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Sample records for caspase activity regulates

  1. Caspase-10 Negatively Regulates Caspase-8-Mediated Cell Death, Switching the Response to CD95L in Favor of NF-κB Activation and Cell Survival

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    Sebastian Horn

    2017-04-01

    Full Text Available Formation of the death-inducing signaling complex (DISC initiates extrinsic apoptosis. Caspase-8 and its regulator cFLIP control death signaling by binding to death-receptor-bound FADD. By elucidating the function of the caspase-8 homolog, caspase-10, we discover that caspase-10 negatively regulates caspase-8-mediated cell death. Significantly, we reveal that caspase-10 reduces DISC association and activation of caspase-8. Furthermore, we extend our co-operative/hierarchical binding model of caspase-8/cFLIP and show that caspase-10 does not compete with caspase-8 for binding to FADD. Utilizing caspase-8-knockout cells, we demonstrate that caspase-8 is required upstream of both cFLIP and caspase-10 and that DISC formation critically depends on the scaffold function of caspase-8. We establish that caspase-10 rewires DISC signaling to NF-κB activation/cell survival and demonstrate that the catalytic activity of caspase-10, and caspase-8, is redundant in gene induction. Thus, our data are consistent with a model in which both caspase-10 and cFLIP coordinately regulate CD95L-mediated signaling for death or survival.

  2. Expression and activation of Daphnia pulex Caspase-3 are involved in regulation of aging.

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    Tong, Qiaoqiong; Zhang, Mengmeng; Cao, Xiao; Xu, Shanliang; Wang, Danli; Zhao, Yunlong

    2017-11-15

    Death-mediating proteases such as Caspases have been implicated in aging. Remarkably, active Caspase-3 can trigger widespread damage and degeneration, playing a key role in causing cell death. In order to explore the relationship between Caspase-3 and aging in Daphnia pulex, we cloned and analyzed the full-length cDNA sequence of its Caspase-3 gene. Both mRNA expression and activity of D. pulex Caspase-3 increased with age. Moreover, different forms of Caspase-3 appeared with aging. The expression of casp3-L was higher and decreased with age, while that of casp3-S was weak and increased with age, consistent with the trend in Caspase-3 activity. Mhc mRNA expression declined over time and was negatively correlated with age and Caspase-3. In situ hybridization results showed that Caspase-3 mRNA was expressed in different growth and reproduction stages, and its expression levels in embryos and larva were lower than that in adult D. pulex. Western blot analysis revealed the presence of Caspase-3 in the form of zymogens with a molecular weight of ~36kDa. Overall, this study explored age-associated gene regulation to provide a basis for the molecular mechanism of D. pulex reproductive conversion. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Differential regulation of caspase-1 activation, pyroptosis, and autophagy via Ipaf and ASC in Shigella-infected macrophages.

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    Toshihiko Suzuki

    2007-08-01

    Full Text Available Shigella infection, the cause of bacillary dysentery, induces caspase-1 activation and cell death in macrophages, but the precise mechanisms of this activation remain poorly understood. We demonstrate here that caspase-1 activation and IL-1beta processing induced by Shigella are mediated through Ipaf, a cytosolic pattern-recognition receptor of the nucleotide-binding oligomerization domain (NOD-like receptor (NLR family, and the adaptor protein apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC. We also show that Ipaf was critical for pyroptosis, a specialized form of caspase-1-dependent cell death induced in macrophages by bacterial infection, whereas ASC was dispensable. Unlike that observed in Salmonella and Legionella, caspase-1 activation induced by Shigella infection was independent of flagellin. Notably, infection of macrophages with Shigella induced autophagy, which was dramatically increased by the absence of caspase-1 or Ipaf, but not ASC. Autophagy induced by Shigella required an intact bacterial type III secretion system but not VirG protein, a bacterial factor required for autophagy in epithelial-infected cells. Treatment of macrophages with 3-methyladenine, an inhibitor of autophagy, enhanced pyroptosis induced by Shigella infection, suggesting that autophagy protects infected macrophages from pyroptosis. Thus, Ipaf plays a critical role in caspase-1 activation induced by Shigella independently of flagellin. Furthermore, the absence of Ipaf or caspase-1, but not ASC, regulates pyroptosis and the induction of autophagy in Shigella-infected macrophages, providing a novel function for NLR proteins in bacterial-host interactions.

  4. Sox11 Reduces Caspase-6 Cleavage and Activity.

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    Elaine Waldron-Roby

    Full Text Available The apoptotic cascade is an orchestrated event, whose final stages are mediated by effector caspases. Regulatory binding proteins have been identified for caspases such as caspase-3, -7, -8, and -9. Many of these proteins belong to the inhibitor of apoptosis (IAP family. By contrast, caspase-6 is not believed to be influenced by IAPs, and little is known about its regulation. We therefore performed a yeast-two-hybrid screen using a constitutively inactive form of caspase-6 for bait in order to identify novel regulators of caspase-6 activity. Sox11 was identified as a potential caspase-6 interacting protein. Sox11 was capable of dramatically reducing caspase-6 activity, as well as preventing caspase-6 self- cleavage. Several regions, including amino acids 117-214 and 362-395 within sox11 as well as a nuclear localization signal (NLS all contributed to the reduction in caspase-6 activity. Furthermore, sox11 was also capable of decreasing other effector caspase activity but not initiator caspases -8 and -9. The ability of sox11 to reduce effector caspase activity was also reflected in its capacity to reduce cell death following toxic insult. Interestingly, other sox proteins also had the ability to reduce caspase-6 activity but to a lesser extent than sox11.

  5. Caspase 1 activation is protective against hepatocyte cell death by up-regulating beclin 1 protein and mitochondrial autophagy in the setting of redox stress.

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    Sun, Qian; Gao, Wentao; Loughran, Patricia; Shapiro, Rick; Fan, Jie; Billiar, Timothy R; Scott, Melanie J

    2013-05-31

    Caspase 1 activation can be induced by oxidative stress, which leads to the release of the proinflammatory cytokines IL1β and IL18 in myeloid cells and a potentially damaging inflammatory response. However, little is known about the role of caspase 1 in non-immune cells, such as hepatocytes, that express and activate the inflammasome but do not produce a significant amount of IL1β/IL18. Here we demonstrate that caspase 1 activation protects against cell death after redox stress induced by hypoxia/reoxygenation in hepatocytes. Mechanistically, we show that caspase 1 reduces mitochondrial respiration and reactive oxygen species by increasing mitochondrial autophagy and subsequent clearance of mitochondria in hepatocytes after hypoxia/reoxygenation. Caspase 1 increases autophagic flux through up-regulating autophagy initiator beclin 1 during redox stress and is an important cell survival factor in hepatocytes. We find that during hemorrhagic shock with resuscitation, an in vivo mouse model associated with severe hepatic redox stress, caspase 1 activation is also protective against liver injury and excessive oxidative stress through the up-regulation of beclin 1. Our findings suggest an alternative role for caspase 1 activation in promoting adaptive responses to oxidative stress and, more specifically, in limiting reactive oxygen species production and damage in cells and tissues where IL1β/IL18 are not highly expressed.

  6. Caspase 1 Activation Is Protective against Hepatocyte Cell Death by Up-regulating Beclin 1 Protein and Mitochondrial Autophagy in the Setting of Redox Stress*

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    Sun, Qian; Gao, Wentao; Loughran, Patricia; Shapiro, Rick; Fan, Jie; Billiar, Timothy R.; Scott, Melanie J.

    2013-01-01

    Caspase 1 activation can be induced by oxidative stress, which leads to the release of the proinflammatory cytokines IL1β and IL18 in myeloid cells and a potentially damaging inflammatory response. However, little is known about the role of caspase 1 in non-immune cells, such as hepatocytes, that express and activate the inflammasome but do not produce a significant amount of IL1β/IL18. Here we demonstrate that caspase 1 activation protects against cell death after redox stress induced by hypoxia/reoxygenation in hepatocytes. Mechanistically, we show that caspase 1 reduces mitochondrial respiration and reactive oxygen species by increasing mitochondrial autophagy and subsequent clearance of mitochondria in hepatocytes after hypoxia/reoxygenation. Caspase 1 increases autophagic flux through up-regulating autophagy initiator beclin 1 during redox stress and is an important cell survival factor in hepatocytes. We find that during hemorrhagic shock with resuscitation, an in vivo mouse model associated with severe hepatic redox stress, caspase 1 activation is also protective against liver injury and excessive oxidative stress through the up-regulation of beclin 1. Our findings suggest an alternative role for caspase 1 activation in promoting adaptive responses to oxidative stress and, more specifically, in limiting reactive oxygen species production and damage in cells and tissues where IL1β/IL18 are not highly expressed. PMID:23589298

  7. Angelica sinensis polysaccharides promotes apoptosis in human breast cancer cells via CREB-regulated caspase-3 activation

    International Nuclear Information System (INIS)

    Zhou, Wei-Jie; Wang, Sheng; Hu, Zhuang; Zhou, Zhen-Yu; Song, Cai-Juan

    2015-01-01

    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 response 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 induced

  8. Angelica sinensis polysaccharides promotes apoptosis in human breast cancer cells via CREB-regulated caspase-3 activation

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    Zhou, Wei-Jie; Wang, Sheng [Department of Breast and Thyroid Surgery, Huaihe Hospital, Henan University, Kaifeng 475000 (China); Hu, Zhuang, E-mail: zhuanghu475000@sina.com [Department of Breast and Thyroid Surgery, Huaihe Hospital, Henan University, Kaifeng 475000 (China); Zhengzhou Center for Disease Control and Prevention, Zhengzhou 475000 (China); Zhou, Zhen-Yu; Song, Cai-Juan [Department of Breast and Thyroid Surgery, Huaihe Hospital, Henan University, Kaifeng 475000 (China); Zhengzhou Center for Disease Control and Prevention, Zhengzhou 475000 (China)

    2015-11-20

    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 response 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 induced

  9. Fas-Induced Apoptosis of Renal Cell Carcinoma is Mediated by Apoptosis Signal-Regulating Kinase 1 via Mitochondrial Damage-Dependent Caspase-8 Activation

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    Mohamed Hassan

    2009-01-01

    Full Text Available Renal cell carcinoma (RCC is a prototype of a chemo refractory tumour. It remains the most lethal of the common urologic cancers and is highly resistant to conventional therapy. Here, we confirmed the efficiency of anti-Fas monoclonal antibody (CH11 as alternative therapeutic approach for the treatment of RCC and investigated the molecular mechanism(s, whereby CH11 induces apoptosis of RCC cells. The present study shows an essential role for apoptosis signal-regulating kinase 1 (ASK1, together with both c-jun-N-terminal kinase (JNK and p38 pathways, and caspase-8 in this process. Furthermore, CH11-dependent induction of the ASK1–JNK/p38 pathways was found to activate the transcription factors AP-1 and ATF-2, and FADD-caspase-8-Bid signalling, resulting in the translocation of both Bax and Bak proteins, and subsequently mitochondrial dysregulation that is characterized by the loss of mitochondrial membrane potential (ΔΨm, cytochrome c release and cleavage of caspase-9, caspase-3 and PARP. Thus, the described molecular mechanisms of CH11-induced apoptosis suggest the reliability of Fas activation as an alternative therapeutic approach for the treatment of patients with advanced renal cell carcinoma.

  10. Cytotoxicity of diacetoxyscirpenol is associated with apoptosis by activation of caspase-8 and interruption of cell cycle progression by down-regulation of cdk4 and cyclin B1 in human Jurkat T cells

    International Nuclear Information System (INIS)

    Jun, Do Youn; Kim, Jun Seok; Park, Hae Sun; Song, Woo Sun; Bae, Young Seuk; Kim, Young Ho

    2007-01-01

    To understand the mechanism underlying T-cell toxicity of diacetoxyscirpenol (DAS) from Fusarium sambucinum, its apoptogenic as well as growth retardation activity was investigated in human Jurkat T cells. Exposure to DAS (0.01-0.15 μM) caused apoptotic DNA fragmentation along with caspase-8 activation, Bid cleavage, mitochondrial cytochrome c release, activation of caspase-9 and caspase-3, and PARP degradation, without any alteration in the levels of Fas or FasL. Under these conditions, necrosis was not accompanied. The cytotoxicity of DAS was not blocked by the anti-Fas neutralizing antibody ZB-4. Although the DAS-induced apoptotic events were completely prevented by overexpression of Bcl-xL, the cells overexpressing Bcl-xL were unable to divide in the presence of DAS, resulting from the failure of cell cycle progression possibly due to down-regulation in the protein levels of cdk4 and cyclin B1. The DAS-mediated apoptosis and activation of caspase-8, -9, and -3 were abrogated by either pan-caspase inhibitor (z-VAD-fmk) or caspase-8 inhibitor (z-IETD-fmk). While the DAS-mediated apoptosis and activation of caspase-9 and caspase-3 were slightly suppressed by the mitochondrial permeability transition pore inhibitor (CsA), both caspase-8 activation and Bid cleavage were not affected by CsA. The activated normal peripheral T cells possessed a similar susceptibility to the cytotoxicity of DAS. These results demonstrate that the T-cell toxicity of DAS is attributable to not only apoptosis initiated by caspase-8 activation and subsequent mitochondrion-dependent or -independent activation of caspase cascades, which can be regulated by Bcl-xL, but also interruption of cell cycle progression caused by down-regulation of cdk4 and cyclin B1 proteins

  11. THE ROLE OF PROTEIN OXIDATIVE MODIFICATION IN REDOX-REGULATION OF CASPASE-3 ACTIVITY IN BLOOD LYMPHOCYTES DURING OXIDATIVE STRESS IN VITRO

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    O. L. Nosareva

    2015-01-01

    Full Text Available The formation of oxidative stress lies at the heart of many frequent and socially-important diseases. Blood lymphocytes are the cells which provide immunological control of our organism. As a result of their function implementation blood lymphocytes contact with different endogenic and exogenic factors, which can lead to active oxygen species production activation, macromolecules oxidative modification and to cell survival alteration. At the present time it is essential to expand and deepen the fundamental knowledge of blood lymphocytes apoptosis regulation peculiarities. The research objective was to establish the interaction among alterations of glutathione system condition, carbonylation level, protein glutathionylation and caspase-3 activity in blood lymphocytes during oxidative stress in vitro.Material and Methods. The material for research was blood lymphocytes cultivated with addition of hydrogen peroxide in final concentration of 0,5 mmol and/or protein SH-group inhibitor N-ethylmaleimide – 5 mmol, protector – 5 mmol – 1,4-dithioerythritol. Reduced, oxidized and protein-bound glutathione concentration was measured by method of spectropho-tometry, additionally, the ratio size of reduced to oxidized thiol fraction was estimated. With help of enzymoimmunoassay the level of protein carbonyl derivatives was evaluated; caspase-3 activity was registered by spectrofluorometric method.Results. Protein SH-group blocking in blood lymphocytes during oxidative stress in vitro was accompanied by protein-bound glutathione concentration rapid decrease in connection with increase of protein carbonyl derivatives content and caspase-3 activity. Protein SH-group protection in blood lymphocytes during oxidative stress in vitro was accompanied by concentration increase of protein-bound glutathione and protein carbonyl derivatives under comparable values of enzyme activity under study.Conclusion. The carried out research shows that caspase-3 and protein

  12. The human papillomavirus (HPV) E6 oncoproteins promotes nuclear localization of active caspase 8

    International Nuclear Information System (INIS)

    Manzo-Merino, Joaquin; Massimi, Paola; Lizano, Marcela; Banks, Lawrence

    2014-01-01

    The HPV-16 E6 and E6 ⁎ proteins have been shown previously to be capable of regulating caspase 8 activity. We now show that the capacity of E6 to interact with caspase 8 is common to diverse HPV types, being also seen with HPV-11 E6, HPV-18 E6 and HPV-18 E6 ⁎ . Unlike most E6-interacting partners, caspase 8 does not appear to be a major proteasomal target of E6, but instead E6 appears able to stimulate caspase 8 activation, without affecting the overall apoptotic activity. This would appear to be mediated in part by the ability of the HPV E6 oncoproteins to recruit active caspase 8 to the nucleus. - Highlights: • Multiple HPV E6 oncoproteins interact with the caspase 8 DED domain. • HPV E6 stimulates activation of caspase 8. • HPV E6 promotes nuclear accumulation of caspase 8

  13. The human papillomavirus (HPV) E6 oncoproteins promotes nuclear localization of active caspase 8

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    Manzo-Merino, Joaquin [Unidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerología, México/Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México. Av. San Fernando No. 22, Col. Sección XVI, Tlalpan 14080 (Mexico); Massimi, Paola [International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34149 Trieste (Italy); Lizano, Marcela, E-mail: lizanosoberon@gmail.com [Unidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerología, México/Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México. Av. San Fernando No. 22, Col. Sección XVI, Tlalpan 14080 (Mexico); Banks, Lawrence, E-mail: banks@icgeb.org [International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34149 Trieste (Italy)

    2014-02-15

    The HPV-16 E6 and E6{sup ⁎} proteins have been shown previously to be capable of regulating caspase 8 activity. We now show that the capacity of E6 to interact with caspase 8 is common to diverse HPV types, being also seen with HPV-11 E6, HPV-18 E6 and HPV-18 E6{sup ⁎}. Unlike most E6-interacting partners, caspase 8 does not appear to be a major proteasomal target of E6, but instead E6 appears able to stimulate caspase 8 activation, without affecting the overall apoptotic activity. This would appear to be mediated in part by the ability of the HPV E6 oncoproteins to recruit active caspase 8 to the nucleus. - Highlights: • Multiple HPV E6 oncoproteins interact with the caspase 8 DED domain. • HPV E6 stimulates activation of caspase 8. • HPV E6 promotes nuclear accumulation of caspase 8.

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

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    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.

  15. Identification of Early Intermediates of Caspase Activation Using Selective Inhibitors and Activity-Based Probes

    NARCIS (Netherlands)

    Berger, Alicia B.; Witte, Martin D.; Denault, Jean-Bernard; Sadaghiani, Amir Masoud; Sexton, Kelly M.B.; Salvesen, Guy S.; Bogyo, Matthew

    2006-01-01

    Caspases are cysteine proteases that are key effectors in apoptotic cell death. Currently, there is a lack of tools that can be used to monitor the regulation of specific caspases in the context of distinct apoptotic programs. We describe the development of highly selective inhibitors and active

  16. Serial killers: ordering caspase activation events in apoptosis.

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    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.

  17. Reconstituted NALP1 inflammasome reveals two-step mechanism of caspase-1 activation.

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    Faustin, Benjamin; Lartigue, Lydia; Bruey, Jean-Marie; Luciano, Frederic; Sergienko, Eduard; Bailly-Maitre, Beatrice; Volkmann, Niels; Hanein, Dorit; Rouiller, Isabelle; Reed, John C

    2007-03-09

    Interleukin (IL)-1beta maturation is accomplished by caspase-1-mediated proteolysis, an essential element of innate immunity. NLRs constitute a recently recognized family of caspase-1-activating proteins, which contain a nucleotide-binding oligomerization domain and leucine-rich repeat (LRR) domains and which assemble into multiprotein complexes to create caspase-1-activating platforms called "inflammasomes." Using purified recombinant proteins, we have reconstituted the NALP1 inflammasome and have characterized the requirements for inflammasome assembly and caspase-1 activation. Oligomerization of NALP1 and activation of caspase-1 occur via a two-step mechanism, requiring microbial product, muramyl-dipeptide, a component of peptidoglycan, followed by ribonucleoside triphosphates. Caspase-1 activation by NALP1 does not require but is enhanced by adaptor protein ASC. The findings provide the biochemical basis for understanding how inflammasome assembly and function are regulated, and shed light on NALP1 as a direct sensor of bacterial components in host defense against pathogens.

  18. Essential roles of caspases and their upstream regulators in rotenone-induced apoptosis

    International Nuclear Information System (INIS)

    Lee Jihjong; Huang, M.-S.; Yang, I-C.; Lai, T.-C.; Wang, J.-L.; Pang, V.F.; Hsiao, M.; Kuo, M.Y.P.

    2008-01-01

    In the present study, we examined whether caspases and their upstream regulators are involved in rotenone-induced cytotoxicity. Rotenone significantly inhibited the proliferation of oral cancer cell lines in a dose-dependent manner compared to normal oral mucosal fibroblasts. Flow cytometric analysis of DNA content showed that rotenone treatment induced apoptosis following G2/M arrest. Western blotting showed activation of both the caspase-8 and caspase-9 pathways, which differed from previous studies conducted in other cell types. Furthermore, p53 protein and its downstream pro-apoptotic target, Bax, were induced in SAS cells after treatment with rotenone. Rotenone-induced apoptosis was inhibited by antioxidants (glutathione, N-acetylcysteine, and tiron). In conclusion, our results demonstrate significant involvement of caspases and their upstream regulators in rotenone-induced cytotoxicity

  19. Metabolic Regulation of CaMKII Protein and Caspases in Xenopus laevis Egg Extracts*

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    McCoy, Francis; Darbandi, Rashid; Chen, Si-Ing; Eckard, Laura; Dodd, Keela; Jones, Kelly; Baucum, Anthony J.; Gibbons, Jennifer A.; Lin, Sue-Hwa; Colbran, Roger J.; Nutt, Leta K.

    2013-01-01

    The metabolism of the Xenopus laevis egg provides a cell survival signal. We found previously that increased carbon flux from glucose-6-phosphate (G6P) through the pentose phosphate pathway in egg extracts maintains NADPH levels and calcium/calmodulin regulated protein kinase II (CaMKII) activity to phosphorylate caspase 2 and suppress cell death pathways. Here we show that the addition of G6P to oocyte extracts inhibits the dephosphorylation/inactivation of CaMKII bound to caspase 2 by protein phosphatase 1. Thus, G6P sustains the phosphorylation of caspase 2 by CaMKII at Ser-135, preventing the induction of caspase 2-mediated apoptotic pathways. These findings expand our understanding of oocyte biology and clarify mechanisms underlying the metabolic regulation of CaMKII and apoptosis. Furthermore, these findings suggest novel approaches to disrupt the suppressive effects of the abnormal metabolism on cell death pathways. PMID:23400775

  20. Atrial natriuretic peptide down-regulates LPS/ATP-mediated IL-1β release by inhibiting NF-kB, NLRP3 inflammasome and caspase-1 activation in THP-1 cells.

    Science.gov (United States)

    Mezzasoma, Letizia; Antognelli, Cinzia; Talesa, Vincenzo Nicola

    2016-02-01

    Atrial natriuretic peptide (ANP) is an hormone/paracrine/autocrine factor regulating cardiovascular homeostasis by guanylyl cyclase natriuretic peptide receptor (NPR-1). ANP plays an important role also in regulating inflammatory and immune systems by altering macrophages functions and cytokines secretion. Interleukin-1β (IL-1β) is a potent pro-inflammatory cytokine involved in a wide range of biological responses, including the immunological one. Unlike other cytokines, IL-1β production is rigorously controlled. Primarily, NF-kB activation is required to produce pro-IL-1β; subsequently, NALP3 inflammasome/caspase-1 activation is required to cleave pro-IL-1β into the active secreted protein. NALP3 is a molecular platform capable of sensing a large variety of signals and a major player in innate immune defense. Due to their pleiotropism, IL-1β and NALP3 dysregulation is a common feature of a wide range of diseases. Therefore, identifying molecules regulating IL-1β/NALP3/caspase-1 expression is an important step in the development of new potential therapeutic agents. The aim of our study was to evaluate the effect of ANP on IL-1β/NALP3/caspase-1 expression in LPS/ATP-stimulated human THP1 monocytes. We provided new evidence of the direct involvement of ANP/NPR-1/cGMP axis on NF-kB/NALP3/caspase-1-mediated IL-1β release and NF-kB-mediated pro-IL-1β production. In particular, ANP inhibited both NF-kB and NALP3/caspase-1 activation leading to pro- and mature IL-1β down-regulation. Our data, pointing out a modulatory role of this endogenous peptide on IL-1β release and on NF-kB/NALP3/caspase-1 activation, indicate an important anti-inflammatory and immunomodulatory effect of ANP via these mechanisms. We suggest a possible employment of ANP for the treatment of inflammatory/immune-related diseases and IL-1β/NALP3-associated disorders, affecting millions of people worldwide.

  1. Caspase-3 activation as a bifurcation point between plasticity and cell death

    Institute of Scientific and Technical Information of China (English)

    Shikha Snigdha; Erica D Smith; G Aleph Prieto; Carl W Cotman

    2012-01-01

    Death-mediating proteases such as caspases and caspase-3 in particular,have been implicated in neurodegenerative processes,aging and Alzheimer's disease.However,emerging evidence suggests that in addition to their classical role in cell death,caspases play a key role in modulating synaptic function.It is remarkable that active caspases-3,which can trigger widespread damage and degeneration,aggregates in structures as delicate as synapses and persists in neurons without causing acute cell death.Here,we evaluate this dichotomy,and discuss the hypothesis that caspase-3 may be a bifurcation point in cellular signaling,able to orient the neuronal response to stress down either pathological/apoptotic pathways or towards physiological cellular remodeling.We propose that temporal,spatial and other regulators of caspase activity are key determinants of the ultimate effect of caspase-3 activation in neurons.This concept has implications for differential roles of caspase-3 activation across the lifespan.Specifically,we propose that limited caspase-3 activation is critical for synaptic function in the healthy adult brain while chronic activation is involved in degenerative processes in the aging brain.

  2. Activation of caspase-3 in radioinduced apoptosis in developing brain

    International Nuclear Information System (INIS)

    Michelin, Severino C.; Perez, Maria del R.; Gisone, Pablo; Dubner, Diana

    2001-01-01

    ICE/ced-3 related proteases (caspases) have been implicated in programmed cell death in a wide variety of cell types. However, their roles in radiation-induced cell death in cultures of mixed, neuronal and glial precursors cells are poorly understood. In order to further elucidate the molecular mechanisms underlying radiation induced death in this system; we have examined the ability of ionizing radiation to induce cell death and the caspase-3 activity. Survival decreased in a dose-dependent manner 24 h after a single 0,1 to 4 Gy dose of ionizing radiation. Irradiation resulted in a significant induction in caspase-3 activity, as measured by increased cleavage of colorimetric caspase substrates. Specific inhibitor of caspase-3, zDEVD-fmk, protected only partially from radiation induced cell death. These results demonstrate that cell death occurred despite of caspase-3 inhibition, and suggest that radio-induced cell death may occur by other mechanisms. (author)

  3. A novel bicistronic sensor vector for detecting caspase-3 activation.

    Science.gov (United States)

    Vagner, Tatyana; Mouravlev, Alexandre; Young, Deborah

    2015-01-01

    Apoptosis is involved in pathological cell death of a wide range of human diseases. One of the most important biochemical markers of apoptosis is activation of caspase-3. Ability to detect caspase-3 activation early in the pathological process is important for determining the timing for interfering with apoptosis initiation and prevention of cell damage. Techniques allowing detection of caspase-3 activity at a single cell level show increased sensitivity, compared to biochemical assays; therefore, we developed a novel bicistronic caspase-3 sensor vector enabling detection of caspase-3 activity in individual cells. We employed green fluorescent protein (GFP) as a reporter for caspase-3 activation in our constructs and assessed the functionality of the generated constructs in transiently transfected Neuro2A and HEK293 cells under basal conditions and following application of okadaic acid (OA) or staurosporine (STS) to induce apoptosis. To ensure responsiveness of the new sensor vector to active caspase-3, we co-transfected the sensor with plasmid(s) overexpressing active caspase-3 and quantified GFP fluorescence using a plate reader. We observed an increase in GFP expression in cells transfected with the new bicistronic caspase-3 sensor in response to both OA and STS. We also showed a significant increase in GFP fluorescence intensity in cells co-expressing the sensor with the plasmid(s) encoding active caspase-3. We generated a novel bicistronic caspase-3 sensor vector which relies on a transcription factor/response element system. The obtained sensor combines high sensitivity of the single cell level detection with the possibility of automated quantification. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. 13-methyltetradecanoic acid exhibits anti-tumor activity on T-cell lymphomas in vitro and in vivo by down-regulating p-AKT and activating caspase-3.

    Directory of Open Access Journals (Sweden)

    Qingqing Cai

    Full Text Available 13-Methyltetradecanoic acid (13-MTD, a saturated branched-chain fatty acid purified from soy fermentation products, induces apoptosis in human cancer cells. We investigated the inhibitory effects and mechanism of action of 13-MTD on T-cell non-Hodgkin's lymphoma (T-NHL cell lines both in vitro and in vivo. Growth inhibition in response to 13-MTD was evaluated by the cell counting kit-8 (CCK-8 assay in three T-NHL cell lines (Jurkat, Hut78, EL4 cells. Flow cytometry analyses were used to monitor the cell cycle and apoptosis. Proteins involved in 13-MTD-induced apoptosis were examined in Jurkat cells by western blotting. We found that 13-MTD inhibited proliferation and induced the apoptosis of T-NHL cell lines. 13-MTD treatment also induced a concentration-dependent arrest of Jurkat cells in the G1-phase. During 13-MTD-induced apoptosis in Jurkat cells, the cleavage of caspase-3 and poly ADP-ribose polymerase (PARP, a caspase enzymolysis product were detected after incubation for 2 h, and increased after extending the incubation time. However, there was no change in the expression of Bcl-2 or c-myc proteins. The appearance of apoptotic Jurkat cells was accompanied by the inhibition of AKT and nuclear factor-kappa B (NF-κB phosphorylation. In addition, 13-MTD could also effectively inhibit the growth of T-NHL tumors in vivo in a xenograft model. The tumor inhibition rate in the experimental group was 40%. These data indicate that 13-MTD inhibits proliferation and induces apoptosis through the down-regulation of AKT phosphorylation followed by caspase activation, which may provide a new approach for treating T-cell lymphomas.

  5. Silver Nanoparticles Biosynthesized Using Achillea biebersteinii Flower Extract: Apoptosis Induction in MCF-7 Cells via Caspase Activation and Regulation of Bax and Bcl-2 Gene Expression

    Directory of Open Access Journals (Sweden)

    Javad Baharara

    2015-02-01

    Full Text Available Silver nanoparticles (Ag-NPs, the most popular nanoparticles, possess unique properties. Achillea biebersteinii is a plant of the Asteraceae family rich in active antitumor components. The aim of this research was the characterization and investigation of the cytotoxic properties of Ag-NPs synthesized using A. biebersteinii flower extract, on a human breast cancer cell line. The Ag-NPs were synthesized after approximately 180 min of reaction at 40 °C, then they were characterized by UV-visible spectroscopy, Fourier transform infrared spectroscopy (FTIR, transmission electron microscopy (TEM and dynamic light scattering (DLS. The anti-apoptosis effect of Ag-NPs on the MCF-7 cell line was investigated by MTT assay, DAPI and acridine orange staining and caspase activity. The transcriptional expression of bax, bcl-2, caspase-3, -8 and -9 were also evaluated by RT-PCR. The TEM images revealed that the Ag-NPs morphology had a different shape. The DLS indicated that the average hydrodynamic diameter of the biosynthesized Ag-NPs was around 12 nm. By UV-visible spectroscopy the strongest absorbance peak was observed at 460 nm. The FTIR results also showed interaction between the plant extract and Ag-NPs due to the similarity in the peak patterns. The EDS results showed that Ag-NPs display an absorption peak at 3 keV, indicating the presence of the element silver. The Ag-NPs caused a dose-dependent decrease in cell viability, fragmentation in nucleic acid, inhibited the proliferation and induction of apoptosis on MCF-7 by suppressing specific cell cycle genes, and simulation programmed cell dead genes. Further investigation is required to establish the potential of this novel and promising approach in cancer therapy.

  6. Feedback regulation of mitochondria by caspase-9 in the B cell receptor-mediated apoptosis.

    Science.gov (United States)

    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.

  7. Regulation of caspase-3 expression to maintain fetal growth in Porphyromonas gingivalis-infected pregnant rats

    Directory of Open Access Journals (Sweden)

    Banun Kusumawardani

    2016-04-01

    Full Text Available Periodontal disease has been involved in a variety of systemic disorders and suspected as a potential risk factor for fetal growth restriction. Periodontal pathogenic bacteria may actively regulate embryonic development, implantation and placental trophoblast cell invasion. This study aimed to analyze the role of TNF-α, IL-10 and caspase-3 to maintain fetal growth in Porphyromonasgingivalis-infected pregnant rats. Female rats were infected with live-Porphyromonas gingivalis at concentration of 2x109 cells/ml into subgingival sulcus area of the maxillary first molar before and during pregnancy. They were sacrificed on gestational day (GD-14 and GD20. The weight and length of placentas and fetuses were evaluated. The expression of TNF-α, IL-10 and caspase-3 in macrophages and trophoblast cells were detected by immunohistochemistry. On GD14, TNF-α (R2=0.416;P=0.000 and IL-10 (R2=0.187;P=0.012 had an important role to increase expression of caspase-3 in the placenta, but only TNF-α (R2=0.393;P=0.000 was able to increase the expression of caspase-3 on GD20. TNF-α and caspase-3 also had an important role (P0.000. The increasing expressions of TNF-α and IL-10 did not only enhance immune protection, but also maintained the trophoblast cells survival by regulating expression of caspase-3. Porphyromonas gingivalis infection in maternal periodontal tissue can lead to decrease in placental weight, fetal weight and fetal length which mediated by increasing expression of TNF-α, IL-10 and caspase-3 in the placenta.

  8. Caspase Activation in Fetal Rat Brain Following Experimental Intrauterine Inflammation

    Science.gov (United States)

    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

  9. Fenretinide-induced caspase-8 activation and apoptosis in an established model of metastatic neuroblastoma

    International Nuclear Information System (INIS)

    Raguénez, Gilda; Mühlethaler-Mottet, Annick; Meier, Roland; Duros, Caroline; Bénard, Jean; Gross, Nicole

    2009-01-01

    Resistance of high-risk metastatic neuroblastoma (HR-NB) to high dose chemotherapy (HD-CT) raises a major therapeutic challenge in pediatric oncology. Patients are treated by maintenance CT. For some patients, an adjuvant retinoid therapy is proposed, such as the synthetic retinoid fenretinide (4-HPR), an apoptotic inducer. Recent studies demonstrated that NB metastasis process is enhanced by the loss of caspase-8 involved in the Integrin-Mediated Death (IMD) process. As the role of caspase-8 appears to be critical in preventing metastasis, we aimed at studying the effect of 4-HPR on caspase-8 expression in metastatic neuroblasts. We used the human IGR-N-91 MYCN-amplified NB experimental model, able to disseminate in vivo from the primary nude mouse tumor xenograft (PTX) into myocardium (Myoc) and bone marrow (BM) of the animal. NB cell lines, i.e., IGR-N-91 and SH-EP, were treated with various doses of Fenretinide (4-HPR), then cytotoxicity was analyzed by MTS proliferation assay, apoptosis by the propidium staining method, gene or protein expressions by RT-PCR and immunoblotting and caspases activity by colorimetric protease assays. The IGR-N-91 parental cells do not express detectable caspase-8. However the PTX cells established from the primary tumor in the mouse, are caspase-8 positive. In contrast, metastatic BM and Myoc cells show a clear down-regulation of the caspase-8 expression. In parallel, the caspases -3, -9, -10, Bcl-2, or Bax expressions were unchanged. Our data show that in BM, compared to PTX cells, 4-HPR up-regulates caspase-8 expression that parallels a higher sensitivity to apoptotic cell death. Stable caspase-8-silenced SH-EP cells appear more resistant to 4-HPR-induced cell death compared to control SH-EP cells. Moreover, 4-HPR synergizes with drugs since apoptosis is restored in VP16- or TRAIL-resistant-BM cells. These results demonstrate that 4-HPR in up-regulating caspase-8 expression, restores and induces apoptotic cell death in

  10. Caspase activation increases beta-amyloid generation independently of caspase cleavage of the beta-amyloid precursor protein (APP).

    Science.gov (United States)

    Tesco, Giuseppina; Koh, Young Ho; Tanzi, Rudolph E

    2003-11-14

    The amyloid precursor protein (APP) undergoes "alternative" proteolysis mediated by caspases. Three major caspase recognition sites have been identified in the APP, i.e. one at the C terminus (Asp720) and two at the N terminus (Asp197 and Asp219). Caspase cleavage at Asp720 has been suggested as leading to increased production of Abeta. Thus, we set out to determine which putative caspase sites in APP, if any, are cleaved in Chinese hamster ovary cell lines concurrently with the increased Abeta production that occurs during apoptosis. We found that cleavage at Asp720 occurred concurrently with caspase 3 activation and the increased production of total secreted Abeta and Abeta1-42 in association with staurosporine- and etoposide-induced apoptosis. To investigate the contribution of caspase cleavage of APP to Abeta generation, we expressed an APP mutant truncated at Asp720 that mimics APP caspase cleavage at the C-terminal site. This did not increase Abeta generation but, in contrast, dramatically decreased Abeta production in Chinese hamster ovary cells. Furthermore, the ablation of caspase-dependent cleavage at Asp720, Asp197, and Asp219 (by site-directed mutagenesis) did not prevent enhanced Abeta production following etoposide-induced apoptosis. These findings indicate that the enhanced Abeta generation associated with apoptosis does not require cleavage of APP at its C-terminal (Asp720) and/or N-terminal caspase sites.

  11. Intracellular cholesterol level regulates sensitivity of glioblastoma cells against temozolomide-induced cell death by modulation of caspase-8 activation via death receptor 5-accumulation and activation in the plasma membrane lipid raft.

    Science.gov (United States)

    Yamamoto, Yutaro; Tomiyama, Arata; Sasaki, Nobuyoshi; Yamaguchi, Hideki; Shirakihara, Takuya; Nakashima, Katsuhiko; Kumagai, Kosuke; Takeuchi, Satoru; Toyooka, Terushige; Otani, Naoki; Wada, Kojiro; Narita, Yoshitaka; Ichimura, Koichi; Sakai, Ryuichi; Namba, Hiroki; Mori, Kentaro

    2018-01-01

    Development of resistance against temozolomide (TMZ) in glioblastoma (GBM) after continuous treatment with TMZ is one of the critical problems in clinical GBM therapy. Intracellular cholesterol regulates cancer cell biology, but whether intracellular cholesterol is involved in TMZ resistance of GBM cells remains unclear. The involvement of intracellular cholesterol in acquired resistance against TMZ in GBM cells was investigated. Intracellular cholesterol levels were measured in human U251 MG cells with acquired TMZ resistance (U251-R cells) and TMZ-sensitive control U251 MG cells (U251-Con cells), and found that the intracellular cholesterol level was significantly lower in U251-R cells than in U251-Con cells. In addition, treatment by intracellular cholesterol remover, methyl-beta cyclodextrin (MβCD), or intracellular cholesterol inducer, soluble cholesterol (Chol), regulated TMZ-induced U251-Con cell death in line with changes in intracellular cholesterol level. Involvement of death receptor 5 (DR5), a death receptor localized in the plasma membrane, was evaluated. TMZ without or with MβCD and/or Chol caused accumulation of DR5 into the plasma membrane lipid raft and formed a complex with caspase-8, an extrinsic caspase cascade inducer, reflected in the induction of cell death. In addition, treatment with caspase-8 inhibitor or knockdown of DR5 dramatically suppressed U251-Con cell death induced by combination treatment with TMZ, MβCD, and Chol. Combined treatment of Chol with TMZ reversed the TMZ resistance of U251-R cells and another GBM cell model with acquired TMZ resistance, whereas clinical antihypercholesterolemia agents at physiological concentrations suppressed TMZ-induced cell death of U251-Con cells. These findings suggest that intracellular cholesterol level affects TMZ treatment of GBM mediated via a DR5-caspase-8 mechanism. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Diatom-derived oxylipins induce cell death in sea urchin embryos activating caspase-8 and caspase 3/7.

    Science.gov (United States)

    Ruocco, Nadia; Varrella, Stefano; Romano, Giovanna; Ianora, Adrianna; Bentley, Matt G; Somma, Domenico; Leonardi, Antonio; Mellone, Stefano; Zuppa, Antonio; Costantini, Maria

    2016-07-01

    Diatoms are an important class of unicellular algae that produce bioactive secondary metabolites with cytotoxic activity collectively termed oxylipins, including polyunsaturated aldehydes (PUAs), hydroxyacids (HEPEs), oxo-acids and epoxyalcohols. Previous results showed that at higher concentrations, the PUA decadienal induced apoptosis on copepods and sea urchin embryos via caspase-3 activation; at lower concentrations decadienal affected the expression levels of the caspase-8 gene in embryos of the sea urchin Paracentrotus lividus. In the present work, we studied the effects of other common oxylipins produced by diatoms: two PUAs (heptadienal and octadienal) and four hydroxyacids (5-, 9- 11- and 15-HEPE) on P. lividus cell death and caspase activities. Our results showed that (i) at higher concentrations PUAs and HEPEs induced apoptosis in sea urchin embryos, detected by microscopic observation and through the activation of caspase-3/7 and caspase-8 measured by luminescent assays; (ii) at low concentrations, PUAs and HEPEs affected the expression levels of caspase-8 and caspase-3/7 (isolated for the first time here in P. lividus) genes, detected by Real Time qPCR. These findings have interesting implications from the ecological point of view, given the importance of diatom blooms in nutrient-rich aquatic environments. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Prostaglandin F2alpha- and FAS-activating antibody-induced regression of the corpus luteum involves caspase-8 and is defective in caspase-3 deficient mice

    Directory of Open Access Journals (Sweden)

    Flavell Richard A

    2003-02-01

    Full Text Available Abstract We recently demonstrated that caspase-3 is important for apoptosis during spontaneous involution of the corpus luteum (CL. These studies tested if prostaglandin F2α (PGF2α or FAS regulated luteal regression, utilize a caspase-3 dependent pathway to execute luteal cell apoptosis, and if the two receptors work via independent or potentially shared intracellular signaling components/pathways to activate caspase-3. Wild-type (WT or caspase-3 deficient female mice, 25–26 days old, were given 10 IU equine chorionic gonadotropin (eCG intraperitoneally (IP followed by 10 IU human chorionic gonadotropin (hCG IP 46 h later to synchronize ovulation. The animals were then injected with IgG (2 micrograms, i.v., the FAS-activating antibody Jo2 (2 micrograms, i.v., or PGF2α (10 micrograms, i.p. at 24 or 48 h post-ovulation. Ovaries from each group were collected 8 h later for assessment of active caspase-3 enzyme and apoptosis (measured by the TUNEL assay in the CL. Regardless of genotype or treatment, CL in ovaries collected from mice injected 24 h after ovulation showed no evidence of active caspase-3 or apoptosis. However, PGF2α or Jo2 at 48 h post-ovulation and collected 8 h later induced caspase-3 activation in 13.2 ± 1.8% and 13.7 ± 2.2 % of the cells, respectively and resulted in 16.35 ± 0.7% (PGF2α and 14.3 ± 2.5% TUNEL-positive cells when compared to 1.48 ± 0.8% of cells CL in IgG treated controls. In contrast, CL in ovaries collected from caspase-3 deficient mice whether treated with PGF2α , Jo2, or control IgG at 48 h post-ovulation showed little evidence of active caspase-3 or apoptosis. CL of WT mice treated with Jo2 at 48 h post-ovulation had an 8-fold increase in the activity of caspase-8, an activator of caspase-3 that is coupled to the FAS death receptor. Somewhat unexpectedly, however, treatment of WT mice with PGF2α at 48 h post-ovulation resulted in a 22-fold increase in caspase-8 activity in the CL, despite the fact

  14. Voltage dependent anion channel-1 regulates death receptor mediated apoptosis by enabling cleavage of caspase-8

    International Nuclear Information System (INIS)

    Chacko, Alex D; Liberante, Fabio; Paul, Ian; Longley, Daniel B; Fennell, Dean A

    2010-01-01

    Activation of the extrinsic apoptosis pathway by tumour necrosis factor related apoptosis inducing ligand (TRAIL) is a novel therapeutic strategy for treating cancer that is currently under clinical evaluation. Identification of molecular biomarkers of resistance is likely to play an important role in predicting clinical anti tumour activity. The involvement of the mitochondrial type 1 voltage dependent anion channel (VDAC1) in regulating apoptosis has been highly debated. To date, a functional role in regulating the extrinsic apoptosis pathway has not been formally excluded. We carried out stable and transient RNAi knockdowns of VDAC1 in non-small cell lung cancer cells, and stimulated the extrinsic apoptotic pathway principally by incubating cells with the death ligand TRAIL. We used in-vitro apoptotic and cell viability assays, as well as western blot for markers of apoptosis, to demonstrate that TRAIL-induced toxicity is VDAC1 dependant. Confocal microscopy and mitochondrial fractionation were used to determine the importance of mitochondria for caspase-8 activation. Here we show that either stable or transient knockdown of VDAC1 is sufficient to antagonize TRAIL mediated apoptosis in non-small cell lung cancer (NSCLC) cells. Specifically, VDAC1 is required for processing of procaspase-8 to its fully active p18 form at the mitochondria. Loss of VDAC1 does not alter mitochondrial sensitivity to exogenous caspase-8-cleaved BID induced mitochondrial depolarization, even though VDAC1 expression is essential for TRAIL dependent activation of the intrinsic apoptosis pathway. Furthermore, expression of exogenous VDAC1 restores the apoptotic response to TRAIL in cells in which endogenous VDAC1 has been selectively silenced. Expression of VDAC1 is required for full processing and activation of caspase-8 and supports a role for mitochondria in regulating apoptosis signaling via the death receptor pathway

  15. Caspase activity and expression of cell death genes during development of human preimplantation embryos.

    Science.gov (United States)

    Spanos, S; Rice, S; Karagiannis, P; Taylor, D; Becker, D L; Winston, R M L; Hardy, K

    2002-09-01

    It has been observed that apoptosis occurs in human blastocysts. In other types of cell, the characteristic morphological changes seen in apoptotic cells are executed by caspases, which are regulated by the BCL-2 family of proteins. This study investigated whether these components of the apoptotic cascade are present throughout human preimplantation development. Developing and arrested two pronucleate embryos at all stages were incubated with a fluorescently tagged caspase inhibitor that binds only to active caspases, fixed, counterstained with 4,6-diamidino-2-phenylindole (DAPI) to assess nuclear morphology and examined using confocal microscopy. Active caspases were detected only after compaction, at the morula and blastocyst stages, and were frequently associated with apoptotic nuclei. Occasional labelling was seen in arrested embryos. Expression of proapoptotic BAX and BAD and anti-apoptotic BCL-2 was examined in single embryos using RT-PCR and immunohistochemistry. BAX and BCL-2 mRNAs were expressed throughout development, whereas BAD mRNA was expressed mainly after compaction. Simultaneous expression of BAX and BCL-2 proteins within individual embryos was confirmed using immunohistochemistry. The onset of caspase activity and BAD expression after compaction correlates with the previously reported appearance of apoptotic nuclei. As in other types of cell, human embryos express common molecular components of the apoptotic cascade, although apoptosis appears to be suppressed before compaction and differentiation.

  16. Hypocapnia induces caspase-3 activation and increases Abeta production.

    Science.gov (United States)

    Xie, Zhongcong; Moir, Robert D; Romano, Donna M; Tesco, Giuseppina; Kovacs, Dora M; Tanzi, Rudolph E

    2004-01-01

    At least half of all cases of early onset (<60) familial Alzheimer's disease (FAD) are caused by any of over 150 mutations in three genes: the amyloid precursor protein (APP), presenilin 1 (PS1), and presenilin 2 (PS2). Mutant forms of PS1 have been shown to sensitize cells to apoptotic cell death. We investigated the effects of hypocapnia, a risk factor for both cognitive and neurodevelopment deficits, on caspase-3 activation, apoptosis, and amyloid beta-protein (Abeta) production, and assessed the influence of the PS1Delta9 FAD mutation on these effects. For this purpose, we exposed stably transfected H4 human neuroglioma cells to conditions consistent with hypocapnia (PCO2<40 mm Hg) and hypocapnia plus hypoxia (PO2<21%). Hypocapnia (20 mm Hg CO2 for 6 h) induced caspase-3 activation and apoptosis; the PS1Delta9 FAD mutation significantly potentiated these effects. Moreover, the combination of hypocapnia (20 mm Hg CO2) and hypoxia (5%O2) induced caspase-3 activation and apoptosis in a synergistic manner. Hypocapnia (5 and 20 mm Hg CO2 for 6 h) also led to an increased Abeta production. The findings suggest that hypocapnia (e.g. during general anesthesia) could exacerbate AD neuropathogenesis. Copyright (c) 2004 S. Karger AG, Basel.

  17. Acute fasting inhibits central caspase-1 activity reducing anxiety-like behavior and increasing novel object and object location recognition.

    Science.gov (United States)

    Towers, Albert E; Oelschlager, Maci L; Patel, Jay; Gainey, Stephen J; McCusker, Robert H; Freund, Gregory G

    2017-06-01

    Inflammation within the central nervous system (CNS) is frequently comorbid with anxiety. Importantly, the pro-inflammatory cytokine most commonly associated with anxiety is IL-1β. The bioavailability and activity of IL-1β are regulated by caspase-1-dependent proteolysis vis-a-vis the inflammasome. Thus, interventions regulating the activation or activity of caspase-1 should reduce anxiety especially in states that foster IL-1β maturation. Male C57BL/6j, C57BL/6j mice treated with the capase-1 inhibitor biotin-YVAD-cmk, caspase-1 knockout (KO) mice and IL-1R1 KO mice were fasted for 24h or allowed ad libitum access to food. Immediately after fasting, caspase-1 activity was measured in brain region homogenates while activated caspase-1 was localized in the brain by immunohistochemistry. Mouse anxiety-like behavior and cognition were tested using the elevated zero maze and novel object/object location tasks, respectively. A 24h fast in mice reduced the activity of caspase-1 in whole brain and in the prefrontal cortex, amygdala, hippocampus, and hypothalamus by 35%, 25%, 40%, 40%, and 40% respectively. A 24h fast also reduced anxiety-like behavior by 40% and increased novel object and object location recognition by 21% and 31%, respectively. IL-1β protein, however, was not reduced in the brain by fasting. ICV administration of YVAD decreased caspase-1 activity in the prefrontal cortex and amygdala by 55%, respectively leading to a 64% reduction in anxiety like behavior. Importantly, when caspase-1 KO or IL1-R1 KO mice are fasted, no fasting-dependent reduction in anxiety-like behavior was observed. Results indicate that fasting decrease anxiety-like behavior and improves memory by a mechanism tied to reducing caspase-1 activity throughout the brain. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Possible involvement of caspase-6 and -7 but not caspase-3 in the regulation of hypoxia-induced apoptosis in tube-forming endothelial cells

    International Nuclear Information System (INIS)

    Eguchi, Ryoji; Tone, Shigenobu; Suzuki, Akio; Fujimori, Yoshihiro; Nakano, Takashi; Kaji, Kazuhiko; Ohta, Toshiro

    2009-01-01

    We recently reported that a broad-spectrum caspase inhibitor zVAD-fmk failed, while p38 inhibitor SB203580 succeeded, to prevent chromatin condensation and nuclear fragmentation induced by hypoxia in tube-forming HUVECs. In this study, we investigated the reasons for zVAD-fmk's inability to inhibit these morphological changes at the molecular level. The inhibitor effectively inhibited DNA ladder formation and activation of caspase-3 and -6, but it surprisingly failed to inhibit caspase-7 activation. On the other hand, SB203580 successfully inhibited all of these molecular events. When zLEHD-fmk, which specifically inhibits initiator caspase-9 upstream of caspase-3, was used, it inhibited caspase-3 activation but failed to inhibit caspase-6 and -7 activation. It also failed to inhibit hypoxia-induced chromatin condensation, nuclear fragmentation and DNA ladder formation. Taken together, our results indicate that, during hypoxia, caspase-7 is responsible for chromatin condensation and nuclear fragmentation while caspase-6 is responsible for DNA ladder formation

  19. Caspase activity and apoptotic signaling in proliferating C2C12 cells following cisplatin or A23187 exposure

    Directory of Open Access Journals (Sweden)

    Darin Bloemberg

    2016-06-01

    Full Text Available Investigating cell death signaling using cell culture is commonly performed to examine the effects of novel pharmaceuticals or to further characterize discrete cellular signaling pathways. Here, we provide data regarding the cell death response to either cisplatin or A23187 in sub-confluent C2C12 cells, by utilizing several concentrations and incubation times for each chemical. These data include an assessment of the activation of the proteolytic enzymes caspase-3, caspase-8, caspase-9, calpain, and cathepsin B/L. Additionally, the expression of the apoptosis-regulating proteins Bax, Bcl2, and p53 are presented.

  20. The Inflammasome Drives GSDMD-Independent Secondary Pyroptosis and IL-1 Release in the Absence of Caspase-1 Protease Activity.

    Science.gov (United States)

    Schneider, Katharina S; Groß, Christina J; Dreier, Roland F; Saller, Benedikt S; Mishra, Ritu; Gorka, Oliver; Heilig, Rosalie; Meunier, Etienne; Dick, Mathias S; Ćiković, Tamara; Sodenkamp, Jan; Médard, Guillaume; Naumann, Ronald; Ruland, Jürgen; Kuster, Bernhard; Broz, Petr; Groß, Olaf

    2017-12-26

    Inflammasomes activate the protease caspase-1, which cleaves interleukin-1β and interleukin-18 to generate the mature cytokines and controls their secretion and a form of inflammatory cell death called pyroptosis. By generating mice expressing enzymatically inactive caspase-1 C284A , we provide genetic evidence that caspase-1 protease activity is required for canonical IL-1 secretion, pyroptosis, and inflammasome-mediated immunity. In caspase-1-deficient cells, caspase-8 can be activated at the inflammasome. Using mice either lacking the pyroptosis effector gasdermin D (GSDMD) or expressing caspase-1 C284A , we found that GSDMD-dependent pyroptosis prevented caspase-8 activation at the inflammasome. In the absence of GSDMD-dependent pyroptosis, the inflammasome engaged a delayed, alternative form of lytic cell death that was accompanied by the release of large amounts of mature IL-1 and contributed to host protection. Features of this cell death modality distinguished it from apoptosis, suggesting it may represent a distinct form of pro-inflammatory regulated necrosis. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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.

  2. Differential splicing of the apoptosis-associated speck like protein containing a caspase recruitment domain (ASC regulates inflammasomes

    Directory of Open Access Journals (Sweden)

    Rojanasakul Yon

    2010-05-01

    Full Text Available Abstract Background The apoptotic speck-like protein containing a caspase recruitment domain (ASC is the essential adaptor protein for caspase 1 mediated interleukin (IL-1β and IL-18 processing in inflammasomes. It bridges activated Nod like receptors (NLRs, which are a family of cytosolic pattern recognition receptors of the innate immune system, with caspase 1, resulting in caspase 1 activation and subsequent processing of caspase 1 substrates. Hence, macrophages from ASC deficient mice are impaired in their ability to produce bioactive IL-1β. Furthermore, we recently showed that ASC translocates from the nucleus to the cytosol in response to inflammatory stimulation in order to promote an inflammasome response, which triggers IL-1β processing and secretion. However, the precise regulation of inflammasomes at the level of ASC is still not completely understood. In this study we identified and characterized three novel ASC isoforms for their ability to function as an inflammasome adaptor. Methods To establish the ability of ASC and ASC isoforms as functional inflammasome adaptors, IL-1β processing and secretion was investigated by ELISA in inflammasome reconstitution assays, stable expression in THP-1 and J774A1 cells, and by restoring the lack of endogenous ASC in mouse RAW264.7 macrophages. In addition, the localization of ASC and ASC isoforms was determined by immunofluorescence staining. Results The three novel ASC isoforms, ASC-b, ASC-c and ASC-d display unique and distinct capabilities to each other and to full length ASC in respect to their function as an inflammasome adaptor, with one of the isoforms even showing an inhibitory effect. Consistently, only the activating isoforms of ASC, ASC and ASC-b, co-localized with NLRP3 and caspase 1, while the inhibitory isoform ASC-c, co-localized only with caspase 1, but not with NLRP3. ASC-d did not co-localize with NLRP3 or with caspase 1 and consistently lacked the ability to function as an

  3. PIDDosome Expression and the Role of Caspase-2 Activation for Chemotherapy-Induced Apoptosis in RCCs

    Directory of Open Access Journals (Sweden)

    Sebastian Heikaus

    2010-01-01

    Full Text Available Background: The importance of caspase-2 activation for mediating apoptosis in cancer is not clear and seems to differ between different tumour types. Furthermore, only few data have been obtained concerning the expression of caspase-2, which can be alternatively spliced into caspase-2L and caspase-2S, and the other PIDDosome members PIDD and RAIDD in human tumours in vivo. We, therefore, investigated their expression in renal cell carcinomas (RCCs of the clear cell type in vivo and analysed the role of caspase-2 in chemotherapy-induced apoptosis in RCCs in vitro.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  6. Construction and analysis of a modular model of caspase activation in apoptosis

    Directory of Open Access Journals (Sweden)

    Ho Kenneth L

    2008-12-01

    Full Text Available Abstract Background A key physiological mechanism employed by multicellular organisms is apoptosis, or programmed cell death. Apoptosis is triggered by the activation of caspases in response to both extracellular (extrinsic and intracellular (intrinsic signals. The extrinsic and intrinsic pathways are characterized by the formation of the death-inducing signaling complex (DISC and the apoptosome, respectively; both the DISC and the apoptosome are oligomers with complex formation dynamics. Additionally, the extrinsic and intrinsic pathways are coupled through the mitochondrial apoptosis-induced channel via the Bcl-2 family of proteins. Results A model of caspase activation is constructed and analyzed. The apoptosis signaling network is simplified through modularization methodologies and equilibrium abstractions for three functional modules. The mathematical model is composed of a system of ordinary differential equations which is numerically solved. Multiple linear regression analysis investigates the role of each module and reduced models are constructed to identify key contributions of the extrinsic and intrinsic pathways in triggering apoptosis for different cell lines. Conclusion Through linear regression techniques, we identified the feedbacks, dissociation of complexes, and negative regulators as the key components in apoptosis. The analysis and reduced models for our model formulation reveal that the chosen cell lines predominately exhibit strong extrinsic caspase, typical of type I cell, behavior. Furthermore, under the simplified model framework, the selected cells lines exhibit different modes by which caspase activation may occur. Finally the proposed modularized model of apoptosis may generalize behavior for additional cells and tissues, specifically identifying and predicting components responsible for the transition from type I to type II cell behavior.

  7. Caspase 3 inactivates biologically active full length interleukin-33 as a classical cytokine but does not prohibit nuclear translocation

    International Nuclear Information System (INIS)

    Ali, Shafaqat; Nguyen, Dang Quan; Falk, Werner; Martin, Michael Uwe

    2010-01-01

    IL-33 is a member of the IL-1 family of cytokines with dual function which either activates cells via the IL-33 receptor in a paracrine fashion or translocates to the nucleus to regulate gene transcription in an intracrine manner. We show that full length murine IL-33 is active as a cytokine and that it is not processed by caspase 1 to mature IL-33 but instead cleaved by caspase 3 at aa175 to yield two products which are both unable to bind to the IL-33 receptor. Full length IL-33 and its N-terminal caspase 3 breakdown product, however, translocate to the nucleus. Finally, bioactive IL-33 is not released by cells constitutively or after activation. This suggests that IL-33 is not a classical cytokine but exerts its function in the nucleus of intact cells and only activates others cells via its receptor as an alarm mediator after destruction of the producing cell.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  10. A quantitative method for the specific assessment of caspase-6 activity in cell culture

    DEFF Research Database (Denmark)

    Ehrnhoefer, Dagmar E; Skotte, Niels H; Savill, Jane

    2011-01-01

    Aberrant activation of caspase-6 has recently emerged as a major contributor to the pathogeneses of neurodegenerative disorders such as Alzheimer's and Huntington disease. Commercially available assays to measure caspase-6 activity commonly use the VEID peptide as a substrate. However these metho...

  11. NPM1 directs PIDDosome-dependent caspase-2 activation in the nucleolus.

    Science.gov (United States)

    Ando, Kiyohiro; Parsons, Melissa J; Shah, Richa B; Charendoff, Chloé I; Paris, Sheré L; Liu, Peter H; Fassio, Sara R; Rohrman, Brittany A; Thompson, Ruth; Oberst, Andrew; Sidi, Samuel; Bouchier-Hayes, Lisa

    2017-06-05

    The PIDDosome (PIDD-RAIDD-caspase-2 complex) is considered to be the primary signaling platform for caspase-2 activation in response to genotoxic stress. Yet studies of PIDD-deficient mice show that caspase-2 activation can proceed in the absence of PIDD. Here we show that DNA damage induces the assembly of at least two distinct activation platforms for caspase-2: a cytoplasmic platform that is RAIDD dependent but PIDD independent, and a nucleolar platform that requires both PIDD and RAIDD. Furthermore, the nucleolar phosphoprotein nucleophosmin (NPM1) acts as a scaffold for PIDD and is essential for PIDDosome assembly in the nucleolus after DNA damage. Inhibition of NPM1 impairs caspase-2 processing, apoptosis, and caspase-2-dependent inhibition of cell growth, demonstrating that the NPM1-dependent nucleolar PIDDosome is a key initiator of the caspase-2 activation cascade. Thus we have identified the nucleolus as a novel site for caspase-2 activation and function. © 2017 Ando et al.

  12. Perfluorononanoic acid-induced apoptosis in rat spleen involves oxidative stress and the activation of caspase-independent death pathway

    International Nuclear Information System (INIS)

    Fang, Xuemei; Feng, Yixing; Wang, Jianshe; Dai, Jiayin

    2010-01-01

    Perfluoroalkyl acid (PFAA)-induced apoptosis has been reported in many cell types. However, minimal information on its mode of action is available. This study explored the possible involvement of apoptotic signaling pathways in a nine-carbon-chain length PFAA-perfluorononanoic acid (PFNA)-induced splenocyte apoptosis. After a 14-day exposure to PFNA, rat spleens showed dose-dependent levels of apoptosis. The production of pro-inflammatory and anti-inflammatory cytokines was significantly increased and decreased, respectively. However, protein levels of tumor necrosis factor receptor 1 (TNFR1), fas-associated protein with death domain (FADD), caspase 8 and caspase 3, which are involved in inflammation-related and caspase-dependent apoptosis, were discordant. Peroxisome proliferator-activated receptors alpha (PPARα) and PPARγ genes expression was up-regulated in rats treated with 3 or 5 mg/kg/day of PFNA, and the level of hydrogen peroxide (H 2 O 2 ) increased concurrently in rats treated with the highest dose. Moreover, superoxide dismutase (SOD) activity and Bcl-2 protein levels were dramatically decreased in spleens after treatment with 3 and 5 mg/kg/day of PFNA. However, protein levels of Bax were unchanged. Apoptosis-inducing factor (AIF), an initiator of caspase-independent apoptosis, was significantly increased in all PFNA-dosed rats. Thus, oxidative stress and the activation of a caspase-independent apoptotic signaling pathway contributed to PFNA-induced apoptosis in rat splenocytes.

  13. ASSESSING THE ROLE OF CASPASE ACTIVITY AND METACASPASE EXPRESSION ON VIRAL SUSCEPTIBILITY OF THE COCCOLITHOPHORE, EMILIANIA HUXLEYI (HAPTOPHYTA).

    Science.gov (United States)

    Bidle, Kay D; Kwityn, Clifford J

    2012-10-01

    As part of their strategy to infect the globally important coccolithophore, Emiliania huxleyi (Lohmann) W.W. Hay & H.P. Mohler, Coccolithoviruses trigger and regulate the host's programmed cell death (PCD) machinery during lytic infection. The induction and recruitment of host metacaspases, specialized, ancestral death proteases that facilitate viral lysis, suggests they may be important subcellular determinants to infection. We examined the "basal" levels and patterns of caspase activity and metacaspase expression in exponentially growing resistant and sensitive E. huxleyi strains and linked them with susceptibility to E. huxleyi virus 1 (EhV1). Resistant E. huxleyi strains were consistently characterized by low caspase specific activity and a relatively simple metacaspase expression profile. In contrast, sensitive E. huxleyi strains had markedly elevated caspase specific activity and consistently expressed more diverse metacaspase proteins. Using pooled data sets from triplicate experiments, we observed statistically significant linear correlations between infectivity, caspase activity, and metacaspase expression, with each strain forming distinct clusters, within a gradient in viral susceptibility. At the same time, we observed positive correlations between the expression of a subset of metacaspase proteins and lower susceptibility, suggestive of potential protective roles. Our findings implicate the importance of subtle differences in the basal physiological regulation of the PCD machinery to viral resistance or sensitivity and cell fate. © 2012 Phycological Society of America.

  14. Caspase inhibitors of the P35 family are more active when purified from yeast than bacteria.

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    Ingo L Brand

    Full Text Available Many insect viruses express caspase inhibitors of the P35 superfamily, which prevent defensive host apoptosis to enable viral propagation. The prototypical P35 family member, AcP35 from Autographa californica M nucleopolyhedrovirus, has been extensively studied. Bacterially purified AcP35 has been previously shown to inhibit caspases from insect, mammalian and nematode species. This inhibition occurs via a pseudosubstrate mechanism involving caspase-mediated cleavage of a "reactive site loop" within the P35 protein, which ultimately leaves cleaved P35 covalently bound to the caspase's active site. We observed that AcP35 purifed from Saccharomyces cerevisae inhibited caspase activity more efficiently than AcP35 purified from Escherichia coli. This differential potency was more dramatic for another P35 family member, MaviP35, which inhibited human caspase 3 almost 300-fold more potently when purified from yeast than bacteria. Biophysical assays revealed that MaviP35 proteins produced in bacteria and yeast had similar primary and secondary structures. However, bacterially produced MaviP35 possessed greater thermal stability and propensity to form higher order oligomers than its counterpart purified from yeast. Caspase 3 could process yeast-purified MaviP35, but failed to detectably cleave bacterially purified MaviP35. These data suggest that bacterially produced P35 proteins adopt subtly different conformations from their yeast-expressed counterparts, which hinder caspase access to the reactive site loop to reduce the potency of caspase inhibition, and promote aggregation. These data highlight the differential caspase inhibition by recombinant P35 proteins purified from different sources, and caution that analyses of bacterially produced P35 family members (and perhaps other types of proteins may underestimate their activity.

  15. O-GlcNAc regulates NEDD4-1 stability via caspase-mediated pathway

    International Nuclear Information System (INIS)

    Jiang, Kuan; Bai, Bingyang; Ta, Yajie; Zhang, Tingling; Xiao, Zikang; Wang, Peng George; Zhang, Lianwen

    2016-01-01

    O-GlcNAc modification of cytosolic and nuclear proteins regulates essential cellular processes such as stress responses, transcription, translation, and protein degradation. Emerging evidence indicates O-GlcNAcylation has a dynamic interplay with ubiquitination in cellular regulation. Here, we report that O-GlcNAc indirectly targets a vital E3 ubiquitin ligase enzyme of NEDD4-1. The protein level of NEDD4-1 is accordingly decreased following an increase of overall O-GlcNAc level upon PUGNAc or glucosamine stimulation. O-GlcNAc transferase (OGT) knockdown, overexpression and mutation results confirm that the stability of NEDD4-1 is negatively regulated by cellular O-GlcNAc. Moreover, the NEDD4-1 degradation induced by PUGNAc or GlcN is significantly inhibited by the caspase inhibitor. Our study reveals a regulation mechanism of NEDD4-1 stability by O-GlcNAcylation. - Highlights: • Reduced NEDD4-1 correlates with increased overall O-GlcNAc level. • OGT negatively regulates NEDD4-1 stability. • O-GlcNAc regulates NEDD4-1 through caspase-mediated pathway.

  16. Bioluminescence determination of active caspase-3 in single apoptotic cells

    Czech Academy of Sciences Publication Activity Database

    Lišková, Marcela; Klepárník, Karel; Matalová, Eva; Hegrová, Jitka; Přikryl, Jan; Švandová, Eva; Foret, František

    2013-01-01

    Roč. 34, č. 12 (2013), s. 1772-1777 ISSN 0173-0835 R&D Projects: GA ČR GAP206/11/2377 Grant - others:GA ČR(CZ) GAP502/12/1285 Program:GA Institutional support: RVO:68081715 ; RVO:67985904 Keywords : apoptosis * bioluminescence * caspase-3 Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.161, year: 2013

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

    Science.gov (United States)

    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.

  18. Caspase-8 regulates the expression of pro- and anti-inflammatory cytokines in human bone marrow-derived mesenchymal stromal cells.

    Science.gov (United States)

    Moen, Siv H; Westhrin, Marita; Zahoor, Muhammad; Nørgaard, Nikolai N; Hella, Hanne; Størdal, Berit; Sundan, Anders; Nilsen, Nadra J; Sponaas, Anne-Marit; Standal, Therese

    2016-09-01

    Mesenchymal stem cells, also called mesenchymal stromal cells, MSCs, have great potential in stem cell therapy partly due to their immunosuppressive properties. How these cells respond to chronic inflammatory stimuli is therefore of importance. Toll-like receptors (TLR)s are innate immune receptors that mediate inflammatory signals in response to infection, stress, and damage. Caspase-8 is involved in activation of NF-kB downstream of TLRs in immune cells. Here we investigated the role of caspase-8 in regulating TLR-induced cytokine production from human bone marrow-derived mesenchymal stromal cells (hBMSCs). Cytokine expression in hBMCs in response to poly(I:C) and LPS was evaluated by PCR, multiplex cytokine assay, and ELISA. TLR3, TRIF, and caspase-8 were silenced using siRNA. Caspase-8 was also inhibited using a caspase-8 inhibitor, z-IEDT. We found that TLR3 agonist poly(I:C) and TLR4 agonist LPS induced secretion of several pro-inflammatory cytokines in a TLR-dependent manner which required the TLR signaling adaptor molecule TRIF. Further, poly(I:C) reduced the expression of anti-inflammatory cytokines HGF and TGFβ whereas LPS reduced HGF expression only. Notably, caspase-8 was involved in the induction of IL- IL-1β, IL-6, CXCL10, and in the inhibition of HGF and TGFβ. Caspase-8 appears to modulate hBMSCs into gaining a pro-inflammatory phenotype. Therefore, inhibiting caspase-8 in hBMSCs might promote an immunosuppressive phenotype which could be useful in clinical applications to treat inflammatory disorders.

  19. Structural Insights into DD-Fold Assembly and Caspase-9 Activation by the Apaf-1 Apoptosome.

    Science.gov (United States)

    Su, Tsung-Wei; Yang, Chao-Yu; Kao, Wen-Pin; Kuo, Bai-Jiun; Lin, Shan-Meng; Lin, Jung-Yaw; Lo, Yu-Chih; Lin, Su-Chang

    2017-03-07

    Death domain (DD)-fold assemblies play a crucial role in regulating the signaling to cell survival or death. Here we report the crystal structure of the caspase recruitment domain (CARD)-CARD disk of the human apoptosome. The structure surprisingly reveals that three 1:1 Apaf-1:procaspase-9 CARD protomers form a novel helical DD-fold assembly on the heptameric wheel-like platform of the apoptosome. The small-angle X-ray scattering and multi-angle light scattering data also support that three protomers could form an oligomeric complex similar to the crystal structure. Interestingly, the quasi-equivalent environment of CARDs could generate different quaternary CARD assemblies. We also found that the type II interaction is conserved in all DD-fold complexes, whereas the type I interaction is found only in the helical DD-fold assemblies. This study provides crucial insights into the caspase activation mechanism, which is tightly controlled by a sophisticated and highly evolved CARD assembly on the apoptosome, and also enables better understanding of the intricate DD-fold assembly. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    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.

  1. CB1R-Mediated Activation of Caspase-3 Causes Epigenetic and Neurobehavioral Abnormalities in Postnatal Ethanol-Exposed Mice

    Directory of Open Access Journals (Sweden)

    Shivakumar Subbanna

    2018-02-01

    Full Text Available Alcohol exposure can affect brain development, leading to long-lasting behavioral problems, including cognitive impairment, which together is defined as fetal alcohol spectrum disorder (FASD. However, the fundamental mechanisms through which this occurs are largely unknown. In this study, we report that the exposure of postnatal day 7 (P7 mice to ethanol activates caspase-3 via cannabinoid receptor type-1 (CB1R in neonatal mice and causes a reduction in methylated DNA binding protein (MeCP2 levels. The developmental expression of MeCP2 in mice is closely correlated with synaptogenesis and neuronal maturation. It was shown that ethanol treatment of P7 mice enhanced Mecp2 mRNA levels but reduced protein levels. The genetic deletion of CB1R prevented, and administration of a CB1R antagonist before ethanol treatment of P7 mice inhibited caspase-3 activation. Additionally, it reversed the loss of MeCP2 protein, cAMP response element binding protein (CREB activation, and activity-regulated cytoskeleton-associated protein (Arc expression. The inhibition of caspase-3 activity prior to ethanol administration prevented ethanol-induced loss of MeCP2, CREB activation, epigenetic regulation of Arc expression, long-term potentiation (LTP, spatial memory deficits and activity-dependent impairment of several signaling molecules, including MeCP2, in adult mice. Collectively, these results reveal that the ethanol-induced CB1R-mediated activation of caspase-3 degrades the MeCP2 protein in the P7 mouse brain and causes long-lasting neurobehavioral deficits in adult mice. This CB1R-mediated instability of MeCP2 during active synaptic maturation may disrupt synaptic circuit maturation and lead to neurobehavioral abnormalities, as observed in this animal model of FASD.

  2. In vivo imaging of hierarchical spatiotemporal activation of caspase-8 during apoptosis.

    Directory of Open Access Journals (Sweden)

    Katsuya Kominami

    Full Text Available BACKGROUND: Activation of caspases is crucial for the execution of apoptosis. Although the caspase cascade associated with activation of the initiator caspase-8 (CASP8 has been investigated in molecular and biochemical detail, the dynamics of CASP8 activation are not fully understood. METHODOLOGY/PRINCIPAL FINDINGS: We have established a biosensor based on fluorescence resonance energy transfer (FRET for visualizing apoptotic signals associated with CASP8 activation at the single-cell level. Our dual FRET (dual-FRET system, comprising a triple fusion fluorescent protein, enabled us to simultaneously monitor the activation of CASP8 and its downstream effector, caspase-3 (CASP3 in single live cells. With the dual-FRET-based biosensor, we detected distinct activation patterns of CASP8 and CASP3 in response to various apoptotic stimuli in mammalian cells, resulting in the positive feedback amplification of CASP8 activation. We reproduced these observations by in vitro reconstitution of the cascade, with a recombinant protein mixture that included procaspases. Furthermore, using a plasma membrane-bound FRET-based biosensor, we captured the spatiotemporal dynamics of CASP8 activation by the diffusion process, suggesting the focal activation of CASP8 is sufficient to propagate apoptotic signals through death receptors. CONCLUSIONS: Our new FRET-based system visualized the activation process of both initiator and effector caspases in a single apoptotic cell and also elucidated the necessity of an amplification loop for full activation of CASP8.

  3. TRAIL Activates a Caspase 9/7-Dependent Pathway in Caspase 8/10-Defective SK-N-SH Neuroblastoma Cells with Two Functional End Points: Induction of Apoptosis and PGE2 Release

    Directory of Open Access Journals (Sweden)

    Giorgio Zauli

    2003-09-01

    Full Text Available Most neuroblastoma cell lines do not express apical caspases 8 and 10, which play a key role in mediating tumor necrosis factor-related apoptosis-inducing ligand (TRAIL cytotoxicity in a variety of malignant cell types. In this study, we demonstrated that TRAIL induced a moderate but significant increase of apoptosis in the caspase 8/10-deficient SK-N-SH neuroblastoma cell line, through activation of a novel caspase 9/7 pathway. Concomitant to the induction of apoptosis, TRAIL also promoted a significant increase of prostaglandin E2 (PGE2 release by SKN-SH cells. Moreover, coadministration of TRAIL plus indomethacin, a pharmacological inhibitor of cyclooxygenase (COX, showed an additive effect on SKN-SH cell death. In spite of the ability of TRAIL to promote the phosphorylation of both ERKi/2 and p38/MAPK, which have been involved in the control of COX expression/activity, neither PD98059 nor SB203580, pharmacological inhibitors of the ERKi/2 and p38/MAPK pathways, respectively, affected either PGE2 production or apoptosis induced by TRAIL. Finally, both induction of apoptosis and PGE2 release were completely abrogated by the broad caspase inhibitor z-VAD4mk, suggesting that both biologic end points were regulated in SK-N-SH cells through a caspase 9/7-dependent pathway.

  4. IQGAP1 is important for activation of caspase-1 in macrophages and is targeted by Yersinia pestis type III effector YopM.

    Science.gov (United States)

    Chung, Lawton K; Philip, Naomi H; Schmidt, Valentina A; Koller, Antonius; Strowig, Till; Flavell, Richard A; Brodsky, Igor E; Bliska, James B

    2014-07-01

    YopM is a leucine-rich repeat (LRR)-containing effector in several Yersinia species, including Yersinia pestis and Y. pseudotuberculosis. Different Yersinia strains encode distinct YopM isoforms with variable numbers of LRRs but conserved C-terminal tails. A 15-LRR isoform in Y. pseudotuberculosis YPIII was recently shown to bind and inhibit caspase-1 via a YLTD motif in LRR 10, and attenuation of YopM(-) YPIII was reversed in mice lacking caspase-1, indicating that caspase-1 inhibition is a major virulence function of YopM(YPIII). To determine if other YopM proteins inhibit caspase-1, we utilized Y. pseudotuberculosis strains natively expressing a 21-LRR isoform lacking the YLTD motif (YopM(32777)) or ectopically expressing a Y. pestis 15-LRR version with a functional (YopM(KIM)) or inactivated (YopM(KIM) D271A) YLTD motif. Results of mouse and macrophage infections with these strains showed that YopM(32777), YopM(KIM), and YopM(KIM) D271A inhibit caspase-1 activation, indicating that the YLTD motif is dispensable for this activity. Analysis of YopM(KIM) deletion variants revealed that LRRs 6 to 15 and the C-terminal tail are required to inhibit caspase-1 activation. YopM(32777), YopM(KIM), and YopM(KIM) deletion variants were purified, and binding partners in macrophage lysates were identified. Caspase-1 bound to YopM(KIM) but not YopM(32777). Additionally, YopM(KIM) bound IQGAP1 and the use of Iqgap1(-/-) macrophages revealed that this scaffolding protein is important for caspase-1 activation upon infection with YopM(-) Y. pseudotuberculosis. Thus, while multiple YopM isoforms inhibit caspase-1 activation, their variable LRR domains bind different host proteins to perform this function and the LRRs of YopM(KIM) target IQGAP1, a novel regulator of caspase-1, in macrophages. Importance: Activation of caspase-1, mediated by macromolecular complexes termed inflammasomes, is important for innate immune defense against pathogens. Pathogens can, in turn, subvert

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

    Science.gov (United States)

    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.

  6. High LET radiation enhances apoptosis in mutated p53 cancer cells through Caspase-9 activation

    International Nuclear Information System (INIS)

    Yamakawa, Nobuhiro; Takahashi, Akihisa; Mori, Eiichiro; Imai, Yuichiro; Ohnishi, Ken; Kirita, Tadaaki; Ohnishi, Takeo; Furusawa, Yoshiya

    2008-01-01

    Although mutations in the p53 gene can lead to resistance to radiotherapy, chemotherapy and thermotherapy, high linear energy transfer (LET) radiation induces apoptosis regardless of p53 gene status in cancer cells. The aim of this study was to clarify the mechanisms involved in high LET radiation-induced apoptosis. Human gingival cancer cells (Ca9-22 cells) containing a mutated p53 (mp53) gene were irradiated with X-rays, C-ion (13-100 KeV/μm), or Fe-ion beams (200 KeV/μm). Cellular sensitivities were determined using colony forming assays. Apoptosis was detected and quantified with Hoechst 33342 staining. The activity of Caspase-3 was analyzed with Western blotting and flow cytometry. Cells irradiated with high LET radiation showed a high sensitivity with a high frequency of apoptosis induction. The relative biological effectiveness (RBE) values for the surviving fraction and apoptosis induction increased in a LET-dependent manner. Both RBE curves reached a peak at 100 KeV/μm, and then decreased at values over 100 KeV/μm. When cells were irradiated with high LET radiation, Caspase-3 was cleaved and activated, leading to poly (ADP-ribose) polymerase (PARP) cleavage. In addition, Caspase-9 inhibitor suppressed Caspase-3 activation and apoptosis induction resulting from high LET radiation to a greater extent than Caspase-8 inhibitor. These results suggest that high LET radiation enhances apoptosis by activation of Caspase-3 through Caspase-9, even in the presence of mp53. (author)

  7. Glutamate-induced apoptosis in primary cortical neurons is inhibited by equine estrogens via down-regulation of caspase-3 and prevention of mitochondrial cytochrome c release

    Directory of Open Access Journals (Sweden)

    Zhang YueMei

    2005-02-01

    absence of 17β-estradiol or Δ8, 17β-estradiol (10 nM-10 μM resulted in the prevention of cell death and was associated with a significant dose-dependent decrease in caspase-3 protein levels, with Δ8, 17β-E2 being more potent than 17β-E2. Protein levels of Fas receptor remained unchanged in the presence of glutamate. In contrast, treatment with glutamate induced, in a time-dependent manner, the release of cytochrome c into the cytosol. Cytosolic cytochrome c increased as early as 1.5 h after glutamate treatment and these levels were 5 fold higher after 6 h, compared to levels in the untreated cells. Concomitant with these changes, the levels of cytochrome c in mitochondria decreased significantly. Both 17β-E2 and Δ8, 17β-E2 reduced the release of cytochrome c from mitochondria into the cytosol and this decrease in cytosolic cytochrome c was associated with inhibition of glutamate-induced cell death. Conclusion In the primary cortical cells, glutamate-induced apoptosis is accompanied by up-regulation of caspase-3 and its activity is blocked by caspase protease inhibitors. These effects of glutamate on caspase-3 appear to be independent of changes in Fas receptor, but are associated with the rapid release of mitochondrial cytochrome c, which precedes changes in caspase-3 protein levels leading to apoptotic cell death. This process was differentially inhibited by estrogens with the novel equine estrogen Δ8, 17β-E2 being more potent than 17β-E2. To our knowledge, this is the first study to demonstrate that equine estrogens can prevent glutamate-induced translocation of cytochrome c from mitochondria to cytosol in rat primary cortical cells.

  8. The Anti-Apoptotic Activity of BAG3 Is Restricted by Caspases and the Proteasome

    OpenAIRE

    Virador, Victoria M.; Davidson, Ben; Czechowicz, Josephine; Mai, Alisha; Kassis, Jareer; Kohn, Elise C.

    2009-01-01

    Background Caspase-mediated cleavage and proteasomal degradation of ubiquitinated proteins are two independent mechanisms for the regulation of protein stability and cellular function. We previously reported BAG3 overexpression protected ubiquitinated clients, such as AKT, from proteasomal degradation and conferred cytoprotection against heat shock. We hypothesized that the BAG3 protein is regulated by proteolysis. Methodology/Principal Findings Staurosporine (STS) was used as a tool to test ...

  9. The anti-apoptotic activity of BAG3 is restricted by caspases and the proteasome.

    Directory of Open Access Journals (Sweden)

    Victoria M Virador

    Full Text Available Caspase-mediated cleavage and proteasomal degradation of ubiquitinated proteins are two independent mechanisms for the regulation of protein stability and cellular function. We previously reported BAG3 overexpression protected ubiquitinated clients, such as AKT, from proteasomal degradation and conferred cytoprotection against heat shock. We hypothesized that the BAG3 protein is regulated by proteolysis.Staurosporine (STS was used as a tool to test for caspase involvement in BAG3 degradation. MDA435 and HeLa human cancer cell lines exposed to STS underwent apoptosis with a concomitant time and dose-dependent loss of BAG3, suggesting the survival role of BAG3 was subject to STS regulation. zVAD-fmk or caspase 3 and 9 inhibitors provided a strong but incomplete protection of both cells and BAG3 protein. Two putative caspase cleavage sites were tested: KEVD (BAG3(E345A/D347A within the proline-rich center of BAG3 (PXXP and the C-terminal LEAD site (BAG3(E516A/D518A. PXXP deletion mutant and BAG3(E345A/D347A, or BAG3(E516A/D518A respectively slowed or stalled STS-mediated BAG3 loss. BAG3, ubiquitinated under basal growth conditions, underwent augmented ubiquitination upon STS treatment, while there was no increase in ubiquitination of the BAG3(E516A/D518A caspase-resistant mutant. Caspase and proteasome inhibition resulted in partial and independent protection of BAG3 whereas inhibitors of both blocked BAG3 degradation. STS-induced apoptosis was increased when BAG3 was silenced, and retention of BAG3 was associated with cytoprotection.BAG3 is tightly controlled by selective degradation during STS exposure. Loss of BAG3 under STS injury required sequential caspase cleavage followed by polyubiquitination and proteasomal degradation. The need for dual regulation of BAG3 in apoptosis suggests a key role for BAG3 in cancer cell resistance to apoptosis.

  10. Caspase-dependant activation of chymotrypsin-like proteases mediates nuclear events during Jurkat T cell apoptosis

    International Nuclear Information System (INIS)

    O'Connell, A.R.; Lee, B.W.; Stenson-Cox, C.

    2006-01-01

    Apoptosis involves a cascade of biochemical and morphological changes resulting in the systematic disintegration of the cell. Caspases are central mediators of this process. Supporting and primary roles for serine proteases as pro-apoptotic mediators have also been highlighted. Evidence for such roles comes largely from the use of pharmacological inhibitors; as a consequence information regarding their apoptotic function and biochemical properties has been limited. Here, we circumvented limitations associated with traditional serine protease inhibitors through use of a fluorescently labelled inhibitor of serine proteases (FLISP) that allowed for analysis of the specificity, regulation and positioning of apoptotic serine proteases within a classical apoptotic cascade. We demonstrate that staurosporine triggers a caspase-dependant induction of chymotrypsin-like activity in the nucleus of apoptotic Jurkat T cells. We show that serine protease activity is required for the generation of late stage nuclear events including condensation, fragmentation and DNA degradation. Furthermore, we reveal caspase-dependant activation of two chymotrypsin-like protein species that we hypothesize mediate cell death-associated nuclear events

  11. Caffeine inhibits erythrocyte membrane derangement by antioxidant activity and by blocking caspase 3 activation.

    Science.gov (United States)

    Tellone, Ester; Ficarra, Silvana; Russo, Annamaria; Bellocco, Ersilia; Barreca, Davide; Laganà, Giuseppina; Leuzzi, Ugo; Pirolli, Davide; De Rosa, Maria Cristina; Giardina, Bruno; Galtieri, Antonio

    2012-02-01

    The aim of this research was to investigate the effect of caffeine on band 3 (the anion exchanger protein), haemoglobin function, caspase 3 activation and glucose-6-phosphate metabolism during the oxygenation-deoxygenation cycle in human red blood cells. A particular attention has been given to the antioxidant activity by using in vitro antioxidant models. Caffeine crosses the erythrocyte membrane and interacts with the two extreme conformational states of haemoglobin (the T and the R-state within the framework of the simple two states allosteric model) with different binding affinities. By promoting the high affinity state (R-state), the caffeine-haemoglobin interaction does enhance the pentose phosphate pathway. This is of benefit for red blood cells since it leads to an increase of NADPH availability. Moreover, caffeine effect on band 3, mediated by haemoglobin, results in an extreme increase of the anion exchange, particularly in oxygenated erythrocytes. This enhances the transport of the endogenously produced CO(2) thereby avoiding the production of dangerous secondary radicals (carbonate and nitrogen dioxide) which are harmful to the cellular membrane. Furthermore caffeine destabilizes the haeme-protein interactions within the haemoglobin molecule and triggers the production of superoxide and met-haemoglobin. However this damaging effect is almost balanced by the surprising scavenger action of the alkaloid with respect to the hydroxyl radical. These experimental findings are supported by in silico docking and molecular dynamics studies and by what we may call the "caspase silence"; in fact, there is no evidence of any caspase 3 activity enhancement; this is likely due to the promotion of positive metabolic conditions which result in an increase of the cellular reducing power. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

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

    Science.gov (United States)

    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.

  13. Cisplatin-induced caspase activation mediates PTEN cleavage in ovarian cancer cells: a potential mechanism of chemoresistance

    International Nuclear Information System (INIS)

    Singh, Mohan; Chaudhry, Parvesh; Fabi, Francois; Asselin, Eric

    2013-01-01

    The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor protein is a central negative regulator of the PI3K/AKT signaling cascade and suppresses cell survival as well as cell proliferation. PTEN is found to be either inactivated or mutated in various human malignancies. In the present study, we have investigated the regulation of PTEN during cisplatin induced apoptosis in A2780, A270-CP (cisplatin resistant), OVCAR-3 and SKOV3 ovarian cancer cell lines. Cells were treated with 10μM of cisplatin for 24h. Transcript and protein levels were analysed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blotting, respectively. Immunofluorescence microscopy was used to assess the intracellular localization of PTEN. Proteasome inhibitor and various caspases inhibitors were used to find the mechanism of PTEN degradation. PTEN protein levels were found to be decreased significantly in A2780 cells; however, there was no change in PTEN protein levels in A2780-CP, OVCAR-3 and SKOV3 cells with cisplatin treatment. The decrease in PTEN protein was accompanied with an increase in the levels of AKT phosphorylation (pAKT) in A2780 cells and a decrease of BCL-2. Cisplatin treatment induced the activation/cleavage of caspase-3, -6, -7, -8, -9 in all cell lines tested in this study except the resistant variant A2780-CP cells. In A2780 cells, restoration of PTEN levels was achieved upon pre-treatment with Z-DEVD-FMK (broad range caspases inhibitor) and not with MG132 (proteasome inhibitor) and by overexpression of BCL-2, suggesting that caspases and BCL-2 are involved in the decrease of PTEN protein levels in A2780 cells. The decrease in pro-apoptotic PTEN protein levels and increase in survival factor pAKT in A2780 ovarian cancer cells suggest that cisplatin treatment could further exacerbate drug resistance in A2780 ovarian cancer cells

  14. Cisplatin-induced caspase activation mediates PTEN cleavage in ovarian cancer cells: a potential mechanism of chemoresistance.

    Science.gov (United States)

    Singh, Mohan; Chaudhry, Parvesh; Fabi, Francois; Asselin, Eric

    2013-05-10

    The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor protein is a central negative regulator of the PI3K/AKT signaling cascade and suppresses cell survival as well as cell proliferation. PTEN is found to be either inactivated or mutated in various human malignancies. In the present study, we have investigated the regulation of PTEN during cisplatin induced apoptosis in A2780, A270-CP (cisplatin resistant), OVCAR-3 and SKOV3 ovarian cancer cell lines. Cells were treated with 10μM of cisplatin for 24h. Transcript and protein levels were analysed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blotting, respectively. Immunofluorescence microscopy was used to assess the intracellular localization of PTEN. Proteasome inhibitor and various caspases inhibitors were used to find the mechanism of PTEN degradation. PTEN protein levels were found to be decreased significantly in A2780 cells; however, there was no change in PTEN protein levels in A2780-CP, OVCAR-3 and SKOV3 cells with cisplatin treatment. The decrease in PTEN protein was accompanied with an increase in the levels of AKT phosphorylation (pAKT) in A2780 cells and a decrease of BCL-2. Cisplatin treatment induced the activation/cleavage of caspase-3, -6, -7, -8, -9 in all cell lines tested in this study except the resistant variant A2780-CP cells. In A2780 cells, restoration of PTEN levels was achieved upon pre-treatment with Z-DEVD-FMK (broad range caspases inhibitor) and not with MG132 (proteasome inhibitor) and by overexpression of BCL-2, suggesting that caspases and BCL-2 are involved in the decrease of PTEN protein levels in A2780 cells. The decrease in pro-apoptotic PTEN protein levels and increase in survival factor pAKT in A2780 ovarian cancer cells suggest that cisplatin treatment could further exacerbate drug resistance in A2780 ovarian cancer cells.

  15. A Yersinia effector with enhanced inhibitory activity on the NF-κB pathway activates the NLRP3/ASC/caspase-1 inflammasome in macrophages.

    Directory of Open Access Journals (Sweden)

    Ying Zheng

    2011-04-01

    Full Text Available A type III secretion system (T3SS in pathogenic Yersinia species functions to translocate Yop effectors, which modulate cytokine production and regulate cell death in macrophages. Distinct pathways of T3SS-dependent cell death and caspase-1 activation occur in Yersinia-infected macrophages. One pathway of cell death and caspase-1 activation in macrophages requires the effector YopJ. YopJ is an acetyltransferase that inactivates MAPK kinases and IKKβ to cause TLR4-dependent apoptosis in naïve macrophages. A YopJ isoform in Y. pestis KIM (YopJ(KIM has two amino acid substitutions, F177L and K206E, not present in YopJ proteins of Y. pseudotuberculosis and Y. pestis CO92. As compared to other YopJ isoforms, YopJ(KIM causes increased apoptosis, caspase-1 activation, and secretion of IL-1β in Yersinia-infected macrophages. The molecular basis for increased apoptosis and activation of caspase-1 by YopJ(KIM in Yersinia-infected macrophages was studied. Site directed mutagenesis showed that the F177L and K206E substitutions in YopJ(KIM were important for enhanced apoptosis, caspase-1 activation, and IL-1β secretion. As compared to YopJ(CO92, YopJ(KIM displayed an enhanced capacity to inhibit phosphorylation of IκB-α in macrophages and to bind IKKβ in vitro. YopJ(KIM also showed a moderately increased ability to inhibit phosphorylation of MAPKs. Increased caspase-1 cleavage and IL-1β secretion occurred in IKKβ-deficient macrophages infected with Y. pestis expressing YopJ(CO92, confirming that the NF-κB pathway can negatively regulate inflammasome activation. K+ efflux, NLRP3 and ASC were important for secretion of IL-1β in response to Y. pestis KIM infection as shown using macrophages lacking inflammasome components or by the addition of exogenous KCl. These data show that caspase-1 is activated in naïve macrophages in response to infection with a pathogen that inhibits IKKβ and MAPK kinases and induces TLR4-dependent apoptosis. This pro

  16. Propofol and magnesium attenuate isoflurane-induced caspase-3 activation via inhibiting mitochondrial permeability transition pore

    Directory of Open Access Journals (Sweden)

    Zhang Yiying

    2012-08-01

    Full Text Available Abstract Background The inhalation anesthetic isoflurane has been shown to open the mitochondrial permeability transition pore (mPTP and induce caspase activation and apoptosis, which may lead to learning and memory impairment. Cyclosporine A, a blocker of mPTP opening might attenuate the isoflurane-induced mPTP opening, lessening its ripple effects. Magnesium and anesthetic propofol are also mPTP blockers. We therefore set out to determine whether propofol and magnesium can attenuate the isoflurane-induced caspase activation and mPTP opening. Methods We investigated the effects of magnesium sulfate (Mg2+, propofol, and isoflurane on the opening of mPTP and caspase activation in H4 human neuroglioma cells stably transfected to express full-length human amyloid precursor protein (APP (H4 APP cells and in six day-old wild-type mice, employing Western blot analysis and flowcytometry. Results Here we show that Mg2+ and propofol attenuated the isoflurane-induced caspase-3 activation in H4-APP cells and mouse brain tissue. Moreover, Mg2+ and propofol, the blockers of mPTP opening, mitigated the isoflurane-induced mPTP opening in the H4-APP cells. Conclusion These data illustrate that Mg2+ and propofol may ameliorate the isoflurane-induced neurotoxicity by inhibiting its mitochondrial dysfunction. Pending further studies, these findings may suggest the use of Mg2+ and propofol in preventing and treating anesthesia neurotoxicity.

  17. Morphometric alterations, steatosis, fibrosis and active caspase-3 detection in carbamate bendiocarb treated rabbit liver

    Czech Academy of Sciences Publication Activity Database

    Petrovová, E.; Purzyc, H.; Mazenský, D.; Luptáková, L.; Torma, N.; Sopoliga, I.; Sedmera, David

    2015-01-01

    Roč. 30, č. 2 (2015), s. 212-222 ISSN 1520-4081 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : bendiocarb * caspase-3 activity * fibrosis * toxicity * rabbit * liver Subject RIV: EA - Cell Biology Impact factor: 2.868, year: 2015

  18. An Allergic Lung Microenvironment Suppresses Carbon Nanotube-Induced Inflammasome Activation via STAT6-Dependent Inhibition of Caspase-1.

    Directory of Open Access Journals (Sweden)

    Kelly A Shipkowski

    pro-fibrogenic cytokine mRNAs.These data indicate that Th2 cytokines suppress MWCNT-induced inflammasome activation via STAT6-dependent down-regulation of pro-caspase-1 and suggest that suppression of inflammasome activation and IL-1β by an allergic lung microenvironment is a mechanism through which MWCNTs exacerbate allergen-induced airway fibrosis.

  19. Ginsenoside Rh2 Induces Human Hepatoma Cell Apoptosisvia Bax/Bak Triggered Cytochrome C Release and Caspase-9/Caspase-8 Activation

    Directory of Open Access Journals (Sweden)

    Xiao-Xi Guo

    2012-11-01

    Full Text Available Ginsenoside Rh2 (G-Rh2 has been shown to induce apoptotic cell death in a variety of cancer cells. However, the details of the signal transduction cascade involved in G-Rh2-induced cell death is unclear. In this manuscript we elucidate the molecular mechanism of G-Rh2-induced apoptosis in human hepatoma SK-HEP-1 cells by demonstrating that G-Rh2 causes rapid and dramatic translocation of both Bak and Bax, which subsequently triggers mitochondrial cytochrome c release and consequent caspase activation. Interestingly, siRNA-based gene inactivation of caspase-8 effectively delays caspase-9 activation and apoptosis induced by G-Rh2, indicating that caspase-8 also plays an important role in the G-Rh2-induced apoptosis program. Taken together, our results indicate that G-Rh2 employs a multi pro-apoptotic pathway to execute cancer cell death, suggesting a potential role for G-Rh2 as a powerful chemotherapeutic agent.

  20. Anesthetic propofol attenuates the isoflurane-induced caspase-3 activation and Aβ oligomerization.

    Directory of Open Access Journals (Sweden)

    Yiying Zhang

    Full Text Available Accumulation and deposition of β-amyloid protein (Aβ are the hallmark features of Alzheimer's disease. The inhalation anesthetic isoflurane has been shown to induce caspase activation and increase Aβ accumulation. In addition, recent studies suggest that isoflurane may directly promote the formation of cytotoxic soluble Aβ oligomers, which are thought to be the key pathological species in AD. In contrast, propofol, the most commonly used intravenous anesthetic, has been reported to have neuroprotective effects. We therefore set out to compare the effects of isoflurane and propofol alone and in combination on caspase-3 activation and Aβ oligomerization in vitro and in vivo. Naïve and stably-transfected H4 human neuroglioma cells that express human amyloid precursor protein, the precursor for Aβ; neonatal mice; and conditioned cell culture media containing secreted human Aβ40 or Aβ42 were treated with isoflurane and/or propofol. Here we show for the first time that propofol can attenuate isoflurane-induced caspase-3 activation in cultured cells and in the brain tissues of neonatal mice. Furthermore, propofol-mediated caspase inhibition occurred when there were elevated levels of Aβ. Finally, isoflurane alone induces Aβ42, but not Aβ40, oligomerization, and propofol can inhibit the isoflurane-mediated oligomerization of Aβ42. These data suggest that propofol may mitigate the caspase-3 activation by attenuating the isoflurane-induced Aβ42 oligomerization. Our findings provide novel insights into the possible mechanisms of isoflurane-induced neurotoxicity that may aid in the development of strategies to minimize potential adverse effects associated with the administration of anesthetics to patients.

  1. Real-time monitoring of caspase cascade activation in living cells.

    Science.gov (United States)

    Zhu, Lei; Huang, Xinglu; Choi, Ki Young; Ma, Ying; Zhang, Fan; Liu, Gang; Lee, Seulki; Chen, Xiaoyuan

    2012-10-10

    We introduce a simple, versatile and robust one-step technique that enables real-time imaging of multiple intracellular caspase activities in living cells without the need for complicated synthetic protocols. Conventional fluorogenic probes or recently reported activatable probes have been designed to target various proteases but are limited to extracellular molecules. Only a few have been applied to image intracellular proteases in living cells because most of these probes have limited cell-permeability. Our platform does not need complicated synthetic processes; instead it involves a straightforward peptide synthesis and a simple mixing step with a commercial transfection agent. The transfection agent efficiently delivered the highly quenched fluorogenic probes, comprised of distinctive pairs of dyes and quenchers, to the initiator caspase-8 and the effector caspase-3 in MDA-MB-435 cells, allowing dual-imaging of the activities of both caspases during the apoptotic process induced by TNF-related apoptosis induced ligand (TRAIL). With the combination of multiple fluorogenic probes, this simple platform can be applied to multiplexed imaging of selected intracellular proteases to study apoptotic processes in pathologies or for cell-based high throughput screening systems for drug discovery. Published by Elsevier B.V.

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

    Science.gov (United States)

    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.

  3. Involvement of major components from Sporothrix schenckii cell wall in the caspase-1 activation, nitric oxide and cytokines production during experimental sporotrichosis.

    Science.gov (United States)

    Gonçalves, Amanda Costa; Maia, Danielle Cardoso Geraldo; Ferreira, Lucas Souza; Monnazzi, Luis Gustavo Silva; Alegranci, Pâmela; Placeres, Marisa Campos Polesi; Batista-Duharte, Alexander; Carlos, Iracilda Zeppone

    2015-02-01

    Sporotrichosis is a chronic infection caused by the dimorphic fungus Sporothrix schenckii, involving all layers of skin and the subcutaneous tissue. The role of innate immune toll-like receptors 2 and 4 in the defense against this fungus has been reported, but so far, there were no studies on the effect of cell wall major components over the cytosolic oligo-merization domain (NOD)-like receptors, important regulators of inflammation and responsible for the maturation of IL-1β and IL-18, whose functions are dependents of the caspase-1 activation, that can participate of inflammasome. It was evaluated the percentage of activation of caspase-1, the production of IL-1β, IL-18, IL-17, IFN-γ and nitric oxide in a Balb/c model of S. schenckii infection. It was observed a decreased activity of caspase-1 during the fourth and sixth weeks of infection accompanied by reduced secretion of the cytokines IL-1β, IL-18 and IL-17 and high production of nitric oxide. IFN-γ levels were elevated during the entire time course of infection. This temporal reduction in caspase-1 activity coincides exactly with the reported period of fungal burden associated with a transitory immunosuppression induced by this fungus and detected in similar infection models. These results indicate the importance of interaction between caspase-1, cytokines IL-1β and IL-18 in the host defense against S. schenckii infection, suggesting a participation the inflammasome in this response.

  4. Synergistic Effect of Subtoxic-dose Cisplatin and TRAIL to Mediate Apoptosis by Down-regulating Decoy Receptor 2 and Up-regulating Caspase-8, Caspase-9 and Bax Expression on NCI-H460 and A549 Cells

    Directory of Open Access Journals (Sweden)

    Xiaoyan Zhang

    2013-05-01

    Full Text Available Objective(s: Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL can selectively induce apoptosis in tumor cells, more than half of tumors including non-small cell lung cancer (NSCLC exhibit TRAIL-resistance. The purpose of this study was to determine whether subtoxic-dose cisplatin and TRAIL could synergistically enhance apoptosis on NSCLC cells and investigate its underlying mechanisms. Materials and Methods:NCI-H460 and A549 cells were treated with TRAIL alone, cisplatin alone or combination treatment in this study. The cytotoxicity was evaluated according to Sulforhodamine B assay, and apoptosis was examined using Hoechst 33342 staining and flow cytometry. The mRNA and protein levels of TRAIL receptors and apoptotic proteins including caspase-8, caspase-9, Bcl-2 and Bax were determined by RT-PCR and Western blotting, respectively. Results:Our results showed that NCI-H460 cells were sensitive to TRAIL, whereas A549 cells were resistant. However, subtoxic-dose cisplatin could enhance the both cells to TRAIL-mediated cell proliferation inhibition and apoptosis. The underlying mechanisms might be associated with the down-regulation of DcR2 and up-regulation of Caspase-8, Caspase-9 and Bax. Conclusion:Subtoxic-dose cisplatin could enhance both TRAIL- sensitive and TRAIL- resistant NSCLC cells to TRAIL-mediated apoptosis. These findings motivated further studies to evaluate such a combinatory therapeutic strategy against NSCLC in the animal models.

  5. Simvastatin induces caspase-independent apoptosis in LPS-activated RAW264.7 macrophage cells

    International Nuclear Information System (INIS)

    Kim, Yong Chan; Song, Seok Bean; Lee, Mi Hee; Kang, Kwang Il; Lee, Hayyoung; Paik, Sang-Gi; Kim, Kyoon Eon; Kim, Young Sang

    2006-01-01

    Macrophages participate in several inflammatory pathologies such as sepsis and arthritis. We examined the effect of simvastatin on the LPS-induced proinflammatory macrophage RAW264.7 cells. Co-treatment of LPS and a non-toxic dose of simvastatin induced cell death in RAW264.7 cells. The cell death was accompanied by disruption of mitochondrial membrane potential (MMP), genomic DNA fragmentation, and caspase-3 activation. Surprisingly, despite caspase-dependent apoptotic cascade being completely blocked by Z-VAD-fmk, a pan-caspase inhibitor, the cell death was only partially repressed. In the presence of Z-VAD-fmk, DNA fragmentation was blocked, but DNA condensation, disruption of MMP, and nuclear translocation of apoptosis inducing factor were obvious. The cell death by simvastatin and LPS was effectively decreased by both the FPP and GGPP treatments as well as mevalonate. Our findings indicate that simvastatin triggers the cell death of LPS-treated RAW264.7 cells through both caspase-dependent and -independent apoptotic pathways, suggesting a novel mechanism of statins for the severe inflammatory disease therapy

  6. Benzene activates caspase-4 and -12 at the transcription level, without an association with apoptosis, in mouse bone marrow cells lacking the p53 gene

    Energy Technology Data Exchange (ETDEWEB)

    Yi, Jung-Yeon; Han, Jeong-Hee; Yoon, Byung-Il [Kangwon National University, School of Veterinary Medicine, Chuncheon, Gangwon (Korea); Hirabayashi, Yoko; Kodama, Yukio; Kanno, Jun [National Institute of Health Sciences, Division of Cellular and Molecular Toxicology, Center for Biological Safety and Research, Tokyo (Japan); Choi, Yang-Kyu [Konkuk University, College of Veterinary Medicine, Seoul (Korea); Inoue, Tohru [National Institute of Health Sciences, Biological Safety and Research Center, Tokyo (Japan)

    2009-08-15

    level of mRNA and proteins. These results indicated that upregulation of caspase-4 and -12 in mice lacking the p53 gene is not associated with cellular apoptosis. In conclusion, caspase-4 and -12 can be activated by benzene treatment without inducing cell apoptosis in mouse bone marrow, which are partly under the regulation of the p53 gene. (orig.)

  7. Fludarabine inhibits STAT1-mediated up-regulation of caspase-3 expression in dexamethasone-induced osteoblasts apoptosis and slows the progression of steroid-induced avascular necrosis of the femoral head in rats.

    Science.gov (United States)

    Feng, Zhenhua; Zheng, Wenhao; Tang, Qian; Cheng, Liang; Li, Hang; Ni, Wenfei; Pan, Xiaoyun

    2017-08-01

    Steroid-induced avascular necrosis of the femoral head (SANFH) is a major limitation of long-term or excessive clinical administration of glucocorticoids. Fludarabine, which is a compound used to treat various hematological malignancies, such as chronic lymphocytic leukemia, acts by down-regulating signal transducer and activator of transcription 1 (STAT1) by inhibiting STAT1 phosphorylation in both normal and cancer cells. This study assessed the effects of fludarabine in vitro (primary murine osteoblasts) and in vivo (rat SANFH model). In vitro, pretreatment with fludarabine significantly inhibited Dexamethasone (Dex)-induced apoptosis in osteoblasts, which was examined by TUNEL staining. Treatment with Dex caused a remarkable decrease in the expression of Bcl-2; an increase in cytochrome c release; activation of BAX, caspase-9, and caspase-3; and an obvious enhancement in STAT1 phosphorylation. However, treatment resulted in the up-regulation of caspase-3 expression. Enhanced P-STAT1 activity and up-regulation of caspase-3 expression were also observed in osteoblasts. In vivo, the subchondral trabeculae in fludarabine-treated rats exhibited less bone loss and a lower ratio of empty lacunae. Taken together, our results suggest that STAT1-mediated up-regulation of caspase-3 is involved in osteoblast apoptosis induced by Dex and indicates that fludarabine may serve as a potential agent for the treatment of SANFH.

  8. Hypothesis for thermal activation of the caspase cascade in apoptotic cell death at elevated temperatures

    Science.gov (United States)

    Pearce, John A.

    2013-02-01

    Apoptosis is an especially important process affecting disease states from HIV-AIDS to auto-immune disease to cancer. A cascade of initiator and executioner capsase functional proteins is the hallmark of apoptosis. When activated the various caspases activate other caspases or cleave structural proteins of the cytoskeleton, resulting in "blebbing" of the plasma membrane forming apoptotic bodies that completely enclose the disassembled cellular components. Containment of the cytosolic components within the apoptotic bodies differentiates apoptosis from necroptosis and necrosis, both of which release fragmented cytosol and other cellular constituents into the intracellular space. Biochemical models of caspase activation reveal the extensive feedback loops characteristic of apoptosis. They clearly explain the failure of Arrhenius models to give accurate predictions of cell survival curves in hyperthermic heating protocols. Nevertheless, each of the individual reaction velocities can reasonably be assumed to follow Arrhenius kinetics. If so, the thermal sensitivity of the reaction velocity to temperature elevation is: ∂k/∂T = Ea [k/RT2]. Particular reaction steps described by higher activation energies, Ea, are likely more thermally-sensitive than lower energy reactions and may initiate apoptosis in the absence of other stress signals. Additionally, while the classical irreversible Arrhenius formulation fails to accurately represent many cell survival and/or dye uptake curves - those that display an early stage shoulder region - an expanded reversible model of the law of mass action equation seems to prove effective and is directly based on a firm theoretical thermodynamic foundation.

  9. Phorate-induced oxidative stress, DNA damage and transcriptional activation of p53 and caspase genes in male Wistar rats

    International Nuclear Information System (INIS)

    Saquib, Quaiser; Attia, Sabry M.; Siddiqui, Maqsood A.; Aboul-Soud, Mourad A.M.; Al-Khedhairy, Abdulaziz A.; Giesy, John P.; Musarrat, Javed

    2012-01-01

    Male Wistar rats exposed to a systemic organophosphorus insecticide, phorate [O,O-diethyl S-[(ethylthio) methyl] phosphorothioate] at varying oral doses of 0.046, 0.092 or 0.184 mg phorate/kg bw for 14 days, exhibited substantial oxidative stress, cellular DNA damage and activation of apoptosis-related p53, caspase 3 and 9 genes. The histopathological changes including the pyknotic nuclei, inflammatory leukocyte infiltrations, renal necrosis, and cardiac myofiber degeneration were observed in the liver, kidney and heart tissues. Biochemical analysis of catalase and glutathione revealed significantly lesser activities of antioxidative enzymes and lipid peroxidation in tissues of phorate exposed rats. Furthermore, generation of intracellular reactive oxygen species and reduced mitochondrial membrane potential in bone marrow cells confirmed phorate-induced oxidative stress. Significant DNA damage was measured through comet assay in terms of the Olive tail moment in bone marrow cells of treated animals as compared to control. Cell cycle analysis also demonstrated the G 2 /M arrest and appearance of a distinctive SubG 1 peak, which signified induction of apoptosis. Up-regulation of tumor suppressor p53 and caspase 3 and 9 genes, determined by quantitative real-time PCR and enzyme-linked immunosorbent assay, elucidated the activation of intrinsic apoptotic pathways in response to cellular stress. Overall, the results suggest that phorate induces genetic alterations and cellular toxicity, which can adversely affect the normal cellular functioning in rats. -- Highlights: ► This is the first report on molecular toxicity of phorate in an in vivo test system. ► Phorate induces biochemical and histological changes in liver, kidney and heart. ► Rats treated with phorate exhibited DNA damage in bone marrow cells. ► Phorate induces apoptosis, oxidative stress and alters mitochondrial fluorescence. ► Phorate induces transcriptional changes and enhanced activities of

  10. Phorate-induced oxidative stress, DNA damage and transcriptional activation of p53 and caspase genes in male Wistar rats

    Energy Technology Data Exchange (ETDEWEB)

    Saquib, Quaiser [Department of Zoology, College of Science, King Saud University, Riyadh (Saudi Arabia); Attia, Sabry M. [Department of Pharmacology, College of Pharmacy, King Saud University, Riyadh (Saudi Arabia); Siddiqui, Maqsood A. [Department of Zoology, College of Science, King Saud University, Riyadh (Saudi Arabia); Aboul-Soud, Mourad A.M. [Department of Zoology, College of Science, King Saud University, Riyadh (Saudi Arabia); Biochemistry Department, Faculty of Agriculture, Cairo University, 12613 Giza (Egypt); Al-Khedhairy, Abdulaziz A. [Department of Zoology, College of Science, King Saud University, Riyadh (Saudi Arabia); Giesy, John P. [Department of Zoology, College of Science, King Saud University, Riyadh (Saudi Arabia); Department of Biomedical and Veterinary Biosciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Canada S7N 5B3 (Canada); Zoology Department and Center for Integrative Toxicology, Michigan State University, East Lansing 48824 (United States); Musarrat, Javed, E-mail: musarratj1@yahoo.com [Department of Zoology, College of Science, King Saud University, Riyadh (Saudi Arabia); Department of Microbiology, Faculty of Agricultural Sciences, AMU, Aligarh (India)

    2012-02-15

    Male Wistar rats exposed to a systemic organophosphorus insecticide, phorate [O,O-diethyl S-[(ethylthio) methyl] phosphorothioate] at varying oral doses of 0.046, 0.092 or 0.184 mg phorate/kg bw for 14 days, exhibited substantial oxidative stress, cellular DNA damage and activation of apoptosis-related p53, caspase 3 and 9 genes. The histopathological changes including the pyknotic nuclei, inflammatory leukocyte infiltrations, renal necrosis, and cardiac myofiber degeneration were observed in the liver, kidney and heart tissues. Biochemical analysis of catalase and glutathione revealed significantly lesser activities of antioxidative enzymes and lipid peroxidation in tissues of phorate exposed rats. Furthermore, generation of intracellular reactive oxygen species and reduced mitochondrial membrane potential in bone marrow cells confirmed phorate-induced oxidative stress. Significant DNA damage was measured through comet assay in terms of the Olive tail moment in bone marrow cells of treated animals as compared to control. Cell cycle analysis also demonstrated the G{sub 2}/M arrest and appearance of a distinctive SubG{sub 1} peak, which signified induction of apoptosis. Up-regulation of tumor suppressor p53 and caspase 3 and 9 genes, determined by quantitative real-time PCR and enzyme-linked immunosorbent assay, elucidated the activation of intrinsic apoptotic pathways in response to cellular stress. Overall, the results suggest that phorate induces genetic alterations and cellular toxicity, which can adversely affect the normal cellular functioning in rats. -- Highlights: ► This is the first report on molecular toxicity of phorate in an in vivo test system. ► Phorate induces biochemical and histological changes in liver, kidney and heart. ► Rats treated with phorate exhibited DNA damage in bone marrow cells. ► Phorate induces apoptosis, oxidative stress and alters mitochondrial fluorescence. ► Phorate induces transcriptional changes and enhanced

  11. Pituitary adenylate cyclase activating polypeptide reduces A-type K+ currents and caspase activity in cultured adult mouse olfactory neurons.

    Science.gov (United States)

    Han, P; Lucero, M T

    2005-01-01

    Pituitary adenylate cyclase activating polypeptide has been shown to reduce apoptosis in neonatal cerebellar and olfactory receptor neurons, however the underlying mechanisms have not been elucidated. In addition, the neuroprotective effects of pituitary adenylate cyclase activating polypeptide have not been examined in adult tissues. To study the effects of pituitary adenylate cyclase activating polypeptide on neurons in apoptosis, we measured caspase activation in adult olfactory receptor neurons in vitro. Interestingly, we found that the protective effects of pituitary adenylate cyclase activating polypeptide were related to the absence of a 4-aminopyridine (IC50=144 microM) sensitive rapidly inactivating potassium current often referred to as A-type current. In the presence of 40 nM pituitary adenylate cyclase activating polypeptide 38, both A-type current and activated caspases were significantly reduced. A-type current reduction by pituitary adenylate cyclase activating polypeptide was blocked by inhibiting the phospholipase C pathway, but not the adenylyl cyclase pathway. Our observation that 5 mM 4-aminopyridine mimicked the caspase inhibiting effects of pituitary adenylate cyclase activating polypeptide indicates that A-type current is involved in apoptosis. This work contributes to our growing understanding that potassium currents are involved with the activation of caspases to affect the balance between cell life and death.

  12. Pinus densiflora leaf essential oil induces apoptosis via ROS generation and activation of caspases in YD-8 human oral cancer cells.

    Science.gov (United States)

    Jo, Jeong-Rang; Park, Ju Sung; Park, Yu-Kyoung; Chae, Young Zoo; Lee, Gyu-Hee; Park, Gy-Young; Jang, Byeong-Churl

    2012-04-01

    The leaf of Pinus (P.) densiflora, a pine tree widely distributed in Asian countries, has been used as a traditional medicine. In the present study, we investigated the anticancer activity of essential oil, extracted by steam distillation, from the leaf of P. densiflora in YD-8 human oral squamous cell carcinoma (OSCC) cells. Treatment of YD-8 cells with P. densiflora leaf essential oil (PLEO) at 60 µg/ml for 8 h strongly inhibited proliferation and survival and induced apoptosis. Notably, treatment with PLEO led to generation of ROS, activation of caspase-9, PARP cleavage, down-regulation of Bcl-2, and phosphorylation of ERK-1/2 and JNK-1/2 in YD-8 cells. Treatment with PLEO, however, did not affect the expression of Bax, XIAP and GRP78. Importantly, pharmaco-logical inhibition studies demonstrated that treatment with vitamin E (an anti-oxidant) or z-VAD-fmk (a pan-caspase inhibitor), but not with PD98059 (an ERK-1/2 inhibitor) or SP600125 (a JNK-1/2 inhibitor), strongly suppressed PLEO-induced apoptosis in YD-8 cells and reduction of their survival. Vitamin E treatment further blocked activation of caspase-9 and Bcl-2 down-regulation induced by PLEO. Thus, these results demonstrate firstly that PLEO has anti-proliferative, anti-survival and pro-apoptotic effects on YD-8 cells and the effects are largely due to the ROS-dependent activation of caspases.

  13. Differential regulation of caspase-9 by ionizing radiation- and UV-induced apoptotic pathways in thymic cells

    Energy Technology Data Exchange (ETDEWEB)

    Okamoto, Mayumi; Koga, Satomi [Department of Life Sciences, Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Hiroshima 727-0023 (Japan); Tatsuka, Masaaki, E-mail: tatsuka@pu-hiroshima.ac.jp [Department of Life Sciences, Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Hiroshima 727-0023 (Japan)

    2010-06-01

    In mouse thymic lymphoma 3SB cells bearing wild type p53, ionizing radiation (IR) and UV light are potent triggers of caspase-3-dependent apoptosis. Although cytochrome c was released from mitochondria as expected, caspase-9 activation was not observed in UV-exposed cells. Laser scanning confocal microscopy analysis showed that caspase-9 is localized in an unusual punctuated pattern in UV-induced apoptotic cells. In agreement with differences in the status of caspase-9 activation between IR and UV, subcellular protein fractionation experiments showed that pro-apoptotic apoptosis protease-activating factor 1 (Apaf-1), normally a part of the apoptosome assembled in response to the release of cytochrome c from mitochondria, and B-cell lymphoma extra long (Bcl-xL), an inhibitor of the change in mitochondrial membrane permeability, were redistributed by the IR-exposure but not by the UV-exposure. Instead of the sequestration of the capase-9/apoptosome activation in UV-induced apoptotic cells, the extrinsic apoptotic signaling generated by caspase-8 activation and consequent activation of B-cell lymphoma extra long (Bid) to release cytochrome c from mitochondria was observed. Thus, the post-mitochondrial apoptotic pathway downstream of cytochrome c release cannot operate the apoptosome function in UV-induced apoptosis in thymic 3SB cells. The intracellular redistribution and sequestration of apoptosis-related proteins upon mitochondrion-based apoptotic signaling was identified as a novel cellular mechanism to respond to DNA damage in an agent type-specific manner. This finding suggests that the kind of the critical ultimate apoptosis-inducing DNA lesion complex form resulting from the agent-specific DNA damage responses is important to determine which of apoptosis signals would be activated.

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

    Science.gov (United States)

    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.

  15. 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.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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 (PEoL-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.

  17. Midazolam induces apoptosis in MA-10 mouse Leydig tumor cells through caspase activation and the involvement of MAPK signaling pathway

    Directory of Open Access Journals (Sweden)

    So EC

    2014-02-01

    Full Text Available Edmund Cheung So,1,2 Yu-Xuan Lin,3 Chi Hao Tseng,1 Bo-Syong Pan,3 Ka-Shun Cheng,2 Kar-Lok Wong,2 Lyh-Jyh Hao,4 Yang-Kao Wang,5 Bu-Miin Huang2 1Department of Anesthesia, Tainan Municipal An Nan Hospital, China Medical University, Tainan, Taiwan; 2Department of Anesthesia, China Medical University, Taichung, Taiwan; 3Department of Cell Biology and Anatomy, National Cheng Kung University, Tainan, Taiwan; 4Department of Internal Medicine, Division of Endocrinology and Metabolism, Kaohsiung Veteran General Hospital Tainan Branch Tainan, Taiwan; 5Graduate Institute of Biomedical Materials and Tissue Engineering, Taipei Medical University, Taipei, Taiwan Purpose: The present study aims to investigate how midazolam, a sedative drug for clinical use with cytotoxicity on neuronal and peripheral tissues, induced apoptosis in MA-10 mouse Leydig tumor cells. Methods: The apoptotic effect and underlying mechanism of midazolam to MA-10 cells were investigated by flow cytometry assay and Western blotting methods. Results: Data showed that midazolam induced the accumulation of the MA-10 cell population in the sub-G1 phase and a reduction in the G2/M phase in a time- and dose-dependent manner, suggesting an apoptotic phenomenon. Midazolam could also induce the activation of caspase-8, -9, and -3 and poly (ADP-ribose polymerase proteins. There were no changes in the levels of Bax and cytochrome-c, whereas Bid was significantly decreased after midazolam treatment. Moreover, midazolam decreased both pAkt and Akt expression. In addition, midazolam stimulated the phosphorylation of p38 and c-Jun NH2-terminal kinase but not extracellular signal-regulated kinase. Conclusion: Midazolam could induce MA-10 cell apoptosis through the activation of caspase cascade, the inhibition of pAkt pathway, and the induction of p38 and c-Jun NH2-terminal kinase pathways. Keywords: midazolam, apoptosis, MA-10 cell, caspase, Akt, MAPKs

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

    Energy Technology Data Exchange (ETDEWEB)

    Williams, C. David [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Koerner, Michael R., E-mail: mkoern2@illinois.edu [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Lampe, Jed N. [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Farhood, Anwar [Department of Pathology, Brackenridge Hospital, Austin, TX 78701 (United States); Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 (United States)

    2011-12-15

    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. 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 caspase

  19. Design and application of a fluorogenic assay for monitoring inflammatory caspase activity.

    Science.gov (United States)

    Ranganathan, Raj; Lenti, Gena; Tassone, Nicholas M; Scannell, Brian J; Southern, Cathrine A; Karver, Caitlin E

    2018-02-15

    Various fluorogenic assays exist for monitoring the activity of inflammatory caspases. However, there are no continuous assays that provide C-terminal substrate sequence specificity for inflammatory caspases. As a first step towards this, we have developed a continuous in vitro assay that relies on monitoring emission from tryptophan after cleavage of a quenching coumarin chromophore. The coumarin can be attached as an amino acid side chain or capping the C-terminus of the peptide. When the coumarin is a side chain, it allows for C-terminal and N-terminal sequence specificities to be explored. Using this assay, we obtained Michaelis-Menten kinetic data for four proof-of-principle peptides: WEHD-AMC (K M  = 15 ± 2 μM), WEHD-MCA (K M  = 93 ± 19 μM), WEHDG-MCA (K M  = 21 ± 6 μM) and WEHDA-MCA (K M  = 151 ± 37 μM), where AMC is 7-amino-4-methylcoumarin and MCA is β-(7-methoxy-coumarin-4-yl)-Ala. The results indicate the viability of this new assay approach in the design of effective fluorogenic substrates for inflammatory caspases. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Large-scale preparation of active caspase-3 in E. coli by designing its thrombin-activatable precursors

    Directory of Open Access Journals (Sweden)

    Park Sung

    2008-12-01

    Full Text Available Abstract Background Caspase-3, a principal apoptotic effector that cleaves the majority of cellular substrates, is an important medicinal target for the treatment of cancers and neurodegenerative diseases. Large amounts of the protein are required for drug discovery research. However, previous efforts to express the full-length caspase-3 gene in E. coli have been unsuccessful. Results Overproducers of thrombin-activatable full-length caspase-3 precursors were prepared by engineering the auto-activation sites of caspase-3 precursor into a sequence susceptible to thrombin hydrolysis. The engineered precursors were highly expressed as soluble proteins in E. coli and easily purified by affinity chromatography, to levels of 10–15 mg from 1 L of E. coli culture, and readily activated by thrombin digestion. Kinetic evaluation disclosed that thrombin digestion enhanced catalytic activity (kcat/KM of the precursor proteins by two orders of magnitude. Conclusion A novel method for a large-scale preparation of active caspase-3 was developed by a strategic engineering to lack auto-activation during expression with amino acid sequences susceptible to thrombin, facilitating high-level expression in E. coli. The precursor protein was easily purified and activated through specific cleavage at the engineered sites by thrombin, generating active caspase-3 in high yields.

  1. Combination of Vorinostat and caspase-8 inhibition exhibits high anti-tumoral activity on endometrial cancer cells.

    Science.gov (United States)

    Bergadà, Laura; Sorolla, Annabel; Yeramian, Andree; Eritja, Nuria; Mirantes, Cristina; Matias-Guiu, Xavier; Dolcet, Xavier

    2013-08-01

    Histone deacetylase inhibitors such as Vorinostat display anti-neoplastic activity against a variety of solid tumors. Here, we have investigated the anti-tumoral activity of Vorinostat on endometrial cancer cells. We have found that Vorinostat caused cell growth arrest, loss of clonogenic growth and apoptosis of endometrial cancer cells. Vorinostat-induced the activation of caspase-8 and -9, the initiators caspases of the extrinsic and the intrinsic apoptotic pathways, respectively. Next, we investigated the role of the extrinsic pathway in apoptosis triggered by Vorinostat. We found that Vorinostat caused a dramatic decrease of FLIP mRNA and protein levels. However, overexpression of the long from of FLIP did not block Vorinostat-induced apoptosis. To further investigate the role of extrinsic apoptotic pathway in Vorinostat-induced apoptosis, we performed an shRNA-mediated knock-down of caspase-8. Surprisingly, downregulation of caspase-8 alone caused a marked decrease in clonogenic ability and reduced the growth of endometrial cancer xenografts in vivo, revealing that targeting caspase-8 may be an attractive target for anticancer therapy on endometrial tumors. Furthermore, combination of caspase-8 inhibition and Vorinostat treatment caused an enhancement of apoptotic cell death and a further decrease of clonogenic growth of endometrial cancer cells. More importantly, combination of Vorinostat and caspase-8 inhibition caused a nearly complete inhibition of tumor xenograft growth. Finally, we demonstrate that cell death triggered by Vorinostat alone or in combination with caspase-8 shRNAs was inhibited by the anti-apoptotic protein Bcl-XL. Our results suggest that combinatory therapies using Vorinostat treatment and caspase-8 inhibition can be an effective treatment for endometrial carcinomas. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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

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    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.

  3. Proteases and caspase-like activity in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Wilkinson, Derek; Ramsdale, Mark

    2011-10-01

    A variety of proteases have been implicated in yeast PCD (programmed cell death) including the metacaspase Mca1 and the separase Esp1, the HtrA-like serine protease Nma111, the cathepsin-like serine carboxypeptideases and a range of vacuolar proteases. Proteasomal activity is also shown to have an important role in determining cell fate, with both pro- and anti-apoptotic roles. Caspase 3-, 6- and 8-like activities are detected upon stimulation of yeast PCD, but not all of this activity is associated with Mca1, implicating other proteases with caspase-like activity in the yeast cell death response. Global proteolytic events that accompany PCD are discussed alongside a consideration of the conservation of the death-related degradome (both at the level of substrate choice and cleavage site). The importance of both gain-of-function changes in the degradome as well as loss-of-function changes are highlighted. Better understanding of both death-related proteases and their substrates may facilitate the design of future antifungal drugs or the manipulation of industrial yeasts for commercial exploitation.

  4. Impact of Procyanidins from Different Berries on Caspase 8 Activation in Colon Cancer

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    Carole Minker

    2015-01-01

    Full Text Available Scope. The aim of this work is to identify which proapoptotic pathway is induced in human colon cancer cell lines, in contact with proanthocyanidins extracted from various berries. Methods and Results. Proanthocyanidins (Pcys extracted from 11 berry species are monitored for proapoptotic activities on two related human colon cancer cell lines: SW480-TRAIL-sensitive and SW620-TRAIL-resistant. Apoptosis induction is monitored by cell surface phosphatidylserine (PS detection. Lowbush blueberry extract triggers the strongest activity. When tested on the human monocytic cell line THP-1, blueberry Pcys are less effective for PS externalisation and DNA fragmentation is absent, highlighting a specificity of apoptosis induction in gut cells. In Pcys-treated gut cell lines, caspase 8 (apoptosis extrinsic pathway but not caspase 9 (apoptosis intrinsic pathway is activated after 3 hours through P38 phosphorylation (90 min, emphasizing the potency of lowbush blueberry Pcys to eradicate gut TRAIL-resistant cancer cells. Conclusion. We highlight here that berries Pcys, especially lowbush blueberry Pcys, are of putative interest for nutritional chemoprevention of colorectal cancer in view of their apoptosis induction in a human colorectal cancer cell lines.

  5. New therapeutic activity of metabolic enhancer piracetam in treatment of neurodegenerative disease: Participation of caspase independent death factors, oxidative stress, inflammatory responses and apoptosis.

    Science.gov (United States)

    Verma, Dinesh Kumar; Gupta, Sonam; Biswas, Joyshree; Joshi, Neeraj; Singh, Abhishek; Gupta, Parul; Tiwari, Shubhangini; Sivarama Raju, K; Chaturvedi, Swati; Wahajuddin, M; Singh, Sarika

    2018-03-16

    Piracetam, a nootropic drug that has been clinically used for decades but remains enigmatic due to no distinct understanding of its mechanism of action. The present study aimed to investigate the role of caspase independent pathway in piracetam mediated neuroprotection. LPS administration caused significant alterations in oxidative stress related parameters like glutathione, glutathione reductase and increased lipid peroxidation. LPS administration also caused augmented expression of inflammatory cytokines and astrocytes activation. Piracetam treatment offered significant protection against LPS induced oxidative and inflammatory parameters and inhibited astrocytes activation. LPS administration caused augmented level of reactive oxygen species and depleted mitochondrial membrane potential which were attenuated with piracetam treatment. This study for the first time demonstrates the role of caspase independent death factors in piracetam induced neuroprotective effects in rat brain. Translocation of mitochondrial resident apoptosis inducing factor and endonuclease G to nucleus through cytosol after LPS administration was significantly blocked with piracetam treatment. Further, LPS induced DNA fragmentation along with up regulated Poly [ADP-ribose] polymerase 1 (PARP1) levels were also inhibited with piracetam treatment. Apoptotic death was confirmed by the cleavage of caspase 3 as well as histological alteration in rat brain regions. LPS administration caused significantly increased level of cleaved caspase 3, altered neuronal morphology and decreased neuronal density which were restored with piracetam treatment. Collectively our findings indicate that piracetam offered protection against LPS induced inflammatory responses and cellular death including its antioxidative antiapoptotic activity with its attenuation against mitochondria mediated caspase independent pathway. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. Immunohistochemical investigation of cell cycle and apoptosis regulators (Survivin, β-Catenin, P53, Caspase 3 in canine appendicular osteosarcoma

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    Bongiovanni Laura

    2012-06-01

    Full Text Available Abstract Background Osteosarcoma (OSA represents the most common canine primary bone tumour. Despite several pathways have been investigated so far, few molecules have been identified as prognostic tools or potential therapeutic targets, and there is still the need to find out molecular pathways with specific influence over OSA progression to facilitate earlier prognosis and treatment. Aims of the present study were to evaluate the immunohistochemical pattern and levels of expression of a panel of molecules (survivin, β-catenin, caspase 3 -inactive and active forms- and p53 involved in cell cycle and apoptosis regulation in canine OSA samples, known to be of interest in the study also of human OSA, and to detect specific relations among them and with histological tumour grade, disease free interval (DFI and overall survival (OS. Results Nuclear β-catenin immunostaining was detected in normal osteoblasts adjacent to the tumour, and in 47% of the cases. Cytoplasmic and/or membranous immunostaining were also observed. Nuclear survivin and p53 positive cells were found in all cases. Moderate/high cytoplasmic β-catenin expression (≥10% positive cells was significantly associated with the development of metastasis (P = 0.014; moderate/high nuclear p53 expression (≥10% positive cells was significantly associated with moderate/high histological grade (P = 0.017 and shorter OS (P = 0.049. Moderate/high nuclear survivin expression (≥15% positive cells showed a tendency toward a longer OS (P = 0,088. Conclusions The present results confirmed p53 as negative prognostic marker, while suggested survivin as a potential positive prognostic indicator, rather than indicative of a poor prognosis. The detection of nuclear β-catenin immunostaining in normal osteoblasts and the absent/low expression in most of the OSAs, suggested that this pathway could not play a major role in oncogenic transformation of canine osteoblasts. Further studies

  7. Colorimetric Detection of Caspase 3 Activity and Reactive Oxygen Derivatives: Potential Early Indicators of Thermal Stress in Corals

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    Mickael Ros

    2016-01-01

    Full Text Available There is an urgent need to develop and implement rapid assessments of coral health to allow effective adaptive management in response to coastal development and global change. There is now increasing evidence that activation of caspase-dependent apoptosis plays a key role during coral bleaching and subsequent mortality. In this study, a “clinical” approach was used to assess coral health by measuring the activity of caspase 3 using a commercial kit. This method was first applied while inducing thermal bleaching in two coral species, Acropora millepora and Pocillopora damicornis. The latter species was then chosen to undergo further studies combining the detection of oxidative stress-related compounds (catalase activity and glutathione concentrations as well as caspase activity during both stress and recovery phases. Zooxanthellae photosystem II (PSII efficiency and cell density were measured in parallel to assess symbiont health. Our results demonstrate that the increased caspase 3 activity in the coral host could be detected before observing any significant decrease in the photochemical efficiency of PSII in the algal symbionts and/or their expulsion from the host. This study highlights the potential of host caspase 3 and reactive oxygen species scavenging activities as early indicators of stress in individual coral colonies.

  8. PKI 166 induced redox signalling and apoptosis through activation of p53, MAP kinase and caspase pathway in epidermoid carcinoma.

    Science.gov (United States)

    Das, Subhasis; Dey, Kaushik Kumar; Bharti, Rashmi; MaitiChoudhury, Sujata; Maiti, Sukumar; Mandal, Mahitosh

    2012-01-01

    Cellular redox changes have emerged as a pivotal and proximal event in cancer. PKI 166 is used to determine the effects of redox sensitive inhibition of EGFR, metastasis and apoptosis in epidermoid carcinoma. Cytotoxicity study of PKI 166 (IC50 1.0 microM) treated A431 cells were performed by MTT assay for 48 and 72 hrs. Morphological analysis of PKI 166 treated A431 cells for 48 hrs. revealed the cell shrinkage, loss of filopodia and lamellipodia by phase contrast and SEM images in dose dependent manner. It has cytotoxic effects through inhibiting cellular proliferation, leads to the induction of apoptosis, as increased fraction of sub-G1 phase of the cell cycle, chromatin condensation and DNA ladder. It inhibited cyclin-D1 and cyclin-E expression and induced p53, p21 expression in dose dependent manner. Consequently, an imbalance of Bax/Bcl-2 ratio triggered caspase cascade and subsequent cleavage of PARP, thereby shifting the balance in favour of apoptosis. PKI 166 treatment actively stimulated reactive oxygen species (ROS) and mitochondrial membrane depolarization. It inhibited some metastatic properties of A431 cells supressing colony formation by soft agar assay and inhibition of MMP 9 activity by gelatin zymography and western blot analysis. PKI 166 inhibited growth factor induced phosphorylation of EGFR, Akt, MAPK, JNK and colony formation in A431 cells. Thus the inhibition of proliferation was associated with redox regulation of the caspase cascade, EGFR, Akt/PI3K, MAPK/ ERK and JNK pathway. On the other hand, increased antioxidant activity leads to decreased ROS generation inhibit the anti-proliferative and apoptotic properties of PKI 166 in A431 cells. These observations indicated PKI 166 induced redox signalling dependent inhibition of cell proliferation, metastatic properties and induction of apoptotic potential in epidermoid carcinoma.

  9. Neurotoxicity of amphetamine derivatives is mediated by caspase pathway activation in rat cerebellar granule cells

    International Nuclear Information System (INIS)

    Jimenez, Andres; Jorda, Elvira G.; Verdaguer, Ester; Pubill, David; Sureda, Francesc X.; Canudas, Anna M.; Escubedo, Elena; Camarasa, Jordi; Camins, Antoni; Pallas, Merce

    2004-01-01

    The neurotoxic action of the abuse drugs methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA) on cerebellar granule neurones (CGNs) culture was examined. Treatment for 48 h with METH or MDMA (1-5 mM) induced a higher decrease in viability than 24 h treatment. z.VAD.fmk (100 μM) but not MK-801 nor NBQX recovered control viability values. In both cases, cell death was characterised as apoptotic rather than necrotic by morphology cell observation. Apoptosis measured by flow cytometry indicated an increase in the hypodiploid population after 48 h treatment with METH and MDMA. Apoptosis was reverted by the presence of z.VAD.fmk (100 μM) but not by 10 μM MK-801 or NBQX. Similar results were obtained by analysing nuclear chromatine condensation. These results ruled out excitotoxic participation in amphetamine derivative-induced neurotoxicity in CGNs. Participation of radical oxygen species (ROS) was evaluated using α-tocopherol (1-15 μM) and cytometric studies. The co-treatment with 4 mM METH or MDMA for 48 h partially reverted neurotoxic action and apoptotic features, indicating ROS implication in CGNs death by amphetamine derivatives. Alteration of mitochondrial function induced cytochrome C (Cyt C) release after 48-h treatment with METH and MDMA (4 mM). There was also indication of caspase-3-like activation, measured by immunoanalysis and biochemically. Finally, neurodegenerative action caused by amphetamine derivatives may be prevented by using caspase inhibitors

  10. Caspase 3 activity in isolated fetal rat lung fibroblasts and rat periodontal ligament fibroblasts: cigarette smoke-induced alterations

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    James Elliot Scott

    2016-03-01

    Full Text Available Background Cigarette smoking is the leading cause of preventable death in the world. It has been implicated in the pathogenesis of pulmonary, oral and systemic diseases. Smoking during pregnancy is clearly a risk factor for the developing fetus and may be a major cause of infant mortality. Moreover, the oral cavity is the first site of exposure to cigarette smoke and may be a possible source for the spread of toxins to other organs of the body. Fibroblasts in general are morphologically heterogeneous connective tissue cells with diverse functions. Apoptosis or programmed cell death is a crucial process during embryogenesis and for the maintenance of homeostasis throughout life. Deregulation of apoptosis has been implicated in abnormal lung development in the fetus and disease progression in adults. Caspases, are proteases which belong to the family of cysteine aspartic acid proteases and are the key components for the downstream amplification of intra-cellular apoptotic signals. Of the 14 caspases known, caspase-3 is the key executioner of apoptosis. Fetal rat lung fibroblasts but not PDL viability is reduced by exposure to CSE. In addition Caspase 3 activity is elevated after CSE exposure in fetal lung fibroblasts but not in PDLs. Expression of caspase 3 is induced in CSE exposed lung fibroblasts but not in PDLs. Caspase 3 was localized to the cytoplasm in both cell types.

  11. A ginseng saponin metabolite-induced apoptosis in HepG2 cells involves a mitochondria-mediated pathway and its downstream caspase-8 activation and Bid cleavage

    International Nuclear Information System (INIS)

    Oh, Seon-Hee; Lee, Byung-Hoon

    2004-01-01

    20-O-(β-D-Glucopyranosyl)-20(S)-protopanaxadiol (IH901), an intestinal bacterial metabolite of ginseng saponin formed from ginsenosides Rb1, Rb2, and Rc, is suggested to be a potential chemopreventive agent. Here, we show that IH901 induces apoptosis in human hepatoblastoma HepG2 cells. IH901 led to an early activation of procaspase-3 (12 h posttreatment), and the activation of caspase-8 became evident only later (18 h posttreatment). Caspase activation was a necessary requirement for apoptosis because caspase inhibitors significantly inhibited cell death by IH901. Treatment of HepG2 cells with IH901 also induced the cleavage of cytosolic factors such as Bid and Bax and translocation of truncated Bid (tBid) to mitochondria. A time-dependent release of cytochrome c from mitochondria was observed, which was accompanied by activation of caspase-9. A broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), and a specific inhibitor for caspase-8, N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone (zIETD-fmk), abrogated Bid processing and translocation, and caspase-3 activation. Cytochrome c release was inhibited by zVAD-fmk, however, the inhibition by zIETD-fmk was not complete. The activation of caspase-8 was inhibited not only by zIETD-fmk but also by zVAD-fmk. The results, together with the kinetic change of caspase activation, indicate that activation of caspase-8 occurred downstream of caspase-3 and -9. Our data suggest that the activation of caspase-8 after early caspase-3 activation might act as an amplification loop necessary for successful apoptosis. Primary hepatocytes isolated from normal Sprague-Dawley rats were not affected by IH901 (0-60 μM). The very low toxicity in normal hepatocytes and high activity in hepatoblastoma HepG2 cells suggest that IH901 is a promising experimental cancer chemopreventive agent

  12. miR-130a activates apoptotic signaling through activation of caspase-8 in taxane-resistant prostate cancer cells.

    Science.gov (United States)

    Fujita, Yasunori; Kojima, Toshio; Kawakami, Kyojiro; Mizutani, Kosuke; Kato, Taku; Deguchi, Takashi; Ito, Masafumi

    2015-10-01

    The acquisition of drug resistance is one of the most malignant phenotypes of cancer and identification of its therapeutic target is a prerequisite for the development of novel therapy. MicroRNAs (miRNAs) have been implicated in various types of cancer and proposed as potential therapeutic targets for patients. In the present study, we aimed to identify miRNA that could serve as a therapeutic target for taxane-resistant prostate cancer. In order to identify miRNAs related to taxane-resistance, miRNA profiling was performed using prostate cancer PC-3 cells and paclitaxel-resistant PC-3 cell lines established from PC-3 cells. Microarray analysis of mRNA expression was also conducted to search for potential target genes of miRNA. Luciferase reporter assay was performed to examine miRNA binding to the 3'-UTR of target genes. The effects of ectopic expression of miRNA on cell growth, tubulin polymerization, drug sensitivity, and apoptotic signaling pathway were investigated in a paclitaxel-resistant PC-3 cell line. The expression of miR-130a was down-regulated in all paclitaxel-resistant cell lines compared with parental PC-3 cells. Based on mRNA microarray analysis and luciferase reporter assay, we identified SLAIN1 as a direct target gene for miR-130a. Transfection of a miR-130a precursor into a paclitaxel-resistant cell line suppressed cell growth and increased the sensitivity to paclitaxel. Lastly, ectopic expression of miR-130a did not affect the polymerized tubulin level, but activated apoptotic signaling through activation of caspase-8. Our results suggested that reduced expression of miR-130a may be involved in the paclitaxel-resistance and that miR-130a could be a therapeutic target for taxane-resistant prostate cancer patients. © 2015 Wiley Periodicals, Inc.

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

    Science.gov (United States)

    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.

  14. Caspase Activation of p21-Activated Kinase 2 Occurs During Cisplatin-Induced Apoptosis of SH-SY5Y Neuroblastoma Cells and in SH-SY5Y Cell Culture Models of Alzheimer’s and Parkinson’s Disease

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    Jerry W. Marlin

    2010-04-01

    Full Text Available p21-activated kinase 2 (PAK-2 appears to have a dual function in the regulation of cell survival and cell death. Activation of full-length PAK-2 by the p21 G-proteins Rac or Cdc42 stimulates cell survival. However, PAK-2 is unique among the PAK family because it is also activated through proteolytic cleavage by caspase 3 or similar caspases to generate the constitutively active PAK-2p34 fragment. Caspase activation of PAK-2 correlates with the induction of apoptosis in response to many stimuli and recombinant expression of PAK-2p34 has been shown to stimulate apoptosis in several human cell lines. Here, we show that caspase activation of PAK-2 also occurs during cisplatin-induced apoptosis of SH-SY5Y neuroblastoma cells as well as in SH-SY5Y cell culture models for Alzheimer’s and Parkinson’s disease. Inhibition of mitochondrial complex I or of ubiquitin/proteasome-mediated protein degradation, which both appear to be involved in Parkinson’s disease, induce apoptosis and caspase activation of PAK-2 in SH-SY5Y cells. Overexpression of the amyloid precursor protein, which results in accumulation and aggregation of β-amyloid peptide, the main component of β-amyloid plaques in Alzheimer’s disease, also induces apoptosis and caspase activation of PAK-2 in SH-SY5Y cells. Expression of the PAK-2 regulatory domain inhibits caspase-activated PAK-2p34 and prevents apoptosis in 293T human embryonic kidney cells, indicating that caspase activation of PAK-2 is directly involved in the apoptotic response. This is the first evidence that caspase activation of PAK-2 correlates with apoptosis in cell culture models of Alzheimer’s and Parkinson’s disease and that selective inhibition of caspase-activated PAK-2p34 could prevent apoptosis.

  15. Several nuclear events during apoptosis depend on caspase-3 activation but do not constitute a common pathway.

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    Lisa Trisciuoglio

    Full Text Available A number of nuclear events occur during apoptosis, including DNA laddering, nuclear lamina breakdown, phosphorylation of histones H2B and histone H2AX, and the tight binding to chromatin of HMGB1 and CAD, the nuclease responsible for DNA laddering. We have performed an epistasis analysis to investigate whether these events cluster together in pathways. We find that all depend directly or indirectly on caspase-3 activation. CAD activation, H2AX phosphorylation and DNA laddering cluster together into a pathway, but all other events appear to be independent of each other downstream of caspase-3, and likely evolved subject to different functional pressures.

  16. 1800MHz Microwave Induces p53 and p53-Mediated Caspase-3 Activation Leading to Cell Apoptosis In Vitro.

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    Fuqiang Xing

    Full Text Available 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.

  17. Malaria-induced NLRP12/NLRP3-dependent caspase-1 activation mediates inflammation and hypersensitivity to bacterial superinfection.

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    Marco A Ataide

    2014-01-01

    Full Text Available Cyclic paroxysm and high fever are hallmarks of malaria and are associated with high levels of pyrogenic cytokines, including IL-1β. In this report, we describe a signature for the expression of inflammasome-related genes and caspase-1 activation in malaria. Indeed, when we infected mice, Plasmodium infection was sufficient to promote MyD88-mediated caspase-1 activation, dependent on IFN-γ-priming and the expression of inflammasome components ASC, P2X7R, NLRP3 and/or NLRP12. Pro-IL-1β expression required a second stimulation with LPS and was also dependent on IFN-γ-priming and functional TNFR1. As a consequence of Plasmodium-induced caspase-1 activation, mice produced extremely high levels of IL-1β upon a second microbial stimulus, and became hypersensitive to septic shock. Therapeutic intervention with IL-1 receptor antagonist prevented bacterial-induced lethality in rodents. Similar to mice, we observed a significantly increased frequency of circulating CD14(+CD16(-Caspase-1(+ and CD14(dimCD16(+Caspase-1(+ monocytes in peripheral blood mononuclear cells from febrile malaria patients. These cells readily produced large amounts of IL-1β after stimulation with LPS. Furthermore, we observed the presence of inflammasome complexes in monocytes from malaria patients containing either NLRP3 or NLRP12 pyroptosomes. We conclude that NLRP12/NLRP3-dependent activation of caspase-1 is likely to be a key event in mediating systemic production of IL-1β and hypersensitivity to secondary bacterial infection during malaria.

  18. Involvement of caspase-2 and caspase-9 in endoplasmic reticulum stress-induced apoptosis: A role for the IAPs

    International Nuclear Information System (INIS)

    Cheung, Herman H.; Lynn Kelly, N.; Liston, Peter; Korneluk, Robert G.

    2006-01-01

    Dysregulation of apoptosis is involved in a wide spectrum of disease ranging from proliferative to degenerative disorders. An emerging area of study in apoptosis is the critical contribution of the endoplasmic reticulum (ER) in both mitochondrial and ER specific apoptosis pathways. Here we show that brefeldin A and tunicamycin-mediated ER stress lead to caspase-dependent apoptosis involving caspase-2. Confocal microscopy and subcellular fractionation indicate that caspase-2 is localized to the ER, and following ER stress, the processing of caspase-2 and -9 is an early event preceding the activation of caspase-3 and -7 and the cleavage of the caspase substrate poly(ADP-ribose) polymerase (PARP). Inhibition and silencing of either caspase-2 or caspase-9 suppress ER stress-induced apoptosis, as demonstrated by annexin V binding. Similarly, transduction with an adenovirus encoding either Inhibitors of Apoptosis (IAP) protein HIAP1/c-IAP2 or HIAP2/c-IAP1 also suppresses ER stress-induced apoptosis. However, among HIAP1, HIAP2 and XIAP, only HIAP2 binds and inhibits caspase-2. Our results thus indicate a novel mechanism by which HIAP2 can regulate ER-initiated apoptosis by modulating the activity of caspase-2

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

    Science.gov (United States)

    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.

  20. Berberine Induces Apoptotic Cell Death via Activation of Caspase-3 and -8 in HL-60 Human Leukemia Cells: Nuclear Localization and Structure-Activity Relationships.

    Science.gov (United States)

    Okubo, Shinya; Uto, Takuhiro; Goto, Aya; Tanaka, Hiroyuki; Nishioku, Tsuyoshi; Yamada, Katsushi; Shoyama, Yukihiro

    2017-01-01

    Berberine (BBR), an isoquinoline alkaloid, is a well-known bioactive compound contained in medicinal plants used in traditional and folk medicines. In this study, we investigated the subcellular localization and the apoptotic mechanisms of BBR were elucidated. First, we confirmed the incorporation of BBR into the cell visually. BBR showed antiproliferative activity and promptly localized to the nucleus from 5[Formula: see text]min to 15[Formula: see text]min after BBR treatment in HL-60 human promyelocytic leukemia cells. Next, we examined the antiproliferative activity of BBR (1) and its biosynthetically related compounds (2-7) in HL-60 cells. BBR exerted strongest antiproliferative activity among 1-7 and the results of structures and activity relation suggested that a methylenedioxyl group in ring A, an [Formula: see text]-alkyl group at C-9 position, and the frame of isoquinoline may be necessary for antiproliferative activity. Moreover, BBR showed the most potent antiproliferative activity in HL-60 cells among human cancer and normal cell lines tested. Next, we examined the effect of BBR on molecular events known as apoptosis induction. In HL-60 cells, BBR induced chromatin condensation and DNA fragmentation, and triggered the activation of PARP, caspase-3 and caspase-8 without the activation of caspase-9. BBR-induced DNA fragmentation was abolished by pretreatment with inhibitors against caspase-3 and caspase-8, but not against caspase-9. ERK and p38 were promptly phosphorylated after 15 min of BBR treatment, and this was correlated with time of localization to the nucleus of BBR. These results demonstrated that BBR translocated into nucleus immediately after treatments and induced apoptotic cell death by activation of caspase-3 and caspase-8.

  1. Polyphenol Compounds of Mahkota Dewa (Phaleria macrocarpa[Scheff.] Boerl Up-regulated Caspase-3 and Apoptosis Index in Balb/c Strain Mice

    Directory of Open Access Journals (Sweden)

    Indranila KS

    2016-04-01

    Full Text Available Background: Polyphenol compounds of Mahkota Dewa (Phaleria macrocarpa[Scheff.] Boerl (PMD can potentially be used as ant cancer treatment by scavanging radical molecules. The effect in vivois still limited to Indonesia. Purpose: This research was aimed to validate the activity of PMD in increasingcaspase-3 expression and apoptosis in Balb/c mice, induced by Benzo(apyrene (BaP. Methods: A posttest control group was implemented and used by 40 Balb/c mice at the age of 1-2 weeks, with the body weight of 20-30 g. The tumor induction was administered to the mice using BaP. The animals were randomized into two groups called the control group and the PMD treatment group, the latter of which was given a dosage of 50mg. Lung tumor growth was assessed through surgery at week 8, 17, and 26. The results of caspase-3expression and apoptotic index from IHC-TUNEL staining were analyzed using Kruskal-Wallis, Mann-Whitney, One-way ANOVA, and Post hoc test LSD with significant levels of p<α (0,05.This research was approved by Ethical Clearance. Results: Oral administration of 50mg PMD significantly increased caspase-3 expression and apoptotic index in the treatment group animals at weeks 8, 17, and 26. Carcinogenesis incidence in the control group were respectively found at2,32±0,26 and 3,93±0,46 at weeks 8 and 26, while those of the treatment group were 1,88±0,38 and 0,88±0,22 (p=0,001. The apoptotic index in the control group was0,00±0,00 at 8 weeksand0,92+0,22at 26 weeks, whereas the indexes of the treatment group were 1,12±0,71 and 2,02±1,05 (p=0,001. In the control group, the caspase-3 expression at weeks 8 and 26 were 0,28±0,17 and 0,56±0,16, while those in the treatment group were 0,60±0,14 at week 8 and 2,52±0,33 at week 26 (p=0,001. Conclusion: The treatment of PMD effectively induced cell apoptosis in the Balb/c mice via up- regulation of the caspase-3 expression, thereby increasing the apoptotic index. This shows that PMD has anticancer

  2. Dopamine-induced programmed cell death is associated with cytochrome c release and caspase-3 activation in snail salivary gland cells.

    Science.gov (United States)

    Pirger, Zsolt; Rácz, Boglárka; Kiss, Tibor

    2009-02-01

    PCD (programmed cell death) is a common mechanism to remove unwanted and excessive cells from organisms. In several exocrine cell types, PCD mode of release of secretory products has been reported. The molecular mechanism of the release, however, is largely unknown. Our aim was to study the molecular mechanism of saliva release from cystic cells, the specific cell type of snail SGs (salivary glands). SG cells in active feeding animals revealed multiple morphological changes characteristic of PCD. Nerve stimulation and DA (dopamine) increased the number of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling)-positive cells both in inactive and feeding animals. The DA-induced PCD was prevented by TEA (tetraethylammonium chloride) and eticlopride, emphasizing the role of K channels and D2 receptors in the PCD of cystic cells. DA enhanced cyto-c (cytochrome c) translocation into the cytosol and methyl-beta-cyclodextrin prevented it, suggesting apoptosome formation and ceramide involvement in the PCD linking of the surface DA receptor to mitochondria. Western blot analysis revealed that the release of cyto-c was under the control of Bcl-2 and Bad. DA also increased the active caspase-3 in gland cells while D2 receptor antagonists and TEA attenuated it. Our results provide evidence for a type of transmitter-mediated pathway that regulates the PCD of secretory cells in a mitochondrial-caspase-dependent manner. The activation of specific molecules, such as K channels, DA receptors, cyto-c, ceramide, Bcl-2 proteins and caspase-3, but not caspase-8, was demonstrated in cells involved in the DA-induced PCD, suggesting that PCD is a physiological method for the release of saliva from SG cells.

  3. The common inhalation anesthetic isoflurane induces caspase activation and increases amyloid beta-protein level in vivo.

    Science.gov (United States)

    Xie, Zhongcong; Culley, Deborah J; Dong, Yuanlin; Zhang, Guohua; Zhang, Bin; Moir, Robert D; Frosch, Matthew P; Crosby, Gregory; Tanzi, Rudolph E

    2008-12-01

    An estimated 200 million patients worldwide have surgery each year. Anesthesia and surgery have been reported to facilitate emergence of Alzheimer's disease. The commonly used inhalation anesthetic isoflurane has previously been reported to induce apoptosis, and to increase levels and aggregation of Alzheimer's disease-associated amyloid beta-protein (Abeta) in cultured cells. However, the in vivo relevance has not been addressed. We therefore set out to determine effects of isoflurane on caspase activation and levels of beta-site amyloid precursor protein-cleaving enzyme (BACE) and Abeta in naive mice, using Western blot, immunohistochemistry, and reverse transcriptase polymerase chain reaction. Here we show for the first time that a clinically relevant isoflurane anesthesia (1.4% isoflurane for 2 hours) leads to caspase activation and modest increases in levels of BACE 6 hours after anesthesia in mouse brain. Isoflurane anesthesia induces caspase activation, and increases levels of BACE and Abeta up to 24 hours after anesthesia. Isoflurane may increase BACE levels by reducing BACE degradation. Moreover, the Abeta aggregation inhibitor, clioquinol, was able to attenuate isoflurane-induced caspase-3 activation in vivo. Given that transient insults to brain may lead to long-term brain damage, these findings suggest that isoflurane may promote Alzheimer's disease neuropathogenesis and, as such, have implications for use of isoflurane in humans, pending human study confirmation.

  4. Epidermal growth factor and active caspase-3 expression in the levator ani muscle of dogs with and without perineal hernia.

    Science.gov (United States)

    Pérez-Gutiérrez, J F; Argüelles, J C; Iglesias-Núñez, M; Oliveira, K S; De La Muela, M Sánchez

    2011-07-01

    To perform a histological and immunohistochemical study of epidermal growth factor, transforming growth factor-alpha and their receptor, as well as the apoptotic signal active caspase-3 in the levator ani muscle of dogs with and without perineal hernia. Biopsy specimens of the levator ani muscle were obtained from 25 dogs with perineal hernia and 4 non-affected dogs and were processed for Masson and immunohistochemical staining. The affected dogs exhibited myopathological features, internalised nuclei, destruction and abnormal size of muscle fibres, which were replaced by collagen. The immunohistochemical study revealed active caspase-3, epidermal growth factor, transforming growth factor-alpha and epidermal growth factor receptor in the levator ani. Compared to the healthy muscle, transforming growth factor-alpha staining intensity was lower in the affected muscle, whereas epidermal growth factor receptor and active caspase-3 staining were higher. Pelvic diaphragm muscle weakening is the leading cause of perineal hernia in the dog. Survival and death signals expressed in these muscles may contribute to the pathogenesis of this disease. This study reports epidermal growth factor, transforming growth factor-alpha and epidermal growth factor receptor immunohistochemical expression in the skeletal muscle and suggests that perineal hernia in the dog is accompanied by levator ani muscle atrophy, increased expression of epidermal growth factor receptor, caspase-3 activation, and decreased expression of transforming growth factor-alpha. © 2011 British Small Animal Veterinary Association.

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

    Science.gov (United States)

    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.

  6. Caspase-3 activation and DNA damage in pig skin organ culture after solar irradiation.

    Science.gov (United States)

    Bacqueville, Daniel; Mavon, Alain

    2008-01-01

    In the present study, a convenient and easy-to-handle skin organ culture was developed from domestic pig ears using polycarbonate Transwell culture inserts in 12-well plate. This alternative model was then tested for its suitability in analyzing the short-term effects of a single solar radiation dose (from 55 to 275 kJ.m(-2)). Differentiation of the pig skin was maintained for up to 48 h in culture, and its morphology was similar to that of fresh human skin. Solar irradiation induced a significant release of the cytosolic enzymes lactate dehydrogenase and extracellular signal-related kinase 2 protein in the culture medium 24 h after exposure. These photocytotoxic effects were associated with the formation of sunburn cells, thymine dimers and DNA strand breaks in both the epidermis and dermis. Interestingly, cell death was dose dependent and associated with p53 protein upregulation and strong caspase-3 activation in the basal epidermis. None of these cellular responses was observed in non-irradiated skin. Finally, topical application of a broad-spectrum UVB + A sunfilter formulation afforded efficient photoprotection in irradiated explants. Thus, the ex vivo pig ear skin culture may be a useful tool in the assessment of solar radiation-induced DNA damage and apoptosis, and for evaluating the efficacy of sunscreen formulations.

  7. Divergent modulation of neuronal differentiation by caspase-2 and -9.

    Directory of Open Access Journals (Sweden)

    Giuseppa Pistritto

    Full Text Available Human Ntera2/cl.D1 (NT2 cells treated with retinoic acid (RA differentiate towards a well characterized neuronal phenotype sharing many features with human fetal neurons. In view of the emerging role of caspases in murine stem cell/neural precursor differentiation, caspases activity was evaluated during RA differentiation. Caspase-2, -3 and -9 activity was transiently and selectively increased in differentiating and non-apoptotic NT2-cells. SiRNA-mediated selective silencing of either caspase-2 (si-Casp2 or -9 (si-Casp9 was implemented in order to dissect the role of distinct caspases. The RA-induced expression of neuronal markers, i.e. neural cell adhesion molecule (NCAM, microtubule associated protein-2 (MAP2 and tyrosine hydroxylase (TH mRNAs and proteins, was decreased in si-Casp9, but markedly increased in si-Casp2 cells. During RA-induced NT2 differentiation, the class III histone deacetylase Sirt1, a putative caspase substrate implicated in the regulation of the proneural bHLH MASH1 gene expression, was cleaved to a ∼100 kDa fragment. Sirt1 cleavage was markedly reduced in si-Casp9 cells, even though caspase-3 was normally activated, but was not affected (still cleaved in si-Casp2 cells, despite a marked reduction of caspase-3 activity. The expression of MASH1 mRNA was higher and occurred earlier in si-Casp2 cells, while was reduced at early time points during differentiation in si-Casp9 cells. Thus, caspase-2 and -9 may perform opposite functions during RA-induced NT2 neuronal differentiation. While caspase-9 activation is relevant for proper neuronal differentiation, likely through the fine tuning of Sirt1 function, caspase-2 activation appears to hinder the RA-induced neuronal differentiation of NT2 cells.

  8. Caspase Activation and Aberrant Cell Growth in a p53+/+ Cell Line from a Li-Fraumeni Syndrome Family

    Directory of Open Access Journals (Sweden)

    Zaki A. Sherif

    2015-01-01

    Full Text Available Wild-type p53 is well known to induce cell cycle arrest and apoptosis to block aberrant cell growth. However, p53’s unique role in apoptosis and cell proliferation in Li-Fraumeni Syndrome (LFS has not been well elucidated. The aim of this study is to characterize the activity of wild-type p53 protein in LFS family dominated by a germline negative mutant p53. As expected, etoposide-treated wild-type p53-containing cell lines, LFS 2852 and control Jurkat, showed a greater rate of caspase- and annexin V-induced apoptotic cell death compared to the p53-mutant LFS 2673 cell line although mitochondrial and nuclear assays could not detect apoptosis in these organelles. The most intriguing part of the observation was the abnormal proliferation rate of the wild-type p53-containing cell line, which grew twice as fast as 2673 and Jurkat cells. This is important because apoptosis inducers acting through the mitochondrial death pathway are emerging as promising drugs against tumors where the role of p53 is not only to target gene regulation but also to block cell proliferation. This study casts a long shadow on the possible dysregulation of p53 mediators that enable cell proliferation. The deregulation of proliferation pathways represents an important anticancer therapeutic strategy for patients with the LFS phenotype.

  9. Essential Oil from Cryptomeria japonica Induces Apoptosis in Human Oral Epidermoid Carcinoma Cells via Mitochondrial Stress and Activation of Caspases

    OpenAIRE

    Cha, Jeong-Dan; Kim, Ji-Young

    2012-01-01

    Cryptomeria japonica D. Don (C. japonica) has been used in traditional medicines from Asia for a variety of indications, including liver ailments, and an antitussive, and for its antiulcer activities. We examined the cell viability and apoptosis of KB cells treated with C. japonica essential oil at several concentrations for 12 h by MTT assay, Hoechst-33258 dye staining, DNA fragmentation, flow cytometry (cell cycle), and Western blotting for mitochondria stress, activation of caspases, and p...

  10. TUG1 promotes lens epithelial cell apoptosis by regulating miR-421/caspase-3 axis in age-related cataract.

    Science.gov (United States)

    Li, Guoxing; Song, Huiyang; Chen, Lei; Yang, Weihua; Nan, Kaihui; Lu, Peirong

    2017-07-01

    Age-related cataract is among the most common chronic disorders of ageing and the apoptosis of lens epithelial cells contributes to non-congenital cataract development. We amid to explore the role of TUG1 and miR-421 in the age-related cataract. The expression level of TUG1, miR-421 and caspase-3 were detected by RT-qPCR. The apoptotic-related protein, caspase-3, Bax and blc-2 were analyzed by western blot. We performed ultraviolet (UV) irradiation to induce SAR01/04 cell apoptosis which was analyzed by flow cytometry. RIP pull-down and luciferase reporter assay were used to verified the combination and regulating among TUG1, miR-421 and caspase-3. Here, we observed that the expression level of TUG1 and caspase-3 in the anterior lens capsules of age-related cataract were significantly higher and miR-421 was significantly lower than that in the normal anterior lens capsules. The apoptosis-related protein, caspase-3, Bax and blc-2 were abnormal expression in the anterior lens capsules of age-related cataract tissue. Our data showed that the expression level of TUG1 and caspase-3 and cell apoptosis rate in SAR01/04 cells treated with UV irradiation was remarkably higher than that in the control. TUG1 negatively regulated miR-421 expression and promoted UV irradiation-induced SAR01/04 cell apoptosis. However, miR-421 inhibitor and pcDNA-caspase-3 could reverse the action of the SRA01/04 cell apoptosis by si-TUG1, which suggested TUG1 promoted UV irradiation-induced apoptosis through downregulating miR-421 expression. Furthermore, this study confirmed TUG1 could been in combination with miR-421, and TUG1 and caspase-3 were both a directly target of miR-421. TUG1 modulated lens epithelial cell apoptosis through miR-421/caspase-3 axis. These findings will offer a novel insight into the pathogenesis of cataract. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Enhanced caspase activity contributes to aortic wall remodeling and early aneurysm development in a murine model of Marfan syndrome.

    Science.gov (United States)

    Emrich, Fabian C; Okamura, Homare; Dalal, Alex R; Penov, Kiril; Merk, Denis R; Raaz, Uwe; Hennigs, Jan K; Chin, Jocelyn T; Miller, Miquell O; Pedroza, Albert J; Craig, Juliana K; Koyano, Tiffany K; Blankenberg, Francis G; Connolly, Andrew J; Mohr, Friedrich W; Alvira, Cristina M; Rabinovitch, Marlene; Fischbein, Michael P

    2015-01-01

    Rupture and dissection of aortic root aneurysms remain the leading causes of death in patients with the Marfan syndrome, a hereditary connective tissue disorder that affects 1 in 5000 individuals worldwide. In the present study, we use a Marfan mouse model (Fbn1(C1039G/+)) to investigate the biological importance of apoptosis during aneurysm development in Marfan syndrome. Using in vivo single-photon emission computed tomographic-imaging and ex vivo autoradiography for Tc99m-annexin, we discovered increased apoptosis in the Fbn1(C1039G/+) ascending aorta during early aneurysm development peaking at 4 weeks. Immunofluorescence colocalization studies identified smooth muscle cells (SMCs) as the apoptotic cell population. As biological proof of concept that early aortic wall apoptosis plays a role in aneurysm development in Marfan syndrome, Fbn1(C1039G/+) mice were treated daily from 2 to 6 weeks with either (1) a pan-caspase inhibitor, Q-VD-OPh (20 mg/kg), or (2) vehicle control intraperitoneally. Q-VD-OPh treatment led to a significant reduction in aneurysm size and decreased extracellular matrix degradation in the aortic wall compared with control mice. In vitro studies using Fbn1(C1039G/+) ascending SMCs showed that apoptotic SMCs have increased elastolytic potential compared with viable cells, mostly because of caspase activity. Moreover, in vitro (1) cell membrane isolation, (2) immunofluorescence staining, and (3) scanning electron microscopy studies illustrate that caspases are expressed on the exterior cell surface of apoptotic SMCs. Caspase inhibition attenuates aneurysm development in an Fbn1(C1039G/+) Marfan mouse model. Mechanistically, during apoptosis, caspases are expressed on the cell surface of SMCs and likely contribute to elastin degradation and aneurysm development in Marfan syndrome. © 2014 American Heart Association, Inc.

  12. Roles of inflammatory caspases during processing of zebrafish interleukin-1β in Francisella noatunensis infection

    Science.gov (United States)

    Vojtech, Lucia N.; Scharping, Nichole; Woodson, James C.; Hansen, John D.

    2012-01-01

    The interleukin-1 family of cytokines are essential for the control of pathogenic microbes but are also responsible for devastating autoimmune pathologies. Consequently, tight regulation of inflammatory processes is essential for maintaining homeostasis. In mammals, interleukin-1 beta (IL-1β) is primarily regulated at two levels, transcription and processing. The main pathway for processing IL-1β is the inflammasome, a multiprotein complex that forms in the cytosol and which results in the activation of inflammatory caspase (caspase 1) and the subsequent cleavage and secretion of active IL-1β. Although zebrafish encode orthologs of IL-1β and inflammatory caspases, the processing of IL-1β by activated caspase(s) has never been examined. Here, we demonstrate that in response to infection with the fish-specific bacterial pathogen Francisella noatunensis, primary leukocytes from adult zebrafish display caspase-1-like activity that results in IL-1β processing. Addition of caspase 1 or pancaspase inhibitors considerably abrogates IL-1β processing. As in mammals, this processing event is concurrent with the secretion of cleaved IL-1β into the culture medium. Furthermore, two putative zebrafish inflammatory caspase orthologs, caspase A and caspase B, are both able to cleave IL-1β, but with different specificities. These results represent the first demonstration of processing and secretion of zebrafish IL-1β in response to a pathogen, contributing to our understanding of the evolutionary processes governing the regulation of inflammation.                   

  13. Cambogin Induces Caspase-Independent Apoptosis through the ROS/JNK Pathway and Epigenetic Regulation in Breast Cancer Cells.

    Science.gov (United States)

    Shen, Kaikai; Xie, Jianling; Wang, Hua; Zhang, Hong; Yu, Mengyuan; Lu, Fangfang; Tan, Hongsheng; Xu, Hongxi

    2015-07-01

    Cambogin is a polycyclic polyprenylated acylphoroglucinol (PPAP) from the Garcinia genus, which has been used traditionally for cancer treatment across Southeastern Asia. In this study, we found that cambogin inhibited breast cancer cell proliferation and induced cell apoptosis in vitro. Cambogin induced the activation of the caspase-independent mitochondrial apoptotic pathway, as indicated by an increase in the ratio of Bax/Bcl-2 and the nuclear translocation of apoptosis inducing factor (AIF). Two-dimensional gel electrophoresis and mass spectrometry revealed that the expression of proteins involving in the radical oxygen species (ROS) pathway was among the most affected upon cambogin treatment. Cambogin enhanced cellular ROS production, and induced the activation of the ASK1-MKK4/MKK7-JNK/SAPK signaling pathway. Pretreatment with ROS scavenger N-acetylcysteine (NAC), an antioxidant, or the JNK inhibitor SP600125 was able to restore cell viability in the presence of cambogin. Importantly, cambogin treatment led to the activation of activating transcription factor-2 (ATF-2) and the trimethylation of histone H3K9 in the activator protein 1 (AP-1) binding region of the Bcl-2 gene promoter. Finally, cambogin exhibited a potential antitumor effect in MCF-7 breast cancer xenografts without apparent toxicity. Taken in conjunction, the present study indicates that cambogin can induce breast adenocarcinoma cell apoptosis and therefore represents therapeutic potential for cancer treatment. ©2015 American Association for Cancer Research.

  14. Paroxetine-induced apoptosis in human osteosarcoma cells: Activation of p38 MAP kinase and caspase-3 pathways without involvement of [Ca2+]i elevation

    International Nuclear Information System (INIS)

    Chou, C.-T.; He Shiping; Jan, C.-R.

    2007-01-01

    Selective serotonin reuptake inhibitors (SSRIs), a group of antidepressants, are generally used for treatment of various mood and anxiety disorders. There has been much research showing the anti-tumor and cytotoxic activities of some antidepressants; but the detailed mechanisms were unclear. In cultured human osteosarcoma cells (MG63), paroxetine reduced cell viability in a concentration- and time-dependent manner. Paroxetine caused apoptosis as assessed by propidium iodide-stained cells and increased caspase-3 activation. Although immunoblotting data revealed that paroxetine could activate the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH 2 -terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), only SB203580 (a p38 MAPK inhibitor) partially prevented cells from apoptosis. Paroxetine also induced [Ca 2+ ] i increases which involved the mobilization of intracellular Ca 2+ stored in the endoplasmic reticulum and Ca 2+ influx from extracellular medium. However, pretreatment with BAPTA/AM, a Ca 2+ chelator, to prevent paroxetine-induced [Ca 2+ ] i increases did not protect cells from death. The results suggest that in MG63 cells, paroxetine caused Ca 2+ -independent apoptosis via inducing p38 MAPK-associated caspase-3 activation

  15. The co-repressor SMRT delays DNA damage-induced caspase activation by repressing pro-apoptotic genes and modulating the dynamics of checkpoint kinase 2 activation.

    Directory of Open Access Journals (Sweden)

    Claudio Scafoglio

    Full Text Available Checkpoint kinase 2 (Chk2 is a major regulator of DNA damage response and can induce alternative cellular responses: cell cycle arrest and DNA repair or programmed cell death. Here, we report the identification of a new role of Chk2 in transcriptional regulation that also contributes to modulating the balance between survival and apoptosis following DNA damage. We found that Chk2 interacts with members of the NCoR/SMRT transcriptional co-regulator complexes and serves as a functional component of the repressor complex, being required for recruitment of SMRT on the promoter of pro-apoptotic genes upon DNA damage. Thus, the co-repressor SMRT exerts a critical protective action against genotoxic stress-induced caspase activation, repressing a functionally important cohort of pro-apoptotic genes. Amongst them, SMRT is responsible for basal repression of Wip1, a phosphatase that de-phosphorylates and inactivates Chk2, thus affecting a feedback loop responsible for licensing the correct timing of Chk2 activation and the proper execution of the DNA repair process.

  16. Gecko proteins induce the apoptosis of bladder cancer 5637 cells by inhibiting Akt and activating the intrinsic caspase cascade.

    Science.gov (United States)

    Kim, Geun-Young; Park, Soon Yong; Jo, Ara; Kim, Mira; Leem, Sun-Hee; Jun, Woo-Jin; Shim, Sang In; Lee, Sang Chul; Chung, Jin Woong

    2015-09-01

    Gecko proteins have long been used as anti-tumor agents in oriental medicine, without any scientific background. Although anti-tumor effects of Gecko proteins on several cancers were recently reported, their effect on bladder cancer has not been investigated. Thus, we explored the anti-tumor effect of Gecko proteins and its cellular mechanisms in human bladder cancer 5637 cells. Gecko proteins significantly reduced the viability of 5637 cells without any cytotoxic effect on normal cells. These proteins increased the Annexin-V staining and the amount of condensed chromatin, demonstrating that the Gecko proteinsinduced cell death was caused by apoptosis. Gecko proteins suppressed Akt activation, and the overexpression of constitutively active form of myristoylated Akt prevented Gecko proteins-induced death of 5637 cells. Furthermore, Gecko proteins activated caspase 9 and caspase 3/7. Taken together, our data demonstrated that Gecko proteins suppressed the Akt pathway and activated the intrinsic caspase pathway, leading to the apoptosis of bladder cancer cells. [BMB Reports 2015; 48(9): 531-536].

  17. JS-K, a nitric oxide prodrug, induces cytochrome c release and caspase activation in HL-60 myeloid leukemia cells.

    Science.gov (United States)

    Udupi, Vidya; Yu, Margaret; Malaviya, Swati; Saavedra, Joseph E; Shami, Paul J

    2006-10-01

    Nitric oxide (NO) induces differentiation and apoptosis in acute myelogenous leukemia (AML) cells. The NO prodrug O2-(2,4-dinitrophenyl)1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate, or JS-K, has potent antileukemic activity. JS-K induces apoptosis in HL-60 cells by a caspase-dependent mechanism. The purpose of this study was to determine the pathway through which JS-K induces apoptosis. We show that JS-K alters mitochondrial membrane potential (DeltaPsim) and induces cytochrome c release from mitochondria into the cytoplasm. Treatment with JS-K resulted in activation of Caspase (Casp) 9, Casp 3 and Casp 8. JS-K constitutes a promising lead for a new class of anti-leukemic agents.

  18. ER-mediated stress induces mitochondrial-dependent caspases activation in NT2 neuron-like cells.

    Science.gov (United States)

    Arduino, Daniela M; Esteves, A Raquel; Domingues, A Filipa; Pereira, Claudia M F; Cardoso, Sandra M; Oliveira, Catarina R

    2009-11-30

    Recent studies have revealed that endoplasmic reticulum (ER) disturbance is involved in the pathophysiology of neurodegenerative disorders, contributing to the activation of the ER stress-mediated apoptotic pathway. Therefore, we investigated here the molecular mechanisms underlying the ER-mitochondria axis, focusing on calcium as a potential mediator of cell death signals. Using NT2 cells treated with brefeldin A or tunicamycin, we observed that ER stress induces changes in the mitochondrial function, impairing mitochondrial membrane potential and distressing mitochondrial respiratory chain complex Moreover, stress stimuli at ER level evoked calcium fluxes between ER and mitochondria. Under these conditions, ER stress activated the unfolded protein response by an overexpression of GRP78, and also caspase-4 and-2, both involved upstream of caspase-9. Our findings show that ER and mitochondria interconnection plays a prominent role in the induction of neuronal cell death under particular stress circumstances.

  19. Inhibition of Prenylation Promotes Caspase 3 Activation, Lamin B Degradation and Loss in Metabolic Cell Viability in Pancreatic β-Cells

    Directory of Open Access Journals (Sweden)

    Khadija G. Syeda

    2017-10-01

    Full Text Available Background/Aims: Lamins are intermediate filament proteins that constitute the main components of the lamina underlying the inner-nuclear membrane and serve to organize chromatin. Lamins (e.g., lamin B undergo posttranslational modifications (e.g., isoprenylation at their C-terminal cysteine residues. Such modifications are thought to render optimal association of lamins with the nuclear envelop. Using human islets, rodent islets, and INS-1 832/13 cells, we recently reported significant metabolic defects under glucotoxic and endoplasmic reticulum (ER stress conditions, including caspase 3 activation and lamin B degradation. The current study is aimed at further understanding the regulatory roles of protein prenylation in the induction of the aforestated metabolic defects. Methods: Subcellular phase partitioning assay was done using Triton X-114. Cell morphology and metabolic cell viability assays were carried out using standard methodologies. Results: We report that exposure of pancreatic β-cells to Simvastatin, an inhibitor of mevalonic acid (MVA biosynthesis, and its downstream isoprenoid derivatives, or FTI-277, an inhibitor of farnesyltransferase that mediates farnesylation of lamins, leads to activation of caspase 3 and lamin B degradation. Furthermore, Simvastatin-treatment increased activation of p38MAPK (a stress kinase and inhibited ERK1/2 (regulator of cell proliferation. Inhibition of farnesylation also resulted in the release of degraded lamin B into the cytosolic fraction and promoted loss in metabolic cell viability. Conclusion: Based on these findings we conclude that protein prenylation plays key roles in islet β-cell function. These findings affirm further support to the hypothesis that defects in prenylation pathway induce caspase-3 activation and nuclear lamin degradation in pancreatic β-cells under the duress of metabolic stress (e.g., glucotoxicity.

  20. Loss of ABCB4 attenuates the caspase-dependent apoptosis regulating resistance to 5-Fu in colorectal cancer.

    Science.gov (United States)

    Hu, Hanqing; Wang, Meng; Guan, Xu; Yuan, Ziming; Liu, Zheng; Zou, Chaoxia; Wang, Guiyu; Gao, Xu; Wang, Xishan

    2018-02-28

    The adenosine triphosphate-binding cassette (ABC) is a large group of proteins involved in material transportation, cellular homeostasis, and closely associated with chemoresistance. ATP-binding cassette protein B4 (ABCB4) is a member of ABCs which has a similar structure to ABCB1, but fewer researches were performed. The present study is aimed to investigate the putative mechanism of ABCB4 in 5-fluorouracil (5-Fu) resistance. Then, we found that ABCB4 was significantly down-regulated in the 5-Fu resistant HCT8 cell lines by polymerase chain reaction (PCR) and Western blot. The knockdown of ABCB4 by small interfering RNA decreased the apoptosis by 5-Fu in resistant HCT8R cell lines without influencing the proliferation. Also, we found a lower expression of cleaved caspase and PARP by Western blot after the knockdown of ABCB4. However, the knockdown of ABCB4 did not influence the proliferation and apoptosis. Furthermore, the histological detection of ABCB4 mRNA level in human colorectal cancer tissues and even in the recurrent tissues after 5-Fu single-agent chemotherapy was employed to provide more concrete evidence that ABCB4 may be a tumor suppressor gene to regulate chemoresistance in colorectal cancer. Moreover, a 109-patient cohort revealed that ABCB4 predicted a poor recurrence-free survival and overall survival. In summary, ABCB4 was down-regulated in the 5-Fu resistant cells and knockdown of ABCB4 alleviated the cell apoptosis and predicts a shorter recurrence-free survival and overall survival. © 2018 The Author(s).

  1. Caspases : more than just killers?

    OpenAIRE

    Los, Marek Jan; Stroh, C.; Janicke, R. U.; Engels, I. H.; Schulze-Osthoff, K.

    2001-01-01

    Proteases of the caspase family constitute the central executioners of apoptosis, Several recent observations suggest that caspases and apoptosis-regulatory molecules exert important functions beyond that of cell death, including the control of T-cell proliferation and cell-cycle progression. Here, Los and colleagues propose a model that directly connects cell suicide mechanisms to the regulation of cell-cycle progression.

  2. Caspases: more than just killers?

    Science.gov (United States)

    Los, M; Stroh, C; Jänicke, R U; Engels, I H; Schulze-Osthoff, K

    2001-01-01

    Proteases of the caspase family constitute the central executioners of apoptosis. Several recent observations suggest that caspases and apoptosis-regulatory molecules exert important functions beyond that of cell death, including the control of T-cell proliferation and cell-cycle progression. Here, Los and colleagues propose a model that directly connects cell suicide mechanisms to the regulation of cell-cycle progression.

  3. Activation of Pro-apoptotic Caspases in Non-apoptotic Cells During Odontogenesis and Related Osteogenesis

    Czech Academy of Sciences Publication Activity Database

    Švandová, Eva; Veselá, Barbora; Tucker, A. S.; Matalová, Eva

    2018-01-01

    Roč. 9, č. 1 (2018), č. článku 174. ISSN 1664-042X Institutional support: RVO:67985904 Keywords : caspase * differentiation * apoptosis Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Developmental biology Impact factor: 4.134, year: 2016

  4. The neurotoxicant PCB-95 by increasing the neuronal transcriptional repressor REST down-regulates caspase-8 and increases Ripk1, Ripk3 and MLKL expression determining necroptotic neuronal death.

    Science.gov (United States)

    Guida, Natascia; Laudati, Giusy; Serani, Angelo; Mascolo, Luigi; Molinaro, Pasquale; Montuori, Paolo; Di Renzo, Gianfranco; Canzoniero, Lorella M T; Formisano, Luigi

    2017-10-15

    Our previous study showed that the environmental neurotoxicant non-dioxin-like polychlorinated biphenyl (PCB)-95 increases RE1-silencing transcription factor (REST) expression, which is related to necrosis, but not apoptosis, of neurons. Meanwhile, necroptosis is a type of a programmed necrosis that is positively regulated by receptor interacting protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain-like (MLKL) and negatively regulated by caspase-8. Here we evaluated whether necroptosis contributes to PCB-95-induced neuronal death through REST up-regulation. Our results demonstrated that in cortical neurons PCB-95 increased RIPK1, RIPK3, and MLKL expression and decreased caspase-8 at the gene and protein level. Furthermore, the RIPK1 inhibitor necrostatin-1 or siRNA-mediated RIPK1, RIPK3 and MLKL expression knockdown significantly reduced PCB-95-induced neuronal death. Intriguingly, PCB-95-induced increases in RIPK1, RIPK3, MLKL expression and decreases in caspase-8 expression were reversed by knockdown of REST expression with a REST-specific siRNA (siREST). Notably, in silico analysis of the rat genome identified a REST consensus sequence in the caspase-8 gene promoter (Casp8-RE1), but not the RIPK1, RIPK3 and MLKL promoters. Interestingly, in PCB-95-treated neurons, REST binding to the Casp8-RE1 sequence increased in parallel with a reduction in its promoter activity, whereas under the same experimental conditions, transfection of siREST or mutation of the Casp8-RE1 sequence blocked PCB-95-induced caspase-8 reduction. Since RIPK1, RIPK3 and MLKL rat genes showed no putative REST binding site, we assessed whether the transcription factor cAMP Responsive Element Binding Protein (CREB), which has a consensus sequence in all three genes, affected neuronal death. In neurons treated with PCB-95, CREB protein expression decreased in parallel with a reduction in binding to the RIPK1, RIPK3 and MLKL gene promoter sequence. Furthermore, CREB overexpression was

  5. Confocal Microscopy and Image Analysis Indicates a Region-Specific Relation between Active Caspases and Cytoplasm in Ejaculated and Epididymal Sperm

    Science.gov (United States)

    García Vazquez, Susana; Aragón Martínez, Andrés; Flores-Alonso, Juan Carlos

    2012-01-01

    Previously, it was suggested a relation between the presence of apoptosis markers with cytoplasm in mammalian sperm. In this work, flow cytometry, confocal microscopy and image analysis were used to analyze the relationship between active caspase-3 and -7 and intracellular esterases expression in ejaculated or epididymal ram sperm. Sperm obtained from ejaculates from the caput, corpus, or cauda of the epididymis were treated with an inhibitor of active caspase-3 and -7 and a marker of cytoplasmic esterases. Additionally, ejaculated sperm were incubated for one, two, or three hours before evaluation for active caspases. Sperm subpopulations positive for active caspases and/or intracellular esterases were detected by flow cytometry and confocal microscopy; however, image analysis of confocal images showed that the correlation between active caspases and cytoplasmic esterases in sperm is region-specific. Lower values of Spearman correlation coefficients were found when whole sperm or head sperm was analyzed; however, a high correlation was observed for midpiece sperm. Incubation of sperm for two or three hours promoted the autoactivation of caspases. It has been suggested that the presence of apoptotic markers in sperm are related with a process of abortive apoptosis and with errors during spermiogenesis. Our results permit us suggest that the origin of the relationship between active caspases and cytoplasmic esterases is due to differentiation errors occurring during spermiogenesis because the percentages of sperm with active caspases are not different in the caput, corpus, or cauda of the epididymis. In this study we demonstrate that existing sperm subpopulations can express active caspases and intracellular esterases and that the correlation between these molecules is high in midpiece sperm. PMID:22530029

  6. Alendronate augments interleukin-1β release from macrophages infected with periodontal pathogenic bacteria through activation of caspase-1

    International Nuclear Information System (INIS)

    Deng Xue; Tamai, Riyoko; Endo, Yasuo; Kiyoura, Yusuke

    2009-01-01

    Nitrogen-containing bisphosphonates (NBPs) are anti-bone-resorptive drugs with inflammatory side effects that include osteomyelitis and osteonecrosis of the jaw. Oral bacteria have been considered to be a trigger for these NBP-associated jaw bone diseases. The present study examined the effects of alendronate (a typical NBP) and clodronate (a non-NBP) on the production of proinflammatory cytokines by macrophages infected with Porphyromonas gingivalis and Tannerella forsythia, which are important pathogens of periodontal diseases. Pretreatment with alendronate augmented IL-1β, but not TNFα, production by macrophages infected with P. gingivalis or T. forsythia. This augmentation of IL-1β production was inhibited by clodronate. Furthermore, caspase-1, a promoter of IL-1β production, was activated by treatment with alendronate, and caspase-1 inhibitor reduced the production of IL-1β induced by alendronate and P. gingivalis. These results suggest that NBPs augment periodontal pathogenic bacteria-induced IL-1β release via caspase-1 activation, and this phenomenon may contribute to the development of NBP-associated inflammatory side effects including jaw osteomyelitis. Co-treatment with clodronate may prevent and/or reduce these inflammatory effects induced by NBPs

  7. Alendronate augments interleukin-1{beta} release from macrophages infected with periodontal pathogenic bacteria through activation of caspase-1

    Energy Technology Data Exchange (ETDEWEB)

    Xue, Deng; Tamai, Riyoko [Division of Oral Bacteriology, Department of Oral Medical Science, Ohu University School of Dentistry, 31-1 Misumido, Tomitamachi, Koriyama, Fukushima 963-8611 (Japan); Endo, Yasuo [Department of Molecular Regulation, Graduate School of Dentistry, Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Kiyoura, Yusuke [Division of Oral Bacteriology, Department of Oral Medical Science, Ohu University School of Dentistry, 31-1 Misumido, Tomitamachi, Koriyama, Fukushima 963-8611 (Japan)

    2009-02-15

    Nitrogen-containing bisphosphonates (NBPs) are anti-bone-resorptive drugs with inflammatory side effects that include osteomyelitis and osteonecrosis of the jaw. Oral bacteria have been considered to be a trigger for these NBP-associated jaw bone diseases. The present study examined the effects of alendronate (a typical NBP) and clodronate (a non-NBP) on the production of proinflammatory cytokines by macrophages infected with Porphyromonas gingivalis and Tannerella forsythia, which are important pathogens of periodontal diseases. Pretreatment with alendronate augmented IL-1{beta}, but not TNF{alpha}, production by macrophages infected with P. gingivalis or T. forsythia. This augmentation of IL-1{beta} production was inhibited by clodronate. Furthermore, caspase-1, a promoter of IL-1{beta} production, was activated by treatment with alendronate, and caspase-1 inhibitor reduced the production of IL-1{beta} induced by alendronate and P. gingivalis. These results suggest that NBPs augment periodontal pathogenic bacteria-induced IL-1{beta} release via caspase-1 activation, and this phenomenon may contribute to the development of NBP-associated inflammatory side effects including jaw osteomyelitis. Co-treatment with clodronate may prevent and/or reduce these inflammatory effects induced by NBPs.

  8. Granzyme B of cytotoxic T cells induces extramitochondrial reactive oxygen species production via caspase-dependent NADPH oxidase activation.

    Science.gov (United States)

    Aguiló, Juan I; Anel, Alberto; Catalán, Elena; Sebastián, Alvaro; Acín-Pérez, Rebeca; Naval, Javier; Wallich, Reinhard; Simon, Markus M; Pardo, Julián

    2010-07-01

    Induction of reactive oxygen species (ROS) is a hallmark of granzyme B (gzmB)-mediated pro-apoptotic processes and target cell death. However, it is unclear to what extent the generated ROS derive from mitochondrial and/or extra-mitochondrial sources. To clarify this point, we have produced a mutant EL4 cell line, termed EL4-rho(0), which lacks mitochondrial DNA, associated with a decreased mitochondrial membrane potential and a defective ROS production through the electron transport chain of oxidative phosphorylation. When incubated with either recombinant gzmB plus streptolysin or ex vivo gzmB(+) cytotoxic T cells, EL4-rho(0) cells showed phosphatydylserine translocation, caspase 3 activation, Bak conformational change, cytochrome c release and apoptotic morphology comparable to EL4 cells. Moreover, EL4-rho(0) cells produced ROS at levels similar to EL4 under these conditions. GzmB-mediated ROS production was almost totally abolished in both cell lines by the pan-caspase inhibitor, Z-VAD-fmk. However, addition of apocynin, a specific inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, led to a significant reduction of ROS production and cell death only in EL4-rho(0) but not EL4 cells. These data suggest that gzmB-induced cell death is accompanied by a caspase-dependent pathway of extra-mitochondrial ROS production, most probably through activation of NADPH oxidase.

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

    Energy Technology Data Exchange (ETDEWEB)

    Gill, R. [Department of Immunology and Microbiology, Wayne State University, Detroit, MI (United States); Jen, K.L. [Department of Nutrition and Food Science, Wayne State University, Detroit, MI (United States); Center for Urban Responses to Environmental Stressors (CURES), Wayne State University, Detroit, MI (United States); McCabe, M.J.J. [Department of Environmental Medicine, University of Rochester, Rochester, NY (United States); Rosenspire, A., E-mail: arosenspire@wayne.edu [Department of Immunology and Microbiology, Wayne State University, Detroit, MI (United States); Center for Urban Responses to Environmental Stressors (CURES), Wayne State University, Detroit, MI (United States)

    2016-10-15

    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 conceptualize 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

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

    International Nuclear Information System (INIS)

    Gill, R.; Jen, K.L.; McCabe, M.J.J.; Rosenspire, A.

    2016-01-01

    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 conceptualize 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

  11. Caspase 3 activation in the primary enamel knot of developing molar tooth

    Czech Academy of Sciences Publication Activity Database

    Matalová, Eva; Kovářů, František; Míšek, Ivan

    2006-01-01

    Roč. 55, 2 (2006), s. 183-188 ISSN 0862-8408 R&D Projects: GA ČR GA304/04/0101; GA AV ČR KJB500450503; GA MŠk OC B23.001 Institutional research plan: CEZ:AV0Z50450515 Keywords : apoptosis * caspase 3 * primary enamel knot Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.093, year: 2006

  12. Active caspase-3 and ultrastructural evidence of apoptosis in spontaneous and induced cell death in bovine in vitro produced pre-implantation embryos

    DEFF Research Database (Denmark)

    Gjørret, Jakob O.; Fabian, Dusan; Avery, Birthe

    2007-01-01

    In this study we investigated chronological onset and involvement of active caspase-3, apoptotic nuclear morphology, and TUNEL-labeling, as well as ultrastructural evidence of apoptosis, in both spontaneous and induced cell death during pre-implantation development of bovine in vitro produced...... microscopy in both treated and untreated blastocysts. Activation of caspase-3 is likely involved in both spontaneous and induced apoptosis in bovine pre-implantation embryos, and immunohistochemical staining of active caspase-3 may be used in combination with other markers to identify apoptosis in pre...... embryos. Pre-implantation embryos (2-cell to Day 8 blastocysts) were cultured with either no supplementation (untreated) or with 10 µM staurosporine for 24 hr (treated). Embryos were subjected to immunohistochemical staining of active caspase-3, TUNEL-reaction for detection of DNA degradation and DAPI...

  13. 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.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  15. The death effector domains of caspase-8 induce terminal differentiation.

    Directory of Open Access Journals (Sweden)

    Ainhoa Mielgo

    2009-11-01

    Full Text Available The differentiation and senescence programs of metazoans play key roles in regulating normal development and preventing aberrant cell proliferation, such as cancer. These programs are intimately associated with both the mitotic and apoptotic pathways. Caspase-8 is an apical apoptotic initiator that has recently been appreciated to coordinate non-apoptotic roles in the cell. Most of these functions are attributed to the catalytic domain, however, the amino-terminal death effector domains (DEDs, which belong to the death domain superfamily of proteins, can also play key roles during development. Here we describe a novel role for caspase-8 DEDs in regulating cell differentiation and senescence. Caspase-8 DEDs accumulate during terminal differentiation and senescence of epithelial, endothelial and myeloid cells; genetic deletion or shRNA suppression of caspase-8 disrupts cell differentiation, while re-expression of DEDs rescues this phenotype. Among caspase-8 deficient neuroblastoma cells, DED expression attenuated tumor growth in vivo and proliferation in vitro via disruption of mitosis and cytokinesis, resulting in upregulation of p53 and induction of differentiation markers. These events occur independent of caspase-8 catalytic activity, but require a critical lysine (K156 in a microtubule-binding motif in the second DED domain. The results demonstrate a new function for the DEDs of caspase-8, and describe an unexpected mechanism that contributes to cell differentiation and senescence.

  16. Zinc-mediated Allosteric Inhibition of Caspase-6*

    Science.gov (United States)

    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

  17. Active caspase-3 detection to evaluate apoptosis induced by Verbena officinalis essential oil and citral in chronic lymphocytic leukaemia cells

    Directory of Open Access Journals (Sweden)

    Laura De Martino

    2011-10-01

    Full Text Available Verbena officinalis L., Verbenaceae, commonly known as vervain, is a plant widely used in medicine. Despite of its widespread use in different traditional practices, the mechanisms of pharmacological actions of the plant and its volatile oil are still unclear. We evaluated the pro-apoptotic activity of V. officinalis essential oil and of its main component, citral, on lymphocytes collected from ten patients with chronic lymphocytic leukaemia (CLL, a disease in which a faulty apoptotic mechanism is still retained one of the primary pathogenic events, by adding to treated mononuclear cells, annexin-V, propidium iodide, and CD19. Apoptosis was also evaluated using anti-active-caspase-3 monoclonal antibody after permeabilization of the cells. Both V. officinalis essential oil and citral were found able to induce apoptosis in CLL cells and to activate caspase-3, which is considered the way by means they active apoptosis in B neoplastic cells. This data further support evidences that indicate natural compounds as possible lead structure to develop new therapeutic agents for CLL.

  18. Active caspase-3 detection to evaluate apoptosis induced by Verbena officinalis essential oil and citral in chronic lymphocytic leukaemia cells

    Directory of Open Access Journals (Sweden)

    Laura De Martino

    2011-05-01

    Full Text Available Verbena officinalis L., Verbenaceae, commonly known as vervain, is a plant widely used in medicine. Despite of its widespread use in different traditional practices, the mechanisms of pharmacological actions of the plant and its volatile oil are still unclear. We evaluated the pro-apoptotic activity of V. officinalis essential oil and of its main component, citral, on lymphocytes collected from ten patients with chronic lymphocytic leukaemia (CLL, a disease in which a faulty apoptotic mechanism is still retained one of the primary pathogenic events, by adding to treated mononuclear cells, annexin-V, propidium iodide, and CD19. Apoptosis was also evaluated using anti-active-caspase-3 monoclonal antibody after permeabilization of the cells. Both V. officinalis essential oil and citral were found able to induce apoptosis in CLL cells and to activate caspase-3, which is considered the way by means they active apoptosis in B neoplastic cells. This data further support evidences that indicate natural compounds as possible lead structure to develop new therapeutic agents for CLL.

  19. siRNA - Mediated LRP/LR knock-down reduces cellular viability of malignant melanoma cells through the activation of apoptotic caspases.

    Science.gov (United States)

    Rebelo, Thalia M; Vania, Leila; Ferreira, Eloise; Weiss, Stefan F T

    2018-07-01

    The 37 kDa/67 kDa laminin receptor (LRP/LR) is over-expressed in tumor cells and has been implicated in several tumourigenic processes such as metastasis and telomerase activation, however, more importantly the focus of the present study is on the maintenance of cellular viability and the evasion of apoptosis. The aim of the study was to investigate the role of LRP/LR on the cellular viability of early (A375) and late stage (A375SM) malignant melanoma cells. Flow cytometry and western blot analysis revealed that A375SM cells contain more cell-surface and total LRP/LR levels in comparison to the A375 cells, respectively. In order to determine the effect of LRP/LR on cell viability and apoptosis, LRP was down-regulated via siRNA technology. MTT assays revealed that LRP knock-down led to significant reductions in the viability of A375 and A375SM cells. Confocal microscopy indicated nuclear morphological changes suggestive of apoptotic induction in both cell lines and Annexin-V FITC/PI assays confirmed this observation. Additionally, caspase-3 activity assays revealed that apoptosis was induced in both cell lines after siRNA-mediated down-regulation of LRP. Caspase-8 and -9 activity assays suggested that post LRP knock-down; A375 cells undergo apoptosis solely via the extrinsic pathway, while A375SM cells undergo apoptosis via the intrinsic pathway. siRNAs mediated LRP knock-down might represent a powerful alternative therapeutic strategy for the treatment of malignant melanoma through the induction of apoptosis. Copyright © 2018. Published by Elsevier Inc.

  20. CYP24A1 exacerbated activity during diabetes contributes to kidney tubular apoptosis via caspase-3 increased expression and activation.

    Directory of Open Access Journals (Sweden)

    Alexandre Tourigny

    Full Text Available Decreases in circulating 25,hydroxyl-vitamin D3 (25 OH D3 and 1,25,dihydroxyl-vitamin D3 (1,25 (OH2 D3 have been extensively documented in patients with type 2 diabetes. Nevertheless, the molecular reasons behind this drop, and whether it is a cause or an effect of disease progression is still poorly understood. With the skin and the liver, the kidney is one of the most important sites for vitamin D metabolism. Previous studies have also shown that CYP24A1 (an enzyme implicated in vitamin D metabolism, might play an important role in furthering the progression of kidney lesions during diabetic nephropathy. In this study we show a link between CYP24A1 increase and senescence followed by apoptosis induction in the renal proximal tubules of diabetic kidneys. We show that CYP24A1 expression was increased during diabetic nephropathy progression. This increase derived from protein kinase C activation and increased H(2O(2 cellular production. CYP24A1 increase had a major impact on cellular phenotype, by pushing cells into senescence, and later into apoptosis. Our data suggest that control of CYP24A1 increase during diabetes has a beneficial effect on senescence induction and caspase-3 increased expression. We concluded that diabetes induces an increase in CYP24A1 expression, destabilizing vitamin D metabolism in the renal proximal tubules, leading to cellular instability and apoptosis, and thereby accelerating tubular injury progression during diabetic nephropathy.

  1. Caspase-1 deficiency reduces intestinal and hepatic triglyceride-rich lipoprotein secretion

    NARCIS (Netherlands)

    Diepen, van Janna A.; Stienstra, Rinke; Hooiveld, Guido; Willems van Dijk, Ko; Rensen, Patrick C.

    2013-01-01

    Background and Aims: Inflammasome-mediated caspase-1 activity regulates the maturation and release of the pro-inflammatory cytokines interleukin (IL)-1ß and IL-18. Recently, we showed that caspase-1 deficiency strongly reduces high fat diet-induced adiposity although the mechanism is still unclear.

  2. Essential Oil from Cryptomeria japonica Induces Apoptosis in Human Oral Epidermoid Carcinoma Cells via Mitochondrial Stress and Activation of Caspases

    Directory of Open Access Journals (Sweden)

    Ji-Young Kim

    2012-03-01

    Full Text Available Cryptomeria japonica D. Don (C. japonica has been used in traditional medicines from Asia for a variety of indications, including liver ailments, and an antitussive, and for its antiulcer activities. We examined the cell viability and apoptosis of KB cells treated with C. japonica essential oil at several concentrations for 12 h by MTT assay, Hoechst-33258 dye staining, DNA fragmentation, flow cytometry (cell cycle, and Western blotting for mitochondria stress, activation of caspases, and poly (ADP-ribose polymerase. The essential oil induced the apoptosis of KB cells in a dose-dependent manner, which was verified by DNA fragmentation, appearance of apoptotic bodies, and the sub-G1 ratio. The essential oil also induced rapid and transient caspase-3 activity and cleavage of PARP of the KB cells. Treating the cells with the oil also caused changes in the mitochondrial level of the Bcl-2 family proteins such as Bcl-2 and Bax, thereby inducing the release of cytochrome c into the cytosol. The essential oil of C. japonica may have potential as a cancer chemopreventive and therapeutic agent.

  3. Dioscin induces caspase-independent apoptosis through activation of apoptosis-inducing factor in breast cancer cells.

    Science.gov (United States)

    Kim, Eun-Ae; Jang, Ji-Hoon; Lee, Yun-Han; Sung, Eon-Gi; Song, In-Hwan; Kim, Joo-Young; Kim, Suji; Sohn, Ho-Yong; Lee, Tae-Jin

    2014-07-01

    Dioscin, a saponin extracted from the roots of Polygonatum zanlanscianense, shows several bioactivities such as antitumor, antifungal, and antiviral properties. Although, dioscin is already known to induce cell death in variety cancer cells, the molecular basis for dioscin-induced cell death was not definitely known in cancer cells. In this study, we found that dioscin treatment induced cell death in dose-dependent manner in breast cancer cells such as MDA-MB-231, MDA-MB-453, and T47D cells. Dioscin decreased expressions of Bcl-2 and cIAP-1 proteins, which were down-regulated at the transcriptional level. Conversely, Mcl-1 protein level was down-regulated by facilitating ubiquitin/proteasome-mediated Mcl-1 degradation in dioscin-treated cells. Pretreatment with z-VAD fails to attenuate dioscin-induced cell death as well as caspase-mediated events such as cleavages of procaspase-3 and PARP. In addition, dioscin treatment increased the population of annexin V positive cells and induced DNA fragmentation in a dose-dependent manner in MDA-MB-231 cells. Furthermore, apoptosis inducing factor (AIF) was released from the mitochondria and translocated to the nucleus. Suppression in AIF expression by siRNA reduced dioscin-induced apoptosis in MDA-MB-231 cells. Taken together, our results demonstrate that dioscin-induced cell death was mediated via AIF-facilitating caspase-independent pathway as well as down-regulating anti-apoptotic proteins such as Bcl-2, cIAP-1, and Mcl-1 in breast cancer cells.

  4. Metabolic Enhancer Piracetam Attenuates the Translocation of Mitochondrion-Specific Proteins of Caspase-Independent Pathway, Poly [ADP-Ribose] Polymerase 1 Up-regulation and Oxidative DNA Fragmentation.

    Science.gov (United States)

    Verma, Dinesh Kumar; Gupta, Sonam; Biswas, Joyshree; Joshi, Neeraj; Sivarama Raju, K; Wahajuddin, Mu; Singh, Sarika

    2018-03-12

    Piracetam, a nootropic drug, has been clinically used for decades; however, its mechanism of action still remains enigmatic. The present study was undertaken to evaluate the role of mitochondrion-specific factors of caspase-independent pathway like apoptotic-inducing factor (AIF) and endonuclease-G (endo-G) in piracetam-induced neuroprotection. N2A cells treated with lipopolysaccharide (LPS) exhibited significant cytotoxicity, impaired mitochondrial activity, and reactive oxygen species generation which was significantly attenuated with piracetam co-treatment. Cells co-treated with LPS and piracetam exhibited significant uptake of piracetam in comparison to only piracetam-treated cells as estimated by liquid chromatography-mass spectrometry (LC-MSMS). LPS treatment caused significant translocation of AIF and endonuclease-G in neuronal N2A cells which were significantly attenuated with piracetam co-treatment. Significant over-expression of proinflammatory cytokines was also observed after treatment of LPS to cells which was inhibited with piracetam co-treatment demonstrating its anti-inflammatory property. LPS-treated cells exhibited significant oxidative DNA fragmentation and poly [ADP-ribose] polymerase-1 (PARP-1) up-regulation in nucleus, both of which were attenuated with piracetam treatment. Antioxidant melatonin but not z-VAD offered the inhibited LPS-induced DNA fragmentation indicating the involvement of oxidative DNA fragmentation. Further, we did not observe the altered caspase-3 level after LPS treatment initially while at a later time point, significantly augmented level of caspase-3 was observed which was not inhibited with piracetam treatment. In total, our findings indicate the interference of piracetam in mitochondrion-mediated caspase-independent pathway, as well as its anti-inflammatory and antioxidative properties. Graphical Abstract Graphical abstract indicating the novel interference of metabolic enhancer piracetam (P) in neuronal death

  5. Loss of mitochondrial transmembrane potential and caspase-9 activation during apoptosis induced by the novel styryl-lactone goniothalamin in HL-60 leukemia cells.

    Science.gov (United States)

    Inayat-Hussain, S H; Annuar, B O; Din, L B; Ali, A M; Ross, D

    2003-08-01

    Styryl-lactones such as goniothalamin represent a new class of compounds with potential anti-cancer properties. In this study, we investigated the mechanisms of goniothalamin (GTN), a plant styryl-lactone induced apoptosis in human promyelocytic leukemia HL-60 cells. This plant extract resulted in apoptosis in HL-60 cells as assessed by the externalisation of phosphatidylserine. Using the mitochondrial membrane dye (DIOC(6)) in conjunction with flow cytometry, we found that GTN treated HL-60 cells demonstrated a loss of mitochondrial transmembrane potential (Deltapsi(m)). Further immunoblotting on these cells showed activation of initiator caspase-9 and the executioner caspases-3 and -7. Pretreatment with the pharmacological caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.FMK) abrogated apoptosis as assessed by all of the apoptotic features in this study. In summary, our results demonstrate that goniothalamin-induced apoptosis occurs via the mitochondrial pathway in a caspase dependent manner.

  6. RBFOX3 Regulates the Chemosensitivity of Cancer Cells to 5-Fluorouracil via the PI3K/AKT, EMT and Cytochrome-C/Caspase Pathways

    Directory of Open Access Journals (Sweden)

    Tianze Liu

    2018-04-01

    Full Text Available Background/Aims: RBFOX3, an RNA-binding fox protein, plays an important role in the differentiation of neuronal development, but its role in the chemosensitivity of hepatocellular carcinoma (HCC to 5-FU is unknown. Methods: In this study, we examined the biological functions of RBFOX3 and its effect on the chemosensitivity of HCC cells to 5-FU in vitro and in a mouse xenograft model. Results: RBFOX3 was found to have elevated expression in HCC cell lines and tissue samples, and its knockdown inhibited HCC cell proliferation. Moreover, knockdown of RBFOX3 improved the inhibitory effect of 5-fluorouracil (5-FU on cell proliferation, migration and invasion, and enhanced the apoptosis induced by 5-FU. However, overexpression of RBFOX3 reduced the inhibitory effect of 5-fluorouracil (5-FU on cell proliferation, migration and invasion, and decreased the apoptosis induced by 5-FU. We further elucidated that RBFOX3 knockdown synergized with 5-FU to inhibit the growth and invasion of HCC cells through PI3K/AKT and epithelial-mesenchymal transition (EMT signaling, and promote apoptosis by activating the cytochrome-c/caspase signaling pathway. Finally, we validated that RBFOX3 regulated 5-FU-mediated cytotoxicity in HCC in mouse xenograft models. Conclusions: The findings from this study indicate that RBFOX3 regulates the chemosensitivity of HCC to 5-FU in vitro and in vivo. Therefore, targeting RBFOX3 may improve the inhibition of HCC growth and progression by 5-FU, and provide a novel potential therapeutic strategy for HCC.

  7. G-CSF prevents caspase 3 activation in Schwann cells after sciatic nerve transection, but does not improve nerve regeneration.

    Science.gov (United States)

    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.

  8. Copper exposure induces toxicity to the antioxidant system via the destruction of Nrf2/ARE signaling and caspase-3-regulated DNA damage in fish muscle: Amelioration by myo-inositol

    International Nuclear Information System (INIS)

    Jiang, Wei-Dan; Liu, Yang; Jiang, Jun; Wu, Pei; Feng, Lin; Zhou, Xiao-Qiu

    2015-01-01

    time that Cu exposure caused oxidative damage to the muscle by decreasing the antioxidant enzyme activities via the down-regulation of the expression of genes related to the disruption of the Nrf2/ARE signaling, and this down-regulation was partially caused by caspase-3-regulated DNA fragmentation. Finally, MI protects fish against Cu toxicity

  9. Copper exposure induces toxicity to the antioxidant system via the destruction of Nrf2/ARE signaling and caspase-3-regulated DNA damage in fish muscle: Amelioration by myo-inositol

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Wei-Dan; Liu, Yang [Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Jiang, Jun [Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Wu, Pei [Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Feng, Lin, E-mail: fenglin@sicau.edu.cn [Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Zhou, Xiao-Qiu, E-mail: zhouxq@sicau.edu.cn [Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Chengdu 611130, Sichuan (China)

    2015-02-15

    time that Cu exposure caused oxidative damage to the muscle by decreasing the antioxidant enzyme activities via the down-regulation of the expression of genes related to the disruption of the Nrf2/ARE signaling, and this down-regulation was partially caused by caspase-3-regulated DNA fragmentation. Finally, MI protects fish against Cu toxicity.

  10. LPS inhibits caspase 3-dependent apoptosis in RAW264.7 macrophages induced by the AMPK activator AICAR

    International Nuclear Information System (INIS)

    Russe, Otto Quintus; Möser, Christine V.; Kynast, Katharina L.; King, Tanya S.; Olbrich, Katrin; Grösch, Sabine; Geisslinger, Gerd; Niederberger, Ellen

    2014-01-01

    Highlights: • AMPK-activation induces caspase 3-dependent apoptosis in macrophages. • Apoptosis is associated with decreased mTOR and increased p21 levels. • All effects can be significantly inhibited by the TLR4 agonist lipopolysaccharide. - Abstract: AMP-activated kinase is a cellular energy sensor which is activated in stages of increased ATP consumption. Its activation has been associated with a number of beneficial effects such as decreasing inflammatory processes and the disease progress of diabetes and obesity, respectively. Furthermore, AMPK activation has been linked with induction of cell cycle arrest and apoptosis in cancer and vascular cells, indicating that it might have a therapeutic impact for the treatment of cancer and atherosclerosis. However, the impact of AMPK on the proliferation of macrophages, which also play a key role in the formation of atherosclerotic plaques and in inflammatory processes, has not been focused so far. We have assessed the influence of AICAR- and metformin-induced AMPK activation on cell viability of macrophages with and without inflammatory stimulation, respectively. In cells without inflammatory stimulation, we found a strong induction of caspase 3-dependent apoptosis associated with decreased mTOR levels and increased expression of p21. Interestingly, these effects could be inhibited by co-stimulation with bacterial lipopolysaccharide (LPS) but not by other proinflammatory cytokines suggesting that AICAR induces apoptosis via AMPK in a TLR4-pathway dependent manner. In conclusion, our results revealed that AMPK activation is not only associated with positive effects but might also contribute to risk factors by disturbing important features of macrophages. The fact that LPS is able to restore AMPK-associated apoptosis might indicate an important role of TLR4 agonists in preventing unfavorable cell death of immune cells

  11. LPS inhibits caspase 3-dependent apoptosis in RAW264.7 macrophages induced by the AMPK activator AICAR

    Energy Technology Data Exchange (ETDEWEB)

    Russe, Otto Quintus, E-mail: quintus@russe.eu; Möser, Christine V., E-mail: chmoeser@hotmail.com; Kynast, Katharina L., E-mail: katharina.kynast@googlemail.com; King, Tanya S., E-mail: tanya.sarah.king@googlemail.com; Olbrich, Katrin, E-mail: Katrin.olbrich@gmx.net; Grösch, Sabine, E-mail: groesch@em.uni-frankfurt.de; Geisslinger, Gerd, E-mail: geisslinger@em.uni-frankfurt.de; Niederberger, Ellen, E-mail: e.niederberger@em.uni-frankfurt.de

    2014-05-09

    Highlights: • AMPK-activation induces caspase 3-dependent apoptosis in macrophages. • Apoptosis is associated with decreased mTOR and increased p21 levels. • All effects can be significantly inhibited by the TLR4 agonist lipopolysaccharide. - Abstract: AMP-activated kinase is a cellular energy sensor which is activated in stages of increased ATP consumption. Its activation has been associated with a number of beneficial effects such as decreasing inflammatory processes and the disease progress of diabetes and obesity, respectively. Furthermore, AMPK activation has been linked with induction of cell cycle arrest and apoptosis in cancer and vascular cells, indicating that it might have a therapeutic impact for the treatment of cancer and atherosclerosis. However, the impact of AMPK on the proliferation of macrophages, which also play a key role in the formation of atherosclerotic plaques and in inflammatory processes, has not been focused so far. We have assessed the influence of AICAR- and metformin-induced AMPK activation on cell viability of macrophages with and without inflammatory stimulation, respectively. In cells without inflammatory stimulation, we found a strong induction of caspase 3-dependent apoptosis associated with decreased mTOR levels and increased expression of p21. Interestingly, these effects could be inhibited by co-stimulation with bacterial lipopolysaccharide (LPS) but not by other proinflammatory cytokines suggesting that AICAR induces apoptosis via AMPK in a TLR4-pathway dependent manner. In conclusion, our results revealed that AMPK activation is not only associated with positive effects but might also contribute to risk factors by disturbing important features of macrophages. The fact that LPS is able to restore AMPK-associated apoptosis might indicate an important role of TLR4 agonists in preventing unfavorable cell death of immune cells.

  12. The activity of the anti-apoptotic fragment generated by the caspase-3/p120 RasGAP stress-sensing module displays strict Akt isoform specificity.

    Science.gov (United States)

    Vanli, Güliz; Peltzer, Nieves; Dubuis, Gilles; Widmann, Christian

    2014-12-01

    The caspase-3/p120 RasGAP module acts as a stress sensor that promotes pro-survival or pro-death signaling depending on the intensity and the duration of the stressful stimuli. Partial cleavage of p120 RasGAP generates a fragment, called fragment N, which protects stressed cells by activating Akt signaling. Akt family members regulate many cellular processes including proliferation, inhibition of apoptosis and metabolism. These cellular processes are regulated by three distinct Akt isoforms: Akt1, Akt2 and Akt3. However, which of these isoforms are required for fragment N mediated protection have not been defined. In this study, we investigated the individual contribution of each isoform in fragment N-mediated cell protection against Fas ligand induced cell death. To this end, DLD1 and HCT116 isogenic cell lines lacking specific Akt isoforms were used. It was found that fragment N could activate Akt1 and Akt2 but that only the former could mediate the protective activity of the RasGAP-derived fragment. Even overexpression of Akt2 or Akt3 could not rescue the inability of fragment N to protect cells lacking Akt1. These results demonstrate a strict Akt isoform requirement for the anti-apoptotic activity of fragment N. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Profiling cellular bioenergetics, glutathione levels, and caspase activities in stomach biopsies of patients with upper gastrointestinal symptoms

    Science.gov (United States)

    Alfazari, Ali S; Al-Dabbagh, Bayan; Al-Dhaheri, Wafa; Taha, Mazen S; Chebli, Ahmad A; Fontagnier, Eva M; Koutoubi, Zaher; Kochiyi, Jose; Karam, Sherif M; Souid, Abdul-Kader

    2015-01-01

    AIM: To measure biochemical parameters in stomach biopsies and test their suitability as diagnostic biomarkers for gastritis and precancerous lesions. METHODS: Biopsies were obtained from the stomachs of two groups of patients (n = 40) undergoing fiber-optic endoscopy due to upper gastrointestinal symptoms. In the first group (n = 17), only the corpus region was examined. Biopsies were processed for microscopic examination and measurement of mitochondrial O2 consumption (cellular respiration), cellular adenosine triphosphate (ATP), glutathione (GSH), and caspase activity. In the second group of patients (n = 23), both corpus and antral regions were studied. Some biopsies were processed for microscopic examination, while the others were used for measurements of cellular respiration and GSH level. RESULTS: Microscopic examinations of gastric corpus biopsies from 17 patients revealed normal mucosae in 8 patients, superficial gastritis in 7 patients, and chronic atrophic gastritis in 1 patient. In patients with normal histology, the rate (mean ± SD) of cellular respiration was 0.17 ± 0.02 μmol/L O2 min-1 mg-1, ATP content was 487 ± 493 pmol/mg, and GSH was 469 ± 98 pmol/mg. Caspase activity was detected in 3 out of 8 specimens. The values of ATP and caspase activity were highly variable. The presence of superficial gastritis had insignificant effects on the measured biomarkers. In the patient with atrophic gastritis, cellular respiration was high and ATP was relatively low, suggesting uncoupling oxidative phosphorylation. In the second cohort of patients, the examined biopsies showed either normal or superficial gastritis. The rate of cellular respiration (O2. μmol/L min-1 mg-1) was slightly higher in the corpus than the antrum (0.18 ± 0.05 vs 0.15 ± 0.04, P = 0.019). The value of GSH was about the same in both tissues (310 ± 135 vs 322 ± 155, P = 0.692). CONCLUSION: The corpus mucosa was metabolically more active than the antrum tissue. The data in this

  14. Docetaxel-induced prostate cancer cell death involves concomitant activation of caspase and lysosomal pathways and is attenuated by LEDGF/p75

    Directory of Open Access Journals (Sweden)

    Leoh Lai

    2009-08-01

    Full Text Available Abstract Background Hormone-refractory prostate cancer (HRPC is characterized by poor response to chemotherapy and high mortality, particularly among African American men when compared to other racial/ethnic groups. It is generally accepted that docetaxel, the standard of care for chemotherapy of HRPC, primarily exerts tumor cell death by inducing mitotic catastrophe and caspase-dependent apoptosis following inhibition of microtubule depolymerization. However, there is a gap in our knowledge of mechanistic events underlying docetaxel-induced caspase-independent cell death, and the genes that antagonize this process. This knowledge is important for circumventing HRPC chemoresistance and reducing disparities in prostate cancer mortality. Results We investigated mechanistic events associated with docetaxel-induced death in HRPC cell lines using various approaches that distinguish caspase-dependent from caspase-independent cell death. Docetaxel induced both mitotic catastrophe and caspase-dependent apoptosis at various concentrations. However, caspase activity was not essential for docetaxel-induced cytotoxicity since cell death associated with lysosomal membrane permeabilization still occurred in the presence of caspase inhibitors. Partial inhibition of docetaxel-induced cytotoxicity was observed after inhibition of cathepsin B, but not inhibition of cathepsins D and L, suggesting that docetaxel induces caspase-independent, lysosomal cell death. Simultaneous inhibition of caspases and cathepsin B dramatically reduced docetaxel-induced cell death. Ectopic expression of lens epithelium-derived growth factor p75 (LEDGF/p75, a stress survival autoantigen and transcription co-activator, attenuated docetaxel-induced lysosomal destabilization and cell death. Interestingly, LEDGF/p75 overexpression did not protect cells against DTX-induced mitotic catastrophe, and against apoptosis induced by tumor necrosis factor related apoptosis inducing ligand (TRAIL

  15. Bioorthogonal cyclization-mediated in situ self-assembly of small-molecule probes for imaging caspase activity in vivo

    Science.gov (United States)

    Ye, Deju; Shuhendler, Adam J.; Cui, Lina; Tong, Ling; Tee, Sui Seng; Tikhomirov, Grigory; Felsher, Dean W.; Rao, Jianghong

    2014-06-01

    Directed self-assembly of small molecules in living systems could enable a myriad of applications in biology and medicine, and already this has been used widely to synthesize supramolecules and nano/microstructures in solution and in living cells. However, controlling the self-assembly of synthetic small molecules in living animals is challenging because of the complex and dynamic in vivo physiological environment. Here we employ an optimized first-order bioorthogonal cyclization reaction to control the self-assembly of a fluorescent small molecule, and demonstrate its in vivo applicability by imaging caspase-3/7 activity in human tumour xenograft mouse models of chemotherapy. The fluorescent nanoparticles assembled in situ were imaged successfully in both apoptotic cells and tumour tissues using three-dimensional structured illumination microscopy. This strategy combines the advantages offered by small molecules with those of nanomaterials and should find widespread use for non-invasive imaging of enzyme activity in vivo.

  16. Human telomerase activity regulation

    OpenAIRE

    Wojtyla, Aneta; Gladych, Marta; Rubis, Blazej

    2010-01-01

    Telomerase has been recognized as a relevant factor distinguishing cancer cells from normal cells. Thus, it has become a very promising target for anticancer therapy. The cell proliferative potential can be limited by replication end problem, due to telomeres shortening, which is overcome in cancer cells by telomerase activity or by alternative telomeres lengthening (ALT) mechanism. However, this multisubunit enzymatic complex can be regulated at various levels, including expression control b...

  17. Tumor necrosis factor-alpha potentiates the cytotoxicity of amiodarone in Hepa1c1c7 cells: roles of caspase activation and oxidative stress.

    Science.gov (United States)

    Lu, Jingtao; Miyakawa, Kazuhisa; Roth, Robert A; Ganey, Patricia E

    2013-01-01

    Amiodarone (AMD), a class III antiarrhythmic drug, causes idiosyncratic hepatotoxicity in human patients. We demonstrated previously that tumor necrosis factor-alpha (TNF-α) plays an important role in a rat model of AMD-induced hepatotoxicity under inflammatory stress. In this study, we developed a model in vitro to study the roles of caspase activation and oxidative stress in TNF potentiation of AMD cytotoxicity. AMD caused cell death in Hepa1c1c7 cells, and TNF cotreatment potentiated its toxicity. Activation of caspases 9 and 3/7 was observed in AMD/TNF-cotreated cells, and caspase inhibitors provided minor protection from cytotoxicity. Intracellular reactive oxygen species (ROS) generation and lipid peroxidation were observed after treatment with AMD and were further elevated by TNF cotreatment. Adding water-soluble antioxidants (trolox, N-acetylcysteine, glutathione, or ascorbate) produced only minor attenuation of AMD/TNF-induced cytotoxicity and did not influence the effect of AMD alone. On the other hand, α-tocopherol (TOCO), which reduced lipid peroxidation and ROS generation, prevented AMD toxicity and caused pronounced reduction in cytotoxicity from AMD/TNF cotreatment. α-TOCO plus a pancaspase inhibitor completely abolished AMD/TNF-induced cytotoxicity. In summary, activation of caspases and oxidative stress were observed after AMD/TNF cotreatment, and caspase inhibitors and a lipid-soluble free-radical scavenger attenuated AMD/TNF-induced cytotoxicity.

  18. Mononuclear Phagocyte-Derived Microparticulate Caspase-1 Induces Pulmonary Vascular Endothelial Cell Injury.

    Directory of Open Access Journals (Sweden)

    Srabani Mitra

    Full Text Available Lung endothelial cell apoptosis and injury occurs throughout all stages of acute lung injury (ALI/ARDS and impacts disease progression. Lung endothelial injury has traditionally been focused on the role of neutrophil trafficking to lung vascular integrin receptors induced by proinflammatory cytokine expression. Although much is known about the pathogenesis of cell injury and death in ALI/ARDS, gaps remain in our knowledge; as a result of which there is currently no effective pharmacologic therapy. Enzymes known as caspases are essential for completion of the apoptotic program and secretion of pro-inflammatory cytokines. We hypothesized that caspase-1 may serve as a key regulator of human pulmonary microvascular endothelial cell (HPMVEC apoptosis in ALI/ARDS. Our recent experiments confirm that microparticles released from stimulated monocytic cells (THP1 induce lung endothelial cell apoptosis. Microparticles pretreated with the caspase-1 inhibitor, YVAD, or pan-caspase inhibitor, ZVAD, were unable to induce cell death of HPMVEC, suggesting the role of caspase-1 or its substrate in the induction of HPMVEC cell death. Neither un-induced microparticles (control nor direct treatment with LPS induced apoptosis of HPMVEC. Further experiments showed that caspase-1 uptake into HPMVEC and the induction of HPMVEC apoptosis was facilitated by caspase-1 interactions with microparticulate vesicles. Altering vesicle integrity completely abrogated apoptosis of HPMVEC suggesting an encapsulation requirement for target cell uptake of active caspase-1. Taken together, we confirm that microparticle centered caspase-1 can play a regulator role in endothelial cell injury.

  19. Bcl-2 and Bcl-xL overexpression inhibits cytochrome c release, activation of multiple caspases, and virus release following coxsackievirus B3 infection

    International Nuclear Information System (INIS)

    Carthy, Christopher M.; Yanagawa, Bobby; Luo Honglin; Granville, David J.; Yang, Decheng; Cheung, Paul; Cheung, Caroline; Esfandiarei, Mitra; Rudin, Charles M.; Thompson, Craig B.; Hunt, David W.C.; McManus, Bruce M.

    2003-01-01

    Coxsackievirus B3, a cytopathic virus in the family Picornaviridae, induces degenerative changes in host cell morphology. Here we demonstrate cytochrome c release and caspases-2, -3, -6, -7, -8, and -9 processing. Enforced Bcl-2 and Bcl-xL expression markedly reduced release of cytochrome c, presentation of the mitochondrial epitope 7A6, and depressed caspase activation following infection. In comparison, cell death using TRAIL ligand caused caspase-8 processing prior to cytochrome c release and executioner caspases and cell death was only partially rescued by Bcl-2 and Bcl-xL overexpression. Disruption of the mitochondrial inner membrane potential following CVB3 infection was not inhibited by zVAD.fmk treatment. Bcl-2 or Bcl-xL overexpression or zVAD.fmk treatment delayed the loss of host cell viability and decreased progeny virus release following infection. Our data suggest that mitochondrial release of cytochrome c may be an important early event in caspase activation in CVB3 infection, and, as such, may contribute to the loss of host-cell viability and progeny virus release

  20. Mercury-Induced Externalization of Phosphatidylserine and Caspase 3 Activation in Human Liver Carcinoma (HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Paul B. Tchounwou

    2006-03-01

    Full Text Available Apoptosis arises from the active initiation and propagation of a series of highly orchestrated specific biochemical events leading to the demise of the cell. It is a normal physiological process, which occurs during embryonic development as well as in the maintenance of tissue homeostasis. Diverse groups of molecules are involved in the apoptosis pathway and it functions as a mechanism to eliminate unwanted or irreparably damaged cells. However, inappropriate induction of apoptosis by environmental agents has broad ranging pathologic implications and has been associated with several diseases including cancer. The toxicity of several heavy metals such as mercury has been attributed to their high affinity to sulfhydryl groups of proteins and enzymes, and their ability to disrupt cell cycle progression and/or apoptosis in various tissues. The aim of this study was to assess the potential for mercury to induce early and late-stage apoptosis in human liver carcinoma (HepG2 cells. The Annexin-V and Caspase 3 assays were performed by flow cytometric analysis to determine the extent of phosphatidylserine externalization and Caspase 3 activation in mercury-treated HepG2 cells. Cells were exposed to mercury for 10 and 48 hours respectively at doses of 0, 1, 2, and 3 μg/mL based on previous cytotoxicity results in our laboratory indicating an LD50 of 3.5 ± 0.6 μg/mL for mercury in HepG2 cells. The study data indicated a dose response relationship between mercury exposure and the degree of early and late-stage apoptosis in HepG2 cells. The percentages of cells undergoing early apoptosis were 0.03 ± 0.03%, 5.19 ± 0.04%, 6.36 ± 0.04%, and 8.84 ± 0.02% for 0, 1, 2, and 3 μg/mL of mercury respectively, indicating a gradual increase in apoptotic cells with increasing doses of mercury. The percentages of Caspase 3 positive cells undergoing late apoptosis were 3.58 ± 0.03%, 17.06 ± 0

  1. A Novel Non-Apoptotic Role of Procaspase-3 in the Regulation of Mitochondrial Biogenesis Activators.

    Science.gov (United States)

    Kim, Ji-Soo; Ha, Ji-Young; Yang, Sol-Ji; Son, Jin H

    2018-01-01

    The executioner caspase-3 has been proposed as a pharmacological intervention target to preserve degenerating dopaminergic (DA) neurons because apoptotic mechanisms involving caspase-3 contribute, at least in part, to the loss of DA neurons in patients and experimental models of Parkinson's disease (PD). Here, we determined that genetic intervention of caspase-3 was sufficient to prevent cell death against oxidative stress (OS), accompanied by unexpected severe mitochondrial dysfunction. Specifically, as we expected, caspase-3-deficient DA neuronal cells were very significantly resistant to OS-induced cell death, while the activation of the initiator caspase-9 by OS was preserved. Moreover, detailed phenotypic characterization of caspase-3-deficient DA cells revealed severe mitochondrial dysfunction, including an accumulation of damaged mitochondria with a characteristic swollen structure and broken cristae, reduced membrane potential, increased levels of reactive oxygen species (ROS), and deficits in mitochondrial oxidative phosphorylation (OXPHOS) enzymes. Of great interest, we found that mitochondrial biogenesis was dramatically decreased in caspase-3-deficient DA cells, whereas their capability of mitophagy was normal. In accordance with this observation, caspase-3 gene knock down (KD) resulted in dramatically decreased expression of the key transcriptional activators of mitochondrial biogenesis, such as Tfam and Nrf-1, implicating a non-apoptotic role of procaspase-3 in mitochondrial biogenesis. Therefore, a prolonged anti-apoptotic intervention targeting caspase-3 should be considered with caution due to the potential adverse effects in mitochondria dynamics resulting from a novel potential functional role of procaspase-3 in mitochondrial biogenesis via regulating the expression of mitochondrial biogenesis activators. J. Cell. Biochem. 119: 347-357, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

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

    Science.gov (United States)

    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.

  3. Oxidative stress, caspase-3 activation and cleavage of ROCK-1 play an essential role in MeHg-induced cell death in primary astroglial cells.

    Science.gov (United States)

    Dos Santos, Alessandra Antunes; López-Granero, Caridad; Farina, Marcelo; Rocha, João B T; Bowman, Aaron B; Aschner, Michael

    2018-03-01

    Methylmercury is a toxic environmental contaminant that elicits significant toxicity in humans. The central nervous system is the primary target of toxicity, and is particularly vulnerable during development. Rho-associated protein kinase 1 (ROCK-1) is a major downstream effector of the small GTPase RhoA and a direct substrate of caspase-3. The activation of ROCK-1 is necessary for membrane blebbing during apoptosis. In this work, we examined whether MeHg could affect the RhoA/ROCK-1 signaling pathway in primary cultures of mouse astrocytes. Exposure of cells with 10 μM MeHg decreased cellular viability after 24 h of incubation. This reduction in viability was preceded by a significant increase in intracellular and mitochondrial reactive oxygen species levels, as well as a reduced NAD + /NADH ratio. MeHg also induced an increase in mitochondrial-dependent caspase-9 and caspase-3, while the levels of RhoA protein expression were reduced or unchanged. We further found that MeHg induced ROCK-1 cleavage/activation and promoted LIMK1 and MYPT1 phosphorylation, both of which are the best characterized ROCK-1 downstream targets. Inhibiting ROCK-1 and caspases activation attenuated the MeHg-induced cell death. Collectively, these findings are the first to show that astrocytes exposed to MeHg showed increased cleavage/activation of ROCK-1, which was independent of the small GTPase RhoA. Copyright © 2018. Published by Elsevier Ltd.

  4. alpha-Toxin is a mediator of Staphylococcus aureus-induced cell death and activates caspases via the intrinsic death pathway independently of death receptor signaling

    NARCIS (Netherlands)

    Bantel, H; Sinha, B; Domschke, W; Peters, Georg; Schulze-Osthoff, K; Jänicke, R U

    2001-01-01

    Infections with Staphylococcus aureus, a common inducer of septic and toxic shock, often result in tissue damage and death of various cell types. Although S. aureus was suggested to induce apoptosis, the underlying signal transduction pathways remained elusive. We show that caspase activation and

  5. Lentiviral-mediated RNAi targeting caspase-3 inhibits apoptosis induced by serum deprivation in rat endplate chondrocytes in vitro

    International Nuclear Information System (INIS)

    Ding, L.; Wu, J.P.; Xu, G.; Zhu, B.; Zeng, Q.M.; Li, D.F.; Lu, W.

    2014-01-01

    Current studies find that degenerated cartilage endplates (CEP) of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA) was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis

  6. Lentiviral-mediated RNAi targeting caspase-3 inhibits apoptosis induced by serum deprivation in rat endplate chondrocytes in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Ding, L.; Wu, J.P. [Fudan University, Jinshan Hospital, Department of Orthopaedics, Shanghai, China, Department of Orthopaedics, Jinshan Hospital, Fudan University, Shanghai (China); Xu, G. [Fudan University, Jinshan Hospital, Center Laboratory, Shanghai, China, Center Laboratory, Jinshan Hospital, Fudan University, Shanghai (China); Zhu, B.; Zeng, Q.M.; Li, D.F.; Lu, W. [Fudan University, Jinshan Hospital, Department of Orthopaedics, Shanghai, China, Department of Orthopaedics, Jinshan Hospital, Fudan University, Shanghai (China)

    2014-05-09

    Current studies find that degenerated cartilage endplates (CEP) of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA) was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis.

  7. Lentiviral-mediated RNAi targeting caspase-3 inhibits apoptosis induced by serum deprivation in rat endplate chondrocytes in vitro

    Directory of Open Access Journals (Sweden)

    L. Ding

    2014-06-01

    Full Text Available Current studies find that degenerated cartilage endplates (CEP of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis.

  8. The role of caspase 3 and BclxL in the action of interleukin 7 (IL-7): a survival factor in activated human T cells

    DEFF Research Database (Denmark)

    Amos, C L; Woetmann, A; Nielsen, M

    1998-01-01

    cells. Both cytokines abrogated the dexamethasone-induced stimulation of Caspase 3 and prevented the cleavage of poly (ADP-ribose) polymerase (PARP), a substrate for the Caspase 3. IL-7 upregulated the expression of Bc1xL and counteracted the downregulation of this anti-apoptotic protein...... by the synthetic glucocorticoid, dexamethasone. Bcl-2 protein expression was uupregulated by IL-7 with or without dexamethasone, but Bc1-2 was expressed at a much lower level than BclxL in these cells. Levels of Bax did not markedly change on either cytokine stimulation or dexamethasone treatment. An unidentified...... 23-kDa band, which was recognized by the anti-Bc1-2 antibody, was induced by dexamthasone and suppressed by IL-7 and IL-2. This protein was subject to independent regulation as compared to the p26 Bc1-2 protein, suggesting that it may be a novel factor, possibly involved in the regulation...

  9. Increases in apoptosis, caspase activity and expression of p53 and bax, and the transition between two types of mitochondrion-rich cells, in the gills of the climbing perch, Anabas testudineus, during a progressive acclimation from freshwater to seawater

    Science.gov (United States)

    Ching, Biyun; Chen, Xiu L.; Yong, Jing H. A.; Wilson, Jonathan M.; Hiong, Kum C.; Sim, Eugene W. L.; Wong, Wai P.; Lam, Siew H.; Chew, Shit F.; Ip, Yuen K.

    2013-01-01

    This study aimed to test the hypothesis that branchial osmoregulatory acclimation involved increased apoptosis and replacement of mitochdonrion-rich cells (MRCs) in the climbing perch, Anabas testudineus, during a progressive acclimation from freshwater to seawater. A significant increase in branchial caspase-3/-7 activity was observed on day 4 (salinity 20), and an extensive TUNEL-positive apoptosis was detected on day 5 (salinity 25), indicating salinity-induced apoptosis had occurred. This was further supported by an up-regulation of branchial mRNA expression of p53, a key regulator of cell cycle arrest and apoptosis, between day 2 (salinity 10) and day 6 (seawater), and an increase in branchial p53 protein abundance on day 6. Seawater acclimation apparently activated both the extrinsic and intrinsic pathways, as reflected by significant increases in branchial caspase-8 and caspase-9 activities. The involvement of the intrinsic pathway was confirmed by the significant increase in branchial mRNA expression of bax between day 4 (salinity 20) and day 6 (seawater). Western blotting results revealed the presence of a freshwater Na+/K+-ATPase (Nka) α-isoform, Nka α1a, and a seawater isoform, Nka α1b, the protein abundance of which decreased and increased, respectively, during seawater acclimation. Immunofluorescence microscopy revealed the presence of two types of MRCs distinctly different in sizes, and confirmed that the reduction in Nka α1a expression, and the prominent increases in expression of Nka α1b, Na+:K+:2Cl− cotransporter 1, and cystic fibrosis transmembrane conductance regulator Cl− channel coincided with the salinity-induced apoptotic event. Since modulation of existing MRCs alone could not have led to extensive salinity-induced apoptosis, it is probable that some, if not all, freshwater-type MRCs could have been removed through increased apoptosis and subsequently replaced by seawater-type MRCs in the gills of A. testudineus during seawater

  10. Pacific Ciguatoxin Induces Excitotoxicity and Neurodegeneration in the Motor Cortex Via Caspase 3 Activation: Implication for Irreversible Motor Deficit.

    Science.gov (United States)

    Asthana, Pallavi; Zhang, Ni; Kumar, Gajendra; Chine, Virendra Bhagawan; Singh, Kunal Kumar; Mak, Yim Ling; Chan, Leo Lai; Lam, Paul Kwan Sing; Ma, Chi Him Eddie

    2018-01-18

    Consumption of fish containing ciguatera toxins or ciguatoxins (CTXs) causes ciguatera fish poisoning (CFP). In some patients, CFP recurrence occurs even years after exposure related to CTXs accumulation. Pacific CTX-1 (P-CTX-1) is one of the most potent natural substances known that causes predominantly neurological symptoms in patients; however, the underlying pathogenies of CFP remain unknown. Using clinically relevant neurobehavioral tests and electromyography (EMG) to assess effects of P-CTX-1 during the 4 months after exposure, recurrent motor strength deficit occurred in mice exposed to P-CTX-1. We detected irreversible motor strength deficits accompanied by reduced EMG activity, demyelination, and slowing of motor nerve conduction, whereas control unexposed mice fully recovered in 1 month after peripheral nerve injury. Finally, to uncover the mechanism underlying CFP, we detected reduction of spontaneous firing rate of motor cortical neurons even 6 months after exposure and increased number of glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes. Increased numbers of motor cortical neuron apoptosis were detected by dUTP-digoxigenin nick end labeling assay along with activation of caspase 3. Taken together, our study demonstrates that persistence of P-CTX-1 in the nervous system induces irreversible motor deficit that correlates well with excitotoxicity and neurodegeneration detected in the motor cortical neurons.

  11. N-terminal region of gelsolin induces apoptosis of activated hepatic stellate cells by a caspase-dependent mechanism.

    Directory of Open Access Journals (Sweden)

    Budhaditya Mazumdar

    Full Text Available Activated hepatic stellate cells (HSCs are the major source for alteration of extracellular matrix in fibrosis and cirrhosis. Conditioned medium (CM collected from immortalized human hepatocytes (IHH have earlier been shown to be responsible for apoptosis of HSCs. In this study, we have shown that antibodies raised against a peptide derived from a linear B-cell epitope in the N-terminal region of gelsolin identified a gelsolin fragment in IHH CM. Analysis of activated stellate cell death by CM collected from Huh7 cells transfected with plasmids encoding gelsolin deletion mutants suggested that the N-terminal half of gelsolin contained sequences which were responsible for stellate cell death. Further analysis determined that this activity was restricted to a region encompassing amino acids 1-70 in the gelsolin sequence; antibody directed to an epitope within this region was able to neutralize stellate cell death. Gelsolin modulation of cell death using this fragment involved upregulation of TRAIL-R1 and TRAIL-R2, and involved caspase 3 activation by extrinsic pathway. The apoptotic activity of N-terminal gelsolin fragments was restricted to activated but not quiescent stellate cells indicating its potential application in therapeutic use as an anti-fibrotic agent. Gelsolin fragments encompassing N-terminal regions in polypeptides of different molecular sizes were detected by N-terminal peptide specific antiserum in IHH CM immunoprecipitated with chronically HCV infected patient sera, suggesting the presence of autoantibodies generated against N-terminal gelsolin fragments in patients with chronic liver disease.

  12. Abrogation of the presenilin 1/beta-catenin interaction and preservation of the heterodimeric presenilin 1 complex following caspase activation.

    Science.gov (United States)

    Tesco, G; Kim, T W; Diehlmann, A; Beyreuther, K; Tanzi, R E

    1998-12-18

    beta-Catenin has previously been shown to interact with presenilin 1 (PS1) in transfected cells. Here we report that beta-catenin co-immunoprecipitates with the endogenous C-terminal fragment of presenilin 1 (PS1-CTF) but not with the endogenous CTF of presenilin 2 (PS2-CTF) in H4 human neuroglioma cells. During staurosporine (STS)-induced cell death, beta-catenin and PS1-CTF undergo a caspase-mediated cleavage. After 12 h of STS treatment, the beta-catenin.PS1-CTF interaction is abrogated. While PS1-CTF immunoprecipitated with all caspase-cleaved species of beta-catenin, beta-catenin holoprotein did not co-immunoprecipitate with the "alternative" caspase-derived PS1-CTF (PS1-aCTF). Thus, the abrogation of the beta-catenin.PS1-CTF complex was due to caspase cleavage of PS1-CTF. beta-Catenin co-immunoprecipitated with PS1-NTF, but only when PS1-NTF was associated with PS1-CTF. Even though PS1-NTF.CTF complex stability was not altered by caspase cleavage, its ability to bind beta-catenin was abolished. Thus, while the PS1-NTF.CTF complex is preserved after caspase cleavage, it may no longer be fully functional.

  13. Caspase cleaved presenilin-1 is part of active gamma-secretase complexes

    DEFF Research Database (Denmark)

    Hansson, Camilla A; Popescu, Bogdan O; Laudon, Hanna

    2006-01-01

    , and Abeta is believed to be central for the molecular pathogenesis of AD. Apoptosis has been implicated as one of the mechanisms behind the neuronal cell loss seen in AD. We have studied preservation and activity of the gamma-secretase complex during apoptosis in neuroblastoma cells (SH-SY5Y) exposed...

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

    Science.gov (United States)

    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.

  15. Mycobacterium tuberculosis-infected human monocytes down-regulate microglial MMP-2 secretion in CNS tuberculosis via TNFα, NFκB, p38 and caspase 8 dependent pathways

    Directory of Open Access Journals (Sweden)

    Elkington Paul T

    2011-05-01

    Full Text Available Abstract Tuberculosis (TB of the central nervous system (CNS is a deadly disease characterized by extensive tissue destruction, driven by molecules such as Matrix Metalloproteinase-2 (MMP-2 which targets CNS-specific substrates. In a simplified cellular model of CNS TB, we demonstrated that conditioned medium from Mycobacterium tuberculosis-infected primary human monocytes (CoMTb, but not direct infection, unexpectedly down-regulates constitutive microglial MMP-2 gene expression and secretion by 72.8% at 24 hours, sustained up to 96 hours (P M.tb-infected monocyte-dependent networks paradoxically involves the pro-inflammatory mediators TNF-α, p38 MAP kinase and NFκB in addition to a novel caspase 8-dependent pathway.

  16. Programmed Cell Death and Caspase Functions During Neural Development.

    Science.gov (United States)

    Yamaguchi, Yoshifumi; Miura, Masayuki

    2015-01-01

    Programmed cell death (PCD) is a fundamental component of nervous system development. PCD serves as the mechanism for quantitative matching of the number of projecting neurons and their target cells through direct competition for neurotrophic factors in the vertebrate peripheral nervous system. In addition, PCD plays roles in regulating neural cell numbers, canceling developmental errors or noise, and tissue remodeling processes. These findings are mainly derived from genetic studies that prevent cells from dying by apoptosis, which is a major form of PCD and is executed by activation of evolutionarily conserved cysteine protease caspases. Recent studies suggest that caspase activation can be coordinated in time and space at multiple levels, which might underlie nonapoptotic roles of caspases in neural development in addition to apoptotic roles. © 2015 Elsevier Inc. All rights reserved.

  17. The Fas-associated death domain protein/caspase-8/c-FLIP signaling pathway is involved in TNF-induced activation of ERK

    International Nuclear Information System (INIS)

    Lueschen, Silke; Falk, Markus; Scherer, Gudrun; Ussat, Sandra; Paulsen, Maren; Adam-Klages, Sabine

    2005-01-01

    The cytokine TNF activates multiple signaling pathways leading to cellular responses ranging from proliferation and survival to apoptosis. While most of these pathways have been elucidated in detail over the past few years, the molecular mechanism leading to the activation of the MAP kinases ERK remains ill defined and is controversially discussed. Therefore, we have analyzed TNF-induced ERK activation in various human and murine cell lines and show that it occurs in a cell-type-specific manner. In addition, we provide evidence for the involvement of the signaling components Fas-associated death domain protein (FADD), caspase-8, and c-FLIP in the pathway activating ERK in response to TNF. This conclusion is based on the following observations: (I) Overexpression of FADD, caspase-8, or a c-FLIP protein containing the death effector domains only leads to enhanced and prolonged ERK activation after TNF treatment. (II) TNF-induced ERK activation is strongly diminished in the absence of FADD. Interestingly, the enzymatic function of caspase-8 is not required for TNF-induced ERK activation. Additional evidence suggests a role for this pathway in the proliferative response of murine fibroblasts to TNF

  18. pEgr-sTRAIL transfer in combination with 60Co γ ray irradiation to induce the apoptosis on HeLa cells and activation of Caspase-3

    International Nuclear Information System (INIS)

    Tian Mei; Guo Zhiying; Zhao Baofeng; Ruan Jianlei; Su Xu

    2009-01-01

    In order to approach the radiosensitivity of TRAIL expression controlled by Egr-1 promotor, the recombinant vector pEgr-sTRAIL was tranfected into HeLa cells, the early apoptosis and Caspase-3 activity were detected after different doses of 60 Co γ-ray irradiation. The results showed that pEgr-sTRAIL transfected in combination with γ-ray irradiation could significantly induce the apoptosis of HeLa cells in a dose-dependent manner. The higher activity of Capase-3 was also found in pEgr-sTRAIL irradiated group by using western blotting and spectrophotometry. Our result demonstrated that the activity of Caspase-3, as the apoptosis executor, play an important role in TRAIL-induced apoptosis and the enhanced TRAIL-induced apoptosis in transfected cells after irradiation can be controlled by radio-sensitive promoter Egr-1. (authors)

  19. A short caspase-3 isoform inhibits chemotherapy-induced apoptosis by blocking apoptosome assembly.

    Directory of Open Access Journals (Sweden)

    Frédérique Végran

    Full Text Available Alternative splicing of caspase-3 produces a short isoform caspase-3s that antagonizes caspase-3 apoptotic activity. However, the mechanism of apoptosis inhibition by caspase-3s remains unknown. Here we show that exogenous caspase-3 sensitizes MCF-7 and HBL100 breast cancers cells to chemotherapeutic treatments such as etoposide and methotrexate whereas co-transfection with caspase-3s strongly inhibits etoposide and methotrexate-induced apoptosis underlying thus the anti-apoptotic role of caspase-3s. In caspase-3 transfected cells, lamin-A and α-fodrin were cleaved when caspase-3 was activated by etoposide or methotrexate. When caspase-3s was co-transfected, this cleavage was strongly reduced. Depletion of caspase-3 by RNA interference in HBL100 containing endogenous caspase-3s caused reduction in etoposide and methotrexate-induced apoptosis, whereas the depletion of caspase-3s sensitized cells to chemotherapy. In the presence of caspase-3s, a lack of interaction between caspase-3 and caspase-9 was observed. Immunoprecipitation assays showed that caspase-3s binds the pro-forms of caspase-3. This result suggested that the absence of interaction with caspase-9 when both variants of caspase-3 are present contribute to block the apoptosome assembly and inhibit apoptosis. These data support that caspases-3s negatively interferes with caspase-3 activation and apoptosis in breast cancer, and that it can play key roles in the modulation of response to chemotherapeutic treatments.

  20. RNase MC2: a new Momordica charantia ribonuclease that induces apoptosis in breast cancer cells associated with activation of MAPKs and induction of caspase pathways.

    Science.gov (United States)

    Fang, Evandro Fei; Zhang, Chris Zhi Yi; Fong, Wing Ping; Ng, Tzi Bun

    2012-04-01

    Ribonucleases (RNases) are ubiquitously distributed nucleases that cleave RNA into smaller pieces. They are promising drugs for different cancers based on their concrete antitumor activities in vitro and in vivo. Here we report for the first time purification and characterization of a 14-kDa RNase, designated as RNase MC2, in the seeds of bitter gourd (Momordica charantia). RNase MC2 manifested potent RNA-cleavage activity toward baker's yeast tRNA, tumor cell rRNA, and an absolute specificity for uridine. RNase MC2 demonstrated both cytostatic and cytotoxic activities against MCF-7 breast cancer cells. Treatment of MCF-7 cells with RNase MC2 caused nuclear damage (karyorrhexis, chromatin condensation, and DNA fragmentation), ultimately resulting in early/late apoptosis. Further molecular studies unveiled that RNase MC2 induced differential activation of MAPKs (p38, JNK and ERK) and Akt. On the other hand, RNase MC2 exposure activated caspase-8, caspase-9, caspase-7, increased the production of Bak and cleaved PARP, which in turn contributed to the apoptotic response. In conclusion, RNase MC2 is a potential agent which can be exploited in the worldwide fight against breast cancer.

  1. [Effects of introduction of short peptides before carotid artery occlusion on behaviour and caspase-3 activity in the brain of old rats].

    Science.gov (United States)

    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.

  2. MeCP2 Promotes Gastric Cancer Progression Through Regulating FOXF1/Wnt5a/β-Catenin and MYOD1/Caspase-3 Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Lingyu Zhao

    2017-02-01

    Full Text Available Methyl-CpG binding protein 2 (MeCP2 has recently been characterized as an oncogene frequently amplified in several types of cancer. However, its precise role in gastric cancer (GC and the molecular mechanism of MeCP2 regulation are still largely unknown. Here we report that MeCP2 is highly expressed in primary GC tissues and the expression level is correlated with the clinicopathologic features of GC. In our experiments, knockdown of MeCP2 inhibited tumor growth. Molecular mechanism of MeCP2 regulation was investigated using an integrated approach with combination of microarray analysis and chromatin immunoprecipitation sequencing (ChIP-Seq. The results suggest that MeCP2 binds to the methylated CpG islands of FOXF1 and MYOD1 promoters and inhibits their expression at the transcription level. Furthermore, we show that MeCP2 promotes GC cell proliferation via FOXF1-mediated Wnt5a/β-Catenin signaling pathway and suppresses apoptosis through MYOD1-mediated Caspase-3 signaling pathway. Due to its high expression level in GC and its critical function in driving GC progression, MeCP2 represents a promising therapeutic target for GC treatment.

  3. Bcl-xL acts downstream of caspase-8 activation by the CD95 death-inducing signaling complex

    NARCIS (Netherlands)

    Medema, J. P.; Scaffidi, C.; Krammer, P. H.; Peter, M. E.

    1998-01-01

    The Bcl-2 family member Bcl-xL has often been correlated with apoptosis resistance. We have shown recently that in peripheral human T cells resistance to CD95-mediated apoptosis is characterized by a lack of caspase-8 recruitment to the CD95 death-inducing signaling complex (DISC) and by increased

  4. A miniaturized device for bioluminescence analysis of caspase-3/7 activity in a single apoptotic cell

    Czech Academy of Sciences Publication Activity Database

    Adamová, Eva; Lišková, M.; Matalová, Eva; Klepárník, K.

    2014-01-01

    Roč. 406, č. 22 (2014), s. 5389-5394 ISSN 1618-2642 R&D Projects: GA ČR GAP304/11/1418 Institutional support: RVO:67985904 Keywords : apoptosis * bioluminiscence * caspase3/7 * single-cell analysis Subject RIV: CE - Biochemistry Impact factor: 3.436, year: 2014

  5. Cr(VI) induces mitochondrial-mediated and caspase-dependent apoptosis through reactive oxygen species-mediated p53 activation in JB6 Cl41 cells

    International Nuclear Information System (INIS)

    Son, Young-Ok; Hitron, J. Andrew; Wang Xin; Chang Qingshan; Pan Jingju; Zhang Zhuo; Liu Jiankang; Wang Shuxia; Lee, Jeong-Chae; Shi Xianglin

    2010-01-01

    Cr(VI) compounds are known to cause serious toxic and carcinogenic effects. Cr(VI) exposure can lead to a severe damage to the skin, but the mechanisms involved in the Cr(VI)-mediated toxicity in the skin are unclear. The present study examined whether Cr(VI) induces cell death by apoptosis or necrosis using mouse skin epidermal cell line, JB6 Cl41 cells. We also investigated the cellular mechanisms of Cr(VI)-induced cell death. This study showed that Cr(VI) induced apoptotic cell death in a dose-dependent manner, as demonstrated by the appearance of cell shrinkage, the migration of cells into the sub-G1 phase, the increase of Annexin V positively stained cells, and the formation of nuclear DNA ladders. Cr(VI) treatment resulted in the increases of mitochondrial membrane depolarization and caspases activation. Electron spin resonance (ESR) and fluorescence analysis revealed that Cr(VI) increased intracellular levels of reactive oxygen species (ROS) such as hydrogen peroxide and superoxide anion radical in dose-dependent manner. Blockage of p53 by si-RNA transfection suppressed mitochondrial changes of Bcl-2 family composition, mitochondrial membrane depolarization, caspase activation and PARP cleavage, leading to the inhibition of Cr(VI)-induced apoptosis. Further, catalase treatment prevented p53 phosphorylation stimulated by Cr(VI) with the concomitant inhibition of caspase activation. These results suggest that Cr(VI) induced a mitochondrial-mediated and caspase-dependent apoptosis in skin epidermal cells through activation of p53, which are mainly mediated by reactive oxidants generated by the chemical.

  6. B1-induced caspase-independent apoptosis in MCF-7 cells is mediated by down-regulation of Bcl-2 via p53 binding to P2 promoter TATA box

    International Nuclear Information System (INIS)

    Liang Xin; Xu Ke; Xu Yufang; Liu Jianwen; Qian Xuhong

    2011-01-01

    The Bcl-2 family contains a panel of proteins which are conserved regulators of apoptosis in mammalian cells, like the anti-apoptotic protein Bcl-2. According to its significant role in altering susceptibility to apoptosis, the deciphering of the mechanism of Bcl-2 expression modulation may be crucial for identifying therapeutics strategies for cancer. Treatment with naphthalimide-based DNA intercalators, including M2-A and R16, generally leads to a decrease in Bcl-2 intracellular amounts. Whereas the interest for these chemotherapeutics is accompanied by advances in the fundamental understanding of their anticancer properties, the molecular mechanism underlying changes in Bcl-2 expression remains poorly understood. We report here that p53 contributes to Bcl-2 down-regulation induced by B1, a novel naphthalimide-based DNA intercalating agent. Indeed, the decrease in Bcl-2 protein levels observed during B1-induced apoptosis was correlated to the decrease in mRNA levels, as a result of the inhibition of Bcl-2 transcription and promoter activity. In this context, we evaluated p53 contribution in the Bcl-2 transcriptional down-regulation. We found a significant increase of p53 binding to P 2 promoter TATA box in MCF7 cells by chromatin immunoprecipitation. These data suggest that B1-induced caspase-independent apoptosis in MCF-7 cells is associated with the activation of p53 and the down-regulation of Bcl-2. Our study strengthens the links between p53 and Bcl-2 at a transcriptional level, upon naphthalimide-based DNA intercalator treatment. - Research highlights: → B1 induced apoptosis in MCF-7 cells, following a transcriptional decrease in Bcl-2. → B1 treatment triggered p53 activation and leads to a p53-dependent down-regulation of Bcl-2. → B1 induced significant increase of p53 binding to Bcl-2 P 2 promoter TATA box.

  7. Anaplasma phagocytophilum inhibits human neutrophil apoptosis via upregulation of bfl-1, maintenance of mitochondrial membrane potential and prevention of caspase 3 activation.

    Science.gov (United States)

    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.

  8. Mycoplasma hyopneumoniae-derived lipid-associated membrane proteins induce apoptosis in porcine alveolar macrophage via increasing nitric oxide production, oxidative stress, and caspase-3 activation.

    Science.gov (United States)

    Bai, Fangfang; Ni, Bo; Liu, Maojun; Feng, Zhixin; Xiong, Qiyan; Xiao, Shaobo; Shao, Guoqing

    2013-09-15

    Mycoplasma hyopneumoniae is the primary etiological agent of enzootic pneumonia in swine. Lipid-associated membrane proteins (LAMP) of mycoplasma are the main pathogenicity factors in mycoplasma diseases. In this study, we investigated the effects of M. hyopneumoniae LAMP on porcine alveolar macrophage (PAM) 3D4/21 cell line. Apoptotic features, such as chromatin condensation and apoptotic bodies, were observed in LAMP-treated PAM 3D4/21 cells. Moreover, LAMP significantly increased the number of TUNEL positive apoptotic cells in PAM 3D4/21 cells compared with the untreated control. In addition, flow cytometric analysis using dual staining with annexin-V-FITC and propidium iodide (PI) showed that LAMP of M. hyopneumoniae induced a time-dependent apoptosis in PAM 3D4/21 cells. Moreover, increased levels of superoxide anion production and activated caspase-3 in PAM 3D4/21 cells were observed after exposure to LAMP. Increased production of nitric oxide (NO) was also confirmed in the cell supernatants. Besides, apoptotic rates increase and caspase-3 activation were suppressed by NOS inhibitor or antioxidant. It is suggested that LAMP of M. hyopneumoniae induced apoptosis in porcine alveolar macrophage via NO production, superoxide anion production, and caspase-3 activation. Copyright © 2013 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Tamayev Robert

    2012-12-01

    Full Text Available Abstract 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.

  10. Equol enhances tamoxifen’s anti-tumor activity by induction of caspase-mediated apoptosis in MCF-7 breast cancer cells

    International Nuclear Information System (INIS)

    Charalambous, Christiana; Pitta, Chara A; Constantinou, Andreas I

    2013-01-01

    Soy phytoestrogens, such as daidzein and its metabolite equol, have been proposed to be responsible for the low breast cancer rate in Asian women. Since the majority of estrogen receptor positive breast cancer patients are treated with tamoxifen, the basic objective of this study is to determine whether equol enhances tamoxifen’s anti-tumor effect, and to identify the molecular mechanisms involved. For this purpose, we examined the individual and combined effects of equol and tamoxifen on the estrogen-dependent MCF-7 breast cancer cells using viability assays, annexin-V/PI staining, cell cycle and western blot analysis. We found that equol (>50 μM) and 4-hydroxy-tamoxifen (4-OHT; >100 nM) significantly reduced the MCF-7 cell viability. Furthermore, the combination of equol (100 μM) and 4-OHT (10 μM) induced apoptosis more effectively than each compound alone. Subsequent treatment of MCF-7 cells with the pan-caspase inhibitor Z-VAD-FMK inhibited equol- and 4-OHT-mediated apoptosis, which was accompanied by PARP and α-fodrin cleavage, indicating that apoptosis is mainly caspase-mediated. These compounds also induced a marked reduction in the bcl-2:bax ratio, which was accompanied by caspase-9 and caspase-7 activation and cytochrome-c release to the cytosol. Taken together, these data support the notion that the combination of equol and tamoxifen activates the intrinsic apoptotic pathway more efficiently than each compound alone. Consequently, equol may be used therapeutically in combination treatments and clinical studies to enhance tamoxifen’s effect by providing additional protection against estrogen-responsive breast cancers

  11. Inhibition of CUG-binding protein 1 and activation of caspases are critically involved in piperazine derivative BK10007S induced apoptosis in hepatocellular carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Ju-Ha Kim

    Full Text Available Though piperazine derivative BK10007S was known to induce apoptosis in pancreatic cancer xenograft model as a T-type CaV3.1 a1G isoform calcium channel blocker, its underlying antitumor mechanism still remains unclear so far. Thus, in the present study, the antitumor mechanism of BK10007S was elucidated in hepatocellular carcinoma cells (HCCs. Herein, BK10007S showed significant cytotoxicity by 3-[4,5-2-yl]-2,5-diphenyltetra-zolium bromide (MTT assay and anti-proliferative effects by colony formation assay in HepG2 and SK-Hep1 cells. Also, apoptotic bodies and terminal deoxynucleotidyl transferase (TdT dUTP Nick End Labeling (TUNEL positive cells were observed in BK10007S treated HepG2 and SK-Hep1 cells by 4',6-diamidino-2-phenylinodole (DAPI staining and TUNEL assay, respectively. Consistently, BK10007S increased sub G1 population in HepG2 and SK-Hep1 cells by cell cycle analysis. Furthermore, Western blotting revealed that BK10007S activated the caspase cascades (caspase 8, 9 and 3, cleaved poly (ADP-ribose polymerase (PARP, and downregulated the expression of cyclin D1, survivin and for CUG-binding protein 1 (CUGBP1 or CELF1 in HepG2 and SK-Hep1 cells. Conversely, overexpression of CUGBP1 reduced cleavages of PARP and caspase 3, cytotoxicity and subG1 population in BK10007S treated HepG2 cells. Overall, these findings provide scientific evidences that BK10007S induces apoptosis via inhibition of CUGBP1 and activation of caspases in hepatocellular carcinomas as a potent anticancer candidate.

  12. Early Activation of Apoptosis and Caspase-independent Cell Death Plays an Important Role in Mediating the Cytotoxic and Genotoxic Effects of WP 631 in Ovarian Cancer Cells.

    Science.gov (United States)

    Gajek, Arkadiusz; Denel-Bobrowska, Marta; Rogalska, Aneta; Bukowska, Barbara; Maszewski, Janusz; Marczak, Agnieszka

    2015-01-01

    The purpose of this study was to provide a detailed explanation of the mechanism of bisanthracycline,?WP 631 in comparison to doxorubicin (DOX), a first generation anthracycline, currently the most widely used pharmaceutical in clinical oncology. Experiments were performed in SKOV-3 ovarian cancer cells which are otherwise resistant to standard drugs such as cis-platinum and adriamycin. As attention was focused on the ability of WP 631 to induce apoptosis, this was examined using a double staining method with Annexin V and propidium iodide probes, with measurement of the level of intracellular calcium ions and cytosolic cytochrome c. The western blotting technique was performed to confirm PARP cleavage. We also investigated the involvement of caspase activation and DNA degradation (comet assay and immunocytochemical detection of phosphorylated H2AX histones) in the development of apoptotic events. WP 631 demonstrated significantly higher effectiveness as a pro-apoptotic drug than DOX. This was evident in the higher levels of markers of apoptosis, such as the externalization of phosphatidylserine and the elevated level of cytochrome c. An extension of incubation time led to an increase in intracellular calcium levels after treatment with DOX. Lower changes in the calcium content were associated with the influence of WP 631. DOX led to the activation of all tested caspases, 8, 9 and 3, whereas WP 631 only induced an increase in caspase 8 activity after 24h of treatment and consequently led to the cleavage of PARP. The lack of active caspase 3 had no outcome on the single and double-stranded DNA breaks. The obtained results show that WP 631 was considerably more genotoxic towards the investigated cell line than DOX. This effect was especially visible after longer times of incubation. The above detailed studies indicate that WP 631 generates early apoptosis and cell death independent of caspase-3, detected at relatively late time points. The observed differences in the

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

    Czech Academy of Sciences Publication Activity Database

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

    2017-01-01

    Roč. 409, č. 1 (2017), s. 269-274 ISSN 1618-2642 R&D Projects: GA ČR(CZ) GA14-28254S Institutional support: RVO:68081715 ; RVO:67985904 Keywords : single-cell analysis * bioluminescence * apoptosis * caspase-3/7 Subject RIV: CB - Analytical Chemistry , Separation; EB - Genetics ; Molecular Biology (UZFG-Y) OBOR OECD: Analytical chemistry ; Developmental biology (UZFG-Y) Impact factor: 3.431, year: 2016

  14. Inhibition of vacuolar ATPase attenuates the TRAIL-induced activation of caspase-8 and modulates the trafficking of TRAIL receptosomes

    Czech Academy of Sciences Publication Activity Database

    Horová, Vladimíra; Hradilová, Naďa; Jelínková, Iva; Koc, Michal; Švadlenka, Jan; Bražina, Jan; Klíma, Martin; Slavík, J.; Vaculová, Alena; Anděra, Ladislav

    2013-01-01

    Roč. 280, č. 14 (2013), s. 3436-3450 ISSN 1742-464X R&D Projects: GA ČR GAP301/10/1971; GA ČR(CZ) GAP301/11/1730; GA MŠk 1M0506 Institutional support: RVO:68378050 ; RVO:68081707 Keywords : acidification * apoptosis * caspase-8 * TRAIL * V- ATPase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.986, year: 2013

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

    Czech Academy of Sciences Publication Activity Database

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

    2017-01-01

    Roč. 409, č. 1 (2017), s. 269-274 ISSN 1618-2642 R&D Projects: GA ČR(CZ) GA14-28254S Institutional support: RVO:68081715 ; RVO:67985904 Keywords : single-cell analysis * bioluminescence * apoptosis * caspase-3/7 Subject RIV: CB - Analytical Chemistry, Separation; EB - Genetics ; Molecular Biology (UZFG-Y) OBOR OECD: Analytical chemistry; Developmental biology (UZFG-Y) Impact factor: 3.431, year: 2016

  16. Inhibition of vacuolar ATPase attenuates the TRAIL-induced activation of caspase-8 and modulates the trafficking of TRAIL receptosomes

    Czech Academy of Sciences Publication Activity Database

    Horová, Vladimíra; Hradilová, Naďa; Jelínková, Iva; Koc, Michal; Švadlenka, Jan; Bražina, Jan; Klíma, Martin; Slavík, J.; Vaculová, Alena; Anděra, Ladislav

    2013-01-01

    Roč. 280, č. 14 (2013), s. 3436-3450 ISSN 1742-464X R&D Projects: GA ČR GAP301/10/1971; GA ČR(CZ) GAP301/11/1730; GA MŠk 1M0506 Institutional support: RVO:68378050 ; RVO:68081707 Keywords : acidification * apoptosis * caspase-8 * TRAIL * V-ATPase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.986, year: 2013

  17. Regulating prefrontal cortex activation

    DEFF Research Database (Denmark)

    Aznar, Susana; Klein, Anders Bue

    2013-01-01

    The prefrontal cortex (PFC) is involved in mediating important higher-order cognitive processes such as decision making, prompting thereby our actions. At the same time, PFC activation is strongly influenced by emotional reactions through its functional interaction with the amygdala...... of emotion-based actions, such as addiction and other impulse-related behaviors. In this review, we give an overview of the 5-HT2A receptor distribution (neuronal, intracellular, and anatomical) along with its functional and physiological effect on PFC activation, and how that relates to more recent findings...... of a regulatory effect of the PFC on the emotional control of our actions....

  18. Diosgenin induces apoptosis in IGF-1-stimulated human thyrocytes through two caspase-dependent pathways

    International Nuclear Information System (INIS)

    Mu, Shumin; Tian, Xingsong; Ruan, Yongwei; Liu, Yuantao; Bian, Dezhi; Ma, Chunyan; Yu, Chunxiao; Feng, Mei; Wang, Furong; Gao, Ling; Zhao, Jia-jun

    2012-01-01

    Highlights: ► Diosgenin induces apoptosis in IGF-1-treated thyrocytes through two caspase pathways. ► Diosgenin inhibits FLIP and activates caspase-8 in FAS related-pathway. ► Diosgenin increases ROS, regulates the ratio of Bax/Bcl-2 in mitochondrial pathway. -- Abstract: Insulin-like growth factor-1 (IGF-1) is a growth factor of the thyroid that has been shown in our previous study to possess proliferative and antiapoptotic effects in FRTL-5 cell lines through the upregulation of cyclin D and Fas-associated death domain-like interleukin-1-converting enzyme (FLICE)-inhibitory protein (FLIP). Diosgenin, a natural steroid sapogenin from plants, has been shown to induce apoptosis in many cell lines, with the exception of thyroid cells. In this report, we investigated the apoptotic effect and mechanism of diosgenin in IGF-1-stimulated primary human thyrocytes. Primary human thyrocytes were preincubated with or without IGF-1 for 24 h and subsequently exposed to varying concentrations of diosgenin for different times. We found that diosgenin induced apoptosis in human thyrocytes pretreated with IGF-1 in a dose-dependent manner through the activation of caspase cascades. Moreover, diosgenin inhibited FLIP and activated caspase-8 in the FAS-related apoptotic pathway. Diosgenin increased the production of ROS, regulated the balance of Bax and Bcl-2 and cleaved caspase-9 in the mitochondrial apoptotic pathway. These results indicate that diosgenin induces apoptosis in IGF-1-stimulated primary human thyrocytes through two caspase-dependent pathways.

  19. Uncovering a Dual Regulatory Role for Caspases During Endoplasmic Reticulum Stress-induced Cell Death.

    Science.gov (United States)

    Anania, Veronica G; Yu, Kebing; Gnad, Florian; Pferdehirt, Rebecca R; Li, Han; Ma, Taylur P; Jeon, Diana; Fortelny, Nikolaus; Forrest, William; Ashkenazi, Avi; Overall, Christopher M; Lill, Jennie R

    2016-07-01

    Many diseases are associated with endoplasmic reticulum (ER) stress, which results from an accumulation of misfolded proteins. This triggers an adaptive response called the "unfolded protein response" (UPR), and prolonged exposure to ER stress leads to cell death. Caspases are reported to play a critical role in ER stress-induced cell death but the underlying mechanisms by which they exert their effect continue to remain elusive. To understand the role caspases play during ER stress, a systems level approach integrating analysis of the transcriptome, proteome, and proteolytic substrate profile was employed. This quantitative analysis revealed transcriptional profiles for most human genes, provided information on protein abundance for 4476 proteins, and identified 445 caspase substrates. Based on these data sets many caspase substrates were shown to be downregulated at the protein level during ER stress suggesting caspase activity inhibits their cellular function. Additionally, RNA sequencing revealed a role for caspases in regulation of ER stress-induced transcriptional pathways and gene set enrichment analysis showed expression of multiple gene targets of essential transcription factors to be upregulated during ER stress upon inhibition of caspases. Furthermore, these transcription factors were degraded in a caspase-dependent manner during ER stress. These results indicate that caspases play a dual role in regulating the cellular response to ER stress through both post-translational and transcriptional regulatory mechanisms. Moreover, this study provides unique insight into progression of the unfolded protein response into cell death, which may help identify therapeutic strategies to treat ER stress-related diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. EspC, an Autotransporter Protein Secreted by Enteropathogenic Escherichia coli, Causes Apoptosis and Necrosis through Caspase and Calpain Activation, Including Direct Procaspase-3 Cleavage

    Directory of Open Access Journals (Sweden)

    Antonio Serapio-Palacios

    2016-06-01

    Full Text Available Enteropathogenic Escherichia coli (EPEC has the ability to antagonize host apoptosis during infection through promotion and inhibition of effectors injected by the type III secretion system (T3SS, but the total number of these effectors and the overall functional relationships between these effectors during infection are poorly understood. EspC produced by EPEC cleaves fodrin, paxillin, and focal adhesion kinase (FAK, which are also cleaved by caspases and calpains during apoptosis. Here we show the role of EspC in cell death induced by EPEC. EspC is involved in EPEC-mediated cell death and induces both apoptosis and necrosis in epithelial cells. EspC induces apoptosis through the mitochondrial apoptotic pathway by provoking (i a decrease in the expression levels of antiapoptotic protein Bcl-2, (ii translocation of the proapoptotic protein Bax from cytosol to mitochondria, (iii cytochrome c release from mitochondria to the cytoplasm, (iv loss of mitochondrial membrane potential, (v caspase-9 activation, (vi cleavage of procaspase-3 and (vii an increase in caspase-3 activity, (viii PARP proteolysis, and (ix nuclear fragmentation and an increase in the sub-G1 population. Interestingly, EspC-induced apoptosis was triggered through a dual mechanism involving both independent and dependent functions of its EspC serine protease motif, the direct cleavage of procaspase-3 being dependent on this motif. This is the first report showing a shortcut for induction of apoptosis by the catalytic activity of an EPEC protein. Furthermore, this atypical intrinsic apoptosis appeared to induce necrosis through the activation of calpain and through the increase of intracellular calcium induced by EspC. Our data indicate that EspC plays a relevant role in cell death induced by EPEC.

  1. Caspase inhibition in select olfactory neurons restores innate attraction behavior in aged Drosophila.

    Directory of Open Access Journals (Sweden)

    Takahiro Chihara

    2014-06-01

    Full Text Available 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.

  2. Caspase inhibition in select olfactory neurons restores innate attraction behavior in aged Drosophila.

    Science.gov (United States)

    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.

  3. Different ways to die: cell death modes of the unicellular chlorophyte Dunaliella viridis exposed to various environmental stresses are mediated by the caspase-like activity DEVDase.

    Science.gov (United States)

    Jiménez, Carlos; Capasso, Juan M; Edelstein, Charles L; Rivard, Christopher J; Lucia, Scott; Breusegem, Sophia; Berl, Tomás; Segovia, María

    2009-01-01

    Programmed cell death is necessary for homeostasis in multicellular organisms and it is also widely recognized to occur in unicellular organisms. However, the mechanisms through which it occurs in unicells, and the enzymes involved within the final response is still the subject of heated debate. It is shown here that exposure of the unicellular microalga Dunaliella viridis to several environmental stresses, induced different cell death morphotypes, depending on the stimulus received. Senescent cells demonstrated classical and unambiguous apoptotic-like characteristics such as chromatin condensation, DNA fragmentation, intact organelles, and blebbing of the cell membrane. Acute heat shock caused general swelling and altered plasma membrane, but the presence of chromatin clusters and DNA strand breaks suggested a necrotic-like event. UV irradiated cells presented changes typical for necrosis, together with apoptotic characteristics resembling an intermediate cell-death phenotype termed aponecrosis-like. Cells subjected to hyperosmotic shock revealed chromatin spotting without DNA fragmentation, and extensive cytoplasmic swelling and vacuolization, comparable to a paraptotic-like cell death phenotype. Nitrogen-starved cells showed pyknosis, blebbing, and cytoplasmic consumption, indicating a similarity to autophagic/vacuolar-like cell death. The caspase-like activity DEVDase was measured by using the fluorescent substrate Ac-DEVD-AMC and antibodies against the human caspase-3 active enzyme cross-reacted with bands, the intensity of which paralleled the activity. All the environmental stresses tested produced a substantial increase in both DEVDase activity and protein levels. The irreversible caspase-3 inhibitor Z-DEVD-FMK completely inhibited the enzymatic activity whereas serine and aspartyl proteases inhibitors did not. These results show that cell death in D. viridis does not conform to a single pattern and that environmental stimuli may produce different types of

  4. Antrodia camphorata Potentiates Neuroprotection against Cerebral Ischemia in Rats via Downregulation of iNOS/HO-1/Bax and Activated Caspase-3 and Inhibition of Hydroxyl Radical Formation

    Directory of Open Access Journals (Sweden)

    Po-Sheng Yang

    2015-01-01

    Full Text Available Antrodia camphorata (A. camphorata is a fungus generally used in Chinese folk medicine for treatment of viral hepatitis and cancer. Our previous study found A. camphorata has neuroprotective properties and could reduce stroke injury in cerebral ischemia animal models. In this study, we sought to investigate the molecular mechanisms of neuroprotective effects of A. camphorata in middle cerebral artery occlusion (MCAO rats. A selective occlusion of the middle cerebral artery (MCA with whole blood clots was used to induce ischemic stroke in rats and they were orally treated with A. camphorata (0.25 and 0.75 g/kg/day alone or combined with aspirin (5 mg/kg/day. To provide insight into the functions of A. camphorata mediated neuroprotection, the expression of Bax, inducible nitric oxide synthase (iNOS, haem oxygenase-1 (HO-1, and activated caspase-3 was determined by Western blot assay. Treatment of aspirin alone significantly reduced the expressions of HO-1 (P<0.001, iNOS (P<0.001, and Bax (P<0.01 in ischemic regions. The reduction of these expressions was more potentiated when rats treated by aspirin combined with A. camphorata (0.75 g/kg/day. Combination treatment also reduced apoptosis as measured by a significant reduction in active caspase-3 expression in the ischemic brain compared to MCAO group (P<0.01. Moreover, treatment of A. camphorata significantly (P<0.05 reduced fenton reaction-induced hydroxyl radical (OH• formation at a dose of 40 mg/mL. Taken together, A. camphorata has shown neuroprotective effects in embolic rats, and the molecular mechanisms may correlate with the downregulation of Bax, iNOS, HO-1, and activated caspase-3 and the inhibition of OH• signals.

  5. Cord Blood CD8+ T Cells Have a Natural Propensity to Express IL-4 in a Fatty Acid Metabolism and Caspase Activation-Dependent Manner

    Directory of Open Access Journals (Sweden)

    Yuxia Zhang

    2018-04-01

    Full Text Available How T cells differentiate in the neonate may critically determine the ability of the infant to cope with infections, respond to vaccines and avert allergies. Previously, we found that naïve cord blood CD4+ T cells differentiated toward an IL-4-expressing phenotype when activated in the presence of TGF-β and monocyte-derived inflammatory cytokines, the latter are more highly secreted by infants who developed food allergy. Here, we show that in the absence of IL-2 or IL-12, naïve cord blood CD8+ T cells have a natural propensity to differentiate into IL-4-producing non-classic TC2 cells when they are activated alone, or in the presence of TGF-β and/or inflammatory cytokines. Mechanistically, non-classic TC2 development is associated with decreased expression of IL-2 receptor alpha (CD25 and glycolysis, and increased fatty acid metabolism and caspase-dependent cell death. Consequently, the short chain fatty acid, sodium propionate (NaPo, enhanced IL-4 expression, but exogenous IL-2 or pan-caspase inhibition prevented IL-4 expression. In children with endoscopically and histologically confirmed non-inflammatory bowel disease and non-infectious pediatric idiopathic colitis, the presence of TGF-β, NaPo, and IL-1β or TNF-α promoted TC2 differentiation in vitro. In vivo, colonic mucosa of children with colitis had significantly increased expression of IL-4 in CD8+ T cells compared with controls. In addition, activated caspase-3 and IL-4 were co-expressed in CD8+ T cells in the colonic mucosa of children with colitis. Thus, in the context of colonic inflammation and limited IL-2 signaling, CD8+ T cells differentiate into non-classic TC2 that may contribute to the pathology of inflammatory/allergic diseases in children.

  6. Blau syndrome-associated mutations in exon 4 of the caspase activating recruitment domain 15 (CARD 15) gene are not found in ethnic Danes with sarcoidosis

    DEFF Research Database (Denmark)

    Milman, Nils; Nielsen, Finn Cilius; Hviid, Thomas Vauvert F

    2007-01-01

    BACKGROUND: Distinct mutations of the caspase activating recruitment domain 15 (CARD15) gene (also known as nucleotide-binding oligomerisation domain protein 2) on chromosome 16q are associated with the chronic granulomatous disease called Blau syndrome. Sarcoidosis is a systemic granulomatous...... disease, which has features in common with Blau syndrome. AIM: The aim of this study was to evaluate whether ethnic Danes with sarcoidosis have CARD15 mutations associated with Blau syndrome. METHODS: Analysis of exon 4 of the CARD15 gene containing mutations associated with Blau syndrome was performed...

  7. Decreased rate of protein synthesis, caspase-3 activity, and ubiquitin-proteasome proteolysis in soleus muscles from growing rats fed a low-protein, high-carbohydrate diet.

    Science.gov (United States)

    Batistela, Emanuele; Pereira, Mayara Peron; Siqueira, Juliany Torres; Paula-Gomes, Silvia; Zanon, Neusa Maria; Oliveira, Eduardo Brandt; Navegantes, Luiz Carlos Carvalho; Kettelhut, Isis C; Andrade, Claudia Marlise Balbinotti; Kawashita, Nair Honda; Baviera, Amanda Martins

    2014-06-01

    The aim of this study was to investigate the changes in the rates of both protein synthesis and breakdown, and the activation of intracellular effectors that control these processes in soleus muscles from growing rats fed a low-protein, high-carbohydrate (LPHC) diet for 15 days. The mass and the protein content, as well as the rate of protein synthesis, were decreased in the soleus from LPHC-fed rats. The availability of amino acids was diminished, since the levels of various essential amino acids were decreased in the plasma of LPHC-fed rats. Overall rate of proteolysis was also decreased, explained by reductions in the mRNA levels of atrogin-1 and MuRF-1, ubiquitin conjugates, proteasome activity, and in the activity of caspase-3. Soleus muscles from LPHC-fed rats showed increased insulin sensitivity, with increased levels of insulin receptor and phosphorylation levels of AKT, which probably explains the inhibition of both the caspase-3 activity and the ubiquitin-proteasome system. The fall of muscle proteolysis seems to represent an adaptive response that contributes to spare proteins in a condition of diminished availability of dietary amino acids. Furthermore, the decreased rate of protein synthesis may be the driving factor to the lower muscle mass gain in growing rats fed the LPHC diet.

  8. Euglycemia in Diabetic Rats Leads to Reduced Liver Weight via Increased Autophagy and Apoptosis through Increased AMPK and Caspase-3 and Decreased mTOR Activities

    Directory of Open Access Journals (Sweden)

    Jun-Ho Lee

    2015-01-01

    Full Text Available Euglycemia is the ultimate goal in diabetes care to prevent complications. However, the benefits of euglycemia in type 2 diabetes are controversial because near-euglycemic subjects show higher mortality than moderately hyperglycemic subjects. We previously reported that euglycemic-diabetic rats on calorie-control lose a critical liver weight (LW compared with hyperglycemic rats. Here, we elucidated the molecular mechanisms underlying the loss of LW in euglycemic-diabetic rats and identified a potential risk in achieving euglycemia by calorie-control. Sprague-Dawley diabetic rats generated by subtotal-pancreatectomy were fed a calorie-controlled diet for 7 weeks to achieve euglycemia using 19 kcal% (19R or 6 kcal% (6R protein-containing chow or fed ad libitum (19AL. The diet in both R groups was isocaloric/kg body weight to the sham-operated group (19S. Compared with 19S and hyperglycemic 19AL, both euglycemic R groups showed lower LWs, increased autophagy, and increased AMPK and caspase-3 and decreased mTOR activities. Though degree of insulin deficiency was similar among the diabetic rats, Akt activity was lower, and PTEN activity was higher in both R groups than in 19AL whose signaling patterns were similar to 19S. In conclusion, euglycemia achieved by calorie-control is deleterious in insulin deficiency due to increased autophagy and apoptosis in the liver via AMPK and caspase-3 activation.

  9. Single-cell analysis of dihydroartemisinin-induced apoptosis through reactive oxygen species-mediated caspase-8 activation and mitochondrial pathway in ASTC-a-1 cells using fluorescence imaging techniques

    Science.gov (United States)

    Lu, Ying-Ying; Chen, Tong-Sheng; Wang, Xiao-Ping; Li, Li

    2010-07-01

    Dihydroartemisinin (DHA), a front-line antimalarial herbal compound, has been shown to possess promising anticancer activity with low toxicity. We have previously reported that DHA induced caspase-3-dependent apoptosis in human lung adenocarcinoma cells. However, the cellular target and molecular mechanism of DHA-induced apoptosis is still poorly defined. We use confocal fluorescence microscopy imaging, fluorescence resonance energy transfer, and fluorescence recovery after photobleaching techniques to explore the roles of DHA-elicited reactive oxygen species (ROS) in the DHA-induced Bcl-2 family proteins activation, mitochondrial dysfunction, caspase cascade, and cell death. Cell Counting Kit-8 assay and flow cytometry analysis showed that DHA induced ROS-mediated apoptosis. Confocal imaging analysis in a single living cell and Western blot assay showed that DHA triggered ROS-dependent Bax translocation, mitochondrial membrane depolarization, alteration of mitochondrial morphology, cytochrome c release, caspase-9, caspase-8, and caspase-3 activation, indicating the coexistence of ROS-mediated mitochondrial and death receptor pathway. Collectively, our findings demonstrate for the first time that DHA induces cell apoptosis by triggering ROS-mediated caspase-8/Bid activation and the mitochondrial pathway, which provides some novel insights into the application of DHA as a potential anticancer drug and a new therapeutic strategy by targeting ROS signaling in lung adenocarcinoma therapy in the future.

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

    Science.gov (United States)

    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.

  11. Dietary n-3 polyunsaturated fatty acid depletion activates caspases and decreases NMDA receptors in the brain of a transgenic mouse model of Alzheimer's disease.

    Science.gov (United States)

    Calon, Frédéric; Lim, Giselle P; Morihara, Takashi; Yang, Fusheng; Ubeda, Oliver; Salem, Norman; Frautschy, Sally A; Cole, Greg M

    2005-08-01

    Epidemiological data indicate that low n-3 polyunsaturated fatty acids (PFA) intake is a readily manipulated dietary risk factor for Alzheimer's disease (AD). Studies in animals confirm the deleterious effect of n-3 PFA depletion on cognition and on dendritic scaffold proteins. Here, we show that in transgenic mice overexpressing the human AD gene APPswe (Tg2576), safflower oil-induced n-3 PFA deficiency caused a decrease in N-methyl-D-aspartate (NMDA) receptor subunits, NR2A and NR2B, in the cortex and hippocampus with no loss of the presynaptic markers, synaptophysin and synaptosomal-associated protein 25 (SNAP-25). n-3 PFA depletion also decreased the NR1 subunit in the hippocampus and Ca2+/calmodulin-dependent protein kinase (CaMKII) in the cortex of Tg2576 mice. These effects of dietary n-3 PFA deficiency were greatly amplified in Tg2576 mice compared to nontransgenic mice. Loss of the NR2B receptor subunit was not explained by changes in mRNA expression, but correlated with p85alpha phosphatidylinositol 3-kinase levels. Most interestingly, n-3 PFA deficiency dramatically increased levels of protein fragments, corresponding to caspase/calpain-cleaved fodrin and gelsolin in Tg2576 mice. This effect was minimal in nontransgenic mice suggesting that n-3 PFA depletion potentiated caspase activation in the Tg2576 mouse model of AD. Dietary supplementation with docosahexaenoic acid (DHA; 22 : 6n-3) partly protected from NMDA receptor subunit loss and accumulation of fodrin and gelsolin fragments but fully prevented CaMKII decrease. The marked effect of dietary n-3 PFA on NMDA receptors and caspase/calpain activation in the cortex of an animal model of AD provide new insights into how dietary essential fatty acids may influence cognition and AD risk.

  12. Docosahexaenoic acid induces apoptosis in MCF-7 cells in vitro and in vivo via reactive oxygen species formation and caspase 8 activation.

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    Ki Sung Kang

    Full Text Available BACKGROUND: The present study sought to further investigate the in vitro and in vivo anticancer effects of a representative omega-3 fatty acid, docosahexaenoic acid (DHA, with a focus on assessing the induction of oxidative stress and apoptosis as an important mechanism for its anticancer actions. METHODOLOGY/PRINCIPAL FINDINGS: In vitro studies showed that DHA strongly reduces the viability and DNA synthesis of MCF-7 human breast cancer cells in culture, and also promotes cell death via apoptosis. Mechanistically, accumulation of reactive oxygen species and activation of caspase 8 contribute critically to the induction of apoptotic cell death. Co-presence of antioxidants or selective inhibition or knockdown of caspase 8 each effectively abrogates the cytotoxic effect of DHA. Using athymic nude mice as an in vivo model, we found that feeding animals the 5% fish oil-supplemented diet for 6 weeks significantly reduces the growth of MCF-7 human breast cancer cells in vivo through inhibition of cancer cell proliferation as well as promotion of cell death. Using 3-nitrotyrosine as a parameter, we confirmed that the fish oil-supplemented diet significantly increases oxidative stress in tumor cells in vivo. Analysis of fatty acid content in plasma and tissues showed that feeding animals a 5% fish oil diet increases the levels of DHA and eicosapentaenoic acid in both normal and tumorous mammary tissues by 329% and 300%, respectively. CONCLUSIONS/SIGNIFICANCE: DHA can strongly induce apoptosis in human MCF-7 breast cancer cells both in vitro and in vivo. The induction of apoptosis in these cells is selectively mediated via caspase 8 activation. These observations call for further studies to assess the effectiveness of fish oil as a dietary supplement in the prevention and treatment of human breast cancer.

  13. Protective mechanism of Korean Red Ginseng in cisplatin-induced ototoxicity through attenuation of nuclear factor-κB and caspase-1 activation.

    Science.gov (United States)

    Kim, Su-Jin; Kwak, Hyun Jeong; Kim, Dae-Seung; Choi, Hyun-Myung; Sim, Jung-Eun; Kim, Sung-Hoon; Um, Jae-Young; Hong, Seung-Heon

    2015-07-01

    Cisplatin is an effective anti-cancer drug; however, one of its side effects is irreversible sensorineural hearing damage. Korean Red Ginseng (KRG) has been used clinically for the treatment of various diseases; however, the underlying mechanism of KRG treatment of ototoxicity has not been studied extensively. The present study aimed to further investigate the mechanism of KRG on cisplatin-induced toxicity in auditory HEI-OC1 cells in vitro, as well as in vivo. The pharmacological effects of KRG on cisplatin-induced changes in the hearing threshold of mice were determined, as well as the effect on the impairment of hair cell arrays. In addition, in order to elucidate the protective mechanisms of KRG, the regulatory effects of KRG on cisplatin-induced apoptosis-associated gene levels and nuclear factor-κB (NF-κB) activation were investigated in auditory cells. The results revealed that KRG prevented cisplatin-induced alterations in the hearing threshold of mice as well as the destruction of hair cell arrays in rat organ of Corti primary explants. In addition, KRG inhibited cisplatin-mediated cell toxicity, reactive oxygen species generation, interleukin-6 production, cytochrome c release and activation of caspases-3 in the HEI-OC1 auditory cell line. Furthermore, the results demonstrated that KRG inhibited the activation of NF-κB and caspase-1. In conclusion, these results provided a model for the pharmacological mechanism of KRG and provided evidence for potential therapies against ototoxicity.

  14. Caspase-1-dependent and -independent cell death pathways in Burkholderia pseudomallei infection of macrophages.

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    Antje Bast

    2014-03-01

    Full Text Available The cytosolic pathogen Burkholderia pseudomallei and causative agent of melioidosis has been shown to regulate IL-1β and IL-18 production through NOD-like receptor NLRP3 and pyroptosis via NLRC4. Downstream signalling pathways of those receptors and other cell death mechanisms induced during B. pseudomallei infection have not been addressed so far in detail. Furthermore, the role of B. pseudomallei factors in inflammasome activation is still ill defined. In the present study we show that caspase-1 processing and pyroptosis is exclusively dependent on NLRC4, but not on NLRP3 in the early phase of macrophage infection, whereas at later time points caspase-1 activation and cell death is NLRC4- independent. In the early phase we identified an activation pathway involving caspases-9, -7 and PARP downstream of NLRC4 and caspase-1. Analyses of caspase-1/11-deficient infected macrophages revealed a strong induction of apoptosis, which is dependent on activation of apoptotic initiator and effector caspases. The early activation pathway of caspase-1 in macrophages was markedly reduced or completely abolished after infection with a B. pseudomallei flagellin FliC or a T3SS3 BsaU mutant. Studies using cells transfected with the wild-type and mutated T3SS3 effector protein BopE indicated also a role of this protein in caspase-1 processing. A T3SS3 inner rod protein BsaK mutant failed to activate caspase-1, revealed higher intracellular counts, reduced cell death and IL-1β secretion during early but not during late macrophage infection compared to the wild-type. Intranasal infection of BALB/c mice with the BsaK mutant displayed a strongly decreased mortality, lower bacterial loads in organs, and reduced levels of IL-1β, myeloperoxidase and neutrophils in bronchoalveolar lavage fluid. In conclusion, our results indicate a major role for a functional T3SS3 in early NLRC4-mediated caspase-1 activation and pyroptosis and a contribution of late caspase-1

  15. Fisetin inhibits growth, induces G2/M arrest and apoptosis of human epidermoid carcinoma A431 cells: Role of mitochondrial membrane potential disruption and consequent caspases activation

    Science.gov (United States)

    Pal, Harish C.; Sharma, Samriti; Elmets, Craig A.; Athar, Mohammad; Afaq, Farrukh

    2013-01-01

    Non-melanoma skin cancers (NMSCs) one of the most common neoplasms causes serious morbidity and mortality. Therefore, identification of non-toxic phytochemicals for prevention/treatment of NMSCs is highly desirable. Fisetin (3,3′,4′,7-tetrahydroxyflavone), a dietary flavonoid, present in fruits and vegetables possesses anti-oxidant and anti-proliferative properties. The aim of this study was to investigate the chemotherapeutic potential of fisetin in cultured human epidermoid carcinoma A431 cells. Treatment of A431 cells with fistein (5-80 μM) resulted in a significant decrease in cell viability in a dose- and time-dependent manner. Employing clonogenic assay, we found that fisetin treatment significantly reduced colony formation in A431 cells. Fisetin treatment of A431 cells resulted in G2/M arrest and induction of apoptosis. Furthermore, treatment of A431 cells with fisetin resulted in (i) decreased expression of anti-apoptotic proteins (Bcl2, Bcl-xL and Mcl-1), (ii) increased expression of pro-apoptotic proteins (Bax, Bak and Bad), (iii) disruption of mitochondrial potential, (iv) release of cytchrome c and Smac/DIABLO from mitochondria, (v) activation of caspases, and (vi) cleavage of PARP protein. Pretreatment of A431 cells with the pan-caspase inhibitor (Z-VAD-FMK) blocked fisetin-induced cleavage of caspases and PARP. Taken together, these data provide evidence that fisetin possesses chemotherapeutic potential against human epidermoid carcinoma A431 cells. Overall, these results suggest that fisetin could be developed as a novel therapeutic agent for the management of NMSCs. PMID:23800058

  16. TGFβ1 induces apoptosis in invasive prostate cancer and bladder cancer cells via Akt-independent, p38 MAPK and JNK/SAPK-mediated activation of caspases

    International Nuclear Information System (INIS)

    Al-Azayzih, Ahmad; Gao, Fei; Goc, Anna; Somanath, Payaningal R.

    2012-01-01

    Highlights: ► TGFβ induced apoptosis in invasive prostate cancer and bladder cancer cells. ► TGFβ inhibited prostate/bladder cancer cell proliferation and colony/foci formation. ► TGFβ induced prostate/bladder cancer cell apoptosis independent of Akt inhibition. ► TGFβ inhibited ERK1/2 phosphorylation in prostate/bladder cancer cells. ► TGFβ induced p38 MAPK and JNK-mediated activation of caspases-9, -8 and -3. -- Abstract: Recent findings indicate that advanced stage cancers shun the tumor suppressive actions of TGFβ and inexplicably utilize the cytokine as a tumor promoter. We investigated the effect of TGFβ1 on the survival and proliferation of invasive prostate (PC3) and bladder (T24) cancer cells. Our study indicated that TGFβ1 decreased cell viability and induced apoptosis in invasive human PC3 and T24 cells via activation of p38 MAPK-JNK-Caspase9/8/3 pathway. Surprisingly, no change in the phosphorylation of pro-survival Akt kinase was observed. We postulate that TGFβ1 pathway may be utilized for specifically targeting urological cancers without inflicting side effects on normal tissues.

  17. Oxcarbazepine causes neurocyte apoptosis and developing brain damage by triggering Bax/Bcl-2 signaling pathway mediated caspase 3 activation in neonatal rats.

    Science.gov (United States)

    Song, Y; Zhong, M; Cai, F-C

    2018-01-01

    Anti-epileptic drugs (AEDs) are the main methods for treatment of neonatal seizures; however, a few AEDs may cause developing brain damage of neonate. This study aims to investigate effects of oxcarbazepine (OXC) on developing brain damage of neonatal rats. Both of neonatal and adult rats were divided into 6 groups, including Control, OXC 187.5 mg/kg, OXC 281.25 mg/kg, OXC 375 mg/kg group, LEV and PHT group. Body weight and brain weight were evaluated. Hematoxylin and eosin (HE) and Nissl staining were used to observe neurocyte morphology and Nissl bodies, respectively. Apoptosis was examined using TUNEL assay, and caspase 8 activity was evaluated using spectrophotometer method. Cytochrome C-release was evaluated using flow cytometry. Western blot was used to examine Bax and Bcl-2 expression. OXC 375 mg/kg treatment significantly decreased brain weight compared to Control group in neonatal rats (P5 rats) (pOxcarbazepine at a concentration of 281.25 mg/kg or more causes neurocyte apoptosis and developing brain damage by triggering Bax/Bcl-2 signaling pathway mediated caspase 3 activation in neonatal rats.

  18. Multicentric osteolysis with nodulosis, arthritis, and cardiac defect syndrome: loss of MMP2 leads to increased apoptosis with alteration of apoptotic regulators and caspases and embryonic lethality

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    Mosig RA

    2014-11-01

    Full Text Available Rebecca A Mosig,1 Richard Schulz,2,3 Zamaneh Kassiri,4 Mitchell B Schaffler,5 John A Martignetti1 1Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA; 2Department of Pharmacology; 3Department of Pediatrics, 4Department of Physiology, Cardiovascular Research Centre, Mazankowski Alberta Heart Institute, University of Alberta, Edmonton, AB, Canada; 5Department of Biomedical Engineering, City College of New York, New York, NY, USA Abstract: Inactivating mutations of matrix metalloproteinase 2 (MMP2 cause multicentric osteolysis with nodulosis and arthritis, one of a group of inherited osteolytic and arthritic disorders. We have previously shown that mice lacking Mmp2 share similar syndromic features with the human disorder, and at the cellular level, Mmp2-/- mouse osteoblasts and osteoclasts have reduced numbers and proliferation rates at critical developmental time points. While previously hypothesized, the effect of MMP2 loss on apoptosis has not been examined in this system. We therefore sought to clarify its role in mediating the developmental defects in Mmp2-/- mice using immunohistochemistry, immunoblot analysis, and quantitative reverse transcription polymerase chain reaction analysis. We also explored the effects of MMP2 inhibition in the osteogenic sarcoma cell line SaOS2. Loss of MMP2 resulted in increased apoptosis and caspase activation both in vitro and in vivo. MMP2-deficient cells had increased Fas expression and reduced levels of the key survival signals p-FAK, p-ERK, cFLIP, and Bcl-2. Notably, and in marked contrast to their original characterization, there was a significant increase in the in utero demise of homozygous Mmp2-/- embryos. Specifically, litters from heterozygous crosses consistently yielded nearly 85% fewer than expected homozygous Mmp2-/- pups. Taken together, our findings highlight a new role for MMP2 in preventing apoptosis during development and growth. Keywords

  19. Tubeimoside-1 induces glioma apoptosis through regulation of Bax/Bcl-2 and the ROS/Cytochrome C/Caspase-3 pathway

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    Jia G

    2015-01-01

    Full Text Available Geng Jia,1,* Qiang Wang,2,* Rong Wang,2,* Danni Deng,2 Lian Xue,2 Naiyuan Shao,1 Yi Zhang,1 Xiwei Xia,1 Feng Zhi,2 Yilin Yang1,2 1Department of Neurosurgery, Third Affiliated Hospital of Soochow University, Jiangsu, People’s Republic of China; 2Modern Medical Research Center, Third Affiliated Hospital of Soochow University, Jiangsu, People’s Republic of China * These authors contributed equally to this workBackground: Tubeimoside-1 (TBMS1 is a natural compound isolated from tubeimoside, which has been widely used as a traditional Chinese herbal medicine. The purpose of the present study is to investigate the anti-tumor effect and the underling mechanism of TBMS1 on glioma cancer cells.Methods: The MTT assay was performed to evaluate the effect of TBMS1 on glioma cell proliferation. The fluorescent microscopy and flow cytometry analysis were performed to evaluate the effect of TBMS1 on glioma cell apoptosis. The Western blot analysis was used to evaluate the protein change.Results: TBMS1 inhibited glioma cancer cell proliferation in a dose- and time-dependent manner. Fluorescent microscopy and flow cytometry analysis demonstrated that TBMS1 induced glioma cell apoptosis in a concentration-dependent manner. Western blotting showed that TBMS1 induced apoptosis by increasing the expression of Bax and downregulating the level of Bcl-2. Furthermore, we found that TBMS1 induced apoptosis by increasing the concentration of reactive oxygen species through the release of Cytochrome C and activation of Caspase-3.Conclusion: These findings indicate that TBMS1 may be developed as a possible therapeutic agent for the management of glioma. Keywords: Tubeimoside-1, glioma, proliferation, apoptosis

  20. New insights into the apoptotic process in mollusks: characterization of caspase genes in Mytilus galloprovincialis.

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    Alejandro Romero

    2011-02-01

    Full Text Available Apoptosis is an essential biological process in the development and maintenance of immune system homeostasis. Caspase proteins constitute the core of the apoptotic machinery and can be categorized as either initiators or effectors of apoptosis. Although the genes encoding caspase proteins have been described in vertebrates and in almost all invertebrate phyla, there are few reports describing the initiator and executioner caspases or the modulation of their expression by different stimuli in different apoptotic pathways in bivalves. In the present work, we characterized two initiator and four executioner caspases in the mussel Mytilus galloprovincialis. Both initiators and executioners showed structural features that make them different from other caspase proteins already described. Evaluation of the genes' tissue expression patterns revealed extremely high expression levels within the gland and gills, where the apoptotic process is highly active due to the clearance of damaged cells. Hemocytes also showed high expression values, probably due to of the role of apoptosis in the defense against pathogens. To understand the mechanisms of caspase gene regulation, hemocytes were treated with UV-light, environmental pollutants and pathogen-associated molecular patterns (PAMPs and apoptosis was evaluated by microscopy, flow cytometry and qPCR techniques. Our results suggest that the apoptotic process could be tightly regulated in bivalve mollusks by overexpression/suppression of caspase genes; additionally, there is evidence of caspase-specific responses to pathogens and pollutants. The apoptotic process in mollusks has a similar complexity to that of vertebrates, but presents unique features that may be related to recurrent exposure to environmental changes, pollutants and pathogens imposed by their sedentary nature.

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

    International Nuclear Information System (INIS)

    Garcia-Rates, Sara; Camarasa, Jordi; Sanchez-Garcia, Ana I.; Gandia, Luis; Escubedo, Elena; Pubill, David

    2010-01-01

    Previous work by our group demonstrated that homomeric α7 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 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 inhibited the response induced by ACh, nicotine, and the specific α7 agonist PNU 282987 with IC 50 values in the low micromolar range. Similarly, MDMA induced inward currents in Xenopus oocytes transfected with human α7 but not with α4β2 nAChR and inhibited ACh-induced currents in both receptors in a concentration-dependent manner. The calcium response was inhibited by methyllycaconitine (MLA) and α-bungarotoxin but not by dihydro-β-erythroidine. These results therefore indicate that MDMA acts as a partial agonist on α7 nAChRs and as an antagonist on the heteromeric subtypes. Subsequently, calcium-induced Ca 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 2+ levels and induced an increase in α-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 α7 nAChR. Sustained calcium entry and calpain activation could favor the activation of Ca 2+ -dependent enzymes such as

  2. BID is cleaved by caspase-8 within a native complex on the mitochondrial membrane

    NARCIS (Netherlands)

    Schug, Z. T.; Gonzalvez, F.; Houtkooper, R. H.; Vaz, F. M.; Gottlieb, E.

    2011-01-01

    Caspase-8 stably inserts into the mitochondrial outer membrane during extrinsic apoptosis. Inhibition of caspase-8 enrichment on the mitochondria impairs caspase-8 activation and prevents apoptosis. However, the function of active caspase-8 on the mitochondrial membrane remains unknown. In this

  3. Both the caspase CSP-1 and a caspase-independent pathway promote programmed cell death in parallel to the canonical pathway for apoptosis in Caenorhabditis elegans.

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    Daniel P Denning

    Full Text Available Caspases are cysteine proteases that can drive apoptosis in metazoans and have critical functions in the elimination of cells during development, the maintenance of tissue homeostasis, and responses to cellular damage. Although a growing body of research suggests that programmed cell death can occur in the absence of caspases, mammalian studies of caspase-independent apoptosis are confounded by the existence of at least seven caspase homologs that can function redundantly to promote cell death. Caspase-independent programmed cell death is also thought to occur in the invertebrate nematode Caenorhabditis elegans. The C. elegans genome contains four caspase genes (ced-3, csp-1, csp-2, and csp-3, of which only ced-3 has been demonstrated to promote apoptosis. Here, we show that CSP-1 is a pro-apoptotic caspase that promotes programmed cell death in a subset of cells fated to die during C. elegans embryogenesis. csp-1 is expressed robustly in late pachytene nuclei of the germline and is required maternally for its role in embryonic programmed cell deaths. Unlike CED-3, CSP-1 is not regulated by the APAF-1 homolog CED-4 or the BCL-2 homolog CED-9, revealing that csp-1 functions independently of the canonical genetic pathway for apoptosis. Previously we demonstrated that embryos lacking all four caspases can eliminate cells through an extrusion mechanism and that these cells are apoptotic. Extruded cells differ from cells that normally undergo programmed cell death not only by being extruded but also by not being engulfed by neighboring cells. In this study, we identify in csp-3; csp-1; csp-2 ced-3 quadruple mutants apoptotic cell corpses that fully resemble wild-type cell corpses: these caspase-deficient cell corpses are morphologically apoptotic, are not extruded, and are internalized by engulfing cells. We conclude that both caspase-dependent and caspase-independent pathways promote apoptotic programmed cell death and the phagocytosis of cell

  4. Increased killing of SCCVII squamous cell carcinoma cells after the combination of Pc 4 photodynamic therapy and dasatinib is associated with enhanced caspase-3 activity and ceramide synthase 1 upregulation

    Science.gov (United States)

    SEPAROVIC, DUSKA; BREEN, PAUL; BOPPANA, NITHIN B.; VAN BUREN, ERIC; JOSEPH, NICHOLAS; KRAVEKA, JACQUELINE M.; RAHMANIYAN, MEHRDAD; LI, LI; GUDZ, TATYANA I.; BIELAWSKA, ALICJA; BAI, AIPING; BIELAWSKI, JACEK; PIERCE, JASON S.; KORBELIK, MLADEN

    2013-01-01

    Photodynamic therapy (PDT) is not always effective as an anticancer treatment, therefore, PDT is combined with other anticancer agents for improved efficacy. The combination of dasatinib and PDT with the silicone phthalocyanine photosensitizer Pc 4 was assessed for increased killing of SCCVII mouse squamous cell carcinoma cells, a preclinical model of head and neck squamous cell carcinoma, using apoptotic markers and colony formation as experimental end-points. Because each of these treatments regulates the metabolism of the sphingolipid ceramide, their effects on mRNA levels of ceramide synthase, a ceramide-producing enzyme, and the sphingolipid profile were determined. PDT + dasatinib induced an additive loss of clonogenicity. Unlike PDT alone or PDT + dasatinib, dasatinib induced zVAD-fmk-dependent cell killing. PDT or dasatinib-induced caspase-3 activation was potentiated after the combination. PDT alone induced mitochondrial depolarization, and the effect was inhibited after the combination. Annexin V+ and propidium iodide+ cells remained at control levels after treatments. In contrast to PDT alone, dasatinib induced upregulation of ceramide synthase 1 mRNA, and the effect was enhanced after the combination. Dasatinib induced a modest increase in C20:1-and C22-ceramide but had no effect on total ceramide levels. PDT increased the levels of 12 individual ceramides and total ceramides, and the addition of dasatinib did not affect these increases. PDT alone decreased substantially sphingosine levels and inhibited the activity of acid ceramidase, an enzyme that converts ceramide to sphingosine. The data suggest that PDT-induced increases in ceramide levels do not correlate with ceramide synthase mRNA levels but rather with inhibition of ceramidase. Cell killing was zVAD-fmk-sensitive after dasatinib but not after either PDT or the combination and enhanced cell killing after the combination correlated with potentiated caspase-3 activation and upregulation of

  5. Structurally related antitumor effects of flavanones in vitro and in vivo: involvement of caspase 3 activation, p21 gene expression, and reactive oxygen species production

    International Nuclear Information System (INIS)

    Shen, S.-C.; Ko, C.H.; Tseng, S.-W.; Tsai, S.-H.; Chen, Y.-C.

    2004-01-01

    Flavonoids exist extensively in plants and Chinese herbs, and several biological effects of flavonoids have been demonstrated. The antitumor effects in colorectal carcinoma cells (HT29, COLO205, and COLO320HSR) of eight flavanones including flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone, 7-OH flavanone, naringenin, nargin, and taxifolin were investigated. Results of the MTT assay indicate that 2'-OH flavanone showed the most potent cytotoxic effect on these three cells, and cell death induced by 2'-OH flavanone was via the occurrence of DNA ladders, apoptotic bodies, and hypodiploid cells, all characteristics of apoptosis. Induction of caspase 3 protein processing and enzyme activity associated with cleavage of poly(ADP-ribose) polymerase (PARP) was identified in 2'-OH flavanone-treated cells, and a peptidyl inhibitor (Ac-DEVD-FMK) of caspase 3 attenuated the cytotoxicity of 2'-OH flavanone in COLO205 and HT-29 cells. Elevation of p21 (but not p53) and a decrease in Mcl-1 protein were found in 2'-OH flavanone-treated COLO205 and HT-29 cells. Elevation of intracellular reactive oxygen species (ROS) was detected in 2'-OH flavanone-treated cells by the 2',7'-dichlorodihydrofluorescein diacetate (DCHF-DA) assay, and ROS scavengers including 4,5-dihydro-1,3-benzene disulfonic acid (tiron), catalase, superoxide dismutase (SOD), and pyrrolidine dithiocarbamate (PDTC) suppressed the 2'-OH flavanone-induced cytotoxic effect. Subcutaneous injection of COLO205 induced tumor formation in nude mice, and 2'-OH flavanone showed a significant inhibitory effect on tumor formation. The appearance of apoptotic cells with H and E staining, and an increase in p21, but not p53, protein by immunohistochemistry were observed in tumor tissues under 2'-OH flavanone treatment. Primary tumor cells (COLO205-X) derived from a tumor specimen elicited by COLO205 were established, and 2'-OH flavanone showed an significant apoptotic effect in COLO205-X cells in accordance with the

  6. N,N-dimethyl phytosphingosine induces caspase-8-dependent cytochrome c release and apoptosis through ROS generation in human leukemia cells

    International Nuclear Information System (INIS)

    Kim, Byeong Mo; Choi, Yun Jung; Han, Youngsoo; Yun, Yeon-Sook; Hong, Sung Hee

    2009-01-01

    N,N-dimethyl phytosphingosine (DMPS) blocks the conversion of sphingosine to sphingosine-1-phosphate (S1P) by the enzyme sphingosine kinase (SK). In this study, we elucidated the apoptotic mechanisms of DMPS action on a human leukemia cell line using functional pharmacologic and genetic approaches. First, we demonstrated that DMPS-induced apoptosis is evidenced by nuclear morphological change, distinct internucleosomal DNA fragmentation, and an increased sub-G1 cell population. DMPS treatment led to the activation of caspase-9 and caspase-3, accompanied by the cleavage of poly(ADP-ribose) polymerase (PARP) and led to cytochrome c release, depolarization of the mitochondrial membrane potential, and downregulation of the anti-apoptotic members of the bcl-2 family. Ectopic expression of bcl-2 and bcl-xL conferred resistance of HL-60 cells to DMPS-induced cell death, suggesting that DMPS-induced apoptosis occurs predominantly through the activation of the intrinsic mitochondrial pathway. We also observed that DMPS activated the caspase-8-Bid-Bax pathway and that the inhibition of caspase-8 by z-IETD-fmk or small interfering RNA suppressed the cleavage of Bid, cytochrome c release, caspase-3 activation, and apoptotic cell death. In addition, cells subjected to DMPS exhibited significantly increased reactive oxygen species (ROS) generation, and ROS scavengers, such as quercetin and Tiron, but not N-acetylcysteine (NAC), inhibited DMPS-induced activations of caspase-8, -3 and subsequent apoptotic cell death, indicating the role of ROS in caspase-8-mediated apoptosis. Taken together, these results indicate that caspase-8 acts upstream of caspase-3, and that the caspase-8-mediated mitochondrial pathway is important in DMPS-induced apoptosis. Our results also suggest that ROS are critical regulators of caspase-8-mediated apoptosis in DMPS-treated leukemia cells.

  7. The Enigmatic Roles of Caspases in Tumor Development

    Energy Technology Data Exchange (ETDEWEB)

    Jäger, Richard; Zwacka, Ralf M., E-mail: ralf.zwacka@nuigalway.ie [National University of Ireland, Galway, National Centre for Biomedical Engineering Science and Apoptosis Research Centre, Molecular Therapeutics Group, Galway (Ireland)

    2010-11-24

    One function ascribed to apoptosis is the suicidal destruction of potentially harmful cells, such as cancerous cells. Hence, their growth depends on evasion of apoptosis, which is considered as one of the hallmarks of cancer. Apoptosis is ultimately carried out by the sequential activation of initiator and executioner caspases, which constitute a family of intracellular proteases involved in dismantling the cell in an ordered fashion. In cancer, therefore, one would anticipate caspases to be frequently rendered inactive, either by gene silencing or by somatic mutations. From clinical data, however, there is little evidence that caspase genes are impaired in cancer. Executioner caspases have only rarely been found mutated or silenced, and also initiator caspases are only affected in particular types of cancer. There is experimental evidence from transgenic mice that certain initiator caspases, such as caspase-8 and -2, might act as tumor suppressors. Loss of the initiator caspase of the intrinsic apoptotic pathway, caspase-9, however, did not promote cellular transformation. These data seem to question a general tumor-suppressive role of caspases. We discuss several possible ways how tumor cells might evade the need for alterations of caspase genes. First, alternative splicing in tumor cells might generate caspase variants that counteract apoptosis. Second, in tumor cells caspases might be kept in check by cellular caspase inhibitors such as c-FLIP or XIAP. Third, pathways upstream of caspase activation might be disrupted in tumor cells. Finally, caspase-independent cell death mechanisms might abrogate the selection pressure for caspase inactivation during tumor development. These scenarios, however, are hardly compatible with the considerable frequency of spontaneous apoptosis occurring in several cancer types. Therefore, alternative concepts might come into play, such as compensatory proliferation. Herein, apoptosis and/or non-apoptotic functions of caspases may

  8. The Enigmatic Roles of Caspases in Tumor Development

    International Nuclear Information System (INIS)

    Jäger, Richard; Zwacka, Ralf M.

    2010-01-01

    One function ascribed to apoptosis is the suicidal destruction of potentially harmful cells, such as cancerous cells. Hence, their growth depends on evasion of apoptosis, which is considered as one of the hallmarks of cancer. Apoptosis is ultimately carried out by the sequential activation of initiator and executioner caspases, which constitute a family of intracellular proteases involved in dismantling the cell in an ordered fashion. In cancer, therefore, one would anticipate caspases to be frequently rendered inactive, either by gene silencing or by somatic mutations. From clinical data, however, there is little evidence that caspase genes are impaired in cancer. Executioner caspases have only rarely been found mutated or silenced, and also initiator caspases are only affected in particular types of cancer. There is experimental evidence from transgenic mice that certain initiator caspases, such as caspase-8 and -2, might act as tumor suppressors. Loss of the initiator caspase of the intrinsic apoptotic pathway, caspase-9, however, did not promote cellular transformation. These data seem to question a general tumor-suppressive role of caspases. We discuss several possible ways how tumor cells might evade the need for alterations of caspase genes. First, alternative splicing in tumor cells might generate caspase variants that counteract apoptosis. Second, in tumor cells caspases might be kept in check by cellular caspase inhibitors such as c-FLIP or XIAP. Third, pathways upstream of caspase activation might be disrupted in tumor cells. Finally, caspase-independent cell death mechanisms might abrogate the selection pressure for caspase inactivation during tumor development. These scenarios, however, are hardly compatible with the considerable frequency of spontaneous apoptosis occurring in several cancer types. Therefore, alternative concepts might come into play, such as compensatory proliferation. Herein, apoptosis and/or non-apoptotic functions of caspases may

  9. Doxorubicin in vivo rapidly alters expression and translation of myocardial electron transport chain genes, leads to ATP loss and caspase 3 activation.

    Directory of Open Access Journals (Sweden)

    Amy V Pointon

    2010-09-01

    Full Text Available Doxorubicin is one of the most effective anti-cancer drugs but its use is limited by cumulative cardiotoxicity that restricts lifetime dose. Redox damage is one of the most accepted mechanisms of toxicity, but not fully substantiated. Moreover doxorubicin is not an efficient redox cycling compound due to its low redox potential. Here we used genomic and chemical systems approaches in vivo to investigate the mechanisms of doxorubicin cardiotoxicity, and specifically test the hypothesis of redox cycling mediated cardiotoxicity.Mice were treated with an acute dose of either doxorubicin (DOX (15 mg/kg or 2,3-dimethoxy-1,4-naphthoquinone (DMNQ (25 mg/kg. DMNQ is a more efficient redox cycling agent than DOX but unlike DOX has limited ability to inhibit gene transcription and DNA replication. This allowed specific testing of the redox hypothesis for cardiotoxicity. An acute dose was used to avoid pathophysiological effects in the genomic analysis. However similar data were obtained with a chronic model, but are not specifically presented. All data are deposited in the Gene Expression Omnibus (GEO. Pathway and biochemical analysis of cardiac global gene transcription and mRNA translation data derived at time points from 5 min after an acute exposure in vivo showed a pronounced effect on electron transport chain activity. This led to loss of ATP, increased AMPK expression, mitochondrial genome amplification and activation of caspase 3. No data gathered with either compound indicated general redox damage, though site specific redox damage in mitochondria cannot be entirely discounted.These data indicate the major mechanism of doxorubicin cardiotoxicity is via damage or inhibition of the electron transport chain and not general redox stress. There is a rapid response at transcriptional and translational level of many of the genes coding for proteins of the electron transport chain complexes. Still though ATP loss occurs with activation caspase 3 and these

  10. Anti-cancer effect of bee venom in prostate cancer cells through activation of caspase pathway via inactivation of NF-κB.

    Science.gov (United States)

    Park, Mi Hee; Choi, Myoung Suk; Kwak, Dong Hoon; Oh, Ki-Wan; Yoon, Do Young; Han, Sang Bae; Song, Ho Sueb; Song, Min Jong; Hong, Jin Tae

    2011-06-01

    Bee venom has been used as a traditional medicine to treat arthritis, rheumatism, back pain, cancerous tumors, and skin diseases. However, the effects of bee venom on the prostate cancer and their action mechanisms have not been reported yet. To determine the effect of bee venom and its major component, melittin on the prostate cancer cells, apoptosis is analyzed by tunnel assay and apoptotic gene expression. For xenograft studies, bee venom was administrated intraperitoneally twice per week for 4 weeks, and the tumor growth was measured and the tumor were analyzed by immunohistochemistry. To investigate whether bee venom and melittin can inactivate nuclear factor kappa B (NF-κB), we assessed NF-κB activity in vitro and in vivo. Bee venom (1-10 µg/ml) and melittin (0.5-2.5 µg/ml) inhibited cancer cell growth through induction of apoptotic cell death in LNCaP, DU145, and PC-3 human prostate cancer cells. These effects were mediated by the suppression of constitutively activated NF-κB. Bee venom and melittin decreased anti-apoptotic proteins but induced pro-apoptotic proteins. However, pan caspase inhibitor abolished bee venom and melittin-induced apoptotic cell death and NF-κB inactivation. Bee venom (3-6 mg/kg) administration to nude mice implanted with PC-3 cells resulted in inhibition of tumor growth and activity of NF-κB accompanied with apoptotic cell death. Therefore, these results indicated that bee venom and melittin could inhibit prostate cancer in in vitro and in vivo, and these effects may be related to NF-κB/caspase signal mediated induction of apoptotic cell death. Copyright © 2010 Wiley-Liss, Inc.

  11. In vitro antiproliferative activity of 2,3-dihydroxy-9,10-anthraquinone induced apoptosis against COLO320 cells through cytochrome c release caspase mediated pathway with PI3K/AKT and COX-2 inhibition.

    Science.gov (United States)

    Balachandran, C; Emi, N; Arun, Y; Yamamoto, N; Duraipandiyan, V; Inaguma, Yoko; Okamoto, Akinao; Ignacimuthu, S; Al-Dhabi, N A; Perumal, P T

    2016-04-05

    The present study investigated the anticancer activity of 2,3-dihydroxy-9,10-anthraquinone against different cancer cells such as MCF-7, COLO320, HepG-2, Skov-3, MOLM-14, NB-4, CEM, K562, Jurkat, HL-60, U937, IM-9 and Vero. 2,3-dihydroxy-9,10-anthraquinone showed good antiproliferative activity against COLO320 cells when compared to other tested cells. The cytotoxicity results showed 79.8% activity at the dose of 2.07 μM with IC50 value of 0.13 μM at 24 h in COLO320 cells. So we chose COLO320 cells for further anticancer studies. mRNA expression was confirmed by qPCR analysis using SYBR green method. Treatment with 2,3-dihydroxy-9,10-anthraquinone was found to trigger intrinsic apoptotic pathway as indicated by down regulation of Bcl-2, Bcl-xl; up regulation of Bim, Bax, Bad; release of cytochrome c and pro-caspases cleaving to caspases. Furthermore, 2,3-dihydroxy-9,10-anthraquinone stopped at G0/G1 phase with modulation in protein levels of cyclins. On the other hand PI3K/AKT signaling plays an important role in cell metabolism. We found that 2,3-dihydroxy-9,10-anthraquinone inhibits PI3K/AKT activity after treatment. Also, COX-2 enzyme plays a major role in colorectal cancer. Our results showed that the treatment significantly reduced COX-2 enzyme in COLO320 cells. These results indicated antiproliferative activity of 2,3-dihydroxy-9,10-anthraquinone involving apoptotic pathways, mitochondrial functions, cell cycle checkpoint and controlling the over expression genes during the colorectal cancer. Molecular docking studies showed that the compound bound stably to the active sites of Bcl-2, COX-2, PI3K and AKT. This is the first report of anticancer mechanism involving 2,3-dihydroxy-9,10-anthraquinone in COLO320 cells. The present results might provide helpful suggestions for the design of antitumor drugs toward colorectal cancer treatment. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  12. Caspase-14 Expression Impairs Retinal Pigment Epithelium Barrier Function: Potential Role in Diabetic Macular Edema

    Directory of Open Access Journals (Sweden)

    Selina Beasley

    2014-01-01

    Full Text Available We recently showed that caspase-14 is a novel molecule in retina with potential role in accelerated vascular cell death during diabetic retinopathy (DR. Here, we evaluated whether caspase-14 is implicated in retinal pigment epithelial cells (RPE dysfunction under hyperglycemia. The impact of high glucose (HG, 30 mM D-glucose on caspase-14 expression in human RPE (ARPE-19 cells was tested, which showed significant increase in caspase-14 expression compared with normal glucose (5 mM D-glucose + 25 mM L-glucose. We also evaluated the impact of modulating caspase-14 expression on RPE cells barrier function, phagocytosis, and activation of other caspases using ARPE-19 cells transfected with caspase-14 plasmid or caspase-14 siRNA. We used FITC-dextran flux assay and electric cell substrate impedance sensing (ECIS to test the changes in RPE cell barrier function. Similar to HG, caspase-14 expression in ARPE-19 cells increased FITC-dextran leakage through the confluent monolayer and decreased the transcellular electrical resistance (TER. These effects of HG were prevented by caspase-14 knockdown. Furthermore, caspase-14 knockdown prevented the HG-induced activation of caspase-1 and caspase-9, the only activated caspases by HG. Phagocytic activity was unaffected by caspase-14 expression. Our results suggest that caspase-14 contributes to RPE cell barrier disruption under hyperglycemic conditions and thus plays a role in the development of diabetic macular edema.

  13. The VPAC2 agonist peptide histidine isoleucine (PHI) up-regulates glutamate transport in the corpus callosum of a rat model of amyotrophic lateral sclerosis (hSOD1G93A) by inhibiting caspase-3 mediated inactivation of GLT-1a.

    Science.gov (United States)

    Goursaud, Stéphanie; Focant, Marylène C; Berger, Julie V; Nizet, Yannick; Maloteaux, Jean-Marie; Hermans, Emmanuel

    2011-10-01

    Degeneration of corpus callosum appears in patients with amyotrophic lateral sclerosis (ALS) before clinical signs of upper motor neuron death. Considering the ALS-associated impairment of astrocytic glutamate uptake, we have characterized the expression and activity of the glutamate transporter isoforms GLT-1a and GLT-1b in the corpus callosum of transgenic rats expressing a mutated form of the human superoxide dismutase 1 (hSOD1(G93A)). We have also studied the effect of peptide histidine isoleucine (PHI), a vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating polypeptide (PACAP) receptor 2 (VPAC(2)) agonist on glutamate transporters both in vivo and in callosal astrocytes. Before the onset of motor symptoms, the expression of both transporter isoforms was correlated with a constitutive activity of caspase-3. This enzyme participates in the down-regulation of GLT-1 in ALS, and here we demonstrated its involvement in the selective degradation of GLT-1a in the white matter. A single stereotactic injection of PHI into the corpus callosum of symptomatic rats decreased caspase-3 activity and promoted GLT-1a expression and uptake activity. Together, with evidence for a reduced expression of prepro-VIP/PHI mRNA in the corpus callosum of transgenic animals, these data shed light on the modulatory role of the VIP/PHI system on the glutamatergic transmission in ALS.

  14. Cirtical role for Salmonella effector SopB in regulating inflammasome activation.

    Science.gov (United States)

    Hu, Gui-Qiu; Song, Pei-Xuan; Chen, Wei; Qi, Shuai; Yu, Shui-Xing; Du, Chong-Tao; Deng, Xu-Ming; Ouyang, Hong-Sheng; Yang, Yong-Jun

    2017-10-01

    Salmonella is known to evolve many mechanisms to avoid or delay inflammasome activation which remain largely unknown. In this study, we investigated whether the SopB protein critical to bacteria virulence capacity was an effector that involved in the regulation of inflammasome activation. BMDMs from NLRC4-, NLRP3-, caspase-1/-11-, IFI16- and AIM2-deficient mice were pretreated with LPS, and subsequently stimulated with a series of SopB-related strains of Salmonella, inflammasome induced cell death, IL-1β secretion, cleaved caspase-1 production and ASC speckle formation were detected. We found that SopB could inhibit host IL-1β secretion, caspase-1 activation and inflammasome induced cell death using a series of SopB-related strains of Salmonella; however the reduction of IL-1β secretion was not dependent on sensor that contain PYD domain, such as NLRP3, AIM2 or IFI16, but dependent on NLRC4. Notably, SopB specifically prevented ASC oligomerization and the enzymatic activity of SopB was responsible for the inflammasome inhibition. Furthermore, inhibition of Akt signaling induced enhanced inflammasome activation. These results revealed a novel role in inhibition of NLRC4 inflammasome for Salmonella effector SopB. Copyright © 2017. Published by Elsevier Ltd.

  15. Protective Effects of Chlorella-Derived Peptide Against UVC-Induced Cytotoxicity through Inhibition of Caspase-3 Activity and Reduction of the Expression of Phosphorylated FADD and Cleaved PARP-1 in Skin Fibroblasts

    Directory of Open Access Journals (Sweden)

    Jong Yuh Cherng

    2012-08-01

    Full Text Available UVC irradiation induces oxidative stress and leads to cell death through an apoptotic pathway. This apoptosis is caused by activation of caspase-3 and formation of poly(ADP-ribose polymerase-1 (PARP-1. In this study, the underlying mechanisms of Chlorella derived peptide (CDP activity against UVC-induced cytotoxicity were investigated. Human skin fibroblasts were treated with CDP, vitamin C, or vitamin E after UVC irradiation for a total energy of 15 J/cm2. After the UVC exposure, cell proliferation and caspase-3 activity were measured at 12, 24, 48, and 72 h later. Expression of phosphorylated FADD and cleaved PARP-1 were measured 16 h later. DNA damage (expressed as pyrimidine (6-4 pyrimidone photoproducts DNA concentration and fragmentation assay were performed 24 h after the UVC exposure. Results showed that UVC irradiation induced cytotoxicity in all groups except those treated with CDP. The caspase-3 activity in CDP-treated cells was inhibited from 12 h onward. Expression of phosphorylated FADD and cleaved PARP-1 were also reduced in CDP-treated cells. Moreover, UVC-induced DNA damage and fragmentation were also prevented by the CDP treatment. This study shows that treatment of CDP provides protective effects against UVC-induced cytotoxicity through the inhibition of caspase-3 activity and the reduction of phosphorylated FADD and cleaved PARP-1 expression.

  16. AKT2 Blocks Nucleus Translocation of Apoptosis-Inducing Factor (AIF and Endonuclease G (EndoG While Promoting Caspase Activation during Cardiac Ischemia

    Directory of Open Access Journals (Sweden)

    Shuai Yang

    2017-03-01

    Full Text Available The AKT (protein kinase B, PKB family has been shown to participate in diverse cellular processes, including apoptosis. Previous studies demonstrated that protein kinase B2 (AKT2−/− mice heart was sensitized to apoptosis in response to ischemic injury. However, little is known about the mechanism and apoptotic signaling pathway. Here, we show that AKT2 inhibition does not affect the development of cardiomyocytes but increases cell death during cardiomyocyte ischemia. Caspase-dependent apoptosis of both the extrinsic and intrinsic pathway was inactivated in cardiomyocytes with AKT2 inhibition during ischemia, while significant mitochondrial disruption was observed as well as intracytosolic translocation of cytochrome C (Cyto C together with apoptosis-inducing factor (AIF and endonuclease G (EndoG, both of which are proven to conduct DNA degradation in a range of cell death stimuli. Therefore, mitochondria-dependent cell death was investigated and the results suggested that AIF and EndoG nucleus translocation causes cardiomyocyte DNA degradation during ischemia when AKT2 is blocked. These data are the first to show a previous unrecognized function and mechanism of AKT2 in regulating cardiomyocyte survival during ischemia by inducing a unique mitochondrial-dependent DNA degradation pathway when it is inhibited.

  17. Caspase-9 has a nonapoptotic function in Xenopus embryonic primitive blood formation.

    Science.gov (United States)

    Tran, Hong Thi; Fransen, Mathias; Dimitrakopoulou, Dionysia; Van Imschoot, Griet; Willemarck, Nicolas; Vleminckx, Kris

    2017-07-15

    Caspases constitute a family of cysteine proteases centrally involved in programmed cell death, which is an integral part of normal embryonic and fetal development. However, it has become clear that specific caspases also have functions independent of cell death. In order to identify novel apoptotic and nonapoptotic developmental caspase functions, we designed and transgenically integrated novel fluorescent caspase reporter constructs in developing Xenopus embryos and tadpoles. This model organism has an external development, allowing direct and continuous monitoring. These studies uncovered a nonapoptotic role for the initiator caspase-9 in primitive blood formation. Functional experiments further corroborated that caspase-9, but possibly not the executioners caspase-3 and caspase-7, are required for primitive erythropoiesis in the early embryo. These data reveal a novel nonapoptotic function for the initiator caspase-9 and, for the first time, implicate nonapoptotic caspase activity in primitive blood formation. © 2017. Published by The Company of Biologists Ltd.

  18. Taurine reverses sodium fluoride-mediated increase in inflammation, caspase-3 activity, and oxidative damage along the brain-pituitary-gonadal axis in male rats.

    Science.gov (United States)

    Adedara, Isaac A; Olabiyi, Bolanle F; Ojuade, TeminiJesu D; Idris, Umar F; Onibiyo, Esther M; Farombi, Ebenezer O

    2017-09-01

    Excessive exposure to fluoride is associated with male reproductive dysfunction in humans and animals. Taurine (2-aminoethane sulfonic acid) is a free intracellular β-amino acid with antioxidant, anti-inflammatory, and neuroprotective properties. However, the effect of taurine on fluoride-induced reproductive toxicity has not been reported. The present study investigated the influence of taurine on sodium fluoride (NaF)-induced functional changes along the brain-pituitary-gonadal axis in male rats. NaF was administered singly in drinking water at 15 mg·L -1 alone or orally co-administered by gavage with taurine at 100 and 200 mg·(kg body mass) -1 for 45 consecutive days. Results showed that taurine significantly prevented NaF-induced increase in oxidative stress indices as well as augmented antioxidant enzymes activities and glutathione level in the brain, testes, and epididymis of the treated rats. Moreover, taurine reversed NaF-induced elevation in inflammatory biomarkers and caspase-3 activity as well as histological damage in the brain, testes, and epididymis of the treated rats. The significant reversal of NaF-induced decreases in testosterone level and testicular activities of acid phosphatase, alkaline phosphatase, and lactate dehydrogenase by taurine was accompanied by enhancement of sperm functional characteristics in the treated rats. Taurine may be a possible chemopreventive candidate against reproductive dysfunction resulting from fluoride exposure.

  19. Diosgenin induces apoptosis in IGF-1-stimulated human thyrocytes through two caspase-dependent pathways

    Energy Technology Data Exchange (ETDEWEB)

    Mu, Shumin [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Hospital Affiliated to Shandong Traditional Chinese Medicine University, Jinan 250011 (China); Institute of Endocrinology, Shandong Academy of Clinical Medicine, Jinan 250021 (China); Tian, Xingsong; Ruan, Yongwei [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Liu, Yuantao [The Second Hospital of Shandong University, Jinan 250033 (China); Bian, Dezhi [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Jining Medical College, Jining 272013 (China); Ma, Chunyan [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Yu, Chunxiao [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Institute of Endocrinology, Shandong Academy of Clinical Medicine, Jinan 250021 (China); Feng, Mei [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Wang, Furong [Shandong University of Traditional Chinese Medicine, Jinan 250011 (China); Gao, Ling [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Institute of Endocrinology, Shandong Academy of Clinical Medicine, Jinan 250021 (China); Zhao, Jia-jun, E-mail: jjzhao@medmail.com.cn [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Institute of Endocrinology, Shandong Academy of Clinical Medicine, Jinan 250021 (China)

    2012-02-10

    Highlights: Black-Right-Pointing-Pointer Diosgenin induces apoptosis in IGF-1-treated thyrocytes through two caspase pathways. Black-Right-Pointing-Pointer Diosgenin inhibits FLIP and activates caspase-8 in FAS related-pathway. Black-Right-Pointing-Pointer Diosgenin increases ROS, regulates the ratio of Bax/Bcl-2 in mitochondrial pathway. -- Abstract: Insulin-like growth factor-1 (IGF-1) is a growth factor of the thyroid that has been shown in our previous study to possess proliferative and antiapoptotic effects in FRTL-5 cell lines through the upregulation of cyclin D and Fas-associated death domain-like interleukin-1-converting enzyme (FLICE)-inhibitory protein (FLIP). Diosgenin, a natural steroid sapogenin from plants, has been shown to induce apoptosis in many cell lines, with the exception of thyroid cells. In this report, we investigated the apoptotic effect and mechanism of diosgenin in IGF-1-stimulated primary human thyrocytes. Primary human thyrocytes were preincubated with or without IGF-1 for 24 h and subsequently exposed to varying concentrations of diosgenin for different times. We found that diosgenin induced apoptosis in human thyrocytes pretreated with IGF-1 in a dose-dependent manner through the activation of caspase cascades. Moreover, diosgenin inhibited FLIP and activated caspase-8 in the FAS-related apoptotic pathway. Diosgenin increased the production of ROS, regulated the balance of Bax and Bcl-2 and cleaved caspase-9 in the mitochondrial apoptotic pathway. These results indicate that diosgenin induces apoptosis in IGF-1-stimulated primary human thyrocytes through two caspase-dependent pathways.

  20. Caspase-1 Deficiency Alleviates Dopaminergic Neuronal Death via Inhibiting Caspase-7/AIF Pathway in MPTP/p Mouse Model of Parkinson's Disease.

    Science.gov (United States)

    Qiao, Chen; Zhang, Lin-Xia; Sun, Xi-Yang; Ding, Jian-Hua; Lu, Ming; Hu, Gang

    2017-08-01

    Caspase family has been recognized to be involved in dopaminergic (DA) neuronal death and to exert an unfavorable role in Parkinson's disease (PD) pathology. Our previous study has revealed that caspase-1, as an important component of NLRP3 inflammasome, induces microglia-mediated neuroinflammation in the pathogenesis of PD. However, the role of caspase-1 in DA neuronal degeneration in the onset of PD remains unclear. Here, we showed that caspase-1 knockout ameliorated DA neuronal loss and dyskinesia in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine/probenecid (MPTP/p)-induced PD model mice. We further found that caspase-1 knockout decreased MPTP/p-induced caspase-7 cleavage, subsequently inhibited nuclear translocation of poly (ADP-ribose) polymerase 1 (PARP1), and reduced the release of apoptosis-inducing factor (AIF). Consistently, we demonstrated that caspase-1 inhibitor suppressed caspase-7/PARP1/AIF-mediated apoptosis pathway by 1-methyl-4-phenylpyridinium ion (MPP + ) stimulation in SH-SY5Y cells. Caspase-7 overexpression reduced the protective effects of caspase-1 inhibitor on SH-SY5Y cell apoptosis. Collectively, our results have revealed that caspase-1 regulates DA neuronal death in the pathogenesis of PD in mice via caspase-7/PARP1/AIF pathway. These findings will shed new insight into the potential of caspase-1 as a target for PD therapy.

  1. Lactoferrin inhibits dexamethasone-induced chondrocyte impairment from osteoarthritic cartilage through up-regulation of extracellular signal-regulated kinase 1/2 and suppression of FASL, FAS, and Caspase 3

    International Nuclear Information System (INIS)

    Tu, Yihui; Xue, Huaming; Francis, Wendy; Davies, Andrew P.; Pallister, Ian; Kanamarlapudi, Venkateswarlu; Xia, Zhidao

    2013-01-01

    Highlights: •Dex exerts dose-dependant inhibition of HACs viability and induction of apoptosis. •Dex-induced impairment of chondrocytes was attenuated by rhLF. •ERK and FASL/FAS signaling are involved in the effects of rhLF. •OA patients with glucocorticoid-induced cartilage damage may benefit from treatment with rhLF. -- Abstract: Dexamethasone (Dex) is commonly used for osteoarthritis (OA) with excellent anti-inflammatory and analgesic effect. However, Dex also has many side effects following repeated use over prolonged periods mainly through increasing apoptosis and inhibiting proliferation. Lactoferrin (LF) exerts significantly anabolic effect on many cells and little is known about its effect on OA chondrocytes. Therefore, the aim of this study is to investigate whether LF can inhibit Dex-induced OA chondrocytes apoptosis and explore its possible molecular mechanism involved in. MTT assay was used to determine the optimal concentration of Dex and recombinant human LF (rhLF) on chondrocytes at different time and dose points. Chondrocytes were then stimulated with Dex in the absence or presence of optimal concentration of rhLF. Cell proliferation and viability were evaluated using MTT and LIVE/DEAD assay, respectively. Cell apoptosis was evaluated by multi-parameter apoptosis assay kit using both confocal microscopy and flow cytometry, respectively. The expression of extracellular signal-regulated kinase (ERK), FAS, FASL, and Caspase-3 (CASP3) at the mRNA and protein levels were examined by real-time polymerase chain reaction (PCR) and immunocytochemistry, respectively. The optimal concentration of Dex (25 μg/ml) and rhLF (200 μg/ml) were chosen for the following experiments. rhLF significantly reversed the detrimental effect of Dex on chondrocytes proliferation, viability, and apoptosis. In addition, rhLF significantly prevented Dex-induced down-regulation of ERK and up-regulation of FAS, FASL, and CASP3. These findings demonstrated that rhLF acts as

  2. Lactoferrin inhibits dexamethasone-induced chondrocyte impairment from osteoarthritic cartilage through up-regulation of extracellular signal-regulated kinase 1/2 and suppression of FASL, FAS, and Caspase 3

    Energy Technology Data Exchange (ETDEWEB)

    Tu, Yihui [Department of Orthopaedics, Yangpu District Central Hospital Affiliated to Tongji University School of Medicine, 450 Tengyue Road, Shanghai (China); Xue, Huaming [Department of Orthopaedics, Yangpu District Central Hospital Affiliated to Tongji University School of Medicine, 450 Tengyue Road, Shanghai (China); Institute of Life Science, College of Medicine, Swansea University, Singleton Park (United Kingdom); Francis, Wendy [Institute of Life Science, College of Medicine, Swansea University, Singleton Park (United Kingdom); Davies, Andrew P. [Department of Orthopaedics and Trauma, Moriston Hospital, Swansea (United Kingdom); Pallister, Ian; Kanamarlapudi, Venkateswarlu [Institute of Life Science, College of Medicine, Swansea University, Singleton Park (United Kingdom); Xia, Zhidao, E-mail: zhidao.xia@gmail.com [Institute of Life Science, College of Medicine, Swansea University, Singleton Park (United Kingdom)

    2013-11-08

    Highlights: •Dex exerts dose-dependant inhibition of HACs viability and induction of apoptosis. •Dex-induced impairment of chondrocytes was attenuated by rhLF. •ERK and FASL/FAS signaling are involved in the effects of rhLF. •OA patients with glucocorticoid-induced cartilage damage may benefit from treatment with rhLF. -- Abstract: Dexamethasone (Dex) is commonly used for osteoarthritis (OA) with excellent anti-inflammatory and analgesic effect. However, Dex also has many side effects following repeated use over prolonged periods mainly through increasing apoptosis and inhibiting proliferation. Lactoferrin (LF) exerts significantly anabolic effect on many cells and little is known about its effect on OA chondrocytes. Therefore, the aim of this study is to investigate whether LF can inhibit Dex-induced OA chondrocytes apoptosis and explore its possible molecular mechanism involved in. MTT assay was used to determine the optimal concentration of Dex and recombinant human LF (rhLF) on chondrocytes at different time and dose points. Chondrocytes were then stimulated with Dex in the absence or presence of optimal concentration of rhLF. Cell proliferation and viability were evaluated using MTT and LIVE/DEAD assay, respectively. Cell apoptosis was evaluated by multi-parameter apoptosis assay kit using both confocal microscopy and flow cytometry, respectively. The expression of extracellular signal-regulated kinase (ERK), FAS, FASL, and Caspase-3 (CASP3) at the mRNA and protein levels were examined by real-time polymerase chain reaction (PCR) and immunocytochemistry, respectively. The optimal concentration of Dex (25 μg/ml) and rhLF (200 μg/ml) were chosen for the following experiments. rhLF significantly reversed the detrimental effect of Dex on chondrocytes proliferation, viability, and apoptosis. In addition, rhLF significantly prevented Dex-induced down-regulation of ERK and up-regulation of FAS, FASL, and CASP3. These findings demonstrated that rhLF acts as

  3. Inhibition of Mitochondrial Cytochrome c Release and Suppression of Caspases by Gamma-Tocotrienol Prevent Apoptosis and Delay Aging in Stress-Induced Premature Senescence of Skin Fibroblasts

    Directory of Open Access Journals (Sweden)

    Suzana Makpol

    2012-01-01

    Full Text Available In this study, we determined the molecular mechanism of γ-tocotrienol (GTT in preventing cellular aging by focusing on its anti-apoptotic effect in stress-induced premature senescence (SIPS model of human diploid fibroblasts (HDFs. Results obtained showed that SIPS exhibited senescent-phenotypic characteristic, increased expression of senescence-associated β-galactosidase (SA β-gal and promoted G0/G1 cell cycle arrest accompanied by shortening of telomere length with decreased telomerase activity. Both SIPS and senescent HDFs shared similar apoptotic changes such as increased Annexin V-FITC positive cells, increased cytochrome c release and increased activation of caspase-9 and caspase-3 (P<0.05. GTT treatment resulted in a significant reduction of Annexin V-FITC positive cells, inhibited cytochrome c release and decreased activation of caspase-9 and caspase-3 (P<0.05. Gene expression analysis showed that GTT treatment down regulated BAX mRNA, up-regulated BCL2A1 mRNA and decreased the ratio of Bax/Bcl-2 protein expression (P<0.05 in SIPS. These findings suggested that GTT inhibits apoptosis by modulating the upstream apoptosis cascade, causing the inhibition of cytochrome c release from the mitochondria with concomitant suppression of caspase-9 and caspase-3 activation. In conclusion, GTT delays cellular senescence of human diploid fibroblasts through the inhibition of intrinsic mitochondria-mediated pathway which involved the regulation of pro- and anti-apoptotic genes and proteins.

  4. Inhibitor specificity of recombinant and endogenous caspase-9.

    Science.gov (United States)

    Ryan, Ciara A; Stennicke, Henning R; Nava, Victor E; Burch, Jennifer B; Hardwick, J Marie; Salvesen, Guy S

    2002-01-01

    Apoptosis triggered through the intrinsic pathway by radiation and anti-neoplastic drugs is initiated by the activation of caspase-9. To elucidate control mechanisms in this pathway we used a range of synthetic and natural reagents. The inhibitory potency of acetyl-Asp-Glu-Val-Asp-aldehyde ('Ac-DEVD-CHO'), benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone ('Z-VAD-FMK') and the endogenous caspase inhibitor X-chromosome-linked inhibitor of apoptosis protein ('XIAP') against recombinant caspase-9 were predictive of the efficacy of these compounds in a cell-free system. However, the viral proteins CrmA and p35, although potent inhibitors of recombinant caspase-9, had almost no ability to block caspase-9 in this system. These findings were also mirrored in cell expression studies. We hypothesize that the viral inhibitors CrmA and p35 are excluded from reacting productively with the natural form of active caspase-9 in vivo, making the potency of inhibitors highly context-dependent. This is supported by survival data from a mouse model of apoptosis driven by Sindbis virus expressing either p35 or a catalytic mutant of caspase-9. These results consolidate previous findings that CrmA is a potent inhibitor of caspase-9 in vitro, yet fails to block caspase-9-mediated cell death. PMID:12067274

  5. Suppression of human T cell proliferation by the caspase inhibitors, z-VAD-FMK and z-IETD-FMK is independent of their caspase inhibition properties

    Energy Technology Data Exchange (ETDEWEB)

    Lawrence, C.P. [Medical Research Council Toxicology Unit, Hodgkin Building, Lancaster Road, University of Leicester, Leicester LE1 9HN (United Kingdom); Chow, S.C., E-mail: chow.sek.chuen@monash.edu [School of Science, Monash University Sunway Campus, Jalan Lagoon Selatan, Bandar Sunway, 46150 Selangor Darul Ehsan (Malaysia)

    2012-11-15

    The caspase inhibitors, benzyloxycarbony (Cbz)-l-Val-Ala-Asp (OMe)-fluoromethylketone (z-VAD-FMK) and benzyloxycarbonyl (Cbz)-Ile-Glu (OMe)-Thr-Asp (OMe)-FMK (z-IETD-FMK) at non-toxic doses were found to be immunosuppressive and inhibit human T cell proliferation induced by mitogens and IL-2 in vitro. Both caspase inhibitors were shown to block NF-κB in activated primary T cells, but have little inhibitory effect on the secretion of IL-2 and IFN-γ during T cell activation. However, the expression of IL-2 receptor α-chain (CD25) in activated T cells was inhibited by both z-VAD-FMK and z-IETD-FMK, whereas the expression of the early activated T cell marker, CD69 was unaffected. During primary T cell activation via the antigen receptor, both caspase-8 and caspase-3 were activated and processed to their respective subunits, but neither caspase inhibitors had any effect on the processing of these two caspases. In sharp contrast both caspase inhibitors readily blocked apoptosis and the activation of caspases during FasL-induced apoptosis in activated primary T cells and Jurkat T cells. Collectively, the results demonstrate that both z-VAD-FMK and z-IETD-FMK are immunosuppressive in vitro and inhibit T cell proliferation without blocking the processing of caspase-8 and caspase-3. -- Highlights: ► Caspase-8 and caspase-3 were activated during T cell activation and proliferation. ► T cell proliferation was blocked by caspase inhibitors. ► Caspase activation during T cell proliferation was not block by caspase inhibitors.

  6. Caspase-3 Inhibition Attenuates the Cytopathic Effects of EV71 Infection

    Directory of Open Access Journals (Sweden)

    Fengmei Song

    2018-04-01

    Full Text Available Previous studies demonstrate that human enterovirus 71 (EV71, a primary causative agent for hand, foot, and mouth disease, activates caspase-3 through the non-structural viral 3C protein to induce host cell apoptosis; however, until now it was unclear how 3C activates caspase-3 and how caspase-3 activation affects viral production. Our results demonstrate that 3C binds caspase-8 and caspase-9 but does not directly bind caspase-3 to activate them, and that the proteolytic activity of 3C is required by the activation of caspase-8, caspase-9, and caspase-3. Inhibition of caspase-3 activity attenuates apoptosis in 3C-transfected cells. Furthermore, caspase-3 inhibitor protects host cells from the cytopathic effect of EV71 infection and prevents cell cycle arrest, which is known to be favored for EV71 viral replication. Inhibition of caspase-3 activity decreases EV71 viral protein expression and viral production, but has no effect on viral entry, replication, even polyprotein translation. Therefore, caspase-3 is exploited functionally by EV71 to facilitate its production, which suggests a novel therapeutic approach for the treatment and prevention of hand, foot, and mouth disease.

  7. Implications of caspase-dependent proteolytic cleavage of cyclin A1 in DNA damage-induced cell death

    Energy Technology Data Exchange (ETDEWEB)

    Woo, Sang Hyeok; Seo, Sung-Keum [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of); An, Sungkwan; Choe, Tae-Boo [Department of Microbiological Engineering, Kon-Kuk University, Gwangjin-gu, Seoul (Korea, Republic of); Hong, Seok-Il [Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of); Lee, Yun-Han, E-mail: yhlee87@yuhs.ac [Department of Radiation Oncology, College of Medicine, Yonsei University, 250 Seongsan-no, Seodaemun-gu, Seoul (Korea, Republic of); Park, In-Chul, E-mail: parkic@kcch.re.kr [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of)

    2014-10-24

    Highlights: • Caspase-1 mediates doxorubicin-induced downregulation of cyclin A1. • Active caspase-1 effectively cleaved cyclin A1 at D165. • Cyclin A1 expression is involved in DNA damage-induced cell death. - Abstract: Cyclin A1 is an A-type cyclin that directly binds to CDK2 to regulate cell-cycle progression. In the present study, we found that doxorubicin decreased the expression of cyclin A1 at the protein level in A549 lung cancer cells, while markedly downregulating its mRNA levels. Interestingly, doxorubicin upregulated caspase-1 in a concentration-dependent manner, and z-YAVD-fmk, a specific inhibitor of caspase-1, reversed the doxorubicin-induced decrease in cyclin A1 in A549 lung cancer and MCF7 breast cancer cells. Active caspase-1 effectively cleaved cyclin A1 at D165 into two fragments, which in vitro cleavage assays showed were further cleaved by caspase-3. Finally, we found that overexpression of cyclin A1 significantly reduced the cytotoxicity of doxorubicin, and knockdown of cyclin A1 by RNA interference enhanced the sensitivity of cells to ionizing radiation. Our data suggest a new mechanism for the downregulation of cyclin A1 by DNA-damaging stimuli that could be intimately involved in the cell death induced by DNA damage-inducing stimuli, including doxorubicin and ionizing radiation.

  8. Dentatin Induces Apoptosis in Prostate Cancer Cells via Bcl-2, Bcl-xL, Survivin Downregulation, Caspase-9, -3/7 Activation, and NF-κB Inhibition

    Directory of Open Access Journals (Sweden)

    Ismail Adam Arbab

    2012-01-01

    Full Text Available This study was set to investigate antiproliferative potential of dentatin (a natural coumarin isolated from Clausena excavata Burm. F against prostate cancer and to delineate the underlying mechanism of action. Treatment with dentatin dose-dependently inhibited cell growth of PC-3 and LNCaP prostate cancer cell lines, whereas it showed less cytotoxic effects on normal prostate epithelial cell line (RWPE-1. The inhibitory effect of dentatin on prostate cancer cell growth was due to induction of apoptosis as evidenced by Annexin V staining and cell shrinkage. We found that dentatin-mediated accumulation of reactive oxygen species (ROS and downregulated expression levels of antiapoptotic molecules (Bcl-2, Bcl-xL, and Survivin, leading to disruption of mitochondrial membrane potential (MMP, cell membrane permeability, and release of cytochrome c from the mitochondria into the cytosol. These effects were associated with induction of caspase-9, -3/7 activities, and subsequent DNA fragmentation. In addition, we found that dentatin inhibited TNF-α-induced nuclear translocation of p65, suggesting dentatin as a potential NF-κB inhibitor. Thus, we suggest that dentatin may have therapeutic value in prostate cancer treatment worthy of further development.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Yang Liu

    Full Text Available 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.

  11. 3-Monochloro-1,2-propanediol (3-MCPD) induces apoptosis via mitochondrial oxidative phosphorylation system impairment and the caspase cascade pathway

    International Nuclear Information System (INIS)

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

    2016-01-01

    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.

  12. 3-Monochloro-1,2-propanediol (3-MCPD) induces apoptosis via mitochondrial oxidative phosphorylation system impairment and the caspase cascade pathway.

    Science.gov (United States)

    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.

  13. Quercetin inhibits the poly(dA:dT)-induced secretion of IL-18 via down-regulation of the expressions of AIM2 and pro-caspase-1 by inhibiting the JAK2/STAT1 pathway in IFN-γ-primed human keratinocytes.

    Science.gov (United States)

    Lee, Kyung-Mi; Kang, Jung Hoon; Yun, Mihee; Lee, Seong-Beom

    2018-06-05

    Quercetin, a polyphenol, belongs to a class of flavonoids that exerts anti-inflammatory effects. Interleukin (IL)-18 is a member of the IL-1 family cytokine that regulates immune responses and is implicated in various inflammatory skin diseases. Absent in melanoma 2 (AIM2) is a cytosolic double-stranded (ds) DNA sensor that recognizes the dsDNA of a microbial or host origin. Binding of dsDNA to AIM2 simulates caspase-1-dependent inflammasome activity, which leads to the production of IL-1β and IL-18. Increased levels of AIM2 have been observed in patients with inflammatory skin diseases. In the current study, we investigated the issue of whether or how Quercetin attenuates poly (dA:dT), a synthetic analog of microbial dsDNA, -induced IL-18 secretion in IFN-γ-primed human keratinocytes. Treatment with 5 and 10 μM of Quercetin inhibited the poly (dA:dT)-induced secretion of IL-18 after IFN-γ priming and before poly (dA:dT)-induced AIM2 activation. In addition, treatment with Quercetin at 10 μM, significantly inhibited the phosphorylation of JAK2 and STAT1, and the nuclear translocation of phosphorylated STAT1 in poly (dA:dT)-treated and IFN-γ-primed keratinocytes. These results suggest that treatment with Quercetin inhibits the poly (dA:dT)-induced secretion of IL-18 via down-regulation of the expressions of AIM2 and pro-caspase-1 by inhibiting the JAK2/STAT1 pathway in IFN-γ-primed keratinocytes. Copyright © 2018 Elsevier Inc. All rights reserved.

  14. THE EXPRESSION OF Bcl-2 AND PRO-CASPASE 3 IN HEAD AND NECK SQUAMOUS CELL CARCINOMA

    Directory of Open Access Journals (Sweden)

    Andrej Cör

    2002-12-01

    Full Text Available Background. Head and neck squamous cell carcinoma (HNSCC is the sixth most common cancer and accounts for 6% of cancers worldwide. A better understanding of its biology could lead to improved treatment options. Generally, the goal of cancer treatment is to abolish cell proliferation and to induce necrotic or aptoptotic cell death. Apoptosis has been recognized as a key mechanism of tumour cell elimination. Different apoptotic signals converge to induce caspase cascade activation. Caspase 3 is the central executioner caspase and is necessary for effective apoptotic cell death. Bcl-2 protein family regulates apoptosis. The Bcl-2 protein itself is a product of a proto-oncogene and has an antiapoptotic action.Methods. In our study, the expression of Bcl-2 and pro-caspase 3 by immunohistochemistry in 28 HNSCC graded into well, moderately and poorly differentiated cancers were investigated.Results. Our results of Bcl-2 expression confirm and extend previous reports in which Bcl-2 over-expression has been recognised as an important parameter in HNSCC biological behaviour. Three of 28 tumours (11% showed significant Bcl-2 expression. Two of them were poorly and one was moderately differentiated. Pro-caspase 3 immunoreactivity was confined mainly to the cytoplasm. Absent or low pro-caspase 3 immunoreactivity was found only in 1 of 6 well differentiated and in 1of 10 moderately differentiated tumours in contrast to 5 of 12 poorly differentiated tumours. In six of 12 poorly differentiated tumours procasapse 3 immunoreactivity was strongly positive. In two cases hyperplastic epithelium was strongly positive in contrast to adjacent HNSCC in the same slide which was completely negative for pro-caspase 3.Conclusions. Our results indicate downregulation of pro-caspase 3 expression, especially in poorly differentiated HNSCC. Further studies are needed to test whether this is related to HNSCC behaviour and predict treatment outcome.

  15. Glutamate-induced apoptosis in neuronal cells is mediated via caspase-dependent and independent mechanisms involving calpain and caspase-3 proteases as well as apoptosis inducing factor (AIF and this process is inhibited by equine estrogens

    Directory of Open Access Journals (Sweden)

    Bhavnani Bhagu R

    2006-06-01

    Full Text Available Abstract Background Glutamate, a major excitatory amino acid neurotransmitter, causes apoptotic neuronal cell death at high concentrations. Our previous studies have shown that depending on the neuronal cell type, glutamate-induced apoptotic cell death was associated with regulation of genes such as Bcl-2, Bax, and/or caspase-3 and mitochondrial cytochrome c. To further delineate the intracellular mechanisms, we have investigated the role of calpain, an important calcium-dependent protease thought to be involved in apoptosis along with mitochondrial apoptosis inducing factor (AIF and caspase-3 in primary cortical cells and a mouse hippocampal cell line HT22. Results Glutamate-induced apoptotic cell death in neuronal cells was associated with characteristic DNA fragmentation, morphological changes, activation of calpain and caspase-3 as well as the upregulation and/or translocation of AIF from mitochondria into cytosol and nuclei. Our results reveal that primary cortical cells and HT22 cells display different patterns of regulation of these genes/proteins. In primary cortical cells, glutamate induces activation of calpain, caspase-3 and translocation of AIF from mitochondria to cytosol and nuclei. In contrast, in HT22 cells, only the activation of calpain and upregulation and translocation of AIF occurred. In both cell types, these processes were inhibited/reversed by 17β-estradiol and Δ8,17β-estradiol with the latter being more potent. Conclusion Depending upon the neuronal cell type, at least two mechanisms are involved in glutamate-induced apoptosis: a caspase-3-dependent pathway and a caspase-independent pathway involving calpain and AIF. Since HT22 cells lack caspase-3, glutamate-induced apoptosis is mediated via the caspase-independent pathway in this cell line. Kinetics of this apoptotic pathway further indicate that calpain rather than caspase-3, plays a critical role in the glutamate-induced apoptosis. Our studies further indicate

  16. Estrous cycle dependent changes in expression and distribution of Fas, Fas ligand, Bcl-2, Bax, and pro- and active caspase-3 in the rat ovary

    NARCIS (Netherlands)

    Slot, K.A.; Voorendt, M.; Boer-Brouwer, de M.; Vugt, van H.H.; Teerds, K.J.

    2006-01-01

    In the present investigation, the localization of proteins involved in ovarian apoptosis were studied throughout the estrous cycle in the presence of fluctuating hormone levels. Fas, Fas ligand, Bcl-2, Bax and caspase-3 mRNA expression and proteins were detected in all ovarian tissue extracts,

  17. Protective effect of lycopene on fluoride-induced ameloblasts apoptosis and dental fluorosis through oxidative stress-mediated Caspase pathways.

    Science.gov (United States)

    Li, Weishan; Jiang, Binghua; Cao, Xianglin; Xie, Yongjiang; Huang, Ting

    2017-01-05

    Fluoride is an environmental toxicant and induces dental fluorosis and oxidative stress. Lycopene (LYC) is an effective antioxidant that is reported to attenuate fluoride toxicity. To determine the effects of LYC on sodium fluoride (NaF) -induced teeth and ameloblasts toxicity, rats were treated with NaF (10 mg/kg) and/or LYC (10 mg/kg) by orally administration for 5 weeks; ameloblasts were treated with NaF (5 mM) and/or LYC (2 μM) for 6 h. We found that the concentrations of fluoride, malondialdehyde (MDA) and reactive oxygen species (ROS), gene expressions and activities of Caspase-9 and Caspase-3, and the gene expressions of Bax were significantly decreased, while the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX), the gene expression of Bcl-2 were significantly increased in the LYC + NaF-treated rats group; concentrations of MDA and ROS, gene expressions and activities of Caspase-9 and Caspase-3, and the gene expression of Bax, and ameloblasts apoptosis rate were significantly decreased, while the activities of SOD and GPX, the gene expression of Bcl-2 were significantly increased in the LYC + NaF-treated ameloblasts group. These results suggest that LYC significantly combated NaF-induced ameloblasts apoptosis and dental fluorosis by attenuation oxidative stress and down-regulation Caspase pathway. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. SAG/ROC-SCFβ-TrCP E3 Ubiquitin Ligase Promotes Pro-Caspase-3 Degradation as a Mechanism of Apoptosis Protection

    Directory of Open Access Journals (Sweden)

    Mingjia Tan

    2006-12-01

    Full Text Available Skp1-cullin-F-box protein (SCF is a multicomponent E3 ubiquitin (Ub ligase that ubiquitinates a number of important biologic molecules such as p27, β-catenin, and lκB for proteasomal degradation, thus regulating cell proliferation and survival. One SCF component, SAG/ROC2/Rbx2/Hrt2, a RING finger protein, was first identified as a redox-inducible protein, which, when overexpressed, inhibited apoptosis both in vitro and in vivo. We report here that sensitive to apoptosis gene (SAG, as well as its family member ROC1/Rbxi, bound to the proinactive form of caspase-3 (pro-caspase-3. Binding was likely mediated through F-box protein, β-transducin repeat-containing protein (β-TrCP, which binds to the first 38 amino acids of pro-caspase-3. Importantly, β-TrCP1 expression significantly shortened the protein half-life of pro-caspase-3, whereas expression of a dominant-negative β-TrCP1 mutant with the F-box domain deleted extended it. An in vitro ubiquitination assay showed that SAG/ROC-SCF -Trcp promoted ubiquitination of pro-caspase-3. Furthermore, endogenous levels of pro-caspase-3 were decreased by overexpression of SAG/ROC-SCFβ-TrCP E3 Ub ligases, but increased on siRNA silencing of SAG, regulator of cullin-1 (ROC1, or β-TrCPs, leading to increased apoptosis by etoposide and TNF-related apoptosis-inducing ligand through increased activation of caspase-3. Thus, pro-caspase-3 appears to be a substrate of SAG/ROC-SCFβ-TrCP E3 Ub ligase, which protects cells from apoptosis through increased apoptosis threshold by reducing the basal level of pro-caspase-3.

  19. Cadmium induces Ca2+ mediated, calpain-1/caspase-3-dependent apoptosis in primary cultured rat proximal tubular cells.

    Science.gov (United States)

    Wang, Hong; Zhai, Nianhui; Chen, Ying; Xu, Haibin; Huang, Kehe

    2017-07-01

    Calcium, as a ubiquitous second messenger, governs a large array of cellular processes and is necessary for cell survival. More recently, it was observed that the cytosolic Ca 2+ concentration ([Ca 2+ ] c ) elevation could induce apoptosis in primary cultured rat proximal tubular (rPT) cells exposed to cadmium (Cd), but the concrete mechanism is still unclear. This study was designed to investigate the signal pathway involved in [Ca 2+ ] c elevation-mediated apoptosis. The results confirmed the elevation of [Ca 2+ ] c by confocal microscopy and enhancement of the apoptosis by Hoechst 33258 staining and flow cytometer when rPT cells were exposed to Cd for 12h. Then we demonstrated that Cd enhanced the protein levels of active calpain-1 and caspase-3 in rPT cells. Pretreatment with a cytosolic Ca 2+ chelator, 1,2-Bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), markedly blocked the up-regulation of active calpain-1 and caspase-3 and inhibited the apoptosis induced by Cd. Further, rPT cells were pretreated with a cell-permeable selective calpain-1 inhibitor, 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606) and caspase-3 inhibitor, N-Acetyl-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO), respectively. PD150606 significantly attenuated the up-regulation of active caspase-3 and the apoptosis induced by Cd. As expected, inhibition of active caspase-3 by Ac-DEVD-CHO decreased the apoptosis induced by Cd. Taken together, it could be concluded that [Ca 2+ ] c elevation did act as a pro-apoptotic signal in Cd-induced cytotoxicity of rPT cells, triggered calpain-1 and caspase-3 activation in turn, and induced apoptosis of rPT cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. A flavonoid isolated from Streptomyces sp. (ERINLG-4) induces apoptosis in human lung cancer A549 cells through p53 and cytochrome c release caspase dependant pathway.

    Science.gov (United States)

    Balachandran, C; Sangeetha, B; Duraipandiyan, V; Raj, M Karunai; Ignacimuthu, S; Al-Dhabi, N A; Balakrishna, K; Parthasarathy, K; Arulmozhi, N M; Arasu, M Valan

    2014-12-05

    The aim of this study was to investigate the anticancer activity of a flavonoid type of compound isolated from soil derived filamentous bacterium Streptomyces sp. (ERINLG-4) and to explore the molecular mechanisms of action. Cytotoxic properties of ethyl acetate extract was carried out against A549 lung cancer cell line using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cytotoxic properties of isolated compound were investigated in A549 lung cancer cell line, COLO320DM cancer cell line and Vero cells. The compound showed potent cytotoxic properties against A549 lung cancer cell line and moderate cytotoxic properties against COLO320DM cancer cell line. Isolated compound showed no toxicity up to 2000 μg/mL in Vero cells. So we have chosen the A549 lung cancer cell line for further anticancer studies. Intracellular visualization was done by using a laser scanning confocal microscope. Apoptosis was measured using DNA fragmentation technique. Treatment of the A549 cancer cells with isolated compound significantly reduced cell proliferation, increased formation of fragmented DNA and apoptotic body. Activation of caspase-9 and caspase-3 indicated that compound may be inducing intrinsic and extrinsic apoptosis pathways. Bcl-2, p53, pro-caspases, caspase-3, caspase-9 and cytochrome c release were detected by western blotting analysis after compound treatment (123 and 164 μM). The activities of pro-caspases-3, caspase-9 cleaved to caspase-3 and caspase-9 gradually increased after the addition of isolated compound. But Bcl-2 protein was down regulated after treatment with isolated compound. Molecular docking studies showed that the compound bound stably to the active sites of caspase-3 and caspase-9. These results strongly suggest that the isolated compound induces apoptosis in A549 cancer cells via caspase activation through cytochrome c release from mitochondria. The present results might provide helpful suggestions for the design of

  1. P2X7 receptor blockade protects against cisplatin-induced nephrotoxicity in mice by decreasing the activities of inflammasome components, oxidative stress and caspase-3

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yuanyuan; Yuan, Fahuan; Cao, Xuejiao [Department of Nephrology, Xinqiao Hospital, PLA, Third Military Medical University, Chongqing 400037 (China); Zhai, Zhifang [Department of Dermatology, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Gang Huang [Department of Medical Genetics, Third Military Medical University, Chongqing 430038 (China); Du, Xiang; Wang, Yiqin; Zhang, Jingbo; Huang, Yunjian; Zhao, Jinghong [Department of Nephrology, Xinqiao Hospital, PLA, Third Military Medical University, Chongqing 400037 (China); Hou, Weiping, E-mail: hwp0518@aliyun.com [Department of Nephrology, Xinqiao Hospital, PLA, Third Military Medical University, Chongqing 400037 (China)

    2014-11-15

    Nephrotoxicity is a common complication of cisplatin chemotherapy and thus limits the use of cisplatin in clinic. The purinergic 2X7 receptor (P2X7R) plays important roles in inflammation and apoptosis in some inflammatory diseases; however, its roles in cisplatin-induced nephrotoxicity remain unclear. In this study, we first assessed the expression of P2X7R in cisplatin-induced nephrotoxicity in C57BL/6 mice, and then we investigated the changes of renal function, histological injury, inflammatory response, and apoptosis in renal tissues after P2X7R blockade in vivo using an antagonist A-438079. Moreover, we measured the changes of nod-like receptor family, pyrin domain containing proteins (NLRP3) inflammasome components, oxidative stress, and proapoptotic genes in renal tissues in cisplatin-induced nephrotoxicity after treatment with A-438079. We found that the expression of P2X7R was significantly upregulated in the renal tubular epithelial cells in cisplatin-induced nephrotoxicity compared with that of the normal control group. Furthermore, pretreatment with A-438079 markedly attenuated the cisplatin-induced renal injury while lightening the histological damage, inflammatory response and apoptosis in renal tissue, and improved the renal function. These effects were associated with the significantly reduced levels of NLRP3 inflammasome components, oxidative stress, p53 and caspase-3 in renal tissues in cisplatin-induced nephrotoxicity. In conclusions, our studies suggest that the upregulated activity of P2X7R might play important roles in the development of cisplatin-induced nephrotoxicity, and P2X7R blockade might become an effective therapeutic strategy for this disease. - Highlights: • The P2X7R expression was markedly upregulated in cisplatin-induced nephrotoxicity. • P2X7R blockade significantly attenuated the cisplatin-induced renal injury. • P2X7R blockade reduced activities of NLRP3 inflammasome components in renal tissue. • P2X7R blockade

  2. Curcumin I mediates neuroprotective effect through attenuation of quinoprotein formation, p-p38 MAPK expression, and caspase-3 activation in 6-hydroxydopamine treated SH-SY5Y cells.

    Science.gov (United States)

    Meesarapee, Benjawan; Thampithak, Anusorn; Jaisin, Yamaratee; Sanvarinda, Pimtip; Suksamrarn, Apichart; Tuchinda, Patoomratana; Morales, Noppawan Phumala; Sanvarinda, Yupin

    2014-04-01

    6-Hydroxydopamine (6-OHDA) selectively enters dopaminergic neurons and undergoes auto-oxidation resulting in the generation of reactive oxygen species and dopamine quinones, subsequently leading to apoptosis. This mechanism mimics the pathogenesis of Parkinson's disease and has been used to induce experimental Parkinsonism in both in vitro and in vivo systems. In this study, we investigated the effects of curcumin I (diferuloylmethane) purified from Curcuma longa on quinoprotein production, phosphorylation of p38 MAPK (p-p38), and caspase-3 activation in 6-OHDA-treated SH-SY5Y dopaminergic cells. Pretreatment of SH-SY5Y with curcumin I at concentrations of 1, 5, 10, and 20 μM, significantly decreased the formation of quinoprotein and reduced the levels of p-p38 and cleaved caspase-3 in a dose-dependent manner. Moreover, the levels of the dopaminergic neuron marker, phospho-tyrosine hydroxylase (p-TH), were also dose-dependently increased upon treatment with curcumin I. Our results clearly demonstrated that curcumin I protects neurons against oxidative damage, as shown by attenuation of p-p38 expression, caspase-3-activation, and toxic quinoprotein formation, together with the restoration of p-TH levels. This study provides evidence for the therapeutic potential of curcumin I in the chemoprevention of oxidative stress-related neurodegeneration. Copyright © 2013 John Wiley & Sons, Ltd.

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

    Science.gov (United States)

    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.

  4. Suppression of survivin expression in glioblastoma cells by the Ras inhibitor farnesylthiosalicylic acid promotes caspase-dependent apoptosis.

    Science.gov (United States)

    Blum, Roy; Jacob-Hirsch, Jasmine; Rechavi, Gideon; Kloog, Yoel

    2006-09-01

    The Ras inhibitor farnesylthiosalicylic acid (FTS) has been shown to induce apoptosis in glioblastoma multiforme, but its mechanism of action was unknown. We show that FTS or dominant-negative Ras, by deregulating extracellular signal-regulated kinase and Akt signaling, decreases survivin gene transcripts in U87 glioblastoma multiforme, leading to disappearance of survivin protein and cell death. FTS affected both Ras-controlled regulators of survivin transcription and Ras-regulated survival signals. Thus, Ras inhibition by FTS resulted in release of the survivin "brake" on apoptosis and in activation of the mitochondrial apoptotic pathway: dephosphorylation of Bad, activation of Bax, release of cytochrome c, and caspase activation. FTS-induced apoptosis of U87 cells was strongly attenuated by forced expression of survivin or by caspase inhibitors. These results show that resistance to apoptosis in glioblastoma multiforme can be abolished by a single Ras inhibitor, which targets both survivin, a critical inhibitor of apoptosis, and the intrinsic mitochondrial apoptotic machinery.

  5. Regulation of p21ras activity

    DEFF Research Database (Denmark)

    Lowy, D R; Zhang, K; DeClue, J E

    1992-01-01

    The ras genes encode GTP/GDP-binding proteins that participate in mediating mitogenic signals from membrane tyrosine kinases to downstream targets. The activity of p21ras is determined by the concentration of GTP-p21ras, which is tightly regulated by a complex array of positive and negative control...... mechanisms. GAP and NF1 can negatively regulate p21ras activity by stimulating hydrolysis of GTP bound to p21ras. Other cellular factors can positively regulate p21ras by stimulating GDP/GTP exchange....

  6. Regulation of ROCK Activity in Cancer

    DEFF Research Database (Denmark)

    Morgan-Fisher, Marie; Wewer, Ulla M; Yoneda, Atsuko

    2013-01-01

    , these findings demonstrate additional modes to regulate ROCK activity. This review describes the molecular mechanisms of ROCK activity regulation in cancer, with emphasis on ROCK isoform-specific regulation and interaction partners, and discusses the potential of ROCKs as therapeutic targets in cancer.......Cancer-associated changes in cellular behavior, such as modified cell-cell contact, increased migratory potential, and generation of cellular force, all require alteration of the cytoskeleton. Two homologous mammalian serine/threonine kinases, Rho-associated protein kinases (ROCK I and II), are key...... regulators of the actin cytoskeleton acting downstream of the small GTPase Rho. ROCK is associated with cancer progression, and ROCK protein expression is elevated in several types of cancer. ROCKs exist in a closed, inactive conformation under quiescent conditions, which is changed to an open, active...

  7. Cytosolic and nuclear caspase-8 have opposite impact on survival after liver resection for hepatocellular carcinoma

    International Nuclear Information System (INIS)

    Koschny, Ronald; Schemmer, Peter; Schirmacher, Peter; Ganten, Tom M; Brost, Sylvia; Hinz, Ulf; Sykora, Jaromir; Batke, Emanuela M; Singer, Stephan; Breuhahn, Kai; Stremmel, Wolfgang; Walczak, Henning

    2013-01-01

    An imbalance between proliferation and apoptosis is one of the main features of carcinogenesis. TRAIL (TNF-related apoptosis-inducing ligand) induces apoptosis upon binding to the TRAIL death receptors, TRAIL receptor 1 (TRAIL-R1) and TRAIL-R2, whereas binding to TRAIL-R3 and TRAIL-R4 might promote cell survival and proliferation. The anti-tumor activity of TRAIL-R1 and TRAIL-R2 agonists is currently investigated in clinical trials. To gain further insight into the regulation of apoptosis in hepatocellular carcinoma (HCC), we investigated the TRAIL pathway and the regulators of apoptosis caspase-8, Bcl-xL and Mcl-1 in patients with HCC regarding patient survival. We analyzed 157 hepatocellular carcinoma patients who underwent partial liver resection or orthotopic liver transplantation and healthy control liver tissue using immunohistochemistry on tissue microarrays for the expression of TRAIL-R1 to TRAIL-R4, caspase-8, Bcl-xL and Mcl-1. Immunohistochemical data were evaluated for potential associations with clinico-pathological parameters and survival. Whereas TRAIL-R1 was downregulated in HCC in comparison to normal liver tissue, TRAIL-R2 and –R4 were upregulated in HCC, especially in G2 and G3 tumors. TRAIL-R1 downregulation and upregulation of TRAIL-R2 and TRAIL-R4 correlated with tumor dedifferentiation (G2/G3). TRAIL-R3, Bcl-xL and Mcl-1 showed no differential expression in tumor tissue compared to normal tissue. The expression levels of TRAIL receptors did not correlate with patient survival after partial hepatectomy. Interestingly, in tumor tissue, but not in normal hepatocytes, caspase-8 showed a strong nuclear staining. Low cytosolic and high nuclear staining intensity of caspase-8 significantly correlated with impaired survival after partial hepatectomy, which, for cytosolic caspase-8, was independent from tumor grade. Assessment of TRAIL-receptor expression patterns may have therapeutic implications for the use of TRAIL receptor agonists in HCC therapy

  8. A genetic screen for modifiers of Drosophila caspase Dcp-1 reveals caspase involvement in autophagy and novel caspase-related genes

    Directory of Open Access Journals (Sweden)

    Ahnn Joohong

    2010-01-01

    Full Text Available Abstract Background Caspases are cysteine proteases with essential functions in the apoptotic pathway; their proteolytic activity toward various substrates is associated with the morphological changes of cells. Recent reports have described non-apoptotic functions of caspases, including autophagy. In this report, we searched for novel modifiers of the phenotype of Dcp-1 gain-of-function (GF animals by screening promoter element- inserted Drosophila melanogaster lines (EP lines. Results We screened ~15,000 EP lines and identified 72 Dcp-1-interacting genes that were classified into 10 groups based on their functions and pathways: 4 apoptosis signaling genes, 10 autophagy genes, 5 insulin/IGF and TOR signaling pathway genes, 6 MAP kinase and JNK signaling pathway genes, 4 ecdysone signaling genes, 6 ubiquitination genes, 11 various developmental signaling genes, 12 transcription factors, 3 translation factors, and 11 other unclassified genes including 5 functionally undefined genes. Among them, insulin/IGF and TOR signaling pathway, MAP kinase and JNK signaling pathway, and ecdysone signaling are known to be involved in autophagy. Together with the identification of autophagy genes, the results of our screen suggest that autophagy counteracts Dcp-1-induced apoptosis. Consistent with this idea, we show that expression of eGFP-Atg5 rescued the eye phenotype caused by Dcp-1 GF. Paradoxically, we found that over-expression of full-length Dcp-1 induced autophagy, as Atg8b-GFP, an indicator of autophagy, was increased in the eye imaginal discs and in the S2 cell line. Taken together, these data suggest that autophagy suppresses Dcp-1-mediated apoptotic cell death, whereas Dcp-1 positively regulates autophagy, possibly through feedback regulation. Conclusions We identified a number of Dcp-1 modifiers that genetically interact with Dcp-1-induced cell death. Our results showing that Dcp-1 and autophagy-related genes influence each other will aid future

  9. The Marine Fungal Metabolite, Dicitrinone B, Induces A375 Cell Apoptosis through the ROS-Related Caspase Pathway

    Directory of Open Access Journals (Sweden)

    Li Chen

    2014-04-01

    Full Text Available Dicitrinone B, a rare carbon-bridged citrinin dimer, was isolated from the marine-derived fungus, Penicillium citrinum. It was reported to have antitumor effects on tumor cells previously; however, the details of the mechanism remain unclear. In this study, we found that dicitrinone B inhibited the proliferation of multiple tumor types. Among them, the human malignant melanoma cell, A375, was confirmed to be the most sensitive. Morphologic evaluation, cell cycle arrest and apoptosis rate analysis results showed that dicitrinone B significantly induced A375 cell apoptosis. Subsequent observation of reactive oxygen species (ROS accumulation and mitochondrial membrane potential (MMP reduction revealed that the apoptosis induced by dicitrinone B may be triggered by over-producing ROS. Further studies indicated that the apoptosis was associated with both intrinsic and extrinsic apoptosis pathways under the regulation of Bcl-2 family proteins. Caspase-9, caspase-8 and caspase-3 were activated during the process, leading to PARP cleavage. The pan-caspase inhibitor, Z-VAD-FMK, could reverse dicitrinone B-induced apoptosis, suggesting that it is a caspase-dependent pathway. Our data for the first time showed that dicitrinone B inhibits the proliferation of tumor cells by inducing cell apoptosis. Moreover, compared with the first-line chemotherapy drug, 5-fluorouracil (5-Fu, dicitrinone B showed much more potent anticancer efficacy, suggesting that it might serve as a potential antitumor agent.

  10. Proteolytic fragmentation of inositol 1,4,5-trisphosphate receptors: a novel mechanism regulating channel activity?

    Science.gov (United States)

    Wang, Liwei; Alzayady, Kamil J; Yule, David I

    2016-06-01

    Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are a family of ubiquitously expressed intracellular Ca(2+) release channels. Regulation of channel activity by Ca(2+) , nucleotides, phosphorylation, protein binding partners and other cellular factors is thought to play a major role in defining the specific spatiotemporal characteristics of intracellular Ca(2+) signals. These properties are, in turn, believed pivotal for the selective and specific physiological activation of Ca(2+) -dependent effectors. IP3 Rs are also substrates for the intracellular cysteine proteases, calpain and caspase. Cleavage of the IP3 R has been proposed to play a role in apoptotic cell death by uncoupling regions important for IP3 binding from the channel domain, leaving an unregulated leaky Ca(2+) pore. Contrary to this hypothesis, we demonstrate following proteolysis that N- and C-termini of IP3 R1 remain associated, presumably through non-covalent interactions. Further, we show that complementary fragments of IP3 R1 assemble into tetrameric structures and retain their ability to be regulated robustly by IP3 . While peptide continuity is clearly not necessary for IP3 -gating of the channel, we propose that cleavage of the IP3 R peptide chain may alter other important regulatory events to modulate channel activity. In this scenario, stimulation of the cleaved IP3 R may support distinct spatiotemporal Ca(2+) signals and activation of specific effectors. Notably, in many adaptive physiological events, the non-apoptotic activities of caspase and calpain are demonstrated to be important, but the substrates of the proteases are poorly defined. We speculate that proteolytic fragmentation may represent a novel form of IP3 R regulation, which plays a role in varied adaptive physiological processes. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  11. DNMT1 and DNMT3b silencing sensitizes human hepatoma cells to TRAIL-mediated apoptosis via up-regulation of TRAIL-R2/DR5 and caspase-8.

    Science.gov (United States)

    Kurita, Satoshi; Higuchi, Hajime; Saito, Yoshimasa; Nakamoto, Nobuhiro; Takaishi, Hiromasa; Tada, Shinichiro; Saito, Hidetsugu; Gores, Gregory J; Hibi, Toshifumi

    2010-06-01

    DNA methylation plays a critical role in chromatin remodeling and gene expression. DNA methyltransferases (DNMTs) are hypothesized to mediate cellular DNA methylation status and gene expression during mammalian development and in malignant diseases. In this study, we examined the role of DNA methyltransferase 1 (DNMT1) and DNMT3b in cell proliferation and survival of hepatocellular carcinoma (HCC) cells. Gene silencing of both DNMT1 and DNMT3b by targeted siRNA knockdown reduces cell proliferation and sensitizes the cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cell death. The proapoptotic protein caspase-8 demonstrated promoter hypermethylation in HCC cells and was up-regulated by knockdown of DNMT1 and DNMT3b both at mRNA and protein levels. In addition, death receptor TRAIL-R2/DR5 (TRAIL receptor 2/death receptor 5) did not exhibit promoter hypermethylation in HCC cells but was also up-regulated by knockdown of DNMT1 and DNMT3b both at mRNA and protein levels. Consistent with this observation, the combined transfection of DNMT1-siRNA plus DNMT3b-siRNA enhanced formation of the TRAIL-death-inducing signaling complex formation in HCC cells. In conclusion, our data suggest that DNA methylation of specific genomic regions maintained by DNMT1 and DNMT3b plays a critical role in survival of HCC cells, and a simultaneous knockdown of both DNMT1 and DNMT3b may be a novel anticancer strategy for the treatment of HCC.

  12. Transient protective effect of caspase inhibitors in RCS rat.

    Science.gov (United States)

    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

  13. Simple route of caspase-3 FRET sensor synthesis using “click chemistry”

    OpenAIRE

    Lišková, M. (Marcela); Křenková, J. (Jana); Klepárník, K. (Karel); Pazdera, P.; Foret, F. (František)

    2015-01-01

    Programmed cell death or apoptosis is regulated process of cell suicide. The central role in apoptosis play cysteine proteases called caspases. Caspases recognize tetra-peptide sequences Asp-Glu-Val-Asp (DEVD) on their substrates and hydrolyze peptide bonds after aspartic acid residues. Various techniques for the determination of caspase-3 are commercially available e.g. Enzyme Linked Immuno-Sorbent Assay (ELISA), Western blotting or flow cytometric analysis. The products of the cleavage can ...

  14. 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.

    Science.gov (United States)

    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.

  15. Nocardia cyriacigeogica from Bovine Mastitis Induced In vitro Apoptosis of Bovine Mammary Epithelial Cells via Activation of Mitochondrial-Caspase Pathway

    Directory of Open Access Journals (Sweden)

    Wei Chen

    2017-05-01

    Full Text Available Nocardia is one of the causing agents of bovine mastitis and increasing prevalence of nocardial mastitis in shape of serious outbreaks has been reported from many countries. However, the mechanisms by which this pathogen damages the bovine mammary epithelial cells (bMECs is not yet studied. Therefore, this study was designed with the aim to evaluate the apoptotic effects elicited by Nocardia and to investigate the pathway by which the Nocardia induce apoptosis in bMECs. Clinical Nocardia cyriacigeorgica strain from bovine mastitis was used to infect the bMECs for different time intervals, viz. 1, 3, 6, 12, and 18 h, and then the induced effects on bMECs were studied using adhesion and invasion assays, release of lactate dehydrogenase (LDH, apoptosis analysis by annexin V and propidium iodide (PI double staining, morphological, and ultrastructural observations under scanning electron microscope (SEM and transmission electron microscope (TEM, mitochondrial transmembrane potential (ΔΨm assay using flow cytometry, and the protein quantification of mitochondrial cytochrome c and caspase-9 and caspase-3 by western blotting. The results of this study showed that N. cyriacigeorgica possessed the abilities of adhesion and invasion to bMECs. N. cyriacigeorgica was found to collapse mitochondrial transmembrane potential, significantly (p < 0.05 release mitochondrial cytochrome c and ultimately induce cell apoptosis. Additionally, it promoted casepase-9 (p < 0.01 and casepase-3 (p < 0.05 levels, significantly (p < 0.01 increased the release of LDH and promoted DNA fragmentation which further confirmed the apoptosis. Furthermore, N. cyriacigeorgica induced apoptosis/necrosis manifested specific ultrastructure features under TEM, such as swollen endoplasmic reticulum, cristae degeneration, and swelling of mitochondria, vesicle formation on the cell surface, rupturing of cell membrane and nuclear membrane, clumping, fragmentation, and margination of

  16. Nocardia cyriacigeogica from Bovine Mastitis Induced In vitro Apoptosis of Bovine Mammary Epithelial Cells via Activation of Mitochondrial-Caspase Pathway.

    Science.gov (United States)

    Chen, Wei; Liu, Yongxia; Zhang, Limei; Gu, Xiaolong; Liu, Gang; Shahid, Muhammad; Gao, Jian; Ali, Tariq; Han, Bo

    2017-01-01

    Nocardia is one of the causing agents of bovine mastitis and increasing prevalence of nocardial mastitis in shape of serious outbreaks has been reported from many countries. However, the mechanisms by which this pathogen damages the bovine mammary epithelial cells (bMECs) is not yet studied. Therefore, this study was designed with the aim to evaluate the apoptotic effects elicited by Nocardia and to investigate the pathway by which the Nocardia induce apoptosis in bMECs. Clinical Nocardia cyriacigeorgica strain from bovine mastitis was used to infect the bMECs for different time intervals, viz . 1, 3, 6, 12, and 18 h, and then the induced effects on bMECs were studied using adhesion and invasion assays, release of lactate dehydrogenase (LDH), apoptosis analysis by annexin V and propidium iodide (PI) double staining, morphological, and ultrastructural observations under scanning electron microscope (SEM) and transmission electron microscope (TEM), mitochondrial transmembrane potential (ΔΨm) assay using flow cytometry, and the protein quantification of mitochondrial cytochrome c and caspase-9 and caspase-3 by western blotting. The results of this study showed that N. cyriacigeorgica possessed the abilities of adhesion and invasion to bMECs. N. cyriacigeorgica was found to collapse mitochondrial transmembrane potential, significantly ( p < 0.05) release mitochondrial cytochrome c and ultimately induce cell apoptosis. Additionally, it promoted casepase-9 ( p < 0.01) and casepase-3 ( p < 0.05) levels, significantly ( p < 0.01) increased the release of LDH and promoted DNA fragmentation which further confirmed the apoptosis. Furthermore, N. cyriacigeorgica induced apoptosis/necrosis manifested specific ultrastructure features under TEM, such as swollen endoplasmic reticulum, cristae degeneration, and swelling of mitochondria, vesicle formation on the cell surface, rupturing of cell membrane and nuclear membrane, clumping, fragmentation, and margination of chromatin

  17. Regulation of ROCK Activity in Cancer

    Science.gov (United States)

    Morgan-Fisher, Marie; Wewer, Ulla M.

    2013-01-01

    Cancer-associated changes in cellular behavior, such as modified cell-cell contact, increased migratory potential, and generation of cellular force, all require alteration of the cytoskeleton. Two homologous mammalian serine/threonine kinases, Rho-associated protein kinases (ROCK I and II), are key regulators of the actin cytoskeleton acting downstream of the small GTPase Rho. ROCK is associated with cancer progression, and ROCK protein expression is elevated in several types of cancer. ROCKs exist in a closed, inactive conformation under quiescent conditions, which is changed to an open, active conformation by the direct binding of guanosine triphosphate (GTP)–loaded Rho. In recent years, a number of ROCK isoform-specific binding partners have been found to modulate the kinase activity through direct interactions with the catalytic domain or via altered cellular localization of the kinases. Thus, these findings demonstrate additional modes to regulate ROCK activity. This review describes the molecular mechanisms of ROCK activity regulation in cancer, with emphasis on ROCK isoform-specific regulation and interaction partners, and discusses the potential of ROCKs as therapeutic targets in cancer. PMID:23204112

  18. Regulation of brain aromatase activity in rats

    International Nuclear Information System (INIS)

    Roselli, C.E.; Ellinwood, W.E.; Resko, J.A.

    1984-01-01

    The distribution and regulation of aromatase activity in the adult rat brain with a sensitive in vitro assay that measures the amount of 3 H 2 O formed during the conversion of [1 beta- 3 H]androstenedione to estrone. The rate of aromatase activity in the hypothalamus-preoptic area (HPOA) was linear with time up to 1 h, and with tissue concentrations up to 5 mgeq/200 microliters incubation mixture. The enzyme demonstrated a pH optimum of 7.4 and an apparent Michaelis-Menten constant (Km) of 0.04 microns. The greatest amount of aromatase activity was found in amygdala and HPOA from intact male rats. The hippocampus, midbrain tegmentum, cerebral cortex, cerebellum, and anterior pituitary all contained negligible enzymatic activity. Castration produced a significant decrease in aromatase activity in the HPOA, but not in the amygdala or cerebral cortex. The HPOAs of male rats contained significantly greater aromatase activity than the HPOAs of female rats. In females, this enzyme activity did not change during the estrous cycle or after ovariectomy. Administration of testosterone to gonadectomized male and female rats significantly enhanced HPOA aromatase activities to levels approximating those found in HPOA from intact males. Therefore, the results suggest that testosterone, or one of its metabolites, is a major steroidal regulator of HPOA aromatase activity in rats

  19. Bacterial lipoprotein delays apoptosis in human neutrophils through inhibition of caspase-3 activity: regulatory roles for CD14 and TLR-2.

    LENUS (Irish Health Repository)

    Power, Colm P

    2012-02-03

    The human sepsis syndrome resulting from bacterial infection continues to account for a significant proportion of hospital mortality. Neutralizing strategies aimed at individual bacterial wall products (such as LPS) have enjoyed limited success in this arena. Bacterial lipoprotein (BLP) is a major constituent of the wall of diverse bacterial forms and profoundly influences cellular function in vivo and in vitro, and has been implicated in the etiology of human sepsis. Delayed polymorphonuclear cell (PMN) apoptosis is a characteristic feature of human sepsis arising from Gram-negative or Gram-positive bacterial infection. Bacterial wall product ligation and subsequent receptor-mediated events upstream of caspase inhibition in neutrophils remain incompletely understood. BLP has been shown to exert its cellular effects primarily through TLR-2, and it is now widely accepted that lateral associations with the TLRs represent the means by which CD14 communicates intracellular messages. In this study, we demonstrate that BLP inhibits neutrophil mitochondrial membrane depolarization with a subsequent reduction in caspase-3 processing, ultimately leading to a significant delay in PMN apoptosis. Pretreatment of PMNs with an anti-TLR-2 mAb or anti-CD14 mAb prevented BLP from delaying PMN apoptosis to such a marked degree. Combination blockade using both mAbs completely prevented the effects of BLP (in 1 and 10 ng\\/ml concentrations) on PMN apoptosis. At higher concentrations of BLP, the antiapoptotic effects were observed, but were not as pronounced. Our findings therefore provide the first evidence of a crucial role for both CD14 and TLR-2 in delayed PMN apoptosis arising from bacterial infection.

  20. Mesenchymal stem cells improve mouse non-heart-beating liver graft survival by inhibiting Kupffer cell apoptosis via TLR4-ERK1/2-Fas/FasL-caspase3 pathway regulation

    Directory of Open Access Journals (Sweden)

    Yang Tian

    2016-10-01

    Full Text Available Abstract Background Liver transplantation is the optimal treatment option for end-stage liver disease, but organ shortages dramatically restrict its application. Donation after cardiac death (DCD is an alternative approach that may expand the donor pool, but it faces challenges such as graft dysfunction, early graft loss, and cholangiopathy. Moreover, DCD liver grafts are no longer eligible for transplantation after their warm ischaemic time exceeds 30 min. Mesenchymal stem cells (MSCs have been proposed as a promising therapy for treatment of certain liver diseases, but the role of MSCs in DCD liver graft function remains elusive. Methods In this study, we established an arterialized mouse non-heart-beating (NHB liver transplantation model, and compared survival rates, cytokine and chemokine expression, histology, and the results of in vitro co-culture experiments in animals with or without MSC infusion. Results MSCs markedly ameliorated NHB liver graft injury and improved survival post-transplantation. Additionally, MSCs suppressed Kupffer cell apoptosis, Th1/Th17 immune responses, chemokine expression, and inflammatory cell infiltration. In vitro, PGE2 secreted by MSCs inhibited Kupffer cell apoptosis via TLR4-ERK1/2-caspase3 pathway regulation. Conclusion Our study uncovers a protective role for MSCs and elucidates the underlying immunomodulatory mechanism in an NHB liver transplantation model. Our results suggest that MSCs are uniquely positioned for use in future clinical studies owing to their ability to protect DCD liver grafts, particularly in patients for whom DCD organs are not an option according to current criteria.

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

    Science.gov (United States)

    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.

  2. Commission of energy regulation. 2004 activity report

    International Nuclear Information System (INIS)

    2004-01-01

    The commission of energy regulation (CRE) is an independent administrative authority in charge of the control of the operation of gas and electricity markets. This document is the fifth activity report of CRE and covers the July 1, 2003 - June 30, 2004 period, which corresponds to the era of opening of energy markets as a consequence of the enforcement of the June 26, 2003 European directive. In the framework of the stakes made by energy markets liberalization, this document presents the situation of the gas and electricity markets during this period (European framework, regulation of both markets, public utility mission..) and describes CRE's means for the monitoring of these markets. (J.S.)

  3. TSA-induced cell death in prostate cancer cell lines is caspase-2 dependent and involves the PIDDosome.

    Science.gov (United States)

    Taghiyev, Agshin F; Guseva, Natalya V; Glover, Rebecca A; Rokhlin, Oskar W; Cohen, Michael B

    2006-09-01

    The histone deacetylase inhibitor Trichostatin A (TSA) has previously been found to induce caspase activity in the human prostate cancer cell lines DU145 and LNCaP. TSA treatment resulted in the release of cytochrome c and Smac/DIABLO from mitochondria in DU145, and activation of caspase-9 in both cell lines. We concluded that TSA mediated its effect via the mitochondrial pathway. The aim of the current study was to determine how TSA initiated the caspase cascade. The results revealed that caspase-2 plays an important role in TSA-induced apoptosis. Inhibition of caspase-2 by siRNA or expression of caspase-2dn substantially decreased caspase activity after TSA treatment in both cell lines, siRNA caspase-2 also inhibited TSA-induced cell death. Caspase-2 acts upstream of caspase-8 and -9 and mediates mitochondrial cytochrome c release. Coimmunoprecipitation experiments show that caspase-2 formed protein complexes with RADD/RAIDD and PIDD. Together, these data indicate that caspase-2 initiates caspase cascade after TSA treatment and involves the formation of the PIDDosome.

  4. Apoptotic block in colon cancer cells may be rectified by lentivirus mediated overexpression of caspase-9.

    Science.gov (United States)

    Xu, D; Wang, C; Shen, X; Yu, Y; Rui, Y; Zhang, D; Zhou, Z

    2013-12-01

    At present, the inhibition of apoptosis during pathogenesis of colorectal cancer is widely recognized while the role of caspase-9 in this process remains controversial. We aimed to investigate the differential expression of caspase-9 and evaluate the therapeutic potential of expression intervention in this study. We first examined the different expression of caspase-9 in normal colon mucosa, adenoma and cancer, investigating the relationship between its expression and clinico-pathological characteristics. Secondly, overexpression of caspase-9 was established in colon cancer cell lines by lentivirus infection to study the changes in growth, proliferation and apoptosis. Compared with normal colon mucosa, the expression of caspase-9 was higher in adenoma while lower in cancer both at mRNA and protein level (P expression is more common in poorly differentiated cancers (P expression of caspase-9, poorer colony formation and slower cell proliferation. In terms of apoptosis related indicators, caspase-9 overexpression leads to higher apoptosis rate and GO/G1 arrest, while up-regulating the expression of caspase-3 (P expression from colon mucosa, adenoma to cancer suggested it may be involved in the carcinogenesis of colon cancer. The overexpression of caspase-9 exhibits an inhibitory role in cancer growth and proliferation while promoting apoptosis. However, a non-apoptotic role of caspase-9 facilitating differentiation was also implied.

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

    Science.gov (United States)

    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.

  6. Human herpesvirus 6A induces apoptosis of primary human fetal astrocytes via both caspase-dependent and -independent pathways

    Directory of Open Access Journals (Sweden)

    Gu Bin

    2011-12-01

    Full Text Available Abstract Background Human herpesvirus 6 (HHV-6 is a T-lymphtropic and neurotropic virus that can infect various types of cells. Sequential studies reported that apoptosis of glia and neurons induced by HHV-6 might act a potential trigger for some central nervous system (CNS diseases. HHV-6 is involved in the pathogenesis of encephalitis, multiple sclerosis (MS and fatigue syndrome. However, the mechanisms responsible for the apoptosis of infected CNS cells induced by HHV-6 are poorly understood. In this study, we investigated the cell death processes of primary human fetal astrocytes (PHFAs during productive HHV-6A infection and the underlying mechanisms. Results HHV-6A can cause productive infection in primary human fetal astrocytes. Annexin V-PI staining and electron microscopic analysis indicated that HHV-6A was an inducer of apoptosis. The cell death was associated with activation of caspase-3 and cleavage of poly (ADP-ribose polymerase (PARP, which is known to be an important substrate for activated caspase-3. Caspase-8 and -9 were also significantly activated in HHV-6A-infected cells. Moreover, HHV-6A infection led to Bax up-regulation and Bcl-2 down-regulation. HHV-6A infection increased the release of Smac/Diablo, AIF and cytochrome c from mitochondria to cytosol, which induced apoptosis via the caspase-dependent and -independent pathways. In addition, we also found that anti-apoptotic factors such as IAPs and NF-κB decreased in HHV-6A infected PHFAs. Conclusion This is the first demonstration of caspase-dependent and -independent apoptosis in HHV-6A-infected glial cells. These findings would be helpful in understanding the mechanisms of CNS diseases caused by HHV-6.

  7. Mast cell activators as novel immune regulators.

    Science.gov (United States)

    Johnson-Weaver, Brandi; Choi, Hae Woong; Abraham, Soman N; Staats, Herman F

    2018-05-26

    Mast cells are an important cell type of the innate immune system that when activated, play a crucial role in generating protective innate host responses after bacterial and viral infection. Additionally, activated mast cells influence lymph node composition to regulate the induction of adaptive immune responses. The recognition that mast cells play a beneficial role in host responses to microbial infection and induction of adaptive immunity has provided the rationale to evaluate mast cell activators for use as antimicrobials or vaccine adjuvants. This review summarizes the role of mast cell activators in antimicrobial responses while also discussing the use of different classes of mast cell activators as potent vaccine adjuvants that enhance the induction of protective immune responses. Copyright © 2018 Elsevier Ltd. All rights reserved.

  8. Src regulates the activity of SIRT2

    International Nuclear Information System (INIS)

    Choi, You Hee; Kim, Hangun; Lee, Sung Ho; Jin, Yun-Hye; Lee, Kwang Youl

    2014-01-01

    Highlights: • Src decreases the protein levels of Sirt2. • Src inhibitor and knockdown of Src increase the protein levels of Sirt2. • Src interacts with and phosphorylates Sirt2. • Src regulate the activity of Sirt2. - Abstract: SIRT2 is a mammalian member of the Sirtuin family of NAD + -dependent protein deacetylases. The tyrosine kinase Src is involved in a variety of cellular signaling pathways, leading to the induction of DNA synthesis, cell proliferation, and cytoskeletal reorganization. The function of SIRT2 is modulated by post-translational modifications; however, the precise molecular signaling mechanism of SIRT2 through interactions with c-Src has not yet been established. In this study, we investigated the potential regulation of SIRT2 function by c-Src. We found that the protein levels of SIRT2 were decreased by c-Src, and subsequently rescued by the addition of a Src specific inhibitor, SU6656, or by siRNA-mediated knockdown of c-Src. The c-Src interacts with and phosphorylates SIRT2 at Tyr104. c-Src also showed the ability to regulate the deacetylation activity of SIRT2. Investigation on the phosphorylation of SIRT2 suggested that this was the method of c-Src-mediated SIRT2 regulation

  9. Markets, prices and regulation in energetic activities

    International Nuclear Information System (INIS)

    Percebois, Jacques

    2015-09-01

    The author first outlines some fundamental characteristics of the different energy world markets (oil, natural gas, coal, electricity). He outlines their availability, locations, and different main geographical areas. Then, he discusses the relationships between costs and prices in which intervene external costs, taxes, feed-in tariffs, national regulations, incentives for consumers. He discusses the issue of regulation of some energy activities, i.e. how State may or may not intervene on the markets, how competition may influence the market, how activities can thus be divided (production, transport, distribution) with implications and consequences for prices. He finally outlines concerns about the future financing of investments required to face tomorrow's needs

  10. Chlamydia abortus Pmp18.1 Induces IL-1β Secretion by TLR4 Activation through the MyD88, NF-κB, and Caspase-1 Signaling Pathways.

    Science.gov (United States)

    Pan, Qing; Zhang, Qiang; Chu, Jun; Pais, Roshan; Liu, Shanshan; He, Cheng; Eko, Francis O

    2017-01-01

    The polymorphic membrane protein D (Pmp18D) is a 160-kDa outer membrane protein that is conserved and plays an important role in Chlamydia abortus pathogenesis. We have identified an N-terminal fragment of Pmp18D (designated Pmp18.1) as a possible subunit vaccine antigen. In this study, we evaluated the vaccine potential of Pmp18.1 by investigating its ability to induce innate immune responses in dendritic cells and the signaling pathway(s) involved in rPmp18.1-induced IL-1β secretion. We next investigated the immunomodulatory impact of VCG, in comparison with the more established Th1-promoting adjuvants, CpG and FL, on rPmp18.1-mediated innate immune activation. Finally, the effect of siRNA targeting TLR4, MyD88, NF-κB p50, and Caspase-1 mRNA in DCs on IL-1β cytokine secretion was also investigated. Bone marrow-derived dendritic cells (BMDCs) were stimulated with rPmp18.1 in the presence or absence of VCG or CpG or FL and the magnitude of cytokines produced was assessed using a multiplex cytokine ELISA assay. Expression of costimulatory molecules and Toll-like receptors (TLRs) was analyzed by flow cytometry. Quantitation of intracellular levels of myeloid differentiation factor 88 (MyD88), nuclear factor kappa beta (NF-κB p50/p65), and Caspase-1 was evaluated by Western immunoblotting analysis while NF-κB p65 nuclear translocation was assessed by confocal microscopy. The results showed DC stimulation with rPmp18.1 provoked the secretion of proinflammatory cytokines and upregulated expression of TLRs and co-stimulatory molecules associated with DC maturation. These responses were significantly ( p ≤ 0.001) enhanced by VCG but not CpG or FL. In addition, rPmp18.1 activated the expression of MyD88, NF-κB p50, and Caspase-1 as well as the nuclear expression of NF-κB p65 in treated DCs. Furthermore, targeting TLR4, MyD88, NF-κB p50, and Caspase-1 mRNA in BMDCs with siRNA significantly reduced their expression levels, resulting in decreased IL-1β cytokine

  11. Chlamydia abortus Pmp18.1 Induces IL-1β Secretion by TLR4 Activation through the MyD88, NF-κB, and Caspase-1 Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Qing Pan

    2017-12-01

    Full Text Available The polymorphic membrane protein D (Pmp18D is a 160-kDa outer membrane protein that is conserved and plays an important role in Chlamydia abortus pathogenesis. We have identified an N-terminal fragment of Pmp18D (designated Pmp18.1 as a possible subunit vaccine antigen. In this study, we evaluated the vaccine potential of Pmp18.1 by investigating its ability to induce innate immune responses in dendritic cells and the signaling pathway(s involved in rPmp18.1-induced IL-1β secretion. We next investigated the immunomodulatory impact of VCG, in comparison with the more established Th1-promoting adjuvants, CpG and FL, on rPmp18.1-mediated innate immune activation. Finally, the effect of siRNA targeting TLR4, MyD88, NF-κB p50, and Caspase-1 mRNA in DCs on IL-1β cytokine secretion was also investigated. Bone marrow-derived dendritic cells (BMDCs were stimulated with rPmp18.1 in the presence or absence of VCG or CpG or FL and the magnitude of cytokines produced was assessed using a multiplex cytokine ELISA assay. Expression of costimulatory molecules and Toll-like receptors (TLRs was analyzed by flow cytometry. Quantitation of intracellular levels of myeloid differentiation factor 88 (MyD88, nuclear factor kappa beta (NF-κB p50/p65, and Caspase-1 was evaluated by Western immunoblotting analysis while NF-κB p65 nuclear translocation was assessed by confocal microscopy. The results showed DC stimulation with rPmp18.1 provoked the secretion of proinflammatory cytokines and upregulated expression of TLRs and co-stimulatory molecules associated with DC maturation. These responses were significantly (p ≤ 0.001 enhanced by VCG but not CpG or FL. In addition, rPmp18.1 activated the expression of MyD88, NF-κB p50, and Caspase-1 as well as the nuclear expression of NF-κB p65 in treated DCs. Furthermore, targeting TLR4, MyD88, NF-κB p50, and Caspase-1 mRNA in BMDCs with siRNA significantly reduced their expression levels, resulting in decreased IL-1

  12. Saikosaponin d induces cell death through caspase-3-dependent, caspase-3-independent and mitochondrial pathways in mammalian hepatic stellate cells

    International Nuclear Information System (INIS)

    Chen, Ming-Feng; Huang, S. Joseph; Huang, Chao-Cheng; Liu, Pei-Shan; Lin, Kun-I; Liu, Ching-Wen; Hsieh, Wen-Chuan; Shiu, Li-Yen; Chen, Chang-Han

    2016-01-01

    Saikosaponin d (SSd) is one of the main active triterpene saponins in Bupleurum falcatum. It has a steroid-like structure, and is reported to have pharmacological activities, including liver protection in rat, cell cycle arrest and apoptosis induction in several cancer cell lines. However, the biological functions and molecular mechanisms of mammalian cells under SSd treatment are still unclear. The cytotoxicity and apoptosis of hepatic stellate cells (HSCs) upon SSd treatment were discovered by MTT assay, colony formation assay and flow cytometry. The collage I/III, caspase activity and apoptotic related genes were examined by quantitative PCR, Western blotting, immunofluorescence and ELISA. The mitochondrial functions were monitored by flow cytometry, MitoTracker staining, ATP production and XF24 bioenergetic assay. This study found that SSd triggers cell death via an apoptosis path. An example of this path might be typical apoptotic morphology, increased sub-G1 phase cell population, inhibition of cell proliferation and activation of caspase-3 and caspase-9. However, the apoptotic effects induced by SSd are partially blocked by the caspase-3 inhibitor, Z-DEVD-FMK, suggesting that SSd may trigger both HSC-T6 and LX-2 cell apoptosis through caspase-3-dependent and independent pathways. We also found that SSd can trigger BAX and BAK translocation from the cytosol to the mitochondria, resulting in mitochondrial function inhibition, membrane potential disruption. Finally, SSd also increases the release of apoptotic factors. The overall analytical data indicate that SSd-elicited cell death may occur through caspase-3-dependent, caspase-3-independent and mitochondrial pathways in mammalian HSCs, and thus can delay the formation of liver fibrosis by reducing the level of HSCs

  13. Restraint of apoptosis during mitosis through interdomain phosphorylation of caspase-2

    Science.gov (United States)

    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

  14. Regulators of Slc4 bicarbonate transporter activity

    Directory of Open Access Journals (Sweden)

    Ian M. Thornell

    2015-06-01

    Full Text Available The Slc4 family of transporters is comprised of anion exchangers (AE1-4, Na-coupled bicarbonate transporters (NCBTs including electrogenic Na/bicarbonate cotransporters (NBCe1 and NBCe2, electroneutral Na/bicarbonate cotransporters (NBCn1 and NBCn2, and the electroneutral Na-driven Cl-bicarbonate exchanger (NDCBE, as well as a borate transporter (BTR1. These transporters regulate intracellular pH (pHi and contribute to steady-state pHi, but are also involved in other physiological processes including CO2 carriage by red blood cells and solute secretion/reabsorption across epithelia. Acid-base transporters function as either acid extruders or acid loaders, with the Slc4 proteins moving HCO3– either into or out of cells. According to results from both molecular and functional studies, multiple Slc4 proteins and/or associated splice variants with similar expected effects on pHi are often found in the same tissue or cell. Such apparent redundancy is likely to be physiologically important. In addition to regulating pHi, a HCO3– transporter contributes to a cell’s ability to fine tune the intracellular regulation of the cotransported/exchanged ion(s (e.g., Na+ or Cl–. In addition, functionally similar transporters or splice variants with different regulatory profiles will optimize pH physiology and solute transport under various conditions or within subcellular domains. Such optimization will depend on activated signaling pathways and transporter expression profiles. In this review, we will summarize and discuss both classical and more recently identified regulators of the Slc4 proteins. Some of these regulators include traditional second messengers, lipids, binding proteins, autoregulatory domains, and less conventional regulators. The material presented will provide insight into the diversity and physiological significance of multiple members within the Slc4 gene family.

  15. Imidacloprid exposure cause the histopathological changes, activation of TNF-α, iNOS, 8-OHdG biomarkers, and alteration of caspase 3, iNOS, CYP1A, MT1 gene expression levels in common carp (Cyprinus carpio L.).

    Science.gov (United States)

    Özdemir, Selçuk; Altun, Serdar; Arslan, Harun

    2018-01-01

    Imidacloprid (IMI) is a neonicotinoid that is widely used for the protection of crops and carnivores from insects and parasites, respectively. It is well known that imidacloprid exposure has a harmful effect on several organisms. However, there is little information about imidacloprid toxicity in aquatic animals, particularly fish. Thus, in the current study, we assessed the histopathological changes; activation of iNOS, 8-OHdG and TNF-α; and expression levels of caspase 3, iNOS, CYP1A and MT1 genes in the common carp exposed to imidacloprid. For this purpose, fish were exposed to either a low dose (140 mg/L) or a high dose (280 mg/L) of imidacloprid for 24 h, 48 h, 72 h and 96 h. After IMI exposure, we detected hyperplasia of secondary lamellar cells and mucous cell hyperplasia in the gills, as well as hydropic degeneration in hepatocytes and necrosis in the liver. Moreover, 8-OHdG, iNOS and TNF-α activation was found particularly in the gills and liver but also moderately in the brain. Transcriptional analysis showed that caspase 3 expression was altered low dose and high doses of IMI for 72 h and 96 h exposure ( p   0.05) except with low and high doses of IMI for 96 h ( p <  0.05), and lastly, MT1 gene expression was up-regulated only in the brain with low doses of IMI for 96 h and high doses of IMI for 48 h, 72 h and 96 h exposure ( p <  0.05, p <  0.01). Our results indicated that acute IMI exposure moderately induce apoptosis in the brain but caused severe histopathological lesions, inflammation, and oxidative stress in the gills, liver, and brain of the common carp.

  16. Regulation of pokemon 1 activity by sumoylation.

    Science.gov (United States)

    Roh, Hee-Eun; Lee, Min-Nyung; Jeon, Bu-Nam; Choi, Won-Il; Kim, Yoo-Jin; Yu, Mi-Young; Hur, Man-Wook

    2007-01-01

    Pokemon 1 is a proto-oncogenic transcriptional regulator that contains a POZ domain at the N-terminus and four Kruppel-like zinc fingers at the C-terminus. Pokemon 1 plays an important role in adipogenesis, osteogenesis, oncogenesis, and transcription of NF-kB responsive genes. Recent reports have shown that biological activities of transcription factors are regulated by sumolylation. We investigated whether Pokemon 1 is post-translationally modified by sumoylation and whether the modification affects Pokemon 1's transcriptional properties. We found that Pokemon 1 is sumoylated in vitro and in vivo. Upon careful analysis of the amino acid sequence of Pokemon 1, we found ten potential sumoylation sites located at lysines 61, 354, 371, 379, 383, 396, 486, 487, 536 and 539. We mutated each of these amino acids into arginine and tested whether the mutation could affect the transcriptional properties of Pokemon 1 on the Pokemon 1 responsive genes, such as ADH5/FDH and pG5-FRE-Luc. Wild-type Pokemon 1 potently represses transcription of ADH5/FDH. Most of the mutants, however, were weaker transcription repressors and repressed transcription 1.3-3.3 fold less effective. Although potential sumoylation sites were located close to the DNA binding domain or the nuclear localization sequence, the mutations did not alter nuclear localization or DNA binding activity. In addition, on the pG5-FRE-Luc test promoter construct, ectopic SUMO-1 repressed transcription in the presence of Pokemon 1. The sumoylation target lysine residue at amino acid 61, which is located in the middle of the POZ-domain, is important because K61R mutation resulted in a much weaker molecular interaction with corepressors. Our data suggest that Pokemon 1's activity as a transcription factor may involve sumoylation, and that sumoylation might be important in the regulation of transcription by Pokemon 1.

  17. NEA activities in safety and regulation

    International Nuclear Information System (INIS)

    Stadie, K.B.

    1983-01-01

    The NEA programme on Safety and Regulations is briefly reviewed. It encompasses four main areas - nuclear safety technology; nuclear licensing; radiation protection; and waste management - with three principal objectives: to promote exchanges of technical information in order to enlarge the data base for national decision making; to improve co-ordination of national R and D activities with emphasis on international standard problem exercises, and to promote international projects; to develop common technical, administrative and legal approaches to improve compatibility of safety and regulatory practices

  18. Prokaryotic caspase homologs: phylogenetic patterns and functional characteristics reveal considerable diversity.

    Directory of Open Access Journals (Sweden)

    Johannes Asplund-Samuelsson

    Full Text Available Caspases accomplish initiation and execution of apoptosis, a programmed cell death process specific to metazoans. The existence of prokaryotic caspase homologs, termed metacaspases, has been known for slightly more than a decade. Despite their potential connection to the evolution of programmed cell death in eukaryotes, the phylogenetic distribution and functions of these prokaryotic metacaspase sequences are largely uncharted, while a few experiments imply involvement in programmed cell death. Aiming at providing a more detailed picture of prokaryotic caspase homologs, we applied a computational approach based on Hidden Markov Model search profiles to identify and functionally characterize putative metacaspases in bacterial and archaeal genomes. Out of the total of 1463 analyzed genomes, merely 267 (18% were identified to contain putative metacaspases, but their taxonomic distribution included most prokaryotic phyla and a few archaea (Euryarchaeota. Metacaspases were particularly abundant in Alphaproteobacteria, Deltaproteobacteria and Cyanobacteria, which harbor many morphologically and developmentally complex organisms, and a distinct correlation was found between abundance and phenotypic complexity in Cyanobacteria. Notably, Bacillus subtilis and Escherichia coli, known to undergo genetically regulated autolysis, lacked metacaspases. Pfam domain architecture analysis combined with operon identification revealed rich and varied configurations among the metacaspase sequences. These imply roles in programmed cell death, but also e.g. in signaling, various enzymatic activities and protein modification. Together our data show a wide and scattered distribution of caspase homologs in prokaryotes with structurally and functionally diverse sub-groups, and with a potentially intriguing evolutionary role. These features will help delineate future characterizations of death pathways in prokaryotes.

  19. Basic principles for regulating nuclear activities

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1996-03-01

    The AECB has developed as its mission statement: `To ensure that the use of nuclear energy in Canada does not pose undue risk to health, safety, security and the environment`. This report proposes eleven qualitative principles for regulating nuclear activities whose achievement would satisfy the broad policy enunciated in the statement. They would further provide a basis for the specific regulatory requirements expressed by the AECB in its Regulations and other documents. They would thus represent a connecting link between the policy enunciated in the mission statement and the requirements. The proposed principles are largely concerned with how the allowable risk should be set for members of the public, for industry workers, for society as a whole, and for the environment. In making these recommendations the risks from normal operation of the licensed facility and those from a possible serious accident are considered separately. The distribution of risk between geographic communities and between generations is also addressed in the proposed principles. These are listed in the final section of the report. 23 refs.

  20. Basic principles for regulating nuclear activities

    International Nuclear Information System (INIS)

    1996-03-01

    The AECB has developed as its mission statement: 'To ensure that the use of nuclear energy in Canada does not pose undue risk to health, safety, security and the environment'. This report proposes eleven qualitative principles for regulating nuclear activities whose achievement would satisfy the broad policy enunciated in the statement. They would further provide a basis for the specific regulatory requirements expressed by the AECB in its Regulations and other documents. They would thus represent a connecting link between the policy enunciated in the mission statement and the requirements. The proposed principles are largely concerned with how the allowable risk should be set for members of the public, for industry workers, for society as a whole, and for the environment. In making these recommendations the risks from normal operation of the licensed facility and those from a possible serious accident are considered separately. The distribution of risk between geographic communities and between generations is also addressed in the proposed principles. These are listed in the final section of the report. 23 refs

  1. Caspase-1 deficiency in mice reduces intestinal triglyceride absorption and hepatic triglyceride secretion

    NARCIS (Netherlands)

    Diepen, J.A. van; Stienstra, R.; Vroegrijk, I.O.C.M.; Berg, S.A.A. van den; Salvatori, D.; Hooiveld, G.J.; Kersten, S.; Tack, C.J.; Netea, M.G.; Smit, J.W.A.; Joosten, L.A.B.; Havekes, L.M.; Dijk, K.W. van; Rensen, P.C.N.

    2013-01-01

    Caspase-1 is known to activate the proinflammatory cytokines IL-1β and IL-18. Additionally, it can cleave other substrates, including proteins involved in metabolism. Recently, we showed that caspase-1 deficiency in mice strongly reduces high-fat diet-induced weight gain, at least partly caused by

  2. Caspase-mediated apoptosis induction in zebrafish cerebellar Purkinje neurons.

    Science.gov (United States)

    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.

  3. RIP3 Inhibits Inflammatory Hepatocarcinogenesis but Promotes Cholestasis by Controlling Caspase-8- and JNK-Dependent Compensatory Cell Proliferation

    Directory of Open Access Journals (Sweden)

    Mihael Vucur

    2013-08-01

    Full Text Available For years, the term “apoptosis” was used synonymously with programmed cell death. However, it was recently discovered that receptor interacting protein 3 (RIP3-dependent “necroptosis” represents an alternative programmed cell death pathway activated in many inflamed tissues. Here, we show in a genetic model of chronic hepatic inflammation that activation of RIP3 limits immune responses and compensatory proliferation of liver parenchymal cells (LPC by inhibiting Caspase-8-dependent activation of Jun-(N-terminal kinase in LPC and nonparenchymal liver cells. In this way, RIP3 inhibits intrahepatic tumor growth and impedes the Caspase-8-dependent establishment of specific chromosomal aberrations that mediate resistance to tumor-necrosis-factor-induced apoptosis and underlie hepatocarcinogenesis. Moreover, RIP3 promotes the development of jaundice and cholestasis, because its activation suppresses compensatory proliferation of cholangiocytes and hepatic stem cells. These findings demonstrate a function of RIP3 in regulating carcinogenesis and cholestasis. Controlling RIP3 or Caspase-8 might represent a chemopreventive or therapeutic strategy against hepatocellular carcinoma and biliary disease.

  4. Pharmacological caspase inhibitors: Research towards therapeutic perspectives

    Czech Academy of Sciences Publication Activity Database

    Kudělová, J.; Fleischmannová, J.; Adamová, E.; Matalová, Eva

    2015-01-01

    Roč. 66, č. 4 (2015), s. 473-482 ISSN 0867-5910 R&D Projects: GA ČR(CZ) GA14-28254S Institutional support: RVO:68081715 Keywords : caspase * caspase inhibitor * apoptosis Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 2.804, year: 2015

  5. Pharmacological caspase inhibitors: Research towards therapeutic perspectives

    Czech Academy of Sciences Publication Activity Database

    Kudělová, J.; Fleischmannová, Jana; Adamová, Eva; Matalová, Eva

    2015-01-01

    Roč. 66, č. 4 (2015), s. 473-482 ISSN 0867-5910 R&D Projects: GA ČR GB14-37368G Institutional support: RVO:67985904 Keywords : caspase * caspase inhibitor * apoptosis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.804, year: 2015

  6. The Role of Sulfur Dioxide in the Regulation of Mitochondrion-Related Cardiomyocyte Apoptosis in Rats with Isopropylarterenol-Induced Myocardial Injury

    Directory of Open Access Journals (Sweden)

    Junbao Du

    2013-05-01

    Full Text Available The authors investigated the regulatory effects of sulfur dioxide (SO2 on myocardial injury induced by isopropylarterenol (ISO hydrochloride and its mechanisms. Wistar rats were divided into four groups: control group, ISO group, ISO plus SO2 group, and SO2 only group. Cardiac function was measured and cardiomyocyte apoptosis was detected. Bcl-2, bax and cytochrome c (cytc expressions, and caspase-9 and caspase-3 activities in the left ventricular tissues were examined in the rats. The opening status of myocardial mitochondrial permeability transition pore (MPTP and membrane potential were analyzed. The results showed that ISO-treated rats developed heart dysfunction and cardiac injury. Furthermore, cardiomyocyte apoptosis in the left ventricular tissues was augmented, left ventricular tissue bcl-2 expression was down-regulated, bax expression was up-regulated, mitochondrial membrane potential was significantly reduced, MPTP opened, cytc release from mitochondrion into cytoplasm was significantly increased, and both caspase-9 and caspase-3 activities were increased. Administration of an SO2 donor, however, markedly improved heart function and relieved myocardial injury of the ISO-treated rats; it lessened cardiomyocyte apoptosis, up-regulated myocardial bcl-2, down-regulated bax expression, stimulated mitochondrial membrane potential, closed MPTP, and reduced cytc release as well as caspase-9 and caspase-3 activities in the left ventricular tissue. Hence, SO2 attenuated myocardial injury in association with the inhibition of apoptosis in myocardial tissues, and the bcl-2/cytc/caspase-9/caspase-3 pathway was possibly involved in this process.

  7. Caspase-11 Plays a Protective Role in Pulmonary Acinetobacter baumannii Infection.

    Science.gov (United States)

    Wang, Wei; Shao, Yue; Li, Shengjun; Xin, Na; Ma, Tingxian; Zhao, Chenghai; Song, Min

    2017-10-01

    Activation of caspase-11 by some Gram-negative bacteria triggers the caspase-1/interleukin 1β (IL-1β) pathway, independent of canonical inflammasomes. Acinetobacter baumannii is a Gram-negative, conditionally pathogenic bacterium that can cause severe pulmonary infection in hospitalized patients. A. baumannii was revealed to activate canonical and noncanonical inflammasome pathways in bone marrow-derived macrophages (BMDMs). Pulmonary infection of caspase-11 -/- mice with A. baumannii showed that caspase-11 deficiency impaired A. baumannii clearance, exacerbated pulmonary pathological changes, and enhanced susceptibility to A. baumannii These data indicate that the caspase-11-mediated innate immune response plays a crucial role in defending against A. baumannii . Copyright © 2017 American Society for Microbiology.

  8. Systemic and cerebral vascular endothelial growth factor levels increase in murine cerebral malaria along with increased Calpain and caspase activity and can be reduced by erythropoietin treatment

    DEFF Research Database (Denmark)

    Hempel, Casper; Hoyer, Nils; Kildemoes, Anna

    2014-01-01

    The pathogenesis of cerebral malaria (CM) includes compromised microvascular perfusion, increased inflammation, cytoadhesion, and endothelial activation. These events cause blood-brain barrier disruption and neuropathology and associations with the vascular endothelial growth factor (VEGF) signal...

  9. Oxidative stress by monosodium urate crystals promotes renal cell apoptosis through mitochondrial caspase-dependent pathway in human embryonic kidney 293 cells: mechanism for urate-induced nephropathy.

    Science.gov (United States)

    Choe, Jung-Yoon; Park, Ki-Yeun; Kim, Seong-Kyu

    2015-01-01

    The aim of this study is to clarify the effect of oxidative stress on monosodium urate (MSU)-mediated apoptosis of renal cells. Quantitative real-time polymerase chain reaction and immunoblotting for Bcl-2, caspase-9, caspase-3, iNOS, cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), IL-18, TNF receptor-associated factor-6 (TRAF-6), and mitogen-activated protein kinases were performed on human embryonic kidney 293 (HEK293) cells, which were stimulated by MSU crystals. Fluorescence-activated cell sorting was performed using annexin V for assessment of apoptosis. Reactive oxygen species (ROS) were measured. IL-1β siRNA was used for blocking IL-1β expression. MSU crystals promoted ROS, iNOS, and COX-2 expression and also increased TRAF-6 and IL-1β expression in HEK293 cells, which was inhibited by an antioxidant ascorbic acid. Caspase-dependent renal cell apoptosis was induced through attenuation of Bcl-2 and enhanced caspase-3 and caspase-9 expression by MSU crystals, which was significantly reversed by ascorbic acid and transfection of IL-1β siRNA to HEK293 cells. Ascorbic acid inhibited phosphorylation of extracellular signal-regulated kinase and Jun N-terminal protein kinase stimulated by MSU crystals. ROS accumulation and iNOS and COX-2 mRNA expression by MSU crystals was also suppressed by transfection with IL-1β siRNA. Oxidative stress generated by MSU crystals promotes renal apoptosis through the mitochondrial caspase-dependent apoptosis pathway.

  10. Regulation of Metabolic Activity by p53

    Directory of Open Access Journals (Sweden)

    Jessica Flöter

    2017-05-01

    Full Text Available Metabolic reprogramming in cancer cells is controlled by the activation of multiple oncogenic signalling pathways in order to promote macromolecule biosynthesis during rapid proliferation. Cancer cells also need to adapt their metabolism to survive and multiply under the metabolically compromised conditions provided by the tumour microenvironment. The tumour suppressor p53 interacts with the metabolic network at multiple nodes, mostly to reduce anabolic metabolism and promote preservation of cellular energy under conditions of nutrient restriction. Inactivation of this tumour suppressor by deletion or mutation is a frequent event in human cancer. While loss of p53 function lifts an important barrier to cancer development by deleting cell cycle and apoptosis checkpoints, it also removes a crucial regulatory mechanism and can render cancer cells highly sensitive to metabolic perturbation. In this review, we will summarise the major concepts of metabolic regulation by p53 and explore how this knowledge can be used to selectively target p53 deficient cancer cells in the context of the tumour microenvironment.

  11. Novel HTS strategy identifies TRAIL-sensitizing compounds acting specifically through the caspase-8 apoptotic axis.

    Directory of Open Access Journals (Sweden)

    Darren Finlay

    Full Text Available Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL is potentially a very important therapeutic as it shows selectivity for inducing apoptosis in cancer cells whilst normal cells are refractory. TRAIL binding to its cognate receptors, Death Receptors-4 and -5, leads to recruitment of caspase-8 and classical activation of downstream effector caspases, leading to apoptosis. As with many drugs however, TRAIL's usefulness is limited by resistance, either innate or acquired. We describe here the development of a novel 384-well high-throughput screening (HTS strategy for identifying potential TRAIL-sensitizing agents that act solely in a caspase-8 dependent manner. By utilizing a TRAIL resistant cell line lacking caspase-8 (NB7 compared to the same cells reconstituted with the wild-type protein, or with a catalytically inactive point mutant of caspase-8, we are able to identify compounds that act specifically through the caspase-8 axis, rather than through general toxicity. In addition, false positive hits can easily be "weeded out" in this assay due to their activity in cells lacking caspase-8-inducible activity. Screening of the library of pharmacologically active compounds (LOPAC was performed as both proof-of-concept and to discover potential unknown TRAIL sensitizers whose mechanism is caspase-8 mediated. We identified known TRAIL sensitizers from the library and identified new compounds that appear to sensitize specifically through caspase-8. In sum, we demonstrate proof-of-concept and discovery of novel compounds with a screening strategy optimized for the detection of caspase-8 pathway-specific TRAIL sensitizers. This screen was performed in the 384-well format, but could easily be further miniaturized, allows easy identification of artifactual false positives, and is highly scalable to accommodate diverse libraries.

  12. Notch inhibition counteracts Paneth cell death in absence of caspase-8.

    Science.gov (United States)

    Jeon, M K; Kaemmerer, E; Schneider, U; Schiffer, M; Klaus, C; Hennings, J; Clahsen, T; Ackerstaff, T; Niggemann, M; Schippers, A; Longerich, T; Sellge, G; Trautwein, C; Wagner, N; Liedtke, C; Gassler, N

    2018-05-16

    Opposing activities of Notch and Wnt signaling regulate mucosal barrier homeostasis and differentiation of intestinal epithelial cells. Specifically, Wnt activity is essential for differentiation of secretory cells including Wnt3-producing Paneth cells, whereas Notch signaling strongly promotes generation of absorptive cells. Loss of caspase-8 in intestinal epithelium (casp8 ∆int ) is associated with fulminant epithelial necroptosis, severe Paneth cell death, secondary intestinal inflammation, and an increase in Notch activity. Here, we found that pharmacological Notch inhibition with dibenzazepine (DBZ) is able to essentially rescue the loss of Paneth cells, deescalate the inflammatory phenotype, and reduce intestinal permeability in casp8 ∆int mice. The secretory cell metaplasia in DBZ-treated casp8 ∆int animals is proliferative, indicating for Notch activities partially insensitive to gamma-secretase inhibition in a casp8 ∆int background. Our data suggest that casp8 acts in the intestinal Notch network.

  13. Regulation of ROS in transmissible gastroenteritis virus-activated apoptotic signaling

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Li [College of Veterinary Medicine, Northwest A and F University, Yangling, Shaanxi 712100 (China); College of Life Sciences, Hainan Normal University, Haikou, Hainan 571158 (China); Zhao, Xiaomin; Huang, Yong; Du, Qian; Dong, Feng; Zhang, Hongling; Song, Xiangjun; Zhang, Wenlong [College of Veterinary Medicine, Northwest A and F University, Yangling, Shaanxi 712100 (China); Tong, Dewen, E-mail: dwtong@nwsuaf.edu.cn [College of Veterinary Medicine, Northwest A and F University, Yangling, Shaanxi 712100 (China)

    2013-12-06

    Highlights: •TGEV infection induced ROS accumulation. •ROS accumulation is involved in TGEV-induced mitochondrial integrity impairment. •ROS is associated with p53 activation and apoptosis occurrence in TGEV-infected cells. -- Abstract: Transmissible gastroenteritis virus (TGEV), an enteropathogenic coronavirus, causes severe lethal watery diarrhea and dehydration in piglets. Previous studies indicate that TGEV infection induces cell apoptosis in host cells. In this study, we investigated the roles and regulation of reactive oxygen species (ROS) in TGEV-activated apoptotic signaling. The results showed that TGEV infection induced ROS accumulation, whereas UV-irradiated TGEV did not promote ROS accumulation. In addition, TGEV infection lowered mitochondrial transmembrane potential in PK-15 cell line, which could be inhibited by ROS scavengers, pyrrolidinedithiocarbamic (PDTC) and N-acetyl-L-cysteine (NAC). Furthermore, the two scavengers significantly inhibited the activation of p38 MAPK and p53 and further blocked apoptosis occurrence through suppressing the TGEV-induced Bcl-2 reduction, Bax redistribution, cytochrome c release and caspase-3 activation. These results suggest that oxidative stress pathway might be a key element in TGEV-induced apoptosis and TGEV pathogenesis.

  14. Legislation on and regulation of nuclear activities

    International Nuclear Information System (INIS)

    1984-05-01

    This work is a compilation of legislative texts and regulations published by the Atomic Energy Commission's Legal Affairs Department (CEA). It provides a comprehensive source of knowledge and information on nuclear energy law. Legislative texts published over the last forty years, are collected and analytically indexed. The publication covers both French regulations and regulations of international organisations such as the International Atomic Energy Agency and Euratom. It is divided into eight different chapters, dealing with regulations relevant to international and national institutions, nuclear installations, third party liability, protection of persons and the environment, etc. A chronological table of the texts of international and national laws is also included in this work. (NEA) [fr

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

    Science.gov (United States)

    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.

  16. Hepatitis C virus infection induces apoptosis through a Bax-triggered, mitochondrion-mediated, caspase 3-dependent pathway.

    Science.gov (United States)

    Deng, Lin; Adachi, Tetsuya; Kitayama, Kikumi; Bungyoku, Yasuaki; Kitazawa, Sohei; Ishido, Satoshi; Shoji, Ikuo; Hotta, Hak

    2008-11-01

    We previously reported that cells harboring the hepatitis C virus (HCV) RNA replicon as well as those expressing HCV NS3/4A exhibited increased sensitivity to suboptimal doses of apoptotic stimuli to undergo mitochondrion-mediated apoptosis (Y. Nomura-Takigawa, et al., J. Gen. Virol. 87:1935-1945, 2006). Little is known, however, about whether or not HCV infection induces apoptosis of the virus-infected cells. In this study, by using the chimeric J6/JFH1 strain of HCV genotype 2a, we demonstrated that HCV infection induced cell death in Huh7.5 cells. The cell death was associated with activation of caspase 3, nuclear translocation of activated caspase 3, and cleavage of DNA repair enzyme poly(ADP-ribose) polymerase, which is known to be an important substrate for activated caspase 3. These results suggest that HCV-induced cell death is, in fact, apoptosis. Moreover, HCV infection activated Bax, a proapoptotic member of the Bcl-2 family, as revealed by its conformational change and its increased accumulation on mitochondrial membranes. Concomitantly, HCV infection induced disruption of mitochondrial transmembrane potential, followed by mitochondrial swelling and release of cytochrome c from mitochondria. HCV infection also caused oxidative stress via increased production of mitochondrial superoxide. On the other hand, HCV infection did not mediate increased expression of glucose-regulated protein 78 (GRP78) or GRP94, which are known as endoplasmic reticulum (ER) stress-induced proteins; this result suggests that ER stress is not primarily involved in HCV-induced apoptosis in our experimental system. Taken together, our present results suggest that HCV infection induces apoptosis of the host cell through a Bax-triggered, mitochondrion-mediated, caspase 3-dependent pathway(s).

  17. Contribution of caspase-3 differs by p53 status in apoptosis induced by X-irradiation

    International Nuclear Information System (INIS)

    Kobayashi, Daisuke; Tokino, Takashi; Watanabe, Naoki

    2001-01-01

    We investigated the effect of p53 status on involvement of caspase-3 activation in cell death induced by X-irradiation, using rat embryonic fibroblasts (REFs) transduced with a temperature-sensitive mutant (mt) p53 gene. Cells with wild-type (wt) p53 showed greater resistance to X-irradiation than cells with mt p53. In cells with wt p53, X-irradiation-induced apoptosis was not inhibited by the caspase-3 inhibitor acetyl-L-aspartyl-L-methionyl-L-glutaminyl-L-aspartyl-aldehyde (Ac-DMQD-CHO) and caspase-3 activity was not elevated following X-irradiation, although induction of p53 and p21/WAF-1 protein was observed. In contrast, irradiated cells with mt p53 showed 89% inhibition of cell death with Ac-DMQD-CHO and 98% inhibition with the antioxidant N-acetyl-L-cysteine (NAC). In cells with mt p53, caspase-3 activity was increased approximately 5 times beyond baseline activity at 24 h after irradiation. This increase was almost completely inhibited by NAC. However, inhibition of caspase-3 by Ac-DMQD-CHO failed to decrease production of reactive oxygen species by cells with mt p53. Differential involvement of caspase-3 is a reason for differences in sensitivity to X-irradiation in cells with different p53 status. Caspase-3 activation appears to occur downstream from generation of reactive oxygen species occurring independently of wt p53 during X-irradiation-induced cell death. (author)

  18. Inner ear dysfunction in caspase-3 deficient mice

    Directory of Open Access Journals (Sweden)

    Woo Minna

    2011-10-01

    Full Text Available Abstract Background Caspase-3 is one of the most downstream enzymes activated in the apoptotic pathway. In caspase-3 deficient mice, loss of cochlear hair cells and spiral ganglion cells coincide closely with hearing loss. In contrast with the auditory system, details of the vestibular phenotype have not been characterized. Here we report the vestibular phenotype and inner ear anatomy in the caspase-3 deficient (Casp3-/- mouse strain. Results Average ABR thresholds of Casp3-/- mice were significantly elevated (P Casp3+/- mice and Casp3+/+ mice at 3 months of age. In DPOAE testing, distortion product 2F1-F2 was significantly decreased (P Casp3-/- mice, whereas Casp3+/- and Casp3+/+ mice showed normal and comparable values to each other. Casp3-/- mice were hyperactive and exhibited circling behavior when excited. In lateral canal VOR testing, Casp3-/- mice had minimal response to any of the stimuli tested, whereas Casp3+/- mice had an intermediate response compared to Casp3+/+ mice. Inner ear anatomical and histological analysis revealed gross hypomorphism of the vestibular organs, in which the main site was the anterior semicircular canal. Hair cell numbers in the anterior- and lateral crista, and utricle were significantly smaller in Casp3-/- mice whereas the Casp3+/- and Casp3+/+ mice had normal hair cell numbers. Conclusions These results indicate that caspase-3 is essential for correct functioning of the cochlea as well as normal development and function of the vestibule.

  19. Proliferation related acidic leucine-rich protein PAL31 functions as a caspase-3 inhibitor

    International Nuclear Information System (INIS)

    Sun Weiyong; Kimura, Hiromichi; Hattori, Naka; Tanaka, Satoshi; Matsuyama, Shigemi; Shiota, Kunio

    2006-01-01

    Proliferation related acidic leucine-rich protein PAL31 (PAL31) is expressed in proliferating cells and consists of 272 amino acids with a tandem structure of leucine-rich repeats in the N-terminus and a highly acidic region with a putative nuclear localization signal in the C-terminus. We previously reported that PAL31 is required for cell cycle progression. In the present study, we found that the antisense oligonucleotide of PAL31 induced apoptosis to the transfected Nb2 cells. Stable transfectants, in which PAL31 was regulated by an inducible promoter, were generated to gain further insight into the signaling role of PAL31 in the regulation of apoptosis. Expression of PAL31 resulted in the marked rescue of Rat1 cells from etoposide and UV radiation-induced apoptosis and the cytoprotection was correlated with the levels of PAL31 protein. Thus, cytoprotection from apoptosis is a physiological function of PAL31. PAL31 can suppress caspase-3 activity but not cytochrome c release in vitro, indicating that PAL31 is a direct caspase-3 inhibitor. In conclusion, PAL31 is a multifunctional protein working as a cell cycle progression factor as well as a cell survival factor

  20. NuMA and nuclear lamins are cleaved during viral infection - inhibition of caspase activity prevents cleavage and rescues HeLa cells from measles virus-induced but not from rhinovirus 1B-induced cell death

    International Nuclear Information System (INIS)

    Taimen, Pekka; Berghaell, Heidi; Vainionpaeae, Raija; Kallajoki, Markku

    2004-01-01

    Nuclear matrix is a structural framework of important nuclear processes. We studied the effect of two different types of viral infections on nuclear matrix. HeLa cells were infected with human rhinovirus 1B (HRV 1B) or measles virus (MV), and Nuclear Mitotic Apparatus protein (NuMA) and lamins A/C and B were used as markers for internal nuclear matrix and peripheral nuclear lamina, respectively. We show that NuMA, lamins, and poly(ADP-ribose) polymerase-1 are cleaved during viral infection in a virus family-specific manner suggesting that these viruses activate different sets of proteases. Morphologically, NuMA was excluded from the condensed chromatin, lamins showed a folded distribution, and both proteins finally remained around the nuclear fragments. A general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-FMK) prevented the nuclear disintegration and the cleavage of the proteins studied. Interestingly, z-VAD-FMK rescued MV-infected but not HRV 1B-infected cells from cell death. These results show for the first time that NuMA and lamins are specific target proteins during virus-induced programmed cell death

  1. Proliferative and anti-proliferative effects of dietary levels of phytoestrogens in rat pituitary GH3/B6/F10 cells - the involvement of rapidly activated kinases and caspases

    International Nuclear Information System (INIS)

    Jeng, Yow-Jiun; Watson, Cheryl S

    2009-01-01

    Phytoestogens are a group of lipophillic plant compounds that can have estrogenic effects in animals; both tumorigenic and anti-tumorigenic effects have been reported. Prolactin-secreting adenomas are the most prevalent form of pituitary tumors in humans and have been linked to estrogen exposures. We examined the proliferative effects of phytoestrogens on a rat pituitary tumor cell line, GH 3 /B 6 /F 10 , originally subcloned from GH 3 cells based on its ability to express high levels of the membrane estrogen receptor-α. We measured the proliferative effects of these phytoestrogens using crystal violet staining, the activation of several mitogen-activated protein kinases (MAPKs) and their downstream targets via a quantitative plate immunoassay, and caspase enzymatic activities. Four phytoestrogens (coumestrol, daidzein, genistein, and trans-resveratrol) were studied over wide concentration ranges. Except trans-resveratrol, all phytoestrogens increased GH 3 /B 6 /F 10 cell proliferation at some concentration relevant to dietary levels. All four phytoestrogens attenuated the proliferative effects of estradiol when administered simultaneously. All phytoestrogens elicited MAPK and downstream target activations, but with time course patterns that often differed from that of estradiol and each other. Using selective antagonists, we determined that MAPKs play a role in the ability of these phytoestrogens to elicit these responses. In addition, except for trans-resveratrol, a serum removal-induced extrinsic apoptotic pathway was blocked by these phytoestrogens. Phytoestrogens can block physiological estrogen-induced tumor cell growth in vitro and can also stimulate growth at high dietary concentrations in the absence of endogenous estrogens; these actions are correlated with slightly different signaling response patterns. Consumption of these compounds should be considered in strategies to control endocrine tumor cell growth, such as in the pituitary

  2. Involvement of caspase-12-dependent apoptotic pathway in ionic radiocontrast urografin-induced renal tubular cell injury

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Cheng Tien [Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Weng, Te I. [Department of Forensic Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Chen, Li Ping [Department of Dentistry, Chang Gang Memorial Hospital, Chang Gang University, Taoyuan, Taiwan (China); Chiang, Chih Kang [Department of Integrated Diagnostics and Therapeutics, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan (China); Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan (China); Liu, Shing Hwa, E-mail: shinghwaliu@ntu.edu.tw [Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Department of Urology, National Taiwan University Hospital, Taipei, Taiwan (China)

    2013-01-01

    Contrast medium (CM) induces a direct toxic effect on renal tubular cells. This toxic effect subjects in the disorder of CM-induced nephropathy. Our previous work has demonstrated that CM shows to activate the endoplasmic reticulum (ER)-related adaptive unfolding protein response (UPR) activators. Glucose-regulated protein 78 (GRP78)/eukaryotic initiation factor 2α (eIF2α)-related pathways play a protective role during the urografin (an ionic CM)-induced renal tubular injury. However, the involvement of ER stress-related apoptotic signals in the urografin-induced renal tubular cell injury remains unclear. Here, we examined by the in vivo and in vitro experiments to explore whether ER stress-regulated pro-apoptotic activators participate in urografin-induced renal injury. Urografin induced renal tubular dilation, tubular cells detachment, and necrosis in the kidneys of rats. The tubular apoptosis, ER stress-related pro-apoptotic transcriptional factors, and kidney injury marker-1 (kim-1) were also conspicuously up-regulated in urografin-treated rats. Furthermore, treatment of normal rat kidney (NRK)-52E tubular cells with urografin augmented the expressions of activating transcription factor-6 (ATF-6), C/EBP homologous protein (CHOP), Bax, caspase-12, JNK, and inositol-requiring enzyme (IRE) 1 signals. Urografin-induced renal tubular cell apoptosis was not reversed by the inhibitors of ATF-6, JNK signals or CHOP siRNA transfection, but it could be partially reversed by the inhibitor of caspase-12. Taken together, the present results and our previous findings suggest that exposure of CM/urografin activates the ER stress-regulated survival- and apoptosis-related signaling pathways in renal tubular cells. Caspase-12-dependent apoptotic pathway may be partially involved in the urografin-induced nephropathy. -- Highlights: ► Ionic contrast medium-urografin induces renal tubular cell apoptosis. ► Urografin induces the ER stress-regulated survival and apoptosis

  3. Effect of PCB3 and its hydroxylated metabolites on estradiol secretion, cell viability, and caspase-3 activity in porcine small folicles

    Energy Technology Data Exchange (ETDEWEB)

    Ptak, A.; Gregoraszczuk, E.L. [Lab. of Reproductive Physiology and Toxicology of Domestic Animals, Inst. of Zoology, Jagiellonian Univ., Krakow (Poland); Ludewig, G.; Lehmler, H.J.; Robertson, L.W. [Dept. of Occupational and Environmental Health, Coll. of Public Health, The Univ. of Iowa, Iowa City, IA (United States)

    2004-09-15

    In general, highly chlorinated PCBs are slowly metabolized and eliminated. Lower chlorinated PCBs on the other hand are hydroxylated in vitro and in vivo. Surprisingly this does not necessarily mean that these hydroxylated PCBs are rapidly excreted, as recent findings of substantial amounts of hydroxylated PCBs in animal and human blood have shown. Therefore it must be assumed that not only the PCBs themselves, but also their metabolites can participate in the toxic effects of PCBs. Indeed, some hydroxylated metabolites of PCBs (OH-PCBs) have significant estrogenic activity through binding to the estrogen receptors. Surprisingly, PCB54 (2,2',6,6'-tetrachlorobiphenyl) has about 10% of the activity of 4-OH-PCB54 in the MCF-7 focus assay, but does not bind to the estrogen receptor, suggesting the possibility of an additional, yet unknown mechanism of estrogenicity. We found that PCBs and their hydroxylated metabolites cause an increase in estrogen secretion from ovarian follicular cells in vitro, with PCB3 < 4-OHPCB3 < 3, 4-OH-PCB3. The most sensitive follicles were those collected during the early stage of their development. In the present study we used this type of follicles to answer the question whether this observed huge stimulatory action of PCB3 and/or its metabolites on estrogen release into the medium is due to the action on cells viability and cell apoptosis.

  4. Bauhinia variegata candida Fraction Induces Tumor Cell Death by Activation of Caspase-3, RIP, and TNF-R1 and Inhibits Cell Migration and Invasion In Vitro

    Directory of Open Access Journals (Sweden)

    K. M. Santos

    2018-01-01

    Full Text Available Metastasis remains the most common cause of death in cancer patients. Inhibition of metalloproteinases (MMPs is an interesting approach to cancer therapy because of their role in the degradation of extracellular matrix (ECM, cell-cell, and cell-ECM interactions, modulating key events in cell migration and invasion. Herein, we show the cytotoxic and antimetastatic effects of the third fraction (FR3 from Bauhinia variegata candida (Bvc stem on human cervical tumor cells (HeLa and human peripheral blood mononuclear cells (PBMCs. FR3 inhibited MMP-2 and MMP-9 activity, indicated by zymogram. This fraction was cytotoxic to HeLa cells and noncytotoxic to PBMCs and decreased HeLa cell migration and invasion. FR3 is believed to stimulate extrinsic apoptosis together with necroptosis, assessed by western blotting. FR3 inhibited MMP-2 activity in the HeLa supernatant, differently from the control. The atomic mass spectrometry (ESI-MS characterization suggested the presence of glucopyranosides, D-pinitol, fatty acids, and phenolic acid. These findings provide insight suggesting that FR3 contains components with potential tumor-selective cytotoxic action in addition to the action on the migration of tumor cells, which may be due to inhibition of MMPs.

  5. Activation and Regulation of Cellular Eicosanoid Biosynthesis

    Directory of Open Access Journals (Sweden)

    Thomas G. Brock

    2007-01-01

    Full Text Available There is a growing appreciation for the wide variety of physiological responses that are regulated by lipid messengers. One particular group of lipid messengers, the eicosanoids, plays a central role in regulating immune and inflammatory responses in a receptor-mediated fashion. These mediators are related in that they are all derived from one polyunsaturated fatty acid, arachidonic acid. However, the various eicosanoids are synthesized by a wide variety of cell types by distinct enzymatic pathways, and have diverse roles in immunity and inflammation. In this review, the major pathways involved in the synthesis of eicosanoids, as well as key points of regulation, are presented.

  6. Stroke promotes survival of nearby transplanted neural stem cells by decreasing their activation of caspase 3 while not affecting their differentiation.

    Science.gov (United States)

    Kosi, Nina; Alić, Ivan; Salamon, Iva; Mitrečić, Dinko

    2018-02-14

    Although transplantation of stem cells improves recovery of the nervous tissue, little is known about the influence of different brain regions on transplanted cells. After we confirmed that cells with uniform differentiation potential can be generated in independent experiments, one million of neural stem cells isolated from B6.Cg-Tg(Thy1-YFP)16Jrs/J mouse embryos were transplanted into the brain 24 h after induction of stroke. The lateral ventricles, the corpus callosum and the striatum were tested. Two and four weeks after the transplantation, the cells transplanted in all three regions have been attracted to the ischemic core. The largest number of attracted cells has been observed after transplantation into the striatum. Their differentiation pattern and expression of neuroligin 1, SynCAM 1, postsynaptic density protein 95 and synapsin 1 followed the same pattern observed during in vitro cultivation and it did not differ among the tested regions. Differentiation pattern of the cells transplanted in the stroke-affected and healthy animals was the same. On the other hand, neural stem cells transplanted in the striatum of the animals affected by stroke exhibited significantly increased survival rates reaching 260 ± 19%, when compared to cells transplanted in their wild type controls. Surprisingly, improved survival two and four weeks after transplantation was not due to increased proliferation of the grafted cells and it was accompanied by decreased levels of activity of Casp3 (19.56 ± 3.1% in the stroke-affected vs. 30.14 ± 2.4% in healthy animals after four weeks). We assume that the decreased levels of Casp3 in cells transplanted near the ischemic region was linked to increased vasculogenesis, synaptogenesis, astrocytosis and axonogenesis detected in the host tissue affected by ischemia. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Interferon-β-induced activation of c-Jun NH2-terminal kinase mediates apoptosis through up-regulation of CD95 in CH31 B lymphoma cells

    International Nuclear Information System (INIS)

    Takada, Eiko; Shimo, Kuniaki; Hata, Kikumi; Abiake, Maira; Mukai, Yasuo; Moriyama, Masami; Heasley, Lynn; Mizuguchi, Junichiro

    2005-01-01

    Type I interferon (IFN)-induced antitumor action is due in part to apoptosis, but the molecular mechanisms underlying IFN-induced apoptosis remain largely unresolved. In the present study, we demonstrate that IFN-β induced apoptosis and the loss of mitochondrial membrane potential (ΔΨm) in the murine CH31 B lymphoma cell line, and this was accompanied by the up-regulation of CD95, but not CD95-ligand (CD95-L), tumor necrosis factor (TNF), or TNF-related apoptosis-inducing ligand (TRAIL). Pretreatment with anti-CD95-L mAb partially prevented the IFN-β-induced loss of ΔΨm, suggesting that the interaction of IFN-β-up-regulated CD95 with CD95-L plays a crucial role in the induction of fratricide. IFN-β induced a sustained activation of c-Jun NH 2 -terminal kinase 1 (JNK1), but not extracellular signal-regulated kinases (ERKs). The IFN-β-induced apoptosis and loss of ΔΨm were substantially compromised in cells overexpressing a dominant-negative form of JNK1 (dnJNK1), and it was slightly enhanced in cells carrying a constitutively active JNK construct, MKK7-JNK1 fusion protein. The IFN-β-induced up-regulation of CD95 together with caspase-8 activation was also abrogated in the dnJNK1 cells while it was further enhanced in the MKK7-JNK1 cells. The levels of cellular FLIP (c-FLIP), competitively interacting with caspase-8, were down-regulated by stimulation with IFN-β but were reversed by the proteasome inhibitor lactacystin. Collectively, the IFN-β-induced sustained activation of JNK mediates apoptosis, at least in part, through up-regulation of CD95 protein in combination with down-regulation of c-FLIP protein

  8. Drosophila spaghetti and doubletime link the circadian clock and light to caspases, apoptosis and tauopathy.

    Directory of Open Access Journals (Sweden)

    John C Means

    2015-05-01

    Full Text Available While circadian dysfunction and neurodegeneration are correlated, the mechanism for this is not understood. It is not known if age-dependent circadian dysfunction leads to neurodegeneration or vice-versa, and the proteins that mediate the effect remain unidentified. Here, we show that the knock-down of a regulator (spag of the circadian kinase Dbt in circadian cells lowers Dbt levels abnormally, lengthens circadian rhythms and causes expression of activated initiator caspase (Dronc in the optic lobes during the middle of the day or after light pulses at night. Likewise, reduced Dbt activity lengthens circadian period and causes expression of activated Dronc, and a loss-of-function mutation in Clk also leads to expression of activated Dronc in a light-dependent manner. Genetic epistasis experiments place Dbt downstream of Spag in the pathway, and Spag-dependent reductions of Dbt are shown to require the proteasome. Importantly, activated Dronc expression due to reduced Spag or Dbt activity occurs in cells that do not express the spag RNAi or dominant negative Dbt and requires PDF neuropeptide signaling from the same neurons that support behavioral rhythms. Furthermore, reduction of Dbt or Spag activity leads to Dronc-dependent Drosophila Tau cleavage and enhanced neurodegeneration produced by human Tau in a fly eye model for tauopathy. Aging flies with lowered Dbt or Spag function show markers of cell death as well as behavioral deficits and shortened lifespans, and even old wild type flies exhibit Dbt modification and activated caspase at particular times of day. These results suggest that Dbt suppresses expression of activated Dronc to prevent Tau cleavage, and that the circadian clock defects confer sensitivity to expression of activated Dronc in response to prolonged light. They establish a link between the circadian clock factors, light, cell death pathways and Tau toxicity, potentially via dysregulation of circadian neuronal remodeling in

  9. Caspase Cleavages of the Lymphocyte-oriented Kinase Prevent Ezrin, Radixin, and Moesin Phosphorylation during Apoptosis*

    Science.gov (United States)

    Leroy, Catherine; Belkina, Natalya V.; Long, Thavy; Deruy, Emeric; Dissous, Colette; Shaw, Stephen; Tulasne, David

    2016-01-01

    The lymphocyte-oriented kinase (LOK), also called serine threonine kinase 10 (STK10), is synthesized mainly in lymphocytes. It is involved in lymphocyte migration and polarization and can phosphorylate ezrin, radixin, and moesin (the ERM proteins). In a T lymphocyte cell line and in purified human lymphocytes, we found LOK to be cleaved by caspases during apoptosis. The first cleavage occurs at aspartic residue 332, located between the kinase domain and the coiled-coil regulation domain. This cleavage generates an N-terminal fragment, p50 N-LOK, containing the kinase domain and a C-terminal fragment, which is further cleaved during apoptosis. Although these cleavages preserve the entire kinase domain, p50 N-LOK displays no kinase activity. In apoptotic lymphocytes, caspase cleavages of LOK are concomitant with a decrease in ERM phosphorylation. When non-apoptotic lymphocytes from mice with homozygous and heterozygous LOK knockout were compared, the latter showed a higher level of ERM phosphorylation, but when apoptosis was induced, LOK−/− and LOK+/− lymphocytes showed the same low level, confirming in vivo that LOK-induced ERM phosphorylation is prevented during lymphocyte apoptosis. Our results demonstrate that cleavage of LOK during apoptosis abolishes its kinase activity, causing a decrease in ERM phosphorylation, crucial to the role of the ERM proteins in linking the plasma membrane to actin filaments. PMID:26945071

  10. Caspase Cleavages of the Lymphocyte-oriented Kinase Prevent Ezrin, Radixin, and Moesin Phosphorylation during Apoptosis.

    Science.gov (United States)

    Leroy, Catherine; Belkina, Natalya V; Long, Thavy; Deruy, Emeric; Dissous, Colette; Shaw, Stephen; Tulasne, David

    2016-05-06

    The lymphocyte-oriented kinase (LOK), also called serine threonine kinase 10 (STK10), is synthesized mainly in lymphocytes. It is involved in lymphocyte migration and polarization and can phosphorylate ezrin, radixin, and moesin (the ERM proteins). In a T lymphocyte cell line and in purified human lymphocytes, we found LOK to be cleaved by caspases during apoptosis. The first cleavage occurs at aspartic residue 332, located between the kinase domain and the coiled-coil regulation domain. This cleavage generates an N-terminal fragment, p50 N-LOK, containing the kinase domain and a C-terminal fragment, which is further cleaved during apoptosis. Although these cleavages preserve the entire kinase domain, p50 N-LOK displays no kinase activity. In apoptotic lymphocytes, caspase cleavages of LOK are concomitant with a decrease in ERM phosphorylation. When non-apoptotic lymphocytes from mice with homozygous and heterozygous LOK knockout were compared, the latter showed a higher level of ERM phosphorylation, but when apoptosis was induced, LOK(-/-) and LOK(+/-) lymphocytes showed the same low level, confirming in vivo that LOK-induced ERM phosphorylation is prevented during lymphocyte apoptosis. Our results demonstrate that cleavage of LOK during apoptosis abolishes its kinase activity, causing a decrease in ERM phosphorylation, crucial to the role of the ERM proteins in linking the plasma membrane to actin filaments. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Function of caspase-14 in trophoblast differentiation

    Directory of Open Access Journals (Sweden)

    Charles Adrian K

    2009-09-01

    Full Text Available Abstract Background Within the human placenta, the cytotrophoblast consists of a proliferative pool of progenitor cells which differentiate to replenish the overlying continuous, multi-nucleated syncytiotrophoblast, which forms the barrier between the maternal and fetal tissues. Disruption to trophoblast differentiation and function may result in impaired fetal development and preeclampsia. Caspase-14 expression is limited to barrier forming tissues. It promotes keratinocyte differentiation by cleaving profilaggrin to stabilise keratin intermediate filaments, and indirectly providing hydration and UV protection. However its role in the trophoblast remains unexplored. Methods Using RNA Interference the reaction of control and differentiating trophoblastic BeWo cells to suppressed caspase-14 was examined for genes pertaining to hormonal, cell cycle and cytoskeletal pathways. Results Transcription of hCG, KLF4 and cytokeratin-18 were increased following caspase-14 suppression suggesting a role for caspase-14 in inhibiting their pathways. Furthermore, hCG, KLF4 and cytokeratin-18 protein levels were disrupted. Conclusion Since expression of these molecules is normally increased with trophoblast differentiation, our results imply that caspase-14 inhibits trophoblast differentiation. This is the first functional study of this unusual member of the caspase family in the trophoblast, where it has a different function than in the epidermis. This knowledge of the molecular underpinnings of trophoblast differentiation may instruct future therapies of trophoblast disease.

  12. Region-specific proteolysis differentially regulates type 1 inositol 1,4,5-trisphosphate receptor activity.

    Science.gov (United States)

    Wang, Liwei; Wagner, Larry E; Alzayady, Kamil J; Yule, David I

    2017-07-14

    The inositol 1,4,5 trisphosphate receptor (IP 3 R) is an intracellular Ca 2+ release channel expressed predominately on the membranes of the endoplasmic reticulum. IP 3 R1 can be cleaved by caspase or calpain into at least two receptor fragments. However, the functional consequences of receptor fragmentation are poorly understood. Our previous work has demonstrated that IP 3 R1 channels, formed following either enzymatic fragmentation or expression of the corresponding complementary polypeptide chains, retain tetrameric architecture and are still activated by IP 3 binding despite the loss of peptide continuity. In this study, we demonstrate that region-specific receptor fragmentation modifies channel regulation. Specifically, the agonist-evoked temporal Ca 2+ release profile and protein kinase A modulation of Ca 2+ release are markedly altered. Moreover, we also demonstrate that activation of fragmented IP 3 R1 can result in a distinct functional outcome. Our work suggests that proteolysis of IP 3 R1 may represent a novel form of modulation of IP 3 R1 channel function and increases the repertoire of Ca 2+ signals achievable through this channel. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    Science.gov (United States)

    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.

  14. Regulation of higher-activity NARM wastes by EPA

    International Nuclear Information System (INIS)

    Bandrowski, M.S.

    1988-01-01

    The US Environmental Protection Agency (EPA) is currently developing standards for the disposal of low-level radioactive waste (LLW). As part of this Standard, EPA is including regulations for the disposal of naturally occurring and accelerator-produced radioactive material (NARM) wastes not covered under the Atomic Energy Act (AEA). The regulations will cover only higher-activity NARM wastes, defined as NARM waste with specific activity exceeding two nanocuries per gram. The proposed regulations will specify that NARM wastes exceeding the above limits, except for specific exempted items, must be disposed of in regulated radioactive waste disposal facilities. The proposed EPA regulations for NARM wastes will be discussed, as well as the costs and benefits of the regulation, how it will be implemented by EPA, and the rationale for covering only higher-activity NARM wastes exceeding two nanocuries per gram

  15. Caspase-12 and the inflammatory response to Yersinia pestis.

    NARCIS (Netherlands)

    Ferwerda, B.; McCall, M.B.B.; Vries, M.C. de; Hopman, J.C.W.; Maiga, B.; Dolo, A.; Doumbo, O.; Daou, M.; Jong, D.J. de; Joosten, L.A.B.; Tissingh, R.A.; Reubsaet, F.A.; Sauerwein, R.W.; Meer, J.W.M. van der; Ven, A.J.A.M. van der; Netea, M.G.

    2009-01-01

    BACKGROUND: 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

  16. IAPs: from caspase inhibitors to modulators of NF-kappaB, inflammation and cancer

    DEFF Research Database (Denmark)

    Gyrd-Hansen, Mads; Meier, Pascal

    2010-01-01

    . The development of such inhibitors has radically changed our knowledge of the signalling processes that are regulated by IAPs. Recent studies indicate that IAPs not only regulate caspases and apoptosis, but also modulate inflammatory signalling and immunity, mitogenic kinase signalling, proliferation and mitosis...

  17. A caspase-2-RFXANK interaction and its implication for MHC class II expression.

    Science.gov (United States)

    Forsberg, Jeremy; Li, Xinge; Akpinar, Birce; Salvatori, Roger; Ott, Martin; Zhivotovsky, Boris; Olsson, Magnus

    2018-01-23

    Despite recent achievements implicating caspase-2 in tumor suppression, the enzyme stands out from the apoptotic caspase family as a factor whose function requires further clarification. To specify enzyme characteristics through the definition of interacting proteins in apoptotic or non-apoptotic settings, a yeast 2-hybrid (Y2H) screen was performed using the full-length protein as bait. The current report describes the analysis of a captured prey and putative novel caspase-2 interacting factor, the regulatory factor X-associated ankyrin-containing protein (RFXANK), previously associated with CIITA, the transactivator regulating cell-type specificity and inducibility of MHC class II gene expression. The interaction between caspase-2 and RFXANK was verified by co-immunoprecipitations using both exogenous and endogenous proteins, where the latter approach suggested that binding of the components occurs in the cytoplasm. Cellular co-localization was confirmed by transfection of fluorescently conjugated proteins. Enhanced caspase-2 processing in RFXANK-overexpressing HEK293T cells treated with chemotherapeutic agents further supported Y2H data. Yet, no distinct differences with respect to MHC class II expression were observed in plasma membranes of antigen-presenting cells derived from wild type and caspase-2 -/- mice. In contrast, increased levels of the total MHC class II protein was evident in protein lysates from caspase-2 RNAi-silenced leukemia cell lines and B-cells isolated from gene-targeted mice. Together, these data identify a novel caspase-2-interacting factor, RFXANK, and indicate a potential non-apoptotic role for the enzyme in the control of MHC class II gene regulation.

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

    Science.gov (United States)

    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 .

  19. Modern aspects of tax regulation of investment activity

    Directory of Open Access Journals (Sweden)

    E.S. Podakov

    2016-03-01

    Full Text Available The article investigates the tax regulation of investment activity in modern conditions. Scientists studied different views about the impact of tax regulations on the investment activity in the country. The author determines that the tax regulation of investment activity involves the use of state mechanisms taxation of certain measures to improve investment conditions. The subject is the state tax regulations, and the object is the investment activity of individual and institutional investors of any form of ownership including organizational and legal forms. Such regulation is performed by using complex special tools. The possible methods of tax stimulation of investment processes are described. The article deals with the current results of tax reform in Ukraine and predicts its possible consequences for agricultural producers. The rating positions of Ukraine according to international organizations are showed. The systematic analysis has been carried out and the impact of differential tax rates, tax exemption for a specified period, reducing the tax base, elimination of double taxation on investment activity in certain areas have been researched. The special instruments of investment activity tax regulation are considered. The options for improving investment activity by introducing effective tax regulation are determined.

  20. Rhein triggers apoptosis via induction of endoplasmic reticulum stress, caspase-4 and intracellular calcium in primary human hepatic HL-7702 cells

    Energy Technology Data Exchange (ETDEWEB)

    KoraMagazi, Arouna [Department of Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu (China); Wang, Dandan [Department of Pharmacology, China Pharmaceutical University, Nanjing, Jiangsu (China); Yousef, Bashir; Guerram, Mounia [Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing, Jiangsu (China); Yu, Feng, E-mail: yufengcpu14@yahoo.com [Department of Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu (China); Department of Pharmacology, China Pharmaceutical University, Nanjing, Jiangsu (China); Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University, Nanjing, Jiangsu (China)

    2016-04-22

    Rhein is an active component of rhubarb; a traditional Chinese medicine reported to induce apoptosis and cause liver toxicity. However, rhein's apoptotic-inducing effects, as well as its molecular mechanisms of action on hepatic cells need to be further explored. In the present study, rhein was found to trigger apoptosis in primary human hepatic HL-7702 cells as showed by annexin V/PI double staining assay and nuclear morphological changes demonstrated by Hoechst 33258 staining. Moreover, it was observed that the mechanism implicated in rhein-induced apoptosis was caspase-dependent, presumably via ER-stress associated pathways, as illustrated by up-regulation of glucose-regulated protein 78 (GRP 78), PKR-like ER kinase (PERK), C-Jun N-terminal kinase (JNK) and CCAAT/enhancer-binding protein homologous protein (CHOP). Meanwhile, caspase-4 as a hallmark of ER-stress, was also showed to be activated following by caspase-3 activation. Furthermore, rhein also promoted intracellular elevation of calcium that contributed in apoptosis induction. Interestingly, pre-treatment with calpain inhibitor I reduced the effects of rhein on apoptosis induction and JNK activation. These data suggested that rhein-induced apoptosis through ER-stress and elevated intracellular calcium level in HL-7702 cells. - Highlights: • Rhein triggers apoptotic cell death on primary human hepatic HL-7702 cells. • Rhein leads to caspase-4 activation in HL-7702 cells. • Rhein induces endoplasmic reticulum stress pathways in HL-7702 cells. • Rhein causes elevation of intracellular calcium concentrations in HL-7702 cells.

  1. Caspase-Dependent Apoptosis Induced by Telomere Cleavage and TRF2 Loss

    Directory of Open Access Journals (Sweden)

    Asha S. Multani

    2000-07-01

    Full Text Available Chromosomal abnormalities involving telomeric associations (TAs often precede replicative senescence and abnormal chromosome configurations. We report here that telomere cleavage following exposure to proapoptotic agents is an early event in apoptosis. Exposure of human and murine cancer cells to a variety of pro-apoptotic stimuli (staurosporine, thapsigargin, anti-Fas antibody, cancer chemotherapeutic agents resulted in telomere cleavage and aggregation, finally their extrusion from the nuclei. Telomere loss was associated with arrest of cells in G2/M phase and preceded DNA fragmentation. Telomere erosion and subsequent large-scale chromatin cleavage were inhibited by overexpression of the anti -apoptotic protein, bcl-2, two peptide caspase inhibitors (BACMK and zVADfmk, indicating that both events are regulated by caspase activation. The results demonstrate that telomere cleavage is an early chromatin alteration detected in various cancer cell lines leading to drug-induced apoptosis, suggest that this event contributes to mitotic catastrophe and induction of cell death. Results also suggest that the decrease of telomeric-repeat binding factor 2 (TRF2 may be the earliest event in the ara-C-induced telomere shortening, induction of endoreduplication and chromosomal fragmentation leading to cell death.

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

    Science.gov (United States)

    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

  3. Caspase-2 associates with FAN through direct interaction and overlapping functionality.

    Science.gov (United States)

    Forsberg, Jeremy; Li, Xinge; Zamaraev, Aleksey V; Panaretakis, Theocharis; Zhivotovsky, Boris; Olsson, Magnus

    2018-05-23

    Caspase-2 has been implicated in diverse cellular processes, and the identification of factors with which it interacts has steadily increased. In the present study, we report a direct interaction between caspase-2 and factor associated with neutral sphingomyelinase activation (FAN) using yeast two-hybrid screening and co-immunoprecipitation. Further, stable suppression of caspase-2 expression in HEK293T and HeLa cells enabled a systematic investigation of putative novel enzyme functionalities, especially with respect to ceramide production, cell migration, IL-6 production and vesicular homeostasis, all of which have been previously reported to be associated with FAN. Lipidomics excluded the involvement of caspase-2 in the generation of ceramide species, but caspase-2-dependent deregulation of IL-6 release, vesicular size and delayed cell relocation supported an association between caspase-2 and FAN. Collectively, these data identify a novel caspase-2-interacting factor, FAN, and expand the role for the enzyme in seemingly non-apoptotic cellular mechanisms. Copyright © 2018 Elsevier Inc. All rights reserved.

  4. Commission for energy regulation - 2012 Activity Report

    International Nuclear Information System (INIS)

    2013-06-01

    After a presentation of the organisation, role and missions of the French Commission for Energy Regulation (CRE), and of its relationship with other institutional actors, this report describes and comments the action of the CRE in the fields of dialogue and transparency. It presents and comments key figures regarding the electricity and gas retail markets. It reports and comments the European reaction to the cold peak of February 2012 (historical peak for consumption and prices, inquiry on the causes of these price peaks, need of a European market). The next part addresses the relationship between electricity grids and territories (solidarity between electricity grids as the basis of the Europe of energy, evolution of French grids to face new needs and to take regional and local dimensions into account). Another part addresses gas infrastructures which are considered as the cornerstone of a good operation for the French market and for the integration of the European energy market (gas world market in 2012, definition of a target model for the gas market by European regulators, evolution of the French market in compliance with the European target model, new tariffs for the use of natural gas transport networks). The report then addresses the development of renewable energies: actions of CRE (bidding, opinion of tariffs), influence of renewable energy development on electricity prices on gross markets, needed evolution of electricity grids. A last part addresses the issues of energy cost, demand management, and struggle against energy poverty

  5. Synergism in regulation of nuclear and radiological activities

    International Nuclear Information System (INIS)

    Buzdugan, A.

    2009-01-01

    In 2006 the reform of nuclear activity regulation in Moldova was initiated. On May 11, 2006, the Parliament of the Republic of Moldova passed the law Nr 111-XVI 'About Safe Accomplishment of Nuclear and Radiological Activity'. On the 23rd of March, 2007 the National Agency for Regulation of Nuclear and Radiological Activities (NARNRA) was founded due to the decree of the Government under the Ministry of Ecology and Natural Resources. Its first objective was elaboration of necessary regulation documents in this field

  6. How is AMPK activity regulated in skeletal muscles during exercise?

    DEFF Research Database (Denmark)

    Jørgensen, Sebastian Beck; Rose, Adam John

    2008-01-01

    AMPK is a metabolic "master" controller activated in skeletal muscle by exercise in a time and intensity dependent manner, and has been implicated in regulating metabolic pathways in muscle during physical exercise. AMPK signaling in skeletal muscle is regulated by several systemic...... and intracellular factors and the regulation of skeletal muscle AMPK in response to exercise is the focus of this review. Specifically, the role of LKB1 and phosphatase PP2C in nucleotide-dependent activation of AMPK, and ionized calcium in CaMKK-dependent activation of AMPK in working muscle is discussed. We also...

  7. Self-regulation as a type of managerial activity.

    Directory of Open Access Journals (Sweden)

    Anna Algazina

    2017-01-01

    Full Text Available УДК 342.9The subject. In the context of the ongoing administrative reform in the Russian Federation the issue of self-regulation is becoming increasingly important.Introduction of Institute of self-regulation is intended to reduce the degree of state intervention in private spheres of professional activity, to eliminate excessive administrative barriers, reduce government expenditures on regulation and control in their respective areas of operation, which is especially important in the current economic conditions.However, in Russian legal science is no recognized definition of "self-regulation", but a unity of views on the question of the relationship between self-regulation and state regulation of business relations.In this regard, the author attempts to examine the concept of "self-regulation" through the prism of knowledge about public administration.The purpose of the article is to identify the essential features and to articulate the concept of self-regulation by comparing it with other varieties of regulation.Methodology. The methodological basis for the study: general scientific methods (analysis, synthesis, comparison, description; private and academic (interpretation, formal-legal.Results, scope. Based on the analysis allocated in the science of administrative law approaches to the system of public administration justifies the conclusion that the notion "regulation" is specific in relation to the generic concept of "management" and is a kind of management, consisting in the drafting of rules of conduct and sanctions for non-compliance or inadequate performance.In addition, the article highlights the problem of the genesis of self-regulation, building a system of principles of self-regulation, comparison of varieties of self-regulatory organizations among themselves.Conclusions. The comparison of self-regulation other types of regulation (such as state regulation and co-regulation highlighted the essential features of this phenomenon

  8. Endogenous Methanol Regulates Mammalian Gene Activity

    Science.gov (United States)

    Komarova, Tatiana V.; Petrunia, Igor V.; Shindyapina, Anastasia V.; Silachev, Denis N.; Sheshukova, Ekaterina V.; Kiryanov, Gleb I.; Dorokhov, Yuri L.

    2014-01-01

    We recently showed that methanol emitted by wounded plants might function as a signaling molecule for plant-to-plant and plant-to-animal communications. In mammals, methanol is considered a poison because the enzyme alcohol dehydrogenase (ADH) converts methanol into toxic formaldehyde. However, the detection of methanol in the blood and exhaled air of healthy volunteers suggests that methanol may be a chemical with specific functions rather than a metabolic waste product. Using a genome-wide analysis of the mouse brain, we demonstrated that an increase in blood methanol concentration led to a change in the accumulation of mRNAs from genes primarily involved in detoxification processes and regulation of the alcohol/aldehyde dehydrogenases gene cluster. To test the role of ADH in the maintenance of low methanol concentration in the plasma, we used the specific ADH inhibitor 4-methylpyrazole (4-MP) and showed that intraperitoneal administration of 4-MP resulted in a significant increase in the plasma methanol, ethanol and formaldehyde concentrations. Removal of the intestine significantly decreased the rate of methanol addition to the plasma and suggested that the gut flora may be involved in the endogenous production of methanol. ADH in the liver was identified as the main enzyme for metabolizing methanol because an increase in the methanol and ethanol contents in the liver homogenate was observed after 4-MP administration into the portal vein. Liver mRNA quantification showed changes in the accumulation of mRNAs from genes involved in cell signalling and detoxification processes. We hypothesized that endogenous methanol acts as a regulator of homeostasis by controlling the mRNA synthesis. PMID:24587296

  9. Dietary methanol regulates human gene activity.

    Directory of Open Access Journals (Sweden)

    Anastasia V Shindyapina

    Full Text Available Methanol (MeOH is considered to be a poison in humans because of the alcohol dehydrogenase (ADH-mediated conversion of MeOH to formaldehyde (FA, which is toxic. Our recent genome-wide analysis of the mouse brain demonstrated that an increase in endogenous MeOH after ADH inhibition led to a significant increase in the plasma MeOH concentration and a modification of mRNA synthesis. These findings suggest endogenous MeOH involvement in homeostasis regulation by controlling mRNA levels. Here, we demonstrate directly that study volunteers displayed increasing concentrations of MeOH and FA in their blood plasma when consuming citrus pectin, ethanol and red wine. A microarray analysis of white blood cells (WBC from volunteers after pectin intake showed various responses for 30 significantly differentially regulated mRNAs, most of which were somehow involved in the pathogenesis of Alzheimer's disease (AD. There was also a decreased synthesis of hemoglobin mRNA, HBA and HBB, the presence of which in WBC RNA was not a result of red blood cells contamination because erythrocyte-specific marker genes were not significantly expressed. A qRT-PCR analysis of volunteer WBCs after pectin and red wine intake confirmed the complicated relationship between the plasma MeOH content and the mRNA accumulation of both genes that were previously identified, namely, GAPDH and SNX27, and genes revealed in this study, including MME, SORL1, DDIT4, HBA and HBB. We hypothesized that human plasma MeOH has an impact on the WBC mRNA levels of genes involved in cell signaling.

  10. Endogenous methanol regulates mammalian gene activity.

    Directory of Open Access Journals (Sweden)

    Tatiana V Komarova

    Full Text Available We recently showed that methanol emitted by wounded plants might function as a signaling molecule for plant-to-plant and plant-to-animal communications. In mammals, methanol is considered a poison because the enzyme alcohol dehydrogenase (ADH converts methanol into toxic formaldehyde. However, the detection of methanol in the blood and exhaled air of healthy volunteers suggests that methanol may be a chemical with specific functions rather than a metabolic waste product. Using a genome-wide analysis of the mouse brain, we demonstrated that an increase in blood methanol concentration led to a change in the accumulation of mRNAs from genes primarily involved in detoxification processes and regulation of the alcohol/aldehyde dehydrogenases gene cluster. To test the role of ADH in the maintenance of low methanol concentration in the plasma, we used the specific ADH inhibitor 4-methylpyrazole (4-MP and showed that intraperitoneal administration of 4-MP resulted in a significant increase in the plasma methanol, ethanol and formaldehyde concentrations. Removal of the intestine significantly decreased the rate of methanol addition to the plasma and suggested that the gut flora may be involved in the endogenous production of methanol. ADH in the liver was identified as the main enzyme for metabolizing methanol because an increase in the methanol and ethanol contents in the liver homogenate was observed after 4-MP administration into the portal vein. Liver mRNA quantification showed changes in the accumulation of mRNAs from genes involved in cell signalling and detoxification processes. We hypothesized that endogenous methanol acts as a regulator of homeostasis by controlling the mRNA synthesis.

  11. Caspase-10 Is the Key Initiator Caspase Involved in Tributyltin-Mediated Apoptosis in Human Immune Cells

    Directory of Open Access Journals (Sweden)

    Harald F. Krug

    2012-01-01

    Full Text Available Tributyltin (TBT is one of the most toxic compounds produced by man and distributed in the environment. A multitude of toxic activities have been described, for example, immunotoxic, neurotoxic, and endocrine disruptive effects. Moreover, it has been shown for many cell types that they undergo apoptosis after treatment with TBT and the cell death of immune cells could be the molecular background of its immunotoxic effect. As low as 200 nM up to 1 μM of TBT induces all signs of apoptosis in Jurkat T cells within 1 to 24 hrs of treatment. When compared to Fas-ligand control stimulation, the same sequence of events occurs: membrane blebbing, phosphatidylserine externalisation, the activation of the “death-inducing signalling complex,” and the following sequence of cleavage processes. In genetically modified caspase-8-deficient Jurkat cells, the apoptotic effects are only slightly reduced, whereas, in FADD-negative Jurkat cells, the TBT effect is significantly diminished. We could show that caspase-10 is recruited by the TRAIL-R2 receptor and apoptosis is totally prevented when caspase-10 is specifically inhibited in all three cell lines.

  12. Cordycepin enhances cisplatin apoptotic effect through caspase/MAPK pathways in human head and neck tumor cells

    Directory of Open Access Journals (Sweden)

    Chen YH

    2013-07-01

    -Jun NH2-terminal kinase, extracellular signal-regulated kinase, and p38 protein phosphorylations. Moreover, cordycepin plus cisplatin cotreatment significantly activated those proteins with much better effects among three cell lines. Conclusion: Cordycepin plus cisplatin have better apoptotic effect by activating caspase activation with possible MAPK pathway involvement in HNSCC cells. Keywords: cordycepin, cisplatin, apoptosis, caspase, MAPK, HNSCC

  13. Regulation of MDM2 Activity by Nucleolin

    Science.gov (United States)

    2007-06-01

    plicated in the binding of HIV particles to CD4 cells. J. Biol. Chem. 273:21988–21997. 8. Carty, M. P., M. Zernik -Kobak, S. McGrath, and K. Dixon. 1994...replication products and not due to repair synthesis (Fig. 3D ). RPARPA2D is therefore functionally active in supporting DNA replication in vitro...Further intermediate RPA2 mutants were designed to roughly follow the phosphor- ylation pathway, as suggested by the data of Zernik -Kobak and colleagues

  14. Expression levels of cleaved caspase-3 and caspase-3 in tumorigenesis and prognosis of oral tongue squamous cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Pei-Feng Liu

    Full Text Available Apoptosis plays a dual role in cancer development and malignancy. The role of apoptosis-related caspases in cancer remains controversial, particularly in oral tongue squamous cell carcinoma (OTSCC. In this study, we examined the protein levels of cleaved caspase-3, caspase-3, caspase-8, and caspase-9 on tissue microarrays consisting of samples from 246 OTSCC patients by immunohistochemistry. Wilcoxon signed-rank test indicated that the protein levels of cleaved caspase-3, caspase-3, caspase-8, and caspase-9 in tumor tissues were significantly higher compared to those in adjacent normal tissues (all p<0.001. The expression level of caspase-8 in tumors was elevated in patients with lymph node invasion. Moreover, positive expression of cleaved caspase-3 was associated with shorter disease-free survival (DFS in OTSCC patients with moderate differentiation and lymph node invasion. Combination of either positive cleaved caspase-3 or higher caspase-3 expression or both was associated with poor DFS. Interestingly, stratification analysis showed that co-expression levels of positive cleaved caspase-3 or/and higher caspase-3 were associated with better disease-specific survival in patients with advanced stages of the disease, such as large tumor size and lymph node invasion, whereas it was associated with poor DFS in OTSCC patients with moderate cell differentiation and small tumor size. Taken together, cleaved caspase-3 and caspase-3/8/9 could be biomarkers for tumorigenesis in OTSCC patients. The co-expression level of cleaved caspase-3 and caspase-3 might be a prognostic biomarker for OTSCC patients, particular in those patients with certain tumor stages and cell differentiation status.

  15. Regulation of Intrinsic and Extrinsic Apoptotic Pathways in Osteosarcoma Cells Following Oleandrin Treatment.

    Science.gov (United States)

    Ma, Yunlong; Zhu, Bin; Yong, Lei; Song, Chunyu; Liu, Xiao; Yu, Huilei; Wang, Peng; Liu, Zhongjun; Liu, Xiaoguang

    2016-11-23

    Our previous study has reported the anti-tumor effect of oleandrin on osteosarcoma (OS) cells. In the current study, we mainly explored its potential regulation on intrinsic and extrinsic apoptotic pathway in OS cells. Cells apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were detected using fluorescence staining and flow cytometry. Caspase-3 activity was detected using a commercial kit. The levels of cytoplasmic cytochrome c, mitochondrial cytochrome c, bcl-2, bax, caspase-9, Fas, FasL, caspase-8 and caspase-3 were detected by Western blotting. z-VAD-fmk was applied to block both intrinsic and extrinsic apoptosis pathways, and cells apoptosis was also tested. Furthermore, we used z-LEHD-fmk and Fas blocking antibody to inhibit intrinsic and extrinsic pathways, separately, and the selectivity of oleandrin on these pathways was explored. Results showed that oleandrin induced the apoptosis of OS cells, which was accompanied by an increase in ROS and a decrease in MMP. Furthermore, cytochrome c level was reduced in mitochondria but elevated in the cytoplasm. Caspase-3 activity was enhanced by oleandrin in a concentration- and time-dependent manner. Oleandrin also down-regulated the expression of bcl-2, but up-regulated bax, caspase-9, Fas, FasL, caspase-8 and caspase-3. In addition, the suppression of both apoptotic pathways by z-VAD-fmk greatly reverted the oleandrin-induced apoptosis. Moreover, the suppression of one pathway by a corresponding inhibitor did not affect the regulation of oleandrin on another pathway. Taken together, we concluded that oleandrin induced apoptosis of OS cells via activating both intrinsic and extrinsic apoptotic pathways.

  16. P53-dependent ceramide generation in response ro ionizing irradiation is caspase-dependent

    International Nuclear Information System (INIS)

    Dbaibo, G.; El-Assaad, W.

    2000-01-01

    Full text.We have previously reported that p53-dependent apoptosis is accompanied by ceramide accumulation. Lack of p53 prevents ceramide accumulation in response to induces such as ionizing irradiation. The mechanisms of ceramide accumulation have not been explored. P53 has been reported to function by inducing the death receptors Fas and DR5 both of which function by initiating a caspase cascade that results in apoptosis. We decided to examine the role of caspases in the elevation of cellular ceramide levels. We treated Molt-4 cells with 5Gy of ionizing irradiation and examined the effects of co-treatment with the general caspase inhibitor z-VAD-fmk at concentration of 50 and 100μM. We found that z-VAD blocked apoptosis induced by irradiation without interfering with p53 accumulation indicating that it was not functioning upstream of p53. However, z-VAD treatment resulted in a significant decrease in ceramide accumulation. Additionally, z-VAD partially blocked the loss of glutathione in response to irradiation. This was important since glutathione has been described as an inhibitor of neutral sphindomyelinase, a major source of cellular ceramide via sphingomyelin hydrolysis. These studies indicate that p53 induces ceramide accumulation in a caspase-dependent manner and that the regulation of cellular glutathione by caspases may be a mechanism by which they regulate ceramide accumulation

  17. Active Power Regulation based on Droop for AC Microgrid

    DEFF Research Database (Denmark)

    Li, Chendan; Coelho, Ernane A. A.; Firoozabadi, Mehdi Savaghebi

    2015-01-01

    In this paper, two different control strategies are proposed to address the active power regulation issue in AC microgrids. The principle of power regulation in the droop controller is firstly introduced. Frequency scheduling and droop gain scheduling on top of droop control is proposed...... to successfully follow the active power command. The limitation of each method is discussed in term of small signal stability and light load sharing, respectively. Discussion on the effects of power command is also given. The simulation is carried out for both the strategies to verify the active power control...

  18. Flavopiridol induces apoptosis in glioma cell lines independent of retinoblastoma and p53 tumor suppressor pathway alterations by a caspase-independent pathway.

    Science.gov (United States)

    Alonso, Michelle; Tamasdan, Cristina; Miller, Douglas C; Newcomb, Elizabeth W

    2003-02-01

    Flavopiridol is a synthetic flavone, which inhibits growth in vitro and in vivo of several solid malignancies such as renal, prostate, and colon cancers. It is a potent cyclin-dependent kinase inhibitor presently in clinical trials. In this study, we examined the effect of flavopiridol on a panel of glioma cell lines having different genetic profiles: five of six have codeletion of p16(INK4a) and p14(ARF); three of six have p53 mutations; and one of six shows overexpression of mouse double minute-2 (MDM2) protein. Independent of retinoblastoma and p53 tumor suppressor pathway alterations, flavopiridol induced apoptosis in all cell lines but through a caspase-independent mechanism. No cleavage products for caspase 3 or its substrate poly(ADP-ribose) polymerase or caspase 8 were detected. The pan-caspase inhibitor Z-VAD-fmk did not inhibit flavopiridol-induced apoptosis. Mitochondrial damage measured by cytochrome c release and transmission electron microscopy was not observed in drug-treated glioma cells. In contrast, flavopiridol treatment induced translocation of apoptosis-inducing factor from the mitochondria to the nucleus. The proteins cyclin D(1) and MDM2 involved in the regulation of retinoblastoma and p53 activity, respectively, were down-regulated early after flavopiridol treatment. Given that MDM2 protein can confer oncogenic properties under certain circumstances, loss of MDM2 expression in tumor cells could promote increased chemosensitivity. After drug treatment, a low Bcl-2/Bax ratio was observed, a condition that may favor apoptosis. Taken together, the data indicate that flavopiridol has activity against glioma cell lines in vitro and should be considered for clinical development in the treatment of glioblastoma multiforme.

  19. Regulated necrosis and its implications in toxicology.

    Science.gov (United States)

    Aki, Toshihiko; Funakoshi, Takeshi; Uemura, Koichi

    2015-07-03

    Recent research developments have revealed that caspase-dependent apoptosis is not the sole form of regulated cell death. Caspase-independent, but genetically regulated, forms of cell death include pyroptosis, necroptosis, parthanatos, and the recently discovered ferroptosis and autosis. Importantly, regulated necrosis can be modulated by small molecule inhibitors/activators, confirming the cell autonomous mechanism of these forms of cell death. The success of small molecule-mediated manipulation of regulated necrosis has produced great changes in the field of cell death research, and has also brought about significant changes in the fields of pharmacology as well as toxicology. In this review, we intend to summarize the modes of regulated cell death other than apoptosis, and discuss their implications in toxicology. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  20. Developmental regulation of aromatase activity in the rat hypothalamus

    International Nuclear Information System (INIS)

    Lephart, E.D.

    1989-01-01

    The brain of all mammalian species studied thus far contain an enzymatic activity (aromatase) that catalyzes the conversion of androgens to estrogens. The activity is highest during prenatal development and contributes to the establishment of sex differences which determine adult gonadotropin secretion patterns and reproductive behavior. The studies presented in this dissertation represent a systematic effort to elucidate the mechanism(s) that control the initiation of and contribute to maintaining rat hypothalamic aromatase activity during pre- and postnatal development. Aromatase enzyme activity was measured by the 3 H 2 O release assay or by traditional estrogen product isolation. Brain aromatase mRNA was detected by hybridization to a cDNA encoding rat aromatase cytochrome P-450. In both males and females the time of puberty was associated with a decline in hypothalamic aromatase activity. This decline may represent a factor underlying the peri-pubertal decrease in the sensitivity to gonadal steroid feedback that accompanies completion of puberty. The results also indicate that androgens regulate brain aromatase levels during both the prepubertal and peri-pubertal stages of sexual development and that this regulation is transiently lost in young adults. Utilizing a hypothalamic organotypic culture system, aromatase activity in vitro was maintained for as long as two days. The results of studies of a variety of hormonal and metabolic regulators suggest that prenatal aromatase activity is regulated by factor(s) that function independently from the classical cyclic AMP and protein kinase C trans-membrane signaling pathways

  1. Activity-regulated genes as mediators of neural circuit plasticity.

    Science.gov (United States)

    Leslie, Jennifer H; Nedivi, Elly

    2011-08-01

    Modifications of neuronal circuits allow the brain to adapt and change with experience. This plasticity manifests during development and throughout life, and can be remarkably long lasting. Evidence has linked activity-regulated gene expression to the long-term structural and electrophysiological adaptations that take place during developmental critical periods, learning and memory, and alterations to sensory map representations in the adult. In all these cases, the cellular response to neuronal activity integrates multiple tightly coordinated mechanisms to precisely orchestrate long-lasting, functional and structural changes in brain circuits. Experience-dependent plasticity is triggered when neuronal excitation activates cellular signaling pathways from the synapse to the nucleus that initiate new programs of gene expression. The protein products of activity-regulated genes then work via a diverse array of cellular mechanisms to modify neuronal functional properties. Synaptic strengthening or weakening can reweight existing circuit connections, while structural changes including synapse addition and elimination create new connections. Posttranscriptional regulatory mechanisms, often also dependent on activity, further modulate activity-regulated gene transcript and protein function. Thus, activity-regulated genes implement varied forms of structural and functional plasticity to fine-tune brain circuit wiring. Copyright © 2011 Elsevier Ltd. All rights reserved.

  2. Caspase-2-dependent dendritic cell death, maturation, and priming of T cells in response to Brucella abortus infection.

    Directory of Open Access Journals (Sweden)

    Xinna Li

    Full Text Available Smooth virulent Brucella abortus strain 2308 (S2308 causes zoonotic brucellosis in cattle and humans. Rough B. abortus strain RB51, derived from S2308, is a live attenuated cattle vaccine strain licensed in the USA and many other countries. Our previous report indicated that RB51, but not S2308, induces a caspase-2-dependent apoptotic and necrotic macrophage cell death. Dendritic cells (DCs are professional antigen presenting cells critical for bridging innate and adaptive immune responses. In contrast to Brucella-infected macrophages, here we report that S2308 induced higher levels of apoptotic and necrotic cell death in wild type bone marrow-derived DCs (WT BMDCs than RB51. The RB51 and S2308-induced BMDC cell death was regulated by caspase-2, indicated by the minimal cell death in RB51 and S2308-infected BMDCs isolated from caspase-2 knockout mice (Casp2KO BMDCs. More S2308 bacteria were taken up by Casp2KO BMDCs than wild type BMDCs. Higher levels of S2308 and RB51 cells were found in infected Casp2KO BMDCs compared to infected WT BMDCs at different time points. RB51-infected wild type BMDCs were mature and activated as shown by significantly up-regulated expression of CD40, CD80, CD86, MHC-I, and MHC-II. RB51 induced the production of cytokines TNF-α, IL-6, IFN-γ and IL12/IL23p40 in infected BMDCs. RB51-infected WT BMDCs also stimulated the proliferation of CD4(+ and CD8(+ T cells compared to uninfected WT BMDCs. However, the maturation, activation, and cytokine secretion are significantly impaired in Casp2KO BMDCs infected with RB51 or Salmonella (control. S2308-infected WT and Casp2KO BMDCs were not activated and could not induce cytokine production. These results demonstrated that virulent smooth strain S2308 induced more apoptotic and necrotic dendritic cell death than live attenuated rough vaccine strain RB51; however, RB51, but not its parent strain S2308, induced caspase-2-mediated DC maturation, cytokine production, antigen

  3. Nascent histamine induces α-synuclein and caspase-3 on human cells

    Energy Technology Data Exchange (ETDEWEB)

    Caro-Astorga, Joaquín; Fajardo, Ignacio; Ruiz-Pérez, María Victoria; Sánchez-Jiménez, Francisca; Urdiales, José Luis, E-mail: jlurdial@uma.es

    2014-09-05

    Highlights: • Nascent histamine alters cyclin expression pattern. • Nascent histamine increases expression of α-synuclein. • Nascent histamine activates caspase-3. - Abstract: Histamine (Hia) is the most multifunctional biogenic amine. It is synthetized by histidine decarboxylase (HDC) in a reduced set of mammalian cell types. Mast cells and histaminergic neurons store Hia in specialized organelles until the amine is extruded by exocytosis; however, other immune and cancer cells are able to produce but not store Hia. The intracellular effects of Hia are still not well characterized, in spite of its physiopathological relevance. Multiple functional relationships exist among Hia metabolism/signaling elements and those of other biogenic amines, including growth-related polyamines. Previously, we obtained the first insights for an inhibitory effect of newly synthetized Hia on both growth-related polyamine biosynthesis and cell cycle progression of non-fully differentiated mammalian cells. In this work, we describe progress in this line. HEK293 cells were transfected to express active and inactive versions of GFP-human HDC fusion proteins and, after cell sorting by flow cytometry, the relative expression of a large number of proteins associated with cell signaling were measured using an antibody microarray. Experimental results were analyzed in terms of protein–protein and functional interaction networks. Expression of active HDC induced a cell cycle arrest through the alteration of the levels of several proteins such as cyclin D1, cdk6, cdk7 and cyclin A. Regulation of α-synuclein and caspase-3 was also observed. The analyses provide new clues on the molecular mechanisms underlying the regulatory effects of intracellular newly synthetized Hia on cell proliferation/survival, cell trafficking and protein turnover. This information is especially interesting for emergent and orphan immune and neuroinflammatory diseases.

  4. Post-translational regulation of Oct4 transcriptional activity.

    Directory of Open Access Journals (Sweden)

    Jonathan P Saxe

    Full Text Available Oct4 is a key component of the molecular circuitry which regulates embryonic stem cell proliferation and differentiation. It is essential for maintenance of undifferentiated, pluripotent cell populations, and accomplishes these tasks by binding DNA in multiple heterodimer and homodimer configurations. Very little is known about how formation of these complexes is regulated, or the mechanisms through which Oct4 proteins respond to complex extracellular stimuli which regulate pluripotency. Here, we provide evidence for a phosphorylation-based mechanism which regulates specific Oct4 homodimer conformations. Point mutations of a putative phosphorylation site can specifically abrogate transcriptional activity of a specific homodimer assembly, with little effect on other configurations. Moreover, we performed bioinformatic predictions to identify a subset of Oct4 target genes which may be regulated by this specific assembly, and show that altering Oct4 protein levels affects transcription of Oct4 target genes which are regulated by this assembly but not others. Finally, we identified several signaling pathways which may mediate this phosphorylation and act in combination to regulate Oct4 transcriptional activity and protein stability. These results provide a mechanism for rapid and reversible alteration of Oct4 transactivation potential in response to extracellular signals.

  5. Triglyceride-induced macrophage cell death is triggered by caspase-1.

    Science.gov (United States)

    Son, Sin Jee; Rhee, Ki-Jong; Lim, Jaewon; Kim, Tae Ue; Kim, Tack-Joong; Kim, Yoon Suk

    2013-01-01

    Triglyceride (TG) induces macrophage cell death which contributes to the development of atherosclerosis. We confirmed that exogenous TG accumulates in human THP-1 macrophages and causes cell death. TG treated THP-1 macrophages exhibited no change in tumor necrosis factor (TNF)-α, interleukin (IL)-18, macrophage inflammatory protein (MIP)-1α, and IL-1R1 receptor mRNA expression. However, there was a marked decrease in IL-1β mRNA expression but an increase in IL-1β protein secretion. Decreased expression of IL-1β mRNA and increased secretion of IL-1β protein was not the direct cause of cell death. Until now, TG was assumed to induce necrotic cell death in macrophages. Since caspase-1 is known to be involved in activation and secretion of IL-1β protein and pyroptotic cell death, next we determined whether caspase-1 is associated with TG-induced macrophage cell death. We found an increase in caspase-1 activity in TG-treated THP-1 macrophages and inhibition of caspase-1 activity using a specific inhibitor partially rescued cell death. These results suggest activation of the pyroptotic pathway by TG. This is the first report implicating the activation of caspase-1 and the triggering of the pyroptosis pathway in TG-induced macrophage cell death.

  6. Ofloxacin induces apoptosis in microencapsulated juvenile rabbit chondrocytes by caspase-8-dependent mitochondrial pathway

    International Nuclear Information System (INIS)

    Sheng Zhiguo; Cao Xiaojuan; Peng Shuangqing; Wang Changyong; Li Qianqian; Wang Yimei; Liu Mifeng

    2008-01-01

    Quinolones (QNs)-induced arthropathy is an important toxic effect in immature animals leading to restriction of their therapeutic use in pediatrics. However, the exact mechanism still remains unclear. Recently, we have demonstrated that ofloxacin, a typical QN, induces apoptosis of alginate microencapsulated juvenile rabbit joint chondrocytes by disturbing the β 1 integrin functions and inactivating the ERK/MAPK signaling pathway. In this study, we extend our initial observations to further elucidate the mechanism(s) of ofloxacin-induced apoptosis by utilizing specific caspase inhibitors. Pretreatment with both caspase-9-specific inhibitor zLEHD-fmk and caspase-8 inhibitor zIETD-fmk attenuated ofloxacin-induced apoptosis and activation of caspase-3 of chondrocyte in a concentration-dependent manner, as determined by fluorescent dye staining, enzyme activity assay and immunoblotting. Furthermore, the activation of caspase-9, -8 and -3 stimulated by ofloxacin was significantly inhibited in the presence of zIETD-fmk while pretreatment with zLEHD-fmk only blocked the activation of caspase-9 and -3. Ofloxacin also stimulated a concentration-dependent translocation of cytochrome c from mitochondria into the cytosol and a decrease of mitochondrial transmembrane potential, which was completely inhibited by zIETD-fmk. In addition, ofloxacin was found to increase the level of Bax, tBid, p53 in a concentration- and time-dependent manner. Taken together, The current results indicate that the caspase-8-dependent mitochondrial pathway is primarily involved in the ofloxacin-induced apoptosis of microencapsulated juvenile rabbit joint chondrocytes

  7. High Ca2+ Influx During Traumatic Brain Injury Leads to Caspase-1-Dependent Neuroinflammation and Cell Death.

    Science.gov (United States)

    Abdul-Muneer, P M; Long, Mathew; Conte, Adriano Andrea; Santhakumar, Vijayalakshmi; Pfister, Bryan J

    2017-08-01

    We investigated the hypothesis that high Ca 2+ influx during traumatic brain injury induces the activation of the caspase-1 enzyme, which triggers neuroinflammation and cell apoptosis in a cell culture model of neuronal stretch injury and an in vivo model of fluid percussion injury (FPI). We first established that stretch injury causes a rapid increase in the intracellular Ca 2+ level, which activates interleukin-converting enzyme caspase-1. The increase in the intracellular Ca 2+ level and subsequent caspase-1 activation culminates into neuroinflammation via the maturation of IL-1β. Further, we analyzed caspase-1-mediated apoptosis by TUNEL staining and PARP western blotting. The voltage-gated sodium channel blocker, tetrodotoxin, mitigated the stretch injury-induced neuroinflammation and subsequent apoptosis by blocking Ca 2+ influx during the injury. The effect of tetrodotoxin was similar to the caspase-1 inhibitor, zYVAD-fmk, in neuronal culture. To validate the in vitro results, we demonstrated an increase in caspase-1 activity, neuroinflammation and neurodegeneration in fluid percussion-injured animals. Our data suggest that neuronal injury/traumatic brain injury (TBI) can induce a high influx of Ca 2+ to the cells that cause neuroinflammation and cell death by activating caspase-1, IL-1β, and intrinsic apoptotic pathways. We conclude that excess IL-1β production and cell death may contribute to neuronal dysfunction and cognitive impairment associated with TBI.

  8. Caspase-12 and the inflammatory response to Yersinia pestis.

    Science.gov (United States)

    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.

  9. Identification of two novel activities of the Wnt signaling regulator Dickkopf 3 and characterization of its expression in the mouse retina

    Directory of Open Access Journals (Sweden)

    Yi Hyun

    2007-12-01

    Full Text Available Abstract Background The Wnt signaling pathway is a cellular communication pathway that plays critical roles in development and disease. A major class of Wnt signaling regulators is the Dickkopf (Dkk family of secreted glycoproteins. Although the biological properties of Dickkopf 1 (Dkk1 and Dickkopf 2 (Dkk2 are well characterized, little is known about the function of the related Dickkopf 3 (Dkk3 protein in vivo or in cell lines. We recently demonstrated that Dkk3 transcripts are upregulated during photoreceptor death in a mouse model of retinal degeneration. In this study, we characterized the activity of Dkk3 in Wnt signaling and cell death. Results Dkk3 was localized to Müller glia and retinal ganglion cells in developing and adult mouse retina. Western blotting confirmed that Dkk3 is secreted from Müller glia cells in culture. We demonstrated that Dkk3 potentiated Wnt signaling in Müller glia and HEK293 cells but not in COS7 cells, indicating that it is a cell-type specific regulator of Wnt signaling. This unique Dkk3 activity was blocked by co-expression of Dkk1. Additionally, Dkk3 displayed pro-survival properties by decreasing caspase activation and increasing viability in HEK293 cells exposed to staurosporine and H2O2. In contrast, Dkk3 did not protect COS7 cells from apoptosis. Conclusion These data demonstrate that Dkk3 is a positive regulator of Wnt signaling, in contrast to its family member Dkk1. Furthermore, Dkk3 protects against apoptosis by reducing caspase activity, suggesting that Dkk3 may play a cytoprotective role in the retina.

  10. The Hepatitis C Virus-induced NLRP3 Inflammasome Activates the Sterol Regulatory Element-binding Protein (SREBP) and Regulates Lipid Metabolism.

    Science.gov (United States)

    McRae, Steven; Iqbal, Jawed; Sarkar-Dutta, Mehuli; Lane, Samantha; Nagaraj, Abhiram; Ali, Naushad; Waris, Gulam

    2016-02-12

    Hepatitis C virus (HCV) relies on host lipids and lipid droplets for replication and morphogenesis. The accumulation of lipid droplets in infected hepatocytes manifests as hepatosteatosis, a common pathology observed in chronic hepatitis C patients. One way by which HCV promotes the accumulation of intracellular lipids is through enhancing de novo lipogenesis by activating the sterol regulatory element-binding proteins (SREBPs). In general, activation of SREBPs occurs during cholesterol depletion. Interestingly, during HCV infection, the activation of SREBPs occurs under normal cholesterol levels, but the underlying mechanisms are still elusive. Our previous study has demonstrated the activation of the inflammasome complex in HCV-infected human hepatoma cells. In this study, we elucidate the potential link between chronic hepatitis C-associated inflammation and alteration of lipid homeostasis in infected cells. Our results reveal that the HCV-activated NLRP3 inflammasome is required for the up-regulation of lipogenic genes such as 3-hydroxy-3-methylglutaryl-coenzyme A synthase, fatty acid synthase, and stearoyl-CoA desaturase. Using pharmacological inhibitors and siRNA against the inflammasome components (NLRP3, apoptosis-associated speck-like protein containing a CARD, and caspase-1), we further show that the activation of the NLRP3 inflammasome plays a critical role in lipid droplet formation. NLRP3 inflammasome activation in HCV-infected cells enables caspase-1-mediated degradation of insulin-induced gene proteins. This subsequently leads to the transport of the SREBP cleavage-activating protein·SREBP complex from the endoplasmic reticulum to the Golgi, followed by proteolytic activation of SREBPs by S1P and S2P in the Golgi. Typically, inflammasome activation leads to viral clearance. Paradoxically, here we demonstrate how HCV exploits the NLRP3 inflammasome to activate SREBPs and host lipid metabolism, leading to liver disease pathogenesis associated with

  11. Activation of ion transport systems during cell volume regulation

    International Nuclear Information System (INIS)

    Eveloff, J.L.; Warnock, D.G.

    1987-01-01

    This review discusses the activation of transport pathways during volume regulation, including their characteristics, the possible biochemical pathways that may mediate the activation of transport pathways, and the relations between volume regulation and transepithelial transport in renal cells. Many cells regulate their volume when exposed to an anisotonic medium. The changes in cell volume are caused by activation of ion transport pathways, plus the accompanying osmotically driven water movement such that cell volume returns toward normal levels. The swelling of hypertonically shrunken cells is termed regulatory volume increase (RVI) and involves an influx of NaCl into the cell via either activation of Na-Cl, Na-K-2Cl cotransport systems, or Na + -H + and Cl - -HCO 3 - exchangers. The reshrinking of hypotonically swollen cells is termed regulatory volume decrease (RVD) and involves an efflux of KCl and water from the cell by activation of either separate K + and Cl - conductances, a K-Cl cotransport system, or parallel K + -H + and Cl - -HCO 3 - exchangers. The biochemical mechanisms involved in the activation of transport systems are largely unknown, however, the phosphoinositide pathway may be implicated in RVI; phorbol esters, cGMP, and Ca 2+ affect the process of volume regulation. Renal tubular cells, as well as the blood cells that transverse the medulla, are subjected to increasing osmotic gradients from the corticomedullary junction to the papillary tip, as well as changing interstitial and tubule fluid osmolarity, depending on the diuretic state of the animal. Medullary cells from the loop of Henle and the papilla can volume regulate by activating Na-K-2Cl cotransport or Na + -H + and Cl - -HCO 3 - exchange systems

  12. Energy Regulation Commission. Activity report. 1 July - 31 December 2008

    International Nuclear Information System (INIS)

    2009-01-01

    After a description of the scope of activities, organisation and operation of the CRE (Commission de Regulation de l'Energie, Energy regulation commission) and of the CorDIS (Comite de reglement des differents et des sanctions de la CRE, CRE's Committee for settlements of controversies and sanctions), this report outlines the importance of the grid manager independence and of the regulation reinforcement for the building up of a domestic energy market. It discusses the role of the regulation authority in the interconnection of European grids, their operation security and supply security, but also in pricing and in investments. It highlights the relationship between the reduction of carbon emission, energy demand management, strengthening of electric grids, financial incentives, and advanced metering systems. It describes how the CRE ensures a good operation of electricity and natural gas markets

  13. Caspase-1 from Human Myeloid-Derived Suppressor Cells Can Promote T Cell-Independent Tumor Proliferation.

    Science.gov (United States)

    Zeng, Qi; Fu, Juan; Korrer, Michael; Gorbounov, Mikhail; Murray, Peter J; Pardoll, Drew; Masica, David L; Kim, Young J

    2018-05-01

    Immunosuppressive myeloid-derived suppressive cells (MDSCs) are characterized by their phenotypic and functional heterogeneity. To better define their T cell-independent functions within the tumor, sorted monocytic CD14 + CD11b + HLA-DR low/- MDSCs (mMDSC) from squamous cell carcinoma patients showed upregulated caspase-1 activity, which was associated with increased IL1β and IL18 expression. In vitro studies demonstrated that mMDSCs promoted caspase-1-dependent proliferation of multiple squamous carcinoma cell lines in both human and murine systems. In vivo , growth rates of B16, MOC1, and Panc02 were significantly blunted in chimeric mice adoptively transferred with caspase-1 null bone marrow cells under T cell-depleted conditions. Adoptive transfer of wild-type Gr-1 + CD11b + MDSCs from tumor-bearing mice reversed this antitumor response, whereas caspase-1 inhibiting thalidomide-treated MDSCs phenocopied the antitumor response found in caspase-1 null mice. We further hypothesized that MDSC caspase-1 activity could promote tumor-intrinsic MyD88-dependent carcinogenesis. In mice with wild-type caspase-1, MyD88-silenced tumors displayed reduced growth rate, but in chimeric mice with caspase-1 null bone marrow cells, MyD88-silenced tumors did not display differential tumor growth rate. When we queried the TCGA database, we found that caspase-1 expression is correlated with overall survival in squamous cell carcinoma patients. Taken together, our findings demonstrated that caspase-1 in MDSCs is a direct T cell-independent mediator of tumor proliferation. Cancer Immunol Res; 6(5); 566-77. ©2018 AACR . ©2018 American Association for Cancer Research.

  14. 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.

    Directory of Open Access Journals (Sweden)

    Philip B Busbee

    Full Text Available 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 (40 mg/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.

  15. Active Inference, homeostatic regulation and adaptive behavioural control.

    Science.gov (United States)

    Pezzulo, Giovanni; Rigoli, Francesco; Friston, Karl

    2015-11-01

    We review a theory of homeostatic regulation and adaptive behavioural control within the Active Inference framework. Our aim is to connect two research streams that are usually considered independently; namely, Active Inference and associative learning theories of animal behaviour. The former uses a probabilistic (Bayesian) formulation of perception and action, while the latter calls on multiple (Pavlovian, habitual, goal-directed) processes for homeostatic and behavioural control. We offer a synthesis these classical processes and cast them as successive hierarchical contextualisations of sensorimotor constructs, using the generative models that underpin Active Inference. This dissolves any apparent mechanistic distinction between the optimization processes that mediate classical control or learning. Furthermore, we generalize the scope of Active Inference by emphasizing interoceptive inference and homeostatic regulation. The ensuing homeostatic (or allostatic) perspective provides an intuitive explanation for how priors act as drives or goals to enslave action, and emphasises the embodied nature of inference. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  16. THE EUROPEAN MODEL OF STATE REGULATION OF TOURISM ACTIVITIES

    Directory of Open Access Journals (Sweden)

    О. Davydova

    2013-11-01

    Full Text Available In the article the existing model of state regulation of the development of tourism. Expediency of the European model of state regulation of tourism development in Ukraine. It is noted that the European model of state regulation of tourism activities based on the coordination of marketing activities and the development of cooperation between the public and private sectors. The basic forms of public-private partnerships and the advantages of using cluster model of development of tourism, namely, contracts, production sharing agreement, lease, joint venture. Promising areas of application of the PPP identified the transport sector, housing and utilities, energy and tourism sector. The features of cluster formations in the country and the prospects for tourism clusters.

  17. Commission for Energy regulation (CRE) - Activity report June 2004

    International Nuclear Information System (INIS)

    2004-01-01

    CRE is the French commission for energy regulation. CRE's remit is to assist in ensuring the proper operation of the electricity and natural gas markets for the benefit of the end-user. In particular, CRE ensures that the conditions of access to electricity and natural gas transmission and distribution systems do not hinder the development of competition. It monitors, for the electricity and natural gas sectors, all transactions made between suppliers, traders and producers, all transactions made on the organised markets and cross-border trading. It ensures that suppliers, traders and producers propose offers that are consistent with their financial and technical constraints. It monitors the implementation of and compliance with regulations giving consumers the right to choose their supplier in a competitive market, and allowing new suppliers to enter the market. This document is the 2004 activity report of CRE. Content: A - Opening of the gas and electricity markets for professional customers on 1 July 2004; B - Regulation of the gas market: Gas markets and players (The European environment, The French gas market); Regulation of the gas market (Implementing regulation, Works planned for the coming year; C - Regulation of the electricity market: The electricity markets and players (The European electricity markets, The French electricity market, Monitoring the electricity market); Regulation of the French electricity market (Access to public grid, Cross-border exchanges, Un-bundled accounting principles); The public electricity service in the regulated market (Content of the public service, Public service charges, Electricity production public service financing, Electricity sales tariffs) D - The working of CRE: How CRE exercises its jurisdiction, Tools; E - Appendices: Glossary, Units and conversions, Council of European Energy Regulators, Index of tables and figures

  18. Commission for Energy regulation (CRE) - Activity report June 2004

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2004-07-01

    CRE is the French commission for energy regulation. CRE's remit is to assist in ensuring the proper operation of the electricity and natural gas markets for the benefit of the end-user. In particular, CRE ensures that the conditions of access to electricity and natural gas transmission and distribution systems do not hinder the development of competition. It monitors, for the electricity and natural gas sectors, all transactions made between suppliers, traders and producers, all transactions made on the organised markets and cross-border trading. It ensures that suppliers, traders and producers propose offers that are consistent with their financial and technical constraints. It monitors the implementation of and compliance with regulations giving consumers the right to choose their supplier in a competitive market, and allowing new suppliers to enter the market. This document is the 2004 activity report of CRE. Content: A - Opening of the gas and electricity markets for professional customers on 1 July 2004; B - Regulation of the gas market: Gas markets and players (The European environment, The French gas market); Regulation of the gas market (Implementing regulation, Works planned for the coming year; C - Regulation of the electricity market: The electricity markets and players (The European electricity markets, The French electricity market, Monitoring the electricity market); Regulation of the French electricity market (Access to public grid, Cross-border exchanges, Un-bundled accounting principles); The public electricity service in the regulated market (Content of the public service, Public service charges, Electricity production public service financing, Electricity sales tariffs) D - The working of CRE: How CRE exercises its jurisdiction, Tools; E - Appendices: Glossary, Units and conversions, Council of European Energy Regulators, Index of tables and figures.

  19. Regulations on environmental data for the petroleum activity

    International Nuclear Information System (INIS)

    1990-01-01

    The publication deals with the regulations on environmental data for the petroleum activity, stipulated by the Norwegian Petroleum Directorate on 1 December 1989 pursuant to Royal Decree of 28 June 1985, cf. Sections 7 and 33, cf. delegation of authority by the Ministry of Local Government and Labour of 28 June 1985. 1 tab

  20. [Immune regulation activity and mechanism of Tibetan Kefir exopolysaccharide fractions].

    Science.gov (United States)

    Meng, Li; Zhang, Lanwei

    2009-12-01

    To investigate the effects and mechanism on immune regulation activity in mice of two Tibetan Kefir exoploysaccharides (EPS) with different molecular weight of 0.1 x 10(5) - 3 x 10(5) (fraction 1) and 1.8 x 10(3) (fraction 2). The immune regulation activity experiment was carried out in vitro based on the Functional Assessment Procedure and Test Methods of Health Food, which was issued by Ministry of Health of China. First, we treated mice subjects with EPS at doses of 40 mg/kg, 80 mg/kg, 120 mg/kg through ig. Then we detected the index of immune organs, the ability of antibody production (tested by HC50), activity of NK cell, delayed type hypersensitivity (DTH) and phagocytosis of macrophage in mice. Finally, we examined the expression of Erk protein in Macrophages by Western Blot assay. Fraction 1 could promote HC50, activity of NK cell and DTH in mice which low dose showed better. Fraction 2 could promote DTH, phagocytosis of macrophage which high dose showed better. The expression of Erk and COX-2 had the same trend with Phagocytic index. We verified the two fractions of Tibetan Kefir EPS could enhance immune functions in mice. Fraction 1 regulated immune function through NK cell and B cell while fraction 2 through macrophage cell and T cell. The effects to macrophage of Tibetan Kefir EPS in mice may realize through extra cellular signal-regulated kinase Erk pathway.

  1. Commission for Energy regulation (CRE) - Activity report june 2008

    International Nuclear Information System (INIS)

    2008-01-01

    CRE is the French commission for energy regulation. CRE's remit is to assist in ensuring the proper operation of the electricity and natural gas markets for the benefit of the end-user. In particular, CRE ensures that the conditions of access to electricity and natural gas transmission and distribution systems do not hinder the development of competition. It monitors, for the electricity and natural gas sectors, all transactions made between suppliers, traders and producers, all transactions made on the organised markets and cross-border trading. It ensures that suppliers, traders and producers propose offers that are consistent with their financial and technical constraints. It monitors the implementation of and compliance with regulations giving consumers the right to choose their supplier in a competitive market, and allowing new suppliers to enter the market. This document is the 2008 activity report of CRE. Content: A - How CRE works: CRE regulatory authority and organisation: Powers, Organisation; Budget resources; Personnel; B - The Standing Committee for Dispute Settlement and Sanctions (CoRDiS) activity: Admissibility, Authority; C - Building a single European energy market: Overview; Organisation and coordination of the main European regulators (Work carried out collectively by European regulators, Regulator organisation and development, CRE's relations with European Community institutions, Development of CEER activities outside the European Union); CRE's European activities (The contribution of European regulators to the Third Energy Package, Integration of gas markets, Integration of electricity markets, Operation of the European interconnected electricity grid and security of supply, Opening up markets to benefit consumers); European Community activities (The European Commission's proposals for the internal energy market: the Third Energy Package, The European Commission's proposals for fighting climate change: the Climate Package, Infringement

  2. RNA silencing of Mcl-1 enhances ABT-737-mediated apoptosis in melanoma: role for a caspase-8-dependent pathway.

    Science.gov (United States)

    Keuling, Angela M; Felton, Kathleen E A; Parker, Arabesque A M; Akbari, Majid; Andrew, Susan E; Tron, Victor A

    2009-08-17

    Malignant melanoma is resistant to almost all conventional forms of chemotherapy. Recent evidence suggests that anti-apoptotic proteins of the Bcl-2 family are overexpressed in melanoma and may contribute to melanoma's striking resistance to apoptosis. ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-xl and Bcl-w, has demonstrated efficacy in several forms of leukemia, lymphoma as well as solid tumors. However, overexpression of Mcl-1, a frequent observance in melanoma, is known to confer ABT-737 resistance. Here we report that knockdown of Mcl-1 greatly reduces cell viability in combination with ABT-737 in six different melanoma cell lines. We demonstrate that the cytotoxic effect of this combination treatment is due to apoptotic cell death involving not only caspase-9 activation but also activation of caspase-8, caspase-10 and Bid, which are normally associated with the extrinsic pathway of apoptosis. Caspase-8 (and caspase-10) activation is abrogated by inhibition of caspase-9 but not by inhibitors of the death receptor pathways. Furthermore, while caspase-8/-10 activity is required for the full induction of cell death with treatment, the death receptor pathways are not. Finally, we demonstrate that basal levels of caspase-8 and Bid correlate with treatment sensitivity. Our findings suggest that the combination of ABT-737 and Mcl-1 knockdown represents a promising, new treatment strategy for malignant melanoma. We also report a death receptor-independent role for extrinsic pathway proteins in treatment response and suggest that caspase-8 and Bid may represent potential markers of treatment sensitivity.

  3. RNA silencing of Mcl-1 enhances ABT-737-mediated apoptosis in melanoma: role for a caspase-8-dependent pathway.

    Directory of Open Access Journals (Sweden)

    Angela M Keuling

    Full Text Available BACKGROUND: Malignant melanoma is resistant to almost all conventional forms of chemotherapy. Recent evidence suggests that anti-apoptotic proteins of the Bcl-2 family are overexpressed in melanoma and may contribute to melanoma's striking resistance to apoptosis. ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-xl and Bcl-w, has demonstrated efficacy in several forms of leukemia, lymphoma as well as solid tumors. However, overexpression of Mcl-1, a frequent observance in melanoma, is known to confer ABT-737 resistance. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that knockdown of Mcl-1 greatly reduces cell viability in combination with ABT-737 in six different melanoma cell lines. We demonstrate that the cytotoxic effect of this combination treatment is due to apoptotic cell death involving not only caspase-9 activation but also activation of caspase-8, caspase-10 and Bid, which are normally associated with the extrinsic pathway of apoptosis. Caspase-8 (and caspase-10 activation is abrogated by inhibition of caspase-9 but not by inhibitors of the death receptor pathways. Furthermore, while caspase-8/-10 activity is required for the full induction of cell death with treatment, the death receptor pathways are not. Finally, we demonstrate that basal levels of caspase-8 and Bid correlate with treatment sensitivity. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that the combination of ABT-737 and Mcl-1 knockdown represents a promising, new treatment strategy for malignant melanoma. We also report a death receptor-independent role for extrinsic pathway proteins in treatment response and suggest that caspase-8 and Bid may represent potential markers of treatment sensitivity.

  4. Paraoxon induces apoptosis in EL4 cells via activation of mitochondrial pathways.

    Science.gov (United States)

    Saleh, A M; Vijayasarathy, C; Masoud, L; Kumar, L; Shahin, A; Kambal, A

    2003-07-01

    The toxicity of organophosphorus compounds, such as paraoxon (POX), is due to their anticholinesterase action. Recently, we have shown that, at noncholinergic doses (1 to 10 nM), POX (the bioactive metabolite of parathion) causes apoptotic cell death in murine EL4 T-lymphocytic leukemia cell line through activation of caspase-3. In this study, by employing caspase-specific inhibitors, we extend our observations to elucidate the sequence of events involved in POX-stimulated apoptosis. Pretreatment of EL4 cells with the caspase-9-specific inhibitor zLEHD-fmk attenuated POX-induced apoptosis in a dose-dependent manner, whereas the caspase-8 inhibitor zIETD-fmk had no effect. Furthermore, the activation of caspase-9, -8, and -3 in response to POX treatment was completely inhibited in the presence of zLEHD-fmk, implicating the involvement of caspase 9-dependent mitochondrial pathways in POX-stimulated apoptosis. Indeed, under both in vitro and in vivo conditions, POX triggered a dose- and time-dependent translocation of cytochrome c from mitochondria into the cytosol, as assessed by Western blot analysis. Investigation of the mechanism of cytochrome c release revealed that POX disrupted mitochondrial transmembrane potential. Neither this effect nor cytchrome c release was dependent on caspase activation, since the general inhibitor of the caspase family zVAD-fmk did not influence both processes. Finally, POX treatment also resulted in a time-dependent up-regulation and translocation of the proapoptotic molecule Bax to mitochondria. Inhibition of this event by zVAD-fmk suggests that the activation and translocation of Bax to mitochondria is subsequent to activation of the caspase cascades. The results indicate that POX induces apoptosis in EL4 cells through a direct effect on mitochondria by disrupting its transmembrane potential, causing the release of cytochrome c into the cytosol and subsequent activation of caspase-9. Inhibition of this specific pathway might provide

  5. Tetraspanin CD9 regulates osteoclastogenesis via regulation of p44/42 MAPK activity

    International Nuclear Information System (INIS)

    Yi, TacGhee; Kim, Hye-Jin; Cho, Je-Yoel; Woo, Kyung Mi; Ryoo, Hyun-Mo; Kim, Gwan-Shik; Baek, Jeong-Hwa

    2006-01-01

    Tetraspanin CD9 has been shown to regulate cell-cell fusion in sperm-egg fusion and myotube formation. However, the role of CD9 in osteoclast, another multinucleated cell type, is not still clear. Therefore, we investigated the role of CD9 in osteoclast differentiation. CD9 was expressed in osteoclast lineage cells and its expression level increased during the progression of RANKL-induced osteoclastogenesis. KMC8, a neutralizing antibody specific to CD9, significantly suppressed RANKL-induced multinucleated osteoclast formation and the mRNA expression of osteoclast differentiation marker genes. To define CD9-regulated osteoclastogenic signaling pathway, MAPK pathways were examined. KMC8 induced long-term phosphorylation of p44/42 MAPK, but not of p38 MAPK. Constitutive activation of p44/42 MAPK by overexpressing constitutive-active mutant of MEK1 almost completely blocked osteoclast differentiation. Taken together, these results suggest that CD9 expressed on osteoclast lineage cells might positively regulate osteoclastogenesis via the regulation of p44/42 MAPK activity

  6. HAMLET triggers apoptosis but tumor cell death is independent of caspases, Bcl-2 and p53.

    Science.gov (United States)

    Hallgren, O; Gustafsson, L; Irjala, H; Selivanova, G; Orrenius, S; Svanborg, C

    2006-02-01

    HAMLET (Human alpha-lactalbumin Made Lethal to Tumor cells) triggers selective tumor cell death in vitro and limits tumor progression in vivo. Dying cells show features of apoptosis but it is not clear if the apoptotic response explains tumor cell death. This study examined the contribution of apoptosis to cell death in response to HAMLET. Apoptotic changes like caspase activation, phosphatidyl serine externalization, chromatin condensation were detected in HAMLET-treated tumor cells, but caspase inhibition or Bcl-2 over-expression did not prolong cell survival and the caspase response was Bcl-2 independent. HAMLET translocates to the nuclei and binds directly to chromatin, but the death response was unrelated to the p53 status of the tumor cells. p53 deletions or gain of function mutations did not influence the HAMLET sensitivity of tumor cells. Chromatin condensation was partly caspase dependent, but apoptosis-like marginalization of chromatin was also observed. The results show that tumor cell death in response to HAMLET is independent of caspases, p53 and Bcl-2 even though HAMLET activates an apoptotic response. The use of other cell death pathways allows HAMLET to successfully circumvent fundamental anti-apoptotic strategies that are present in many tumor cells.

  7. Staurosporine induces necroptotic cell death under caspase-compromised conditions in U937 cells.

    Directory of Open Access Journals (Sweden)

    Zsuzsanna A Dunai

    Full Text Available 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-ribosepolymerase (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.

  8. Regulation of p53, nuclear factor κB and cyclooxygenase-2 expression by bromelain through targeting mitogen-activated protein kinase pathway in mouse skin

    International Nuclear Information System (INIS)

    Kalra, Neetu; Bhui, Kulpreet; Roy, Preeti; Srivastava, Smita; George, Jasmine; Prasad, Sahdeo; Shukla, Yogeshwer

    2008-01-01

    Bromelain is a pharmacologically active compound, present in stems and immature fruits of pineapples (Ananas cosmosus), which has been shown to have anti-edematous, anti-inflammatory, anti-thrombotic and anti-metastatic properties. In the present study, antitumorigenic activity of bromelain was recorded in 7,12-dimethylbenz(a)anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted 2-stage mouse skin model. Results showed that bromelain application delayed the onset of tumorigenesis and reduced the cumulative number of tumors, tumor volume and the average number of tumors/mouse. To establish a cause and effect relationship, we targeted the proteins involved in the cell death pathway. Bromelain treatment resulted in upregulation of p53 and Bax and subsequent activation of caspase 3 and caspase 9 with concomitant decrease in antiapoptotic protein Bcl-2 in mouse skin. Since persistent induction of cyclooxygenase-2 (Cox-2) is frequently implicated in tumorigenesis and is regulated by nuclear factor-kappa B (NF-κB), we also investigated the effect of bromelain on Cox-2 and NF-κB expression. Results showed that bromelain application significantly inhibited Cox-2 and inactivated NF-κB by blocking phosphorylation and subsequent degradation of IκBα. In addition, bromelain treatment attenuated DMBA-TPA-induced phosphorylation of extracellular signal-regulated protein kinase (ERK1/2), mitogen-activated protein kinase (MAPK) and Akt. Taken together, we conclude that bromelain induces apoptosis-related proteins along with inhibition of NF-κB-driven Cox-2 expression by blocking the MAPK and Akt/protein kinase B signaling in DMBA-TPA-induced mouse skin tumors, which may account for its anti-tumorigenic effects

  9. Ras activation by SOS: Allosteric regulation by altered fluctuation dynamics

    Science.gov (United States)

    Iversen, Lars; Tu, Hsiung-Lin; Lin, Wan-Chen; Christensen, Sune M.; Abel, Steven M.; Iwig, Jeff; Wu, Hung-Jen; Gureasko, Jodi; Rhodes, Christopher; Petit, Rebecca S.; Hansen, Scott D.; Thill, Peter; Yu, Cheng-Han; Stamou, Dimitrios; Chakraborty, Arup K.; Kuriyan, John; Groves, Jay T.

    2014-01-01

    Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras–guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average. PMID:24994643

  10. Measuring T cell-mediated cytotoxicity using fluorogenic caspase substrates.

    Science.gov (United States)

    Chahroudi, A; Silvestri, G; Feinberg, M B

    2003-10-01

    Cytotoxic T lymphocytes (CTLs) play a major role in the immune response against viruses and other intracellular pathogens. In addition, CTLs are implicated in the control of tumor cells in certain settings. Accurate measures of CTL function are of critical importance to study the pathogenesis of infectious diseases and to evaluate the efficacy of new vaccines and immunotherapies. To this end, we have recently developed a flow cytometry-based CTL (FCC) assay that measures the CTL-induced caspase activation within target cells using cell permeable fluorogenic caspase substrates. This novel assay reliably detects, by flow cytometry or fluorescence/confocal microscopy, antigen-specific CTLs in a wide variety of human and murine systems, and is safer and more informative than the standard 51Cr-release assay. In addition, the flow cytometric CTL (FCC) assay provides an alternative method that is often more sensitive and physiologically informative when compared to previously described FCC assays, as it measures a biological indicator of apoptosis within the target cell. The FCC assay may thus represent a useful tool to further understand the molecular and cellular mechanisms that underlie CTL-mediated killing during tumorigenesis or following infection with viruses or other intracellular pathogens.

  11. Commission for Energy regulation (CRE) - Activity report June 2007

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2007-07-01

    CRE is the French commission for energy regulation. CRE's remit is to assist in ensuring the proper operation of the electricity and natural gas markets for the benefit of the end-user. In particular, CRE ensures that the conditions of access to electricity and natural gas transmission and distribution systems do not hinder the development of competition. It monitors, for the electricity and natural gas sectors, all transactions made between suppliers, traders and producers, all transactions made on the organised markets and cross-border trading. It ensures that suppliers, traders and producers propose offers that are consistent with their financial and technical constraints. It monitors the implementation of and compliance with regulations giving consumers the right to choose their supplier in a competitive market, and allowing new suppliers to enter the market. This document is the 2007 activity report of CRE. Content: A - Towards a single European energy market: Birth of a single European energy market (Origins of Europe of Energy, Emergence of a European energy policy); Main European Community guiding lines (European governance as regards energy, Guiding principles for the internal energy market); European Community activities (European Commission reports, Electricity and gas Regional Initiatives); Organisation and coordination of European regulators (Joint organisation of European regulators, CRE's relations with European Community institutions); CRE's European activities (Regional integration of gas markets, Regional integration of electricity markets, Operation of the European interconnected electricity grid and security of supply, CRE's other European activities); B - CRE action at national level: Grids/networks and infrastructures (General information, Electricity grids, Regulation of gas networks and infrastructures); Markets (Changes in the regulatory and legislative contexts of electricity and natural gas markets, Electricity markets

  12. Commission for Energy regulation (CRE) - Activity report June 2007

    International Nuclear Information System (INIS)

    2007-01-01

    CRE is the French commission for energy regulation. CRE's remit is to assist in ensuring the proper operation of the electricity and natural gas markets for the benefit of the end-user. In particular, CRE ensures that the conditions of access to electricity and natural gas transmission and distribution systems do not hinder the development of competition. It monitors, for the electricity and natural gas sectors, all transactions made between suppliers, traders and producers, all transactions made on the organised markets and cross-border trading. It ensures that suppliers, traders and producers propose offers that are consistent with their financial and technical constraints. It monitors the implementation of and compliance with regulations giving consumers the right to choose their supplier in a competitive market, and allowing new suppliers to enter the market. This document is the 2007 activity report of CRE. Content: A - Towards a single European energy market: Birth of a single European energy market (Origins of Europe of Energy, Emergence of a European energy policy); Main European Community guiding lines (European governance as regards energy, Guiding principles for the internal energy market); European Community activities (European Commission reports, Electricity and gas Regional Initiatives); Organisation and coordination of European regulators (Joint organisation of European regulators, CRE's relations with European Community institutions); CRE's European activities (Regional integration of gas markets, Regional integration of electricity markets, Operation of the European interconnected electricity grid and security of supply, CRE's other European activities); B - CRE action at national level: Grids/networks and infrastructures (General information, Electricity grids, Regulation of gas networks and infrastructures); Markets (Changes in the regulatory and legislative contexts of electricity and natural gas markets, Electricity markets, Natural gas

  13. Commission for Energy regulation (CRE) - Activity report June 2005

    International Nuclear Information System (INIS)

    2005-01-01

    CRE is the French commission for energy regulation. CRE's remit is to assist in ensuring the proper operation of the electricity and natural gas markets for the benefit of the end-user. In particular, CRE ensures that the conditions of access to electricity and natural gas transmission and distribution systems do not hinder the development of competition. It monitors, for the electricity and natural gas sectors, all transactions made between suppliers, traders and producers, all transactions made on the organised markets and cross-border trading. It ensures that suppliers, traders and producers propose offers that are consistent with their financial and technical constraints. It monitors the implementation of and compliance with regulations giving consumers the right to choose their supplier in a competitive market, and allowing new suppliers to enter the market. This document is the 2005 activity report of CRE. Content: A - The opening of the markets in France and in Europe: The opening of the markets one year after 1 July 2004 (An especially important step, Electricity and gas: a common framework with structural differences, The coexistence of market prices and regulated tariffs); The European texts of 26 June 2003 (Texts to give new impetus, Texts to harmonize the role and powers of national regulators, Texts to guarantee the independence of system operators, Texts to ensure transparent and non-discriminatory access to networks, Texts providing for strengthening of interconnections); The outlook for 2007, a fully open market (1 July 2007: a date set by the directives, Priority given to informing and protecting consumers); B - Regulation of the natural gas market: The gas market in the European context (Europe's dependency on imports is increasing, Gas prices increased considerably across the whole of Europe in 2004, The European gas scene continues to be dominated by a small number of players, Gas infrastructures need to be developed in Europe, The new European

  14. TAF15 and the leukemia-associated fusion protein TAF15-CIZ/NMP4 are cleaved by caspases-3 and -7

    Energy Technology Data Exchange (ETDEWEB)

    Alves, Juliano, E-mail: jalves@gnf.org [Department of Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037 (United States); Wurdak, Heiko [Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037 (United States); Garay-Malpartida, Humberto M. [Department of Pharmacology, Institute of Biomedical Sciences, University of Sao Paulo, Av. Lineu Prestes 1524, Sao Paulo, SP, CEP 05508-900 (Brazil); Harris, Jennifer L. [Department of Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037 (United States); Protease Biochemistry, Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121 (United States); Occhiucci, Joao M.; Belizario, Jose E. [Department of Pharmacology, Institute of Biomedical Sciences, University of Sao Paulo, Av. Lineu Prestes 1524, Sao Paulo, SP, CEP 05508-900 (Brazil); Li, Jun, E-mail: jli2@gnf.org [Protease Biochemistry, Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121 (United States)

    2009-07-10

    Caspases are central players in proteolytic pathways that regulate cellular processes such as apoptosis and differentiation. To accelerate the discovery of novel caspase substrates we developed a method combining in silico screening and in vitro validation. With this approach, we identified TAF15 as a novel caspase substrate in a trial study. We find that TAF15 was specifically cleaved by caspases-3 and -7. Site-directed mutagenesis revealed the consensus sequence {sup 106}DQPD/Y{sup 110} as the only site recognized by these caspases. Surprisingly, TAF15 was cleaved at more than one site in staurosporine-treated Jurkat cells. In addition, we generated two oncogenic TAF15-CIZ/NMP4-fused proteins which have been found in acute myeloid leukemia and demonstrate that caspases-3 and -7 cleave the fusion proteins at one single site. Broad application of this combination approach should expedite identification of novel caspase-interacting proteins and provide new insights into the regulation of caspase pathways leading to cell death in normal and cancer cells.

  15. Summary of regulation applicable to the gamma-graphy activities

    International Nuclear Information System (INIS)

    2004-04-01

    The regulations relative to the gamma radiography activities concerns in one hand the radioactive sources, and on the other hand the radiation protection of the workers. The necessity of having a person competent in radiation protection and the tasks he or she has to do are described. The training of operators, the medical surveillance, the dosimetry are such obligations. The maintenance of equipment is an other obligation. The last point concerns the transport of radioactive materials. (N.C.)

  16. Sphingosine-1-Phosphate Is a Novel Regulator of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR Activity.

    Directory of Open Access Journals (Sweden)

    Firhan A Malik

    Full Text Available The cystic fibrosis transmembrane conductance regulator (CFTR attenuates sphingosine-1-phosphate (S1P signaling in resistance arteries and has emerged as a prominent regulator of myogenic vasoconstriction. This investigation demonstrates that S1P inhibits CFTR activity via adenosine monophosphate-activated kinase (AMPK, establishing a potential feedback link. In Baby Hamster Kidney (BHK cells expressing wild-type human CFTR, S1P (1μmol/L attenuates forskolin-stimulated, CFTR-dependent iodide efflux. S1P's inhibitory effect is rapid (within 30 seconds, transient and correlates with CFTR serine residue 737 (S737 phosphorylation. Both S1P receptor antagonism (4μmol/L VPC 23019 and AMPK inhibition (80μmol/L Compound C or AMPK siRNA attenuate S1P-stimluated (i AMPK phosphorylation, (ii CFTR S737 phosphorylation and (iii CFTR activity inhibition. In BHK cells expressing the ΔF508 CFTR mutant (CFTRΔF508, the most common mutation causing cystic fibrosis, both S1P receptor antagonism and AMPK inhibition enhance CFTR activity, without instigating discernable correction. In summary, we demonstrate that S1P/AMPK signaling transiently attenuates CFTR activity. Since our previous work positions CFTR as a negative S1P signaling regulator, this signaling link may positively reinforce S1P signals. This discovery has clinical ramifications for the treatment of disease states associated with enhanced S1P signaling and/or deficient CFTR activity (e.g. cystic fibrosis, heart failure. S1P receptor/AMPK inhibition could synergistically enhance the efficacy of therapeutic strategies aiming to correct aberrant CFTR trafficking.

  17. After the Slippery Slope: Dutch Experiences on Regulating Active Euthanasia

    OpenAIRE

    Boer, Th.A.

    2003-01-01

    “When a country legalizes active euthanasia, it puts itself on a slippery slope from where it may well go further downward.” If true, this is a forceful argument in the battle of those who try to prevent euthanasia from becoming legal. The force of any slippery-slope argument, however, is by definition limited by its reference to future developments which cannot empirically be sustained. Experience in the Netherlands—where a law regulating active euthanasia was accepted in April 2001—may shed...

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

    Science.gov (United States)

    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.

  19. The inhibition of human T cell proliferation by the caspase inhibitor z-VAD-FMK is mediated through oxidative stress

    Energy Technology Data Exchange (ETDEWEB)

    Rajah, T.; Chow, S.C., E-mail: chow.sek.chuen@monash.edu

    2014-07-15

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