immunoexpression in hyperplastic, potentially malignant disorder (PMD), and malignant tumors in intraoral and lower lip sites. A retrospective study using paraffin blocks with tissues from patients with inflammatory fibrous hyperplasia (IFH), actinic cheilitis, oral leukoplakia, lower lip and intraoral squamous cell carcinoma (SCC) was performed. The tissues were evaluated by immunohistochemical analysis with anti-cleaved caspase-3 antibody. Apoptotic area index was then correlated with lesion type. From 120 lesions assessed, 55 (46%) were cleaved caspase-3-positive. The SCC samples (n=40) had the highest apoptotic area indices (n=35; 87.5%). Significant differences were detected between SCCs and PMDs (p=0.0003), as well as SCCs and IFHs (p=0.001), regarding caspase-3 immunopositivity. Carcinomas of the lower lip had lower apoptotic area indices than intraoral cancer (p=0.0015). Cleaved caspase-3 immunoexpression showed differences in oral SCCs and PMDs and demonstrated a distinct role of apoptosis in carcinogenesis of intraoral and lower lip cancer. In future, the expression of cleaved caspase-3 with other target molecules in oral cancer may be helpful in delineating the prognosis and treatment of these tumors.

RIPK3 regulates p62-LC3 complex formation via the caspase-8-dependent cleavage of p62.

PubMed

Matsuzawa, Yu; Oshima, Shigeru; Nibe, Yoichi; Kobayashi, Masanori; Maeyashiki, Chiaki; Nemoto, Yasuhiro; Nagaishi, Takashi; Okamoto, Ryuichi; Tsuchiya, Kiichiro; Nakamura, Tetsuya; Watanabe, Mamoru

2015-01-02

RIPK3 is a key molecule for necroptosis, initially characterized by necrotic cell death morphology and the activation of autophagy. Cell death and autophagic signaling are believed to tightly regulate each other. However, the associated recruitment of signaling proteins remains poorly understood. p62/sequestosome-1 is a selective autophagy substrate and a selective receptor for ubiquitinated proteins. In this study, we illustrated that both mouse and human RIPK3 mediate p62 cleavage and that RIPK3 interacts with p62, resulting in complex formation. In addition, RIPK3-dependent p62 cleavage is restricted by the inhibition of caspases, especially caspase-8. Moreover, overexpression of A20, a ubiquitin-editing enzyme and an inhibitor of caspase-8 activity, inhibits RIPK3-dependent p62 cleavage. To further investigate the potential role of RIPK3 in selective autophagy, we analyzed p62-LC3 complex formation, revealing that RIPK3 prevents the localization of LC3 and ubiquitinated proteins to the p62 complex. In addition, RIPK3-dependent p62-LC3 complex disruption is regulated by caspase inhibition. Taken together, these results demonstrated that RIPK3 interacts with p62 and regulates p62-LC3 complex formation. These findings suggested that RIPK3 serves as a negative regulator of selective autophagy and provides new insights into the mechanism by which RIPK3 regulates autophagic signaling. Copyright © 2014 Elsevier Inc. All rights reserved.

Isoflurane-Induced Caspase-3 Activation Is Dependent on Cytosolic Calcium and Can Be Attenuated by Memantine

PubMed Central

Zhang, Guohua; Dong, Yuanlin; Zhang, Bin; Ichinose, Fumito; Wu, Xu; Culley, Deborah J.; Crosby, Gregory

2008-01-01

Increasing evidence indicates that caspase activation and apoptosis are associated with a variety of neurodegenerative disorders, including Alzheimer's disease. We reported that anesthetic isoflurane can induce apoptosis, alter processing of the amyloid precursor protein (APP), and increase amyloid-β protein (Aβ) generation. However, the mechanism by which isoflurane induces apoptosis is primarily unknown. We therefore set out to assess effects of extracellular calcium concentration on isoflurane-induced caspase-3 activation in H4 human neuroglioma cells stably transfected to express human full-length APP (H4-APP cells). In addition, we tested effects of RNA interference (RNAi) silencing of IP3 receptor, NMDA receptor, and endoplasmic reticulum (ER) calcium pump, sacro-/ER calcium ATPase (SERCA1). Finally, we examined the effects of the NMDA receptor partial antagonist, memantine, in H4-APP cells and brain tissue of naive mice. EDTA (10 mm), BAPTA (10 μm), and RNAi silencing of IP3 receptor, NMDA receptor, or SERCA1 attenuated capase-3 activation. Memantine (4 μm) inhibited isoflurane-induced elevations in cytosolic calcium levels and attenuated isoflurane-induced caspase-3 activation, apoptosis, and cell viability. Memantine (20 mg/kg, i.p.) reduced isoflurane-induced caspase-3 activation in brain tissue of naive mice. These results suggest that disruption of calcium homeostasis underlies isoflurane-induced caspase activation and apoptosis. We also show for the first time that the NMDA receptor partial antagonist, memantine, can prevent isoflurane-induced caspase-3 activation and apoptosis in vivo and in vitro. These findings, indicating that isoflurane-induced caspase activation and apoptosis are dependent on cytosolic calcium levels, should facilitate the provision of safer anesthesia care, especially for Alzheimer's disease and elderly patients. PMID:18434534

Sca-1(+) mesenchymal stromal cells inhibit splenic marginal zone B lymphocytes commitment through Caspase-3.

PubMed

Chen, Yaozhen; Yang, Jialei; Zhang, Hui-Jie; Fan, Hong; An, Ning; Xin, Jiajia; Li, Na; Xu, Jinmei; Yin, Wen; Wu, Zhongliang; Hu, Xingbin

2016-05-01

Mesenchymal stromal cells (MSCs) have been characterized as an important component of hematopoietic niche, which are capable of modulating the immune system through interaction with a wide range of immune cells. Marginal zone B cells, one main type of mature B lymphocytes, play a central role in eliciting antibody response against pathogens. However, how MSCs and its subpopulations regulate marginal zone B cells commitment is unknown yet. In this study, we assessed the contribution of Sca-1(+) MSCs on marginal zone B cells commitment. Our results showed that Sca-1(+) MSCs inhibit the commitment of marginal zone B lymphocytes. The inhibition was exerted through lowered Caspase-3 expression. Furthermore, we found marginal zone B lymphocytes in spleen of Caspase-3 knockout mice decreased and Caspase-3 knockout Sca-1(+) MSCs accounted for the MZB lymphocytes decrease. In conclusion, our investigation provided clues about Sca-1(+) MSCs regulation on the commitment of marginal zone B cells through Caspase-3 gene. © 2016 International Federation for Cell Biology.

Immunoexpression of cleaved caspase-3 shows lower apoptotic area indices in lip carcinomas than in intraoral cancer

PubMed Central

LEITE, Ana Flávia Schueler de Assumpção; BERNARDO, Vagner Gonçalves; BUEXM, Luisa Aguirre; da FONSECA, Eliene Carvalho; da SILVA, Licínio Esmeraldo; BARROSO, Danielle Resende Camisasca; LOURENÇO, Simone de Queiroz Chaves

2016-01-01

ABSTRACT Objective This study aimed to evaluate apoptosis by assessing cleaved caspase-3 immunoexpression in hyperplastic, potentially malignant disorder (PMD), and malignant tumors in intraoral and lower lip sites. Material and Methods A retrospective study using paraffin blocks with tissues from patients with inflammatory fibrous hyperplasia (IFH), actinic cheilitis, oral leukoplakia, lower lip and intraoral squamous cell carcinoma (SCC) was performed. The tissues were evaluated by immunohistochemical analysis with anti-cleaved caspase-3 antibody. Apoptotic area index was then correlated with lesion type. Results From 120 lesions assessed, 55 (46%) were cleaved caspase-3-positive. The SCC samples (n=40) had the highest apoptotic area indices (n=35; 87.5%). Significant differences were detected between SCCs and PMDs (p=0.0003), as well as SCCs and IFHs (p=0.001), regarding caspase-3 immunopositivity. Carcinomas of the lower lip had lower apoptotic area indices than intraoral cancer (p=0.0015). Conclusions Cleaved caspase-3 immunoexpression showed differences in oral SCCs and PMDs and demonstrated a distinct role of apoptosis in carcinogenesis of intraoral and lower lip cancer. In future, the expression of cleaved caspase-3 with other target molecules in oral cancer may be helpful in delineating the prognosis and treatment of these tumors. PMID:27556207

Caspase-3-independent pathways proceeding in bystander effect of HSV-tk/GCV system

NASA Astrophysics Data System (ADS)

Lin, Juqiang; Ma, Yan; Zeng, Shaoqun; Zhang, Zhihong

2008-02-01

HSV-tk/GCV system, which is the virus-directed enzyme/prodrug therapy of herpes simplex virus (HSV) thymidine kinase (tk) gene / the anti-viral reagent ganciclovir (GCV), is one of the promising approaches in the rapidly growing area of gene therapy. As gene therapy of cancer such as suicide gene therapy has entered the clinic, another therapy effect which is called 'bystander effect' was reported. Bystander effect can lead to killing of non-transduced tumor cells in the immediate vicinity of GCV-treated HSV-TK-positive cells. Now the magnitude of 'bystander effect' is an essential factor for this anti-tumor approach in vivo. However, the mechanism which HSV-tk/ACV brings "bystander effect" is poorly understood. In this study, we monitor the activation of caspase-3 in HSV-tk/GCV system by a FRET probe CD3, a FRET-based indicator for activity of caspase3, which is composed of an enhanced cyan fluorescent protein, a caspase-sensitive linker, and a red fluorescent protein from Discosoma with efficient maturation property. Through application of CD3 we have visualized the activation of caspase-3 in tk gene positive human adenoid cystic carcinoma (ACC-M) cells but not in bystander effect of HSV-tk/GCV system induced by GCV. This finding provides needed information for understanding the mechanisms by which suicide gene approaches actually kill cancer cells, and may prove to be helpful for the clinical treatment of cancers.

Application of the FRET method for monitoring the dynamics of caspase-3 activation during apoptosis in living cells

NASA Astrophysics Data System (ADS)

Chen, Tongsheng; Xing, Da

2005-01-01

Activation of caspase-3 is a central event in apoptosis. A fluorescence techniques, fluorescence resonance energy transfer (FRET), was used to study the dynamic of caspase-3 activation during apoptosis induced by tumor necrosis factor TNF-α in living cells. The FRET probe consists a CFP (cyan fluorescent protein) and a Venus (YFP mutant, yellow fluorescent protein) with a specialized linker containing the caspase-3 cleavage sequence: DEVD (Luo et al., 2001). Human lung adenocarcinoma cell line (ASTC-a-1) were stably expressed with the FRET probe and then were treated by TNF-α, respectively. Experimental results showed that FRET could monitor more insensitively the dynamic of caspase-3 activation in real-time in vivo, and this technique will be highly useful for correlating the caspase-3 activation with other apoptotic events and for rapid-screening of potential drugs that may target the apoptotic process.

Imaging of activated caspase-3 in living cell by fluorescence resonance energy transfer during photosensitization-induced apoptosis

NASA Astrophysics Data System (ADS)

Wu, Yunxia; Xing, Da; Chen, Qun; Tang, Yonghong

2005-01-01

Photodynamic therapy (PDT) is a novel and promising cancer treatment that employs a combination of a photosensitizing chemical and visible light, induces apoptosis in cell, and activation of caspase-3 is considered to be the final step in many apoptosis pathways. The changes of caspase-3 activation in cell during TNFα- and photodynamic therapy-induced apoptosis was measured by fluorescence resonance energy transfer (FRET) analysis. FRET probe consisting of fusions of an enhanced cyan fluorescent protein (ECFP), Venus and a linker peptide containing the caspase-3 cleavage sequence DEVD was utilized. Therefore, activated caspase-3 cleaved the linker peptide of FRET probe and disrupted the FRET signal. Human lung adenocarcinoma cell line (ASTC-a-1) were stably transfected with the plasmid (ECFP-DEVD-Venus) and then were treated by TNF-α and PDT, respectively. Experimental results indicated that caspase-3 activation resulted in cleavage of linker peptide and subsequent disruption of the FRET signal during TNFα- and photodynamic therapy-induced apoptosis, and that the activation of caspase-3 induced by photodynamic therapy was faster than that induce by TNF-α. The study supports that using FRET technique and different recombinant substrates as FRET probes could be used to detect the process of PDT-induced apoptosis and provide a new means to investigate apoptotic mechanism of PDT.

Caspase-3 inhibitor prevents the apoptosis of brain tissue in rats with acute cerebral infarction.

PubMed

Sun, Yuhua; Xu, Yuming; Geng, Lijiao

2015-07-01

The aim of the present study was to investigate the effect of the caspase-3 inhibitor z-DEVD-fmk on the apoptosis of the brain tissues of rats with acute cerebral infarction. Middle cerebral artery occlusion was used to establish a rat model of infarction, and the rats were randomly divided into a sham group (n=15), model group (n=15) and treatment group (n=15). z-DEVD-fmk (2.5 µg/kg) was injected into the intracranial artery of rats in the treatment group, while the same volume of phosphate-buffered saline solution was administered to the rats of the sham and model groups. After 48 h, all rats were sacrificed and their brain tissues were removed. The caspase-3 mRNA level, protein level and activity, brain cell apoptosis index and infarction scope of the three groups were analyzed. Neurological impairment was also assessed. At 48 h after model establishment, the caspase-3 mRNA and protein levels in the brain tissues of the model group were significantly higher than those of the sham group, and those in the treatment group were significantly lower than those in the model group (P<0.05); however, they remained significantly higher than those in the sham group. Caspase-3 activity in the model group was significantly higher than that in the sham group, and treatment with the caspase-3 inhibitor significantly reduced caspase-3 activity compared with that in the model group (P<0.05). The apoptosis index and infarction scope in the model and treatment groups were significantly increased compared with those in the sham group, and were significantly lower in the treatment group than in the model group (P<0.05). The neurological impairment of rats in the model and treatment groups was increased significantly compared with that in the sham group, and the treatment group exhibited a significantly lower level of neurological impairment than the model group (P<0.05). In conclusion, the caspase-3 inhibitor z-DEVD-fmk effectively inhibited apoptosis and delayed the necrosis of

XIAP inhibits caspase-3 and -7 using two binding sites: evolutionarily conserved mechanism of IAPs

PubMed Central

Scott, Fiona L; Denault, Jean-Bernard; Riedl, Stefan J; Shin, Hwain; Renatus, Martin; Salvesen, Guy S

2005-01-01

The X-linked inhibitor of apoptosis protein (XIAP) uses its second baculovirus IAP repeat domain (BIR2) to inhibit the apoptotic executioner caspase-3 and -7. Structural studies have demonstrated that it is not the BIR2 domain itself but a segment N-terminal to it that directly targets the activity of these caspases. These studies failed to demonstrate a role of the BIR2 domain in inhibition. We used site-directed mutagenesis of BIR2 and its linker to determine the mechanism of executioner caspase inhibition by XIAP. We show that the BIR2 domain contributes substantially to inhibition of executioner caspases. A surface groove on BIR2, which also binds to Smac/DIABLO, interacts with a neoepitope generated at the N-terminus of the caspase small subunit following activation. Therefore, BIR2 uses a two-site interaction mechanism to achieve high specificity and potency for inhibition. Moreover, for caspase-7, the precise location of the activating cleavage is critical for subsequent inhibition. Since apical caspases utilize this cleavage site differently, we predict that the origin of the death stimulus should dictate the efficiency of inhibition by XIAP. PMID:15650747

PAK2 is cleaved and activated during hyperosmotic shock-induced apoptosis via a caspase-dependent mechanism: evidence for the involvement of oxidative stress.

PubMed

Chan, W H; Yu, J S; Yang, S D

1999-03-01

Hyperosmotic shock elicits a stress response in mammalian cells and can lead to apoptotic cell death. In the present study, we report that hyperosmotic shock can induce activation of a 36 kDa kinase detected by an in-gel kinase assay in several cell types, including mouse Balb/c 3T3 fibroblasts, and human Hep 3B and A431 cells. This 36 kDa kinase can be recognized by an antibody against the C-terminal region of a family of p21Cdc42/Rac-activated kinases (PAKs) on immunoblot. Further studies with this antibody and a PAK2-specific antibody against the N-terminal region of PAK2 demonstrate that hyperosmotic shock can induce cleavage of PAK2 to generate a 36 kDa C-terminal catalytic fragment in cells. The cleavage and activation of PAK2 was found to be closely associated with both DNA fragmentation and activation of an ICE/CED-3 family cysteine protease termed caspase-3 in hyperosmotically shocked cells. Furthermore, pretreating the cells with two caspase inhibitors (Ac-DEVD-cho and Ac-YVAD-cmk) could inhibit both cleavage/activation of PAK2 and DNA fragmentation induced by hyperosmotic shock. Moreover, all these hyperosmotic shock-induced changes (i.e., activation of caspase-3, cleavage/activation of PAK2, and DNA fragmentation) in cells could be blocked by antioxidants such as ascorbic acid (vitamine C), alpha-tocopherol (vitamine E), dithiothreitol, beta-mercaptoethanol, and glutathione. Taken together, our results show that PAK2 is cleaved and activated via a caspase-dependent mechanism during hyperosmotic shock-induced apoptosis and suggest the involvement of antioxidant-preventable oxidative stress in inducing this process.

Shikonin Suppresses NLRP3 and AIM2 Inflammasomes by Direct Inhibition of Caspase-1

PubMed Central

Zorman, Jernej; Sušjan, Petra; Hafner-Bratkovič, Iva

2016-01-01

Shikonin is a highly lipophilic naphtoquinone found in the roots of Lithospermum erythrorhizon used for its pleiotropic effects in traditional Chinese medicine. Based on its reported antipyretic and anti-inflammatory properties, we investigated whether shikonin suppresses the activation of NLRP3 inflammasome. Inflammasomes are cytosolic protein complexes that serve as scaffolds for recruitment and activation of caspase-1, which, in turn, results in cleavage and secretion of proinflammatory cytokines IL-1β and IL-18. NLRP3 inflammasome activation involves two steps: priming, i.e. the activation of NF-κB pathway, and inflammasome assembly. While shikonin has previously been reported to suppress the priming step, we demonstrated that shikonin also inhibits the second step of inflammasome activation induced by soluble and particulate NLRP3 instigators in primed immortalized murine bone marrow-derived macrophages. Shikonin decreased NLRP3 inflammasome activation in response to nigericin more potently than acetylshikonin. Our results showed that shikonin also inhibits AIM2 inflammasome activation by double stranded DNA. Shikonin inhibited ASC speck formation and caspase-1 activation in murine macrophages and suppressed the activity of isolated caspase-1, demonstrating that it directly targets caspase-1. Complexing shikonin with β-lactoglobulin reduced its toxicity while preserving the inhibitory effect on NLRP3 inflammasome activation, suggesting that shikonin with improved bioavailability might be interesting for therapeutic applications in inflammasome-mediated conditions. PMID:27467658

Role of caspase-9 in the effector caspases and genome expressions, and growth of bovine skeletal myoblasts.

PubMed

Van Ba, Hoa; Hwang, Inho

2014-02-01

Caspase-9 has been reported as the key regulator of apoptosis, however, its role in skeletal myoblast development and molecular involvements during cell growth still remains unknown. The current study aimed to present the key role of caspase-9 in the expressions of apoptotic caspases and genome, and cell viability during myoblast growth using RNA interference mediated silencing. Three small interference RNA sequences (siRNAs) targeting caspase-9 gene was designed and ligated into pSilencer plasmid vector to construct shRNA expression constructs. Cells were transfected with the constructs for 48 h. Results indicated that all three siRNAs could silence the caspase-9 mRNA expression significantly. Particularly, the mRNA expression level of caspase-9 in the cells transfected by shRNA1, shRNA2 and shRNA3 constructs were reduced by 37.85%, 68.20% and 58.14%, respectively. Suppression of caspase-9 led to the significant increases in the mRNA and protein expressions of effector caspase-3, whereas the reduction in mRNA and protein expressions of caspase-7. The microarray results showed that the suppression of caspase-9 resulted in significant upregulations of cell proliferation-, adhesion-, growth-, development- and division-regulating genes, whereas the reduction in the expressions of cell death program- and stress response-regulating genes. Furthermore, cell viability was significantly increased following the transfection. These data suggest that caspase-9 could play an important role in the control of cell growth, and knockdown of caspase-9 may have genuine potential in the treatment of skeletal muscle atrophy. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

MicroRNAs Targeting Caspase-3 and -7 in PANC-1 Cells.

PubMed

Park, Jong Kook; Doseff, Andrea I; Schmittgen, Thomas D

2018-04-16

MicroRNAs (miRNAs), a critical part of the RNA silencing machinery, are known to play important regulatory roles in cancer. However, the consequence of miRNA deregulation in cancer is unknown for many miRNAs. Here, we define that miRNAs, miR-17-5p, miR-132-3p/-212-3p, and miR-337-3p are significantly up-regulated in the pancreatic ductal adenocarcinomas (PDAC) compared to the normal and benign tissues. Furthermore, by using PANC-1 cells, we demonstrate that overexpressed miR-337-3p and miR-17-5p/miR-132-3p/-212-3p can regulate executioner caspases-3 and -7, respectively. In addition, over-expression of miRNAs, especially miR-337-3p, attenuates tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity in PANC-1 cells. Our findings unveil an important biological function for miRNAs up-regulated in PDAC in coordinately regulating caspases, potentially contributing to the malignant progression of PDAC.

Human caspase-4 detects tetra-acylated LPS and cytosolic Francisella and functions differently from murine caspase-11.

PubMed

Lagrange, Brice; Benaoudia, Sacha; Wallet, Pierre; Magnotti, Flora; Provost, Angelina; Michal, Fanny; Martin, Amandine; Di Lorenzo, Flaviana; Py, Bénédicte F; Molinaro, Antonio; Henry, Thomas

2018-01-16

Caspase-4/5 in humans and caspase-11 in mice bind hexa-acylated lipid A, the lipid moeity of lipopolysaccharide (LPS), to induce the activation of non-canonical inflammasome. Pathogens such as Francisella novicida express an under-acylated lipid A and escape caspase-11 recognition in mice. Here, we show that caspase-4 drives inflammasome responses to F. novicida infection in human macrophages. Caspase-4 triggers F. novicida-mediated, gasdermin D-dependent pyroptosis and activates the NLRP3 inflammasome. Inflammasome activation could be recapitulated by transfection of under-acylated LPS from different bacterial species or synthetic tetra-acylated lipid A into cytosol of human macrophage. Our results indicate functional differences between human caspase-4 and murine caspase-11. We further establish that human Guanylate-binding proteins promote inflammasome responses to under-acylated LPS. Altogether, our data demonstrate a broader reactivity of caspase-4 to under-acylated LPS than caspase-11, which may have important clinical implications for management of sepsis.

Members of the bcl-2 and caspase families regulate nuclear degeneration during chick lens fibre differentiation.

PubMed

Wride, M A; Parker, E; Sanders, E J

1999-09-01

The optical clarity of the lens is ensured by the programmed removal of nuclei and other organelles from the lens fibre cells during development. The morphology of the degenerating nuclei is similar to that observed during apoptosis and is accompanied by DNA fragmentation. Proteins encoded by the bcl-2 proto-oncogene family are important in either promoting or inhibiting apoptosis, and caspases are involved in downstream proteolytic events. Here, the expression of bcl-2 family members (bcl-2, bax, bad, and bcl-x(s/l)) and caspases-1, -2, -3, -4, and -6 was investigated through a range of stages of chick lens development using immunocytochemistry, Western blotting, and affinity labelling for caspases using biotinylated caspase inhibitors. Using differentiating lens epithelial cell cultures, it was demonstrated that the addition to cultures of synthetic peptide inhibitors of caspases -1, -2, -4, -6, and -9 brought about a 50-70% reduction in the number of degenerating nuclei per unit area of culture, as assessed by image analysis. These effects were comparable to those seen when general inhibitors of caspases were added to cultures. On the other hand, inhibitors of caspases-3 and -8 were not effective in significantly reducing the number of TUNEL-labelled nuclei. Expression of the caspase substrates poly(ADP-ribose) polymerase (PARP) and the 45-kDa subunit of DNA fragmentation factor (DFF 45) was also observed in the developing lens. Western blots of cultures to which caspase inhibitors were added revealed alterations in the PARP cleavage pattern, but not in that of DFF. These results demonstrate a role for members of the bcl-2 family and caspases in the degeneration of lens fibre cell nuclei during chick secondary lens fibre development and support the proposal that this process has many characteristics in common with apoptosis. Copyright 1999 Academic Press.

Dihydroartemisinin (DHA) induces caspase-3-dependent apoptosis in human lung adenocarcinoma ASTC-a-1 cells

PubMed Central

Lu, Ying-Ying; Chen, Tong-Sheng; Qu, Jun-Le; Pan, Wen-Liang; Sun, Lei; Wei, Xun-Bin

2009-01-01

Background Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, isolated from the traditional Chinese herb Artemisia annua, is recommended as the first-line anti-malarial drug with low toxicity. DHA has been shown to possess promising anticancer activities and induce cancer cell death through apoptotic pathways, although the molecular mechanisms are not well understood. Methods In this study, cell counting kit (CCK-8) assay was employed to evaluate the survival of DHA-treated ASTC-a-1 cells. The induction of apoptosis was detected by Hoechst 33258 and PI staining as well as flow cytometry analysis. Collapse of mitochondrial transmembrane potential (ΔΨm) was measured by dynamic detection under a laser scanning confocal microscope and flow cytometry analysis using Rhodamine123. Caspase-3 activities measured with or without Z-VAD-fmk (a broad spectrum caspase inhibitor) pretreatment by FRET techniques, caspase-3 activity measurement, and western blotting analysis. Results Our results indicated that DHA induced apoptotic cell death in a dose- and time-dependent manner, which was accompanied by mitochondrial morphology changes, the loss of ΔΨm and the activation of caspase-3. Conclusion These results show for the first time that DHA can inhibit proliferation and induce apoptosis via caspase-3-dependent mitochondrial death pathway in ASTC-a-1 cells. Our work may provide evidence for further studies of DHA as a possible anticancer drug in the clinical treatment of lung adenocarcinoma. PMID:19272183

TLR3 mediates release of IL-1β and cell death in keratinocytes in a caspase-4 dependent manner.

PubMed

Grimstad, Øystein; Husebye, Harald; Espevik, Terje

2013-10-01

Inflammation and timely cell death are important elements in host defence and healing processes. Keratinocytes express high levels of Toll-like receptor 3 (TLR3), and stimulation of the receptor with its ligand polyinosinic-polycytidylic acid (polyI:C) is a powerful signal for release of a variety of proinflammatory cytokines. Caspase-4 is required for maturation of pro-IL-1β through activation of caspase-1 in keratinocytes. TLR3 in keratinocytes was stimulated with polyI:C. Induction of messenger RNA of pro-IL-1β and inflammasomal components was measured using quantitative polymerase chain reaction methodology. Protein expression of IL-1β was analysed with ELISA and Western blot techniques. Activation of apoptotic caspases was measured with flow cytometry, and cytotoxicity was determined. TLR3 induced release of substantial amounts of pro-IL-1β in keratinocytes. NLRP3 or ASC dependent processing of IL-1β into its cleaved bioactive form was found to be minimal. The release of IL-1β was due to polyI:C induced cell death that occurred through a caspase-4 dependent manner. Caspase-1 did not seem to be involved in the polyI:C induced cytotoxicity despite that TLR3 stimulation induced activation of caspase-1. In addition, the apoptotic caspases -8, -9 and -3/7 were activated by polyI:C. TLR3 stimulation in keratinocytes induces a caspase-4 dependent release of pro-IL-1β, but further processing to active IL-1β is limited. Furthermore, TLR3 stimulation results in pyroptotic- and apoptotic cell death. Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

NIR-triggered high-efficient photodynamic and chemo-cascade therapy using caspase-3 responsive functionalized upconversion nanoparticles.

PubMed

Zhao, Na; Wu, Baoyan; Hu, Xianglong; Xing, Da

2017-10-01

Stimuli-responsive nanoparticles with multiple therapeutic/diagnostic functions are highly desirable for effective tumor treatment. Herein novel caspase-3 responsive functionalized upconversion nanoparticles (CFUNs) were fabricated with three-in-one functional integration: near-infrared (NIR) triggered photodynamic damage along with caspase-3 activation, subsequent caspase-3 responsive drug release, and cascade chemotherapeutic activation. CFUNs were formulated from the self-assembly of caspase-3 responsive doxorubicin (DOX) prodrug tethered with DEVD peptide (DEVD-DOX), upconversion nanoparticles (UCNP), a photosensitizer (pyropheophorbide-a methyl ester, MPPa), and tumor-targeting cRGD-PEG-DSPE to afford multifunctional CFUNs, MPPa/UCNP-DEVD-DOX/cRGD. Upon cellular uptake and NIR irradiation, the visible light emission of UCNP could excite MPPa to produce reactive oxygen species for photodynamic therapy (PDT) along with the activation of caspase-3, which further cleaved DEVD peptide to release DOX within tumor cells, thus accomplishing NIR-triggered PDT and cascade chemotherapy. CFUNs presented silent therapeutic potency and negligible cytotoxicity in the dark, whereas in vitro and in vivo experiments demonstrated the NIR-triggered cascade therapeutic activation and tumor inhibition due to consecutive PDT and chemotherapy. Current NIR-activated cascade tumor therapy with two distinct mechanisms is significantly favorable to overcome multidrug resistance and tumor heterogeneity for persistent tumor treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Parallel single-cell analysis of active caspase-3/7 in apoptotic and non-apoptotic cells.

PubMed

Ledvina, Vojtěch; Janečková, Eva; Matalová, Eva; Klepárník, Karel

2017-01-01

Analysing the chemical content of individual cells has already been proven to reveal unique information on various biological processes. Single-cell analysis provides more accurate and reliable results for biology and medicine than analyses of extracts from cell populations, where a natural heterogeneity is averaged. To meet the requirements in the research of important biologically active molecules, such as caspases, we have developed a miniaturized device for simultaneous analyses of individual cells. A stainless steel body with a carousel holder enables high-sensitivity parallel detections in eight microvials. The holder is mounted in front of a photomultiplier tube with cooled photocathode working in photon counting mode. The detection of active caspase-3/7, central effector caspases in apoptosis, in single cells is based on the bioluminescence chemistry commercially available as Caspase-Glo ® 3/7 reagent developed by Promega. Individual cells were captured from a culture medium under microscope and transferred by micromanipulator into detection microvial filled with the reagent. As a result of testing, the limits of detection and quantification were determined to be 0.27/0.86 of active caspase-3/7 content in an average apoptotic cell and 0.46/2.92 for non-apoptotic cells. Application potential of this technology in laboratory diagnostics and related medical research is discussed. Graphical abstract Miniaturized device for simultaneous analyses of individual cells.

Anticancer Effect of Ursodeoxycholic Acid in Human Oral Squamous Carcinoma HSC-3 Cells through the Caspases

PubMed Central

Pang, Liang; Zhao, Xin; Liu, Weiwei; Deng, Jiang; Tan, Xiaotong; Qiu, Lihua

2015-01-01

Bear bile was used as a traditional medicine or tonic in East Asia, and ursodeoxycholic acid (UDCA) is the most important compound in bear bile. Further, synthetic UDCA is also used in modern medicine and nutrition; therefore, its further functional effects warrant research, in vitro methods could be used for the fundamental research of its anticancer effects. In this study, the apoptotic effects of UDCA in human oral squamous carcinoma HSC-3 cells through the activation of caspases were observed by the experimental methods of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay, DAPI (4’,6-diamidino-2-phenylindole) staining, flow cytometry analysis, RT-PCR (reverse transcription-polymerase chain reaction) assay and Western blot assay after HSC-3 cells were treated by different concentrations of UDCA. With 0 to 400 μg/mL UDCA treatment, UDCA had strong growth inhibitory effects in HSC-3 cells, but had almost no effect in HOK normal oral cells. At concentrations of 100, 200 and 400 μg/mL, UDCA could induce apoptosis compared to untreated control HSC-3 cells. Treatment of 400 μg/mL UDCA could induce more apoptotic cancer cells than 100 and 200 μg/mL treatment; the sub-G1 DNA content of 400 μg/mL UDCA treated cancer cells was 41.3% versus 10.6% (100 μg/mL) and 22.4% (200 μg/mL). After different concentrations of UDCA treatment, the mRNA and protein expressions of caspase-3, caspase-8, caspase-9, Bax, Fas/FasL (Fas ligand), TRAIL (TNF-related apoptosis-inducing ligand), DR4 (death receptor 4) and DR5 (death receptor 5) were increased in HSC-3 cells, and mRNA and protein expressions of Bcl-2 (B-cell lymphoma 2), Bcl-xL (B-cell lymphoma-extra large), XIAP (X-linked inhibitor of apoptosis protein), cIAP-1 (cellular inhibitor of apoptosis 1), cIAP-2 (cellular inhibitor of apoptosis 2) and survival were decreased. Meanwhile, at the highest concentration of 400 μg/mL, caspase-3, caspase-8, caspase-9, Bax, Fas/FasL, TRAIL, DR4, DR5, and Iκ

Activity of Uncleaved Caspase-8 Controls Anti-bacterial Immune Defense and TLR-Induced Cytokine Production Independent of Cell Death.

PubMed

Philip, Naomi H; DeLaney, Alexandra; Peterson, Lance W; Santos-Marrero, Melanie; Grier, Jennifer T; Sun, Yan; Wynosky-Dolfi, Meghan A; Zwack, Erin E; Hu, Baofeng; Olsen, Tayla M; Rongvaux, Anthony; Pope, Scott D; López, Carolina B; Oberst, Andrew; Beiting, Daniel P; Henao-Mejia, Jorge; Brodsky, Igor E

2016-10-01

Caspases regulate cell death programs in response to environmental stresses, including infection and inflammation, and are therefore critical for the proper operation of the mammalian immune system. Caspase-8 is necessary for optimal production of inflammatory cytokines and host defense against infection by multiple pathogens including Yersinia, but whether this is due to death of infected cells or an intrinsic role of caspase-8 in TLR-induced gene expression is unknown. Caspase-8 activation at death signaling complexes results in its autoprocessing and subsequent cleavage and activation of its downstream apoptotic targets. Whether caspase-8 activity is also important for inflammatory gene expression during bacterial infection has not been investigated. Here, we report that caspase-8 plays an essential cell-intrinsic role in innate inflammatory cytokine production in vivo during Yersinia infection. Unexpectedly, we found that caspase-8 enzymatic activity regulates gene expression in response to bacterial infection as well as TLR signaling independently of apoptosis. Using newly-generated mice in which caspase-8 autoprocessing is ablated (Casp8DA/DA), we now demonstrate that caspase-8 enzymatic activity, but not autoprocessing, mediates induction of inflammatory cytokines by bacterial infection and a wide variety of TLR stimuli. Because unprocessed caspase-8 functions in an enzymatic complex with its homolog cFLIP, our findings implicate the caspase-8/cFLIP heterodimer in control of inflammatory cytokines during microbial infection, and provide new insight into regulation of antibacterial immune defense.

Analysis of caspase-3 in ASTC-a-1 cells treated with mitomycin C using acceptor photobleaching techniques

NASA Astrophysics Data System (ADS)

Wang, Huiying; Chen, Tongsheng; Sun, Lei

2008-02-01

Caspase-3 is a key activated death protease, which catalyzes the specific cleavage of many cellular proteins and induces DNA cleavage eventually. In this report, cells were treated with mitomycin C (MMC) at different concentration and its activity was detected by cell counting kit (CCK-8). Based on results of CCK-8, cells were treated with 10μg/mL MMC and Hoechst 33258 has been used to observe cell apoptosis. Fluorescence resonance energy transfer (FRET) and confocal microscopy have been used to the effect of MMC on the caspase3 activation in living cells. Human lung adenocarcinoma cells (ASTC-a-1) was transfected with plasmid SCAT3 (pSCAT3)/CKAR FRET receptor. Acceptor photobleaching techniques of FRET plasmid has been used to destruct fluorophore of cells stably expressing SCAT3 reporter on a fluorescence confocal microscope. The activity of caspase3 can be analyzed by FRET dynamics of SCAT3 in living cells. Our results show that MM C can induce ASTC-a-1 cell apoptosis through activation of caspase3.

High-fat diet feeding causes rapid, non-apoptotic cleavage of caspase-3 in astrocytes.

PubMed

Guyenet, Stephan J; Nguyen, Hong T; Hwang, Bang H; Schwartz, Michael W; Baskin, Denis G; Thaler, Joshua P

2013-05-28

Astrocytes respond to multiple forms of central nervous system (CNS) injury by entering a reactive state characterized by morphological changes and a specific pattern of altered protein expression. Termed astrogliosis, this response has been shown to strongly influence the injury response and functional recovery of CNS tissues. This pattern of CNS inflammation and injury associated with astrogliosis has recently been found to occur in the energy homeostasis centers of the hypothalamus during diet-induced obesity (DIO) in rodent models, but the characterization of the astrocyte response remains incomplete. Here, we report that astrocytes in the mediobasal hypothalamus respond robustly and rapidly to purified high-fat diet (HFD) feeding by cleaving caspase-3, a protease whose cleavage is often associated with apoptosis. Although obesity develops in HFD-fed rats by day 14, caspase-3 cleavage occurs by day 3, prior to the development of obesity, suggesting the possibility that it could play a causal role in the hypothalamic neuropathology and fat gain observed in DIO. Caspase-3 cleavage is not associated with an increase in the rate of apoptosis, as determined by TUNEL staining, suggesting it plays a non-apoptotic role analogous to the response to excitotoxic neuron injury. Our results indicate that astrocytes in the mediobasal hypothalamus respond rapidly and robustly to HFD feeding, activating caspase-3 in the absence of apoptosis, a process that has the potential to influence the course of DIO. Copyright © 2013 Elsevier B.V. All rights reserved.

Apoptosis induction in prostate cancer cells by a novel gene product, pHyde, involves caspase-3.

PubMed

Zhang, X; Steiner, M S; Rinaldy, A; Lu, Y

2001-09-20

A novel gene, pHyde, was recently cloned from Dunning rat prostate cancer cells. A recombinant adenovirus containing pHyde cDNA gene (AdpHyde) was generated to investigate the biological function of pHyde protein. AdpHyde inhibited the growth of human prostate cancer cells. Apoptosis was induced in AdpHyde transduced cells as demonstrated by DAPI (4', 6-diamino-2-phenylindole), TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling) staining, and flow cytometry assays. Apoptosis was also induced in human xenograft prostate cancer tumors growing in nude mice following treatment with AdpHyde. AdpHyde transduction resulted in a dose-dependent stimulation of caspase-3 activity in DU145 cells which was blocked by DEVD (succinyl-Asp-Glu-Val-Asp-aldehyde) and VAD (benzyloxycarbonyl - Val - Ala - Asp -fluoromethylketone), inhibitors specifically against caspase-3. Moreover, cancer cells that lacked expression of endogenous caspase-3 were not or barely inhibited by pHyde. These results taken together suggest that pHyde inhibits cancer growth by inducing apoptosis through a caspase-3 dependent pathway.

Analysis of expression patterns of IGF-1, caspase-3 and HSP-70 in developing human tooth germs.

PubMed

Kero, Darko; Kalibovic Govorko, Danijela; Medvedec Mikic, Ivana; Vukojevic, Katarina; Cigic, Livia; Saraga-Babic, Mirna

2015-10-01

To analyze expression patterns of IGF-1, caspase-3 and HSP-70 in human incisor and canine tooth germs during the late bud, cap and bell stages of odontogenesis. Head areas or parts of jaw containing teeth from 10 human fetuses aged between 9th and 20th developmental weeks were immunohistochemically analyzed using IGF-1, active caspase-3 and HSP-70 markers. Semi-quantitative analysis of each marker's expression pattern was also performed. During the analyzed period, IGF-1 and HSP-70 were mostly expressed in enamel organ. As development progressed, expression of IGF-1 and HSP-70 became more confined to differentiating tissues in the future cusp tip area, as well as in highly proliferating cervical loops. Few apoptotic bodies highly positive to active caspase-3 were observed in enamel organ and dental papilla from the cap stage onward. However, both enamel epithelia moderately expressed active caspase-3 throughout the investigated period. Expression patterns of IGF-1, active caspase-3 and HSP-70 imply importance of these factors for early human tooth development. IGF-1 and HSP-70 have versatile functions in control of proliferation, differentiation and anti-apoptotic protection of epithelial parts of human enamel organ. Active caspase-3 is partially involved in formation and apoptotic removal of primary enamel knot, although present findings might reflect its ability to perform other non-death functions such as differentiation of hard dental tissues secreting cells and guidance of ingrowth of proliferating cervical loops. Copyright © 2015 Elsevier Ltd. All rights reserved.

Tunable allosteric library of caspase-3 identifies coupling between conserved water molecules and conformational selection

PubMed Central

Maciag, Joseph J.; Mackenzie, Sarah H.; Tucker, Matthew B.; Schipper, Joshua L.; Swartz, Paul; Clark, A. Clay

2016-01-01

The native ensemble of caspases is described globally by a complex energy landscape where the binding of substrate selects for the active conformation, whereas targeting an allosteric site in the dimer interface selects an inactive conformation that contains disordered active-site loops. Mutations and posttranslational modifications stabilize high-energy inactive conformations, with mostly formed, but distorted, active sites. To examine the interconversion of active and inactive states in the ensemble, we used detection of related solvent positions to analyze 4,995 waters in 15 high-resolution (<2.0 Å) structures of wild-type caspase-3, resulting in 450 clusters with the most highly conserved set containing 145 water molecules. The data show that regions of the protein that contact the conserved waters also correspond to sites of posttranslational modifications, suggesting that the conserved waters are an integral part of allosteric mechanisms. To test this hypothesis, we created a library of 19 caspase-3 variants through saturation mutagenesis in a single position of the allosteric site of the dimer interface, and we show that the enzyme activity varies by more than four orders of magnitude. Altogether, our database consists of 37 high-resolution structures of caspase-3 variants, and we demonstrate that the decrease in activity correlates with a loss of conserved water molecules. The data show that the activity of caspase-3 can be fine-tuned through globally desolvating the active conformation within the native ensemble, providing a mechanism for cells to repartition the ensemble and thus fine-tune activity through conformational selection. PMID:27681633

A Crohn's disease variant in Atg16l1 enhances its degradation by caspase 3

NASA Astrophysics Data System (ADS)

Murthy, Aditya; Li, Yun; Peng, Ivan; Reichelt, Mike; Katakam, Anand Kumar; Noubade, Rajkumar; Roose-Girma, Merone; Devoss, Jason; Diehl, Lauri; Graham, Robert R.; van Lookeren Campagne, Menno

2014-02-01

Crohn's disease is a debilitating inflammatory bowel disease (IBD) that can involve the entire digestive tract. A single-nucleotide polymorphism (SNP) encoding a missense variant in the autophagy gene ATG16L1 (rs2241880, Thr300Ala) is strongly associated with the incidence of Crohn's disease. Numerous studies have demonstrated the effect of ATG16L1 deletion or deficiency; however, the molecular consequences of the Thr300Ala (T300A) variant remains unknown. Here we show that amino acids 296-299 constitute a caspase cleavage motif in ATG16L1 and that the T300A variant (T316A in mice) significantly increases ATG16L1 sensitization to caspase-3-mediated processing. We observed that death-receptor activation or starvation-induced metabolic stress in human and murine macrophages increased degradation of the T300A or T316A variants of ATG16L1, respectively, resulting in diminished autophagy. Knock-in mice harbouring the T316A variant showed defective clearance of the ileal pathogen Yersinia enterocolitica and an elevated inflammatory cytokine response. In turn, deletion of the caspase-3-encoding gene, Casp3, or elimination of the caspase cleavage site by site-directed mutagenesis rescued starvation-induced autophagy and pathogen clearance, respectively. These findings demonstrate that caspase 3 activation in the presence of a common risk allele leads to accelerated degradation of ATG16L1, placing cellular stress, apoptotic stimuli and impaired autophagy in a unified pathway that predisposes to Crohn's disease.

Tunable allosteric library of caspase-3 identifies coupling between conserved water molecules and conformational selection.

PubMed

Maciag, Joseph J; Mackenzie, Sarah H; Tucker, Matthew B; Schipper, Joshua L; Swartz, Paul; Clark, A Clay

2016-10-11

The native ensemble of caspases is described globally by a complex energy landscape where the binding of substrate selects for the active conformation, whereas targeting an allosteric site in the dimer interface selects an inactive conformation that contains disordered active-site loops. Mutations and posttranslational modifications stabilize high-energy inactive conformations, with mostly formed, but distorted, active sites. To examine the interconversion of active and inactive states in the ensemble, we used detection of related solvent positions to analyze 4,995 waters in 15 high-resolution (<2.0 Å) structures of wild-type caspase-3, resulting in 450 clusters with the most highly conserved set containing 145 water molecules. The data show that regions of the protein that contact the conserved waters also correspond to sites of posttranslational modifications, suggesting that the conserved waters are an integral part of allosteric mechanisms. To test this hypothesis, we created a library of 19 caspase-3 variants through saturation mutagenesis in a single position of the allosteric site of the dimer interface, and we show that the enzyme activity varies by more than four orders of magnitude. Altogether, our database consists of 37 high-resolution structures of caspase-3 variants, and we demonstrate that the decrease in activity correlates with a loss of conserved water molecules. The data show that the activity of caspase-3 can be fine-tuned through globally desolvating the active conformation within the native ensemble, providing a mechanism for cells to repartition the ensemble and thus fine-tune activity through conformational selection.

[Changes in Ca(2+)concentration and caspase-3 expression and their relationship in Raji cells exposed to electromagnetic radiation].

PubMed

Wang, Wei; Liu, Huan-xin; Wang, De-wen; Zuo, Hong-yan; Peng, Rui-yun

2013-02-01

To study the effects of electromagnetic pulse (EMP), S-band high power microwave (S-HPM), and X-band high power microwave (X-HPM) on the Ca(2+) concentration and caspase-3 expression in Raji cells and the relationship between Ca(2+) concentration and caspase-3 expression, and to investigate the regulatory mechanism of electromagnetic radiation damage. Raji cells were cultured conventionally. Some cells were irradiated by EMP, S-HPM, and X-HPM in the logarithmic growth phase for 6 hours and then collected; others received sham irradiation as a control. The Ca(2+) concentration in the cells was measured by laser scanning confocal microscope; the caspase-3 expression in the cells was evaluated by Western blot. Compared with the control group (Ca(2+) fluorescence intensity = 43.08 ± 2.08; caspase-3 expression level = 0.444 ± 0.13), the EMP,S-HPM, and X-HPM groups had significantly increased Ca(2+) concentrations, with Ca(2+) fluorescence intensities of 69.56 ± 1.71, 50.06 ± 1.89, and 70.68 ± 1.59, respectively (P < 0.01), and had upregulated caspase-3 expression, with expression levels of 0.964 ± 0.12, 0.586 ± 0.16, and 0.970 ± 0.07, respectively (P < 0.01). Each of the EMP and X-HPM groups had significantly higher Ca(2+) fluorescence intensity and caspase-3 expression level than the S-HPM group (P < 0.01), but there were no significant differences between the EMP and X-HPM groups. The linear regression analysis showed that the caspase-3 expression was upregulated as the Ca(2+) concentration increased, with a positive correlation between them (P < 0.01). EMP, S-HPM, and X-HPM cause damage probably by increasing the Ca(2+) concentration in cells and in turn inducing caspase-3 overexpression.

Role of HIF-1α and CASPASE-3 in cystogenesis of odontogenic cysts and tumors.

PubMed

da Costa, Natacha M M; de Siqueira, Adriane S; Ribeiro, André L R; da Silva Kataoka, Maria S; Jaeger, Ruy G; de Alves-Júnior, Sérgio M; Smith, Andrew M; de Jesus Viana Pinheiro, João

2018-01-01

Odontogenic cysts and tumors are the most relevant lesions that affect the gnathic bones. These lesions have in common the formation of cystic areas and this common feature may suggest involvement of similar mechanisms. The hypoxia inducible factor 1 alpha (HIF-1α), a responsive protein to hypoxia and caspase-3, an irreversible apoptosis marker, may contribute to cyst formation. Thus, this study aimed to investigate the immunoexpression of these proteins in odontogenic cysts and tumors. Twenty cases of ameloblastoma, keratocystic odontogenic tumor (KOT) (n = 20), radicular cyst (RC) (n = 18), dentigerous cyst (DC) (n = 11), calcifying cystic odontogenic tumor (n = 8), and dental follicle (DF) (n = 10) were used to investigate HIF-1α and caspase-3 expression in sequential serial cuts by immunohistochemistry. HIF-1α was overexpressed in RC, DC, and ameloblastoma when compared with DF. The basal and sometimes the lower suprabasal layer showed no or very low expression in DC, KOT, and ameloblastoma, the last also showing strong expression in solid epithelial areas and initial cystic formation regions. Caspase-3 was found to be overexpressed in all lesions, with the highest expression in odontogenic cysts compared to tumors. HIF-1α and caspase-3 were localized in similar areas of the same lesions, especially in the epithelium surrounding cystic formations. This study showed distinct immunoexpression of HIF-1α and caspase-3 in odontogenic cyst and tumors, with higher expression observed in odontogenic cysts. These findings suggest a possible correlation between hypoxia, apoptosis, and cystogenesis, leading to understand the mechanisms responsible to cystic formation in odontogenic lesions.

Balance between senescence and apoptosis is regulated by telomere damage-induced association between p16 and caspase-3.

PubMed

Panneer Selvam, Shanmugam; Roth, Braden M; Nganga, Rose; Kim, Jisun; Cooley, Marion A; Helke, Kristi L; Smith, Charles D; Ogretmen, Besim

2018-05-10

Telomerase activation protects cells from telomere damage by delaying senescence and inducing cell immortalization, whereas telomerase inhibition mediates rapid senescence or apoptosis. However, the cellular mechanisms that determine telomere damage-dependent senescence versus apoptosis induction are largely unknown. Here, we demonstrate that telomerase instability mediated by silencing of sphingosine kinase 2 (SPHK2) and sphingosine 1-phosphate (S1P), which binds and stabilizes telomerase, induces telomere damage-dependent caspase-3 activation and apoptosis, but not senescence, in p16-deficient lung cancer cells or tumors. These outcomes were prevented by knockdown of a tumor-suppressor protein, transcription factor 21 (TCF21), or by ectopic expression of WT human telomerase reverse transcriptase (hTERT), but not mutant hTERT with altered S1P binding. Interestingly, SphK2-deficient mice exhibited accelerated aging and telomerase instability that increased telomere damage and senescence via p16 activation especially in testes tissues, but not in apoptosis. Moreover, p16 silencing in SphK2-/- mouse embryonic fibroblasts activated caspase-3 and apoptosis without inducing senescence. Further, ectopic WT p16 expression in p16-deficient A549 lung cancer cells prevented TCF21 and caspase-3 activation, and resulted in senescence in response to SphK2/S1P inhibition and telomere damage. Mechanistically, a p16 mutant with impaired [MS2] caspase-3 association did not prevent telomere damage-induced apoptosis, indicating that an association between p16 and caspase-3 proteins forces senescence induction by inhibiting caspase-3 activation and apoptosis.[MS3]  These results suggest that p16 plays a direct role in telomere damage-dependent senescence by limiting apoptosis via binding to caspase-3, revealing a direct link between telomere damage-dependent senescence and apoptosis with regards to aging and cancer. Published under license by The American Society for Biochemistry

Long-term fluorescence lifetime imaging of a genetically encoded sensor for caspase-3 activity in mouse tumor xenografts

NASA Astrophysics Data System (ADS)

Zherdeva, Victoria; Kazachkina, Natalia I.; Shcheslavskiy, Vladislav; Savitsky, Alexander P.

2018-03-01

Caspase-3 is known for its role in apoptosis and programmed cell death regulation. We detected caspase-3 activation in vivo in tumor xenografts via shift of mean fluorescence lifetimes of a caspase-3 sensor. We used the genetically encoded sensor TR23K based on the red fluorescent protein TagRFP and chromoprotein KFP linked by 23 amino acid residues (TagRFP-23-KFP) containing a specific caspase cleavage DEVD motif to monitor the activity of caspase-3 in tumor xenografts by means of fluorescence lifetime imaging-Forster resonance energy transfer. Apoptosis was induced by injection of paclitaxel for A549 lung adenocarcinoma and etoposide and cisplatin for HEp-2 pharynx adenocarcinoma. We observed a shift in lifetime distribution from 1.6 to 1.9 ns to 2.1 to 2.4 ns, which indicated the activation of caspase-3. Even within the same tumor, the lifetime varied presumably due to the tumor heterogeneity and the different depth of tumor invasion. Thus, processing time-resolved fluorescence images allows detection of both the cleaved and noncleaved states of the TR23K sensor in real-time mode during the course of several weeks noninvasively. This approach can be used in drug screening, facilitating the development of new anticancer agents as well as improvement of chemotherapy efficiency and its adaptation for personal treatment.

The effects of propofol on hippocampal caspase-3 and Bcl-2 expression following forebrain ischemia-reperfusion in rats.

PubMed

Li, Jun; Han, Baoqing; Ma, Xuesong; Qi, Sihua

2010-10-14

Transient cerebral ischemia may result in neuronal apoptosis. During this process, several apoptosis-regulatory genes are induced in apoptotic cells. Among these genes, cysteinyl aspartate-specific protease-3 (caspase-3) and B-cell leukemia-2 (Bcl-2) are the most effective apoptotic regulators because they play a decisive role in the occurrence of apoptosis. Research has shown that propofol, which is an intravenous anesthetic agent, exhibits neuroprotective effects against cerebral ischemia-reperfusion injury, although the neuroprotective mechanism is still unclear. In this study, we examined the effects of propofol in rats after forebrain ischemia-reperfusion. We assessed the expression of hippocampal caspase-3, which acts as an apoptotic activator, and Bcl-2, which acts as an apoptotic suppressor. Forebrain ischemia was induced in hypotensive rats by clamping the bilateral common carotid arteries for 10 min. Propofol was administered via a lateral cerebral ventricle injection using a microsyringe after the induction of ischemia. Neuronal damage was determined by histological examination of brain sections at the level of the dorsal hippocampus. Caspase-3 and Bcl-2 expression in the hippocampus were detected using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. We also used an immunohistochemical method after ischemia-reperfusion. In the hippocampus, caspase-3 and Bcl-2 mRNA were dramatically increased at 24h after forebrain ischemia. Following 6-24h of reperfusion, forebrain ischemia for 10 min induced a gradual increase in the expression of caspase-3 and Bcl-2 protein in the rat hippocampus, which peaked at 24h. In the propofol (1.0mg/kg) intervention group, the hippocampal expression of caspase-3 mRNA decreased significantly in rats 24h after ischemia; Bcl-2 mRNA was increased at the same time point. During the 24-h reperfusion period and after treatment with propofol, the level of caspase-3 protein expression

Identification of Caspase Cleavage Sites in KSHV Latency-Associated Nuclear Antigen and Their Effects on Caspase-Related Host Defense Responses.

PubMed

Davis, David A; Naiman, Nicole E; Wang, Victoria; Shrestha, Prabha; Haque, Muzammel; Hu, Duosha; Anagho, Holda A; Carey, Robert F; Davidoff, Katharine S; Yarchoan, Robert

2015-07-01

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causative agent of three hyperproliferative disorders: Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman's disease. During viral latency a small subset of viral genes are produced, including KSHV latency-associated nuclear antigen (LANA), which help the virus thwart cellular defense responses. We found that exposure of KSHV-infected cells to oxidative stress, or other inducers of apoptosis and caspase activation, led to processing of LANA and that this processing could be inhibited with the pan-caspase inhibitor Z-VAD-FMK. Using sequence, peptide, and mutational analysis, two caspase cleavage sites within LANA were identified: a site for caspase-3 type caspases at the N-terminus and a site for caspase-1 and-3 type caspases at the C-terminus. Using LANA expression plasmids, we demonstrated that mutation of these cleavage sites prevents caspase-1 and caspase-3 processing of LANA. This indicates that these are the principal sites that are susceptible to caspase cleavage. Using peptides spanning the identified LANA cleavage sites, we show that caspase activity can be inhibited in vitro and that a cell-permeable peptide spanning the C-terminal cleavage site could inhibit cleavage of poly (ADP-ribose) polymerase and increase viability in cells undergoing etoposide-induced apoptosis. The C-terminal peptide of LANA also inhibited interleukin-1 beta (IL-1β) production from lipopolysaccharide-treated THP-1 cells by more than 50%. Furthermore, mutation of the two cleavage sites in LANA led to a significant increase in IL-1β production in transfected THP-1 cells; this provides evidence that these sites function to blunt the inflammasome, which is known to be activated in latently infected PEL cells. These results suggest that specific caspase cleavage sites in KSHV LANA function to blunt apoptosis as well as interfere with the caspase-1-mediated inflammasome

Endoplasmic Reticulum Stress Activates the Inflammasome via NLRP3- and Caspase-2-Driven Mitochondrial Damage.

PubMed

Bronner, Denise N; Abuaita, Basel H; Chen, Xiaoyun; Fitzgerald, Katherine A; Nuñez, Gabriel; He, Yongqun; Yin, Xiao-Ming; O'Riordan, Mary X D

2015-09-15

Endoplasmic reticulum (ER) stress is observed in many human diseases, often associated with inflammation. ER stress can trigger inflammation through nucleotide-binding domain and leucine-rich repeat containing (NLRP3) inflammasome, which might stimulate inflammasome formation by association with damaged mitochondria. How ER stress triggers mitochondrial dysfunction and inflammasome activation is ill defined. Here we have used an infection model to show that the IRE1α ER stress sensor regulates regulated mitochondrial dysfunction through an NLRP3-mediated feed-forward loop, independently of ASC. IRE1α activation increased mitochondrial reactive oxygen species, promoting NLRP3 association with mitochondria. NLRP3 was required for ER stress-induced cleavage of caspase-2 and the pro-apoptotic factor, Bid, leading to subsequent release of mitochondrial contents. Caspase-2 and Bid were necessary for activation of the canonical inflammasome by infection-associated or general ER stress. These data identify an NLRP3-caspase-2-dependent mechanism that relays ER stress to the mitochondria to promote inflammation, integrating cellular stress and innate immunity. Copyright © 2015 Elsevier Inc. All rights reserved.

Prognostic Value of Cd95, Active Caspase-3, and Bcl-2 Expression in Adult Patients With De Novo Acute Lymphoblastic Leukemia.

PubMed

Montiel-Cervantes, Laura Arcelia; Reyes-Maldonado, Elba; Garcia-Chavez, Jaime; Hernandez-Caballero, Alvaro; Molina-Aguilar, Rubiraida; Garcia-Ruiz Esparza, Miriam America; Vela-Ojeda, Jorge

2018-04-18

Acute lymphoblastic leukemia is an aggressive malignant disease with high mortality rates in adults. The expression levels of CD95, active caspase-3, and Bcl-2 were determined in 111 patients with de novo acute lymphoblastic leukemia (ALL) and correlated with overall survival (OS) and disease-free survival (DFS). The immunophenotyped ok leukemia and the expression of CD95, active caspase-3, and Bcl-2, were determined by flow cytometry. Apoptotic variables were correlated by Spearman test, and survival by Kaplan-Meier method. Log-rank test was used to compare survival curves. From a total of 111 patients, 56 cases were B-ALL, 16 T-ALL, 16 B-ALL/CD33 + , and 23 ambiguous lineage-AL (AmbLin-AL). The median expression of CD95 (61.5%) and active-caspase-3 (19.4%) was higher in T-ALL (p < 0.05), whereas Bcl-2 was lower in T-ALL (p < 0.038). There was a highly significant correlation in B-ALL, B-ALL/CD33 + and AmbLin-AL between CD95 and Bcl-2, CD95-Active caspase-3, and Bcl-2-Active caspase-3; while in T-ALL, there was only a correlation between CD95-Active caspase-3, and Bcl-2-Active caspase-3. OS and DFS were better for T-ALL than the other groups, especially in patients having higher values of CD95 and active caspase 3, and lower values of Bcl-2. The worse survival rates were observed in patients with B-ALL/CD33 + , and AmbLin-AL. The prognosis of ALL in adults is influenced by the expression levels of Bcl-2, active-caspase-3, and CD95. Copyright © 2018 IMSS. Published by Elsevier Inc. All rights reserved.

Inhibition of Stat3 activation suppresses caspase-3 and the ubiquitin-proteasome system, leading to preservation of muscle mass in cancer cachexia.

PubMed

Silva, Kleiton Augusto Santos; Dong, Jiangling; Dong, Yanjun; Dong, Yanlan; Schor, Nestor; Tweardy, David J; Zhang, Liping; Mitch, William E

2015-04-24

Cachexia occurs in patients with advanced cancers. Despite the adverse clinical impact of cancer-induced muscle wasting, pathways causing cachexia are controversial, and clinically reliable therapies are not available. A trigger of muscle protein loss is the Jak/Stat pathway, and indeed, we found that conditioned medium from C26 colon carcinoma (C26) or Lewis lung carcinoma cells activates Stat3 (p-Stat3) in C2C12 myotubes. We identified two proteolytic pathways that are activated in muscle by p-Stat3; one is activation of caspase-3, and the other is p-Stat3 to myostatin, MAFbx/Atrogin-1, and MuRF-1 via CAAT/enhancer-binding protein δ (C/EBPδ). Using sequential deletions of the caspase-3 promoter and CHIP assays, we determined that Stat3 activation increases caspase-3 expression in C2C12 cells. Caspase-3 expression and proteolytic activity were stimulated by p-Stat3 in muscles of tumor-bearing mice. In mice with cachexia caused by Lewis lung carcinoma or C26 tumors, knock-out of p-Stat3 in muscle or with a small chemical inhibitor of p-Stat3 suppressed muscle mass losses, improved protein synthesis and degradation in muscle, and increased body weight and grip strength. Activation of p-Stat3 stimulates a pathway from C/EBPδ to myostatin and expression of MAFbx/Atrogin-1 and increases the ubiquitin-proteasome system. Indeed, C/EBPδ KO decreases the expression of MAFbx/Atrogin-1 and myostatin, while increasing muscle mass and grip strength. In conclusion, cancer stimulates p-Stat3 in muscle, activating protein loss by stimulating caspase-3, myostatin, and the ubiquitin-proteasome system. These results could lead to novel strategies for preventing cancer-induced muscle wasting. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Inhibition of Stat3 Activation Suppresses Caspase-3 and the Ubiquitin-Proteasome System, Leading to Preservation of Muscle Mass in Cancer Cachexia*

PubMed Central

Silva, Kleiton Augusto Santos; Dong, Jiangling; Dong, Yanjun; Dong, Yanlan; Schor, Nestor; Tweardy, David J.; Zhang, Liping; Mitch, William E.

2015-01-01

Cachexia occurs in patients with advanced cancers. Despite the adverse clinical impact of cancer-induced muscle wasting, pathways causing cachexia are controversial, and clinically reliable therapies are not available. A trigger of muscle protein loss is the Jak/Stat pathway, and indeed, we found that conditioned medium from C26 colon carcinoma (C26) or Lewis lung carcinoma cells activates Stat3 (p-Stat3) in C2C12 myotubes. We identified two proteolytic pathways that are activated in muscle by p-Stat3; one is activation of caspase-3, and the other is p-Stat3 to myostatin, MAFbx/Atrogin-1, and MuRF-1 via CAAT/enhancer-binding protein δ (C/EBPδ). Using sequential deletions of the caspase-3 promoter and CHIP assays, we determined that Stat3 activation increases caspase-3 expression in C2C12 cells. Caspase-3 expression and proteolytic activity were stimulated by p-Stat3 in muscles of tumor-bearing mice. In mice with cachexia caused by Lewis lung carcinoma or C26 tumors, knock-out of p-Stat3 in muscle or with a small chemical inhibitor of p-Stat3 suppressed muscle mass losses, improved protein synthesis and degradation in muscle, and increased body weight and grip strength. Activation of p-Stat3 stimulates a pathway from C/EBPδ to myostatin and expression of MAFbx/Atrogin-1 and increases the ubiquitin-proteasome system. Indeed, C/EBPδ KO decreases the expression of MAFbx/Atrogin-1 and myostatin, while increasing muscle mass and grip strength. In conclusion, cancer stimulates p-Stat3 in muscle, activating protein loss by stimulating caspase-3, myostatin, and the ubiquitin-proteasome system. These results could lead to novel strategies for preventing cancer-induced muscle wasting. PMID:25787076

Caspase-3 Is Transiently Activated without Cell Death during Early Antigen Driven Expansion of CD8+ T Cells In Vivo

PubMed Central

McComb, Scott; Mulligan, Rebecca; Sad, Subash

2010-01-01

Background CD8+ T cell responses develop rapidly during infection and are swiftly reduced during contraction, wherein >90% of primed CD8+ T cells are eliminated. The role of apoptotic mechanisms in controlling this rapid proliferation and contraction of CD8+ T cells remains unclear. Surprisingly, evidence has shown non-apoptotic activation of caspase-3 to occur during in vitro T-cell proliferation, but the relevance of these mechanisms to in vivo CD8+ T cell responses has yet to be examined. Methods and Findings We have evaluated the activity of caspase-3, a key downstream inducer of apoptosis, throughout the entirety of a CD8+ T cell response. We utilized two infection models that differ in the intensity, onset and duration of antigen-presentation and inflammation. Expression of cleaved caspase-3 in antigen specific CD8+ T cells was coupled to the timing and strength of antigen presentation in lymphoid organs. We also observed coordinated activation of additional canonical apoptotic markers, including phosphatidylserine exposure. Limiting dilution analysis directly showed that in the presence of IL7, very little cell death occurred in both caspase-3hi and caspase-3low CD8+ T cells. The expression of active caspase-3 peaked before effector phenotype (CD62Llow) CD8+ T cells emerged, and was undetectable in effector-phenotype cells. In addition, OVA-specific CD8+ cells remained active caspase-3low throughout the contraction phase. Conclusions Our results specifically implicate antigen and not inflammation in driving activation of apoptotic mechanisms without cell death in proliferating CD8+ T cells. Furthermore, the contraction of CD8+ T cell response following expansion is likely not mediated by the key downstream apoptosis inducer, caspase-3. PMID:21203525

alpha-Amino-3-hydroxy-5-methyl-4-isoxazole propionate attenuates glutamate-induced caspase-3 cleavage via regulation of glycogen synthase kinase 3beta.

PubMed

Nishimoto, Takaaki; Kihara, Takeshi; Akaike, Akinori; Niidome, Tetsuhiro; Sugimoto, Hachiro

2008-04-01

Preconditioning of sublethal ischemia exhibits neuroprotection against subsequent ischemia-induced neuronal death. It has been indicated that glutamate, an excitatory amino acid, is involved in the pathogenesis of ischemia-induced neuronal death or neurodegeneration. To elucidate whether prestimulation of glutamate receptor could counter ischemia-induced neuronal death or neurodegeneration, we examined the effect of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), an ionotropic subtype of glutamate receptor, on excess glutamate-induced excitotoxicity using primary cortical neuronal cultures. We found that AMPA exerted a neuroprotective effect in a time- and concentration-dependent manner. A blocker of phosphatidylinositol-3 kinase (PI3K), LY294002 (10 microM), significantly attenuated AMPA-induced protection. In addition, Ser473 of Akt/PKB, a downstream target of PI3K, was phosphorylated by AMPA administration (10 microM). Glycogen synthase kinase 3beta (GSK3beta), which has been reported to be inactivated by Akt, was phosphorylated at Ser9 by AMPA. Ser9-phosphorylated GSK3beta or inactivated form would be a key molecule for neuroprotection, insofar as lithium chloride (100 microM) and SB216763 (10 microM), inhibitors of GSK3beta, also induced phosphorylation of GSK3beta at Ser9 and exerted neuroprotection, respectively. Glutamate (100 microM) increased cleaved caspase-3, an apoptosis-related cysteine protease, and caspase-3 inhibitor (Ac-DEVD-CHO; 1 microM) blocked glutamate-induced excitotoxicity in our culture. AMPA (10 microM, 24 hr) and SB216763 (10 microM) prominently decreased glutamate-induced caspase-3 cleavage. These findings suggest that AMPA activates PI3K-Akt and subsequently inhibits GSK3beta and that inactivated GSK3beta attenuates glutamate-induced caspase-3 cleavage and neurotoxicity.

Memantine Can Reduce Ethanol-Induced Caspase-3 Activity and Apoptosis in H4 Cells by Decreasing Intracellular Calcium.

PubMed

Wang, Xiaolong; Chen, Jiajun; Wang, Hongbo; Yu, Hao; Wang, Changliang; You, Jiabin; Wang, Pengfei; Feng, Chunmei; Xu, Guohui; Wu, Xu; Zhao, Rui; Zhang, Guohua

2017-08-01

Caspase-3 activation and apoptosis are associated with various neurodegenerative disorders. Calcium activation is an important factor in promoting apoptosis. We, therefore, assessed the role of intracellular calcium in ethanol-induced activation of caspase-3 in H4 human neuroglioma cells and the protective effect of the NMDA receptor antagonist, memantine, on ethanol-induced apoptosis in H4 cells. H4 cells were treated with 100 mM EtOH (in culture medium) for 2 days. For interaction studies, cells were treated with memantine (4 μM), EDTA (1 mM), or BAPTA-AM (10 μM) before treatment with EtOH. Knockdown of the gene encoding the NR1 subunit of the NMDA receptor was performed using RNAi. Apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry. Cell viability was detected using an MTS cell proliferation kit. Fluorescence dual wavelength spectrophotometry was used to determine the intracellular calcium concentration. The levels of NR1, caspase-3, IP3R1, and SERCA1 proteins were detected by western blotting. NR1, IP3R1, and SERCA1 mRNA levels were detected by qPCR. We observed increased expression of NR1, IP3R1, SERCA1, and increased intracellular levels of calcium ions in H4 cells exposed to ethanol. In addition, the calcium chelators, EDTA and BAPTA, and RNAi disruption of the NMDA receptor reduced ethanol-induced caspase-3 activation in H4 cells. Memantine treatment reduced the ethanol-induced increase of intracellular calcium, caspase-3 activation, apoptosis, and the ethanol-induced decrease in cell viability. Our results indicate that ethanol-induced caspase-3 activation and apoptosis are likely to be dependent on cytosolic calcium levels and that they can be reduced by memantine treatment.

Serial killers: ordering caspase activation events in apoptosis.

PubMed

Slee, E A; Adrain, C; Martin, S J

1999-11-01

Caspases participate in the molecular control of apoptosis in several guises; as triggers of the death machinery, as regulatory elements within it, and ultimately as a subset of the effector elements of the machinery itself. The mammalian caspase family is steadily growing and currently contains 14 members. At present, it is unclear whether all of these proteases participate in apoptosis. Thus, current research in this area is focused upon establishing the repertoire and order of caspase activation events that occur during the signalling and demolition phases of cell death. Evidence is accumulating to suggest that proximal caspase activation events are typically initiated by molecules that promote caspase aggregation. As expected, distal caspase activation events are likely to be controlled by caspases activated earlier in the cascade. However, recent data has cast doubt upon the functional demarcation of caspases into signalling (upstream) and effector (downstream) roles based upon their prodomain lengths. In particular, caspase-3 may perform an important role in propagating the caspase cascade, in addition to its role as an effector caspase within the death programme. Here, we discuss the apoptosis-associated caspase cascade and the hierarchy of caspase activation events within it.

Targeting caspase-3 as dual therapeutic benefits by RNAi facilitating brain-targeted nanoparticles in a rat model of Parkinson's disease.

PubMed

Liu, Yang; Guo, Yubo; An, Sai; Kuang, Yuyang; He, Xi; Ma, Haojun; Li, Jianfeng; Lu, Jing; Lv, Jing; Zhang, Ning; Jiang, Chen

2013-01-01

The activation of caspase-3 is an important hallmark in Parkinson's disease. It could induce neuron death by apoptosis and microglia activation by inflammation. As a result, inhibition the activation of caspase-3 would exert synergistic dual effect in brain in order to prevent the progress of Parkinson's disease. Silencing caspase-3 genes by RNA interference could inhibit the activation of caspase-3. We developed a brain-targeted gene delivery system based on non-viral gene vector, dendrigraft poly-L-lysines. A rabies virus glycoprotein peptide with 29 amino-acid linked to dendrigraft poly-L-lysines could render gene vectors the ability to get across the blood brain barrier by specific receptor mediated transcytosis. The resultant brain-targeted vector was complexed with caspase-3 short hairpin RNA coding plasmid DNA, yielding nanoparticles. In vivo imaging analysis indicated the targeted nanoparticles could accumulate in brain more efficiently than non-targeted ones. A multiple dosing regimen by weekly intravenous administration of the nanoparticles could reduce activated casapse-3 levels, significantly improve locomotor activity and rescue dopaminergic neuronal loss and in Parkinson's disease rats' brain. These results indicated the rabies virus glycoprotein peptide modified brain-targeted nanoparticles were promising gene delivery system for RNA interference to achieve anti-apoptotic and anti-inflammation synergistic therapeutic effects by down-regulation the expression and activation of caspase-3.

Apoptosis in acute shigellosis is associated with increased production of Fas/Fas ligand, perforin, caspase-1, and caspase-3 but reduced production of Bcl-2 and interleukin-2.

PubMed

Raqib, Rubhana; Ekberg, Caroline; Sharkar, Protim; Bardhan, Pradip K; Zychlinsky, Arturo; Sansonetti, Philippe J; Andersson, Jan

2002-06-01

Shigella dysenteriae type 1-induced apoptotic cell death in rectal tissues from patients infected with Shigella dysenteriae type 1 was studied by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) technique and annexin V staining. Expression of proteins and cytokines participating in the apoptotic process (caspase-1, caspase-3, Fas [CD95], Fas ligand [Fas-L], perforin, granzyme A, Bax, WAF-1, Bcl-2, interleukin-2 [IL-2], IL-18, and granulocyte-macrophage colony-stimulating factor) in tissue in the acute and convalescent stages of dysentery was quantified at the single-cell level by in situ immunostaining. Apoptotic cell death in the lamina propria was markedly up-regulated at the acute stage (P < 0.05), where an increased number of necrotic cells were also seen. Phenotypic analysis of apoptotic cells revealed that 43% of T cells (CD3), 10% of granulocytes (CD15), and 5% of macrophages (CD56) underwent apoptosis. Increased activity of caspase-1 persisted in the rectum up to 1 month after onset. More-extensive expression of Fas, Fas-L, perforin, caspase-3, and IL-18, but not IL-2, at the acute stage than at the convalescent stage was observed. Increased expression of caspase-3 and IL-18 in tissues with severe inflammation compared to expression in those with mild inflammation was evident, implying a possible role in the perpetuation of inflammation. Significantly reduced cell death during convalescence was associated with a significant up-regulation of Bcl-2, Bax, and WAF-1 expression in the rectum compared to that in the acute phase of infection. Thus, induction of apoptosis at the local site in the early phase of S. dysenteriae type 1 infection was associated with a significant up-regulation of Fas/Fas-L and perforin and granzyme A expression and a down-regulation of Bcl-2 and IL-2, which promote cell survival.

Highly sensitive detection of caspase-3 activities via a nonconjugated gold nanoparticle-quantum dot pair mediated by an inner-filter effect.

PubMed

Li, Jingwen; Li, Xinming; Shi, Xiujuan; He, Xuewen; Wei, Wei; Ma, Nan; Chen, Hong

2013-10-09

We describe here a simple fluorometric assay for the highly sensitive detection of caspase-3 activities on the basis of the inner-filter effect of gold nanoparticles (AuNPs) on CdTe quantum dots (QDs). The method takes advantage of the high molar absorptivity of the plasmon band of gold nanoparticles as well as the large absorption band shift from 520 to 680 nm upon nanoparticle aggregation. When labeled with a peptide possessing the caspase-3 cleavage sequence (DEVD), the monodispersed Au-Ps (peptide-modified AuNPs) exhibited a tendency to aggregate when exposed to caspase-3, which induced the absorption band transition from 520 to 680 nm and turned on the fluorescence of the CdTe QDs for caspase-3 sensing. Under optimum conditions, a high sensitivity towards caspase-3 was achieved with a detection limit as low as 18 pM, which was much lower than the corresponding assays based on absorbance or other approaches. Overall, we demonstrated a facile and sensitive approach for caspase-3 detection, and we expected that this method could be potentially generalized to design more fluorescent assays for sensing other bioactive entities.

Complementary optical and nuclear imaging of caspase-3 activity using combined activatable and radio-labeled multimodality molecular probe.

PubMed

Lee, Hyeran; Akers, Walter J; Cheney, Philip P; Edwards, W Barry; Liang, Kexian; Culver, Joseph P; Achilefu, Samuel

2009-01-01

Based on the capability of modulating fluorescence intensity by specific molecular events, we report a new multimodal optical-nuclear molecular probe with complementary reporting strategies. The molecular probe (LS498) consists of tetraazacyclododecanetetraacetic acid (DOTA) for chelating a radionuclide, a near-infrared fluorescent dye, and an efficient quencher dye. The two dyes are separated by a cleavable peptide substrate for caspase-3, a diagnostic enzyme that is upregulated in dying cells. LS498 is radiolabeled with (64)Cu, a radionuclide used in positron emission tomography. In the native form, LS498 fluorescence is quenched until caspase-3 cleavage of the peptide substrate. Enzyme kinetics assay shows that LS498 is readily cleaved by caspase-3, with excellent enzyme kinetic parameters k(cat) and K(M) of 0.55+/-0.01 s(-1) and 1.12+/-0.06 microM, respectively. In mice, the initial fluorescence of LS498 is ten-fold less than control. Using radiolabeled (64)Cu-LS498 in a controlled and localized in-vivo model of caspase-3 activation, a time-dependent five-fold NIR fluorescence enhancement is observed, but radioactivity remains identical in caspase-3 positive and negative controls. These results demonstrate the feasibility of using radionuclide imaging for localizing and quantifying the distribution of molecular probes and optical imaging for reporting the functional status of diagnostic enzymes.

Complementary optical and nuclear imaging of caspase-3 activity using combined activatable and radio-labeled multimodality molecular probe

NASA Astrophysics Data System (ADS)

Lee, Hyeran; Akers, Walter J.; Cheney, Philip P.; Edwards, W. Barry; Liang, Kexian; Culver, Joseph P.; Achilefu, Samuel

2009-07-01

Based on the capability of modulating fluorescence intensity by specific molecular events, we report a new multimodal optical-nuclear molecular probe with complementary reporting strategies. The molecular probe (LS498) consists of tetraazacyclododecanetetraacetic acid (DOTA) for chelating a radionuclide, a near-infrared fluorescent dye, and an efficient quencher dye. The two dyes are separated by a cleavable peptide substrate for caspase-3, a diagnostic enzyme that is upregulated in dying cells. LS498 is radiolabeled with 64Cu, a radionuclide used in positron emission tomography. In the native form, LS498 fluorescence is quenched until caspase-3 cleavage of the peptide substrate. Enzyme kinetics assay shows that LS498 is readily cleaved by caspase-3, with excellent enzyme kinetic parameters kcat and KM of 0.55+/-0.01 s-1 and 1.12+/-0.06 μM, respectively. In mice, the initial fluorescence of LS498 is ten-fold less than control. Using radiolabeled 64Cu-LS498 in a controlled and localized in-vivo model of caspase-3 activation, a time-dependent five-fold NIR fluorescence enhancement is observed, but radioactivity remains identical in caspase-3 positive and negative controls. These results demonstrate the feasibility of using radionuclide imaging for localizing and quantifying the distribution of molecular probes and optical imaging for reporting the functional status of diagnostic enzymes.

Insight into the mechanism of action and selectivity of caspase-3 reversible inhibitors through in silico studies

NASA Astrophysics Data System (ADS)

Minini, Lucía; Ferraro, Florencia; Cancela, Saira; Merlino, Alicia

2017-11-01

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder worldwide for which there is currently no cure. Recently, caspase-3 has been proposed as a potential therapeutic target for treating AD. Since this enzyme is overexpressed in brains from AD patients its selective modulation by non-covalent inhibitors becomes an interesting strategy in the search of potential drugs against this neuropathology. With this in mind, we have combined molecular docking, molecular dynamics simulations and QM calculations of unliganded caspase-3 and caspase-7 and in complex with a series of known inhibitors of caspase-3 described in the literature in order to assess the structural features responsible for good inhibitory activity and selectivity against this potential target. This work has allowed us to identify hotspots for drug binding as well as the importance of shape and charge distribution for interacting into the substrate binding cleft or into the dimer interface in each enzyme. Our results showed that most selective compounds against caspsase-3 bind into the substrate binding cleft acting as competitive inhibitors whereas in caspase-7 they bind close to an allosteric site at the dimer interface but since they are weakly bound their presence would not be affecting enzyme dynamics or function. In addition, for both enzymes we have found evidence indicating that differences in shape and accessibility exist between the substrate binding site of each monomer which could be modulating the binding affinity of non-covalent molecules.

Confinement of caspase-12 proteolytic activity to autoprocessing

PubMed Central

Roy, Sophie; Sharom, Jeffrey R.; Houde, Caroline; Loisel, Thomas P.; Vaillancourt, John P.; Shao, Wei; Saleh, Maya; Nicholson, Donald W.

2008-01-01

Caspase-12 is a dominant-negative regulator of caspase-1 (IL-1β-converting enzyme) and an attenuator of cytokine responsiveness to septic infections. This molecular role for caspase-12 appears to be akin to the role of cFLIP in regulating caspase-8 in the extrinsic cell death pathway; however, unlike cFLIP/Usurpin, we demonstrate here that caspase-12 is catalytically competent. To examine these catalytic properties, rat caspase-12 was cloned, and the recombinant enzyme was used to examine the cleavage of macromolecular and synthetic fluorogenic substrates. Although caspase-12 could mediate autoproteolytic maturation of its own proenzyme, in both cis and trans, it was not able to cleave any other polypeptide substrate, including other caspase proenzymes, apoptotic substrates, cytokine precursors, or proteins in the endoplasmic reticulum that normally undergo caspase-mediated proteolysis. The dearth of potential substrates for caspase-12 also was confirmed by whole-cell diagonal-gel analysis. Autolytic cleavage within the caspase-12 proenzyme was mapped to a single site at the large–small subunit junction, ATAD319, and this motif was recognized by caspase-12 when incorporated into synthetic fluorogenic substrates. The specific activity of caspase-12 with these substates was several orders of magnitude lower than caspases-1 and -3, highlighting its relative catalytic paucity. In intact cells, caspase-12 autoproteolysis occurred in the inhibitory complex containing caspase-1. We propose that the proteolytic activity of caspase-12 is confined to its own proenzyme and that autocleavage within the caspase-1 complex may be a means for temporal limitation of the inhibitory effects of caspase-12 on proinflammatory cytokine maturation. PMID:18332441

Cleavage by Caspase 8 and Mitochondrial Membrane Association Activate the BH3-only Protein Bid during TRAIL-induced Apoptosis*

PubMed Central

Huang, Kai; Zhang, Jingjing; O'Neill, Katelyn L.; Gurumurthy, Channabasavaiah B.; Quadros, Rolen M.; Tu, Yaping; Luo, Xu

2016-01-01

The BH3-only protein Bid is known as a critical mediator of the mitochondrial pathway of apoptosis following death receptor activation. However, since full-length Bid possesses potent apoptotic activity, the role of a caspase-mediated Bid cleavage is not established in vivo. In addition, due to the fact that multiple caspases cleave Bid at the same site in vitro, the identity of the Bid-cleaving caspase during death receptor signaling remains uncertain. Moreover, as Bid maintains its overall structure following its cleavage by caspase 8, it remains unclear how Bid is activated upon cleavage. Here, Bid-deficient (Bid KO) colon cancer cells were generated by gene editing, and were reconstituted with wild-type or mutants of Bid. While the loss of Bid blocked apoptosis following treatment by TNF-related apoptosis inducing ligand (TRAIL), this blockade was relieved by re-introduction of the wild-type Bid. In contrast, the caspase-resistant mutant BidD60E and a BH3 defective mutant BidG94E failed to restore TRAIL-induced apoptosis. By generating Bid/Bax/Bak-deficient (TKO) cells, we demonstrated that Bid is primarily cleaved by caspase 8, not by effector caspases, to give rise to truncated Bid (tBid) upon TRAIL treatment. Importantly, despite the presence of an intact BH3 domain, a tBid mutant lacking the mitochondrial targeting helices (α6 and α7) showed diminished apoptotic activity. Together, these results for the first time establish that cleavage by caspase 8 and the subsequent association with the outer mitochondrial membrane are two critical events that activate Bid during death receptor-mediated apoptosis. PMID:27053107

CASC2/miR-24/miR-221 modulates the TRAIL resistance of hepatocellular carcinoma cell through caspase-8/caspase-3.

PubMed

Jin, Xiaoxin; Cai, Lifeng; Wang, Changfa; Deng, Xiaofeng; Yi, Shengen; Lei, Zhao; Xiao, Qiangsheng; Xu, Hongbo; Luo, Hongwu; Sun, Jichun

2018-02-23

Hepatocellular carcinoma is one of the most common solid tumors in the digestive system. The prognosis of patients with hepatocellular carcinoma is still poor due to the acquisition of multi-drug resistance. TNF Related Apoptosis Inducing Ligand (TRAIL), an attractive anticancer agent, exerts its effect of selectively inducing apoptosis in tumor cells through death receptors and the formation of the downstream death-inducing signaling complex, which activates apical caspases 3/8 and leads to apoptosis. However, hepatocellular carcinoma cells are resistant to TRAIL. Non-coding RNAs, including long non-coding RNAs (lncRNAs) and miRNAs have been regarded as major regulators of normal development and diseases, including cancers. Moreover, lncRNAs and miRNAs have been reported to be associated with multi-drug resistance. In the present study, we investigated the mechanism by which TRAIL resistance of hepatocellular carcinoma is affected from the view of non-coding RNA regulation. We selected and validated candidate miRNAs, miR-24 and miR-221, that regulated caspase 3/8 expression through direct targeting, and thereby affecting TRAIL-induced tumor cell apoptosis TRAIL resistance of hepatocellular carcinoma. In addition, we revealed that CASC2, a well-established tumor suppressive long non-coding RNA, could serve as a "Sponge" of miR-24 and miR-221, thus modulating TRAIL-induced tumor cell apoptosis TRAIL resistance of hepatocellular carcinoma. Taken together, we demonstrated a CASC2/miR-24/miR-221 axis, which can affect the TRAIL resistance of hepatocellular carcinoma through regulating caspase 3/8; through acting as a "Sponge" of miR-24 and miR-221, CASC2 may contribute to improving hepatocellular carcinoma TRAIL resistance, and finally promoting the treatment efficiency of TRAIL-based therapies.

Effects of apoptosis-related proteins caspase-3, Bax and Bcl-2 on cerebral ischemia rats

PubMed Central

LIU, GUANGYI; WANG, TAO; WANG, TINGING; SONG, JINMING; ZHOU, ZHEN

2013-01-01

Neuron apoptosis is known to mediate a change of ethology following cerebral ischemia-reperfusion injury in rats. Additionally, Bcl-2, Bax and caspase-3 proteins may exert a significant effect on neuron injury. The aim of this study was to investigate the role, mechanism of action and clinical significance of these proteins in neuron apoptosis and functional impairment following cerebral ischemia-reperfusion injury in rats. Sixty male healthy adult Wistar rats were randomly assigned into control (n=6), sham operation (n=6) and experimental (n=48) groups. The model of rat cerebral ischemia-reperfusion injury was set up according to the method of Zea-Longa. Eight subsets of 6 rats-subset were designed according to time points (at 3, 6, 12, 24 and 48 h and at 3, 7 and 14 days). Nerve functional injury was evaluated and graded using nerve function score, balance, coordination function detection and measurement of forelimb placing. The neurons expressing caspase-3, Bax and Bcl-2 in the cortical area, CA3, CA1, stratum lucidum (Slu) and molecular layer of the dentate gyrus (MoDG) of the hippocampus were detected using immunohistochemistry or the TUNEL method. The expression of caspase-3, Bax and Bcl-2 genes was detected by the reverse transcriptase polymerase chain reaction (RT-PCR). The results indicated that, compared to the sham operation group, the score of nerve function and balance beam walking were distinctly higher (P<0.01) and the percentage of rat foreleg touching the angle or margin of the table was significantly lower in the experimental rat group (P<0.01) at 3 h following reperfusion. The expression of TUNEL-positive neurons was high in the cortical area and the CA3 region of the hippocampus (P<0.01), caspase-3 was at peak value in the cortical area and the CA1 region of the hippocampus (P<0.01), Bax was increased in the cortical area and the Slu of the hippocampus (P<0.01) and Bcl-2 was low in the cortical area and the MoDG of the hippocampus (P<0.01) in

Effects of apoptosis-related proteins caspase-3, Bax and Bcl-2 on cerebral ischemia rats.

PubMed

Liu, Guangyi; Wang, Tao; Wang, Tinging; Song, Jinming; Zhou, Zhen

2013-11-01

Neuron apoptosis is known to mediate a change of ethology following cerebral ischemia-reperfusion injury in rats. Additionally, Bcl-2, Bax and caspase-3 proteins may exert a significant effect on neuron injury. The aim of this study was to investigate the role, mechanism of action and clinical significance of these proteins in neuron apoptosis and functional impairment following cerebral ischemia-reperfusion injury in rats. Sixty male healthy adult Wistar rats were randomly assigned into control (n=6), sham operation (n=6) and experimental (n=48) groups. The model of rat cerebral ischemia-reperfusion injury was set up according to the method of Zea-Longa. Eight subsets of 6 rats-subset were designed according to time points (at 3, 6, 12, 24 and 48 h and at 3, 7 and 14 days). Nerve functional injury was evaluated and graded using nerve function score, balance, coordination function detection and measurement of forelimb placing. The neurons expressing caspase-3, Bax and Bcl-2 in the cortical area, CA3, CA1, stratum lucidum (Slu) and molecular layer of the dentate gyrus (MoDG) of the hippocampus were detected using immunohistochemistry or the TUNEL method. The expression of caspase-3, Bax and Bcl-2 genes was detected by the reverse transcriptase polymerase chain reaction (RT-PCR). The results indicated that, compared to the sham operation group, the score of nerve function and balance beam walking were distinctly higher (P<0.01) and the percentage of rat foreleg touching the angle or margin of the table was significantly lower in the experimental rat group (P<0.01) at 3 h following reperfusion. The expression of TUNEL-positive neurons was high in the cortical area and the CA3 region of the hippocampus (P<0.01), caspase-3 was at peak value in the cortical area and the CA1 region of the hippocampus (P<0.01), Bax was increased in the cortical area and the Slu of the hippocampus (P<0.01) and Bcl-2 was low in the cortical area and the MoDG of the hippocampus (P<0.01) in

α-Santalol, a derivative of sandalwood oil, induces apoptosis in human prostate cancer cells by causing caspase-3 activation.

PubMed

Bommareddy, Ajay; Rule, Brittny; VanWert, Adam L; Santha, Sreevidya; Dwivedi, Chandradhar

2012-06-15

The anticancer effects of α-santalol, a major component of sandalwood oil, have been reported against the development of certain cancers such as skin cancer both in vitro and in vivo. The primary objectives of the current study were to investigate the cancer preventive properties of α-santalol on human prostate cancer cells PC-3 (androgen independent and P-53 null) and LNCaP (androgen dependent and P-53 wild-type), and determine the possible mechanisms of its action. The effect of α-santalol on cell viability was determined by trypan blue dye exclusion assay. Apoptosis induction was confirmed by analysis of cytoplasmic histone-associated DNA fragmentation using both an apoptotic ELISA kit and a DAPI fluorescence assay. Caspase-3 activity was determined using caspase-3 (active) ELISA kit. PARP cleavage was analyzed using immunoblotting. α-Santalol at 25-75 μM decreased cell viability in both cell lines in a concentration and time dependent manner. Treatment of prostate cancer cells with α-santalol resulted in induction of apoptosis as evidenced by DNA fragmentation and nuclear staining of apoptotic cells by DAPI. α-Santalol treatment also resulted in activation of caspase-3 activity and PARP cleavage. The α-santalol-induced apoptotic cell death and activation of caspase-3 was significantly attenuated in the presence of pharmacological inhibitors of caspase-8 and caspase-9. In conclusion, the present study reveals the apoptotic effects of α-santalol in inhibiting the growth of human prostate cancer cells. Copyright © 2012 Elsevier GmbH. All rights reserved.

Impact of caspase-1/11, -3, -7, or IL-1β/IL-18 deficiency on rabies virus-induced macrophage cell death and onset of disease.

PubMed

Kip, E; Nazé, F; Suin, V; Vanden Berghe, T; Francart, A; Lamoral, S; Vandenabeele, P; Beyaert, R; Van Gucht, S; Kalai, M

2017-01-01

Rabies virus is a highly neurovirulent RNA virus, which causes about 59000 deaths in humans each year. Previously, we described macrophage cytotoxicity upon infection with rabies virus. Here we examined the type of cell death and the role of specific caspases in cell death and disease development upon infection with two laboratory strains of rabies virus: Challenge Virus Standard strain-11 (CVS-11) is highly neurotropic and lethal for mice, while the attenuated Evelyn-Rotnycki-Abelseth (ERA) strain has a broader cell tropism, is non-lethal and has been used as an oral vaccine for animals. Infection of Mf4/4 macrophages with both strains led to caspase-1 activation and IL-1 β and IL-18 production, as well as activation of caspases-3, -7, -8, and -9. Moreover, absence of caspase-3, but not of caspase-1 and -11 or -7, partially inhibited virus-induced cell death of bone marrow-derived macrophages. Intranasal inoculation with CVS-11 of mice deficient for either caspase-1 and -11 or -7 or both IL-1 β and IL-18 led to general brain infection and lethal disease similar to wild-type mice. Deficiency of caspase-3, on the other hand, significantly delayed the onset of disease, but did not prevent final lethal outcome. Interestingly, deficiency of caspase-1/11, the key executioner of pyroptosis, aggravated disease severity caused by ERA virus, whereas wild-type mice or mice deficient for either caspase-3, -7, or both IL-1 β and IL-18 presented the typical mild symptoms associated with ERA virus. In conclusion, rabies virus infection of macrophages induces caspase-1- and caspase-3-dependent cell death. In vivo caspase-1/11 and caspase-3 differently affect disease development in response to infection with the attenuated ERA strain or the virulent CVS-11 strain, respectively. Inflammatory caspases seem to control attenuated rabies virus infection, while caspase-3 aggravates virulent rabies virus infection.

Cleavage by Caspase 8 and Mitochondrial Membrane Association Activate the BH3-only Protein Bid during TRAIL-induced Apoptosis.

PubMed

Huang, Kai; Zhang, Jingjing; O'Neill, Katelyn L; Gurumurthy, Channabasavaiah B; Quadros, Rolen M; Tu, Yaping; Luo, Xu

2016-05-27

The BH3-only protein Bid is known as a critical mediator of the mitochondrial pathway of apoptosis following death receptor activation. However, since full-length Bid possesses potent apoptotic activity, the role of a caspase-mediated Bid cleavage is not established in vivo In addition, due to the fact that multiple caspases cleave Bid at the same site in vitro, the identity of the Bid-cleaving caspase during death receptor signaling remains uncertain. Moreover, as Bid maintains its overall structure following its cleavage by caspase 8, it remains unclear how Bid is activated upon cleavage. Here, Bid-deficient (Bid KO) colon cancer cells were generated by gene editing, and were reconstituted with wild-type or mutants of Bid. While the loss of Bid blocked apoptosis following treatment by TNF-related apoptosis inducing ligand (TRAIL), this blockade was relieved by re-introduction of the wild-type Bid. In contrast, the caspase-resistant mutant Bid(D60E) and a BH3 defective mutant Bid(G94E) failed to restore TRAIL-induced apoptosis. By generating Bid/Bax/Bak-deficient (TKO) cells, we demonstrated that Bid is primarily cleaved by caspase 8, not by effector caspases, to give rise to truncated Bid (tBid) upon TRAIL treatment. Importantly, despite the presence of an intact BH3 domain, a tBid mutant lacking the mitochondrial targeting helices (α6 and α7) showed diminished apoptotic activity. Together, these results for the first time establish that cleavage by caspase 8 and the subsequent association with the outer mitochondrial membrane are two critical events that activate Bid during death receptor-mediated apoptosis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Sub-lethal oxidative stress induces lysosome biogenesis via a lysosomal membrane permeabilization-cathepsin-caspase 3-transcription factor EB-dependent pathway.

PubMed

Leow, San Min; Chua, Shu Xian Serene; Venkatachalam, Gireedhar; Shen, Liang; Luo, Le; Clement, Marie-Veronique

2017-03-07

Here we provide evidence to link sub-lethal oxidative stress to lysosome biogenesis. Exposure of cells to sub-lethal concentrations of exogenously added hydrogen peroxide resulted in cytosol to nuclear translocation of the Transcription Factor EB (TFEB), the master controller of lysosome biogenesis and function. Nuclear translocation of TFEB was dependent upon the activation of a cathepsin-caspase 3 signaling pathway, downstream of lysosomal membrane permeabilization and accompanied by a significant increase in lysosome numbers as well as induction of TFEB-dependent lysosome-associated genes expression such as Ctsl, Lamp2 and its spliced variant Lamp2a, Neu1and Ctsb and Sqstm1 and Atg9b. The effects of sub-lethal oxidative stress on lysosomal gene expression and biogenesis were rescued upon gene silencing of caspase 3 and TFEB. Notably, caspase 3 activation was not associated with phenotypic hallmarks of apoptosis, evidenced by the absence of caspase 3 substrate cleavage, such as PARP, Lamin A/C or gelsolin. Taken together, these data demonstrate for the first time an unexpected and non-canonical role of a cathepsin-caspase 3 axis in the nuclear translocation of TFEB leading to lysosome biogenesis under conditions of sub-lethal oxidative stress.

Relationship between intracellular pH, metabolic co-factors and caspase-3 activation in cancer cells during apoptosis.

PubMed

Sergeeva, Tatiana F; Shirmanova, Marina V; Zlobovskaya, Olga A; Gavrina, Alena I; Dudenkova, Varvara V; Lukina, Maria M; Lukyanov, Konstantin A; Zagaynova, Elena V

2017-03-01

A complex cascade of molecular events occurs in apoptotic cells but cell-to-cell variability significantly complicates determination of the order and interconnections between different processes. For better understanding of the mechanisms of programmed cell death, dynamic simultaneous registration of several parameters is required. In this paper we used multiparameter fluorescence microscopy to analyze energy metabolism, intracellular pH and caspase-3 activation in living cancer cells in vitro during staurosporine-induced apoptosis. We performed metabolic imaging of two co-factors, NAD(P)H and FAD, and used the genetically encoded pH-indicator SypHer1 and the FRET-based sensor for caspase-3 activity, mKate2-DEVD-iRFP, to visualize these parameters by confocal fluorescence microscopy and two-photon fluorescence lifetime imaging microscopy. The correlation between energy metabolism, intracellular pH and caspase-3 activation and their dynamic changes were studied in CT26 cancer cells during apoptosis. Induction of apoptosis was accompanied by a switch to oxidative phosphorylation, cytosol acidification and caspase-3 activation. We showed that alterations in cytosolic pH and the activation of oxidative phosphorylation are relatively early events associated with the induction of apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Dietary flavonoid fisetin targets caspase-3-deficient human breast cancer MCF-7 cells by induction of caspase-7-associated apoptosis and inhibition of autophagy.

PubMed

Yang, Pei-Ming; Tseng, Ho-Hsing; Peng, Chih-Wen; Chen, Wen-Shu; Chiu, Shu-Jun

2012-02-01

The outcome of producing apoptotic defects in cancer cells is the primary obstacle that limits the therapeutic efficacy of anticancer agents, and hence the development of novel agents targeting novel non-canonical cell death pathways has become an imperative mission for clinical research. Fisetin (3,3',4',7-tetrahydroxyflavone) is a naturally occurring flavonoid commonly found in fruits and vegetables. In this study, we investigated the potential anticancer effects of fisetin on breast cancer cells. The result showed fisetin induced higher cytotoxicity in human breast cancer MCF-7 than in MDA-MB-231 cells otherwise it did not exert any detectable cytotoxicity in non-tumorigenic MCF-10A cells. We found fisetin can trigger a novel form of atypical apoptosis in caspase-3-deficient MCF-7 cells, which was characterized by several apoptotic features, including plasma membrane rupture, mitochondrial depolarization, activation of caspase-7, -8 and -9, and PARP cleavage; however, neither DNA fragmentation and phosphotidylserine (PS) externalization was observed. Although p53 was also activated by fisetin, the fisetin-induced apoptosis was not rescued by the p53 inhibitor pifithrin-α. In contrast, the fisetin-induced apoptosis was abrogated by pan-caspase inhibitor z-VAD-fmk. Furthermore, inhibition of autophagy by fisetin was shown as additional route to prompt anticancer activity in MCF-7 cells. These data allow us to propose that fisetin appears as a new potential anticancer agent which can be applied to develop a clinical protocol of human breast cancers.

Apoptosis in Acute Shigellosis Is Associated with Increased Production of Fas/Fas Ligand, Perforin, Caspase-1, and Caspase-3 but Reduced Production of Bcl-2 and Interleukin-2

PubMed Central

Raqib, Rubhana; Ekberg, Caroline; Sharkar, Protim; Bardhan, Pradip K.; Zychlinsky, Arturo; Sansonetti, Philippe J.; Andersson, Jan

2002-01-01

Shigella dysenteriae type 1-induced apoptotic cell death in rectal tissues from patients infected with Shigella dysenteriae type 1 was studied by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) technique and annexin V staining. Expression of proteins and cytokines participating in the apoptotic process (caspase-1, caspase-3, Fas [CD95], Fas ligand [Fas-L], perforin, granzyme A, Bax, WAF-1, Bcl-2, interleukin-2 [IL-2], IL-18, and granulocyte-macrophage colony-stimulating factor) in tissue in the acute and convalescent stages of dysentery was quantified at the single-cell level by in situ immunostaining. Apoptotic cell death in the lamina propria was markedly up-regulated at the acute stage (P < 0.05), where an increased number of necrotic cells were also seen. Phenotypic analysis of apoptotic cells revealed that 43% of T cells (CD3), 10% of granulocytes (CD15), and 5% of macrophages (CD56) underwent apoptosis. Increased activity of caspase-1 persisted in the rectum up to 1 month after onset. More-extensive expression of Fas, Fas-L, perforin, caspase-3, and IL-18, but not IL-2, at the acute stage than at the convalescent stage was observed. Increased expression of caspase-3 and IL-18 in tissues with severe inflammation compared to expression in those with mild inflammation was evident, implying a possible role in the perpetuation of inflammation. Significantly reduced cell death during convalescence was associated with a significant up-regulation of Bcl-2, Bax, and WAF-1 expression in the rectum compared to that in the acute phase of infection. Thus, induction of apoptosis at the local site in the early phase of S. dysenteriae type 1 infection was associated with a significant up-regulation of Fas/Fas-L and perforin and granzyme A expression and a down-regulation of Bcl-2 and IL-2, which promote cell survival. PMID:12011015

SfDredd, a Novel Initiator Caspase Possessing Activity on Effector Caspase Substrates in Spodoptera frugiperda

PubMed Central

Liu, Hao; Wu, Andong; Mei, Long; Liu, Qingzhen

2016-01-01

Sf9, a cell line derived from Spodoptera frugiperda, is an ideal model organism for studying insect apoptosis. The first notable study that attempted to identify the apoptotic pathway in Sf9 was performed in 1997 and included the discovery of Sf-caspase-1, an effector caspase of Sf9. However, it was not until 2013 that the first initiator caspase in Sf9, SfDronc, was discovered, and the apoptotic pathway in Sf9 became clearer. In this study, we report another caspase of Sf9, SfDredd. SfDredd is highly similar to insect initiator caspase Dredd homologs. Experimentally, recombinant SfDredd underwent autocleavage and exhibited different efficiencies in cleavage of synthetic caspase substrates. This was attributed to its caspase activity for the predicted active site mutation blocked the above autocleavage and synthetic caspase substrates cleavage activity. SfDredd was capable of not only cleaving Sf-caspase-1 in vitro but also cleaving Sf-caspase-1 and inducing apoptosis when it was co-expressed with Sf-caspase-1 in Sf9 cells. The protein level of SfDredd was increased when Sf9 cells were treated by Actinomycin D, whereas silencing of SfDredd reduced apoptosis and Sf-caspase-1 cleavage induced by Actinomycin D treatment. These results clearly indicate that SfDredd functioned as an apoptotic initiator caspase. Apoptosis induced in Sf9 cells by overexpression of SfDredd alone was not as obvious as that induced by SfDronc alone, and the cleavage sites of Sf-caspase-1 for SfDredd and SfDronc are different. In addition, despite sharing a sequence homology with initiator caspases and possessing weak activity on initiator caspase substrates, SfDredd showed strong activity on effector caspase substrates, making it the only insect caspase reported so far functioning similar to human caspase-2 in this aspect. We believe that the discovery of SfDredd, and its different properties from SfDronc, will improve the understanding of apoptosis pathway in Sf9 cells. PMID:26977926

SfDredd, a Novel Initiator Caspase Possessing Activity on Effector Caspase Substrates in Spodoptera frugiperda.

PubMed

Yang, Zhouning; Zhou, Ke; Liu, Hao; Wu, Andong; Mei, Long; Liu, Qingzhen

2016-01-01

Sf9, a cell line derived from Spodoptera frugiperda, is an ideal model organism for studying insect apoptosis. The first notable study that attempted to identify the apoptotic pathway in Sf9 was performed in 1997 and included the discovery of Sf-caspase-1, an effector caspase of Sf9. However, it was not until 2013 that the first initiator caspase in Sf9, SfDronc, was discovered, and the apoptotic pathway in Sf9 became clearer. In this study, we report another caspase of Sf9, SfDredd. SfDredd is highly similar to insect initiator caspase Dredd homologs. Experimentally, recombinant SfDredd underwent autocleavage and exhibited different efficiencies in cleavage of synthetic caspase substrates. This was attributed to its caspase activity for the predicted active site mutation blocked the above autocleavage and synthetic caspase substrates cleavage activity. SfDredd was capable of not only cleaving Sf-caspase-1 in vitro but also cleaving Sf-caspase-1 and inducing apoptosis when it was co-expressed with Sf-caspase-1 in Sf9 cells. The protein level of SfDredd was increased when Sf9 cells were treated by Actinomycin D, whereas silencing of SfDredd reduced apoptosis and Sf-caspase-1 cleavage induced by Actinomycin D treatment. These results clearly indicate that SfDredd functioned as an apoptotic initiator caspase. Apoptosis induced in Sf9 cells by overexpression of SfDredd alone was not as obvious as that induced by SfDronc alone, and the cleavage sites of Sf-caspase-1 for SfDredd and SfDronc are different. In addition, despite sharing a sequence homology with initiator caspases and possessing weak activity on initiator caspase substrates, SfDredd showed strong activity on effector caspase substrates, making it the only insect caspase reported so far functioning similar to human caspase-2 in this aspect. We believe that the discovery of SfDredd, and its different properties from SfDronc, will improve the understanding of apoptosis pathway in Sf9 cells.

1800MHz Microwave Induces p53 and p53-Mediated Caspase-3 Activation Leading to Cell Apoptosis In Vitro

PubMed Central

Xing, Fuqiang; Zhan, Qiuqiang; He, Yiduo; Cui, Jiesheng; He, Sailing; Wang, Guanyu

2016-01-01

Recent studies have reported that exposure of mammalian cells to microwave radiation may have adverse effects such as induction of cell apoptosis. However, the molecular mechanisms underlying microwave induced mammalian cell apoptosis are not fully understood. Here, we report a novel mechanism: exposure to 1800MHz microwave radiation induces p53-dependent cell apoptosis through cytochrome c-mediated caspase-3 activation pathway. We first measured intensity of microwave radiation from several electronic devices with an irradiation detector. Mouse NIH/3T3 and human U-87 MG cells were then used as receivers of 1800MHz electromagnetic radiation (EMR) at a power density of 1209 mW/m2. Following EMR exposure, cells were analyzed for viability, intracellular reactive oxygen species (ROS) generation, DNA damage, p53 expression, and caspase-3 activity. Our analysis revealed that EMR exposure significantly decreased viability of NIH/3T3 and U-87 MG cells, and increased caspase-3 activity. ROS burst was observed at 6 h and 48 h in NIH/3T3 cells, while at 3 h in U-87 MG cells. Hoechst 33258 staining and in situ TUNEL assay detected that EMR exposure increased DNA damage, which was significantly restrained in the presence of N-acetyl-L-cysteine (NAC, an antioxidant). Moreover, EMR exposure increased the levels of p53 protein and p53 target gene expression, promoted cytochrome c release from mitochondrion, and increased caspase-3 activity. These events were inhibited by pretreatment with NAC, pifithrin-α (a p53 inhibitor) and caspase inhibitor. Collectively, our findings demonstrate, for the first time, that 1800MHz EMR induces apoptosis-related events such as ROS burst and more oxidative DNA damage, which in turn promote p53-dependent caspase-3 activation through release of cytochrome c from mitochondrion. These findings thus provide new insights into physiological mechanisms underlying microwave-induced cell apoptosis. PMID:27689798

AGEs Induce Apoptosis in Rat Osteoblast Cells by Activating the Caspase-3 Signaling Pathway Under a High-Glucose Environment In Vitro.

PubMed

Liu, Jiaqiang; Mao, Jing; Jiang, Yi; Xia, Lunguo; Mao, Lixia; Wu, Yong; Ma, Pan; Fang, Bing

2016-03-01

Advanced glycation end products (AGEs) accumulate under high-glucose conditions and affect the healing of bone damage through various pathways; however, the detail mechanisms underlying these changes are unknown. In this study, we investigated the effects of AGEs on the apoptosis of in vitro-cultured rat osteoblasts under high-glucose conditions and explored the underlying mechanisms of these effects. First, we cultured rat osteoblasts and determined the accumulation of AGEs in the culture medium under high-glucose conditions. Then, we cultured rat osteoblasts under a high glucose concentration (35 mM), a normal glucose concentration (5.5 mM), and a normal glucose concentration (5.5 mM) in the presence of AGEs. We examined the effects of high glucose and AGEs on the apoptosis of rat osteoblasts at different time points and further analyzed the activity and changes in the levels of procaspase-3, caspase-3, and the caspase-3 substrate poly ADP-ribose polymerase (PARP). Finally, we added sRAGE (soluble RAGE) (an AGE inhibitor) or DEVD (a caspase-3 inhibitor) to each culture group and examined apoptosis under each culture condition and the changes in the levels of procaspase-3, caspase-3, and its substrate PARP. The results showed that the high-glucose condition and the addition of AGEs increased the apoptosis of rat osteoblast cells and simultaneously increased the activity and quantity of caspase-3. These increases could be inhibited by the AGE inhibitor sRAGE or the caspase-3 inhibitor DEVD. The above results demonstrate that high-glucose conditions lead to the accumulation of AGEs and activation of the caspase-3 signaling pathway, resulting in the increased apoptosis of cultured rat osteoblast cells.

Death wins against life in a spatially extended model of the caspase-3/8 feedback loop.

PubMed

Daub, M; Waldherr, S; Allgöwer, F; Scheurich, P; Schneider, G

2012-01-01

Apoptosis is an important physiological process which enables organisms to remove unwanted or damaged cells. A mathematical model of the extrinsic pro-apoptotic signaling pathway has been introduced by Eissing et al. (2007) and a bistable behavior with a stable death state and a stable life state of the reaction system has been established. In this paper, we consider a spatial extension of the extrinsic pro-apoptotic signaling pathway incorporating diffusion terms and make a model-based, numerical analysis of the apoptotic switch in the spatial dimension. For the parameter regimes under consideration it turns out that for this model diffusion homogenizes rapidly the concentrations which afterward are governed by the original reaction system. The activation of effector-caspase 3 depends on the space averaged initial concentration of pro-caspase 8 and pro-caspase 3 at the beginning of the process. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Blocking caspase-3-dependent pathway preserves hair cells from salicylate-induced apoptosis in the guinea pig cochlea.

PubMed

Feng, Hao; Yin, Shi-Hua; Tang, An-Zhou

2011-07-01

In the present study, we aim to explore whether the caspase-3-dependent pathway is involved in the apoptotic cell death that occurs in the hair cells (HCs) of guinea pig cochlea following a salicylate treatment. Guinea pigs received sodium salicylate (Na-SA), at a dose of 200 mg·kg(-1)·d(-1) i.p., as a vehicle for 5 consecutive days. In some experiments, N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (zDEVD-FMK), a specific apoptosis inhibitor, was directly applied into the cochlea via the round window niche (RWN) prior to salicylate treatment for determination of caspase-3 activation. Alterations in auditory function were evaluated with auditory brainstem responses (ABR) thresholds. Caspase-3 activity was determined by measuring the proteolytic cleavage product of caspase-3 (N-terminated peptide substrate). DNA fragmentation within the nuclei was examined with a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. Ultrastructure variation in the target cell was assessed by electron microscopy (EM). Salicylate treatment initiated an obvious elevation in ABR thresholds with a maximum average shift of 60 dB sound pressure level (SPL), and caused significant apoptosis in both inner (IHCs) and outer (OHCs) hair cells resulted from an evident increasing in immunoreactivity to caspase-3 protease. Transmission electron microscopy (TEM) displayed chromatin condensation and nucleus margination accompanied by cell body shrinkage in the OHCs, but not in the IHCs. Scanning electron microscopy (SEM) showed breakdown, fusion, and loss in the stereociliary bundles at the apex of OHCs rather than IHCs. zDEVD-FMK pretreatment prior to salicylate injection substantially attenuated an expression of the apoptotic protease and protected HCs against apoptotic death, followed by a moderate relief in the thresholds of ABR, an alleviation in the submicroscopic structure was also identified. In particular, disorientation and insertion in the

Caspase enzymology and activation mechanisms.

PubMed

Mace, Peter D; Riedl, Stefan J; Salvesen, Guy S

2014-01-01

Apical caspases 8, 9, and 10 are only active as dimers. These dimers are unstable, and to characterize their activity they need to be maintained in vitro in a dimeric state. We provide updated methods for those looking to characterize various aspects of caspase function. We describe full methods for those looking to activate caspases in vitro using kosmotropic reagents, an essential step in characterizing upstream (apical) caspases. We detail methods for fusion of caspase domains to engineered dimerization domains as an alternative method to trigger regulated dimerization of caspases. We also describe methods to determine caspase activity profiles in cells and provide methods for studying the ability of SMAC-mimetic reagents to release inhibition of caspases by IAPs. © 2014 Elsevier Inc. All rights reserved.

Caspase-1 Inflammasome Activation Mediates Homocysteine-Induced Pyrop-Apoptosis in Endothelial Cells

PubMed Central

Xi, Hang; Zhang, Yuling; Xu, Yanjie; Yang, William Y; Jiang, Xiaohua; Sha, Xiaojin; Cheng, Xiaoshu; Wang, Jingfeng; Qin, Xuebin; Yu, Jun; Ji, Yong; Yang, Xiaofeng; Wang, Hong

2016-01-01

Rationale Endothelial injury is an initial mechanism mediating cardiovascular disease. Objective Here, we investigated the effect of hyperhomocysteinemia (HHcy) on programed cell death in endothelial cells (EC). Methods and Results We established a novel flow-cytometric gating method to define pyrotosis (Annexin V−/Propidium iodide+). In cultured human EC, we found that: 1). Hcy and Lipopolysaccharide (LPS) individually and synergistically induced inflammatory pyroptotic and non-inflammatory apoptotic cell death. 2). Hcy/LPS induced caspase-1 activation prior to caspase-8, -9, -3 activations. 3). Caspase-1/3 inhibitors rescued Hcy/LPS-induced pyroptosis/apoptosis, but caspase-8/9 inhibitors had differential rescue effect. 4). Hcy/LPS induced NLRP3 protein, caused NLRP3-containing inflammasome assembly, caspase-1 activation and IL-1β cleavage/activation. 5). Hcy/LPS elevated intracellular reactive oxidative species (ROS). 6). Intracellular oxidative gradient determined cell death destiny as intermediate intracellular ROS levels are associated with pyroptosis, whereas, high ROS corresponded to apoptosis. 7). Hcy/LPS induced mitochondrial membrane potential collapse and cytochrome-c release, and increased Bax/Bcl-2 ratio which were attenuated by antioxidants and caspase-1 inhibitor. 8). Antioxidants extracellular superoxide dismutase and catalase prevented Hcy/LPS-induced caspase-1 activation, mitochondrial dysfunction and pyroptosis/apoptosis. In cystathionine β-synthase deficient (Cbs−/−) mice, severe HHcy induced caspase-1 activation in isolated lung EC and caspase-1 expression in aortic endothelium, and elevated aortic caspase-1,9 protein/activity and Bax/Bcl-2 ratio in Cbs−/− aorta and HUVEC. Finally, Hcy-induced DNA fragmentation was reversed in caspase-1−/− EC. HHcy-induced aortic endothelial dysfunction was rescued in caspase-1−/− and NLRP3−/− mice. Conclusion HHcy preferentially induces EC pyroptosis via caspase-1-dependent

miR-98 and its host gene Huwe1 target Caspase-3 in Silica nanoparticles-treated male germ cells

NASA Astrophysics Data System (ADS)

Xu, Bo; Mao, Zhilei; Ji, Xiaoli; Yao, Mengmeng; Chen, Minjian; Zhang, Xuemei; Hang, Bo; Liu, Yi; Tang, Wei; Tang, Qiusha; Xia, Yankai

2015-08-01

Silica nanoparticles (NP) is one of the most commonly used nanomaterials with potential health hazards. However, the effects of Silica NP on germ cells and the underlying mechanisms are still unclear. In this study, GC-2 and TM-4, which are two different types of male germ cells were exposed to Silica NP for 24h, and then general cytotoxicity and multi-parameter cytotoxicity were evaluated. Our results showed that Silica NP could induce apoptosis in GC-2 cells. Transmission electron microscopy (TEM) results showed that Silica NP was localized in the lysosomes of GC-2 cells. High content screening (HCS) showed that Silica NP exposure could increased cell permeabilization and decreased mitochondrial membrane potential in GC-2 cells. The mRNA and protein levels of apoptosis markers (Bax, Caspase-3, Caspase-9) in GC-2 cells were significantly increased, while Bcl-2 was decreased. Accordingly, the expression level of miR-98, which can regulate Caspase-3, was significantly decreased. Huwe1, the host gene of miR-98, was positively associated with miR-98 expression after Silica NP exposure. Dual luciferase reporter assay suggested that miR-98 directly targets Caspase-3. These results suggest that Silica NP induces apoptosis via loss of mitochondrial membrane potential and Caspase-3 activation, while miR-98 plays key role in modulating this effect.

Dietary n-3 PUFAs augment caspase 8 activation in Staphylococcal aureus enterotoxin B stimulated T-cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gill, R.

Epidemiological studies have linked consumption of n-3 PUFAs with a variety of beneficial health benefits, particularly with respect to putative anti-inflammatory effects. Unfortunately, many of these results remain somewhat controversial because in most instances there has not been a linkage to specific molecular mechanisms. For instance, dietary exposure to low levels of mercury has been shown to be damaging to neural development, but concomitant ingestion of n-3 PUFAs as occurs during consumption of fish, has been shown to counteract the detrimental effects. As the mechanisms mediating the neurotoxicity of environmental mercury are not fully delineated, it is difficult to conceptualizemore » a testable molecular mechanism explaining how n-3 PUFAs negate its neurotoxic effects. However, environmental exposure to mercury also has been linked to increased autoimmunity. By way of a molecular understanding of this immuno-toxic association, disruption of CD95 signaling is well established as a triggering factor for autoimmunity, and we have previously shown that environmentally relevant in vitro and dietary exposures to mercury interfere with CD95 signaling. In particular we have shown that activation of caspase 8, as well as downstream activation of caspase 3, in response to CD95 agonist stimulation is depressed by mercury. More recently we have shown in vitro that the n-3 PUFA docosahexaenoic acid counteracts the negative effect of mercury on CD95 signaling by restoring caspase activity. We hypothesized that concomitant ingestion of n-3 PUFAs with mercury might be protective from the immuno-toxic effects of mercury, as it is with mercury's neuro-toxic effects, and in the case of immuno-toxicity this would be related to restoration of CD95 signal strength. We now show that dietary ingestion of n-3 PUFAs generally promotes CD95 signaling by upregulating caspase 8 activation. Apart from accounting for the ability of n-3 PUFAs to specifically counteract autoimmune

Correlation of Hsp110 expression with caspase-3 and -9 during apoptosis induced by in vivo embryonic exposition to retinoic acid or irradiation in early mouse craniofacial development.

PubMed

Gashegu, J; Vanmuylder, N; Philippson, C; Choa-Duterre, M; Rooze, M; Louryan, S

2006-05-01

To analyze the expression and role of three proteins (HSP110, caspase-3 and caspase-9) during craniofacial development. Seven pregnant C57Bl/6J mice received, by force-feeding at gestation day 9 (E9), 80 mg/kg of all-trans retinoic acid mixed to sesame oil. Seven pregnant NMRI mice received two grays irradiation at the same gestation day. Control mice of both strains (seven mice for each strain) were not submitted to any treatment. Embryos were obtained at various stages after exposition (3, 6, 12 and 24 h), fixed, dehydrated and embedded. Coronal sections (5 microm) were made. Slide staining occurred alternatively using anti-Hsp110, anti-caspase-3 and anti-caspase-9 immunohistochemistry. Expression of HSP110, caspase-3 and caspase-9 was found in cells of well-known locations of programmed cell death. After retinoic acid exposure, expressions were increased especially in neural crest cells of mandibular and hyoid arches. Quantification of positive cells shows that caspase-9 and Hsp110 were expressed before caspase-3. After irradiation, the expression of the three proteins quickly increased with a maximum 3 h after irradiation. For all three models of apoptosis (physiological, retinoic-induced and irradiation-induced) HSP110 positive cells were more numerous than caspase-3 positive cells. Caspase-3 positive cells were more numerous than caspase-9 positive cells especially in mesectodermal irradiation-induced apoptotic cells. The findings show a potential function of HSP110 in apoptosis during embryo development. Caspase-3-expressing cells are more numerous than cells expressing caspase-9, especially irradiation-induced apoptotic neural crest cells. This suggests that other caspases, still to be identified, may activate caspase-3 in this model.

AMPA receptor-mediated toxicity in oligodendrocyte progenitors involves free radical generation and activation of JNK, calpain and caspase 3.

PubMed

Liu, Hsueh-Ning; Giasson, Benoit I; Mushynski, Walter E; Almazan, Guillermina

2002-07-01

The molecular mechanisms underlying AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) receptor-mediated excitotoxicity were characterized in rat oligodendrocyte progenitor cultures. Activation of AMPA receptors, in the presence of cyclothiazide to selectively block desensitization, produced a massive Ca(2+) influx and cytotoxicity which were blocked by the antagonists CNQX and GYKI 52466. A role for free radical generation in oligodendrocyte progenitor cell death was deduced from three observations: (i) treatment with AMPA agonists decreased intracellular glutathione; (ii) depletion of intracellular glutathione with buthionine sulfoximine potentiated cell death; and (iii) the antioxidant N -acetylcysteine replenished intracellular glutathione and protected cultures from AMPA receptor-mediated toxicity. Cell death displayed some characteristics of apoptosis, including DNA fragmentation, chromatin condensation and activation of caspase-3 and c-Jun N-terminal kinase (JNK). A substrate of calpain and caspase-3, alpha-spectrin, was cleaved into characteristic products following treatment with AMPA agonists. In contrast, inhibition of either caspase-3 by DEVD-CHO or calpain by PD 150606 protected cells from excitotoxicity. Our results indicate that overactivation of AMPA receptors causes apoptosis in oligodendrocyte progenitors through mechanisms involving Ca(2+) influx, depletion of glutathione, and activation of JNK, calpain, and caspase-3.

Caspase vinyl sulfone small molecule inhibitors prevent axonal degeneration in human neurons and reverse cognitive impairment in Caspase-6-overexpressing mice.

PubMed

Pakavathkumar, Prateep; Noël, Anastasia; Lecrux, Clotilde; Tubeleviciute-Aydin, Agne; Hamel, Edith; Ahlfors, Jan-Eric; LeBlanc, Andrea C

2017-02-28

The activation of the aspartate-specific cysteinyl protease, Caspase-6, is proposed as an early pathogenic event of Alzheimer disease (AD) and Huntington's disease. Caspase-6 inhibitors could be useful against these neurodegenerative diseases but most Caspase-6 inhibitors have been exclusively studied in vitro or show acute liver toxicity in humans. Here, we assessed vinyl sulfone small molecule peptide caspase inhibitors for potential use in vivo. The IC 50 of NWL vinyl sulfone small molecule caspase inhibitors were determined on Caspase-1 to 10, and Caspase-6-transfected human colon carcinoma HCT116 cells. Inhibition of Caspase-6-mediated axonal degeneration was assessed in serum-deprived or amyloid precursor protein-transfected primary human CNS neurons. Cellular toxicity was measured by phase contrast microscopy, mitochondrial and lactate dehydrogenase colorimetric activity assays, or flow cytometry. Caspase inhibition was measured by fluorogenic activity assays, fluorescence microscopy, and western blot analyses. The effect of inhibitors on age-dependent cognitive deficits in Caspase-6 transgenic mice was assessed by the novel object recognition task. Liquid chromatography coupled to tandem mass spectrometry assessed the blood-brain barrier permeability of inhibitors in Caspase-6 mice. Vinyl sulfone NWL-117 caspase inhibitor has a higher selectivity against Caspase-6, -4, -8, -9, and -10 whereas NWL-154 has higher selectivity against Caspase-6, -8, and -10. The half-maximal inhibitory concentrations (IC 50 ) of NWL-117 and NWL-154 is 192 nM and 100 nM against Caspase-6 in vitro, and 4.82 μM and 3.63 μM in Caspase-6-transfected HCT116 cells, respectively. NWL inhibitors are not toxic to HCT116 cells or to human primary neurons. NWL-117 and NWL-154 inhibit serum deprivation-induced Caspase-6 activity and prevent amyloid precursor protein-mediated neurite degeneration in human primary CNS neurons. NWL-117 crosses the blood brain barrier and reverses age

Caspase-9 Mediates Photoreceptor Death After Blunt Ocular Trauma

PubMed Central

Blanch, Richard J.; Ahmed, Zubair; Thompson, Adam R.; Akpan, Nsikan; Snead, David R. J.; Berry, Martin; Troy, Carol M.; Scott, Robert A. H.; Logan, Ann

2014-01-01

Purpose. Ocular trauma is common in civilian and military populations. Commotio retinae involves acute disruption of photoreceptor outer segments after blunt ocular trauma, with subsequent photoreceptor apoptosis causing permanent visual impairment. The mechanisms of photoreceptor death in commotio retinae have not previously been described, although caspase-dependent death is important in other nontraumatic retinal degenerations. We assessed the role of caspase-9 as a mediator of photoreceptor death in a rat model of ballistic ocular trauma causing commotio retinae. Methods. Bilateral commotio retinae was induced in rats by ballistic ocular trauma. Caspase-9 activity was assessed by immunohistochemistry, Western blotting, and bVAD-fmk active caspase capture. Caspase-9 was inhibited by unilateral intravitreal injection of highly specific X-linked inhibitor of apoptosis (IAP) baculoviral IAP repeat 3 (XBIR3) domain linked to the cell transduction peptide penetratin 1 (Pen-1) after ballistic injury, and the affected eyes were compared with control eyes treated with Pen-1 injection alone, and retinal function was assessed by electroretinogram a-wave amplitude and photoreceptor survival by outer nuclear layer thickness. Results. Increased levels of cleaved caspase-9 were shown in photoreceptors 5 hours after injury, and catalytically active full-length caspase-9 was isolated from retinas. Photoreceptor death after commotio retinae was reduced by caspase-9 inhibition by using Pen-1–XBIR3, and electroretinographic measurements of photoreceptor function was preserved, providing structural and functional neuroprotection. Conclusions. The time course of caspase-9 activation and the neuroprotective effects of inhibition suggest that caspase-9 initiates cell death in a proportion of photoreceptors after blunt ocular trauma and that an intravitreally delivered biologic inhibitor may be an effective translational treatment strategy. PMID:25190658

Comparison of activated caspase detection methods in the gentamicin-treated chick cochlea

PubMed Central

Kaiser, Christina L.; Chapman, Brittany J.; Guidi, Jessica L.; Terry, Caitlin E.; Mangiardi, Dominic A.; Cotanche, Douglas A.

2008-01-01

Aminoglycoside antibiotics induce caspase-dependent apoptotic death in cochlear hair cells. Apoptosis, a regulated form of cell death, can be induced by many stressors, which activate signaling pathways that result in the controlled dismantling of the affected cell. The caspase family of proteases is activated in the apoptotic signaling pathway and is responsible for cellular destruction. The initiator caspase-9 and the effector caspase-3 are both activated in chick cochlear hair cells following aminoglycoside exposure. We have analyzed caspase activation in the avian cochlea during gentamicin-induced hair cell death to compare two different methods of caspase detection: caspase antibodies and CaspaTag kits. Caspase antibodies bind to the cleaved activated form of caspase-9 or caspase-3 in specific locations in fixed tissue. CaspaTag is a fluorescent inhibitor that binds to a reactive cysteine residue on the large subunit of the caspase heterodimer in unfixed tissue. To induce cochlear hair cell loss, 1-2 week-old chickens received a single injection of gentamicin (300 mg/kg). Chicks were sacrificed 24, 30, 42, 48, 72, or 96 h after injection. Cochleae were dissected and labeled for activated caspase-9 or caspase-3 using either caspase-directed antibodies or CaspaTag kits. Ears were co-labeled with either phalloidin or myosin VI to visualize hair cells and to determine the progression of cochlear damage. The timing of caspase activation was similar for both assays; however, caspase-9 and caspase-3 antibodies labeled only those cells currently undergoing apoptotic cell death. Conversely, CaspaTag-labeled all the cells that have undergone apoptotic cell death and ejection from the sensory epithelium, in addition to those that are currently in the cell death process. This makes CaspaTag ideal for showing an overall pattern or level of cell death over a period of time, while caspase antibodies provide a snapshot of cell death at a specific time point. PMID:18487027

Flavonoids of Rosa roxburghii Tratt exhibit radioprotection and anti-apoptosis properties via the Bcl-2(Ca(2+))/Caspase-3/PARP-1 pathway.

PubMed

Xu, Ping; Cai, Xinhua; Zhang, Wenbo; Li, Yana; Qiu, Peiyong; Lu, Dandan; He, Xiaoyang

2016-10-01

The objective of our study was to assess the radioprotective effect of flavonoids extracted from Rosa roxburghii Tratt (FRT) and investigate the role of Bcl-2(Ca(2+))/Caspase-3/PARP-1 pathway in radiation-induced apoptosis. Cells and mice were exposed to (60)Co γ-rays at a dose of 6 Gy. The radiation treatment induced significant effects on tissue pathological changes, apoptosis, Ca(2+), ROS, DNA damage, and expression levels of Bcl-2, Caspase-3 (C-Caspase-3), and PARP-1. The results showed that FRT acted as an antioxidant, reduced DNA damage, corrected the pathological changes of the tissue induced by radiation, promoted the formation of spleen nodules, resisted sperm aberration, and protected the thymus. FRT significantly reduced cell apoptosis compared with the irradiation group. The expression of Ca(2+) and C-Caspase-3 was decreased after FRT treatment compared with the radiation-treated group. At the same time, expression of prototype PARP-1 and Bcl-2 increased, leading to a decrease in the percentage of apoptosis cells in FRT treatment groups. We conclude that FRT acts as a radioprotector. Apoptosis signals were activated via the Bcl-2(Ca(2+))/Caspase-3/PARP-1 pathway in irradiated cells and FRT inhibited this pathway of apoptosis by down-regulation of C-Caspase-3 and Ca(2+) and up-regulation of prototype PARP-1 and Bcl-2.

NLRP3 Inflammasome and Caspase-1/11 Pathway Orchestrate Different Outcomes in the Host Protection Against Trypanosoma cruzi Acute Infection.

PubMed

Paroli, Augusto F; Gonzalez, Patricia V; Díaz-Luján, Cintia; Onofrio, Luisina I; Arocena, Alfredo; Cano, Roxana C; Carrera-Silva, Eugenio A; Gea, Susana

2018-01-01

Infection with protozoan parasite Trypanosoma cruzi results in activation of nucleotide-binding domain and leucine-rich repeat containing receptors (NLRs). NLR activation leads to inflammasome formation, the activation of caspase-1, and the subsequent cleavage of IL-1β and IL-18. Considering that inflammasome activation and IL-1β induction by macrophages are key players for an appropriate T cell response, we investigated the relevance of NLR pyrin domain-containing 3 (NLRP3) and caspase-1/11 to elucidate their roles in the induction of different T cell phenotypes and the relationship with parasite load and hepatic inflammation during T. cruzi- Tulahuen strain acute infection. We demonstrated that infected nlrp3-/- and C57BL/6 wild type (WT) mice exhibited similar parasitemia and survival, although the parasite load was higher in the livers of nlrp3-/- mice than in those of WT mice. Increased levels of transaminases and pro-inflammatory cytokines were found in the plasma of WT and nlrp3-/- mice indicating that NLRP3 is dispensable to control the parasitemia but it is required for a better clearance of parasites in the liver. Importantly, we have found that NLRP3 and caspase-1/11-deficient mice differentially modulate T helper (Th1, Th2, and Th17) and cytotoxic T lymphocyte phenotypes. Strikingly, caspase-1/11-/- mice showed the most dramatic reduction in the number of IFN-γ- and IL-17-producing CD4+ and CD8+ T cells associated with higher parasitemia and lower survival. Additionally, caspase-1/11-/- mice demonstrated significantly reduced liver inflammation with the lowest alanine aminotransferase (ALT) levels but the highest hepatic parasitic load. These results unequivocally demonstrate that caspase-1/11 pathway plays an important role in the induction of liver adaptive immunity against this parasite infection as well as in hepatic inflammation.

Elevated extracellular [K+] inhibits death-receptor- and chemical-mediated apoptosis prior to caspase activation and cytochrome c release.

PubMed Central

Thompson, G J; Langlais, C; Cain, K; Conley, E C; Cohen, G M

2001-01-01

Efflux of intracellular K(+) and cell shrinkage are features of apoptosis in many experimental systems, and a regulatory role has been proposed for cytoplasmic [K(+)] in initiating apoptosis. We have investigated this in both death-receptor-mediated and chemical-induced apoptosis. Using Jurkat T cells pre-loaded with the K(+) ion surrogate (86)Rb(+), we have demonstrated an efflux of intracellular K(+) during apoptosis that was concomitant with, but did not precede, other apoptotic changes, including phosphatidylserine externalization, mitochondrial depolarization and cell shrinkage. To further clarify the role of K(+) ions in apoptosis, cytoprotection by elevated extracellular [K(+)] was studied. Induction of apoptosis by diverse death-receptor and chemical stimuli in two cell lines was inhibited prior to phosphatidylserine externalization, mitochondrial depolarization, cytochrome c release and caspase activation. Using a cell-free system, we have demonstrated a novel mechanism by which increasing [K(+)] inhibited caspase activation. In control dATP-activated lysates, Apaf-1 oligomerized to a biologically active caspase processing approximately 700 kDa complex and an inactive approximately 1.4 MDa complex. Increasing [K(+)] inhibited caspase activation by preventing formation of the approximately 700 kDa complex, but not of the inactive complex. Thus intracellular and extracellular [K(+)] markedly affect caspase activation and the initiation of apoptosis induced by both death-receptor ligation and chemical stress. PMID:11415444

Intrinsic-mediated caspase activation is essential for cardiomyocyte hypertrophy

PubMed Central

Putinski, Charis; Abdul-Ghani, Mohammad; Stiles, Rebecca; Brunette, Steve; Dick, Sarah A.; Fernando, Pasan; Megeney, Lynn A.

2013-01-01

Cardiomyocyte hypertrophy is the cellular response that mediates pathologic enlargement of the heart. This maladaptation is also characterized by cell behaviors that are typically associated with apoptosis, including cytoskeletal reorganization and disassembly, altered nuclear morphology, and enhanced protein synthesis/translation. Here, we investigated the requirement of apoptotic caspase pathways in mediating cardiomyocyte hypertrophy. Cardiomyocytes treated with hypertrophy agonists displayed rapid and transient activation of the intrinsic-mediated cell death pathway, characterized by elevated levels of caspase 9, followed by caspase 3 protease activity. Disruption of the intrinsic cell death pathway at multiple junctures led to a significant inhibition of cardiomyocyte hypertrophy during agonist stimulation, with a corresponding reduction in the expression of known hypertrophic markers (atrial natriuretic peptide) and transcription factor activity [myocyte enhancer factor-2, nuclear factor kappa B (NF-κB)]. Similarly, in vivo attenuation of caspase activity via adenoviral expression of the biologic effector caspase inhibitor p35 blunted cardiomyocyte hypertrophy in response to agonist stimulation. Treatment of cardiomyocytes with procaspase 3 activating compound 1, a small-molecule activator of caspase 3, resulted in a robust induction of the hypertrophy response in the absence of any agonist stimulation. These results suggest that caspase-dependent signaling is necessary and sufficient to promote cardiomyocyte hypertrophy. These results also confirm that cell death signal pathways behave as active remodeling agents in cardiomyocytes, independent of inducing an apoptosis response. PMID:24101493

Natural Indoles, Indole-3-Carbinol (I3C) and 3,3’-Diindolylmethane (DIM), Attenuate Staphylococcal Enterotoxin B-Mediated Liver Injury by Downregulating miR-31 Expression and Promoting Caspase-2-Mediated Apoptosis

PubMed Central

Busbee, Philip B.; Nagarkatti, Mitzi; Nagarkatti, Prakash S.

2015-01-01

Staphylococcal enterotoxin B (SEB) is a potent superantigen capable of inducing inflammation characterized by robust immune cell activation and proinflammatory cytokine release. Exposure to SEB can result in food poisoning as well as fatal conditions such as toxic shock syndrome. In the current study, we investigated the effect of natural indoles including indole-3-carbinol (I3C) and 3,3’-diindolylmethane (DIM) on SEB-mediated liver injury. Injection of SEB into D-galactosamine-sensitized female C57BL/6 mice resulted in liver injury as indicated by an increase in enzyme aspartate transaminase (AST) levels, induction of inflammatory cytokines, and massive infiltration of immune cells into the liver. Administration of I3C and DIM (40mg/kg), by intraperitonal injection, attenuated SEB-induced acute liver injury, as evidenced by decrease in AST levels, inflammatory cytokines and cellular infiltration in the liver. I3C and DIM triggered apoptosis in SEB-activated T cells primarily through activation of the intrinsic mitochondrial pathway. In addition, inhibitor studies involving caspases revealed that I3C and DIM-mediated apoptosis in these activated cells was dependent on caspase-2 but independent of caspase-8, 9 and 3. In addition, I3C and DIM caused a decrease in Bcl-2 expression. Both compounds also down-regulated miR-31, which directly targets caspase-2 and influences apoptosis in SEB-activated cells. Our data demonstrate for the first time that indoles can effectively suppress acute hepatic inflammation caused by SEB and that this may be mediated by decreased expression of miR-31 and consequent caspase-2-dependent apoptosis in T cells. PMID:25706292

Caspase-3 mediated release of SAC domain containing fragment from Par-4 is necessary for the sphingosine-induced apoptosis in Jurkat cells

PubMed Central

2013-01-01

Background Prostate apoptosis response-4 (Par-4) is a tumor-suppressor protein that selectively activates and induces apoptosis in cancer cells, but not in normal cells. The cancer specific pro-apoptotic function of Par-4 is encoded in its centrally located SAC (Selective for Apoptosis induction in Cancer cells) domain (amino acids 137–195). The SAC domain itself is capable of nuclear entry, caspase activation, inhibition of NF-κB activity, and induction of apoptosis in cancer cells. However, the precise mechanism(s) of how the SAC domain is released from Par-4, in response to apoptotic stimulation, is not well explored. Results In this study, we demonstrate for the first time that sphingosine (SPH), a member of the sphingolipid family, induces caspase-dependant cleavage of Par-4, leading to the release of SAC domain containing fragment from it. Par-4 is cleaved at the EEPD131G site on incubation with caspase-3 in vitro, and by treating cells with several anti-cancer agents. The caspase-3 mediated cleavage of Par-4 is blocked by addition of the pan-caspase inhibitor z-VAD-fmk, caspase-3 specific inhibitor Ac-DEVD-CHO, and by introduction of alanine substitution for D131 residue. Moreover, suppression of SPH-induced Akt dephosphorylation also abrogated the caspase dependant cleavage of Par-4. Conclusion Evidence provided here shows that Par-4 is cleaved by caspase-3 during SPH-induced apoptosis. Cleavage of Par-4 leads to the generation of SAC domain containing fragment which may possibly be essential and sufficient to induce or augment apoptosis in cancer cells. PMID:23442976

Chronic sleep restriction induces changes in the mandibular condylar cartilage of rats: roles of Akt, Bad and Caspase-3.

PubMed

Zhu, Yong; Wu, Gaoyi; Zhu, Guoxiong; Ma, Chuan; Zhao, Huaqiang

2014-01-01

The aim of the present study was to observe changes in the temporomandibular joint (TMJ) of rats that had been subjected to chronic sleep restriction and to investigate whether Akt, Bad and Caspase3 play a role in the mechanism underlying the changes. One hundred and eighty male Wistar rats were randomly divided into three groups (n = 60 in each): cage control group, large-platform control group, and sleep restriction group. Each group was divided into three subgroups (n = 20 in each) of three different time points (7, 14 and 21 days), respectively. The modified multiple platform method was used to induce chronic sleep restriction. The TMJ tissue histology was studied by staining with haematoxylin and eosin. The expression of Akt, p-Aktser473, Bad, p-Badser136 and Caspase3 proteins was detected by immunohistochemistry and western blotting. The expression of Akt, Bad and Caspase3 mRNAs was measured by real-time quantitative polymerase chain reaction (RT-qPCR). Compared with the large-platform and cage control groups, condylar cartilage pathological alterations were found in the sleep restriction group. There were significantly decreased expression levels of Akt, p-Aktser473 and p-Badser136 and significantly increased expression levels of Bad and Caspase3 after sleep restriction. These data suggest that sleep restriction may induce pathological alterations in the condylar cartilage of rats. Alterations in Akt, Bad and Caspase3 may be associated with the potential mechanism by which chronic sleep restriction influences the condylar cartilage.

Chronic sleep restriction induces changes in the mandibular condylar cartilage of rats: roles of Akt, Bad and Caspase-3

PubMed Central

Zhu, Yong; Wu, Gaoyi; Zhu, Guoxiong; Ma, Chuan; Zhao, Huaqiang

2014-01-01

Aims: The aim of the present study was to observe changes in the temporomandibular joint (TMJ) of rats that had been subjected to chronic sleep restriction and to investigate whether Akt, Bad and Caspase3 play a role in the mechanism underlying the changes. Main methods: One hundred and eighty male Wistar rats were randomly divided into three groups (n = 60 in each): cage control group, large-platform control group, and sleep restriction group. Each group was divided into three subgroups (n = 20 in each) of three different time points (7, 14 and 21 days), respectively. The modified multiple platform method was used to induce chronic sleep restriction. The TMJ tissue histology was studied by staining with haematoxylin and eosin. The expression of Akt, p-Aktser473, Bad, p-Badser136 and Caspase3 proteins was detected by immunohistochemistry and western blotting. The expression of Akt, Bad and Caspase3 mRNAs was measured by real-time quantitative polymerase chain reaction (RT-qPCR). Key findings: Compared with the large-platform and cage control groups, condylar cartilage pathological alterations were found in the sleep restriction group. There were significantly decreased expression levels of Akt, p-Aktser473 and p-Badser136 and significantly increased expression levels of Bad and Caspase3 after sleep restriction. Significance: These data suggest that sleep restriction may induce pathological alterations in the condylar cartilage of rats. Alterations in Akt, Bad and Caspase3 may be associated with the potential mechanism by which chronic sleep restriction influences the condylar cartilage. PMID:25356113

Caspase-3/-8/-9, Bax and Bcl-2 expression in the cerebellum, lymph nodes and leukocytes of dogs naturally infected with canine distemper virus.

PubMed

Del Puerto, H L; Martins, A S; Moro, L; Milsted, A; Alves, F; Braz, G F; Vasconcelos, A C

2010-01-26

Canine distemper is an immunosuppressive disease caused by the canine distemper virus (CDV). Pathogenesis mainly involves the central nervous system and immunosuppression. Dogs naturally infected with CDV develop apoptotic cells in lymphoid tissues and the cerebellum, but this apoptotic mechanism is not well characterized. To better understand this process, we evaluated the expression of Bax, Bcl-2, and caspase-3, -8 and -9, by evaluating mRNA levels in the peripheral blood, lymph nodes and cerebellum of CDV-infected (CDV+) and uninfected (CDV-) dogs by real-time polymerase chain reaction (PCR). Blood samples from 12 CDV+ and 8 CDV- dogs, diagnosed by reverse transcription-PCR, were subjected to hematological analysis and apoptotic gene expression was evaluated using real-time-PCR. Tissues from the cerebellum and lymph nodes of four CDV+ and three CDV-dogs were also subjected to real time-PCR. No significant differences were found between CDV+ and CDV- dogs in the hemotological results or in the expression of caspase-3, -8, -9, Bax, and Bcl-2 in the peripheral blood. However, expression of Bax, caspase-3, -8 and -9 was significantly higher in the cerebellum of CDV+ compared to CDV- dogs. Expression of caspase-3 and -8 was significantly higher in the lymph nodes of CDV+ compared to CDV- dogs. We concluded that infection with CDV induces apoptosis in the cerebellum and lymph nodes in different ways. Lymph node apoptosis apparently occurs via caspase-3 activation, through the caspase-8 pathway, and cerebellum apoptosis apparently occurs via caspase-3 activation, through the caspase-8 and mitochondrial pathways.

[Effect of Electroacupuncture on Cerebro-cortex Caspase-3 Expression and Blood Lipid Levels in Hyperlipemia Rats with Cerebral Ischemia].

PubMed

Wang, Zhuo-Yu; Ma, Jia-Jia; Guan, Han-Yu; Tian, Yao; Ren, Xiu-Jun; Ma, Hui-Fang

2017-04-25

To observe the effect of electroacupuncture (EA) stimulation of "Fenglong" (ST 40), "Sanyinjiao" (SP 6) plus manual acupuncture (MA) stimulation of "Shuigou" (GV 26) and "Baihui" (GV 20) on Caspase-3 protein expression in the cerebral cortex of rats with hyperlipemia and cerebral ischemia(HL-CI),so as to reveal its mechanisms underlying improvement of HL-CI. Forty-five rats were randomly divided into normal control,sham operation,model,EA group I(EA+MA was given for 14 days, i.e., 7 days before CI, and 7 days more after HL-CI)and EA group Ⅱ (EA+MA was given for only 7 days after HL-CI),with 9 rats being in each group. The HL-CI model was established by feeding the animals with high fat forage for 6 weeks and then making an occlusion of the unilateral middle cerebral artery by regional application of quantitative paper adsorbing 50% FeCl 3 solution (10 μL). Rats of the sham operation group were treated with the same procedures only without application of FeCl 3 solution. For rats of the EA group I,EA (1-3 mA, 2 Hz/100 Hz) was applied to bilateral acupoints SP 6 and ST 40 (for 20 min),and MA stimulation applied to GV 26 and GV 20. EA was conducted once daily for 7 days after 6 weeks' high fat fo-rage feeding, and EA+MA intervention was conducted once daily for 7 days after CI modeling. For rats in the EA group Ⅱ, EA+MA was applied to the same 4 acupoints once a day for 7 days only after CI modeling. The neurological impairment was assessed by Zea Longa's scoring. The blood sample was taken from the abdominal aorta for measuring the contents of serum cholesterol (CHO),triglyceride (TG) and high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C). Pathological changes of the cerebral cortex were observed after H.E. staining, and the expression of cerebro-cortex Caspase-3 was analyzed by immunohistochemistry. Following modeling,the neurological score,CHO, TG and LDL-C contents, and the number of Caspase-3 positive cells as well

Methylene Blue Inhibits Caspases by Oxidation of the Catalytic Cysteine.

PubMed

Pakavathkumar, Prateep; Sharma, Gyanesh; Kaushal, Vikas; Foveau, Bénédicte; LeBlanc, Andrea C

2015-09-24

Methylene blue, currently in phase 3 clinical trials against Alzheimer Disease, disaggregates the Tau protein of neurofibrillary tangles by oxidizing specific cysteine residues. Here, we investigated if methylene blue can inhibit caspases via the oxidation of their active site cysteine. Methylene blue, and derivatives, azure A and azure B competitively inhibited recombinant Caspase-6 (Casp6), and inhibited Casp6 activity in transfected human colon carcinoma cells and in serum-deprived primary human neuron cultures. Methylene blue also inhibited recombinant Casp1 and Casp3. Furthermore, methylene blue inhibited Casp3 activity in an acute mouse model of liver toxicity. Mass spectrometry confirmed methylene blue and azure B oxidation of the catalytic Cys163 cysteine of Casp6. Together, these results show a novel inhibitory mechanism of caspases via sulfenation of the active site cysteine. These results indicate that methylene blue or its derivatives could (1) have an additional effect against Alzheimer Disease by inhibiting brain caspase activity, (2) be used as a drug to prevent caspase activation in other conditions, and (3) predispose chronically treated individuals to cancer via the inhibition of caspases.

Methylene Blue Inhibits Caspases by Oxidation of the Catalytic Cysteine

PubMed Central

Pakavathkumar, Prateep; Sharma, Gyanesh; Kaushal, Vikas; Foveau, Bénédicte; LeBlanc, Andrea C.

2015-01-01

Methylene blue, currently in phase 3 clinical trials against Alzheimer Disease, disaggregates the Tau protein of neurofibrillary tangles by oxidizing specific cysteine residues. Here, we investigated if methylene blue can inhibit caspases via the oxidation of their active site cysteine. Methylene blue, and derivatives, azure A and azure B competitively inhibited recombinant Caspase-6 (Casp6), and inhibited Casp6 activity in transfected human colon carcinoma cells and in serum-deprived primary human neuron cultures. Methylene blue also inhibited recombinant Casp1 and Casp3. Furthermore, methylene blue inhibited Casp3 activity in an acute mouse model of liver toxicity. Mass spectrometry confirmed methylene blue and azure B oxidation of the catalytic Cys163 cysteine of Casp6. Together, these results show a novel inhibitory mechanism of caspases via sulfenation of the active site cysteine. These results indicate that methylene blue or its derivatives could (1) have an additional effect against Alzheimer Disease by inhibiting brain caspase activity, (2) be used as a drug to prevent caspase activation in other conditions, and (3) predispose chronically treated individuals to cancer via the inhibition of caspases. PMID:26400108

Interferon-induced RIP1/RIP3-mediated necrosis requires PKR and is licensed by FADD and caspases

PubMed Central

Thapa, Roshan J.; Nogusa, Shoko; Chen, Peirong; Maki, Jenny L.; Lerro, Anthony; Andrake, Mark; Rall, Glenn F.; Degterev, Alexei; Balachandran, Siddharth

2013-01-01

Interferons (IFNs) are cytokines with powerful immunomodulatory and antiviral properties, but less is known about how they induce cell death. Here, we show that both type I (α/β) and type II (γ) IFNs induce precipitous receptor-interacting protein (RIP)1/RIP3 kinase-mediated necrosis when the adaptor protein Fas-associated death domain (FADD) is lost or disabled by phosphorylation, or when caspases (e.g., caspase 8) are inactivated. IFN-induced necrosis proceeds via progressive assembly of a RIP1–RIP3 “necrosome” complex that requires Jak1/STAT1-dependent transcription, but does not need the kinase activity of RIP1. Instead, IFNs transcriptionally activate the RNA-responsive protein kinase PKR, which then interacts with RIP1 to initiate necrosome formation and trigger necrosis. Although IFNs are powerful activators of necrosis when FADD is absent, these cytokines are likely not the dominant inducers of RIP kinase-driven embryonic lethality in FADD-deficient mice. We also identify phosphorylation on serine 191 as a mechanism that disables FADD and collaborates with caspase inactivation to allow IFN-activated necrosis. Collectively, these findings outline a mechanism of IFN-induced RIP kinase-dependent necrotic cell death and identify FADD and caspases as negative regulators of this process. PMID:23898178

Caspase Activation in Fetal Rat Brain Following Experimental Intrauterine Inflammation

PubMed Central

Sharangpani, Aditi; Takanohashi, Asako; Bell, Michael J.

2009-01-01

Intrauterine inflammation has been implicated in developmental brain injuries, including the development of periventricular leukomalacia (PVL) and cerebral palsy (CP). Previous studies in our rat model of intrauterine inflammation demonstrated apoptotic cell death in fetal brains within the first 5 days after lipopolysaccharide (LPS) administration to mothers and eventual dysmyelination. Cysteine-containing, aspartate-specific proteases, or caspases, are proteins involved with apoptosis through both intracellular (intrinsic pathway) and extracellular (extrinsic pathway) mechanisms. We hypothesized that cell death in our model would occur mainly via activation of the extrinsic pathway. We further hypothesized that Fas, a member of the tumor necrosis factor receptor (TNFR) superfamily, would be increased and the death inducing signaling complex (DISC) would be detectable. Pregnant rats were injected intracervically with LPS at E15 and immunoblotting, immunohistochemical and immunoprecipitation analyses were performed. The presence of the activated form of the effector caspase (caspase-3) was observed 24 h after LPS administration. Caspase activity assays demonstrated rapid increases in (i) caspases-9 and -10 within 1 h, (ii) caspase-8 at 2 h and (iii) caspase-3 at 4 h. At 24 h after LPS, activated caspase-3+/Fas+ cells were observed within the developing white matter. Lastly, the DISC complex (caspase-8, Fas and Fas-associated Death Domain (FADD)) was observed within 30 min by immunoprecipitation. Apoptosis in our model occurs via both extrinsic and intrinsic pathways, and activation of Fas may play a role. Understanding the mechanisms of cell death in models of intrauterine inflammation may affect development of future strategies to mitigate these injuries in children. PMID:18289516

Coactivation of the PI3K/Akt and ERK signaling pathways in PCB153-induced NF-κB activation and caspase inhibition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Changjiang; Key Lab of Birth Defects and Reproductive Health of National Health and Family Planning Commission, Chongqing Population and Family Planning Science and Technology Research Institute, Chongqing 400020; Yang, Jixin

2014-06-15

Polychlorinated biphenyls (PCBs) are a group of persistent and widely distributed environmental pollutants that have various deleterious effects, e.g., neurotoxicity, endocrine disruption and reproductive abnormalities. In order to verify the hypothesis that the PI3K/Akt and MAPK pathways play important roles in hepatotoxicity induced by PCBs, Sprague–Dawley (SD) rats were dosed with PCB153 intraperitoneally at 0, 4, 16 and 32 mg/kg for five consecutive days; BRL cells (rat liver cell line) were treated with PCB153 (0, 1, 5, and 10 μM) for 24 h. Results indicated that the PI3K/Akt and ERK pathways were activated in vivo and in vitro after exposuremore » to PCB153, and protein levels of phospho-Akt and phospho-ERK were significantly increased. Nuclear factor-κB (NF-κB) activation and caspase-3, -8 and -9 inhibition caused by PCB153 were also observed. Inhibiting the ERK pathway significantly attenuated PCB153-induced NF-κB activation, whereas inhibiting the PI3K/Akt pathway hardly influenced phospho-NF-κB level. However, inhibiting the PI3K/Akt pathway significantly elevated caspase-3, -8 and -9 activities, while the ERK pathway only synergistically regulated caspase-9. Proliferating cell nuclear antigen (PCNA), a reliable indicator of cell proliferation, was also induced. Moreover, PCB153 led to hepatocellular hypertrophy and elevated liver weight. Taken together, PCB153 leads to aberrant proliferation and apoptosis of hepatocytes through NF-κB activation and caspase inhibition, and coactivated PI3K/Akt and ERK pathways play critical roles in PCB153-induced hepatotoxicity. - Highlights: • PCB153 led to hepatotoxicity through NF-κB activation and caspase inhibition. • The PI3K/Akt and ERK pathways were coactivated in vivo and in vitro by PCB153. • The ERK pathway regulated levels of phospho-NF-κB and caspase-9. • The PI3K/Akt pathway regulated levels of caspase-3, -8 and -9.« less

WISP-3/CCN6 inhibits apoptosis by regulating caspase pathway after hyperoxia in lung epithelial cells.

PubMed

Wei, Shuquan; Wang, Kangwei; Zhao, Zhuxiang; Huang, Xiaomei; Tang, Wanna; Zhao, Ziwen

2018-06-16

Cell death is a normal phenomenon in the course of biological development, moreover, which is also a prominent feature in lung exposed to hyperoxia. Severe hypoxia occurs in ALI/ARDS patients, who generally require high concentration oxygen therapy assisted by mechanical ventilation. Nevertheless, high oxygen can cause excessive reactive oxygen species (ROS), leading to apoptosis in lung epithelial cells, which has been reported in our previous study. Herein, the correlation between increments of ROS and CCN6 expression was negative in CCN6-mediated the mitochondria dependent, intrinsic apoptotic pathway. Our latest research explained that CCN6 can inhibit caspase-8 mediated extrinsic apoptotic pathway to protect cells from hyperoxia-induced apoptosis. As demonstrated by Western Blot Analysis, Caspase 8 cleavage and Caspase 3 cleavage in CCN6-depleted cells exceeded the control group treated with high oxygen (48 h). And deletion of CCN6 enhanced caspase-8 activation after hyperoxia shown by Flow Cytometry. Although, it is unclear how CCN6 participated in the regulation of apoptotic pathways, the future targeted therapy drugs inhibiting CCN6 may be useful in the treatment of ALI/ARDS. Copyright © 2018. Published by Elsevier B.V.

Caspase blockade induces RIP3-mediated programmed necrosis in Toll-like receptor-activated microglia.

PubMed

Kim, S J; Li, Jianrong

2013-07-11

Microglia are the resident immune cells in the central nervous system and key players against pathogens and injury. However, persistent microglial activation often exacerbates pathological damage and has been implicated in many neurological diseases. Despite their pivotal physiological and pathophysiological roles, how the survival and death of activated microglia is regulated remains poorly understood. We report here that microglia activated through Toll-like receptors (TLRs) undergo RIP1/RIP3-dependent programmed necrosis (necroptosis) when exposed to the pan caspase inhibitor zVAD-fmk. Although zVAD-fmk and the caspase-8 inhibitor IETD-fmk had no effect on unstimulated primary microglia, they markedly sensitized microglia to TLR1/2,3,4,7/8 ligands or TNF treatment, triggering programmed necrosis that was completely blocked by R1P1 kinase inhibitor necrostatin-1. Interestingly, necroptosis induced by TLR ligands and zVAD was restricted to microglial cells and was not observed in astrocytes, neurons or oligodendrocytes even though they are known to express certain TLRs. Deletion of genes encoding TNF or TNFR1 failed to prevent lipopolysaccharide- and poly(I:C)-induced microglial necroptosis, unveiling a TNF-independent programmed necrosis pathway in TLR3- and TLR4-activated microglia. Microglia from mice lacking functional TRIF were fully protected against TLR3/4 activation and zVAD-fmk-induced necrosis, and genetic deletion of rip3 also prevented microglia necroptosis. Activation of c-jun N-terminal kinase and generation of specific reactive oxygen species were downstream signaling events required for microglial cell death execution. Taken together, this study reveals a robust RIP3-dependent necroptosis signaling pathway in TLR-activated microglia upon caspase blockade and suggests that TLR signaling and programmed cell death pathways are closely linked in microglia, which could contribute to neuropathology and neuroinflammation when dysregulated.

Caspase-8 modulates Dectin-1 and CR3 driven IL-1β production in response to β-glucans and the fungal pathogen, Candida albicans1

PubMed Central

Ganesan, Sandhya; Rathinam, Vijay A. K.; Bossaller, Lukas; Army, Kelly; Kaiser, William J.; Mocarski, Edward S.; Dillon, Christopher P.; Green, Douglas R.; Mayadas, Tanya N.; Levitz, Stuart M.; Hise, Amy G.

2014-01-01

Inflammasomes are central mediators of host defense to a wide range of microbial pathogens. The NLRP3 inflammasome plays a key role in triggering caspase-1 dependent IL-1β maturation and resistance to fungal dissemination in Candida albicans infection. β-glucans are major components of fungal cell walls that trigger IL-1β secretion in both murine and human immune cells. In this study, we sought to determine the contribution of β-glucans to C. albicans-induced inflammasome responses in mouse dendritic cells. We show that the NLRP3-ASC-caspase-1 inflammasome is absolutely critical for IL-1β production in response to β-glucans. Interestingly, we also found that both Complement Receptor 3 (CR3/Mac-1) and dectin-1 play a crucial role in coordinating β-glucan-induced IL-1β processing as well as a cell death response. In addition to the essential role of caspase-1, we identify an important role for the pro-apoptotic protease caspase-8 in promoting β-glucan-induced cell death and NLRP3 inflammasome-dependent IL-1β maturation. A strong requirement for Complement Receptor 3 and caspase-8 was also found for NLRP3 dependent IL-1β production in response to heat killed Candida albicans. Together, these results define the importance of dectin-1, CR3 and caspase-8, in addition to the canonical NLRP3 inflammasome, in mediating β-glucan and C. albicans induced innate responses in dendritic cells. Collectively, these findings establish a novel link between β-glucan recognition receptors and the inflammatory proteases caspase-8 and caspase-1 in coordinating cytokine secretion and cell death in response to immunostimulatory fungal components. PMID:25063877

Cellular maturity and apoptosis in human sperm: creatine kinase, caspase-3 and Bcl-XL levels in mature and diminished maturity sperm.

PubMed

Cayli, Sevil; Sakkas, Denny; Vigue, Lynne; Demir, Ramazan; Huszar, Gabor

2004-05-01

The relationship between human sperm maturity and apoptosis is of interest because of the persistence of immature sperm in ejaculates in spite of various apoptotic processes during spermatogenesis. We assessed sperm maturity by HspA2 chaperone levels, and plasma membrane maturity by sperm binding to immobilized hyaluronic acid (HA). We also utilized objective morphometry. Sperm were stained with three antibody combinations: active caspase-3/creatine kinase (CK, a marker of cytoplasmic retention), caspase-3/the antiapoptotic Bcl-(XL), and CK/Bcl-(XL). In semen, 13% of sperm stained with CK, caspase-3 or Bcl-(XL), and 28% had stained with two markers. In the mature HA-bound sperm fraction, <4% were single- or double-stained. Regarding sperm regions, CK staining, whether alone or as double staining, occurred in the head and midpiece (15-20%), whereas caspase-3 and Bcl-(XL) were primarily (>80% of sperm) in the midpiece. Morphometrical attributes of clear, single- and double-stained sperm, in line with their more pronounced maturation arrest, showed an incremental increase in head size (due to cytoplasmic retention) and shorter tail length. We hypothesize that during faulty sperm development, three alternatives may occur: (i) elimination of aberrant germ cells by apoptosis; (ii) in surviving immature cells, caspase-3 is activated, and in response the antiapoptotic Bcl-(XL), and perhaps HspA2, provide protection; (iii) in a third type of immature sperm, in addition to the CK, caspase-3 and Bcl-(XL) expression, there are related manifestations of increased head size and shorter tail length. Thus, immature sperm may vary in the type of developmental arrest and in protection mechanisms for apoptosis. These variations are likely to explain the persistence of immature sperm in the ejaculate.

ROFA INCREASES CASPASE-3 ACTIVITY IN HUMAN ALVEOLAR MACRAPHAGE

EPA Science Inventory

Exposure to air pollution particles produces pulmonary inflammation and injury, but the mechanisms of this injury are unclear. Apoptosis, involving activation of caspases, may be one potential mechanism. In this study, we hypothesized that ROFA, a constituent of air pollution...

Valproic acid treatment attenuates caspase-3 activation and improves survival after lethal burn injury in a rodent model.

PubMed

Luo, Hong-Min; Hu, Sen; Bai, Hui-Ying; Wang, Hai-Bin; Du, Ming-Hua; Lin, Zhi-Long; Ma, Li; Wang, Huan; Lv, Yi; Sheng, Zhi-Yong

2014-01-01

Burn injury may result in multiple organ dysfunction partially because of apoptotic cell death. The authors have previously shown that valproic acid (VPA) improves survival in a dog burn model. The aim of this study is to examine whether a VPA improves survival in a rodent burn model and whether this was because of inhibition of cell apoptosis. Rats were subjected to third-degree 55% TBSA burns and randomized to treatment with a VPA (300 mg/kg) or normal saline. One group of animals was monitored for 12 hours for survival analysis; another group was killed at 6 hours after injury, and brains, hearts, and blood samples were harvested for examination. Plasma creatine kinase (CK)-MB activities and neuron-specific enolase (NSE) levels were measured to evaluate the cardiac and brain damages. The effects of a VPA on acetylation of histone H3 and caspase-3 activation were also evaluated. Major burn injury resulted in a significant decrease in the acetylation of histone H3, and there was an increase in plasma CK-MB activities, NSE concentrations, and tissue levels of activated caspase-3. A VPA treatment significantly increased the acetylation of histone H3 and survival of the animals after major burn injury. In addition, a VPA treatment significantly attenuated the plasma CK-MB activities, an NSE concentrations, and inhibited caspase-3 activation after major burn injury. These results indicate that a VPA can attenuate cardiac and brain injury, and can improve survival in a rodent model of lethal burn injury. These protective effects may be mediated in part through the inhibition of caspase-3 activation.

Effect of folic acid and vitamin B12 on the expression of PPARγ, caspase-3 and caspase-8 mRNA in the abdominal aortas of rats with hyperlipidemia

PubMed Central

LV, FENG-HUA; GAO, JIAN-ZHI; TENG, QING-LEI; ZHANG, JIN-YING

2013-01-01

Hyperlipidemia may lead to endothelial injury, due to its effects on homocysteine and vascular endothelial growth factor in the serum, and the mRNA expression levels of peroxisome proliferator-activated receptor-γ (PPARγ), and caspase-3 and -8 in the vascular wall. In order to prevent and mitigate the high-fat state that results from endothelial injury, this study examined the effect of folic acid (FA) and vitamin B12 (VB12) on the expression of PPARγ and caspase-3 and -8 mRNA in the abdominal aortas of rats with hyperlipidemia. Sixty 4-week-old healthy male Sprague Dawley rats were randomly divided into five groups (each n=12): the normal control (NC), high-fat diet (HL), FA, VB12 and FA+VB12 groups. Following one week of adaptive feeding, the FA, VB12 and FA+VB12 groups were subject to the intraperitoneal injection of FA (0.5 mg/day), VB12 (0.05 mg/day) and FA+VB12 (0.5 mg/day and 0.05 mg/day), respectively, while fed a high-fat diet. The rats in the NC group were injected intraperitoneally with 0.9% NaCl solution (0.5 ml/day) and fed a normal diet, whereas those in the HL group were fed a high-fat diet only. A reverse transcription-polymerase chain reaction (RT-PCR) assay demonstrated that at the end of week 12, the FA treatment had effectively increased the PPARγ mRNA level, while reducing the caspase-3 and -8 mRNA levels, compared with the high-fat diet treatment (P<0.05). The effect of FA on the expression of PPARγ and caspase-3 and -8 was enhanced when used in combination with VB12 (P<0.05). These results revealed that the application of FA, alone or in combination with VB12, improves and mitigates the high-fat state that results from endothelial injury. PMID:23935743

Caspase-1 inhibitor regulates humoral responses in experimental autoimmune myasthenia gravis via IL-6- dependent inhibiton of STAT3.

PubMed

Wang, Cong-Cong; Zhang, Min; Li, Heng; Li, Xiao-Li; Yue, Long-Tao; Zhang, Peng; Liu, Ru-Tao; Chen, Hui; Li, Yan-Bin; Duan, Rui-Sheng

2017-08-24

We have previously demonstrated that Cysteinyl aspartate-specific proteinase-1 (caspase-1) inhibitor ameliorates experimental autoimmune myasthenia gravis (EAMG) by inhibited cellular immune response, via suppressing DC IL-1 β, CD4 + T and γdT cells IL-17 pathways. In this study, we investigated the effect of caspase-1 inhibitor on humoral immune response of EAMG and further explore the underlying mechanisms. An animal model of MG was induced by region 97-116 of the rat AChR α subunit (R97-116 peptide) in Lewis rats. Rats were treated with caspase-1 inhibitor Ac-YVAD-cmk intraperitoneally (i.p.) every second day from day 13 after the first immunization. Flow cytometry, western blot, immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) were performed to evaluate the neuroprotective effect of caspase-1 inhibitor on humoral immune response of EAMG. The results showed that caspase-1 inhibitor reduced the relative affinity of anti-R97-116 IgG, suppressed germinal center response, decreased follicular helper T cells, and increased follicular regulatory T cells and regulatory B cells. In addition, we found that caspase-1 inhibitor inhibited humoral immunity response in EAMG rats via suppressing IL-6-STAT3-Bcl-6 pathways. These results suggest that caspase-1 inhibitor ameliorates EAMG by regulating humoral immune response, thus providing new insights into the development of myasthenia gravis and other autoimmune diseases therapies. Copyright © 2017 Elsevier B.V. All rights reserved.

Synergistic effect of fisetin combined with sorafenib in human cervical cancer HeLa cells through activation of death receptor-5 mediated caspase-8/caspase-3 and the mitochondria-dependent apoptotic pathway.

PubMed

Lin, Ming-Te; Lin, Chia-Liang; Lin, Tzu-Yu; Cheng, Chun-Wen; Yang, Shun-Fa; Lin, Chu-Liang; Wu, Chih-Chien; Hsieh, Yi-Hsien; Tsai, Jen-Pi

2016-05-01

Combining antitumor agents with bioactive compounds is a potential strategy for improving the effect of chemotherapy on cancer cells. The goal of this study was to elucidate the antitumor effect of the flavonoid, fisetin, combined with the multikinase inhibitor, sorafenib, against human cervical cancer cells in vitro and in vivo. The combination of fisetin and sorafenib synergistically induced apoptosis in HeLa cells, which is accompanied by a marked increase in loss of mitochondrial membrane potential. Apoptosis induction was achieved by caspase-3 and caspase-8 activation which increased the ratio of Bax/Bcl-2 and caused the subsequent cleavage of PARP level while disrupting the mitochondrial membrane potential in HeLa cells. Decreased Bax/Bcl-2 ratio level and mitochondrial membrane potential were also observed in siDR5-treated HeLa cells. In addition, in vivo studies revealed that the combined fisetin and sorafenib treatment was clearly superior to sorafenib treatment alone using a HeLa xenograft model. Our study showed that the combination of fisetin and sorafenib exerted better synergistic effects in vitro and in vivo than either agent used alone against human cervical cancer, and this synergism was based on apoptotic potential through a mitochondrial- and DR5-dependent caspase-8/caspase-3 signaling pathway. This combined fisetin and sorafenib treatment represents a novel therapeutic strategy for further clinical developments in advanced cervical cancer.

Highly Sensitive Detection of Caspase-3/7 Activity in Living Mice Using Enzyme-Responsive 19F MRI Nanoprobes.

PubMed

Akazawa, Kazuki; Sugihara, Fuminori; Nakamura, Tatsuya; Mizukami, Shin; Kikuchi, Kazuya

2018-05-16

Highly sensitive imaging of enzymatic activities in the deep tissues of living mammals provides useful information about their biological functions and for developing new drugs; however, such imaging is challenging. 19 F magnetic resonance imaging (MRI) is suitable for noninvasive visualization of enzymatic activities without endogenous background signals. Although various enzyme-responsive 19 F MRI probes have been developed, most cannot be used for in vivo imaging because of their low sensitivity. Recently, we developed unique nanoparticles, called FLAMEs, that are composed of a liquid perfluorocarbon core and a robust silica shell, and demonstrated their outstanding sensitivity in vivo. Here, we report a highly functionalized nanoprobe, FLAME-DEVD 2, with an OFF/ON 19 F MRI switch for detecting caspase-3/7 activity based on the paramagnetic relaxation enhancement effect. To improve the cleavage efficiency of peptides by caspase-3, we designed a novel Gd 3+ complex-conjugated peptide, DEVD X ( X = 1, 2), which is a substrate peptide sequence tandemly repeated X times, and demonstrated that DEVD 2 showed faster cleavage kinetics than DEVD 1. By incorporating this novel concept into a signal activation strategy, FLAME-DEVD 2 showed a high 19 F MRI signal enhancement rate in response to caspase-3 activity. After intravenous injection of FLAME-DEVD 2 and an apoptosis-inducing reagent, caspase-3/7 activity in the spleen of a living mouse was successfully imaged by 19 F MRI. This imaging platform shows great potential for highly sensitive detection of enzymatic activities in vivo.

Activation of caspase-1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function

PubMed Central

Sokolovska, Anna; Becker, Christine E.; Eddie Ip, WK; Rathinam, Vijay A.K.; Brudner, Matthew; Paquette, Nicholas; Tanne, Antoine; Vanaja, Sivapriya K.; Moore, Kathryn J.; Fitzgerald, Katherine A.; Lacy-Hulbert, Adam; Stuart, Lynda M.

2013-01-01

Phagocytosis is a fundamental cellular process that is pivotal for immunity as it coordinates microbial killing, innate immune activation and antigen presentation. An essential step in this process is phagosome acidification, which regulates a number of functions of these organelles that allow them to participate in processes essential to both innate and adaptive immunity. Here we report that acidification of phagosomes containing Gram-positive bacteria is regulated by the NLRP3-inflammasome and caspase-1. Active caspase-1 accumulates on phagosomes and acts locally to control the pH by modulating buffering by the NADPH oxidase NOX2. These data provide insight into a mechanism by which innate immune signals can modify cellular defenses and establish a new function for the NLRP3-inflammasome and caspase-1 in host defense. PMID:23644505

Combined fluorimetric caspase 3/7 assay and bradford protein determination for assessment of polycation-mediated cytotoxicity.

PubMed

Larsen, Anna K; Hall, Arnaldur; Lundsgart, Henrik; Moghimi, S Moein

2013-01-01

Cationic polyplexes and lipoplexes are widely used as artificial systems for nucleic acid delivery into the cells, but they can also induce cell death. Mechanistic understanding of cell toxicity and biological side effects of these cationic entities is essential for optimization strategies and design of safe and efficient nucleic acid delivery systems. Numerous methods are presently available to detect and delineate cytotoxicity and cell death-mediated signals in cell cultures. Activation of caspases is part of the classical apoptosis program and increased caspase activity is therefore a well-established hallmark of programmed cell death. Additional methods to monitor cell death-related signals must, however, also be carried out to fully define the type of cell toxicity in play. These may include methods that detect plasma membrane damage, loss of mitochondrial membrane potential, phosphatidylserine exposure, and cell morphological changes (e.g., membrane blebbing, nuclear changes, cytoplasmic swelling, cell rounding). Here we describe a 96-well format protocol for detection of capsase-3/7 activity in cell lysates, based on a fluorescent caspase-3 assay, combined with a method to simultaneously determine relative protein contents in the individual wells.

Histomorphometry and expression of CDC-47 and caspase-3 in mammary glands of pregnant female rats with artificial hyperthyroidism.

PubMed

Leite, Eveline Dias; de Freitas, Edmilson Santos; de Almeida Souza, Cintia; de Melo Ocarino, Natalia; Cassali, Geovanni Dantas; Serakides, Rogéria

2008-01-01

The purpose of this study was to evaluate the effect of hyperthyroidism on mammary gland development and expression of two protein markers, CDC-47 for proliferation and caspase-3 for apoptosis in pregnant female rats. Thirty-six adult female Wistar rats were used in two groups: hyperthyroid and control. Rats were mated 60 days after the onset of thyroxine administration. Six animals/group were sacrificed on gestation days 7, 14, and 19. Artificial hyperthyroidism was induced by daily administration of thyroxine in the drinking water until the end of gestation. At the end of each period, rats were sacrificed, and their inguinal mammary glands were collected and processed for morphometric analysis. The percentages of epithelium, stroma, adipose tissue, and lacteal secretion were determined. Immunohistochemical analysis was also carried out using anti-CDC-47 and anti-caspase-3 antibodies to study proliferation and apoptosis, respectively. On the 19th day of gestation, thyroxine treatment significantly increased the percentage of mammary epithelium. Hyperthyroidism, however, did not change CDC-47 expression. The hyperthyroid group presented early lactogenesis and significantly larger lacteal secretion on the 19th day of gestation. There was no significant difference in caspase-3 expression between groups in any period. We may conclude that hyperthyroidism accelerates mammary gland development and increases lacteal secretion during gestation without increasing the proliferation rate and the expression of caspase-3.

Lactobacillus plantarum 299v Prevents Caspase-Dependent Apoptosis In Vitro.

PubMed

Dykstra, Natalie S; Hyde, Lucie; MacKenzie, Alexander; Mack, David R

2011-03-01

Selective microbes used as probiotics can enhance epithelial cell protection. We have previously shown that a Lactobacillus plantarum strain 299v (Lp299v) has the ability to induce mucin genes. In the current study, we utilized a cytokine model of inflammation in cell culture to study the modulation of apoptosis by this probiotic. HT-29 cells were pre-incubated with the Lp299v or L. plantarum strain adh- (Lpadh-), a non-adherent derivative of Lp299v. Cells were challenged with a mixture of cytokines (TNF-α, IFN-γ, and IL-1a) to imitate conditions of inflammation. To assess for cell death, we evaluated TUNEL, multi-caspase, and caspase-3 and caspase-7 activity assays. There was a marked decrease in apoptosis as measured by TUNEL(+) cells in samples pre-treated with Lp299v (18.7 ± 4.1%, p < 0.01) and Lpadh- (16.6 ± 3.2%, p < 0.05) prior to cytokine exposure when compared to cells (43.6 ± 6.2%) exposed to the cytokine mixture. Lp299v pre-incubation with HT-29 cells reduced caspase(+) cells in the multi-caspase activity assay (3.6 ± 0.6%, p < 0.05) compared to cells exposed to cytokines (68.9 ± 5.1%) whereas Lpadh- did not (46.8 ± 17.5%, p > 0.05). Similarly, caspase-3, caspase-7 activity was also reduced by Lp299v. Selected probiotics may confer an exogenous protective effect at the mucosal-luminal interface for intestinal epithelial cells via alteration of caspase-dependent apoptotic pathways.

Development of the terminally differentiated state sensitizes epiphyseal chondrocytes to apoptosis through caspase-3 activation.

PubMed

Pucci, Bruna; Adams, Christopher S; Fertala, Jolanta; Snyder, Bradley C; Mansfield, Kyle D; Tafani, Marco; Freeman, Theresa; Shapiro, Irving M

2007-03-01

The maturation of epiphyseal chondrocytes is accompanied by dramatic changes in energy metabolism and shifts in proteins concerned with the induction of apoptosis. We evaluated the role of mitochondria in this process by evaluating the membrane potential (Delta psi m) of chondrocytes of embryonic tibia and the epiphyseal growth plate. We observed that there was a maturation-dependent change in fluorescence, indicating a fall in the Delta psi m. The level of mitochondrial Bcl-2 was decreased during maturation, while in the same time period there was an obvious increase in Bax levels in the mitochondrial fraction of the terminally differentiated chondrocytes. Bcl(xL), another anti-apoptotic protein, was also robustly expressed in the mitochondrial fraction, but its expression was not dependent on the maturation status of the chondrocytes. We found that caspase-3 was present throughout the growth plate and in hypertrophic cells in culture. We blocked caspase-3 activity and found that alkaline phosphatase staining and mineral formation was decreased, and the cells had lost their characteristic shape. Moreover, we noted that the undifferentiated cells were insensitive to elevated concentrations of inorganic phosphate (Pi). It is concluded that during hypertrophy, the change in membrane potential, the increased binding of a pro-apoptotic protein to mitochondria, and the activation of caspase-3 serve to prime cells for apoptosis. Only when the terminally differentiated chondrocytes are challenged with low levels of apoptogens there is activation of apoptosis. Copyright 2006 Wiley-Liss, Inc.

Activation of caspase-3 noninvolved in the bystander effect of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system.

PubMed

Zhang, Zhihong; Lin, Juqiang; Chu, Jun; Ma, Yan; Zeng, Shaoqun; Luo, Qingming

2008-01-01

Use of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system is one of the promising approaches in the rapidly growing area of gene therapy. The "bystander effect," a phenomenon in which HSV-tk+ cells exposed to GCV are toxic to adjacent HSV-tk- cells, was reported to play an important role in suicide gene therapy. However, the mechanism by which HSV-tk/GCV induces the bystander effect is poorly understood. We monitored the activation of caspase-3 in living cells induced by the HSV-tk/GCV system using a genetically encoded fluorescence resonance energy transfer (FRET) probe CD3, , a caspase-3 recognition site fused with a cyan fluorescent protien (CFP) and a red fluorescent protein (DsRed) which we reported and named in a previous paper. Fluorescence protein (FP)-based multicolor cellular labeling, combined with the multichannel fluorescence imaging and FRET imaging techniques, provides a novel and improved approach to directly determine whether the activation of caspase-3 involved in the HSV-tk/GCV system induces cell apoptosis in tk gene-expressing cells and their neighboring cells. FRET ratio images of CD3, and fluorescence images of the fusion protein of thymidine kinase linked with green fluorescent protein (TK-GFP), indicated that HSV-tk/GCV system-induced apoptosis in human adenoid cystic carcinoma (ACC-M) cells was via a caspase-3 pathway, and the activation of caspase-3 was not involved in the bystander effect of HSV-tk/GCV system.

Nicotinamide Inhibits Ethanol-Induced Caspase-3 and PARP-1 Over-activation and Subsequent Neurodegeneration in the Developing Mouse Cerebellum.

PubMed

Ieraci, Alessandro; Herrera, Daniel G

2018-06-01

Fetal alcohol spectrum disorder (FASD) is the principal preventable cause of mental retardation in the western countries resulting from alcohol exposure during pregnancy. Ethanol-induced massive neuronal cell death occurs mainly in immature neurons during the brain growth spurt period. The cerebellum is one of the brain areas that are most sensitive to ethanol neurotoxicity. Currently, there is no effective treatment that targets the causes of these disorders and efficient treatments to counteract or reverse FASD are desirable. In this study, we investigated the effects of nicotinamide on ethanol-induced neuronal cell death in the developing cerebellum. Subcutaneous administration of ethanol in postnatal 4-day-old mice induced an over-activation of caspase-3 and PARP-1 followed by a massive neurodegeneration in the developing cerebellum. Interestingly, treatment with nicotinamide, immediately or 2 h after ethanol exposure, diminished caspase-3 and PARP-1 over-activation and reduced ethanol-induced neurodegeneration. Conversely, treatment with 3-aminobenzadine, a specific PARP-1 inhibitor, was able to completely block PARP-1 activation, but not caspase-3 activation or ethanol-induced neurodegeneration in the developing cerebellum. Our results showed that nicotinamide reduces ethanol-induced neuronal cell death and inhibits both caspase-3 and PARP-1 alcohol-induced activation in the developing cerebellum, suggesting that nicotinamide might be a promising and safe neuroprotective agent for treating FASD and other neurodegenerative disorders in the developing brain that shares similar cell death pathways.

Synthetic activation of caspases: Artificial death switches

PubMed Central

MacCorkle, Rebecca A.; Freeman, Kevin W.; Spencer, David M.

1998-01-01

The development of safe vectors for gene therapy requires fail-safe mechanisms to terminate therapy or remove genetically altered cells. The ideal “suicide switch” would be nonimmunogenic and nontoxic when uninduced and able to trigger cell death independent of tissue type or cell cycle stage. By using chemically induced dimerization, we have developed powerful death switches based on the cysteine proteases, caspase-1 ICE (interleukin-1β converting enzyme) and caspase-3 YAMA. In both cases, aggregation of the target protein is achieved by a nontoxic lipid-permeable dimeric FK506 analog that binds to the attached FK506-binding proteins, FKBPs. We find that intracellular cross-linking of caspase-1 or caspase-3 is sufficient to trigger rapid apoptosis in a Bcl-xL-independent manner, suggesting that these conditional proapoptotic molecules can bypass intracellular checkpoint genes, such as Bcl-xL, that limit apoptosis. Because these chimeric molecules are derived from autologous proteins, they should be nonimmunogenic and thus ideal for long-lived gene therapy vectors. These properties should also make chemically induced apoptosis useful for developmental studies, for treating hyperproliferative disorders, and for developing animal models to a wide variety of diseases. PMID:9520421

Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death.

PubMed

Shi, Jianjin; Zhao, Yue; Wang, Kun; Shi, Xuyan; Wang, Yue; Huang, Huanwei; Zhuang, Yinghua; Cai, Tao; Wang, Fengchao; Shao, Feng

2015-10-29

Inflammatory caspases (caspase-1, -4, -5 and -11) are critical for innate defences. Caspase-1 is activated by ligands of various canonical inflammasomes, and caspase-4, -5 and -11 directly recognize bacterial lipopolysaccharide, both of which trigger pyroptosis. Despite the crucial role in immunity and endotoxic shock, the mechanism for pyroptosis induction by inflammatory caspases is unknown. Here we identify gasdermin D (Gsdmd) by genome-wide clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 nuclease screens of caspase-11- and caspase-1-mediated pyroptosis in mouse bone marrow macrophages. GSDMD-deficient cells resisted the induction of pyroptosis by cytosolic lipopolysaccharide and known canonical inflammasome ligands. Interleukin-1β release was also diminished in Gsdmd(-/-) cells, despite intact processing by caspase-1. Caspase-1 and caspase-4/5/11 specifically cleaved the linker between the amino-terminal gasdermin-N and carboxy-terminal gasdermin-C domains in GSDMD, which was required and sufficient for pyroptosis. The cleavage released the intramolecular inhibition on the gasdermin-N domain that showed intrinsic pyroptosis-inducing activity. Other gasdermin family members were not cleaved by inflammatory caspases but shared the autoinhibition; gain-of-function mutations in Gsdma3 that cause alopecia and skin defects disrupted the autoinhibition, allowing its gasdermin-N domain to trigger pyroptosis. These findings offer insight into inflammasome-mediated immunity/diseases and also change our understanding of pyroptosis and programmed necrosis.

Truncation of the TAR DNA-binding protein 43 is not a prerequisite for cytoplasmic relocalization, and is suppressed by caspase inhibition and by introduction of the A90V sequence variant

PubMed Central

Brandon, Nicholas J.; Moss, Stephen J.

2017-01-01

The RNA-binding and -processing protein TAR DNA-binding protein 43 (TDP-43) is heavily linked to the underlying causes and pathology of neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. In these diseases, TDP-43 is mislocalized, hyperphosphorylated, ubiquitinated, aggregated and cleaved. The importance of TDP-43 cleavage in the disease pathogenesis is still poorly understood. Here we detail the use of D-sorbitol as an exogenous stressor that causes TDP-43 cleavage in HeLa cells, resulting in a 35 kDa truncated product that accumulates in the cytoplasm within one hour of treatment. We confirm that the formation of this 35 kDa cleavage product is mediated by the activation of caspases. Inhibition of caspases blocks the cleavage of TDP-43, but does not prevent the accumulation of full-length protein in the cytoplasm. Using D-sorbitol as a stressor and caspase activator, we also demonstrate that the A90V variant of TDP-43, which lies adjacent to the caspase cleavage site within the nuclear localization sequence of TDP-43, confers partial resistance against caspase-mediated generation of the 35 kDa cleavage product. PMID:28510586

p75 Neurotrophin Receptor Signaling Activates Sterol Regulatory Element-binding Protein-2 in Hepatocyte Cells via p38 Mitogen-activated Protein Kinase and Caspase-3.

PubMed

Pham, Dan Duc; Do, Hai Thi; Bruelle, Céline; Kukkonen, Jyrki P; Eriksson, Ove; Mogollón, Isabel; Korhonen, Laura T; Arumäe, Urmas; Lindholm, Dan

2016-05-13

Nerve growth factor (NGF) influences the survival and differentiation of a specific population of neurons during development, but its role in non-neuronal cells has been less studied. We observed here that NGF and its pro-form, pro-NGF, are elevated in fatty livers from leptin-deficient mice compared with controls, concomitant with an increase in low density lipoprotein receptors (LDLRs). Stimulation of mouse primary hepatocytes with NGF or pro-NGF increased LDLR expression through the p75 neurotrophin receptor (p75NTR). Studies using Huh7 human hepatocyte cells showed that the neurotrophins activate the sterol regulatory element-binding protein-2 (SREBP2) that regulates genes involved in lipid metabolism. The mechanisms for this were related to stimulation of p38 mitogen-activated protein kinase (p38 MAPK) and activation of caspase-3 and SREBP2 cleavage following NGF and pro-NGF stimulations. Cell fractionation experiments showed that caspase-3 activity was increased particularly in the membrane fraction that harbors SREBP2 and caspase-2. Experiments showed further that caspase-2 interacts with pro-caspase-3 and that p38 MAPK reduced this interaction and caused caspase-3 activation. Because of the increased caspase-3 activity, the cells did not undergo cell death following p75NTR stimulation, possibly due to concomitant activation of nuclear factor-κB (NF-κB) pathway by the neurotrophins. These results identify a novel signaling pathway triggered by ligand-activated p75NTR that via p38 MAPK and caspase-3 mediate the activation of SREBP2. This pathway may regulate LDLRs and lipid uptake particularly after injury or during tissue inflammation accompanied by an increased production of growth factors, including NGF and pro-NGF. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Proteolytic cleavage of polymeric tau protein by caspase-3: implications for Alzheimer disease.

PubMed

Jarero-Basulto, Jose J; Luna-Muñoz, Jose; Mena, Raul; Kristofikova, Zdena; Ripova, Daniela; Perry, George; Binder, Lester I; Garcia-Sierra, Francisco

2013-12-01

Truncated tau protein at Asp(421) is associated with neurofibrillary pathology in Alzheimer disease (AD); however, little is known about its presence in the form of nonfibrillary aggregates. Here, we report immunohistochemical staining of the Tau-C3 antibody, which recognizes Asp(421)-truncated tau, in a group of AD cases with different extents of cognitive impairment. In the hippocampus, we found distinct nonfibrillary aggregates of Asp(421)-truncated tau. Unlike Asp(421)-composed neurofibrillary tangles, however, these nonfibrillary pathologies did not increase significantly with respect to the Braak staging and, therefore, make no significant contribution to cognitive impairment. On the other hand, despite in vitro evidence that caspase-3 cleaves monomeric tau at Asp(421), to date, this truncation has not been demonstrated to be executed by this protease in polymeric tau entities. We determined that Asp(421) truncation can be produced by caspase-3 in oligomeric and multimeric complexes of recombinant full-length tau in isolated native tau filaments in vitro and in situ in neurofibrillary tangles analyzed in fresh brain slices from AD cases. Our data suggest that generation of this pathologic Asp(421) truncation of tau in long-lasting fibrillary structures may produce further permanent toxicity for neurons in the brains of patients with AD.

Specificity of a protein-protein interface: local dynamics direct substrate recognition of effector caspases.

PubMed

Fuchs, Julian E; von Grafenstein, Susanne; Huber, Roland G; Wallnoefer, Hannes G; Liedl, Klaus R

2014-04-01

Proteases are prototypes of multispecific protein-protein interfaces. Proteases recognize and cleave protein and peptide substrates at a well-defined position in a substrate binding groove and a plethora of experimental techniques provide insights into their substrate recognition. We investigate the caspase family of cysteine proteases playing a key role in programmed cell death and inflammation, turning caspases into interesting drug targets. Specific ligand binding to one particular caspase is difficult to achieve, as substrate specificities of caspase isoforms are highly similar. In an effort to rationalize substrate specificity of two closely related caspases, we investigate the substrate promiscuity of the effector Caspases 3 and 7 by data mining (cleavage entropy) and by molecular dynamics simulations. We find a strong correlation between binding site rigidity and substrate readout for individual caspase subpockets explaining more stringent substrate readout of Caspase 7 via its narrower conformational space. Caspase 3 subpockets S3 and S4 show elevated local flexibility explaining the more unspecific substrate readout of that isoform in comparison to Caspase 7. We show by in silico exchange mutations in the S3 pocket of the proteases that a proline residue in Caspase 7 contributes to the narrowed conformational space of the binding site. These findings explain the substrate specificities of caspases via a mechanism of conformational selection and highlight the crucial importance of binding site local dynamics in substrate recognition of proteases. Proteins 2014; 82:546-555. © 2013 Wiley Periodicals, Inc. Copyright © 2013 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

[Effect of high frequency electrotherapy on caspase-3 and ultra microstructure of hippocampus in rats following cerebral ischemia/reperfusion].

PubMed

Fan, Yongmei; Wang, Rumi; Zhang, Changjie; Peng, Wenna; Yin, Jing; Hu, Zhiping

2017-01-28

To investigate the effect of high frequency electrotherapy (HFE) on rat hippocampus after cerebral ischemia/reperfusion (I/R).
 Methods: A rat model of cerebral I/R injury was established. The rats were randomly divided into a sham group, an I/R group and an HFE group. The HFE group received thearapy daily for different sessions for 1, 3, 7 d. Neuronal deficit score,neuron ultra microstructure in the hippocampus and caspase-3 protein expression were measured on 1 st, 3 th and 7th d.
 Results: Compared with the I/R group, the HFE group showed the decreased neurological deficit scores, with significant differences between the 2 groups (P<0.05). The injury in HFE group was reduced compared with that in the I/R group based on the electron microscope test, with significant difference. Caspase-3 protein in brain tissue in the HFE group also downregulated compared with that in the I/R group (P<0.05).
 Conclusion: High frequency electrotherapy can improve neural function, suppress caspase-3 expression and apoptosis in nerve cells and improve the ultra microstructure of neurons, displaying a protective effect on cerebral I/R injury in rats.

Huangzhi Oral Liquid Prevents Arrhythmias by Upregulating Caspase-3 and Apoptosis Network Proteins in Myocardial Ischemia-Reperfusion Injury in Rats

PubMed Central

Ran, Xu; Sun, Xue Gang; Wang, Ming; An, Hui; Huang, Guo Qiang; Zhao, Xiao Shan; Zhou, Feng Hua; Yang, Yun Gao; Miao, Can Ming

2015-01-01

To study the effect of Huangzhi oral liquid (HZOL) on I/R after 2 h and 4 h and determine its regulatory function on caspase-3 and protein networks. 70 SD male rats were randomly divided into seven groups and established myocardial I/R injury model by ligating the left anterior descending coronary artery. Myocardial infarction model was defined by TTC staining and color of the heart. The levels of CK-MB, CTnI, C-RPL, SOD, and MDA were tested at 2 h and 4 h after reperfusion. HE staining and ultramicrostructural were used to observe the pathological changes. The apoptotic index (AI) of cardiomyocyte was marked by TUNEL. The expression levels of caspase-3, p53, fas, Bcl-2, and Bax were tested by immunohistochemistry and western blot. HZOL corrected arrhythmia, improved the pathologic abnormalities, decreased CK-MB, CTnI, C-RPL, MDA, AI, caspase-3, p53, fas, and Bax, and increased SOD ans Bcl-2 with different times of myocardial reperfusion; this result was similar to the ISMOC (P > 0.05). HZOL could inhibit arrhythmia at 2 and 4 h after I/R and ameliorate cardiac function, which was more significant at 4 h after reperfusion. This result may be related to decreased expression of caspase-3, p53, and fas and increased Bcl-2/Bax ratio. PMID:26074995

Protective effect of caspase inhibition on compression-induced muscle damage

PubMed Central

Teng, Bee T; Tam, Eric W; Benzie, Iris F; Siu, Parco M

2011-01-01

Abstract There are currently no effective therapies for treating pressure-induced deep tissue injury. This study tested the efficacy of pharmacological inhibition of caspase in preventing muscle damage following sustained moderate compression. Adult Sprague–Dawley rats were subjected to prolonged moderate compression. Static pressure of 100 mmHg compression was applied to an area of 1.5 cm2 in the tibialis region of the right limb of the rats for 6 h each day for two consecutive days. The left uncompressed limb served as intra-animal control. Rats were randomized to receive either vehicle (DMSO) as control treatment (n = 8) or 6 mg kg−1 of caspase inhibitor (z-VAD-fmk; n = 8) prior to the 6 h compression on the two consecutive days. Muscle tissues directly underneath the compression region of the compressed limb and the same region of control limb were harvested after the compression procedure. Histological examination and biochemical/molecular measurement of apoptosis and autophagy were performed. Caspase inhibition was effective in alleviating the compression-induced pathohistology of muscle. The increases in caspase-3 protease activity, TUNEL index, apoptotic DNA fragmentation and pro-apoptotic factors (Bax, p53 and EndoG) and the decreases in anti-apoptotic factors (XIAP and HSP70) observed in compressed muscle of DMSO-treated animals were not found in animals treated with caspase inhibitor. The mRNA content of autophagic factors (Beclin-1, Atg5 and Atg12) and the protein content of LC3, FoxO3 and phospho-FoxO3 that were down-regulated in compressed muscle of DMSO-treated animals were all maintained at their basal level in the caspase inhibitor treated animals. Our data provide evidence that caspase inhibition attenuates compression-induced muscle apoptosis and maintains the basal autophagy level. These findings demonstrate that pharmacological inhibition of caspase/apoptosis is effective in alleviating muscle damage as induced by prolonged compression

Androstenediol inhibits the trauma-hemorrhage-induced increase in caspase-3 by downregulating the inducible nitric oxide synthase pathway.

PubMed

Kiang, Juliann G; Peckham, Russell M; Duke, Leah E; Shimizu, Tomoharu; Chaudry, Irshad H; Tsokos, George C

2007-03-01

Soft tissue trauma and hemorrhage (T-H) diminishes various aspects of liver function, while it increases hepatic nitrate/nitrite, inducible nitric oxide synthase (iNOS), and endothelin-1 levels. Treatment with androstenediol (AED) inhibits the T-H-induced alterations of the above parameters. We sought to identify the molecular events underlying the beneficial effect of AED. Exposure of rats to T-H significantly increased the caspase-3 activity and protein, whereas treatment with AED significantly limited these increases. AED treatment also suppressed the T-H-induced increase in iNOS by effectively altering the levels of key transcription factors involved in the regulation of iNOS expression. Immunoprecipitation and immunoblotting analyses indicate that T-H increased apoptosome formation, and AED treatment significantly decreased it. Modulating the iNOS protein by transfecting cells with iNOS gene or small interfering RNA further confirmed the correlation between iNOS and caspase-3. Our data indicate that AED limits caspase-3 expression by suppressing the expression of transcription factors involved in the production of iNOS, resulting in decreased apoptosome. AED can potentially be a useful adjuvant for limiting liver apoptosis following T-H shock.

SipA Activation of Caspase-3 Is a Decisive Mediator of Host Cell Survival at Early Stages of Salmonella enterica Serovar Typhimurium Infection

PubMed Central

McIntosh, Anne; Meikle, Lynsey M.; Ormsby, Michael J.; McCormick, Beth A.; Christie, John M.; Brewer, James M.; Roberts, Mark

2017-01-01

ABSTRACT Salmonella invasion protein A (SipA) is a dual-function effector protein that plays roles in both actin polymerization and caspase-3 activation in intestinal epithelial cells. To date its function in other cell types has remained largely unknown despite its expression in multiple cell types and its extracellular secretion during infection. Here we show that in macrophages SipA induces increased caspase-3 activation early in infection. This activation required a threshold level of SipA linked to multiplicity of infection and may be a limiting factor controlling bacterial numbers in infected macrophages. In polymorphonuclear leukocytes, SipA or other Salmonella pathogenicity island 1 effectors had no effect on induction of caspase-3 activation either alone or in the presence of whole bacteria. Tagging of SipA with the small fluorescent phiLOV tag, which can pass through the type three secretion system, allowed visualization and quantification of caspase-3 activation by SipA-phiLOV in macrophages. Additionally, SipA-phiLOV activation of caspase-3 could be tracked in the intestine through multiphoton laser scanning microscopy in an ex vivo intestinal model. This allowed visualization of areas where the intestinal epithelium had been compromised and demonstrated the potential use of this fluorescent tag for in vivo tracking of individual effectors. PMID:28630067

Modulation of caspases and their non-apoptotic functions by Legionella pneumophila.

PubMed

Amer, Amal O

2010-02-01

Legionella pneumophila has become a model system to decipher the non-apoptotic functions of caspases and their role in immunity. In permissive cells, the L. pneumophila-containing vacuole evades endosomal traffic and is remodelled by the endoplasmic reticulum. Evasion of the endosomes is mediated by the Dot/Icm type IV secretion system. Upon L. pneumophila infection of genetically restrictive cells such as wild-type (WT) C57Bl/6J murine macrophages, flagellin is sensed by the NOD-like receptor Nlrc4 leading to caspase-1 activation by the inflammasome complex. Then, caspase-7 is activated downstream of the Nlrc4 inflammasome, promoting non-apoptotic functions such as L. pneumophila-containing phagosome maturation and bacterial degradation. Interestingly, caspase-3 is activated in permissive cells during early stages of infection. However, caspase-3 activation does not lead to apoptosis until late stages of infection because it is associated with potent Dot/Icm-mediated anti-apoptotic stimuli that render the infected cells resistant to external apoptotic inducers. Therefore, the role of caspase-1 and non-apoptotic functions of executioner caspases are temporally and spatially modulated during infection by L. pneumophila, which determine permissiveness to intracellular bacterial proliferation. This review will examine the novel activation pathways of caspases by L. pneumophila and discuss their role in genetic restriction and permissiveness to infection.

Solution structure of Apaf-1 CARD and its interaction with caspase-9 CARD: a structural basis for specific adaptor/caspase interaction.

PubMed

Zhou, P; Chou, J; Olea, R S; Yuan, J; Wagner, G

1999-09-28

Direct recruitment and activation of caspase-9 by Apaf-1 through the homophilic CARD/CARD (Caspase Recruitment Domain) interaction is critical for the activation of caspases downstream of mitochondrial damage in apoptosis. Here we report the solution structure of the Apaf-1 CARD domain and its surface of interaction with caspase-9 CARD. Apaf-1 CARD consists of six tightly packed amphipathic alpha-helices and is topologically similar to the RAIDD CARD, with the exception of a kink observed in the middle of the N-terminal helix. By using chemical shift perturbation data, the homophilic interaction was mapped to the acidic surface of Apaf-1 CARD centered around helices 2 and 3. Interestingly, a significant portion of the chemically perturbed residues are hydrophobic, indicating that in addition to the electrostatic interactions predicted previously, hydrophobic interaction is also an important driving force underlying the CARD/CARD interaction. On the basis of the identified functional residues of Apaf-1 CARD and the surface charge complementarity, we propose a model of CARD/CARD interaction between Apaf-1 and caspase-9.

Fipronil induces apoptosis through caspase-dependent mitochondrial pathways in Drosophila S2 cells.

PubMed

Zhang, Baoyan; Xu, Zhiping; Zhang, Yixi; Shao, Xusheng; Xu, Xiaoyong; Cheng, Jiaogao; Li, Zhong

2015-03-01

Fipronil is the first phenylpyrazole insecticide widely used in controlling pests, including pyrethroid, organophosphate and carbamate insecticides. It is generally accepted that fipronil elicits neurotoxicity via interactions with GABA and glutamate receptors, although alternative mechanisms have recently been proposed. This study evaluates the genotoxicity of fipronil and its likely mode of action in Drosophila S2 cells, as an in vitro model. Fipronil administrated the concentration- and time-dependent S2 cell proliferation. Intracellular biochemical assays showed that fipronil-induced S2 cell apoptosis coincided with a decrease in the mitochondrial membrane potential and an increase reactive oxygen species generation, a significant decrease of Bcl-2 and DIAP1, and a marked augmentation of Cyt c and caspase-3. Because caspase-3 is the major executioner caspase downstream of caspase-9 in Drosophila, enzyme activity assays were used to determine the activities of caspase-3 and caspase-9. Our results indicated that fipronil effectively induced apoptosis in Drosophila S2 cells through caspase-dependent mitochondrial pathways. Copyright © 2015 Elsevier Inc. All rights reserved.

Synthesis of Novel Caspase Inhibitors for Characterization of the Active Caspase Proteome in Vitro and in Vivo

PubMed Central

Henzing, Alexander J.; Dodson, Helen; Reid, Joel M.; Kaufmann, Scott H.; Baxter, Robert L.; Earnshaw, William C.

2008-01-01

Caspases are cysteine proteases that are essential for cytokine maturation and apoptosis. To facilitate the dissection of caspase function in vitro and in vivo, we have synthesized irreversible caspase inhibitors with biotin attached via linker arms of various lengths (12a–d) and a 2,4-dinitrophenyl labeled inhibitor (13). Affinity labeling of apoptotic extracts followed by blotting reveals that these affinity probes detect active caspases. Using the strong affinity of avidin for biotin, we have isolated affinity-labeled caspase-6 from apoptotic cytosolic extracts of cells overexpressing procaspase 6 by treatment with 12c, which contains biotin attached to the Nε-lysine of the inhibitor by a 22.5 Å linker arm, followed by affinity purification on monomeric avidin-Sepharose beads. 13 has proven sufficiently cell permeable to rescue cells from apoptotic execution. These novel caspase inhibitors should provide powerful probes for the study of the active caspase proteome during apoptosis both in vitro and in vivo. PMID:17181147

Transient protective effect of caspase inhibitors in RCS rat.

PubMed

Perche, O; Doly, M; Ranchon-Cole, I

2008-03-01

In most retinal degenerations in humans and in animal models, photoreceptor cells die by apoptosis. Although the biochemical features are similar in all apoptotic cells, different molecular events lead the cell to death. In the present study we used a rat model of inherited retinal degeneration, the RCS rats, to investigate the involvement of the proteases, caspases and/or calpains, in photoreceptor apoptosis. In the first experiments, rats were untreated or injected intravitreally at post natal day 27 (P27) with the large broad spectrum caspase inhibitor, ZVAD, the calpain inhibitor, MuhPhe, or with the vehicle, DMSO. Retinal status was evaluated at P35 and P42 by electroretinography, morphometry and apoptotic nuclei detection. DMSO and MuhPhe had no effect on RCS retinas as evidenced by equivalent loss of function and equivalent number of apoptotic cells than in untreated group. ZVAD transiently reduced apoptotic cells and preserved photoreceptor function at P35 but not at P42. These results suggest that caspases but not calpains are involved in retinal degeneration in the RCS. In the second experiments, RCS rats were injected twice at P27 and P35 with ZVAD or DMSO. Although ZVAD-treated retinas were preserved at P35 compared to the DMSO controls, the second injection of ZVAD did not extend the preserving effect to P42. Moreover, a single injection of ZVAD at P35 had no preserving effect at P42. All these data taken together suggest that caspases do not play a pivotal role after P35. In a fourth set of experiments, we used specific caspase inhibitors to elucidate which caspase was activated. The caspase-1/4 inhibitor (YVAD) or the caspase-3/7 inhibitor (DEVD) were injected intravitreally at P27 and retinal status was evaluated at P35 and P42. Electroretinograms and apoptotic nuclei detection demonstrated that YVAD and DEVD preserved photoreceptors at P35 but not at P42. These results suggest that both caspase-1/4 and caspase-3/7 play a major role in the apoptotic

Increased S-Nitrosylation and Proteasomal Degradation of Caspase-3 during Infection Contribute to the Persistence of Adherent Invasive Escherichia coli (AIEC) in Immune Cells

PubMed Central

Dunne, Karl A.; Allam, Amr; McIntosh, Anne; Houston, Stephanie A.; Cerovic, Vuk; Goodyear, Carl S.; Roe, Andrew J.; Beatson, Scott A.; Milling, Simon W.; Walker, Daniel; Wall, Daniel M.

2013-01-01

Adherent invasive Escherichia coli (AIEC) have been implicated as a causative agent of Crohn’s disease (CD) due to their isolation from the intestines of CD sufferers and their ability to persist in macrophages inducing granulomas. The rapid intracellular multiplication of AIEC sets it apart from other enteric pathogens such as Salmonella Typhimurium which after limited replication induce programmed cell death (PCD). Understanding the response of infected cells to the increased AIEC bacterial load and associated metabolic stress may offer insights into AIEC pathogenesis and its association with CD. Here we show that AIEC persistence within macrophages and dendritic cells is facilitated by increased proteasomal degradation of caspase-3. In addition S-nitrosylation of pro- and active forms of caspase-3, which can inhibit the enzymes activity, is increased in AIEC infected macrophages. This S-nitrosylated caspase-3 was seen to accumulate upon inhibition of the proteasome indicating an additional role for S-nitrosylation in inducing caspase-3 degradation in a manner independent of ubiquitination. In addition to the autophagic genetic defects that are linked to CD, this delay in apoptosis mediated in AIEC infected cells through increased degradation of caspase-3, may be an essential factor in its prolonged persistence in CD patients. PMID:23861899

Caspase selective reagents for diagnosing apoptotic mechanisms.

PubMed

Poreba, Marcin; Groborz, Katarzyna; Navarro, Mario; Snipas, Scott J; Drag, Marcin; Salvesen, Guy S

2018-05-10

Apical caspases initiate and effector caspases execute apoptosis. Reagents that can distinguish between caspases, particularly apical caspases-8, 9, and 10 are scarce and generally nonspecific. Based upon a previously described large-scale screen of peptide-based caspase substrates termed HyCoSuL, we sought to develop reagents to distinguish between apical caspases in order to reveal their function in apoptotic cell death paradigms. To this end, we selected tetrapeptide-based sequences that deliver optimal substrate selectivity and converted them to inhibitors equipped with a detectable tag (activity-based probes-ABPs). We demonstrate a strong relationship between substrate kinetics and ABP kinetics. To evaluate the utility of selective substrates and ABPs, we examined distinct apoptosis pathways in Jurkat T lymphocyte and MDA-MB-231 breast cancer lines triggered to undergo cell death via extrinsic or intrinsic apoptosis. We report the first highly selective substrate appropriate for quantitation of caspase-8 activity during apoptosis. Converting substrates to ABPs promoted loss-of-activity and selectivity, thus we could not define a single ABP capable of detecting individual apical caspases in complex mixtures. To overcome this, we developed a panel strategy utilizing several caspase-selective ABPs to interrogate apoptosis, revealing the first chemistry-based approach to uncover the participation of caspase-8, but not caspase-9 or -10 in TRAIL-induced extrinsic apoptosis. We propose that using select panels of ABPs can provide information regarding caspase-8 apoptotic signaling more faithfully than can single, generally nonspecific reagents.

Caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress.

PubMed

Zhang, Qiang; Liu, Jianing; Chen, Shulan; Liu, Jing; Liu, Lijuan; Liu, Guirong; Wang, Fang; Jiang, Wenxin; Zhang, Caixia; Wang, Shuangyu; Yuan, Xiao

2016-04-01

It is well recognized that mandibular growth, which is caused by a variety of functional appliances, is considered to be the result of both neuromuscular and skeletal adaptations. Accumulating evidence has demonstrated that apoptosis plays an important role in the adaptation of skeletal muscle function. However, the underlying mechanism of apoptosis that is induced by stretch continues to be incompletely understood. Endoplasmic reticulum stress (ERS), a newly defined signaling pathway, initiates apoptosis. This study seeks to determine if caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress in myoblast and its underlying mechanism. Apoptosis was assessed by Hochest staining, DAPI staining and annexin V binding and PI staining. ER chaperones, such as GRP78, CHOP and caspase-12, were determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Furthermore, caspase-12 inhibitor was used to value the mechanism of the caspase-12 pathway. Apoptosis of myoblast, which is subjected to cyclic stretch, was observed in a time-dependent manner. We found that GRP78 mRNA and protein were significantly increased and CHOP and caspase-12 were activated in myoblast that was exposed to cyclic stretch. Caspase-12 inhibition reduced stretch-induced apoptosis, and caspase-12 activated caspase-3 to induce apoptosis. We concluded that caspase-12 played an important role in stretch-induced apoptosis that is associated by endoplasmic reticulum stress by activating caspase-3.

Cordycepin induced eryptosis in mouse erythrocytes through a Ca2+-dependent pathway without caspase-3 activation.

PubMed

Lui, Julian C K; Wong, Judy W Y; Suen, Y K; Kwok, T T; Fung, K P; Kong, S K

2007-12-01

Cordyceps sinensis is a prized traditional Chinese medicine and its major component cordycepin is found to have anti-leukemia activities. However, its cytotoxicity in erythrocytes was unclear. To examine the effect of cordycepin on the induction of eryptosis (an apoptosis-like process in enucleated erythrocytes), flow cytometric assays based on membrane integrity and asymmetry were employed. For comparison, analyses were performed in parallel with two other anti-leukemia agents, indirubin 3'-monoxime (IDM) and As2O3. We found that at the IC50 against leukemia HL-60, cordycepin elicited eryptosis while IDM and As2O3 showed no erythrotoxicity in mouse erythrocytes. Mechanistically, cordycepin increased the [Ca2+]i and activated mu-calpain protease in a dose-dependent manner. Yet, no caspase-3 activation was observed in the cordycepin-treated erythrocytes. When extracellular Ca2+ was depleted, both the cordycepin-induced eryptosis and mu-calpain cleavage were suppressed. Our study therefore demonstrated for the first time that cordycepin induces eryptosis through a calcium-dependent pathway in the absence of mitochondria and caspase-3 activation.

A primer on caspase mechanisms.

PubMed

Ramirez, Monica L Gonzalez; Salvesen, Guy S

2018-01-12

Caspases belong to a diverse clan of proteolytic enzymes known as clan CD with highly disparate functions in cell signaling. The caspase members of this clan are only found in animals, and most of them orchestrate the demise of cells by the highly distinct regulated cell death phenotypes known as apoptosis and pyroptosis. This review looks at the mechanistic distinctions between the activity and activation mechanisms of mammalian caspases compared to other members of clan CD. We also compare and contrast the role of different caspase family members that program anti-inflammatory and pro-inflammatory cell death pathways. Copyright © 2018. Published by Elsevier Ltd.

Molecular cloning of sea bass (Dicentrarchus labrax L.) caspase-8 gene and its involvement in Photobacterium damselae ssp. piscicida triggered apoptosis.

PubMed

Reis, Marta I R; Costa-Ramos, Carolina; do Vale, Ana; dos Santos, Nuno M S

2010-07-01

Caspase-8 is an initiator caspase that plays a crucial role in some cases of apoptosis by extrinsic and intrinsic pathways. Caspase-8 structure and function have been extensively studied in mammals, but in fish the characterization of that initiator caspase is still scarce. In this work, the sea bass counterpart of mammalian caspase-8 was sequenced and characterized, and its involvement in the apoptogenic activity of a toxin from a fish pathogen was assessed. A 2472 bp cDNA of sea bass caspase-8 was obtained, consisting of 1455 bp open reading frame coding for 484 amino acids and with a predicted molecular weight of 55.2 kDa. The sea bass caspase-8 gene has 6639 bp and is organized in 11 introns and 12 exons. Several distinctive features of sea bass caspase-8 were identified, which include two death effector domains, the caspase family domains p20 and p10, the caspase-8 active-site pentapeptide and potential aspartic acid cleavage sites. The sea bass caspase-8 sequence revealed a significant degree of similarity to corresponding sequences from several vertebrate taxonomic groups. A low expression of sea bass caspase-8 was detected in various tissues of non-stimulated sea bass. Furthermore, it is shown that stimulation of sea bass with mid-exponential phase culture supernatants from Photobacterium damselae ssp. piscicida (Phdp), known to induce selective apoptosis of macrophages and neutrophils, resulted in an increased expression of caspase-8 in the spleen, one of the main affected organs by Phdp infection. 2010 Elsevier Ltd. All rights reserved.

Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling.

PubMed

Kayagaki, Nobuhiko; Stowe, Irma B; Lee, Bettina L; O'Rourke, Karen; Anderson, Keith; Warming, Søren; Cuellar, Trinna; Haley, Benjamin; Roose-Girma, Merone; Phung, Qui T; Liu, Peter S; Lill, Jennie R; Li, Hong; Wu, Jiansheng; Kummerfeld, Sarah; Zhang, Juan; Lee, Wyne P; Snipas, Scott J; Salvesen, Guy S; Morris, Lucy X; Fitzgerald, Linda; Zhang, Yafei; Bertram, Edward M; Goodnow, Christopher C; Dixit, Vishva M

2015-10-29

Intracellular lipopolysaccharide from Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Burkholderia thailandensis activates mouse caspase-11, causing pyroptotic cell death, interleukin-1β processing, and lethal septic shock. How caspase-11 executes these downstream signalling events is largely unknown. Here we show that gasdermin D is essential for caspase-11-dependent pyroptosis and interleukin-1β maturation. A forward genetic screen with ethyl-N-nitrosourea-mutagenized mice links Gsdmd to the intracellular lipopolysaccharide response. Macrophages from Gsdmd(-/-) mice generated by gene targeting also exhibit defective pyroptosis and interleukin-1β secretion induced by cytoplasmic lipopolysaccharide or Gram-negative bacteria. In addition, Gsdmd(-/-) mice are protected from a lethal dose of lipopolysaccharide. Mechanistically, caspase-11 cleaves gasdermin D, and the resulting amino-terminal fragment promotes both pyroptosis and NLRP3-dependent activation of caspase-1 in a cell-intrinsic manner. Our data identify gasdermin D as a critical target of caspase-11 and a key mediator of the host response against Gram-negative bacteria.

Caspase-responsive smart gadolinium-based contrast agent for magnetic resonance imaging of drug-induced apoptosis.

PubMed

Ye, Deju; Shuhendler, Adam J; Pandit, Prachi; Brewer, Kimberly D; Tee, Sui Seng; Cui, Lina; Tikhomirov, Grigory; Rutt, Brian; Rao, Jianghong

2014-10-01

Non-invasive detection of caspase-3/7 activity in vivo has provided invaluable predictive information regarding tumor therapeutic efficacy and anti-tumor drug selection. Although a number of caspase-3/7 targeted fluorescence and positron emission tomography (PET) imaging probes have been developed, there is still a lack of gadolinium (Gd)-based magnetic resonance imaging (MRI) probes that enable high spatial resolution detection of caspase-3/7 activity in vivo . Here we employ a self-assembly approach and develop a caspase-3/7 activatable Gd-based MRI probe for monitoring tumor apoptosis in mice. Upon reduction and caspase-3/7 activation, the caspase-sensitive nano-aggregation MR probe (C-SNAM: 1 ) undergoes biocompatible intramolecular cyclization and subsequent self-assembly into Gd-nanoparticles (GdNPs). This results in enhanced r 1 relaxivity-19.0 (post-activation) vs. 10.2 mM -1 s -1 (pre-activation) at 1 T in solution-and prolonged accumulation in chemotherapy-induced apoptotic cells and tumors that express active caspase-3/7. We demonstrate that C-SNAM reports caspase-3/7 activity by generating a significantly brighter T 1 -weighted MR signal compared to non-treated tumors following intravenous administration of C-SNAM, providing great potential for high-resolution imaging of tumor apoptosis in vivo .

Involvement of caspase-dependent and -independent apoptotic pathways in cisplatin-induced apoptosis

NASA Astrophysics Data System (ADS)

Liu, Lei; Zhang, Yingjie; Wang, Xianwang

2009-02-01

Cisplatin, an efficient anticancer agent, can trigger multiple apoptotic pathways in cancer cells. However, the signal transduction pathways in response to cisplatin-based chemotherapy are complicated, and the mechanism is not fully understood. In current study, we showed that, during cisplatin-induced apoptosis of human lung adenocarcinoma cells, both the caspase-dependent and -independent pathways were activated. Herein, we reported that after cisplatin treatment, the activities of caspase-9/-3 were sharply increased; pre-treatment with Z-LEHD-fmk (inhibitor of caspase-9), Z-DEVD-fmk (inhibitor of caspase-3), and Z-VAD-fmk (a pan-caspase inhibitor) increased cell viability and decreased apoptosis, suggesting that caspase-mediated apoptotic pathway was activated following cisplatin treatment. Confocal imaging of the cells transfected with AIF-GFP demonstrated that AIF release occurred about 9 h after cisplatin treatment. The event proceeded progressively over time, coinciding with a nuclear translocation and lasting for more than 2 hours. Down-regulation of AIF by siRNA also significantly increased cell viability and decreased apoptosis, these results suggested that AIF-mediated caspase-independent apoptotic pathway was involved in cispatin-induced apoptosis. In conclusion, the current study demonstrated that both caspase-dependent and -independent apoptotic pathways were involved in cisplatin-induced apoptosis in human lung adenocarcinoma cells.

The Effect of K+ on Caspase Activity of Corneal Epithelial Cells Exposed to UVB

PubMed Central

Leerar, John R.; Glupker, Courtney D.; Schotanus, Mark P.; Ubels, John L.

2016-01-01

Exposure of human corneal limbal epithelial (HCLE) cells to UVB triggers rapid loss of K+ and apoptosis via activation of caspases-9, -8 and -3. It has been shown that preventing loss of intracellular K+ can inhibit apoptosis. The goal of this study was to investigate the effect of K+ on the UVB-induced caspase activity. HCLE cells were exposed to 150 mJ/cm2 UVB, followed by measurement of caspase activity in cell lysates. Caspase activity was measured in the presence and absence of 100 mM K+ in the reaction buffer. UVB-induced activity of caspases-9, -8 and -3 all decreased in the presence of 100 mM K+. These results suggest that a role of high [K+] in the cell is to inhibit caspase activity. Therefore, when cells lose K+ in response to UVB, caspases are activated and cells go into apoptosis. This supports our hypothesis that K+ inhibits caspase activity. PMID:27456133

Extensive peptide and natural protein substrate screens reveal that mouse caspase-11 has much narrower substrate specificity than caspase-1

PubMed Central

Ramirez, Monica L. Gonzalez; Poreba, Marcin; Snipas, Scott J.; Groborz, Katarzyna; Drag, Marcin; Salvesen, Guy S.

2018-01-01

Inflammatory cell death, or pyroptosis, is triggered by pathogenic infections or events. It is executed by caspase-1 (in the canonical pyroptosis pathway) or caspase-11 (noncanonical pathway), each via production of a cell-lytic domain from the pyroptosis effector protein gasdermin D through specific and limited proteolysis. Pyroptosis is accompanied by the release of inflammatory mediators, including the proteolytically processed forms of interleukin-1β (IL-1β) and IL-18. Given the similar inflammatory outcomes of the canonical and noncanonical pyroptosis pathways, we hypothesized that caspase-1 and -11 should have very similar activities and substrate specificities. To test this hypothesis, we purified recombinant murine caspases and analyzed their primary specificities by massive hybrid combinatorial substrate library (HyCoSuL) screens. We correlated the substrate preferences of each caspase with their activities on the recombinant natural substrates IL-1β, IL-18, and gasdermin D. Although we identified highly selective and robust peptidyl substrates for caspase-1, we were unable to do so for caspase-11, because caspase-1 cleaved even the best caspase-11 substrates equally well. Caspase-1 rapidly processed pro-IL-1β and -18, but caspase-11 processed these two pro-ILs extremely poorly. However, both caspase-1 and -11 efficiently produced the cell-lytic domain from the gasdermin D precursor. We hypothesize that caspase-11 may have evolved a specific exosite to selectively engage pyroptosis without directly activating pro-IL-1β or -18. In summary, comparing the activities of caspase-1 and -11 in HyCoSuL screens and with three endogenous protein substrates, we conclude that caspase-11 has highly restricted substrate specificity, preferring gasdermin D over all other substrates examined. PMID:29414788

Extensive peptide and natural protein substrate screens reveal that mouse caspase-11 has much narrower substrate specificity than caspase-1.

PubMed

Ramirez, Monica L Gonzalez; Poreba, Marcin; Snipas, Scott J; Groborz, Katarzyna; Drag, Marcin; Salvesen, Guy S

2018-05-04

Inflammatory cell death, or pyroptosis, is triggered by pathogenic infections or events. It is executed by caspase-1 (in the canonical pyroptosis pathway) or caspase-11 (noncanonical pathway), each via production of a cell-lytic domain from the pyroptosis effector protein gasdermin D through specific and limited proteolysis. Pyroptosis is accompanied by the release of inflammatory mediators, including the proteolytically processed forms of interleukin-1β (IL-1β) and IL-18. Given the similar inflammatory outcomes of the canonical and noncanonical pyroptosis pathways, we hypothesized that caspase-1 and -11 should have very similar activities and substrate specificities. To test this hypothesis, we purified recombinant murine caspases and analyzed their primary specificities by massive hybrid combinatorial substrate library (HyCoSuL) screens. We correlated the substrate preferences of each caspase with their activities on the recombinant natural substrates IL-1β, IL-18, and gasdermin D. Although we identified highly selective and robust peptidyl substrates for caspase-1, we were unable to do so for caspase-11, because caspase-1 cleaved even the best caspase-11 substrates equally well. Caspase-1 rapidly processed pro-IL-1β and -18, but caspase-11 processed these two pro-ILs extremely poorly. However, both caspase-1 and -11 efficiently produced the cell-lytic domain from the gasdermin D precursor. We hypothesize that caspase-11 may have evolved a specific exosite to selectively engage pyroptosis without directly activating pro-IL-1β or -18. In summary, comparing the activities of caspase-1 and -11 in HyCoSuL screens and with three endogenous protein substrates, we conclude that caspase-11 has highly restricted substrate specificity, preferring gasdermin D over all other substrates examined. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

The NLRP3-Caspase 1 Inflammasome Negatively Regulates Autophagy via TLR4-TRIF in Prion Peptide-Infected Microglia

PubMed Central

Lai, Mengyu; Yao, Hao; Shah, Syed Zahid Ali; Wu, Wei; Wang, Di; Zhao, Ying; Wang, Lu; Zhou, Xiangmei; Zhao, Deming; Yang, Lifeng

2018-01-01

Prion diseases are neurodegenerative disorders characterized by the accumulation of misfolded prion protein, spongiform changes in the brain, and brain inflammation as a result of the wide-spread activation of microglia. Autophagy is a highly conserved catabolic process for the clearance of cytoplasmic components, including protein aggregates and damaged organelles; this process also eliminates pathological PrPSc as it accumulates during prion infection. The NALP3 inflammasome is a multiprotein complex that is a component of the innate immune system and is responsible for the release of pro-inflammatory cytokines. Our previous study showed that the neurotoxic prion peptide PrP106-126 induces NALP3 inflammasome activation and subsequent IL-1β release in microglia. Autophagy is involved in the regulation of the immune responses and inflammation in many diseases including neurodegenerative diseases. However, the relationship between autophagy and NALP3 inflammasome in prion diseases has not been investigated. In this study, we demonstrated that the processing and release of mature IL-1β is significantly enhanced by the inhibition of autophagy. Conversely, gene-silencing of the NALP3 inflammasome promotes autophagy. Suppression of TRIF or TLR4 by siRNA attenuated PrP106-126-induced autophagy, which is indicating that the TLR4-TRIF signaling pathway is involved in PrP106-26-induced autophagy. Caspase 1 directly cleaved TRIF to diminish TLR-4-TRIF mediated autophagy. Our findings suggest that the inhibition of autophagy by NALP3 inflammasome is probably mediated by activated Caspase-1-induced TRIF cleavage. This is the first study reporting that the NALP3 inflammasome complex negatively regulates autophagy in response to PrP106-126 stimulation in microglia, and partly explains the mechanism of autophagy inhibition by Caspase-1 in PrP106-126-induced BV2 cell activation. Our findings suggest that autophagy up-regulation and inhibition of Caspase-1 may protect against

Norcantharidin Induces Human Melanoma A375-S2 Cell Apoptosis through Mitochondrial and Caspase Pathways

PubMed Central

An, Wei-wei; Wang, Min-wei; Tashiro, Shin-ichi; Onodera, Satoshi

2004-01-01

Norcantharidin (NCTD) is the demethylated form of cantharidin, which is the active substance of mylabris. To examine the pathway of NCTD-induced A375-S2 cell death, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-dipheyltetrazolium bromide (MTT) assay, photomicroscopical observation, DNA agarose gel electrophoresis, caspase activity assay and Western blot analysis were carried out. A375-S2 cells treated with NCTD exhibited several typical characteristics of apoptosis. The inhibitory effect of NCTD on human melanoma, A375-S2 cells, was partially reversed by the inhibitors of pan-caspase, caspase-3 and caspase-9. The activities of caspase-3 and -9 were significantly increased after treatment with NCTD at different time. The expression of inhibitor of caspase-activated DNase was decreased in a time-dependent manner, simultaneously, the ratio of Bcl-2/Bax or Bcl-xL/Bax was decreased and the expression ratio of proteins could be reversed by caspase-3 inhibitor. The expression of cytochrome c in cytosol was increased after NCTD treatment and caspase-3 inhibitor had no significant effect on the up-regulation of cytochrom c. These results suggest that NCTD induced A375-S2 cell apoptosis and the activation of caspase and mitochondrial pathway were involved in the process of NCTD-induced A375-S2 cell apoptosis. PMID:15308848

Caspases and Kinases in a Death Grip

PubMed Central

Kurokawa, Manabu; Kornbluth, Sally

2011-01-01

The complex process of apoptosis is orchestrated by caspases, a family of cysteine proteases with unique substrate specificities. Accumulating evidence suggests that cell death pathways are finely tuned by multiple signaling events, including direct phosphorylation of caspases, whereas kinases are often substrates of active caspases. Importantly, caspase-mediated cleavage of kinases can terminate prosurvival signaling or generate proapoptotic peptide fragments that help to execute the death program and facilitate packaging of the dying cells. Here, we review caspases as kinase substrates and kinases as caspase substrates and discuss how the balance between cell survival and cell death can be shifted through crosstalk between these two enzyme families. PMID:19737514

Real-time detection of caspase-2 activation in a single living HeLa cell during cisplatin-induced apoptosis

NASA Astrophysics Data System (ADS)

Lin, Juqiang; Zhang, Zhihong; Yang, Jie; Zeng, Shaoqun; Liu, Bifeng; Luo, Qingming

2006-03-01

Caspase-2 is important for the mitochondrial apoptotic pathway, however, the mechanism by which caspase-2 executes apoptosis remains obscure. We carry out the first measurements of the dynamics of caspase-2 activation in a single living cell by a FRET (fluorescence resonance energy transfer) probe. Two FRET probes are constructed that each encoded a CRS (caspase-2 or caspase-3 recognition site) fused with a cyan fluorescent protein (CFP) and a red fluorescent protein (DsRed) (CFP-CRS-DsRed). Using these probes, we found that during cisplatin-induced apoptosis, caspase-2 activation occurred more slowly than did activation of caspase-3; additionally, caspase-2 activation was initiated much earlier than that of caspase-3.

[Effect of qihuang decoction on mRNA expressions of Bcl-2, Bax, and Caspase-3, 9 in intestinal mucosa epithelium of ischemia/reperfusion injured rats].

PubMed

Yu, Qing-Sheng; Yu, Hong-Liang; Pan, Jin-Fang

2011-02-01

To observe the effect of Qihuang Decoction (QHD) on mRNA expression of apoptosis genes Bcl-2, Bax, and signal transduction molecules Caspase-3, 9 in intestinal mucosa epithelium of ischemia/ reperfusion (I/R) injured rats. Forty Wistar rats were randomized equally into 4 groups, the control group, the model group, the glutamine group, and the QHD group. Rats in the latter two groups were gastric infused with glutamine and QHD respectively for 3 days, but saline was infused instead to rats in the control group and model group. After then, except those in the control group intervened only by sham operation, rats were made into I/R injured model by 45 min occlusion of superior mesenteric artery followed by 1 h reperfusion. Immediately after modeling, mRNA expressions of Bcl-2, Bax, Caspase-3, and Caspase-9 in intestinal mucosa epithelium of rats were detected by reverse transcription-polymerase chain reaction (RT-PCR). Compared with the control group, mRNA expressions of Bcl-2, Bax, Caspase-3 and Caspase-9 were higher in the other three groups (P < 0.05). Compared with the model group, Bcl-2 mRNA expression was higher, while the expressions of the other three indices were lower in both the glutamine group and the QHD group (P < 0.05); and comparisons between the glutamine group and the QHD group showed a more depressed Bax mRNA expression (0.281 +/- 0.087 vs 0.350 +/- 0.053) and higher Bcl-2/Bax ratio (1.648 vs 1. 374) in the QHD group. QHD can reduce the I/R injury in the intestinal mucosa epithelium by inhibiting the cell apoptosis. The mechanism may be correlated with increased Bcl-2 mRNA expressions and decreased mRNA expressions of Bax, Caspase-3 and Caspase-9.

Auraptene Induces Apoptosis via Myeloid Cell Leukemia 1-Mediated Activation of Caspases in PC3 and DU145 Prostate Cancer Cells.

PubMed

Lee, Jae Chul; Shin, Eun Ah; Kim, Bonglee; Kim, Bo-Im; Chitsazian-Yazdi, Mahsa; Iranshahi, Mehrdad; Kim, Sung-Hoon

2017-06-01

Although auraptene, a prenyloxy coumarin from Citrus species, was known to have anti-oxidant, anti-bacterial, antiinflammatory, and anti-tumor activities, the underlying anti-tumor mechanism of auraptene in prostate cancers is not fully understood to date. Thus, in the present study, we have investigated the anti-tumor mechanism of auraptene mainly in PC3 and DU145 prostate cancer cells, because auraptene suppressed the viability of androgen-independent PC3 and DU145 prostate cancer cells better than androgen-sensitive LNCaP cells. Also, auraptene notably increased sub-G1 cell population and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells as features of apoptosis in two prostate cancer cells compared with untreated control. Consistently, auraptene cleaved poly(ADP-ribose) polymerase, activated caspase-9 and caspase-3, suppressed the expression of anti-apoptotic proteins, including Bcl-2 and myeloid cell leukemia 1 (Mcl-1), and also activated pro-apoptotic protein Bax in both prostate cancer cells. However, Mcl-1 overexpression reversed the apoptotic effect of auraptene to increase sub-G1 population and induce caspase-9/3 in both prostate cancer cells. Taken together, the results support scientific evidences that auraptene induces apoptosis in PC3 and DU145 prostate cancer cells via Mcl-1-mediated activation of caspases as a potent chemopreventive agent for prostate cancer prevention and treatment. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Radiation inhibits salivary gland function by promoting STIM1 cleavage by caspase-3 and loss of SOCE through a TRPM2-dependent pathway

PubMed Central

Liu, Xibao; Gong, Baijuan; de Souza, Lorena Brito; Ong, Hwei Ling; Subedi, Krishna P.; Cheng, Kwong Tai; Swaim, William; Zheng, Changyu; Mori, Yasuo; Ambudkar, Indu S.

2017-01-01

Store-operated Ca2+ entry (SOCE) is critical for salivary gland fluid secretion. We report that radiation treatment caused persistent salivary gland dysfunction by activating a TRPM2-dependent mitochondrial pathway, leading to caspase-3–mediated cleavage of stromal interaction molecule 1 (STIM1) and loss of SOCE. After irradiation, acinar cells from the submandibular glands of TRPM2+/+, but not those from TRPM2−/− mice, displayed an increase in the concentrations of mitochondrial Ca2+ and reactive oxygen species, a decrease in mitochondrial membrane potential, and activation of caspase-3, which was associated with a sustained decrease in STIM1 abundance and attenuation of SOCE. In a salivary gland cell line, silencing the mitochondrial Ca2+ uniporter or caspase-3 or treatment with inhibitors of TRPM2 or caspase-3 prevented irradiation-induced loss of STIM1 and SOCE. Expression of exogenous STIM1 in the salivary glands of irradiated mice increased SOCE and fluid secretion. We suggest that targeting the mechanisms underlying the loss of STIM1 would be a potentially useful approach for preserving salivary gland function after radiation therapy. PMID:28588080

Detection of Mitochondrial Caspase Activity in Real Time In Situ in Live Cells

NASA Astrophysics Data System (ADS)

Zhang, Yingpei; Haskins, Catherine; Lopez-Cruzan, Marisa; Zhang, Jianhua; Centonze, Victoria E.; Herman, Brian

2004-08-01

Apoptosis plays an important role in many physiological and pathological processes. The initiation and execution of the cell death program requires activation of multiple caspases in a stringently temporal order. Here we describe a method that allows real-time observation of caspase activation in situ in live cells based on fluorescent resonance energy transfer (FRET) measurement using the prism and reflector imaging spectroscopy system (PARISS). When a fusion protein consisting of CFP connected to YFP via an intervening caspase substrate that has been targeted to a specific subcellular location is excited with a light source whose wavelength matches the cyan fluorescent protein (CFP) excitation peak, the energy absorbed by the CFP fluorophore is not emitted as fluorescence. Instead, the excitation energy is absorbed by the nearby yellow fluorescent protein (YFP) fluorophore that is covalently linked to CFP through a short peptide containing the caspase substrate. Cleavage of the linker peptide by caspases results in loss of FRET due to the separation of CFP and YFP fluorophores. Using a mitochondrially targeted CFP caspase 3 substrate YFP construct (mC3Y), we demonstrate for the first time that there is caspase-3-like activity in the mitochondrial matrix of some cells at very late stage of apoptosis.

p62/sequestosome-1 up-regulation promotes ABT-263-induced caspase-8 aggregation/activation on the autophagosome.

PubMed

Huang, Shengbing; Okamoto, Koichi; Yu, Chunrong; Sinicrope, Frank A

2013-11-22

Autophagy and apoptosis regulate cancer cell viability in response to cytotoxic stress; however, their functional relationship remains unclear. p62/sequestosome 1 is a multifunctional protein and a signaling hub that shuttles ubiquitinated proteins to the lysosome during autophagy. Autophagy inhibition up-regulates p62, and prior data suggest that p62 may mediate apoptosis. Here, we demonstrate that p62 can regulate a caspase-8-dependent apoptosis in response to the BH3 mimetic agent, ABT-263. Up-regulation of p62 was shown to enhance ABT-263-induced caspase-8 activation that was Bax-dependent and resulted from mitochondrial amplification. Dependence upon caspase-8 was confirmed using caspase-8-deficient cells and by caspase-8 siRNA. Ectopic wild-type p62, but not p62 mutants with loss of ability to promote apoptosis, was shown to co-localize with caspase-8 and to promote its self-aggregation in ABT-263-treated cells, shown using a bimolecular fluorescence complementation assay. Endogenous p62 co-localized with caspase-8 in the presence of ABT-263 plus an autophagy inhibitor. Caspase-8 was shown to interact and co-localize with the autophagosome marker, LC3II. Knockdown of p62 attenuated binding between caspase-8 and LC3II, whereas p62 overexpression enhanced the co-localization of caspase-8 aggregates with LC3. LC3 knockdown did not affect interaction between caspase-8 and p62, suggesting that p62 may facilitate caspase-8 translocation to the autophagosomal membrane. A direct activator of caspase-8, i.e., TRAIL, alone or combined with ABT-263, induced caspase-8 aggregation and co-localization with p62 that was associated with a synergistic drug interaction. Together, these results demonstrate that up-regulation of p62 can mediate apoptosis via caspase-8 in the setting of autophagy inhibition.

p62/Sequestosome-1 Up-regulation Promotes ABT-263-induced Caspase-8 Aggregation/Activation on the Autophagosome*

PubMed Central

Huang, Shengbing; Okamoto, Koichi; Yu, Chunrong; Sinicrope, Frank A.

2013-01-01

Autophagy and apoptosis regulate cancer cell viability in response to cytotoxic stress; however, their functional relationship remains unclear. p62/sequestosome 1 is a multifunctional protein and a signaling hub that shuttles ubiquitinated proteins to the lysosome during autophagy. Autophagy inhibition up-regulates p62, and prior data suggest that p62 may mediate apoptosis. Here, we demonstrate that p62 can regulate a caspase-8-dependent apoptosis in response to the BH3 mimetic agent, ABT-263. Up-regulation of p62 was shown to enhance ABT-263-induced caspase-8 activation that was Bax-dependent and resulted from mitochondrial amplification. Dependence upon caspase-8 was confirmed using caspase-8-deficient cells and by caspase-8 siRNA. Ectopic wild-type p62, but not p62 mutants with loss of ability to promote apoptosis, was shown to co-localize with caspase-8 and to promote its self-aggregation in ABT-263-treated cells, shown using a bimolecular fluorescence complementation assay. Endogenous p62 co-localized with caspase-8 in the presence of ABT-263 plus an autophagy inhibitor. Caspase-8 was shown to interact and co-localize with the autophagosome marker, LC3II. Knockdown of p62 attenuated binding between caspase-8 and LC3II, whereas p62 overexpression enhanced the co-localization of caspase-8 aggregates with LC3. LC3 knockdown did not affect interaction between caspase-8 and p62, suggesting that p62 may facilitate caspase-8 translocation to the autophagosomal membrane. A direct activator of caspase-8, i.e., TRAIL, alone or combined with ABT-263, induced caspase-8 aggregation and co-localization with p62 that was associated with a synergistic drug interaction. Together, these results demonstrate that up-regulation of p62 can mediate apoptosis via caspase-8 in the setting of autophagy inhibition. PMID:24121507

Mitomycin-C induces the apoptosis of human Tenon's capsule fibroblast by activation of c-Jun N-terminal kinase 1 and caspase-3 protease.

PubMed

Seong, Gong Je; Park, Channy; Kim, Chan Yoon; Hong, Young Jae; So, Hong-Seob; Kim, Sang-Duck; Park, Raekil

2005-10-01

To investigate whether mitochondrial dysfunction and mitogen-activated protein kinase family proteins are implicated in apoptotic signaling of human Tenon's capsule fibroblasts (HTCFs) by mitomycin-C. Apoptosis was determined by Hoechst nuclei staining, agarose gel electrophoresis, and flow cytometry in HTCFs treated with 0.4 mg/mL mitomycin-C for 5 minutes. Enzymatic digestion of florigenic biosubstrate assessed the catalytic activity of caspase proteases, including caspase-3, caspase-8, and caspase-9. Phosphotransferase activity of c-Jun N-terminal kinase (JNK) 1 was measured by in vitro immune complex kinase assay using c-Jun(1-79) protein as a substrate. Mitochondrial membrane potential transition (MPT) was measured by flow cytometric analysis of JC-1 staining. Mitomycin-C (0.4 mg/mL) induced the apoptosis of HTCFs, which was characterized as nucleic acid and genomic DNA fragmentation, chromatin condensation, and sub-G(0)/G(1) fraction of cell cycle increase. The catalytic activity of caspase-3 and caspase-9 was significantly increased and was accompanied by cytosolic release of cytochrome c and MPT in response to mitomycin-C. Treatment with mitomycin-C resulted in the increased expression of Fas, FasL, Bad, and phosphorylated p53 and a decreased level of phosphorylated AKT. Treatment with mitomycin-C also increased the phosphotransferase activity and tyrosine phosphorylation of JNK1, whose inhibitor significantly suppressed the cytotoxicity of mitomycin-C. Mitomycin-C induced the apoptosis of HTCFs through the activation of intrinsic and extrinsic caspase cascades with mitochondrial dysfunction. It also activated Fas-mediated apoptotic signaling of fibroblasts. Furthermore, the activation of JNK1 played a major role in the cytotoxicity of mitomycin-C.

Curcumin induces Apaf-1-dependent, p21-mediated caspase activation and apoptosis

PubMed Central

Zhang, Honghao; Jones, Anthony; Verone, Alissa; Pitarresi, Jason; Jandhyam, Sirisha; Prabhu, Varun; Black, Jennifer D

2011-01-01

Previous studies have demonstrated that curcumin induces mitochondria-mediated apoptosis. However, understanding of the molecular mechanisms underlying curcumin-induced cell death remains limited. In this study, we demonstrate that curcumin treatment of cancer cells caused dose- and time-dependent caspase 3 activation, which is required for apoptosis as confirmed using the pan-caspase inhibitor, z-VAD. Knockdown experiments and knockout cells excluded a role for caspase 8 in curcumin-induced caspase 3 activation. In contrast, Apaf-1 deficiency or silencing inhibited the activity of caspase 3, pointing to a requisite role of Apaf-1 in curcumin-induced apoptotic cell death. Curcumin treatment led to Apaf-1 upregulation, both at the protein and mRNA levels. Cytochrome c release from mitochondria to the cytosol in curcumin-treated cells was associated with upregulation of pro-apoptotic proteins, such as Bax, Bak, Bid and Bim. Cross-linking experiments demonstrated Bax oligomerization during curcumin-induced apoptosis, suggesting that induced expression of Bax, Bid and Bim causes Bax channel formation on the mitochondrial membrane. The release of cytochrome c was unaltered in p53-deficient cells, whereas absence of p21 blocked cytochrome c release, caspase activation and apoptosis. Importantly, p21 deficiency resulted in reduced expression of Apaf-1 during curcumin treatment, indicating a requirement for p21 in Apaf-1-dependent caspase activation and apoptosis. Together, our findings identify Apaf-1, Bax and p21 as novel potential targets for curcumin or curcumin-based anticancer agents. PMID:22101335

PARP-1 regulates the expression of caspase-11

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoo, Lang; Hong, Seokheon; Shin, Ki Soon

2011-05-13

Highlights: {yields} Knockdown of PARP-1 suppresses the LPS-induced expression of caspase-11. {yields} Knockdown of PARP-1 suppresses the caspase-11 promoter activity following LPS stimulation. {yields} PARP-1 is recruited to the caspase-11 promoter region containing NF-{kappa}B-binding sites following LPS stimulation. {yields} PARP-1 inhibitors cannot suppress the caspase-11 induction. {yields} PARP-1 does not suppress IFN-{gamma}-induced expression of caspase-11. -- Abstract: Poly(ADP-ribose) polymerase-1 (PARP-1) is a multifunctional enzyme that regulates DNA repair, cell death and transcription of inflammatory proteins. In the present study, we present evidence that PARP-1 regulates the expression of caspase-11 following lipopolysaccharide (LPS) stimulation. Knockdown of PARP-1 suppressed the LPS-induced expressionmore » of caspase-11 at both mRNA and protein levels as well as caspase-11 promoter activity. Importantly, PARP-1 was recruited to the caspase-11 promoter region containing predicted nuclear factor (NF)-{kappa}B-binding sites when examined by chromatin immunoprecipitation assay. However, knockdown of PARP-1 did not suppress the expression of caspase-11 induced by interferon-{gamma} that activates signal transducer and activator of transcription 1 but not NF-{kappa}B. PARP-1 enzymatic activity was not required for the caspase-11 upregulation since pharmacological inhibitors of PARP-1 did not suppress the induction of caspase-11. Our results suggest that PARP-1, as a transcriptional cofactor for NF-{kappa}B, regulates the induction of caspase-11 at a transcriptional level.« less

A methylene chloride fraction of Saururus chinensis induces apoptosis through the activation of caspase-3 in prostate and breast cancer cells.

PubMed

Kim, Han-Young; Choi, Tae Won; Kim, Hyun Jung; Kim, Sung-Moo; Park, Kyung-Ran; Jang, Hyeung-Jin; Lee, Eun Ha; Kim, Chul Young; Jung, Sang Hoon; Shim, Bum Sang; Ahn, Kwang Seok

2011-05-15

The aerial parts of Saururus chinensis (SC) have been used for the treatment of edema, fever, jaundice, and inflammatory diseases in Korean folk medicine for centuries. However, the mechanism by which SC exerts these anti-tumorigenic activities in human prostate and breast cancer cells has not yet been fully understood. In this study, we report on the methylene chloride fraction from SC exerting cytotoxicity against prostate and breast cancer cells in a dose-dependent manner. Specifically, SC exerted the most potent cytotoxicity in LNCaP and MCF-7 cells. SC was shown to down-regulate various angiogenetic (VEGF), proliferative (Cyclin D₁, anti-apoptotic (Bcl-2) gene products in these cells. SC also increased the number of annexin V-positive apoptotic bodies and the sub-G1 DNA contents of the cell cycle undergoing apoptosis through caspase-3 activation in both LNCaP and MCF-7 cells. We further confirmed that caspase-3 plays an important role in SC-induced apoptosis in LNCaP and MCF-7 cells through the use of the caspase-3 inhibitor. Moreover, we observed that SC potentiated paclitaxel-induced apoptosis in MCF-7 cells and sauchinone is a major active constituent of SC, which could induce apoptosis in the cells. Taken together, our data provide the evidence that SC induces apoptosis depending on caspase-3 activation and overcomes the natural biological resistance to chemotherapy found in human prostate and breast cancer cells. Copyright © 2010 Elsevier GmbH. All rights reserved.

Brain caspase-3 and intestinal FABP responses in preterm and term rats submitted to birth asphyxia.

PubMed

Figueira, R L; Gonçalves, F L; Simões, A L; Bernardino, C A; Lopes, L S; Castro E Silva, O; Sbragia, L

2016-06-23

Neonatal asphyxia can cause irreversible injury of multiple organs resulting in hypoxic-ischemic encephalopathy and necrotizing enterocolitis (NEC). This injury is dependent on time, severity, and gestational age, once the preterm babies need ventilator support. Our aim was to assess the different brain and intestinal effects of ischemia and reperfusion in neonate rats after birth anoxia and mechanical ventilation. Preterm and term neonates were divided into 8 subgroups (n=12/group): 1) preterm control (PTC), 2) preterm ventilated (PTV), 3) preterm asphyxiated (PTA), 4) preterm asphyxiated and ventilated (PTAV), 5) term control (TC), 6) term ventilated (TV), 7) term asphyxiated (TA), and 8) term asphyxiated and ventilated (TAV). We measured body, brain, and intestine weights and respective ratios [(BW), (BrW), (IW), (BrW/BW) and (IW/BW)]. Histology analysis and damage grading were performed in the brain (cortex/hippocampus) and intestine (jejunum/ileum) tissues, as well as immunohistochemistry analysis for caspase-3 and intestinal fatty acid-binding protein (I-FABP). IW was lower in the TA than in the other terms (P<0.05), and the IW/BW ratio was lower in the TA than in the TAV (P<0.005). PTA, PTAV and TA presented high levels of brain damage. In histological intestinal analysis, PTAV and TAV had higher scores than the other groups. Caspase-3 was higher in PTAV (cortex) and TA (cortex/hippocampus) (P<0.005). I-FABP was higher in PTAV (P<0.005) and TA (ileum) (P<0.05). I-FABP expression was increased in PTAV subgroup (P<0.0001). Brain and intestinal responses in neonatal rats caused by neonatal asphyxia, with or without mechanical ventilation, varied with gestational age, with increased expression of caspase-3 and I-FABP biomarkers.

Brain caspase-3 and intestinal FABP responses in preterm and term rats submitted to birth asphyxia

PubMed Central

Figueira, R.L.; Gonçalves, F.L.; Simões, A.L.; Bernardino, C.A.; Lopes, L.S.; Castro e Silva, O.; Sbragia, L.

2016-01-01

Neonatal asphyxia can cause irreversible injury of multiple organs resulting in hypoxic-ischemic encephalopathy and necrotizing enterocolitis (NEC). This injury is dependent on time, severity, and gestational age, once the preterm babies need ventilator support. Our aim was to assess the different brain and intestinal effects of ischemia and reperfusion in neonate rats after birth anoxia and mechanical ventilation. Preterm and term neonates were divided into 8 subgroups (n=12/group): 1) preterm control (PTC), 2) preterm ventilated (PTV), 3) preterm asphyxiated (PTA), 4) preterm asphyxiated and ventilated (PTAV), 5) term control (TC), 6) term ventilated (TV), 7) term asphyxiated (TA), and 8) term asphyxiated and ventilated (TAV). We measured body, brain, and intestine weights and respective ratios [(BW), (BrW), (IW), (BrW/BW) and (IW/BW)]. Histology analysis and damage grading were performed in the brain (cortex/hippocampus) and intestine (jejunum/ileum) tissues, as well as immunohistochemistry analysis for caspase-3 and intestinal fatty acid-binding protein (I-FABP). IW was lower in the TA than in the other terms (P<0.05), and the IW/BW ratio was lower in the TA than in the TAV (P<0.005). PTA, PTAV and TA presented high levels of brain damage. In histological intestinal analysis, PTAV and TAV had higher scores than the other groups. Caspase-3 was higher in PTAV (cortex) and TA (cortex/hippocampus) (P<0.005). I-FABP was higher in PTAV (P<0.005) and TA (ileum) (P<0.05). I-FABP expression was increased in PTAV subgroup (P<0.0001). Brain and intestinal responses in neonatal rats caused by neonatal asphyxia, with or without mechanical ventilation, varied with gestational age, with increased expression of caspase-3 and I-FABP biomarkers. PMID:27356106

Caspase-1 but Not Caspase-11 Is Required for NLRC4-Mediated Pyroptosis and Restriction of Infection by Flagellated Legionella Species in Mouse Macrophages and In Vivo.

PubMed

Cerqueira, Daiane M; Pereira, Marcelo S F; Silva, Alexandre L N; Cunha, Larissa D; Zamboni, Dario S

2015-09-01

Gram-negative bacteria from the Legionella genus are intracellular pathogens that cause a severe form of pneumonia called Legionnaires' disease. The bacteria replicate intracellularly in macrophages, and the restriction of bacterial replication by these cells is critical for host resistance. The activation of the NAIP5/NLRC4 inflammasome, which is readily triggered in response to bacterial flagellin, is essential for the restriction of bacterial replication in murine macrophages. Once activated, this inflammasome induces pore formation and pyroptosis and facilitates the restriction of bacterial replication in macrophages. Because investigations related to the NLRC4-mediated restriction of Legionella replication were performed using mice double deficient for caspase-1 and caspase-11, we assessed the participation of caspase-1 and caspase-11 in the functions of the NLRC4 inflammasome and the restriction of Legionella replication in macrophages and in vivo. By using several species of Legionella and mice singly deficient for caspase-1 or caspase-11, we demonstrated that caspase-1 but not caspase-11 was required for pore formation, pyroptosis, and restriction of Legionella replication in macrophages and in vivo. By generating F1 mice in a mixed 129 × C57BL/6 background deficient (129 × Casp-11(-/-) ) or sufficient (129 × C57BL/6) for caspase-11 expression, we found that caspase-11 was dispensable for the restriction of Legionella pneumophila replication in macrophages and in vivo. Thus, although caspase-11 participates in flagellin-independent noncanonical activation of the NLRP3 inflammasome, it is dispensable for the activities of the NLRC4 inflammasome. In contrast, functional caspase-1 is necessary and sufficient to trigger flagellin/NLRC4-mediated restriction of Legionella spp. infection in macrophages and in vivo. Copyright © 2015 by The American Association of Immunologists, Inc.

Inhibition of caspase-1 or gasdermin-D enable caspase-8 activation in the Naip5/NLRC4/ASC inflammasome

PubMed Central

Pereira, Marcelo S. F.; Manin, Graziele Z.; Cunha, Larissa D.

2017-01-01

Legionella pneumophila is a Gram-negative, flagellated bacterium that survives in phagocytes and causes Legionnaires’ disease. Upon infection of mammalian macrophages, cytosolic flagellin triggers the activation of Naip/NLRC4 inflammasome, which culminates in pyroptosis and restriction of bacterial replication. Although NLRC4 and caspase-1 participate in the same inflammasome, Nlrc4-/- mice and their macrophages are more permissive to L. pneumophila replication compared with Casp1/11-/-. This feature supports the existence of a pathway that is NLRC4-dependent and caspase-1/11-independent. Here, we demonstrate that caspase-8 is recruited to the Naip5/NLRC4/ASC inflammasome in response to flagellin-positive bacteria. Accordingly, caspase-8 is activated in Casp1/11-/- macrophages in a process dependent on flagellin, Naip5, NLRC4 and ASC. Silencing caspase-8 in Casp1/11-/- cells culminated in macrophages that were as susceptible as Nlrc4-/- for the restriction of L. pneumophila replication. Accordingly, macrophages and mice deficient in Asc/Casp1/11-/- were more susceptible than Casp1/11-/- and as susceptible as Nlrc4-/- for the restriction of infection. Mechanistically, we found that caspase-8 activation triggers gasdermin-D-independent pore formation and cell death. Interestingly, caspase-8 is recruited to the Naip5/NLRC4/ASC inflammasome in wild-type macrophages, but it is only activated when caspase-1 or gasdermin-D is inhibited. Our data suggest that caspase-8 activation in the Naip5/NLRC4/ASC inflammasome enable induction of cell death when caspase-1 or gasdermin-D is suppressed. PMID:28771586

Inhibition of caspase-1 or gasdermin-D enable caspase-8 activation in the Naip5/NLRC4/ASC inflammasome.

PubMed

Mascarenhas, Danielle P A; Cerqueira, Daiane M; Pereira, Marcelo S F; Castanheira, Fernanda V S; Fernandes, Talita D; Manin, Graziele Z; Cunha, Larissa D; Zamboni, Dario S

2017-08-01

Legionella pneumophila is a Gram-negative, flagellated bacterium that survives in phagocytes and causes Legionnaires' disease. Upon infection of mammalian macrophages, cytosolic flagellin triggers the activation of Naip/NLRC4 inflammasome, which culminates in pyroptosis and restriction of bacterial replication. Although NLRC4 and caspase-1 participate in the same inflammasome, Nlrc4-/- mice and their macrophages are more permissive to L. pneumophila replication compared with Casp1/11-/-. This feature supports the existence of a pathway that is NLRC4-dependent and caspase-1/11-independent. Here, we demonstrate that caspase-8 is recruited to the Naip5/NLRC4/ASC inflammasome in response to flagellin-positive bacteria. Accordingly, caspase-8 is activated in Casp1/11-/- macrophages in a process dependent on flagellin, Naip5, NLRC4 and ASC. Silencing caspase-8 in Casp1/11-/- cells culminated in macrophages that were as susceptible as Nlrc4-/- for the restriction of L. pneumophila replication. Accordingly, macrophages and mice deficient in Asc/Casp1/11-/- were more susceptible than Casp1/11-/- and as susceptible as Nlrc4-/- for the restriction of infection. Mechanistically, we found that caspase-8 activation triggers gasdermin-D-independent pore formation and cell death. Interestingly, caspase-8 is recruited to the Naip5/NLRC4/ASC inflammasome in wild-type macrophages, but it is only activated when caspase-1 or gasdermin-D is inhibited. Our data suggest that caspase-8 activation in the Naip5/NLRC4/ASC inflammasome enable induction of cell death when caspase-1 or gasdermin-D is suppressed.

[Construction of autocatalytic caspase-3 driven by amplified human telomerase reverse transcriptase promoter and its enhanced efficacy of inducing apoptosis in human ovarian carcinoma].

PubMed

Song, Yue; Shen, Keng; He, Chun-Xia

2007-09-01

To construct recombinant adenoviral vector expressing autocatalysis caspase-3 driven by human telomerase reverse transcriptase promoter amplified by two-step transcription amplification (hTERTp-TSTA), and investigate its antitumor effect in ovarian cancer in vitro and in vivo. Recombinant adenoviruses expressing autocatalytic caspase-3 (rev-caspase-3) driven by hTERTp-TSTA were prepared, which were named as AdHTVP2G5-rev-casp3. AdHT-rev-casp3, Ad-rev-casp3 and AdHTVP2G5-EGEP, which express rev-caspase-3 driven by hTERTp, cytomegalovirus promoter (CMVp) and enhanced green fluorescent protein (EGFP), respectively, were used as controls. Western blot, cell counting kit (CCK-8), flow cytometry (FCM) and TdT-mediated dUTP-biotin nick end labeling (TUNEL) were used to detect the expression of p17, active subunit of caspase-3, and p85, and to measure cell survival rates, apoptotic rates and cell cycle distribution in ovarian cell line AO and normal human umbilical vein endothelial cell line HUVEC, following treatments of AdHTVP2G5-rev-casp3. subcutaneous tumor models and abdominally spread tumor models of human ovarian carcinoma using AO cells in BALB/c nude mice were established. Following treatments of AdHTVP2G5-rev-casp3, western blot was used to detect the expression of active caspase-3 in abdominally spread tumors and liver tissues, respectively, and the mouse survival rates and the volume of tumor nodules were measured, and the serum level of alanine transaminase (ALT) and aspartate transaminase (AST) were analyzed to monitor liver damages and HE staining was used to detect the histopathological changes of various organs. The levels of p17 expression in AdHTVP2G5-rev-casp3-treated AO cells were significantly higher than that in Ad-rev-casp3 or AdHT-rev-casp3 treated AO cells, while no expression was observed in AdHTVP2G5-rev-casp3-treated HUVEC. There was strong cell killing of AdHTVP2G5-rev-casp3 of hTERT positive AO cells, but not of the hTERT-negative HUVEC cells

Metabolic Regulation of Caspase 2 in Breast Cancer

DTIC Science & Technology

2011-04-01

S. The apoptosome: physiological, developmental, and pathological modes of regulation. Dev Cell 10, 549-61 ( 2006 ). 3. Baliga, B.C., Read, S.H...Mol Biol Cell 17, 2150-7 ( 2006 ). 9. Bergeron, L. et al. Defects in regulation of apoptosis in caspase-2-deficient mice. Genes Dev 12, 1304-14 (1998...Warburg, O. On the origin of cancer cells. Science 123, 309-14 (1956). 17. Lassus, P., Opitz- Araya , X. & Lazebnik, Y. Requirement for caspase-2 in

A Caspase-Resistant Form of Bcl-XL, but Not Wild Type Bcl-XL, Promotes Clonogenic Survival After Ionizing Radiation

PubMed Central

Rehemtulla, Alnawaz; Hamilton, A Christin; Taneja, Neelam; Fridman, Jordan; Juan, Todd SC; Maybaum, Jonathan; Chinnaiyan, Arul

1999-01-01

Abstract Bcl-2 and Bcl-XL belong to a family of proteins overexpressed in a variety of human cancers which inhibit apoptosis in response to a number of stimuli including chemotherapeutic agents and ionizing radiation. To better understand the role of these polypeptides in modulating the response of cancer cells to ionizing radiation we used cell lines that were engineered to overexpress the two polypeptides. Although Bcl-2 and Bcl-XL overexpression resulted in inhibition of radiation-induced apoptosis, it did not result in enhanced clonogenic survival. Consistent with this was the observation that Bcl-2 and Bcl-XL protected cells from DNA fragmentation, loss of mitochondrial membrane potential, and caspase activation for up to 72 hours after irradiation. Beyond 72 hours, there was a rapid loss in the ability of Bcl-2 and Bcl-XL to inhibit these markers of apoptosis. When Bcl-XL was analyzed at 72 hours after irradiation and beyond, a rapid accumulation of a 16-kDa form of Bcl-XL was observed. To test the hypothesis that cleavage of the 29-kDa form of Bcl-XL by caspases to a 16-kDa polypeptide results in its inability to inhibit apoptosis beyond 72 hours, we constructed a cell line that overexpressed a caspase-resistant form of Bcl-XL Bcl-XLΔloop. Cells overexpressing Bcl-XL-Δloop were resistant to apoptosis beyond 72 hours after irradiation and did not contain the 16-kDa form at these time points. In addition, Bcl-XL-Δloop overexpression resulted in enhanced clonogenic survival compared with control or Bcl-XL overexpressing cells. These results provide a molecular basis for the observation that expression of Bcl-2 or Bcl-XL is not a prognostic marker of tumor response to cancer therapy. PMID:10935471

Nitric oxide reversibly inhibits seven members of the caspase family via S-nitrosylation.

PubMed

Li, J; Billiar, T R; Talanian, R V; Kim, Y M

1997-11-17

The caspases are a family of at least 10 human cysteine proteases that participate in cytokine maturation and in apoptotic signal transduction and execution mechanisms. Peptidic inhibitors of these enzymes are capable of blocking cytokine maturation and apoptosis, demonstrating their crucial roles in these processes. We have recently discovered that nitric oxide (NO), produced either extracellularly by NO donors or intracellularly by the inducible nitric oxide synthase, prevented apoptosis in hepatocytes. Caspase-3-like activity was found to be inhibited under these conditions. To investigate further the interaction between NO and caspases, we utilized purified human recombinant caspases and examined the effect of NO on enzymatic activities of different caspases. We report here that of the seven caspases studied, all were reversibly inhibited by NO. Dithiothreitol was able to reverse the NO inhibition, indicating direct S-nitrosylation of caspase catalytic cysteine residue by NO. Our results support the concept that NO is an endogenous regulator of caspase activity.

Caspase 3 Activity and Lipoperoxidative Status in Raw Semen Predict the Outcome of Cryopreservation of Stallion Spermatozoa.

PubMed

Muñoz, Patricia Martín; Ferrusola, Cristina Ortega; Lopez, Luis Anel; Del Petre, Chiara; Garcia, Mercedes Alvarez; de Paz Cabello, Paulino; Anel, Luis; Peña, Fernando J

2016-09-01

Stallion-to-stallion variability in the quality of cryopreserved ejaculates postthaw affects the commercial acceptability of frozen semen and thus is a major constraint for the equine industry. In recent years, the molecular mechanisms associated with sperm damage during cryopreservation have become better understood. Identification of the freezability of the ejaculates before the freezing process is initiated will have a major impact on the equine industry. We studied three markers of oxidative stress in sperm, including 8-iso-PGF2alpha, 8-OH guanosine, and 4-hydroxynonenal (4-HNE); the presence of active caspase 3; and their changes after sperm cryopreservation. Although 4-HNE levels increased after cryopreservation (from 7% to 33%, P < 0.001), 8OH-guanosine and 8-ISO-PGF2alpha levels decreased after cryopreservation (from 130 to 35 arbitrary fluorescence units, P < 0.01, and from 1280 to 1233, P < 0.01, respectively). Postthaw sperm quality was classified as poor, average, or good using the 25th and 75th percentiles of all assays of sperm quality studied (motility, velocity, membrane functionality, and thiol content) as thresholds. Using these values, a sperm postthaw quality index was proposed. Receiver operating characteristic curves and the Youden J statistic were used to investigate the value of the measured parameters in fresh sperm as predictors of potential freezability. Using these techniques, we identified markers of bad freezers (percentages of caspase 3-positive dead sperm [area under the curve (AUC) = 0.820, P < 0.05] and percentages of caspase 3- and 4-HNE-positive sperm [AUC = 0.872, P < 0.05]) and good freezers (percentages of caspase 3-negative live sperm [AUC = 0.815, P < 0.05], percentages of live sperm with high thiol content [AUC = 0.907, P < 0.01], and percentages of 8-ISO-PGF2alpha-positive sperm [AUC = 0.900, P < 0.01]. Moreover, we described for the first time the presence of 8-ISO-PGF2alpha in stallion spermatozoa and revealed the

Cnidium officinale Makino extract induces apoptosis through activation of caspase-3 and p53 in human liver cancer HepG2 cells

PubMed Central

Hong, Heeok; An, Jeong Cheol; de La Cruz, Joseph F.; Hwang, Seong-Gu

2017-01-01

A number of diverse studies have reported the anticancer properties of Cnidium officinale Makino (CO). However, the apoptotic effect of this traditional medicinal herb in human hepatocellular carcinoma cells (HepG2) remains to be elucidated. Therefore, the present study investigated the ability of CO to reduce cell viability through apoptotic pathways. Cell viability was determined using the 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide assay. CO extract-induced apoptosis in HepG2 cells was assessed by Hoechst 33258 staining. The cell cycle was monitored using fluorescence-activated cell sorting analysis with propidium iodide staining. Furthermore, the present study explored whether various signaling molecules associated with HepG2 cell death were affected by CO treatment, including caspase-3, B-cell lymphoma 2 (Bcl-2), tumor protein p53 (p53), cyclin-dependent kinase 4 (CDK4) and cyclin D. The expression levels of these genes were examined by reverse-transcription polymerase chain reaction and western blotting. The expression levels of caspase-3 and p53 were upregulated with CO extract treatment, whereas those of Bcl-2, CDK4 and cyclin D were significantly downregulated. Cleaved caspase-3 expression was upregulated following treatment with CO extract in a dose-dependent manner. Collectively, the data suggest that CO extract has the potential to induce apoptosis of HepG2 cells and may act by suppressing the cell cycle, which leads to caspase-3 cleavage and p53 signaling. PMID:28966688

Melatonin partially protects 661W cells from H2O2-induced death by inhibiting Fas/FasL-caspase-3.

PubMed

Sánchez-Bretaño, Aída; Baba, Kenkichi; Janjua, Uzair; Piano, Ilaria; Gargini, Claudia; Tosini, Gianluca

2017-01-01

Previous studies have shown that melatonin (MEL) signaling is involved in the modulation of photoreceptor viability during aging. Recent work by our laboratory suggested that MEL may protect cones by modulating the Fas/FasL-caspase-3 pathway. In this study, we first investigated the presence of MEL receptors (MT 1 and MT 2 ) in 661W cells, then whether MEL can prevent H 2 O 2 -induced cell death, and last, through which pathway MEL confers protection. The mRNA and proteins of the MEL receptors were detected with quantitative PCR (q-PCR) and immunocytochemistry, respectively. To test the protective effect of MEL, 661W cells were treated with H 2 O 2 for 2 h in the presence or absence of MEL, a MEL agonist, and an antagonist. To study the pathways involved in H 2 O 2 -mediated cell death, a Fas/FasL antagonist was used before the exposure to H 2 O 2 . Finally, Fas/FasL and caspase-3 mRNA was analyzed with q-PCR and immunocytochemistry in cells treated with H 2 O 2 and/or MEL. Cell viability was analyzed by using Trypan Blue. Both MEL receptors (MT 1 and MT 2 ) were detected at the mRNA and protein levels in 661W cells. MEL partially prevented H 2 O 2 -mediated cell death (20-25%). This effect was replicated with IIK7 (a melatonin receptor agonist) when used at a concentration of 1 µM. Preincubation with luzindole (a melatonin receptor antagonist) blocked MEL protection. Kp7-6, an antagonist of Fas/FasL, blocked cell death caused by H 2 O 2 similarly to what was observed for MEL. Fas, FasL, and caspase-3 expression was increased in cells treated with H 2 O 2 , and this effect was prevented by MEL. Finally, MEL treatment partially prevented the activation of caspase-3 caused by H 2 O 2 . The results demonstrate that MEL receptors are present and functional in 661W cells. MEL can prevent photoreceptor cell death induced by H 2 O 2 via the inhibition of the proapoptotic pathway Fas/FasL-caspase-3.

Fish oil-derived lipid emulsion induces RIP1-dependent and caspase 8-licensed necroptosis in IEC-6 cells through overproduction of reactive oxygen species.

PubMed

Yan, Jun-Kai; Yan, Wei-Hui; Cai, Wei

2018-06-23

Excessive cell death of enterocytes has been demonstrated to be partially associated with the intravenously-administrated lipid emulsions (LEs) during parenteral nutrition (PN) support. However, as a new generation of LE, the effect of fish oil-derived lipid emulsion (FOLE) on the death of enterocytes remains elusive. Intestinal epithelial cells (IEC-6 cell line) were treated with FOLE (0.25-1%) for 24 h. Cell survival was measured by CCK-8 assay, and morphological changes were monitored by time-lapse live cell imaging. The expression of receptor-interacting protein 1/3 (RIP1/3) and caspase 8 was assessed by westernblot, and the formation of necrosome (characterized by the assembly of RIP1/3 complex along with the dissociation of caspase 8) was examined by immunoprecipitation. Additionally, the production of intracellular reactive oxygen species (ROS) was detected by using a ROS detection kit with an oxidation-sensitive probe (DCFH-DA). FOLE dose-dependently induced non-apoptotic, but programmed necroctic cell death (necroptosis) within 4-8 h after treatment. The assembly of RIP1/3 complex along with the dissociation of caspase 8 from RIP1 was observed in FOLE-treated cells. Moreover, FOLE-induced cell death was significantly alleviated by inhibiting RIP1, and was further aggravated by inhibiting caspase 8. In addition, prior to cell death the accumulation of intracellular ROS was significantly increased in FOLE-treated cells (increased by approximately 5-fold versus control, p < 0.001), which could be attenuated by inhibiting RIP1 (decreased by approximately 35% versus FOLE, p < 0.05). FOLE induces RIP1-dependent and caspase 8-licensed necroptosis through overproduction of ROS in vitro. Our findings may provide novel insights into the clinical applications of FOLE during PN support.

Membrane receptor-mediated apoptosis and caspase activation in the differentiated EoL-1 eosinophilic cell line.

PubMed

Al-Rabia, Mohammed W; Blaylock, Morgan G; Sexton, Darren W; Walsh, Garry M

2004-06-01

Caspases are key molecules in the control of apoptosis, but relatively little is known about their contribution to eosinophil apoptosis. We examined caspase-3, -8, and -9 activities in receptor ligation-dependent apoptosis induction in the differentiated human eosinophilic cell line EoL-1. Differentiated EoL-1 exhibited bi-lobed nuclei, eosinophil-associated membrane receptors, and basic granule proteins. Annexin-V fluorescein isothiocyanate binding to EoL-1 revealed significant (P<0.01) apoptosis induction in cells cultured for 20 h with monoclonal antibodies (mAb) specific for CD45 (71%+/-4.3), CD45RA (58%+/-2.3), CD45RB (68%+/-2.4), CD95 (47%+/-2.6), and CD69 (52%+/-2.1) compared with control (23%+/-1.6) or CD45RO mAb (27%+/-3.9). The pan-caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (fmk) and inhibitors of caspase-8 (Z-Ile-Glu-Thr-Asp-fmk) and caspase-9 (Z-Leu-Glu-His-Asp-fmk) significantly inhibited mAb-induced apoptosis of EoL-1 but had no effect on constitutive (baseline) apoptosis at 16 and 20 h. Caspase activity was analyzed using the novel CaspaTag trade mark technique and flow cytometry. EoL-1 treated with pan-CD45, CD45RA, CD45RB, and CD95 mAb exhibited caspase-3 and -9 activation at 12 h post-treatment, which increased at 16 and 20 h. Activated caspase-8 was detected 12 and 16 h after ligation with CD45, CD45RA, CD45RB, and CD95 mAb followed by a trend toward basal levels at 20 h. CD69 ligation resulted in caspase-3 activation, a modest but significant activation of caspase-8, and a loss in mitochondrial transmembrane potential but had no significant effect on activation of caspase-9. Thus, the intrinsic and extrinsic caspase pathways are involved in controlling receptor ligation-mediated apoptosis induction in human eosinophils, findings that may aid the development of a more targeted, anti-inflammatory therapy for asthma.

5-s-Cysteinyl-conjugates of catecholamines induce cell damage, extensive DNA base modification and increases in caspase-3 activity in neurons.

PubMed

Spencer, Jeremy P E; Whiteman, Matthew; Jenner, Peter; Halliwell, Barry

2002-04-01

A decrease in reduced glutathione levels in dopamine containing nigral cells in Parkinson's disease may result from the formation of cysteinyl-adducts of catecholamines, which in turn exert toxicity on nigral cells. We show that exposure of neurons (CSM 14.1) to 5-S-cysteinyl conjugates of dopamine, L-DOPA, DOPAC or DHMA causes neuronal damage, increases in oxidative DNA base modification and an elevation of caspase-3 activity in cells. Damage to neurons was apparent 12-48 h of post-exposure and there were increases in caspase-3 activity in neurons after 6 h. These changes were paralleled by large increases in pyrimidine and purine base oxidation products, such as 8-OH-guanine suggesting that 5-S-cysteinyl conjugates of catecholamines are capable of diffusing into cells and stimulating the formation of reactive oxygen species (ROS), which may then lead to a mechanism of cell damage involving caspase-3. Indeed, intracellular ROS were observed to rise sharply on exposure to the conjugates. These results suggest one mechanism by which oxidative stress may occur in the substantia nigra in Parkinson's disease.

Emission spectral analysis of caspase-3 activation during artesunate (ART)-induced apoptosis of human lung adenocarcinoma cell

NASA Astrophysics Data System (ADS)

Pan, Wen-liang; Chen, Tong-sheng; Qu, Junle

2009-02-01

Artesunate (ART), a semi-synthetic derivative of the sesquiterpene artemisinin extracted from the Chinese herb Artemisia annua, exerts a broad spectrum of clinical activity against human cancers. Artemisinin-derivative combination chemotherapy is recommended by WHO since it acts rapidly and is well tolerated and particularly effective. In present investigation, we used CKK-8 assay to assess the inhibitory effects of ART on human lung adenocarcinoma (ASTC-a-1) cells. Apoptotic activity of ART in ASTC-a-1 cells was detected by means of nuclear staining with Hoechst33258. In order to monitor the activity of caspase-3 during ART-induced ASTC-a-1 cells apoptosis, the dynamical emission spectra of SCAT3, a FRET plasmid based on GFPs, were performed inside living cell expressed stably with SCAT3 after ART treatment. The results showed that (1) ART could inhibit ASTC-a-1 cells proliferation in a dose-dependent manner; (2) chromatin condensation was observed after ART treatment for 48 h; (3) the SCAT3 inside living cells were cleaved after ART treatment for 48 h, implying that caspase-3 was involved in the ART-induced apoptosis.

Monitoring cleaved caspase-3 activity and apoptosis of immortalized oligodendroglial cells using live-cell imaging and cleaveable fluorogenic-dye substrates following potassium-induced membrane depolarization.

PubMed

Smith, Graham S T; Voyer-Grant, Janine A M; Harauz, George

2012-01-13

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a reduction in neuronal population and function. Damaged axons can release excitatory molecules including potassium or glutamate into the extracellular matrix, which in turn, can produce further insult and injury to the supporting glial cells including astrocytes and oligodendrocytes. If the insult persists, cells will undergo programmed cell death (apoptosis), which is regulated and activated by a number of well-established signal transduction cascades. Apoptosis and tissue necrosis can occur after traumatic brain injury, cerebral ischemia, and seizures. A classical example of apoptotic regulation is the family of cysteine-dependent aspartate-directed proteases, or caspases. Activated proteases including caspases have also been implicated in cell death in response to chronic neurodegenerative diseases including Alzheimer's, Huntington's, and Multiple Sclerosis. In this protocol we describe the use of the NucView 488 caspase-3 substrate to measure the rate of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte (OLG) cell cultures, following exposure to different extracellular stresses such as high concentrations of potassium or glutamate. The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience), and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation. We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein). Here, the N19-OLG cell cultures were treated with either 80 mM potassium chloride or 100 mM sodium glutamate to mimic axonal leakage into the extracellular matrix to induce apoptosis

Caspase-Independent Apoptosis Induced by Reperfusion Following Ischemia without Bile Duct Occlusion in Rat Liver.

PubMed

Matsui, Nobuaki; Yoshioka, Rie; Nozawa, Asako; Kobayashi, Naonobu; Shichijo, Yukari; Yoshikawa, Tadatoshi; Akagi, Masaaki

2017-01-01

The contribution of caspases to hepatic ischemia/reperfusion (I/R)-induced apoptosis has not been completely understood yet. Several studies have demonstrated increased caspase activity during I/R and the protective effect of caspase inhibitors against I/R injuries. However, reports with opposing results also exist. Herein, we examined the contribution of caspases to the I/R-induced hepatic apoptosis in rats using caspase inhibitors and specific substrates of caspases. Hepatic I/R was induced via a 2-h occlusion of the portal vein and the hepatic artery, without conducting bile duct occlusion. DNA laddering and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL)-positive cells were increased at 3 h after reperfusion. Pretreatment with caspase inhibitors (Z-Asp-2,6-dichlorobenzoyloxymethylketone (Z-Asp-cmk) 2 or 10 mg/kg intravenously (i.v.), 20 mg/kg intraperitoneally (i.p.), Z-Val-Ala-Asp(OMe)-fluoromethylketone (Z-VAD-fmk) 3 mg/kg i.v.) failed to reduce apoptosis induced by I/R. Interestingly, apoptosis induced by the portal triad (hepatic artery, portal vein, and bile duct) occlusion/reperfusion could be marginally attenuated using Z-Asp-cmk (2 mg/kg i.v.). The cleavage activity for Ac-DEVD-α-(4-methylcoumaryl-7-amide) (MCA), a caspase-3/7/8/9 substrate, was significantly increased by I/R. Conversely, the cleavage activities for Ac-DNLD-MCA and MCA-VDQVDGW[K-DNP]-NH 2 , specific substrates for caspase-3 and -7 respectively, were decreased by I/R. Protein expression of the cellular inhibitor of apoptosis protein 2 (c-IAP2), an endogenous caspase inhibitor, was increased by ischemia. Nuclear translocation of the apoptosis-inducing factor (AIF), an initiator protein of caspase-independent apoptosis, was also increased during I/R. These results suggest that caspases are inhibited by c-IAP2 induced during ischemia and that AIF may be involved in initiation of apoptosis induced by hepatic I/R without

Carbon ion beam triggers both caspase-dependent and caspase-independent pathway of apoptosis in HeLa and status of PARP-1 controls intensity of apoptosis.

PubMed

Ghorai, Atanu; Sarma, Asitikantha; Bhattacharyya, Nitai P; Ghosh, Utpal

2015-04-01

High linear energy transfer (LET) carbon ion beam (CIB) is becoming very promising tool for various cancer treatments and is more efficient than conventional low LET gamma or X-rays to kill malignant or radio-resistant cells, although detailed mechanism of cell death is still unknown. Poly (ADP-ribose) polymerase-1 (PARP-1) is a key player in DNA repair and its inhibitors are well-known as radio-sensitizer for low LET radiation. The objective of our study was to find mechanism(s) of induction of apoptosis by CIB and role of PARP-1 in CIB-induced apoptosis. We observed overall higher apoptosis in PARP-1 knocked down HeLa cells (HsiI) compared with negative control H-vector cells after irradiation with CIB (0-4 Gy). CIB activated both intrinsic and extrinsic pathways of apoptosis via caspase-9 and caspase-8 activation respectively, followed by caspase-3 activation, apoptotic body, nucleosomal ladder formation and sub-G1 accumulation. Apoptosis inducing factor translocation into nucleus in H-vector but not in HsiI cells after CIB irradiation contributed caspase-independent apoptosis. Higher p53 expression was observed in HsiI cells compared with H-vector after exposure with CIB. Notably, we observed about 37 % fall of mitochondrial membrane potential, activation of caspase-9 and caspase-3 and mild activation of caspase-8 without any detectable apoptotic body formation in un-irradiated HsiI cells. We conclude that reduction of PARP-1 expression activates apoptotic signals via intrinsic and extrinsic pathways in un-irradiated cells. CIB irradiation further intensified both intrinsic and extrinsic pathways of apoptosis synergistically along with up-regulation of p53 in HsiI cells resulting overall higher apoptosis in HsiI than H-vector.

Hexavalent chromium targets mitochondrial respiratory chain complex I to induce reactive oxygen species-dependent caspase-3 activation in L-02 hepatocytes.

PubMed

Xiao, Fang; Li, Yanhong; Dai, Lu; Deng, Yuanyuan; Zou, Yue; Li, Peng; Yang, Yuan; Zhong, Caigao

2012-09-01

Hexavalent chromium [Cr(VI)], which is used for various industrial applications, such as leather tanning and chroming, can cause a number of human diseases including inflammation and cancer. Cr(VI) exposure leads to severe damage to the liver, but the mechanisms involved in Cr(VI)-mediated toxicity in the liver are unclear. The present study provides evidence that Cr(VI) enhances reactive oxygen species (ROS) accumulation by inhibiting the mitochondrial respiratory chain complex (MRCC) I. Cr(VI) did not affect the expression levels of antioxidative proteins such as superoxide dismutase (SOD), catalase and thioredoxin (Trx), indicating that the antioxidative system was not involved in Cr(VI)-induced ROS accumulation. We found that ROS mediated caspase-3 activation partially depends on the downregulation of the heat shock protein (HSP) 70 and 90. In order to confirm our hypothesis that ROS plays a key role in Cr(VI)-mediated cytotoxicity, we used N-acetylcysteine (NAC) to inhibit the accumulation of ROS. NAC successfully blocked the inhibition of HSP70 and HSP90 as well as the activation of caspase-3, suggesting that ROS is essential in Cr(VI)-induced caspase-3 activation. By applying different MRCC substrates as electron donors, we also confirmed that Cr(VI) could accept the electrons leaked from MRCC I and the reduction occurs at MRCC I. In conclusion, the present study demonstrates that Cr(VI) induces ROS-dependent caspase-3 activation by inhibiting MRCC I activity, and MRCC I has been identified as a new target and a new mechanism for the apoptosis-inducing activity displayed by Cr(VI).

UV-A Irradiation Activates Nrf2-Regulated Antioxidant Defense and Induces p53/Caspase3-Dependent Apoptosis in Corneal Endothelial Cells.

PubMed

Liu, Cailing; Vojnovic, Dijana; Kochevar, Irene E; Jurkunas, Ula V

2016-04-01

To examine whether Nrf2-regulated antioxidant defense and p53 are activated in human corneal endothelial cells (CEnCs) by environmental levels of ultraviolet A (UV-A), a known stimulator of oxidative stress. Immortalized human CEnCs (HCEnCi) were exposed to UV-A fluences of 2.5, 5, 10, or 25 J/cm2, then allowed to recover for 3 to 24 hours. Control HCEnCi did not receive UV-A. Reactive oxygen species (ROS) were measured using H2DCFDA. Cell cytotoxicity was evaluated by lactate dehydrogenase (LDH) release. Levels of Nrf2, HO-1, NQO-1, p53, and caspase3 were detected by immunnoblotting or real-time PCR. Activated caspase3 was measured by immunoblotting and a fluorescence assay. Exposure of HCEnCi to 5, 10, and 25 J/cm2 UV-A increased ROS levels compared with controls. Nrf2, HO-1, and NQO-1 mRNA increased 1.7- to 3.2-fold at 3 and 6 hours after irradiation with 2.5 and 5 J/cm2 UV-A. At 6 hours post irradiation, UV-A (5 J/cm2) enhanced nuclear Nrf2 translocation. At 24 hours post treatment, UV-A (5, 10, and 25 J/cm2) produced a 1.8- to 2.8-fold increase in phospho-p53 and a 2.6- to 6.0-fold increase in activated caspase3 compared with controls, resulting in 20% to 42% cell death. Lower fluences of UV-A induce Nrf2-regulated antioxidant defense and higher fluences activate p53 and caspase3, indicating that even near-environmental levels of UV-A may affect normal CEnCs. This data suggest that UV-A may especially damage cells deficient in antioxidant defense, and thus may be involved in the etiology of Fuchs' endothelial corneal dystrophy (FECD).

UV-A Irradiation Activates Nrf2-Regulated Antioxidant Defense and Induces p53/Caspase3-Dependent Apoptosis in Corneal Endothelial Cells

PubMed Central

Liu, Cailing; Vojnovic, Dijana; Kochevar, Irene E.; Jurkunas, Ula V.

2016-01-01

Purpose To examine whether Nrf2-regulated antioxidant defense and p53 are activated in human corneal endothelial cells (CEnCs) by environmental levels of ultraviolet A (UV-A), a known stimulator of oxidative stress. Methods Immortalized human CEnCs (HCEnCi) were exposed to UV-A fluences of 2.5, 5, 10, or 25 J/cm2, then allowed to recover for 3 to 24 hours. Control HCEnCi did not receive UV-A. Reactive oxygen species (ROS) were measured using H2DCFDA. Cell cytotoxicity was evaluated by lactate dehydrogenase (LDH) release. Levels of Nrf2, HO-1, NQO-1, p53, and caspase3 were detected by immunnoblotting or real-time PCR. Activated caspase3 was measured by immunoblotting and a fluorescence assay. Results Exposure of HCEnCi to 5, 10, and 25 J/cm2 UV-A increased ROS levels compared with controls. Nrf2, HO-1, and NQO-1 mRNA increased 1.7- to 3.2-fold at 3 and 6 hours after irradiation with 2.5 and 5 J/cm2 UV-A. At 6 hours post irradiation, UV-A (5 J/cm2) enhanced nuclear Nrf2 translocation. At 24 hours post treatment, UV-A (5, 10, and 25 J/cm2) produced a 1.8- to 2.8-fold increase in phospho-p53 and a 2.6- to 6.0-fold increase in activated caspase3 compared with controls, resulting in 20% to 42% cell death. Conclusions Lower fluences of UV-A induce Nrf2-regulated antioxidant defense and higher fluences activate p53 and caspase3, indicating that even near-environmental levels of UV-A may affect normal CEnCs. This data suggest that UV-A may especially damage cells deficient in antioxidant defense, and thus may be involved in the etiology of Fuchs' endothelial corneal dystrophy (FECD). PMID:27127932

Tanshinone IIA attenuates the cerebral ischemic injury-induced increase in levels of GFAP and of caspases-3 and -8.

PubMed

Zhou, L; Bondy, S C; Jian, L; Wen, P; Yang, F; Luo, H; Li, W; Zhou, Jun

2015-03-12

Tanshinone IIA (TSA) is a lipid soluble agent derived from the root of Salvia miltiorrhiza (Danshen). This plant is a traditional Chinese herb, which has been used widely in China especially for enhancing circulation. However mechanisms underlying its efficacy remain poorly understood. The present study was designed to illuminate events that may underlie the apparently neuroprotective effects of TSA following ischemic insult. Adult Sprague-Dawley rats were subjected to transient focal cerebral ischemia by use of a middle cerebral artery occlusion model. They were then randomly divided into a sham-operated control group, and cerebral ischemia/reperfusion groups receiving a two-hour occlusion. Further subsets of groups received the same durations of occlusion or were sham-operated but then received daily i.p. injections of high or low doses of TSA, for seven or 15days. Hematoxylin and eosin staining revealed lesions in the entorhinal cortex of both rats subject to ischemia and to a lesser extent to those receiving TSA after surgery. Levels of glial fibrillary acidic protein (GFAP), caspase-3 and caspase-8, were quantified by both immunohistochemistry and Western blotting. TSA treatment after middle cerebral artery occlusion, markedly reduced infarct size, and reduced the expression of caspase-3 and caspase-8. These changes were considered protective and were generally proportional to the dose of TSA used. These results suggest that TSA may effect neuroprotection by way of reduction of the extent of cell inflammation and death within affected regions. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Interactions of HIPPI, a molecular partner of Huntingtin interacting protein HIP1, with the specific motif present at the putative promoter sequence of the caspase-1, caspase-8 and caspase-10 genes.

PubMed

Majumder, P; Choudhury, A; Banerjee, M; Lahiri, A; Bhattacharyya, N P

2007-08-01

To investigate the mechanism of increased expression of caspase-1 caused by exogenous Hippi, observed earlier in HeLa and Neuro2A cells, in this work we identified a specific motif AAAGACATG (- 101 to - 93) at the caspase-1 gene upstream sequence where HIPPI could bind. Various mutations in this specific sequence compromised the interaction, showing the specificity of the interactions. In the luciferase reporter assay, when the reporter gene was driven by caspase-1 gene upstream sequences (- 151 to - 92) with the mutation G to T at position - 98, luciferase activity was decreased significantly in green fluorescent protein-Hippi-expressing HeLa cells in comparison to that obtained with the wild-type caspase-1 gene 60 bp upstream sequence, indicating the biological significance of such binding. It was observed that the C-terminal 'pseudo' death effector domain of HIPPI interacted with the 60 bp (- 151 to - 92) upstream sequence of the caspase-1 gene containing the motif. We further observed that expression of caspase-8 and caspase-10 was increased in green fluorescent protein-Hippi-expressing HeLa cells. In addition, HIPPI interacted in vitro with putative promoter sequences of these genes, containing a similar motif. In summary, we identified a novel function of HIPPI; it binds to specific upstream sequences of the caspase-1, caspase-8 and caspase-10 genes and alters the expression of the genes. This result showed the motif-specific interaction of HIPPI with DNA, and indicates that it could act as transcription regulator.

Role of Caspase-3 Cleaved IP3R1 on Ca2+ Homeostasis and Developmental Competence of Mouse Oocytes and Eggs

PubMed Central

Zhang, Nan; Fissore, Rafael. A.

2014-01-01

Apoptosis in most cell types is accompanied by altered Ca2+ homeostasis. During apoptosis, caspase-3 mediated cleavage of the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) generates a 95-kDa C-terminal fragment (C-IP3R1), which represents the channel domain of the receptor. Aged mouse eggs display abnormal Ca2+ homeostasis and express C-IP3R1, although whether or not C-IP3R1 expression contributes to Ca2+ misregulation or a decrease in developmental competency is unknown. We sought to answer these questions by injecting in mouse oocytes and eggs cRNAs encoding CIP3R1. We found that: 1) expression of C-IP3R1 in eggs lowered the Ca2+ content of the endoplasmic reticulum (ER), although, as C-IP3R1 is quickly degraded at this stage, its expression did not impair pre-implantation embryo development; 2) expression of CIP3R1 in eggs enhanced fragmentation associated with aging; 3) endogenous IP3R1 is required for aging associated apoptosis, as its down-regulation prevented fragmentation, and expression of C-IP3R1 in eggs with downregulated IP3R1 partly restored fragmentation; 4) C-IP3R1 expression in GV oocytes resulted in persistent levels of protein, which abolished the increase in the ER releasable Ca2+ pool that occurs during maturation, undermined the Ca2+ oscillatory ability of matured eggs and their activation potential. Collectively, this study supports a role for IP3R1 and C-IP3R1 in regulating Ca2+ homeostasis and the ER Ca2+ content during oocyte maturation. Nevertheless, the role of C-IP3R1 on Ca2+ homeostasis in aged eggs seems minor, as in MII eggs the majority of endogenous IP3R1 remains intact and C-IP3R1 undergoes rapid turnover. PMID:24692207

Caspase-mediated cleavage of Beclin1 inhibits autophagy and promotes apoptosis induced by S1 in human ovarian cancer SKOV3 cells.

PubMed

Li, Xiaoning; Su, Jing; Xia, Meihui; Li, Hongyan; Xu, Ye; Ma, Chunhui; Ma, Liwei; Kang, Jingsong; Yu, Huimei; Zhang, Zhichao; Sun, Liankun

2016-02-01

S1, a novel BH3 mimetic, can induce apoptosis dependent on Bax/Bak through inhibition of Bcl-2 in various tumors. S1 also induces autophagy through interrupting the interaction of Bcl-2 and Beclin1. Our results showed that S1 induces apoptosis in human ovarian cancer SKOV3 cells in a time- and dose-dependent manner. Autophagy precedes apoptosis, in SKOV3 cells treated with S1 (6 μmol/L), autophagy reached the maximum peak at 12 h after treatment and decreased to 24 h. In SKOV3 cells treated with different concentrations of S1 for 24 h, the highest level of autophagy was observed with 5 μmol/L and decreased to 10 μmol/L. Autophagy inhibitors 3-MA and CQ enhanced apoptosis induced by S1 in SKOV3 cells. However, overactivation of caspases in apoptosis induced by S1 may inhibit the autophagy-inducing function of Beclin1. Because the pan-caspase inhibitor Z-VAD recovered the autophagy-inducing function of Beclin1 through reduction of activated caspase-mediated cleavage of Beclin1. Furthermore, the Beclin1 cleavage products could further increase apoptosis induced by S1 in SKOV3 cells. This indicates that apoptosis induced by high doses and long exposure of S1 causes the overactivation of caspases and subsequent cleavage of Beclin1, and inhibits the protection of autophagy. Moreover, the cleaved product of Beclin1 further promotes apoptosis induced by S1 in SKOV3 cells. Our results suggest this may be a molecular mechanism for enhancing the sensitivity of cancer cells to apoptosis induced by small molecular compound targeting Bcl-2.

Lactate dehydrogenase and caspase activity in nasopharyngeal secretions are predictors of bronchiolitis severity.

PubMed

Mehta, Reena; Scheffler, Margaret; Tapia, Lorena; Aideyan, Letisha; Patel, Kirtida D; Jewell, Alan M; Avadhanula, Vasanthi; Mei, Minghua; Garofalo, Roberto P; Piedra, Pedro A

2014-11-01

Bronchiolitis is the leading cause of hospitalization in infants. Biomarkers of disease severity might help in clinical management. To determine the clinical predictiveness of NW-LDH, NW-caspase 3/7, and NW-LDH/NW-caspase 3/7 ratio in bronchiolitis. Previously healthy children less than 24 months of age with bronchiolitis were recruited from the Texas Children's emergency room and intensive care unit from October 2010 to April 2011. Demographic, clinical information, and NW samples were obtained at enrollment. NW samples were analyzed for respiratory viruses, caspase 3/7, and LDH. A viral pathogen was detected in 91·6% of 131 children, with the most common being respiratory syncytial virus and human rhinovirus. A single infection was found in 61·8% of subjects and co-infection in 29·8%. Children admitted to ICU had significantly higher NW-LDH than children sent home from the ER or admitted to the general floor (P = 0·02). Children infected with RSV had the highest NW-LDH concentration (P = 0·03) compared with other viral infections. NW-LDH and NW-caspase were significantly correlated (r = 0·77, P < 0·0001). The univariate models showed NW-LDH and NW-LDH/NW- caspase 3/7 ratio were directly associated with hospitalization. Mutivariate regression analyses suggested a complex interaction between the biomarkers, demographics, and disposition. NW-LDH, NW-caspase 3/7 and NW-LDH/NW-caspase 3/7 ratio and their interactions with demographic factors are predictive of bronchiolitis severity and can help distinguish children requiring ICU-level care from those admitted to the general floor, or discharged home from the emergency center. © 2014 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

Lactate dehydrogenase and caspase activity in nasopharyngeal secretions are predictors of bronchiolitis severity

PubMed Central

Mehta, Reena; Scheffler, Margaret; Tapia, Lorena; Aideyan, Letisha; Patel, Kirtida D; Jewell, Alan M; Avadhanula, Vasanthi; Mei, Minghua; Garofalo, Roberto P; Piedra, Pedro A

2014-01-01

Background Bronchiolitis is the leading cause of hospitalization in infants. Biomarkers of disease severity might help in clinical management. Objective To determine the clinical predictiveness of NW-LDH, NW-caspase 3/7, and NW-LDH/NW-caspase 3/7 ratio in bronchiolitis. Methods Previously healthy children less than 24 months of age with bronchiolitis were recruited from the Texas Children's emergency room and intensive care unit from October 2010 to April 2011. Demographic, clinical information, and NW samples were obtained at enrollment. NW samples were analyzed for respiratory viruses, caspase 3/7, and LDH. Results A viral pathogen was detected in 91·6% of 131 children, with the most common being respiratory syncytial virus and human rhinovirus. A single infection was found in 61·8% of subjects and co-infection in 29·8%. Children admitted to ICU had significantly higher NW-LDH than children sent home from the ER or admitted to the general floor (P = 0·02). Children infected with RSV had the highest NW-LDH concentration (P = 0·03) compared with other viral infections. NW-LDH and NW-caspase were significantly correlated (r = 0·77, P < 0·0001). The univariate models showed NW-LDH and NW-LDH/NW- caspase 3/7 ratio were directly associated with hospitalization. Mutivariate regression analyses suggested a complex interaction between the biomarkers, demographics, and disposition. Conclusions NW-LDH, NW-caspase 3/7 and NW-LDH/NW-caspase 3/7 ratio and their interactions with demographic factors are predictive of bronchiolitis severity and can help distinguish children requiring ICU-level care from those admitted to the general floor, or discharged home from the emergency center. PMID:25132512

Histomorphometry and expression of Cdc47 and caspase-3 in hyperthyroid rat uteri and placentas during gestation and postpartum associated with fetal development.

PubMed

Freitas, E S; Leite, E D; Souza, C A; Ocarino, N M; Ferreira, E; Cassali, G D; Gomes, M G; Serakides, R

2007-01-01

In two different experiments, the effects of hyperthyroidism on the histomorphometry and expression of Cdc47 and caspase-3 were evaluated in the uteri and placentas during gestation and postpartum. Fetal development was also evaluated during gestation. In the first experiment, 36 adult female Wistar rats were divided into two groups of 18 animals each: (1) hyperthyroid; and (2) euthyroid (control). Female rats were mated and killed at 7, 14 and 19 days of gestation. Uteri and placentas were weighed and subjected to histomorphometric and immunohistochemical evaluation to determine the expression of Cdc47 and caspase-3. Ovaries were also evaluated for weight and subjected to morphometric analysis. Fetuses were quantified and weighed individually. In the second experiment, 12 adult female Wistar rats were divided into two groups of six animals each: (1) hyperthyroid; and (2) euthyroid (control). Female rats were mated and killed 2 days postpartum. Uteri were evaluated in the same way as for the first experiment. Hyperthyroidism increased ovulation and conception rates without disturbing the size and viability of the fetuses. In the pregnant uteri, hyperthyroidism did not change the thickness of the layers or the expression of Cdc47 and caspase-3. However, in the placentas, hyperthyroidism increased the medium diameter of trophoblast cells, as well as the thickness and the expression of Cdc47 of spongiotrophoblast cells, at 14 days of gestation. During uterine involution, hyperthyroidism significantly increased the expression of Cdc47 and reduced the expression of caspase-3 in the uterine layers. In conclusion, hyperthyroidism increased the conception rate because of an ovulation gain, induced significant placental changes during pregnancy and, in the uterus, increased Cdc47 expression and decreased caspase-3 expression after parturition.

Characterization of three caspases and their pathogen-induced expression pattern in Portunus trituberculatus.

PubMed

Ren, Xianyun; Yu, Xuan; Gao, Baoquan; Liu, Ping; Li, Jian

2017-07-01

Caspases are a family of proteases involved in many important biological processes including apoptosis and inflammation. In this study, we analyzed the expression patterns and effects on immune response in various tissues of the edible crab Portunus trituberculatus. PtCas 2, PtCas 3 and PtCas 4 share overall sequence identities of 55.88%-74.86%, 8.47%-46.54% and 20.11%-50.87%, respectively, with their other crustacean species. PtCas 2, PtCas 3 and PtCas 4 have the same caspase domain and catalytic site found in known caspases. The expression levels of the three caspases differed between tissues. Following bacterial and viral infection, the expression levels of the three caspases reached a maximum level at 24 h post-infection (hpi) in case of bacteria, whereas it was 48 hpi in virus. Moreover, the WSSV, Vibrio alginolyticus or V. parahaemolyticus induced the activities of PtCas 2-4 in a time-dependent manner. These results indicate an involvement of caspases in bacterial and viral induced immune response and demonstrate for the first time that PtCas 2, PtCas 3 and PtCas 4 are essential for optimal response to bacterial and virus infection in crabs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Memantine attenuates cell apoptosis by suppressing the calpain-caspase-3 pathway in an experimental model of ischemic stroke.

PubMed

Chen, Bin; Wang, Guoxiang; Li, Weiwei; Liu, Weilin; Lin, Ruhui; Tao, Jing; Jiang, Min; Chen, Lidian; Wang, Yun

2017-02-15

Ischemic stroke, the second leading cause of death worldwide, leads to excessive glutamate release, over-activation of N-methyl-D-aspartate receptor (NMDAR), and massive influx of calcium (Ca 2+ ), which may activate calpain and caspase-3, resulting in cellular damage and death. Memantine is an uncompetitive NMDAR antagonist with low-affinity/fast off-rate. We investigated the potential mechanisms through which memantine protects against ischemic stroke in vitro and in vivo. Middle cerebral artery occlusion-reperfusion (MCAO) was performed to establish an experimental model of ischemic stroke. The neuroprotective effects of memantine on ischemic rats were evaluated by neurological deficit scores and infarct volumes. The activities of calpain and caspase-3, and expression levels of microtubule-associated protein-2 (MAP2) and postsynaptic density-95 (PSD95) were determined by Western blotting. Additionally, Nissl staining and immunostaining were performed to examine brain damage, cell apoptosis, and neuronal loss induced by ischemia. Our results show that memantine could significantly prevent ischemic stroke-induced neurological deficits and brain infarct, and reduce ATP depletion-induced neuronal death. Moreover, memantine markedly suppressed the activation of the calpain-caspase-3 pathway and cell apoptosis, and consequently, attenuated brain damage and neuronal loss in MCAO rats. These results provide a molecular basis for the role of memantine in reducing neuronal apoptosis and preventing neuronal damage, suggesting that memantine may be a promising therapy for stroke patients. Copyright © 2017 Elsevier Inc. All rights reserved.

Autocorrelation descriptor improvements for QSAR: 2DA_Sign and 3DA_Sign

NASA Astrophysics Data System (ADS)

Sliwoski, Gregory; Mendenhall, Jeffrey; Meiler, Jens

2016-03-01

Quantitative structure-activity relationship (QSAR) is a branch of computer aided drug discovery that relates chemical structures to biological activity. Two well established and related QSAR descriptors are two- and three-dimensional autocorrelation (2DA and 3DA). These descriptors encode the relative position of atoms or atom properties by calculating the separation between atom pairs in terms of number of bonds (2DA) or Euclidean distance (3DA). The sums of all values computed for a given small molecule are collected in a histogram. Atom properties can be added with a coefficient that is the product of atom properties for each pair. This procedure can lead to information loss when signed atom properties are considered such as partial charge. For example, the product of two positive charges is indistinguishable from the product of two equivalent negative charges. In this paper, we present variations of 2DA and 3DA called 2DA_Sign and 3DA_Sign that avoid information loss by splitting unique sign pairs into individual histograms. We evaluate these variations with models trained on nine datasets spanning a range of drug target classes. Both 2DA_Sign and 3DA_Sign significantly increase model performance across all datasets when compared with traditional 2DA and 3DA. Lastly, we find that limiting 3DA_Sign to maximum atom pair distances of 6 Å instead of 12 Å further increases model performance, suggesting that conformational flexibility may hinder performance with longer 3DA descriptors. Consistent with this finding, limiting the number of bonds in 2DA_Sign from 11 to 5 fails to improve performance.

Molecular and functional characterization of caspase-8 from the big-belly seahorse (Hippocampus abdominalis).

PubMed

Oh, Minyoung; Elvitigala, Don Anushka Sandaruwan; Bathige, S D N K; Lee, Seongdo; Kim, Myoung-Jin; Lee, Jehee

2016-11-01

Apoptosis is a physiological process that can also participate in host immune defense mechanisms, including tumor growth suppression along with homeostasis and maturation of immune cells. Caspases are known to be involved in cellular apoptotic signaling; among them, caspase-8 plays an important role in the initiation phase of the apoptotic death cascade. In the current study, we molecularly characterized a caspase-8 homolog (designated as HaCasp-8) from Hippocampus abdominalis. The HaCasp-8 gene harbors a 1476 bp open reading frame (ORF) that codes for a protein of 492 amino acids (aa) with a predicted molecular mass of 55 kDa. HaCasp-8 houses the typical domain architecture of known initiator caspases, including the death effector domain and the carboxyl-terminal catalytic domain. As expected, phylogenetic analysis reflected a closer evolutionary relationship of HaCasp-8 with its teleostean similitudes. The results of our qPCR assays confirmed the ubiquitous expression of HaCasp-8 in physiologically important tissues examined, with pronounced expression levels in ovary tissues, followed by blood cells. HaCasp-8 expression at the mRNA level was found to be significantly modulated by lipopolysaccharide, polyinosinic:polycytidylic acid, Streptococcus iniae, and Edwardsiella tarda injection. Overexpression of HaCasp-8 could trigger a significant level of cell death in HEK293T cells, suggesting its putative role in cell death. Taken together, our findings suggest that HaCasp-8 is an important component in the caspase cascade, and its expression can be significantly modulated under pathogen stress conditions in the big-belly seahorse. Copyright © 2016 Elsevier Ltd. All rights reserved.

Nuclear envelope disruption involving host caspases plays a role in the parvovirus replication cycle.

PubMed

Cohen, Sarah; Marr, Alexandra K; Garcin, Pierre; Panté, Nelly

2011-05-01

Parvoviruses are small, nonenveloped, single-stranded DNA viruses which replicate in the nucleus of the host cell. We have previously found that early during infection the parvovirus minute virus of mice (MVM) causes small, transient disruptions of the nuclear envelope (NE). We have now investigated the mechanism used by MVM to disrupt the NE. Here we show that the viral phospholipase A2, the only known enzymatic domain on the parvovirus capsid, is not involved in causing NE disruption. Instead, the virus utilizes host cell caspases, which are proteases involved in causing NE breakdown during apoptosis, to facilitate these nuclear membrane disruptions. Studies with pharmacological inhibitors indicate that caspase-3 in particular is involved. A caspase-3 inhibitor prevents nuclear lamin cleavage and NE disruption in MVM-infected mouse fibroblast cells and reduces nuclear entry of MVM capsids and viral gene expression. Caspase-3 is, however, not activated above basal levels in MVM-infected cells, and other aspects of apoptosis are not triggered during early MVM infection. Instead, basally active caspase-3 is relocalized to the nuclei of infected cells. We propose that NE disruption involving caspases plays a role in (i) parvovirus entry into the nucleus and (ii) alteration of the compartmentalization of host proteins in a way that is favorable for the virus.

Caspase 7: increased expression and activation after traumatic brain injury in rats.

PubMed

Larner, Stephen F; McKinsey, Deborah M; Hayes, Ronald L; W Wang, Kevin K

2005-07-01

Caspases, a cysteine proteinase family, are required for the initiation and execution phases of apoptosis. It has been suggested that caspase 7, an apoptosis executioner implicated in cell death proteolysis, is redundant to the main executioner caspase 3 and it is generally believed that it is not present in the brain or present in only minute amounts with highly restricted activity. Here we report evidence that caspase 7 is up-regulated and activated after traumatic brain injury (TBI) in rats. TBI disrupts homeostasis resulting in pathological apoptotic activation. After controlled cortical impact TBI of adult male rats we observed, by semiquantitative real-time PCR, increased mRNA levels within the traumatized cortex and hippocampus peaking in the former about 5 days post-injury and in the latter within 6-24 h of trauma. The activation of caspase 7 protein after TBI, demonstrated by immunoblot by the increase of the active form of caspase 7 peaking 5 days post-injury in the cortex and hippocampus, was found to be up-regulated in both neurons and astrocytes by immunohistochemistry. These findings, the first to document the up-regulation of caspase 7 in the brain after acute brain injury in rats, suggest that caspase 7 activation could contribute to neuronal cell death on a scale not previously recognized.

Caspase-12 and the inflammatory response to Yersinia pestis.

PubMed

Ferwerda, Bart; McCall, Matthew B B; de Vries, Maaike C; Hopman, Joost; Maiga, Boubacar; Dolo, Amagana; Doumbo, Ogobara; Daou, Modibo; de Jong, Dirk; Joosten, Leo A B; Tissingh, Rudi A; Reubsaet, Frans A G; Sauerwein, Robert; van der Meer, Jos W M; van der Ven, André J A M; Netea, Mihai G

2009-09-01

Caspase-12 functions as an antiinflammatory enzyme inhibiting caspase-1 and the NOD2/RIP2 pathways. Due to increased susceptibility to sepsis in individuals with functional caspase-12, an early-stop mutation leading to the loss of caspase-12 has replaced the ancient genotype in Eurasia and a significant proportion of individuals from African populations. In African-Americans, it has been shown that caspase-12 inhibits the pro-inflammatory cytokine production. We assessed whether similar mechanisms are present in African individuals, and whether evolutionary pressures due to plague may have led to the present caspase-12 genotype population frequencies. No difference in cytokine induction through the caspase-1 and/or NOD2/RIP2 pathways was observed in two independent African populations, among individuals with either an intact or absent caspase-12. In addition, stimulations with Yersinia pestis and two other species of Yersinia were preformed to investigate whether caspase-12 modulates the inflammatory reaction induced by Yersinia. We found that caspase-12 did not modulate cytokine production induced by Yersinia spp. Our experiments demonstrate for the first time the involvement of the NOD2/RIP2 pathway for recognition of Yersinia. However, caspase-12 does not modulate innate host defense against Y. pestis and alternative explanations for the geographical distribution of caspase-12 should be sought.

Bcl-2 and caspase-3 are major regulators in Agaricus blazei-induced human leukemic U937 cell apoptosis through dephoshorylation of Akt.

PubMed

Jin, Cheng-Yun; Moon, Dong-Oh; Choi, Yung Hyun; Lee, Jae-Dong; Kim, Gi-Young

2007-08-01

Agaricus blazei is a medicinal mushroom that possesses antimetastatic, antitumor, antimutagenic, and immunostimulating effects. However, the molecular mechanisms involved in A. blazei-mediated apoptosis remain unclear. In the present study, to elucidate the role of the Bcl-2 in A. blazei-mediated apoptosis, U937 cells were transfected with either empty vector (U937/vec) or vector containing cDNA encoding full-length Bcl-2 (U937/Bcl-2). As compared with U937/vec, U937/Bcl-2 cells exhibited a 4-fold greater expression of Bcl-2. Treatment of U937/vec with 1.0-4.0 mg/ml of A. blazei extract (ABE) for 24 h resulted in a significant induction of morphologic features indicative of apoptosis. In contrast, U937/Bcl-2 exposed to the same ABE treatment only exhibited a slight induction of apoptotic features. ABE-induced apoptosis was accompanied by downregulation of antiapoptotic proteins such as X-linked inhibitor of apoptosis protein (XIAP), inhibitor of apoptosis protein (cIAP)-2 and Bcl-2, activation of caspase-3, and cleavage of poly(ADP-ribose)polymerase (PARP). Ectopic expression of Bcl-2 was associated with significantly induced expression of antiapoptotic proteins, such as cIAP-2 and Bcl-2, but not XIAP. Ectopic expression of Bcl-2 also reduced caspase-3 activation and PARP cleavage in ABE treated U937 cells. Furthermore, treatment with the caspase-3 inhibitor z-DEVD-fmk was sufficient to restore cell viability following ABE treatment. This increase in viability was ascribed to downregulation of caspase-3 and blockage of PARP and PLC-gamma cleavage. ABE also triggered the downregulation of Akt, and combined treatment with LY294002 (an inhibitor of Akt) significantly decreased cell viability. The results indicated that major regulators of ABE-induced apoptosis in human leukemic U937 cells are Bcl-2 and caspase-3, which are associated with dephosphorylation of the Akt signal pathway.

Mechanism of Siglec-8-induced human eosinophil apoptosis: role of caspases and mitochondrial injury.

PubMed

Nutku, Esra; Hudson, Sherry A; Bochner, Bruce S

2005-10-28

Sialic acid binding immunoglobulin like lectin (Siglec)-8 crosslinking with specific antibodies causes human eosinophil apoptosis. Mechanisms by which Siglec-8 crosslinking induces apoptosis are not known. Peripheral blood eosinophils were examined for caspase, mitochondria and reactive oxygen species (ROS) involvement after incubating the cells with anti-Siglec-8 crosslinking Abs or control Abs, in the presence or absence of selective inhibitors. Siglec-8 crosslinking induced rapid cleavage of caspase-3, caspase-8, and caspase-9 in eosinophils. Selective caspase-8 and/or caspase-9 inhibitors inhibited this apoptosis. Siglec-8 crosslinking on eosinophils increased dissipation of mitochondrial membrane potential upstream of caspase activation. Rotenone and antimycin, inhibitors of mitochondrial respiratory chain components, completely inhibited apoptosis. Additional experiments with an inhibitor of ROS, diphenyleneiodonium, demonstrated that ROS was also essential for Siglec-8-mediated apoptosis and preceded Siglec-8-mediated mitochondrial dissipation. These experiments show that Siglec-8-induced apoptosis occurs through the sequential production of ROS, followed by induction of mitochondrial injury and caspase cleavage.

Functional PAK-2 knockout and replacement with a caspase cleavage-deficient mutant in mice reveals differential requirements of full-length PAK-2 and caspase-activated PAK-2p34.

PubMed

Marlin, Jerry W; Chang, Yu-Wen E; Ober, Margaret; Handy, Amy; Xu, Wenhao; Jakobi, Rolf

2011-06-01

p21-Activated protein kinase 2 (PAK-2) has both anti- and pro-apoptotic functions depending on its mechanism of activation. Activation of full-length PAK-2 by the monomeric GTPases Cdc42 or Rac stimulates cell survival, whereas caspase activation of PAK-2 to the PAK-2p34 fragment is involved in the apoptotic response. In this study we use functional knockout of PAK-2 and gene replacement with the caspase cleavage-deficient PAK-2D212N mutant to differentiate the biological functions of full-length PAK-2 and caspase-activated PAK-2p34. Knockout of PAK-2 results in embryonic lethality at early stages before organ development, whereas replacement with the caspase cleavage-deficient PAK-2D212N results in viable and healthy mice, indicating that early embryonic lethality is caused by deficiency of full-length PAK-2 rather than lack of caspase activation to the PAK-2p34 fragment. However, deficiency of caspase activation of PAK-2 decreased spontaneous cell death of primary mouse embryonic fibroblasts and increased cell growth at high cell density. In contrast, stress-induced cell death by treatment with the anti-cancer drug cisplatin was not reduced by deficiency of caspase activation of PAK-2, but switched from an apoptotic to a nonapoptotic, caspase-independent mechanism. Homozygous PAK-2D212N primary mouse embryonic fibroblasts that lack the ability to generate the proapoptotic PAK-2p34 show less activation of the effector caspase 3, 6, and 7, indicating that caspase activation of PAK-2 amplifies the apoptotic response through a positive feedback loop resulting in more activation of effector caspases.

Cas IIgly Induces Apoptosis in Glioma C6 Cells In Vitro and In Vivo through Caspase-Dependent and Caspase-Independent Mechanisms1

PubMed Central

Trejo-Solís, Cristina; Palencia, Guadalupe; Zúñiga, Sergio; Rodríguez-Ropon, Andrea; Osorio-Rico, Laura; Torres Luvia, Sanchez; Gracia-Mora, Isabel; Marquez-Rosado, Lucrecia; Sánchez, Aurora; Moreno-García, Miguel E; Cruz, Arturo; Bravo-Gómez, María Elena; Ruiz-Ramírez, Lena; Rodríguez-Enriquez, Sara; Sotelo, Julio

2005-01-01

Abstract In this work, we investigated the effects of Casiopeina II-gly (Cas IIgly)—a new copper compound exhibiting antineoplastic activity—on glioma C6 cells under both in vitro and in vivo conditions, as an approach to identify potential therapeutic agents against malignant glioma. The exposure of C6 cells to Cas IIgly significantly inhibited cell proliferation, increased reactive oxygen species (ROS) formation, and induced apoptosis in a dose-dependent manner. In cultured C6 cells, Cas IIgly caused mitochondrio-nuclear translocation of apoptosis induction factor (AIF) and endonuclease G at all concentrations tested; in contrast, fragmentation of nucleosomal DNA, cytochrome c release, and caspase-3 activation were observed at high concentrations. Administration of N-acetyl-l-cystein, an antioxidant, resulted in significant inhibition of AIF translocation, nucleosomal DNA fragmentation, and caspase-3 activation induced by Cas IIgly. These results suggest that caspase-dependent and caspase-independent pathways both participate in apoptotic events elicited by Cas IIgly. ROS formation induced by Cas IIgly might also be involved in the mitochondrio-nuclear translocation of AIF and apoptosis. In addition, treatment of glioma C6-positive rats with Cas IIgly reduced tumor volume and mitotic and cell proliferation indexes, and increased apoptotic index. Our findings support the use of Cas IIgly for the treatment of malignant gliomas. PMID:16036107

Lunasin, with an arginine-glycine-aspartic acid motif, causes apoptosis to L1210 leukemia cells by activation of caspase-3.

PubMed

de Mejia, Elvira Gonzalez; Wang, Wenyi; Dia, Vermont P

2010-03-01

Lunasin is a novel chemopreventive peptide featuring a cell adhesion motif composed of arginine-glycine-aspartate (RGD) which has been associated to cytotoxicity to established cell lines. The objectives of this study were to determine the effect of lunasin on the viability of L1210 leukemia cells and to understand the underlying mechanisms involved. Pure lunasin and lunasin enriched soy flour (LES) caused cytotoxicity to L1210 leukemia cells with IC(50) of 14 and 16 microM (lunasin equivalent), respectively. Simulated gastrointestinal digestion showed that 25% of the original amount of lunasin survived 3 h of pepsin digestion and 3% of lunasin remained after sequential pepsin-pancreatin digestion for a total of 6 h. Cell cycle analysis showed that lunasin caused a dose-dependent G2 cell cycle arrest and apoptosis. Treatment of L1210 leukemia cells with 1 mg/mL of LES for 18 h led to an increase in the amount of apoptotic cells from 2 to 40%. Compared to untreated cells, treatment with 1 mg/mL LES showed a 6-fold increase on the expressions of caspases-8 and -9, and and a 12-fold increase on the expression of caspase-3. These results showed for the first time that lunasin, a naturally occurring peptide containing an RGD motif, caused apoptosis to L1210 leukemia cells through caspase-3 activation.

NF-κB Regulates Caspase-4 Expression and Sensitizes Neuroblastoma Cells to Fas-Induced Apoptosis

PubMed Central

Yang, Hai-Jie; Wang, Mian; Wang, Lei; Cheng, Bin-Feng; Lin, Xiao-Yu; Feng, Zhi-Wei

2015-01-01

Found in neurons and neuroblastoma cells, Fas-induced apoptosis and accompanied activation of NF-κB signaling were thought to be associated with neurodegenerative diseases. However, the detailed functions of NF-κB activation in Fas killing and the effect of NF-κB activation on its downstream events remain unclear. Here, we demonstrated that agonistic Fas antibody induces cell death in a dose-dependent way and NF-κB signaling is activated as well, in neuroblastoma cells SH-EP1. Unexpectedly, NF-κB activation was shown to be pro-apoptotic, as suggested by the reduction of Fas-induced cell death with either a dominant negative form of IκBα (DN-IκBα) or an IκB kinase-specific inhibitor. To our interest, when analyzing downstream events of NF-κB signaling, we found that DN-IκBα only suppressed the expression of caspase-4, but not other caspases. Vice versa, enhancement of NF-κB activity by p65 (RelA) overexpression increased the expression of caspase-4 at both mRNA and protein levels. More directly, results from dual luciferase reporter assay demonstrated the regulation of caspase-4 promoter activity by NF-κB. When caspase-4 activity was blocked by its dominant negative (DN) form, Fas-induced cell death was substantially reduced. Consistently, the cleavage of PARP and caspase-3 induced by Fas was also reduced. In contrast, the cleavage of caspase-8 remained unaffected in caspase-4 DN cells, although caspase-8 inhibitor could rescue Fas-induced cell death. Collectively, these data suggest that caspase-4 activity is required for Fas-induced cell apoptosis and caspase-4 may act upstream of PARP and caspase-3 and downstream of caspase-8. Overall, we demonstrate that NF-κB can mediate Fas-induced apoptosis through caspase-4 protease, indicating that caspase-4 is a new mediator of NF-κB pro-apoptotic pathway in neuroblastoma cells. PMID:25695505

Unnatural amino acids increase sensitivity and provide for the design of highly selective caspase substrates

PubMed Central

Poreba, M; Kasperkiewicz, P; Snipas, S J; Fasci, D; Salvesen, G S; Drag, M

2014-01-01

Traditional combinatorial peptidyl substrate library approaches generally utilize natural amino acids, limiting the usefulness of this tool in generating selective substrates for proteases that share similar substrate specificity profiles. To address this limitation, we synthesized a Hybrid Combinatorial Substrate Library (HyCoSuL) with the general formula of Ac-P4-P3-P2-Asp-ACC, testing the approach on a family of closely related proteases – the human caspases. The power of this library for caspase discrimination extends far beyond traditional PS-SCL approach, as in addition to 19 natural amino acids we also used 110 diverse unnatural amino acids that can more extensively explore the chemical space represented by caspase-active sites. Using this approach we identified and employed peptide-based substrates that provided excellent discrimination between individual caspases, allowing us to simultaneously resolve the individual contribution of the apical caspase-9 and the executioner caspase-3 and caspase-7 in the development of cytochrome-c-dependent apoptosis for the first time. PMID:24832467

Evaluation of differential representative values between Chinese hamster cells and human lymphocytes in mitomycin C-induced cytogenetic assays and caspase-3 activity.

PubMed

Liao, Pei-Hu; Lin, Ruey-Hseng; Yang, Ming-Ling; Li, Yi-Ching; Kuan, Yu-Hsiang

2012-03-01

Chinese hamster ovary (CHO) cells, its lung fibroblasts (V79), and human lymphocytes are routinely used in in vitro cytogenetic assays, which include micronuclei (MN), sister chromatid exchange (SCE), and chromosome aberration (CA) assays. Mitomycin C (MMC), a DNA cross-link alkylating agent, is both an anticancer medicine and a carcinogen. To study the differential representative values of cell types in MMC-treated cytogenetic assays and its upstream factor, cysteine aspartic acid-specific protease (caspase)-3. Among the chosen cell types, lymphocytes expressed the highest sensitivity in all three MMC-induced assays, whereas CHO and V79 showed varied sensitivity in different assays. In MN assay, the sensitivity of CHO is higher than or equal to V79; in SCE assay, the sensitivity of CHO is the same as V79; and in CA assay, the sensitivity of CHO is higher than V79. In-depth analysis of CA revealed that in chromatid breaks and dicentrics formation, lymphocyte was the most sensitive of all and CHO was more sensitive than V79; and in acentrics and interchanges formation, lymphocyte was much more sensitive than the others. Furthermore, we found caspase-3 activity plays an important role in MMC-induced cytogenetic assays, with MMC-induced caspase-3 activity resulting in more sensitivity in lymphocytes than in CHO and V79. Based on these findings, lymphocyte will make a suitable predictive or representative control reference in cytogenetic assays and caspase-3 activity with its high specificity, positive predictive value, and sensitivity.

Zinc-mediated Allosteric Inhibition of Caspase-6*

PubMed Central

Velázquez-Delgado, Elih M.; Hardy, Jeanne A.

2012-01-01

Zinc and caspase-6 have independently been implicated in several neurodegenerative disorders. Depletion of zinc intracellularly leads to apoptosis by an unknown mechanism. Zinc inhibits cysteine proteases, including the apoptotic caspases, leading to the hypothesis that zinc-mediated inhibition of caspase-6 might contribute to its regulation in a neurodegenerative context. Using inductively coupled plasma optical emission spectroscopy, we observed that caspase-6 binds one zinc per monomer, under the same conditions where the zinc leads to complete loss of enzymatic activity. To understand the molecular details of zinc binding and inhibition, we performed an anomalous diffraction experiment above the zinc edge. The anomalous difference maps showed strong 5σ peaks, indicating the presence of one zinc/monomer bound at an exosite distal from the active site. Zinc was not observed bound to the active site. The zinc in the exosite was liganded by Lys-36, Glu-244, and His-287 with a water molecule serving as the fourth ligand, forming a distorted tetrahedral ligation sphere. This exosite appears to be unique to caspase-6, as the residues involved in zinc binding were not conserved across the caspase family. Our data suggest that binding of zinc at the exosite is the primary route of inhibition, potentially locking caspase-6 into the inactive helical conformation. PMID:22891250

Propolis ameliorates tumor nerosis factor-α, nitric oxide levels, caspase-3 and nitric oxide synthase activities in kainic acid mediated excitotoxicity in rat brain.

PubMed

Swamy, Mummedy; Suhaili, Dian; Sirajudeen, K N S; Mustapha, Zulkarnain; Govindasamy, Chandran

2014-01-01

Increased nitric oxide (NO), neuronal inflammation and apoptosis have been proposed to be involved in excitotoxicity plays a part in many neurodegenerative diseases. To understand the neuro-protective effects of propolis, activities of Nitric oxide synthase (NOS) and caspase-3 along with NO and tumor necrosis factor-α (TNF-α) levels were studied in cerebral cortex (CC), cerebellum (CB) and brain stem (BS) in rats supplemented with propolis prior to excitotoxic injury with kainic acid (KA). Male Sprague-Dawley rats were divided into four groups (n=6 rats per group) as Control, KA, Propolis and KA+Propolis. The control group and KA group have received vehicle and saline. Propolis group and propolis + KA group were orally administered with propolis (150 mg/kg body weight), five times every 12 hours. KA group and propolis +KA group were injected subcutaneously with kainic acid (15 mg/kg body weight) and were sacrificed after 2 hrs. CC, CB and BS were separated, homogenized and used for estimation of NOS, caspase-3, NO and TNF-α by commercial kits. Results were analyzed by one way ANOVA, reported as mean + SD (n=6 rats), and p<0.05 was considered statistically significant. The concentration of NO, TNF-α, NOS and caspase-3 activity were increased significantly (p<0.001) in all the three brain regions tested in KA group compared to the control. Propolis supplementation significantly (p<0.001) prevented the increase in NOS, NO, TNF-α and caspase-3 due to KA. Results of this study clearly demonstrated that the propolis supplementation attenuated the NOS, caspase-3 activities, NO, and TNF-α concentration and in KA mediated excitotoxicity. Hence propolis can be a possible potential protective agent against excitotoxicity and neurodegenerative disorders.

A bioluminescent caspase-1 activity assay rapidly monitors inflammasome activation in cells.

PubMed

O'Brien, Martha; Moehring, Danielle; Muñoz-Planillo, Raúl; Núñez, Gabriel; Callaway, Justin; Ting, Jenny; Scurria, Mike; Ugo, Tim; Bernad, Laurent; Cali, James; Lazar, Dan

2017-08-01

Inflammasomes are protein complexes induced by diverse inflammatory stimuli that activate caspase-1, resulting in the processing and release of cytokines, IL-1β and IL-18, and pyroptosis, an immunogenic form of cell death. To provide a homogeneous method for detecting caspase-1 activity, we developed a bioluminescent, plate-based assay that combines a substrate, Z-WEHD-aminoluciferin, with a thermostable luciferase in an optimized lytic reagent added directly to cultured cells. Assay specificity for caspase-1 is conferred by inclusion of a proteasome inhibitor in the lytic reagent and by use of a caspase-1 inhibitor to confirm activity. This approach enables a specific and rapid determination of caspase-1 activation. Caspase-1 activity is stable in the reagent thereby providing assay convenience and flexibility. Using this assay system, caspase-1 activation has been determined in THP-1 cells following treatment with α-hemolysin, LPS, nigericin, gramicidin, MSU, R848, Pam3CSK4, and flagellin. Caspase-1 activation has also been demonstrated in treated J774A.1 mouse macrophages, bone marrow-derived macrophages (BMDMs) from mice, as well as in human primary monocytes. Caspase-1 activity was not detected in treated BMDMs derived from Casp1 -/- mice, further confirming the specificity of the assay. Caspase-1 activity can be measured directly in cultured cells using the lytic reagent, or caspase-1 activity released into medium can be monitored by assay of transferred supernatant. The caspase-1 assay can be multiplexed with other assays to monitor additional parameters from the same cells, such as IL-1β release or cell death. The caspase-1 assay in combination with a sensitive real-time monitor of cell death allows one to accurately establish pyroptosis. This assay system provides a rapid, convenient, and flexible method to specifically and quantitatively monitor caspase-1 activation in cells in a plate-based format. This will allow a more efficient and effective

LDR reverses DDP resistance in ovarian cancer cells by affecting ERCC-1, Bcl-2, Survivin and Caspase-3 expressions.

PubMed

Ju, Xingyan; Yu, Hongsheng; Liang, Donghai; Jiang, Tao; Liu, Yuanwei; Chen, Ling; Dong, Qing; Liu, Xiaoran

2018-06-01

Ovarian cancer is the most frequent cause of death resulting from malignant gynecological tumors. After surgical intervention, cisplatin (DDP) is a major chemotherapy drug for ovarian cancer, but the ovarian cancer cells tend to develop DDP resistance in the clinical setting. Tumor cells are sensitive to low-dose radiation (LDR). However, how the LDR therapy improves the effects of chemotherapy drugs on ovarian cancer is not well understood. This study aimed to explore this issue. The SKOV3/DDP cells were divided into 3 groups, including low-dose group, conventional-dose group, and control group (no radiation). Cell counting kit-8 assay was performed to measure cell proliferation. Flow cytometric analysis was then utilized to quantify the apoptosis with classical Annexin V/propidium iodide co-staining. And Real-time quantitative PCR and western blot were eventually used to analyze the mRNA and protein levels of excision repair cross complementing-group 1 (ERCC1), B-cell lymphoma 2 (Bcl-2), Survivin and Caspase-3, respectively. The IC50 value of DDP in the low-dose group was significantly lower compared with the other two groups. Compared with the conventional-dose group and control group, LDR treatment resulted in significantly more apoptosis. Besides, LDR treatment significantly decreased the mRNA and protein expression of ERCC1, Bcl-2, and Survivin, and enhanced the mRNA and protein expression of Caspase-3 compared with the other two groups. LDR reversed DDP resistance in SKOV3/DDP cells possibly by suppressing ERCC1, Bcl-2, and Survivin expressions, and increasing Caspase-3 expression. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

The effects of 3,4-methylenedioxymethamphetamine (MDMA) on nicotinic receptors: Intracellular calcium increase, calpain/caspase 3 activation, and functional upregulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garcia-Rates, Sara; Camarasa, Jordi; Sanchez-Garcia, Ana I.

2010-05-01

Previous work by our group demonstrated that homomeric alpha7 nicotinic acetylcholine receptors (nAChR) play a role in the neurotoxicity induced by 3,4-methylenedioxymethamphetamine (MDMA), as well as the binding affinity of this drug to these receptors. Here we studied the effect of MDMA on the activation of nAChR subtypes, the consequent calcium mobilization, and calpain/caspase 3 activation because prolonged Ca{sup 2+} increase could contribute to cytotoxicity. As techniques, we used fluorimetry in Fluo-4-loaded PC12 cells and electrophysiology in Xenopus oocytes. MDMA produced a rapid and sustained increase in calcium without reaching the maximum effect induced by ACh. It also concentration-dependently inhibitedmore » the response induced by ACh, nicotine, and the specific alpha7 agonist PNU 282987 with IC{sub 50} values in the low micromolar range. Similarly, MDMA induced inward currents in Xenopus oocytes transfected with human alpha7 but not with alpha4beta2 nAChR and inhibited ACh-induced currents in both receptors in a concentration-dependent manner. The calcium response was inhibited by methyllycaconitine (MLA) and alpha-bungarotoxin but not by dihydro-beta-erythroidine. These results therefore indicate that MDMA acts as a partial agonist on alpha7 nAChRs and as an antagonist on the heteromeric subtypes. Subsequently, calcium-induced Ca{sup 2+} release from the endoplasmic reticulum and entry through voltage-operated calcium channels are also implicated as proved using specific antagonists. In addition, treatment with MDMA for 24 h significantly increased basal Ca{sup 2+} levels and induced an increase in alpha-spectrin breakdown products, which indicates that calpain and caspase 3 were activated. These effects were inhibited by pretreatment with MLA. Moreover, pretreatment with MDMA induced functional upregulation of calcium responses to specific agonists of both heteromeric and alpha7 nAChR. Sustained calcium entry and calpain activation could favor

Caspase-2-mediated cleavage of Mdm2 creates p53-induced positive feedback loop

PubMed Central

Oliver, Trudy G.; Meylan, Etienne; Chang, Gregory P.; Xue, Wen; Burke, James R.; Humpton, Timothy J.; Hubbard, Diana; Bhutkar, Arjun; Jacks, Tyler

2011-01-01

SUMMARY Caspase-2 is an evolutionarily conserved caspase, yet its biological function and cleavage targets are poorly understood. Caspase-2 is activated by the p53 target gene product PIDD (also known as LRDD) in a complex called the Caspase-2-PIDDosome. We show that PIDD expression promotes growth arrest and chemotherapy resistance by a mechanism that depends on Caspase-2 and wild-type p53. PIDD-induced Caspase-2 directly cleaves the E3 ubiquitin ligase Mdm2 at Asp 367, leading to loss of the C-terminal RING domain responsible for p53 ubiquitination. As a consequence, N-terminally truncated Mdm2 binds p53 and promotes its stability. Upon DNA damage, p53 induction of the Caspase-2-PIDDosome creates a positive feedback loop that inhibits Mdm2 and reinforces p53 stability and activity, contributing to cell survival and drug resistance. These data establish Mdm2 as a cleavage target of Caspase-2 and provide insight into a mechanism of Mdm2 inhibition that impacts p53 dynamics upon genotoxic stress. PMID:21726810

Caspase-2 Is Localized at the Golgi Complex and Cleaves Golgin-160 during Apoptosis

PubMed Central

Mancini, Marie; Machamer, Carolyn E.; Roy, Sophie; Nicholson, Donald W.; Thornberry, Nancy A.; Casciola-Rosen, Livia A.; Rosen, Antony

2000-01-01

Caspases are an extended family of cysteine proteases that play critical roles in apoptosis. Animals deficient in caspases-2 or -3, which share very similar tetrapeptide cleavage specificities, exhibit very different phenotypes, suggesting that the unique features of individual caspases may account for distinct regulation and specialized functions. Recent studies demonstrate that unique apoptotic stimuli are transduced by distinct proteolytic pathways, with multiple components of the proteolytic machinery clustering at distinct subcellular sites. We demonstrate here that, in addition to its nuclear distribution, caspase-2 is localized to the Golgi complex, where it cleaves golgin-160 at a unique site not susceptible to cleavage by other caspases with very similar tetrapeptide specificities. Early cleavage at this site precedes cleavage at distal sites by other caspases. Prevention of cleavage at the unique caspase-2 site delays disintegration of the Golgi complex after delivery of a pro-apoptotic signal. We propose that the Golgi complex, like mitochondria, senses and integrates unique local conditions, and transduces pro-apoptotic signals through local caspases, which regulate local effectors. PMID:10791974

Mouse strain-dependent caspase activation during acetaminophen hepatotoxicity does not result in apoptosis or modulation of inflammation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Williams, C. David; Koerner, Michael R., E-mail: mkoern2@illinois.edu; Lampe, Jed N.

The mechanisms of acetaminophen (APAP)-mediated hepatic oncotic necrosis have been extensively characterized. However, it was recently demonstrated that fed CD-1 mice have a transient caspase activation which initiates apoptosis. To evaluate these findings in more detail, outbred (Swiss Webster, SW) and inbred (C57BL/6) mice were treated with APAP with or without pan-caspase inhibitor and compared to the apoptosis model of galactosamine (GalN)/endotoxin (ET). Fasted or fed APAP-treated C57BL/6 mice showed no evidence of caspase-3 processing or activity. Interestingly, a minor, temporary increase in caspase-3 processing and activity (150% above baseline) was observed after APAP treatment only in fed SW mice.more » The degree of caspase-3 activation in SW mice after APAP was minor compared to that observed in GalN/ET-treated mice (1600% above baseline). The pancaspase inhibitor attenuated caspase activation and resulted in increased APAP-induced injury (plasma ALT, necrosis scoring). The caspase inhibitor did not affect apoptosis because regardless of treatment only < 0.5% of hepatocytes showed consistent apoptotic morphology after APAP. In contrast, > 20% apoptotic cells were observed in GalN/ET-treated mice. Presence of the caspase inhibitor altered hepatic glutathione levels in SW mice, which could explain the exacerbation of injury. Additionally, the infiltration of hepatic neutrophils was not altered by the fed state of either mouse strain. Conclusion: Minor caspase-3 activation without apoptotic cell death can be observed only in fed mice of some outbred strains. These findings suggest that although the severity of APAP-induced liver injury varies between fed and fasted animals, the mechanism of cell death does not fundamentally change. -- Highlights: Black-Right-Pointing-Pointer During acetaminophen overdose caspase-3 can be activated in fed mice of certain outbred strains. Black-Right-Pointing-Pointer Hepatic ATP levels are not the determining factor for

[Effects of Chinese herbal medicine Yiqi Huayu Recipe on apoptosis of dorsal root ganglion neurons and expression of caspase-3 in rats with lumbar nerve root compression].

PubMed

Xu, Le-qin; Li, Xiao-feng; Zhang, You-wei; Shu, Bing; Shi, Qi; Wang, Yong-jun; Zhou, Chong-jian

2010-12-01

To observe the effects of Yiqi Huayu Recipe, a Chinese compound herbal medicine, on apoptosis of dorsal root ganglion (DRG) neurons and expression of caspase-3 in rats after lumbar nerve root compression injury. A total of 40 male Sprague-Dawley rats were randomly allocated into 4 groups: control group, untreated group, Methylcobal group and Yiqi Huayu Recipe group. Surgery was performed on rats of untreated group, Methylcobal group and Yiqi Huayu Recipe group to place a micro-silica gel on right L₄ DRG, while control group received skin and paravertebral muscle incision only. Rats in Methylcobal group and Yiqi Huayu Recipe group were given Methylcobal by intramuscular injection and Yiqi Huayu Recipe intragastrically respectively. Rats in control group and untreated group received saline intragastrically as equal amount as Yiqi Huayu Recipe group. The compressed nerve roots were harvested at the 10th day after treatment. Apoptosis of DRG neurons was detected by terminal deoxynucleotidyl transferase-mediated nick-end labeling. Caspase-3 activity and mRNA expression in compressed nerve roots were detected with spectrophotography and real-time polymerase chain reaction respectively. Apoptosis of DRG neurons was significantly increased in the rat model. The apoptosis index of untreated group was higher than that of control group (P<0.01). Yiqi Huayu Recipe and Methylcobal could reduce the apoptosis of DRG neurons, and both groups showed a lower apoptosis index than untreated group (P<0.01). Caspase-3 activity and its gene expression were significantly increased in untreated group. The levels of caspase-3 activity and its gene expression in untreated group were higher than those in control group (P<0.05 or P<0.01). Yiqi Huayu Recipe and Methylcobal could reduce the overexpression of caspase-3 mRNA, and statistically significant differences were found between the untreated group and Yiqi Huayu Recipe group or Methylcobal group (P<0.01). Lumbar nerve root compression

Phenyl Saligenin Phosphate Induced Caspase-3 and c-Jun N-Terminal Kinase Activation in Cardiomyocyte-Like Cells.

PubMed

Felemban, Shatha G; Garner, A Christopher; Smida, Fathi A; Boocock, David J; Hargreaves, Alan J; Dickenson, John M

2015-11-16

At present, little is known about the effect(s) of organophosphorous compounds (OPs) on cardiomyocytes. In this study, we have investigated the effects of phenyl saligenin phosphate (PSP), two organophosphorothioate insecticides (diazinon and chlorpyrifos), and their acutely toxic metabolites (diazoxon and chlorpyrifos oxon) on mitotic and differentiated H9c2 cardiomyoblasts. OP-induced cytotoxicity was assessed by monitoring MTT reduction, LDH release, and caspase-3 activity. Cytotoxicity was not observed with diazinon, diazoxon, or chlorpyrifos oxon (48 h exposure; 200 μM). Chlorpyrifos-induced cytotoxicity was only evident at concentrations >100 μM. In marked contrast, PSP displayed pronounced cytotoxicity toward mitotic and differentiated H9c2 cells. PSP triggered the activation of JNK1/2 but not ERK1/2, p38 MAPK, or PKB, suggesting a role for this pro-apoptotic protein kinase in PSP-induced cell death. The JNK1/2 inhibitor SP 600125 attenuated PSP-induced caspase-3 and JNK1/2 activation, confirming the role of JNK1/2 in PSP-induced cytotoxicity. Fluorescently labeled PSP (dansylated PSP) was used to identify novel PSP binding proteins. Dansylated PSP displayed cytotoxicity toward differentiated H9c2 cells. 2D-gel electrophoresis profiles of cells treated with dansylated PSP (25 μM) were used to identify proteins fluorescently labeled with dansylated PSP. Proteomic analysis identified tropomyosin, heat shock protein β-1, and nucleolar protein 58 as novel protein targets for PSP. In summary, PSP triggers cytotoxicity in differentiated H9c2 cardiomyoblasts via JNK1/2-mediated activation of caspase-3. Further studies are required to investigate whether the identified novel protein targets of PSP play a role in the cytotoxicity of this OP, which is usually associated with the development of OP-induced delayed neuropathy.

JNK signaling pathway regulates sorbitol-induced Tau proteolysis and apoptosis in SH-SY5Y cells by targeting caspase-3.

PubMed

Olivera Santa-Catalina, Marta; Caballero Bermejo, Montaña; Argent, Ricardo; Alonso, Juan C; Centeno, Francisco; Lorenzo, María J

2017-12-15

Growing evidence suggests that Diabetes Mellitus increases the risk of developing Alzheimer's disease. It is well known that hyperglycemia, a key feature of Diabetes Mellitus, may induce plasma osmolarity disturbances. Both hyperglycemia and hyperosmolarity promote the altered post-translational regulation of microtubule-associated protein Tau. Interestingly, abnormal hyperphosphorylation and cleavage of Tau have been proven to lead to the genesis of filamentous structures referred to as neurofibrillary tangles, the main pathological hallmark of Alzheimer's disease. We have previously described that hyperosmotic stress induced by sorbitol promotes Tau proteolysis and apoptosis in SH-SY5Y cells via caspase-3 activation. In order to gain insights into the regulatory mechanisms of such processes, in this work we explored the intracellular signaling pathways that regulate these events. We found that sorbitol treatment significantly enhanced the activation of conventional families of MAPK in SH-SY5Y cells. Tau proteolysis was completely prevented by JNK inhibition but not affected by either ERK1/2 or p38 MAPK blockade. Moreover, inhibition of JNK, but not ERK1/2 or p38 MAPK, efficiently prevented sorbitol-induced apoptosis and caspase-3 activation. In summary, we provide evidence that JNK signaling pathway is an upstream regulator of hyperosmotic stress-induced Tau cleavage and apoptosis in SH-SY5Y through the control of caspase-3 activation. Copyright © 2017 Elsevier Inc. All rights reserved.

Higher Caspase-like activity in symptomatic isolates of Blastocystis spp

PubMed Central

2014-01-01

Background Biochemical evidence of a caspase-like execution pathway has been demonstrated in a variety of protozoan parasites, including Blastocystis spp. The distinct differences in the phenotypic characterization reported previously have prompted us to compare the rate of apoptosis in Blastocystis spp. isolated from individuals who were symptomatic and asymptomatic. In the current study, we analysed the caspase activation involved in PCD mediated by a cytotoxic drug, (metronidazole) in both symptomatic & asymptomatic isolates. Methods Apoptosis was induced in Blastocystis spp. by treating cultures of symptomatic and asymptomatic isolates of 3 sub-types namely 1, 3 and 5 with two different concentrations, 0.1 and 0.0001 mg/ml of metronidazole (with and without pre-treatment with a pan-caspase inhibitor, zVAD.fmk). The experiment was repeated to assess the number of apoptotic cells in all the isolates of both conditions. Results Symptomatic isolates of subtype 3 (without pre-treatment with a pan-caspase inhibitor, zVAD.fmk) showed high fluorescence intensity for active caspase-like proteases [0.0001 mg/ml, 88% (p < 0.001) at 0.1 mg/ml, 70% (p < 0.001)] at the 72nd hour in vitro culture in comparison with asymptomatic isolates [0.0001 mg/ml, 65%, at 0.1 mg/ml, 55%]. The number of apoptotic cells was higher [0.0001 mg/ml, 89% (p < 0.001) and at 0.1 mg/ml, 70% (p < 0.001)] at the 72nd hour of in vitro culture in comparison with asymptomatic isolates [0.0001 mg/ml, 66% (p < 0.001) and at 0.1 mg/ml, 45% (p < 0.01)]. Cells treated with metronidazole in the presence of zVAD.fmk showed less than 10% caspase activation. Conclusion The high number of symptomatic cells expressing active caspase-like proteases and becoming apoptotic compared to asymptomatic cells clearly demonstrates that the response to metronidazole treatment is isolate dependent. Hence this justifies the conflicting reports on the curative success rates when treated with

Zinc oxide nanoparticles induce rat retinal ganglion cell damage through bcl-2, caspase-9 and caspase-12 pathways.

PubMed

Guo, Dadong; Bi, Hongsheng; Wu, Qiuxin; Wang, Daoguang; Cui, Yan

2013-06-01

Nanomaterials, including zinc oxide (ZnO) nanoparticles, are being developed for a variety of commercial products. Recent reports showed that cells exposed to ZnO nanoparticles produced severe cytotoxicity accompanied by oxidative stress and genotoxicity. To understand the possible mechanism underlying oxidative stress of ZnO nanoparticles, the present investigation focused on the direct bioactivity of ZnO nanoparticles using a rat retinal ganglion cell (RGC-5) culture. At concentrations relevant to those used in vitro exposure of RGC-5 cells to ZnO nanoparticles, it was found that ZnO nanoparticles could inhibit cell proliferation in time- and concentration-dependent manners. Meanwhile, cell cycle arrest of S and G2/M phases occurred in RGC-5 cells induced by ZnO nanoparticles. Moreover, our results also demonstrated that the overproduction of reactive oxygen species (ROS) and elevated level of caspase-12 as well as decreased levels of bcl-2 and caspase-9 occurred after treatment with different concentrations of ZnO nanoparticles when compared to those in untreated cells. In summary, our findings suggest that ZnO nanoparticles could lead to the over generations of ROS and caspase-12 as well as decreased levels of bcl-2 and caspase-9. These results indicate that bcl-2, caspase-9 and caspase-12 may play significant roles in ZnO nanoparticle-induced RGC-5 cell damage.

Caspase-mediated apoptosis induction in zebrafish cerebellar Purkinje neurons.

PubMed

Weber, Thomas; Namikawa, Kazuhiko; Winter, Barbara; Müller-Brown, Karina; Kühn, Ralf; Wurst, Wolfgang; Köster, Reinhard W

2016-11-15

The zebrafish is a well-established model organism in which to study in vivo mechanisms of cell communication, differentiation and function. Existing cell ablation methods are either invasive or they rely on the cellular expression of prokaryotic enzymes and the use of antibiotic drugs as cell death-inducing compounds. We have recently established a novel inducible genetic cell ablation system based on tamoxifen-inducible Caspase 8 activity, thereby exploiting mechanisms of cell death intrinsic to most cell types. Here, we prove its suitability in vivo by monitoring the ablation of cerebellar Purkinje cells (PCs) in transgenic zebrafish that co-express the inducible caspase and a fluorescent reporter. Incubation of larvae in tamoxifen for 8 h activated endogenous Caspase 3 and cell death, whereas incubation for 16 h led to the near-complete loss of PCs by apoptosis. We observed synchronous cell death autonomous to the PC population and phagocytosing microglia in the cerebellum, reminiscent of developmental apoptosis in the forebrain. Thus, induction of apoptosis through targeted activation of caspase by tamoxifen (ATTAC TM ) further expands the repertoire of genetic tools for conditional interrogation of cellular functions. © 2016. Published by The Company of Biologists Ltd.

Arsenic induced progesterone production in a caspase-3-dependent manner and changed redox status in preovulatory granulosa cells.

PubMed

Yuan, Xiao-Hua; Lu, Cai-Ling; Yao, Nan; An, Li-Sha; Yang, Bai-Qing; Zhang, Chuan-Ling; Ma, Xu

2012-01-01

Arsenic contamination is a principal environmental health threat throughout the world. However, little is known about the effect of arsenic on steroidogenesis in granulosa cells (GCs). We found that the treatment of preovulatory GCs with arsenite stimulated progesterone production. A significant increase in serum level of progesterone was observed in female Sprague-Dawley rats following arsenite treatment at a dose of 10 mg/L/rat/day for 7 days. Further experiments demonstrated that arsenite treatment did not change the level of intracellular cyclic AMP (cAMP) or phosphorylated ERK1/2 in preovulatory GCs; however, progesterone production was significantly decreased when cAMP-dependent protein kinase (PKA) or ERK1/2 pathway was inhibited. This implied that the effect of arsenite on progesterone production may require cAMP/PKA and ERK1/2 signaling but not depend on them. Furthermore, we found that arsenite decreased intracellular reactive oxygen species (ROS) but increased the antioxidant glutathione (GSH) levels and mitochondrial membrane potential (ΔΨm) in parallel to the changes in progesterone production. Progesterone antagonist blocked the arsenic-stimulated increase of GSH levels. Arsenite treatment induced caspase-3 activation, although no apoptosis was observed. Inhibition of caspase-3 activity significantly decreased progesterone production stimulated by arsenite or follicle-stimulating hormone (FSH). GSH depletion with buthionine sulfoximine led to cell apoptosis in response to arsenite treatment. Collectively, this study demonstrated for the first time that arsenite stimulates progesterone production through cleaved/active caspase-3-dependent pathway, and the increase of GSH level promoted by progesterone production may protect GCs against apoptosis and maintain the steroidogenesis of GCs in response to arsenite treatment. Copyright © 2011 Wiley Periodicals, Inc.

Granuloma formation and host defense in chronic Mycobacterium tuberculosis infection requires PYCARD/ASC but not NLRP3 or caspase-1.

PubMed

McElvania Tekippe, Erin; Allen, Irving C; Hulseberg, Paul D; Sullivan, Jonathan T; McCann, Jessica R; Sandor, Matyas; Braunstein, Miriam; Ting, Jenny P-Y

2010-08-20

The NLR gene family mediates host immunity to various acute pathogenic stimuli, but its role in chronic infection is not known. This paper addressed the role of NLRP3 (NALP3), its adaptor protein PYCARD (ASC), and caspase-1 during infection with Mycobacterium tuberculosis (Mtb). Mtb infection of macrophages in culture induced IL-1beta secretion, and this requires the inflammasome components PYCARD, caspase-1, and NLRP3. However, in vivo Mtb aerosol infection of Nlrp3(-/-), Casp-1(-/-), and WT mice showed no differences in pulmonary IL-1beta production, bacterial burden, or long-term survival. In contrast, a significant role was observed for Pycard in host protection during chronic Mtb infection, as shown by an abrupt decrease in survival of Pycard(-/-) mice. Decreased survival of Pycard(-/-) animals was associated with defective granuloma formation. These data demonstrate that PYCARD exerts a novel inflammasome-independent role during chronic Mtb infection by containing the bacteria in granulomas.

Caspase-1 Is Hepatoprotective during Trauma and Hemorrhagic Shock by Reducing Liver Injury and Inflammation

PubMed Central

Menzel, Christoph L; Sun, Qian; Loughran, Patricia A; Pape, Hans-Christoph; Billiar, Timothy R; Scott, Melanie J

2011-01-01

Adaptive immune responses are induced in liver after major stresses such as hemorrhagic shock (HS) and trauma. There is emerging evidence that the inflammasome, the multiprotein platform that induces caspase-1 activation and promotes interleukin (IL)-1β and IL-18 processing, is activated in response to cellular oxidative stress, such as after hypoxia, ischemia and HS. Additionally, damage-associated molecular patterns, such as those released after injury, have been shown to activate the inflammasome and caspase-1 through the NOD-like receptor (NLR) NLRP3. However, the role of the inflammasome in organ injury after HS and trauma is unknown. We therefore investigated inflammatory responses and end-organ injury in wild-type (WT) and caspase-1−/−mice in our model of HS with bilateral femur fracture (HS/BFF). We found that caspase-1−/− mice had higher levels of systemic inflammatory cytokines than WT mice. This result corresponded to higher levels of liver damage, cell death and neutrophil influx in caspase-1−/− liver compared with WT, although there was no difference in lung damage between experimental groups. To determine if hepatoprotection also depended on NLRP3, we subjected NLRP3−/− mice to HS/BFF, but found inflammatory responses and liver damage in these mice was similar to WT. Hepatoprotection was also not due to caspase-1–dependent cytokines, IL-1β and IL-18. Altogether, these data suggest that caspase-1 is hepatoprotective, in part through regulation of cell death pathways in the liver after major trauma, and that caspase-1 activation after HS/BFF does not depend on NLRP3. These findings may have implications for the treatment of trauma patients and may lead to progress in prevention or treatment of multiple organ failure (MOF). PMID:21666957

Stretch-induced uterine myocyte differentiation during rat pregnancy: involvement of caspase activation.

PubMed

Shynlova, Oksana; Dorogin, Anna; Lye, Stephen J

2010-06-01

Proliferation, differentiation, and apoptosis are three major processes by which the pregnant uterus maintains homeostasis to accommodate the growing fetus. We demonstrated previously that caspase activation in the pregnant rat myometrium at midgestation coincides with the transition from uterine hyperplasia to hypertrophy. We hypothesized that this transition was induced by stasis of myometrial blood flow (and subsequent hypoxia/ischaemia insult) resulting from acute myometrial stretch induced by a growing embryo. Therefore, we measured the expression of active caspase 3 and two hypoxia markers (transcription factor HIF1A and pimonidazole hydrochloride) in pregnant rat myometrium. To investigate the effect of gravidity we used unilaterally pregnant rats. Caspase 3 was activated only in the gravid horn of the unilaterally pregnant animals on Gestational Days 12-15. This activation was associated with high levels of HIF1A and pimonidazole immunostaining, which were limited to the circular myometrial layer of the gravid horn, indicative of hypoxia within this tissue. To isolate the effect of myometrial stretch applied by the growing fetus, we inserted an expandable polymer tube (intra-uterine expandable tube [IUET]) into the empty horn of Day 13 and Day 20 unilaterally pregnant rats. Tissue was collected 2, 14, and 24 h later. In the IUET-stretched empty horn, cleaved caspase 3 was activated at midgestation (Day 14), but not at late gestation (Day 21). We speculate that hypoxia resulting from mechanical stretch may activate caspase 3 within the pregnant myometrium only in the context of a specific endocrine environment.

Nuclear Envelope Disruption Involving Host Caspases Plays a Role in the Parvovirus Replication Cycle ▿

PubMed Central

Cohen, Sarah; Marr, Alexandra K.; Garcin, Pierre; Panté, Nelly

2011-01-01

Parvoviruses are small, nonenveloped, single-stranded DNA viruses which replicate in the nucleus of the host cell. We have previously found that early during infection the parvovirus minute virus of mice (MVM) causes small, transient disruptions of the nuclear envelope (NE). We have now investigated the mechanism used by MVM to disrupt the NE. Here we show that the viral phospholipase A2, the only known enzymatic domain on the parvovirus capsid, is not involved in causing NE disruption. Instead, the virus utilizes host cell caspases, which are proteases involved in causing NE breakdown during apoptosis, to facilitate these nuclear membrane disruptions. Studies with pharmacological inhibitors indicate that caspase-3 in particular is involved. A caspase-3 inhibitor prevents nuclear lamin cleavage and NE disruption in MVM-infected mouse fibroblast cells and reduces nuclear entry of MVM capsids and viral gene expression. Caspase-3 is, however, not activated above basal levels in MVM-infected cells, and other aspects of apoptosis are not triggered during early MVM infection. Instead, basally active caspase-3 is relocalized to the nuclei of infected cells. We propose that NE disruption involving caspases plays a role in (i) parvovirus entry into the nucleus and (ii) alteration of the compartmentalization of host proteins in a way that is favorable for the virus. PMID:21367902

Protection of Hippocampal CA1 Neurons Against Ischemia/Reperfusion Injury by Exercise Preconditioning via Modulation of Bax/Bcl-2 Ratio and Prevention of Caspase-3 Activation.

PubMed

Aboutaleb, Nahid; Shamsaei, Nabi; Rajabi, Hamid; Khaksari, Mehdi; Erfani, Sohaila; Nikbakht, Farnaz; Motamedi, Pezhman; Shahbazi, Ali

2016-01-01

Ischemia leads to loss of neurons by apoptosis in specific brain regions, especially in the hippocampus. The purpose of this study was investigating the effects of exercise preconditioning on expression of Bax, Bcl-2, and caspase-3 proteins in hippocampal CA1 neurons after induction of cerebral ischemia. Male rats weighing 260-300 g were randomly allocated into three groups (sham, exercise, and ischemia). The rats in exercise group were trained to run on a treadmill 5 days a week for 4 weeks. Ischemia was induced by the occlusion of both common carotid arteries (CCAs) for 20 min. Levels of expression of Bax, Bcl-2, and caspase-3 proteins in CA1 area of hippocampus were determined by immunohistochemical staining . The number of active caspase-3-positive neurons in CA1 area were significantly increased in ischemia group, compared to sham-operated group (P<0.001), and exercise preconditioning significantly reduced the ischemia/reperfusion-induced caspase-3 activation, compared to the ischemia group (P<0.05). Also, results indicated a significant increase in Bax/Bcl-2 ratio in ischemia group, compared to sham-operated group (P<0.001). This study indicated that exercise has a neuroprotective effects against cerebral ischemia when used as preconditioning stimuli.

Pathological apoptosis by xanthurenic acid, a tryptophan metabolite: activation of cell caspases but not cytoskeleton breakdown

PubMed Central

Malina, Halina Z; Richter, Christoph; Mehl, Martin; Hess, Otto M

2001-01-01

Background A family of aspartate-specific cysteinyl proteases, named caspases, mediates programmed cell death, apoptosis. In this function, caspases are important for physiological processes such as development and maintenance of organ homeostasis. Caspases are, however, also engaged in aging and disease development. The factors inducing age-related caspase activation are not known. Xanthurenic acid, a product of tryptophan degradation, is present in blood and urine, and accumulates in organs with aging. Results Here, we report triggering of apoptotic key events by xanthurenic acid in vascular smooth muscle and retinal pigment epithelium cells. Upon exposure of these cells to xanthurenic acid a degradation of ICAD/DFF45, poly(ADP-ribose) polymerase, and gelsolin was observed, giving a pattern of protein cleavage characteristic for caspase-3 activity. Active caspase-3, -8 and caspase-9 were detected by Western blot analysis and immunofluorescence. In the presence of xanthurenic acid the amino-terminal fragment of gelsolin bound to the cytoskeleton, but did not lead to the usually observed cytoskeleton breakdown. Xanthurenic acid also caused mitochondrial migration, cytochrome C release, and destruction of mitochondria and nuclei. Conclusions These results indicate that xanthurenic acid is a previously not recognized endogenous cell death factor. Its accumulation in cells may lead to accelerated caspase activation related to aging and disease development. PMID:11459518

The intracellular sensor NLRP3 mediates key innate and healing responses to influenza A virus via the regulation of caspase-1.

PubMed

Thomas, Paul G; Dash, Pradyot; Aldridge, Jerry R; Ellebedy, Ali H; Reynolds, Cory; Funk, Amy J; Martin, William J; Lamkanfi, Mohamed; Webby, Richard J; Boyd, Kelli L; Doherty, Peter C; Kanneganti, Thirumala-Devi

2009-04-17

Virus-induced interlukin-1beta (IL-1beta) and IL-18 production in macrophages are mediated via caspase-1 pathway. Multiple microbial components, including viral RNA, are thought to trigger assembly of the cryopyrin inflammasome resulting in caspase-1 activation. Here, we demonstrated that Nlrp3(-/-) and Casp1(-/-) mice were more susceptible than wild-type mice after infection with a pathogenic influenza A virus. This enhanced morbidity correlated with decreased neutrophil and monocyte recruitment and reduced cytokine and chemokine production. Despite the effect on innate immunity, cryopyrin-deficiency was not associated with any obvious defect in virus control or on the later emergence of the adaptive response. Early epithelial necrosis was, however, more severe in the infected mutants, with extensive collagen deposition leading to later respiratory compromise. These findings reveal a function of the cryopyrin inflammasome in healing responses. Thus, cryopyrin and caspase-1 are central to both innate immunity and to moderating lung pathology in influenza pneumonia.

Caspase-9 mediates synaptic plasticity and memory deficits of Danish dementia knock-in mice: caspase-9 inhibition provides therapeutic protection.

PubMed

Tamayev, Robert; Akpan, Nsikan; Arancio, Ottavio; Troy, Carol M; D'Adamio, Luciano

2012-12-10

Mutations in either Aβ Precursor protein (APP) or genes that regulate APP processing, such as BRI2/ITM2B and PSEN1/PSEN2, cause familial dementias. Although dementias due to APP/PSEN1/PSEN2 mutations are classified as familial Alzheimer disease (FAD) and those due to mutations in BRI2/ITM2B as British and Danish dementias (FBD, FDD), data suggest that these diseases have a common pathogenesis involving toxic APP metabolites. It was previously shown that FAD mutations in APP and PSENs promote activation of caspases leading to the hypothesis that aberrant caspase activation could participate in AD pathogenesis. Here, we tested whether a similar mechanism applies to the Danish BRI2/ITM2B mutation. We have generated a genetically congruous mouse model of FDD, called FDD(KI), which presents memory and synaptic plasticity deficits. We found that caspase-9 is activated in hippocampal synaptic fractions of FDD(KI) mice and inhibition of caspase-9 activity rescues both synaptic plasticity and memory deficits. These data directly implicate caspase-9 in the pathogenesis of Danish dementia and suggest that reducing caspase-9 activity is a valid therapeutic approach to treating human dementias.

A Krebs Cycle Component Limits Caspase Activation Rate through Mitochondrial Surface Restriction of CRL Activation.

PubMed

Aram, Lior; Braun, Tslil; Braverman, Carmel; Kaplan, Yosef; Ravid, Liat; Levin-Zaidman, Smadar; Arama, Eli

2016-04-04

How cells avoid excessive caspase activity and unwanted cell death during apoptotic caspase-mediated removal of large cellular structures is poorly understood. We investigate caspase-mediated extrusion of spermatid cytoplasmic contents in Drosophila during spermatid individualization. We show that a Krebs cycle component, the ATP-specific form of the succinyl-CoA synthetase β subunit (A-Sβ), binds to and activates the Cullin-3-based ubiquitin ligase (CRL3) complex required for caspase activation in spermatids. In vitro and in vivo evidence suggests that this interaction occurs on the mitochondrial surface, thereby limiting the source of CRL3 complex activation to the vicinity of this organelle and reducing the potential rate of caspase activation by at least 60%. Domain swapping between A-Sβ and the GTP-specific SCSβ (G-Sβ), which functions redundantly in the Krebs cycle, show that the metabolic and structural roles of A-Sβ in spermatids can be uncoupled, highlighting a moonlighting function of this Krebs cycle component in CRL activation. Copyright © 2016 Elsevier Inc. All rights reserved.

Generation and characterization of antibodies specific for caspase-cleaved neo-epitopes: a novel approach

PubMed Central

Ai, X; Butts, B; Vora, K; Li, W; Tache-Talmadge, C; Fridman, A; Mehmet, H

2011-01-01

Apoptosis research has been significantly aided by the generation of antibodies against caspase-cleaved peptide neo-epitopes. However, most of these antibodies recognize the N-terminal fragment and are specific for the protein in question. The aim of this project was to create antibodies, which could identify caspase-cleaved proteins without a priori knowledge of the cleavage sites or even the proteins themselves. We hypothesized that many caspase-cleavage products might have a common antigenic shape, given that they must all fit into the same active site of caspases. Rabbits were immunized with the eight most prevalent exposed C-terminal tetrapeptide sequences following caspase cleavage. After purification of the antibodies we demonstrated (1) their specificity for exposed C-terminal (but not internal) peptides, (2) their ability to detect known caspase-cleaved proteins from apoptotic cell lysates or supernatants from apoptotic cell culture and (3) their ability to detect a caspase-cleaved protein whose tetrapeptide sequence differs from the eight tetrapeptides used to generate the antibodies. These antibodies have the potential to identify novel neo-epitopes produced by caspase cleavage and so can be used to identify pathway-specific caspase cleavage events in a specific cell type. Additionally this methodology may be applied to generate antibodies against products of other proteases, which have a well-defined and non-promiscuous cleavage activity. PMID:21881607

Antiapoptotic Activity of the Herpesvirus Saimiri-Encoded Bcl-2 Homolog: Stabilization of Mitochondria and Inhibition of Caspase-3-Like Activity

PubMed Central

Derfuss, Tobias; Fickenscher, Helmut; Kraft, Michael S.; Henning, Golo; Lengenfelder, Doris; Fleckenstein, Bernhard; Meinl, Edgar

1998-01-01

Viruses have evolved different strategies to interfere with host cell apoptosis. Herpesvirus saimiri (HVS) and other lymphotropic herpesviruses code for proteins that are homologous to the cellular antiapoptotic Bcl-2. In this study HVS-Bcl-2 was stably expressed in the human leukemia cell line Jurkat and in the murine T-cell hybridoma DO to assess its antiapoptotic spectrum and to gain further insight into its mode of action. HVS- Bcl-2 prevented apoptosis that occurs as a result of a disturbance of intracellular homeostasis by, for example, DNA damage or menadione, which gives rise to oxygen radicals. In Jurkat cells, HVS-Bcl-2 also inhibited apoptosis mediated by the death receptor CD95. In DO cells, HVS-Bcl-2 did not interfere with CD95-mediated apoptosis but blocked dexamethasone-induced cell death. Mitochondrial damage is a central coordinating event in apoptosis induced by different stimuli. To assess the integrity of mitochondria, we used rhodamine 123, which is released upon disturbance of the mitochondrial membrane potential, and determined the release of cytochrome c into the cytosol. Both signs of mitochondrial damage were prevented by HVS-Bcl-2. This viral protein also inhibited the generation of caspase-3-like DEVDase activity and blocked the cleavage of poly(ADP-ribose) polymerase, a natural substrate of caspase-3-like proteases. In conclusion, HVS-Bcl-2 protects against a great variety of apoptotic stimuli, stabilizes mitochondria, and acts upstream of the generation of caspase-3-like activity. PMID:9621051

Stabilization of HIF-1α modulates VEGF and Caspase-3 in the hippocampus of rats following transient global ischemia induced by asphyxial cardiac arrest.

PubMed

Liu, Xiao-Liang; Lu, Jian; Xing, Jihong

2016-04-15

Hypoxia inducible factor-1 (HIF-1) contributes to pathophysiological changes of homeostasis under conditions of oxygen deprivation as well as ischemia. In this study, we examined protein expression of subtype HIF-1α and its downstream product, namely vascular endothelial growth factor (VEGF) in the rat hippocampus after transient global ischemia induced by asphyxial cardiac arrest (CA) followed by cardiopulmonary resuscitation (CPR). We also examined the effects of stabilization of HIF-1α by systemic administration of dimethyloxalylglycine (DMOG) and ML228 on expression of VEGF receptor subtype 2 (VEGFR-2), Caspase-3 and NF-kB in the hippocampus. Ninety-six adult Sprague-Dawley rats were used in this study. The animals surviving from CPR were sacrificed 0, 3, 6 and 24h following CPR and the protein levels of HIF-1α and VEGF in the hippocampus were determined. VEGFR-2, Caspase-3 and NF-kB were also examined in control rats, and rats that survived for 24h after CPR and were given with DMOG/ML228. Moreover, neurological functions were estimated in control rats and rats with DMOG/ML228. Our results show that HIF-1α and VEGF were significantly increased in the hippocampus 3-24h after CA. Significant increases in VEGFR-2, Caspase-3 and NF-κB were observed in the hippocampus 24h after CA (P<0.05 vs. control group). Nonetheless, DMOG and ML228 significantly augmented VEGFR-2, attenuated Caspase-3 and neuronal apoptosis, and improved neurological Severity Score and tissue edema (P<0.05 vs. saline group), without affecting expression of NF-κB. Our data revealed specific signaling pathways in alleviating CA-evoked global cerebral ischemia by elucidating that HIF-1α plays an important role in regulating expression of VEGFR-2 and Caspase-3 as well as improving neurological functions and neuronal edema. The subsequent induction of HIF-1α and its target signal pathways is likely a part of the intrinsic neuroprotective effects aimed at attenuating damage as a result of

Reduced cellular redox status induces 4-hydroxynonenal-mediated caspase 3 activation leading to erythrocyte death during chronic arsenic exposure in rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Biswas, Debabrata; Sen, Gargi; Biswas, Tuli, E-mail: tulibiswas@iicb.res.i

2010-05-01

Chronic exposure to arsenic in rats led to gradual accumulation of the toxicant in erythrocytes causing oxidative stress in these cells. 4-Hydroxynonenal (4-HNE), a major aldehyde product of lipid peroxidation, contributed significantly to the cytopathological events observed during oxidative stress in the erythrocytes of exposed rats. 4-HNE triggered death signal cascade that was initiated with the formation of HNE-protein adducts in cytosol. HNE-protein adduct formation resulted in depletion of cytosolic antioxidants followed by increased generation of ROS. Results showed accumulation of hydrogen peroxide (H{sub 2}O{sub 2}) from the early stages of arsenic exposure, while superoxide (O{sub 2}{sup c}entre dot{sup -})more » and hydroxyl radical ({sup c}entre dotOH) also contributed to the oxidative stress during longer period of exposure. Suppression of antioxidant system coupled with increased generation of ROS eventually led to activation of caspase 3 during arsenic exposure. Attenuation of HNE-mediated activation of caspase 3 in presence of N-acetylcysteine (NAC) indicated the involvement of GSH in the process. Prevention of HNE-mediated degradation of membrane proteins in presence of Z-DEVD-FMK identified caspase 3 as the principal mediator of HNE-induced cellular damage during arsenic exposure. Degradation of band 3 followed by its aggregation on the red cell surface promoted immunologic recognition of redistributed band 3 by autologous IgG with subsequent attachment of C3b. Finally, the formation of C3b-IgG-band 3 immune complex accelerated the elimination of affected cells from circulation and led to the decline of erythrocyte life span during chronic arsenic toxicity.« less

Phosphorylated AKT preserves stallion sperm viability and motility by inhibiting caspases 3 and 7.

PubMed

Gallardo Bolaños, Juan M; Balao da Silva, Carolina M; Martín Muñoz, Patricia; Morillo Rodríguez, Antolín; Plaza Dávila, María; Rodríguez-Martínez, Heriberto; Aparicio, Inés M; Tapia, José A; Ortega Ferrusola, Cristina; Peña, Fernando J

2014-08-01

AKT, also referred to as protein kinase B (PKB or RAC), plays a critical role in controlling cell survival and apoptosis. To gain insights into the mechanisms regulating sperm survival after ejaculation, the role of AKT was investigated in stallion spermatozoa using a specific inhibitor and a phosphoflow approach. Stallion spermatozoa were washed and incubated in Biggers-Whitten-Whittingham medium, supplemented with 1% polyvinyl alcohol (PVA) in the presence of 0 (vehicle), 10, 20 or 30 μM SH5, an AKT inhibitor. SH5 treatment reduced the percentage of sperm displaying AKT phosphorylation, with inhibition reaching a maximum after 1 h of incubation. This decrease in phosphorylation was attributable to either dephosphorylation or suppression of the active phosphorylation pathway. Stallion spermatozoa spontaneously dephosphorylated during in vitro incubation, resulting in a lack of a difference in AKT phosphorylation between the SH5-treated sperm and the control after 4 h of incubation. AKT inhibition decreased the proportion of motile spermatozoa (total and progressive) and the sperm velocity. Similarly, AKT inhibition reduced membrane integrity, leading to increased membrane permeability and reduced the mitochondrial membrane potential concomitantly with activation of caspases 3 and 7. However, the percentage of spermatozoa exhibiting oxidative stress, the production of mitochondrial superoxide radicals, DNA oxidation and DNA fragmentation were not affected by AKT inhibition. It is concluded that AKT maintains the membrane integrity of ejaculated stallion spermatozoa, presumably by inhibiting caspases 3 and 7, which prevents the progression of spermatozoa to an incomplete form of apoptosis. © 2014 Society for Reproduction and Fertility.

Garlic (Allium sativum) Fresh Juice Induces Apoptosis in Human Oral Squamous Cell Carcinoma: The Involvement of Caspase-3, Bax and Bcl-2

PubMed Central

Farhadi, Farrokh; Jahanpour, Salar; Hazem, Kameliya; Aghbali, Amirala; Baradran, Behzad; Vahid Pakdel, Seyyed Mahdi

2015-01-01

Background and aims. There is no report on the apoptotic impact of Allium sativum L.(Garlic) on the oral squamous cell carcinoma (KB); hence, this study was designed to survey the apoptotic effects of garlic fresh juice (GFJ) on the KB cells. Materials and methods. MTTassay (MicrocultureTetrazolium Assay) was carried out to evaluate the cytotoxicity of GFJ on KB cells. Furthermore, TUNEL(Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling)and DNA fragmentation tests were performed to determine if GFJ is able to induce apoptosis in KB cells. Also a standard kit was used to assess caspase-3 activity in KB cells. Also western blotting was employed to evaluate the effect of GFJ on Bax:Bcl-2 ratio. Results. Significant cytotoxic effects were observed for the minimum used concentration (1μg/mL) as calculated to be 77.97±2.3% for 24 h and 818±3.1% for 36h of incubation (P < 0.001). Furthermore, TUNEL and DNA fragmentation tests corroborated the apoptosis inducing activity of GFJ. Consistently, after treating KB cells with GFJ(1μg/mL), caspase-3 activity and Bax:Bcl-2 ratio were raised by 7.3±0.6 and (P <0.001) folds, respectively. Conclusion. The results of this study advanced that GFJ induces apoptosis in the KB cells through increasing caspase-3 activity and Bax:Bcl2 ratio which could be attributed to its organo-sulfurcomponents. PMID:26889365

Caspase-9 mediates synaptic plasticity and memory deficits of Danish dementia knock-in mice: caspase-9 inhibition provides therapeutic protection

PubMed Central

2012-01-01

Background Mutations in either Aβ Precursor protein (APP) or genes that regulate APP processing, such as BRI2/ITM2B and PSEN1/PSEN2, cause familial dementias. Although dementias due to APP/PSEN1/PSEN2 mutations are classified as familial Alzheimer disease (FAD) and those due to mutations in BRI2/ITM2B as British and Danish dementias (FBD, FDD), data suggest that these diseases have a common pathogenesis involving toxic APP metabolites. It was previously shown that FAD mutations in APP and PSENs promote activation of caspases leading to the hypothesis that aberrant caspase activation could participate in AD pathogenesis. Results Here, we tested whether a similar mechanism applies to the Danish BRI2/ITM2B mutation. We have generated a genetically congruous mouse model of FDD, called FDDKI, which presents memory and synaptic plasticity deficits. We found that caspase-9 is activated in hippocampal synaptic fractions of FDDKI mice and inhibition of caspase-9 activity rescues both synaptic plasticity and memory deficits. Conclusion These data directly implicate caspase-9 in the pathogenesis of Danish dementia and suggest that reducing caspase-9 activity is a valid therapeutic approach to treating human dementias. PMID:23217200

Caspase-mediated pro-apoptotic interaction of panaxadiol and irinotecan in human colorectal cancer cells.

PubMed

Du, Guang-Jian; Wang, Chong-Zhi; Zhang, Zhi-Yu; Wen, Xiao-Dong; Somogyi, Jacqueline; Calway, Tyler; He, Tong-Chuan; Du, Wei; Yuan, Chun-Su

2012-05-01

Panaxadiol is a purified sapogenin of ginseng saponins that exhibits anticancer activity. Irinotecan is a second-line anticancer drug, but clinical treatment with irinotecan is limited due to its side effects. In this study, we have investigated the possible synergistic anticancer effects of panaxadiol and irinotecan on human colorectal cancer cells and explored the potential role of apoptosis in their synergistic activity. The combination of panaxadiol and irinotecan significantly enhanced antiproliferative effects in HCT-116 cells (P< 0.05). Cell cycle analysis demonstrated that combining irinotecan treatment with panaxadiol significantly increased the G1-phase fractions of cells, compared with irinotecan treatment alone. In apoptotic assays, the combination of panaxadiol and irinotecan significantly increased the percentage of apoptotic cells compared with irinotecan alone (P<0.01). Increased activity of caspase-3 and caspase-9 was observed after treating with panaxadiol and irinotecan. The synergistic apoptotic effects were supported by docking analysis, which demonstrated that panaxadiol and irinotecan bound two different chains of the caspase-3 protein. Data from this study suggested that caspase-3- and caspase-9-mediated apoptosis may play an important role in the panaxadiol enhanced antiproliferative effects of irinotecan on human colorectal cancer cells. © 2012 The Authors. JPP © 2012 Royal Pharmaceutical Society.

miR-874 regulates myocardial necrosis by targeting caspase-8

PubMed Central

Wang, K; Liu, F; Zhou, L-Y; Ding, S-L; Long, B; Liu, C-Y; Sun, T; Fan, Y-Y; Sun, L; Li, P-F

2013-01-01

Cardiomyocyte death is an important reason for the cardiac syndromes, such as heart failure (HF) and myocardial infarction (MI). In the heart diseases, necrosis is one of the main forms of cell death. MicroRNAs (miRNAs) are a class of small non-coding RNAs that mediate post-transcriptional gene silencing. Hitherto, it is not yet clear whether miRNA can regulate necrosis in cardiomyocyte. In this work, we performed a microarray to detect miRNAs in response to H2O2 treatment, and the results showed that miR-874 was substantially increased. We further studied the function of miR-874, and observed that knockdown of miR-874 attenuated necrosis in the cellular model and also MI in the animal model. We searched for the downstream mediator of miR-874 and identified that caspase-8 was a target of miR-874. Caspase-8 was able to antagonize necrosis. When suppressed by miR-874, caspase-8 lost the ability to repress necrotic program. In exploring the molecular mechanism by which miR-874 expression is regulated, we identified that Foxo3a could transcriptionally repress miR-874 expression. Foxo3a transgenic or knockout mice exhibited a low or high expression level of miR-874, and a reduced or enhanced necrosis and MI. Our present study reveals a novel myocardial necrotic regulating model, which is composed of Foxo3a, miR-874 and caspase-8. Modulation of their levels may provide a new approach for tackling myocardial necrosis. PMID:23828572

Peroxynitrite induces apoptosis of mouse cochlear hair cells via a Caspase-independent pathway in vitro.

PubMed

Cao, Zhixin; Yang, Qianqian; Yin, Haiyan; Qi, Qi; Li, Hongrui; Sun, Gaoying; Wang, Hongliang; Liu, Wenwen; Li, Jianfeng

2017-11-01

Peroxynitrite (ONOO - ) is a potent and versatile oxidant implicated in a number of pathophysiological processes. The present study was designed to investigate the effect of ONOO - on the cultured cochlear hair cells (HCs) of C57BL/6 mice in vitro as well as the possible mechanism underlying the action of such an oxidative stress. The in vitro primary cultured cochlear HCs were subjected to different concentrations of ONOO - , then, the cell survival and morphological changes were examined by immunofluorescence and transmission electron microscopy (TEM), the apoptosis was determined by Terminal deoxynucleotidyl transferase dUNT nick end labeling (TUNEL) assay, the mRNA expressions of Caspase-3, Caspase-8, Caspase-9, Apaf1, Bcl-2, and Bax were analyzed by RT-PCR, and the protein expressions of Caspase-3 and AIF were assessed by immunofluorescence. This work demonstrated that direct exposure of primary cultured cochlear HCs to ONOO - could result in a base-to-apex gradient injury of HCs in a concentration-dependent manner. Furthermore, ONOO - led to much more losses of outer hair cells than inner hair cells mainly through the induction of apoptosis of HCs as evidenced by TEM and TUNEL assays. The mRNA expressions of Caspase-8, Caspase-9, Apaf1, and Bax were increased and, meanwhile, the mRNA expression of Bcl-2 was decreased in response to ONOO - treatment. Of interesting, the expression of Caspase-3 had no significant change, whereas, the expression alteration of AIF was observed. These results suggested that ONOO - can effectively damage the survival of cochlear HCs via triggering the apoptotic pathway. The findings from this work suggest that ONOO - -induced apoptosis is mediated, at least in part, via a Caspase-independent pathway in cochlear HCs.

Temporal regulation of Drosophila IAP1 determines caspase functions in sensory organ development

PubMed Central

Koto, Akiko; Kuranaga, Erina

2009-01-01

The caspases comprise a family of cysteine proteases that function in various cellular processes, including apoptosis. However, how the balance is struck between the caspases’ role in cell death and their nonapoptotic functions is unclear. To address this issue, we monitored the protein turnover of an endogenous caspase inhibitor, Drosophila IAP1 (DIAP1). DIAP1 is an E3 ubiquitin ligase that promotes the ubiquitination of caspases and thereby prevents caspase activation. For this study, we developed a fluorescent probe to monitor DIAP1 turnover in the external sensory organ precursor (SOP) lineage of living Drosophila. The SOP divides asymmetrically to make the shaft, socket, and sheath cells, and the neuron that comprise each sensory organ. We found that the quantity of DIAP1 changed dramatically depending on the cell type and maturity, and that the temporal regulation of DIAP1 turnover determines whether caspases function nonapoptotically in cellular morphogenesis or cause cell death. PMID:19822670

Fluid shear stress inhibits TNF-alpha-induced apoptosis in osteoblasts: a role for fluid shear stress-induced activation of PI3-kinase and inhibition of caspase-3

NASA Technical Reports Server (NTRS)

Pavalko, Fredrick M.; Gerard, Rita L.; Ponik, Suzanne M.; Gallagher, Patricia J.; Jin, Yijun; Norvell, Suzanne M.

2003-01-01

In bone, a large proportion of osteoblasts, the cells responsible for deposition of new bone, normally undergo programmed cell death (apoptosis). Because mechanical loading of bone increases the rate of new bone formation, we hypothesized that mechanical stimulation of osteoblasts might increase their survival. To test this hypothesis, we investigated the effects of fluid shear stress (FSS) on osteoblast apoptosis using three osteoblast cell types: primary rat calvarial osteoblasts (RCOB), MC3T3-E1 osteoblastic cells, and UMR106 osteosarcoma cells. Cells were treated with TNF-alpha in the presence of cyclohexamide (CHX) to rapidly induce apoptosis. Osteoblasts showed significant signs of apoptosis within 4-6 h of exposure to TNF-alpha and CHX, and application of FSS (12 dyne/cm(2)) significantly attenuated this TNF-alpha-induced apoptosis. FSS activated PI3-kinase signaling, induced phosphorylation of Akt, and inhibited TNF-alpha-induced activation of caspase-3. Inhibition of PI3-kinase, using LY294002, blocked the ability of FSS to rescue osteoblasts from TNF-alpha-induced apoptosis and blocked FSS-induced inhibition of caspase-3 activation in osteoblasts treated with TNF-alpha. LY294002 did not, however, prevent FSS-induced phosphorylation of Akt suggesting that activation of Akt alone is not sufficient to rescue cells from apoptosis. This result also suggests that FSS can activate Akt via a PI3-kinase-independent pathway. These studies demonstrate for the first time that application of FSS to osteoblasts in vitro results in inhibition of TNF-alpha-induced apoptosis through a mechanism involving activation of PI3-kinase signaling and inhibition of caspases. FSS-induced activation of PI3-kinase may promote cell survival through a mechanism that is distinct from the Akt-mediated survival pathway. Copyright 2002 Wiley-Liss, Inc.

A polysaccharide from Sanguisorbae radix induces caspase-dependent apoptosis in human leukemia HL-60 cells.

PubMed

Wu, Zhigang; Sun, Honghui; Li, Jingzhong; Ma, Chijiao; Zhao, Siqiao; Guo, Zheng; Lin, Yao; Lin, Yaping; Liu, Li

2014-09-01

One polysaccharide (SRP) was purified from Sanguisorbae radix by DEAE-cellulose-52 anion-exchange and Sephacryl S-400 gel filtration chromatography. The aim of this study was to evaluate the anticancer efficacy of SRP on human leukemia HL-60 cells in vitro and unveil the underlying mechanisms. Our results showed that SRP was able to suppress the proliferation of HL-60 cells in a dose-dependent manner by the mechanism involved in the induction of apoptosis. The increase in SRP-induced apoptosis was correlated with a rapid and sustained loss of mitochondrial transmembrane potential (ΔΨm) and a release of cytochrome c from the mitochondria into the cytosol. Furthermore, Western blot and RT-PCR analysis revealed that the protein and mRNA levels of antiapoptotic Bcl-2 were downregulated, whereas those of pro-apoptotic Bax were upregulated. Besides, caspase-9 and caspase-3 were activated, while caspase-8 was intact. Additionally, the apoptotic cells by SRP were significantly inhibited by a caspase-3 inhibitor (z-DEVD-fmk) or a caspase-9 inhibitor (Z-LETD-FMK), demonstrating the important role of caspase-9 and -3 in the process. Taken together, these findings provided evidence that SRP induced the apoptosis of HL-60 cells through an intrinsic mitochondria-mediated signaling pathway and SRP may be a promising chemotherapeutic agent for treatment of leukemia. Copyright © 2014 Elsevier B.V. All rights reserved.

[Study on the relationship between renal apoptosis and expression of caspase protein in fluoride induced rat].

PubMed

Gao, Jiping; Song, Guohua; Liu, Maolin; Wang, Yu; Yang, Xia

2014-01-01

To study the relationship between death receptor pathway, mitochondrion pathway and fluoride-induced apoptosis of renal cell. Male Sprague-Dawley rats were divided randomly into four groups (control, low-fluoride, medium-fluoride,and high-fluoride) and administered 0, 50, 100, and 200 mg/L of sodium fluoride, respectively, via drinking water for 120 days. The incidence of dental fluorosis were observed, the body weights and urine fluoride levels were measured. Apoptosis was detected by the Flow Cytometry (FCM). The expressions of protein of Caspase-3, Caspase-8, Caspase-9, Cyt C were detectedby immunohistoehemistry. The apoptosis rate in the fluoride exposed low does group,middle dose group and high dose group increased significantly as compared with control group. The average optical density value of Caspase-3, Caspase-8, Caspase-9 and Cyt C were higher in the fluoride exposed middle dose group and high dose group than those in the control group (P < 0.05). Death receptor pathway and mitochondrion pathway may participate in the process of fluoride-induced apoptosis of renal cell.

Levels of Hepatic Activating Transcription Factor 6 and Caspase-3 Are Downregulated in Mice after Excessive Training.

PubMed

Pinto, Ana P; da Rocha, Alisson L; Oliveira, Luciana da C; Morais, Gustavo P; de Vicente, Larissa G; Cintra, Dennys E; Pauli, José R; Moura, Leandro P; Ropelle, Eduardo R; da Silva, Adelino S R

2017-01-01

Recently, we demonstrated that different running overtraining (OT) protocols with the same external load, but performed downhill (OTR/down), uphill (OTR/up), and without inclination (OTR), led to hepatic fat accumulation. As the disruption of endoplasmic reticulum (ER) homeostasis is linked to animal models of fatty liver disease, we investigated the effects of these OT models on the proteins related to ER stress (i.e., BiP, inositol-requiring enzyme 1, protein kinase RNA-like endoplasmic reticulum kinase, eIF2alpha, ATF6beta, and glucose-regulated protein 94) and apoptosis (C/EBP-homologous protein, Caspase-3, 4, and 12, Bax, and tumor necrosis factor receptor-associated factor 2) in livers of C57BL/6 mice. Also, aerobic training can attenuate cardiac ER stress and improve exercise capacity. Therefore, we investigated whether the decrease in performance induced by our OT protocols is linked to ER stress and apoptosis in mouse hearts. The rodents were divided into six groups: naïve (N, sedentary mice), control (CT, sedentary mice submitted to the performance evaluations), trained (TR), OTR/down, OTR/up, and OTR groups. Rotarod, incremental load, exhaustive, and grip force tests were used to evaluate performance. After the grip force test, the livers and cardiac muscles (i.e., left ventricle) were removed and used for immunoblotting. All of the OT protocols led to similar responses in the performance parameters and displayed significantly lower hepatic ATF6beta values compared to the N group. The OTR/down group exhibited lower liver cleaved caspase-3 values compared to the CT group. However, the other proteins related to ER stress and apoptosis were not modulated. Also, the cardiac proteins related to ER stress and apoptosis were not modulated in the experimental groups. In conclusion, the OT protocols decreased the levels of hepatic ATF6beta, and the OTR/down group decreased the levels of hepatic cleaved caspase-3. Also, the decrease in performance induced by our

Caspase inhibition in select olfactory neurons restores innate attraction behavior in aged Drosophila.

PubMed

Chihara, Takahiro; Kitabayashi, Aki; Morimoto, Michie; Takeuchi, Ken-ichi; Masuyama, Kaoru; Tonoki, Ayako; Davis, Ronald L; Wang, Jing W; Miura, Masayuki

2014-06-01

Sensory and cognitive performance decline with age. Neural dysfunction caused by nerve death in senile dementia and neurodegenerative disease has been intensively studied; however, functional changes in neural circuits during the normal aging process are not well understood. Caspases are key regulators of cell death, a hallmark of age-related neurodegeneration. Using a genetic probe for caspase-3-like activity (DEVDase activity), we have mapped age-dependent neuronal changes in the adult brain throughout the lifespan of Drosophila. Spatio-temporally restricted caspase activation was observed in the antennal lobe and ellipsoid body, brain structures required for olfaction and visual place memory, respectively. We also found that caspase was activated in an age-dependent manner in specific subsets of Drosophila olfactory receptor neurons (ORNs), Or42b and Or92a neurons. These neurons are essential for mediating innate attraction to food-related odors. Furthermore, age-induced impairments of neural transmission and attraction behavior could be reversed by specific inhibition of caspase in these ORNs, indicating that caspase activation in Or42b and Or92a neurons is responsible for altering animal behavior during normal aging.

Angelica sinensis polysaccharides promotes apoptosis in human breast cancer cells via CREB-regulated caspase-3 activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Wei-Jie; Wang, Sheng; Hu, Zhuang, E-mail: zhuanghu475000@sina.com

Angelica sinensis polysaccharide (ASP) is purified from the fresh roots of Angelica sinensis (AS). This traditional Chinese medicine has been used for thousands of years for treating gynecological diseases and used in functional foods for the prevention and treatment of various diseases, such as inflammation and cancer. The antitumor activity of ASP is related to its biological activities, because it suppresses a variety of pro-proliferative or anti-apoptotic factors that are dramatically expressed in cancer cells of given types. In this study, we show that angelica sinensis polysaccharide induced apoptosis in breast cancer cells of T47D over-expressing the Cyclic AMP responsemore » element binding protein (CREB), inducing apoptosis-related signaling pathway activity. The result also found that ASP caused cell death was linked to caspase activity, accompanied by the loss of mitochondrial membrane potential, cytochrome c release, and Bax translocation from the cytosol to the mitochondria. We found that ASP significantly affected the poly-ADP-ribose polymerase (PARP), Bcl-2 Associated X Protein (Bax), Bcl-2, Bcl-xL and apoptotic protease activating facter-1 (Apaf1) protein expression in a dose- and time-dependent manner. DAPI staining and Flow cytometry were used to analyze apoptosis. The nude mice xenograft model was used to evaluate the antitumor effect of ASP in vivo. ASP has profound antitumor effect on T47D cells, probably by inducing apoptosis through CREB signaling pathway. Thus, these results suggest that ASP would be a promising therapeutic agent for breast cancer. - Highlights: • CREB and Caspase-3 signaling pathways are involved in the ASP induced breast cancer cells apoptosis. • ROCK1/Mlc signaling pathway plays a critical role in this ASP-mediated apoptosis. • Angelica sinensis polysaccharide (ASP) affected the PARP, Bax, Bcl-2, Bcl-xL and Apaf1 protein expression. • The activation of CREB and ROCK1 promotes caspase-3 activation and apoptosis

Effects of vitamin C on pathology and caspase-3 activity of kidneys with subacute endosulfan toxicity.

PubMed

Ozmen, O; Mor, F

2015-01-01

Endosulfan is an insecticide that is composed of two stereoisomers: α- and β- endosulfan in an approximate ratio of 70:30. Owing to its widespread use, poisoning of both humans and animals is possible. We examined the toxic effects of endosulfan on New Zealand white rabbit kidneys. Rabbit kidneys were examined histopathologically and caspase-3 activity was detected using immunohistochemistry. Animals were divided into four groups: Group 1 was given a sublethal dose of endosulfan in corn oil by oral gavage daily for 6 weeks, Group 2 was given endosulfan + vitamin C during the same period, Group 3 was given corn oil daily and vitamin C on alternate days, Group 4 was given only corn oil daily throughout the experiment. By the end of experimental period, the concentration of α-endosulfan was greater than the β-endosulfan concentration in the kidneys of both of endosulfan treated groups (Groups 1 and 2). Decreased accumulation of α- and β-endosulfan was observed in Group 2, possibly because of the antioxidant effect of the vitamin C. Histopathological examination revealed hemorrhages, tubule cell necrosis, glomerular infiltration, glomerulosclerosis and proteinaceous material in the tubules, and Bowman spaces in the kidneys of Group 1. Caspase-3 reaction was stronger in Group 1 than in the other groups. Apoptotic activity was most frequent in proximal tubule cells. Endosulfan is toxic to rabbit kidneys. Vitamin C treatment reduced the accumulation of endosulfan in kidneys and reduced its toxicity.

Nickel chloride-induced apoptosis via mitochondria- and Fas-mediated caspase-dependent pathways in broiler chickens.

PubMed

Guo, Hongrui; Cui, Hengmin; Fang, Jing; Zuo, Zhicai; Deng, Junliang; Wang, Xun; Zhao, Ling; Wu, Bangyuan; Chen, Kejie; Deng, Jie

2016-11-29

Ni, a metal with industrial and commercial uses, poses a serious hazard to human and animal health. In the present study, we used flow cytometry, immunohistochemistry and qRT-PCR to investigate the mechanisms of NiCl2-induced apoptosis in kidney cells. After treating 280 broiler chickens with 0, 300, 600 or 900 mg/kg NiCl2 for 42 days, we found that two caspase-dependent pathways were involved in the induced renal tubular cell apoptosis. In the mitochondria-mediated caspase-dependent apoptotic pathway, cyt-c, HtrA2/Omi, Smac/Diablo, apaf-1, PARP, and caspase-9, 3, 6 and 7 were all increased, while. XIAP transcription was decreased. Concurrently, in the Fas-mediated caspase-dependent apoptotic pathway, Fas, FasL, caspase-8, caspase-10 and Bid levels were all increased. These results indicate that dietary NiCl2 at 300+ mg/kg induces renal tubular cell apoptosis in broiler chickens, involving both mitochondrial and Fas-mediated caspase-dependent apoptotic pathways. Our results provide novel insight into Ni and Ni-compound toxicology evaluated in vitro and in vivo.

Nickel chloride-induced apoptosis via mitochondria- and Fas-mediated caspase-dependent pathways in broiler chickens

PubMed Central

Guo, Hongrui; Cui, Hengmin; Fang, Jing; Zuo, Zhicai; Deng, Junliang; Wang, Xun; Zhao, Ling; Wu, Bangyuan; Chen, Kejie; Deng, Jie

2016-01-01

Ni, a metal with industrial and commercial uses, poses a serious hazard to human and animal health. In the present study, we used flow cytometry, immunohistochemistry and qRT-PCR to investigate the mechanisms of NiCl2-induced apoptosis in kidney cells. After treating 280 broiler chickens with 0, 300, 600 or 900 mg/kg NiCl2 for 42 days, we found that two caspase-dependent pathways were involved in the induced renal tubular cell apoptosis. In the mitochondria-mediated caspase-dependent apoptotic pathway, cyt-c, HtrA2/Omi, Smac/Diablo, apaf-1, PARP, and caspase-9, 3, 6 and 7 were all increased, while. XIAP transcription was decreased. Concurrently, in the Fas-mediated caspase-dependent apoptotic pathway, Fas, FasL, caspase-8, caspase-10 and Bid levels were all increased. These results indicate that dietary NiCl2 at 300+ mg/kg induces renal tubular cell apoptosis in broiler chickens, involving both mitochondrial and Fas-mediated caspase-dependent apoptotic pathways. Our results provide novel insight into Ni and Ni-compound toxicology evaluated in vitro and in vivo. PMID:27806327

Feedback regulation of mitochondria by caspase-9 in the B cell receptor-mediated apoptosis.

PubMed

Eeva, J; Nuutinen, U; Ropponen, A; Mättö, M; Eray, M; Pellinen, R; Wahlfors, J; Pelkonen, J

2009-12-01

During the germinal centre reaction (GC), B cells with non-functional or self-reactive antigen receptors are negatively selected by apoptosis to generate B cell repertoire with appropriate antigen specificities. We studied the molecular mechanism of Fas/CD95- and B cell receptor (BCR)-induced apoptosis to shed light on the signalling events involved in the negative selection of GC B cells. As an experimental model, we used human follicular lymphoma (FL) cell line HF1A3, which originates from a GC B cell, and transfected HF1A3 cell lines overexpressing Bcl-x(L), c-FLIP(long) or dominant negative (DN) caspase-9. Fas-induced apoptosis was dependent on the caspase-8 activation, since the overexpression of c-FLIP(long), a natural inhibitor of caspase-8 activation, blocked apoptosis induced by Fas. In contrast, caspase-9 activation was not involved in Fas-induced apoptosis. BCR-induced apoptosis showed the typical characteristics of mitochondria-dependent (intrinsic) apoptosis. Firstly, the activation of caspase-9 was involved in BCR-induced DNA fragmentation, while caspase-8 showed only marginal role. Secondly, overexpression of Bcl-x(L) could block all apoptotic changes induced by BCR. As a novel finding, we demonstrate that caspase-9 can enhance the cytochrome-c release and collapse of mitochondrial membrane potential (DeltaPsi(m)) during BCR-induced apoptosis. The requirement of different signalling pathways in apoptosis induced by BCR and Fas may be relevant, since Fas- and BCR-induced apoptosis can thus be regulated independently, and targeted to different subsets of GC B cells.

[Effects of introduction of short peptides before carotid artery occlusion on behaviour and caspase-3 activity in the brain of old rats].

PubMed

Mendzheritskiĭ, A M; Karantysh, G V; Ivonina, K O

2011-01-01

The comparative research of effect of Pinealon and Cortexin on behavior and activity of caspase-3 in a brain of old rats in a model of carotid arteries occlusion was conducted. It is shown that introduction of short peptides promotes a survival rate of the animals that have modeled occlusion of carotid arteries. Under Pinealon before occlusion of carotid arteries, behavioral dream has been increased and a position-finding behavior, a motivational behavior and a motor performance have been reduced. The rats that were introduced Cortexin before carotid arteries occlusion demonstrated the raise of behavioral dream time. At introduction of Pinealon activity of caspase-3 moderately raises in false-operated animals and in a model of occlusion of carotid arteries.

Liquiritin (LT) exhibits suppressive effects against the growth of human cervical cancer cells through activating Caspase-3 in vitro and xenograft mice in vivo.

PubMed

He, She-Hong; Liu, Hong-Gai; Zhou, Yu-Fei; Yue, Qing-Fen

2017-08-01

Cervical cancer is one of the most common female malignancies worldwide. Liquiritin (LT), a major constituent of Glycyrrhiza Radix, possesses a variety of pharmacological activities, including anti-cancer, anti-oxidative, anti-inflammatory and neuro-protective effects. However, its role in human cervical cancer remains to be elusive. In our study, we found that LT suppressed cervical cancer cell migration, invasion and cloning ability with little cytotoxicity to human normal cells. In addition, apoptosis was induced by LT in cervical cancer cells through activation of Caspase-3 and poly ADP-ribose polymerase (PARP) cleavage. LT-triggered apoptosis was dependent on extrinsic and intrinsic pathways, which were relied on Fas-associated protein with death domain (FADD)- and Bcl-2/Bax-regulated pathways, leading to Caspase-8 and Caspase-9 cleavage, respectively. LT was found to increase FADD expression, while reduce Bcl-2 expression, contributing to Caspase-3 cleavage. And tumor suppressors, p21 and p53, were enhanced after LT treatment, inhibiting the growth of cervical cancer cells in vitro. Significantly, in vivo study suggested that tumor growth was impeded by LT in a dose-dependent manner through enhancing apoptosis. Together, the data here revealed that LT was an effective and promising candidate for preventing human cervical cancer progression via apoptosis enhancement. Copyright © 2017. Published by Elsevier Masson SAS.

Staurosporine Induces Necroptotic Cell Death under Caspase-Compromised Conditions in U937 Cells

PubMed Central

Dunai, Zsuzsanna A.; Imre, Gergely; Barna, Gabor; Korcsmaros, Tamas; Petak, Istvan; Bauer, Pal I.; Mihalik, Rudolf

2012-01-01

For a long time necrosis was thought to be an uncontrolled process but evidences recently have revealed that necrosis can also occur in a regulated manner. Necroptosis, a type of programmed necrosis is defined as a death receptor-initiated process under caspase-compromised conditions. The process requires the kinase activity of receptor-interacting protein kinase 1 and 3 (RIPK1 and RIPK3) and mixed lineage kinase domain-like protein (MLKL), as a substrate of RIPK3. The further downstream events remain elusive. We applied known inhibitors to characterize the contributing enzymes in necroptosis and their effect on cell viability and different cellular functions were detected mainly by flow cytometry. Here we report that staurosporine, the classical inducer of intrinsic apoptotic pathway can induce necroptosis under caspase-compromised conditions in U937 cell line. This process could be hampered at least partially by the RIPK1 inhibitor necrotstin-1 and by the heat shock protein 90 kDa inhibitor geldanamycin. Moreover both the staurosporine-triggered and the classical death ligand-induced necroptotic pathway can be effectively arrested by a lysosomal enzyme inhibitor CA-074-OMe and the recently discovered MLKL inhibitor necrosulfonamide. We also confirmed that the enzymatic role of poly(ADP-ribose)polymerase (PARP) is dispensable in necroptosis but it contributes to membrane disruption in secondary necrosis. In conclusion, we identified a novel way of necroptosis induction that can facilitate our understanding of the molecular mechanisms of necroptosis. Our results shed light on alternative application of staurosporine, as a possible anticancer therapeutic agent. Furthermore, we showed that the CA-074-OMe has a target in the signaling pathway leading to necroptosis. Finally, we could differentiate necroptotic and secondary necrotic processes based on participation of PARP enzyme. PMID:22860037

High glucose derived endothelial microparticles increase active caspase-3 and reduce microRNA-Let-7a expression in endothelial cells.

PubMed

Bammert, Tyler D; Hijmans, Jamie G; Reiakvam, Whitney R; Levy, Ma'ayan V; Brewster, Lillian M; Goldthwaite, Zoe A; Greiner, Jared J; Stockelman, Kelly A; DeSouza, Christopher A

2017-11-18

The experimental aim of this study was to determine the effects of high glucose-induced endothelial microparticles (EMPs) on endothelial cell susceptibility to apoptosis. Human umbilical vein endothelial cells (HUVECs) were cultured (3rd passage) and plated in 6-well plates at a density of 5.0 × 10 5  cells/condition. Cells were incubated with media containing 25 mM d-glucose (concentration representing a diabetic glycemic state) or 5 mM d-glucose (normoglycemic condition) for 48 h to generate EMPs. EMP identification (CD144 + expression) and concentration was determined by flow cytometry. HUVECs (3 × 10 6  cells/condition) were treated with EMPs generated from either the normal or high glucose conditions for 24 h. Intracellular concentration of active caspase-3 was determined by enzyme immunoassay. Cellular expression of miR-Let7a, an anti-apoptotic microRNA, was determined by RT-PCR using the ΔΔCT normalized to RNU6. High glucose-derived EMPs significantly increased both basal (1.5 ± 0.1 vs 1.0 ± 0.1 ng/mL) and staurosporine-stimulated (2.2 ± 0.2 vs 1.4 ± 0.1 ng/mL) active caspase-3 compared with normal glucose EMPs. Additionally, the expression of miR-Let-7a was markedly reduced (∼140%) by high glucose EMPs (0.43 ± 0.17 fold vs control). These results demonstrate that hyperglycemic-induced EMPs increase endothelial cell active caspase-3. This apoptotic effect may be mediated, at least in part, by a reduction in miR-Let-7a expression. Copyright © 2017 Elsevier Inc. All rights reserved.

Thymoquinone Induces Cell Death in Human Squamous Carcinoma Cells via Caspase Activation-Dependent Apoptosis and LC3-II Activation-Dependent Autophagy

PubMed Central

Yu, Cheng-Chia; Lai, Yi-Yeh; Chen, Pei-Ni

2014-01-01

Background Thymoquinone (TQ), an active component of Nigella sativa or black cumin, elicits cytotoxic effects on various cancer cell lines. However, the anti-cancer effects of TQ on head and neck squamous cell carcinoma (HNSCC) remain unclear. Methodology/Principal Findings In this study, TQ elicited a strong cytotoxic effect on SASVO3, a highly malignant HNSCC cell line. The mechanisms of this cytotoxic effect were concentration dependent. TQ also induced apoptotic cell death in SASVO3 cells as indicated by an increase in Bax expression and caspase-9 activation. Apoptosis was possibly caspase-9 dependent because the exposure of cells to a caspase-9 inhibitor partially prevented cell death. The exposed cells also showed increased levels of autophagic vacuoles and LC3-II proteins, which are specific autophagy markers. Cell viability assay results further revealed that bafilomycin-A1, an autophagy inhibitor, enhanced TQ cytotoxicity; by comparison, Annexin V and propidium-iodide staining assay results showed that this inhibitor did not promote apoptosis. TQ treatment also increased the accumulation of autophagosomes. Using a lentivirus-shRNA system for LC3 silencing, we found that cell viability was eradicated in autophagy-defective cells. An in vivo BALB/c nude mouse xenograft model further showed that TQ administered by oral gavage reduced tumor growth via induced autophagy and apoptosis. Conclusions These findings indicated that TQ induced cell death in oral cancer cells via two distinct anti-neoplastic activities that can induce apoptosis and autophagy. Therefore, TQ is a promising candidate in phytochemical-based, mechanistic, and pathway-targeted cancer prevention strategies. PMID:25000169

Annonaceous acetogenin mimic AA005 induces cancer cell death via apoptosis inducing factor through a caspase-3-independent mechanism.

PubMed

Han, Bing; Wang, Tong-Dan; Shen, Shao-Ming; Yu, Yun; Mao, Chan; Yao, Zhu-Jun; Wang, Li-Shun

2015-03-18

Annonaceous acetogenins are a family of natural products with antitumor activities. Annonaceous acetogenin mimic AA005 reportedly inhibits mammalian mitochondrial NADH-ubiquinone reductase (Complex I) and induces gastric cancer cell death. However, the mechanisms underlying its cell-death-inducing activity are unclear. We used SW620 colorectal adenocarcinoma cells to study AA005 cytotoxic activity. Cell deaths were determined by Trypan blue assay and flow cytometry, and related proteins were characterized by western blot. Immunofluorescence and subcellular fractionation were used to evaluate AIF nuclear translocation. Reactive oxygen species were assessed by using redox-sensitive dye DCFDA. AA005 induces a unique type of cell death in colorectal adenocarcinoma cells, characterized by lack of caspase-3 activation or apoptotic body formation, sensitivity to poly (ADP-ribose) polymerase inhibitor Olaparib (AZD2281) but not pan-caspase inhibitor Z-VAD.fmk, and dependence on apoptosis-inducing factor (AIF). AA005 treatment also reduced expression of mitochondrial Complex I components, and leads to accumulation of intracellular reactive oxygen species (ROS) at the early stage. Blocking ROS formation significantly suppresses AA005-induced cell death in SW620 cells. Moreover, blocking activation of RIP-1 by necroptosis inhibitor necrotatin-1 inhibits AIF translocation and partially suppresses AA005-induced cell death in SW620 cells demonstrating that RIP-1 protein may be essential for cell death. AA005 may trigger the cell death via mediated by AIF through caspase-3 independent pathway. Our work provided new mechanisms for AA005-induced cancer cell death and novel clues for cancer treatment via AIF dependent cell death.

Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis.

PubMed

Brannelly, Laura A; Roberts, Alexandra A; Skerratt, Lee F; Berger, Lee

2018-05-16

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which infects and disrupts frog epidermis; however, pathological changes have not been explicitly characterized. Apoptosis (programmed cell death) can be used by pathogens to damage host tissue, but can also be a host mechanism of disease resistance for pathogen removal. In this study, we quantify epidermal cell death of infected and uninfected animals using two different assays: terminal transferase-mediated dUTP nick end-labelling (TUNEL), and caspase 3/7. Using ventral, dorsal, and thigh skin tissue in the TUNEL assay, we observe cell death in the epidermal cells in situ of clinically infected animals and compare cell death with uninfected animals using fluorescent microscopy. In order to determine how apoptosis levels in the epidermis change over the course of infection we remove toe-tip samples fortnightly over an 8-week period, and use a caspase 3/7 assay with extracted proteins to quantify activity within the samples. We then correlate caspase 3/7 activity with infection load. The TUNEL assay is useful for localization of cell death in situ, but is expensive and time intensive per sample. The caspase 3/7 assay is efficient for large sample sizes and time course experiments. However, because frog toe tip biopsies are small there is limited extract available for sample standardization via protein quantification methods, such as the Bradford assay. Therefore, we suggest estimating skin surface area through photographic analysis of toe biopsies to avoid consuming extracts during sample standardization.

The caspase-3/p120 RasGAP stress-sensing module reduces liver cancer incidence but does not affect overall survival in gamma-irradiated and carcinogen-treated mice.

PubMed

Vanli, Güliz; Sempoux, Christine; Widmann, Christian

2017-06-01

Activation of oncogenes is the initial step in cellular transformation. Oncogenes favor aberrant proliferation, which, at least initially, induces cellular stress. This oncogenic stress can act as a safeguard mechanism against further transformation by inducing senescence or apoptosis. Yet, the few premalignant cells that tolerate and escape these senescent or apoptotic responses are those that will ultimately generate tumors. The caspase-3/p120 RasGAP module is a stress-sensing device that promotes survival under mild stress conditions. A point mutation in RasGAP that prevents its cleavage by caspase-3 inactivates the pro-survival capacity of the device. When the mice homozygous for this mutation (D455A knock-in mice) are patho-physiologically challenged, they experience much stronger cellular damage than their wild-type counterparts and the affected organs rapidly lose their functionality. We reasoned that the caspase-3/p120 RasGAP module could help premalignant cells to cope with oncogenic stress and hence favor the development of tumors. Using gamma-irradiation and N-ethyl-N-nitrosourea (ENU) as tumor initiators, we assessed the survival advantage that the caspase-3/p120 RasGAP module could provide to premalignant cells. No difference in overall mortality between wild-type and D455A knock-in mice were observed. However, the number of ENU-induced liver tumors in the knock-in mice was higher than in control mice. These results indicate that the caspase-3/p120 RasGAP stress-sensing module impacts on carcinogen-induced liver cancer incidence but not sufficiently so as to affect overall survival. Hence, gamma irradiation and ENU-induced tumorigenesis processes do not critically rely on a survival mechanism that contributes to the maintenance of organ homeostasis in stressed healthy tissues. © 2017 Wiley Periodicals, Inc.

Caspase-3 dependent nitrergic neuronal apoptosis following cavernous nerve injury is mediated via RhoA and ROCK activation in major pelvic ganglion.

PubMed

Hannan, Johanna L; Matsui, Hotaka; Sopko, Nikolai A; Liu, Xiaopu; Weyne, Emmanuel; Albersen, Maarten; Watson, Joseph W; Hoke, Ahmet; Burnett, Arthur L; Bivalacqua, Trinity J

2016-07-08

Axonal injury due to prostatectomy leads to Wallerian degeneration of the cavernous nerve (CN) and erectile dysfunction (ED). Return of potency is dependent on axonal regeneration and reinnervation of the penis. Following CN injury (CNI), RhoA and Rho-associated protein kinase (ROCK) increase in penile endothelial and smooth muscle cells. Previous studies indicate that nerve regeneration is hampered by activation of RhoA/ROCK pathway. We evaluated the role of RhoA/ROCK pathway in CN regulation following CNI using a validated rat model. CNI upregulated gene and protein expression of RhoA/ROCK and caspase-3 mediated apoptosis in the major pelvic ganglion (MPG). ROCK inhibitor (ROCK-I) prevented upregulation of RhoA/ROCK pathway as well as activation of caspase-3 in the MPG. Following CNI, there was decrease in the dimer to monomer ratio of neuronal nitric oxide synthase (nNOS) protein and lowered NOS activity in the MPG, which were prevented by ROCK-I. CNI lowered intracavernous pressure and impaired non-adrenergic non-cholinergic-mediated relaxation in the penis, consistent with ED. ROCK-I maintained the intracavernous pressure and non-adrenergic non-cholinergic-mediated relaxation in the penis following CNI. These results suggest that activation of RhoA/ROCK pathway mediates caspase-3 dependent apoptosis of nitrergic neurons in the MPG following CNI and that ROCK-I can prevent post-prostatectomy ED.

Anaplasma phagocytophilum inhibits human neutrophil apoptosis via upregulation of bfl-1, maintenance of mitochondrial membrane potential and prevention of caspase 3 activation.

PubMed

Ge, Yan; Yoshiie, Kiyotaka; Kuribayashi, Futoshi; Lin, Mingqun; Rikihisa, Yasuko

2005-01-01

The inhibition of neutrophil apoptosis plays a central role in human granulocytic anaplasmosis. Intracellular signalling pathways through which the obligatory intracellular bacterium Anaplasma phagocytophilum inhibits the spontaneous apoptosis of human peripheral blood neutrophils were investigated. bfl-1 mRNA levels in uninfected neutrophils after 12 h in culture were reduced to approximately 5-25% of 0 h levels, but remained high in infected neutrophils. The eukaryotic RNA synthesis inhibitor, actinomycin D, prevented the maintenance of bfl-1 mRNA levels by A. phagocytophilum. Differences in mcl-1, bax, bcl-w, bad or bak mRNA levels in infected versus uninfected neutrophils were not remarkable. By using mitochondrial fluorescent dyes, Mitotracker Red and JC-1, it was found that most uninfected neutrophils lost mitochondrial membrane potential after 10-12 h incubation, whereas A. phagocytophilum-infected neutrophils maintained high membrane potential. Caspase 3 activity and the degree of apoptosis were lower in dose-dependent manner in A. phagocytophilum-infected neutrophils at 16 h post infection, as compared to uninfected neutrophils. Anti-active caspase 3 antibody labelling showed less positively stained population in infected neutrophils compared to those in uninfected neutrophils after 12 h incubation. These results suggest that A. phagocytophilum inhibits human neutrophil apoptosis via transcriptional upregulation of bfl-1 and inhibition of mitochondria-mediated activation of caspase 3.

Nampt/PBEF/visfatin exerts neuroprotective effects against ischemia/reperfusion injury via modulation of Bax/Bcl-2 ratio and prevention of caspase-3 activation.

PubMed

Erfani, Sohaila; Khaksari, Mehdi; Oryan, Shahrbanoo; Shamsaei, Nabi; Aboutaleb, Nahid; Nikbakht, Farnaz

2015-05-01

Nicotinamide phosphoribosyl transferase/pre-B cell colony-enhancing factor/visfatin (Nampt/PBEF/visfatin) is an adipocytokine. By synthesizing nicotinamide adenine dinucleotide (NAD(+)), Nampt/PBEF/visfatin functions to maintain an energy supply that has critical roles in cell survival. Cerebral ischemia leads to energy depletion and eventually neuronal death by apoptosis in specific brain regions specially the hippocampus. However, the role of Nampt/PBEF/visfatin in brain and cerebral ischemia remains to be investigated. This study investigated the role of administration Nampt/PBEF/visfatin in hippocampal CA3 area using a transient global cerebral ischemia model. Both common carotid arteries were occluded for 20 min followed by reperfusion. Saline as a vehicle and Nampt/PBEF/visfatin at a dose of 100 ng were injected intracerebroventricularly (ICV) at the time of cerebral reperfusion. To investigate the underlying mechanisms of Nampt/PBEF/visfatin neuroprotection, levels of expression of apoptosis-related proteins (caspase-3 activation, Bax protein levels, and Bcl-2 protein levels) 96 h after ischemia were determined by immunohistochemical staining. The number of active caspase-3-positive neurons in CA3 was significantly increased in the ischemia group, compared with the sham group (P < 0.001), and treatment with Nampt/PBEF/visfatin significantly reduced the ischemia/reperfusion-induced caspase-3 activation, compared to the ischemia group (P < 0.05). Also, results indicated a significant increase in Bax/Bcl-2 ratio in the ischemia group, compared with the sham group (P < 0.01). However, treatment with Nampt/PBEF/visfatin significantly attenuated the ischemia/reperfusion-induced increase in Bax/Bcl-2 ratio, compared with the ischemia group (P < 0.05). This study has indicated that Nampt/PBEF/visfatin entails neuroprotective effects against ischemia injury when used at the time of cerebral reperfusion. These neuroprotective mechanisms of Nampt

Evidence of cellular stress and caspase-3 resulting from a combined two-frequency signal in the cerebrum and cerebellum of Sprague-dawley rats

PubMed Central

López-Furelos, Alberto; Leiro-Vidal, José Manuel; Salas-Sánchez, Aarón Ángel; Ares-Pena, Francisco José; López-Martín, María Elena

2016-01-01

Multiple simultaneous exposures to electromagnetic signals induced adjustments in mammal nervous systems. In this study, we investigated the non-thermal SAR (Specific Absorption Rate) in the cerebral or cerebellar hemispheres of rats exposed in vivo to combined electromagnetic field (EMF) signals at 900 and 2450 MHz. Forty rats divided into four groups of 10 were individually exposed or not exposed to radiation in a GTEM chamber for one or two hours. After radiation, we used the Chemiluminescent Enzyme-Linked Immunosorbent Assay (ChELISA) technique to measure cellular stress levels, indicated by the presence of heat shock proteins (HSP) 90 and 70, as well as caspase-3-dependent pre-apoptotic activity in left and right cerebral and cerebellar hemispheres of Sprague Dawley rats. Twenty-four hours after exposure to combined or single radiation, significant differences were evident in HSP 90 and 70 but not in caspase 3 levels between the hemispheres of the cerebral cortex at high SAR levels. In the cerebellar hemispheres, groups exposed to a single radiofrequency (RF) and high SAR showed significant differences in HSP 90, 70 and caspase-3 levels compared to control animals. The absorbed energy and/or biological effects of combined signals were not additive, suggesting that multiple signals act on nervous tissue by a different mechanism. PMID:27589837

Evidence of cellular stress and caspase-3 resulting from a combined two-frequency signal in the cerebrum and cerebellum of sprague-dawley rats.

PubMed

López-Furelos, Alberto; Leiro-Vidal, José Manuel; Salas-Sánchez, Aarón Ángel; Ares-Pena, Francisco José; López-Martín, María Elena

2016-10-04

Multiple simultaneous exposures to electromagnetic signals induced adjustments in mammal nervous systems. In this study, we investigated the non-thermal SAR (Specific Absorption Rate) in the cerebral or cerebellar hemispheres of rats exposed in vivo to combined electromagnetic field (EMF) signals at 900 and 2450 MHz.Forty rats divided into four groups of 10 were individually exposed or not exposed to radiation in a GTEM chamber for one or two hours. After radiation, we used the Chemiluminescent Enzyme-Linked Immunosorbent Assay (ChELISA) technique to measure cellular stress levels, indicated by the presence of heat shock proteins (HSP) 90 and 70, as well as caspase-3-dependent pre-apoptotic activity in left and right cerebral and cerebellar hemispheres of Sprague Dawley rats.Twenty-four hours after exposure to combined or single radiation, significant differences were evident in HSP 90 and 70 but not in caspase 3 levels between the hemispheres of the cerebral cortex at high SAR levels. In the cerebellar hemispheres, groups exposed to a single radiofrequency (RF) and high SAR showed significant differences in HSP 90, 70 and caspase-3 levels compared to control animals. The absorbed energy and/or biological effects of combined signals were not additive, suggesting that multiple signals act on nervous tissue by a different mechanism.

Induction of caspase-dependent extrinsic apoptosis by apigenin through inhibition of signal transducer and activator of transcription 3 (STAT3) signalling in HER2-overexpressing BT-474 breast cancer cells.

PubMed

Seo, Hye-Sook; Jo, Jae Kyung; Ku, Jin Mo; Choi, Han-Seok; Choi, Youn Kyung; Woo, Jong-Kyu; Kim, Hyo In; Kang, Soo-Yeon; Lee, Kang Min; Nam, Koong Won; Park, Namkyu; Jang, Bo-Hyoung; Shin, Yong Cheol; Ko, Seong-Gyu

2015-10-23

Phytoestrogen intake is known to be beneficial to decrease breast cancer incidence and progression. But its molecular mechanisms of action are still unknown. The present study aimed to examine the effect of apigenin on proliferation and apoptosis in HER2-expressing breast cancer cells. In our experiments, apigenin inhibited the proliferation of BT-474 cells in a dose- and time-dependent manner. Apigenin also inhibited clonogenic survival (anchorage-dependent and -independent) of BT-474 cells in a dose-dependent manner. These growth inhibitions were accompanied with an increase in sub-G0/G1 apoptotic populations. Apigenin-induced extrinsic a caspase-dependent apoptosis up-regulating the levels of cleaved caspase-8 and cleaved caspase-3, and inducing the cleavage of poly (ADP-ribose) polymerase (PARP). Whereas, apigenin did not induce apoptosis via intrinsic mitochondrial apoptosis pathway since this compound did not decrease mitochondrial membrane potential without affecting the levels of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (BAX). Apigenin reduced the expression of phospho-JAK1, phospho-JAK2 and phospho-STAT3 and decreased signal transducer and activator of transcription 3 (STAT3) dependent luciferase reporter gene activity in BT-474 cells. Apigenin inhibited CoCl2-induced VEGF secretion and decreased the nuclear translocation of STAT3. Our study indicates that apigenin induces apoptosis through inhibition of STAT3 signalling and could serve as a useful compound to prevent or treat HER2-overexpressing breast cancer. © 2015 Authors.

Induction of caspase-dependent extrinsic apoptosis by apigenin through inhibition of signal transducer and activator of transcription 3 (STAT3) signalling in HER2-overexpressing BT-474 breast cancer cells

PubMed Central

Seo, Hye-Sook; Jo, Jae Kyung; Ku, Jin Mo; Choi, Han-Seok; Choi, Youn Kyung; Woo, Jong-Kyu; in Kim, Hyo; Kang, Soo-yeon; Lee, Kang min; Nam, Koong Won; Park, Namkyu; Jang, Bo-Hyoung; Shin, Yong Cheol; Ko, Seong-Gyu

2015-01-01

Phytoestrogen intake is known to be beneficial to decrease breast cancer incidence and progression. But its molecular mechanisms of action are still unknown. The present study aimed to examine the effect of apigenin on proliferation and apoptosis in HER2-expressing breast cancer cells. In our experiments, apigenin inhibited the proliferation of BT-474 cells in a dose- and time-dependent manner. Apigenin also inhibited clonogenic survival (anchorage-dependent and -independent) of BT-474 cells in a dose-dependent manner. These growth inhibitions were accompanied with an increase in sub-G0/G1 apoptotic populations. Apigenin-induced extrinsic a caspase-dependent apoptosis up-regulating the levels of cleaved caspase-8 and cleaved caspase-3, and inducing the cleavage of poly (ADP-ribose) polymerase (PARP). Whereas, apigenin did not induce apoptosis via intrinsic mitochondrial apoptosis pathway since this compound did not decrease mitochondrial membrane potential without affecting the levels of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (BAX). Apigenin reduced the expression of phospho-JAK1, phospho-JAK2 and phospho-STAT3 and decreased signal transducer and activator of transcription 3 (STAT3) dependent luciferase reporter gene activity in BT-474 cells. Apigenin inhibited CoCl2-induced VEGF secretion and decreased the nuclear translocation of STAT3. Our study indicates that apigenin induces apoptosis through inhibition of STAT3 signalling and could serve as a useful compound to prevent or treat HER2-overexpressing breast cancer. PMID:26500281

Ultrafine particles of Ulmus davidiana var. japonica induce apoptosis of gastric cancer cells via activation of caspase and endoplasmic reticulum stress.

PubMed

Ahn, Joungjwa; Lee, Jong Suk; Yang, Kyung Mi

2014-06-01

Small-sized particles are more suitable for targeted delivery and are therapeutically more effective than large-sized particles. In this study, we investigated the anticancer effects of ultrafine particles of Ulmus davidiana var. japonica (ufUJ) on human gastric cancer cell lines SNU-1, SNU-216, and SNU-484. ufUJ induced apoptosis by the proteolytic activation of caspase-9, caspase-6, and caspase-3 and cleavage of poly (ADP-ribose) polymerase. The expression levels of the endoplasmic reticulum stress-related protein BiP markedly increased after ufUJ treatment. BiP knockdown decreased ufUJ-induced cell death. ufUJ-induced apoptosis was inhibited by the caspase-3 inhibitor z-DEVD-fmk, caspase-6 inhibitor z-VEID-fmk, and caspase-9 inhibitor z-LEHD-fmk, and by siRNAs against caspases 3, 6, and 9. Gastric cancer cells did not show anchorage-independent growth in the presence of ufUJ. However, cells treated with caspase inhibitors showed an enhanced colony-forming ability. These findings may be helpful in the prevention of gastric cancer and in the development of functional foods.

E-Cigarette Vapor Induces an Apoptotic Response in Human Gingival Epithelial Cells Through the Caspase-3 Pathway.

PubMed

Rouabhia, Mahmoud; Park, Hyun Jin; Semlali, Abdelhabib; Zakrzewski, Andrew; Chmielewski, Witold; Chakir, Jamila

2017-06-01

Electronic cigarettes represent an increasingly significant proportion of today's consumable tobacco products. E-cigarettes contain several chemicals which may promote oral diseases. The aim of this study was to investigate the effect of e-cigarette vapor on human gingival epithelial cells. Results show that e-cigarette vapor altered the morphology of cells from small cuboidal form to large undefined shapes. Both single and multiple exposures to e-cigarette vapor led to a bulky morphology with large faint nuclei and an enlarged cytoplasm. E-cigarette vapor also increased L-lactate dehydrogenase (LDH) activity in the targeted cells. This activity was greater with repeated exposures. Furthermore, e-cigarette vapor increased apoptotic/necrotic epithelial cell percentages compared to that observed in the control. Epithelial cell apoptosis was confirmed by TUNEL assay showing that exposure to e-cigarette vapor increased apoptotic cell numbers, particularly after two and three exposures. This negative effect involved the caspase-3 pathway, the activity of which was greater with repeated exposure and which decreased following the use of caspase-3 inhibitor. The adverse effects of e-cigarette vapor on gingival epithelial cells may lead to dysregulated gingival cell function and result in oral disease. J. Cell. Physiol. 232: 1539-1547, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Substrate-Induced Conformational Changes Occur in All Cleaved Forms of Caspase-6

DOE Office of Scientific and Technical Information (OSTI.GOV)

S Vaidya; E Velazquez-Delgado; G Abbruzzese

2011-12-31

Caspase-6 is an apoptotic cysteine protease that also governs disease progression in Huntington's and Alzheimer's diseases. Caspase-6 is of great interest as a target for treatment of these neurodegenerative diseases; however, the molecular basis of caspase-6 function and regulation remains poorly understood. In the recently reported structure of caspase-6, the 60's and 130's helices at the base of the substrate-binding groove extend upward, in a conformation entirely different from that of any other caspase. Presently, the central question about caspase-6 structure and function is whether the extended conformation is the catalytically competent conformation or whether the extended helices must undergomore » a large conformational rearrangement in order to bind substrate. We have generated a series of caspase-6 cleavage variants, including a novel constitutively two-chain form, and determined crystal structures of caspase-6 with and without the intersubunit linker. This series allows evaluation of the role of the prodomain and intersubunit linker on caspase-6 structure and function before and after substrate binding. Caspase-6 is inherently more stable than closely related caspases. Cleaved caspase-6 with both the prodomain and the linker present is the most stable, indicating that these two regions act in concert to increase stability, but maintain the extended conformation in the unliganded state. Moreover, these data suggest that caspase-6 undergoes a significant conformational change upon substrate binding, adopting a structure that is more like canonical caspases.« less

Restraint of apoptosis during mitosis through interdomain phosphorylation of caspase-2

PubMed Central

Andersen, Joshua L; Johnson, Carrie E; Freel, Christopher D; Parrish, Amanda B; Day, Jennifer L; Buchakjian, Marisa R; Nutt, Leta K; Thompson, J Will; Moseley, M Arthur; Kornbluth, Sally

2009-01-01

The apoptotic initiator caspase-2 has been implicated in oocyte death, in DNA damage- and heat shock-induced death, and in mitotic catastrophe. We show here that the mitosis-promoting kinase, cdk1–cyclin B1, suppresses apoptosis upstream of mitochondrial cytochrome c release by phosphorylating caspase-2 within an evolutionarily conserved sequence at Ser 340. Phosphorylation of this residue, situated in the caspase-2 interdomain, prevents caspase-2 activation. S340 was susceptible to phosphatase 1 dephosphorylation, and an interaction between phosphatase 1 and caspase-2 detected during interphase was lost in mitosis. Expression of S340A non-phosphorylatable caspase-2 abrogated mitotic suppression of caspase-2 and apoptosis in various settings, including oocytes induced to undergo cdk1-dependent maturation. Moreover, U2OS cells treated with nocodazole were found to undergo mitotic catastrophe more readily when endogenous caspase-2 was replaced with the S340A mutant to lift mitotic inhibition. These data demonstrate that for apoptotic stimuli transduced by caspase-2, cell death is prevented during mitosis through the inhibitory phosphorylation of caspase-2 and suggest that under conditions of mitotic arrest, cdk1–cyclin B1 activity must be overcome for apoptosis to occur. PMID:19730412

Detergent sclerosants at sub-lytic concentrations induce endothelial cell apoptosis through a caspase dependent pathway.

PubMed

Cooley-Andrade, Osvaldo; Cheung, Kelvin; Chew, An-Ning; Connor, David Ewan; Parsi, Kurosh

2016-07-01

To investigate the apoptotic effects of detergent sclerosants sodium tetradecylsulphate (STS) and polidocanol (POL) on endothelial cells at sub-lytic concentrations. Human umbilical vein endothelial cells (HUVECs) were isolated and labelled with antibodies to assess for apoptosis and examined with confocal microscopy and flow cytometry. Isolated HUVECs viability was assessed using propidium iodide staining. Early apoptosis was determined by increased phosphatidylserine exposure by lactadherin binding. Caspase 3, 8, 9 and Bax activation as well as inhibitory assays with Pan Caspase (Z-VAD-FMK) and Bax (BI-6C9) were assessed to identify apoptotic pathways. Porimin activation was used to assess cell membrane permeability. Cell lysis reached almost 100 % with STS at 0.3 % and with POL at 0.6 %. Apoptosis was seen with both STS and POL at concentrations ranging from 0.075 to 0.15 %. PS exposure increased with both STS and POL and exhibited a dose-dependent trend. Active Caspase 3, 8 and 9 but not Bax were increased in HUVECs stimulated with low concentrations of both STS and POL. Inhibitory assays demonstrated Caspase 3, 8, 9 inhibition at low concentrations (0.075 to 0.6 %) with both STS and POL. Both agents increased the activation of porimin at all concentrations. Both sclerosants induced endothelial cell (EC) apoptosis at sub-lytic concentrations through a caspase-dependant pathway. Both agents induced EC oncosis.

G-CSF prevents caspase 3 activation in Schwann cells after sciatic nerve transection, but does not improve nerve regeneration.

PubMed

Frost, Hanna K; Kodama, Akira; Ekström, Per; Dahlin, Lars B

2016-10-15

Exogenous granulocyte-colony stimulating factor (G-CSF) has emerged as a drug candidate for improving the outcome after peripheral nerve injuries. We raised the question if exogenous G-CSF can improve nerve regeneration following a clinically relevant model - nerve transection and repair - in healthy and diabetic rats. In short-term experiments, distance of axonal regeneration and extent of injury-induced Schwann cell death was quantified by staining for neurofilaments and cleaved caspase 3, respectively, seven days after repair. There was no difference in axonal outgrowth between G-CSF-treated and non-treated rats, regardless if healthy Wistar or diabetic Goto-Kakizaki (GK) rats were examined. However, G-CSF treatment caused a significant 13% decrease of cleaved caspase 3-positive Schwann cells at the lesion site in healthy rats, but only a trend in diabetic rats. In the distal nerve segments of healthy rats a similar trend was observed. In long-term experiments of healthy rats, regeneration outcome was evaluated at 90days after repair by presence of neurofilaments, wet weight of gastrocnemius muscle, and perception of touch (von Frey monofilament testing weekly). The presence of neurofilaments distal to the suture line was similar in G-CSF-treated and non-treated rats. The weight ratio of ipsi-over contralateral gastrocnemius muscles, and perception of touch at any time point, were likewise not affected by G-CSF treatment. In addition, the inflammatory response in short- and long-term experiments was studied by analyzing ED1 stainable macrophages in healthy rats, but in neither case was any attenuation seen at the injury site or distal to it. G-CSF can prevent caspase 3 activation in Schwann cells in the short-term, but does not detectably affect the inflammatory response, nor improve early or late axonal outgrowth or functional recovery. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

Specific caspase inhibitor Q-VD-OPh prevents neonatal stroke in P7 rat: a role for gender.