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Sample records for caspase degradome prediction

  1. Pripper: prediction of caspase cleavage sites from whole proteomes

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    Salmi Jussi

    2010-06-01

    Full Text Available Abstract Background Caspases are a family of proteases that have central functions in programmed cell death (apoptosis and inflammation. Caspases mediate their effects through aspartate-specific cleavage of their target proteins, and at present almost 400 caspase substrates are known. There are several methods developed to predict caspase cleavage sites from individual proteins, but currently none of them can be used to predict caspase cleavage sites from multiple proteins or entire proteomes, or to use several classifiers in combination. The possibility to create a database from predicted caspase cleavage products for the whole genome could significantly aid in identifying novel caspase targets from tandem mass spectrometry based proteomic experiments. Results Three different pattern recognition classifiers were developed for predicting caspase cleavage sites from protein sequences. Evaluation of the classifiers with quality measures indicated that all of the three classifiers performed well in predicting caspase cleavage sites, and when combining different classifiers the accuracy increased further. A new tool, Pripper, was developed to utilize the classifiers and predict the caspase cut sites from an arbitrary number of input sequences. A database was constructed with the developed tool, and it was used to identify caspase target proteins from tandem mass spectrometry data from two different proteomic experiments. Both known caspase cleavage products as well as novel cleavage products were identified using the database demonstrating the usefulness of the tool. Pripper is not restricted to predicting only caspase cut sites, but it gives the possibility to scan protein sequences for any given motif(s and predict cut sites once a suitable cut site prediction model for any other protease has been developed. Pripper is freely available and can be downloaded from http://users.utu.fi/mijopi/Pripper. Conclusions We have developed Pripper, a tool for

  2. SVM-based prediction of caspase substrate cleavage sites

    OpenAIRE

    Wee, Lawrence JK; Tan, Tin Wee; Ranganathan, Shoba

    2006-01-01

    Background Caspases belong to a class of cysteine proteases which function as critical effectors in apoptosis and inflammation by cleaving substrates immediately after unique sites. Prediction of such cleavage sites will complement structural and functional studies on substrates cleavage as well as discovery of new substrates. Recently, different computational methods have been developed to predict the cleavage sites of caspase substrates with varying degrees of success. As the support vector...

  3. Caspase cleavage sites in the human proteome: CaspDB, a database of predicted substrates.

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    Sonu Kumar

    Full Text Available Caspases are enzymes belonging to a conserved family of cysteine-dependent aspartic-specific proteases that are involved in vital cellular processes and play a prominent role in apoptosis and inflammation. Determining all relevant protein substrates of caspases remains a challenging task. Over 1500 caspase substrates have been discovered in the human proteome according to published data and new substrates are discovered on a daily basis. To aid the discovery process we developed a caspase cleavage prediction method using the recently published curated MerCASBA database of experimentally determined caspase substrates and a Random Forest classification method. On both internal and external test sets, the ranking of predicted cleavage positions is superior to all previously developed prediction methods. The in silico predicted caspase cleavage positions in human proteins are available from a relational database: CaspDB. Our database provides information about potential cleavage sites in a verified set of all human proteins collected in Uniprot and their orthologs, allowing for tracing of cleavage motif conservation. It also provides information about the positions of disease-annotated single nucleotide polymorphisms, and posttranslational modifications that may modulate the caspase cleaving efficiency.

  4. Caspase cleavage sites in the human proteome: CaspDB, a database of predicted substrates.

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    Kumar, Sonu; van Raam, Bram J; Salvesen, Guy S; Cieplak, Piotr

    2014-01-01

    Caspases are enzymes belonging to a conserved family of cysteine-dependent aspartic-specific proteases that are involved in vital cellular processes and play a prominent role in apoptosis and inflammation. Determining all relevant protein substrates of caspases remains a challenging task. Over 1500 caspase substrates have been discovered in the human proteome according to published data and new substrates are discovered on a daily basis. To aid the discovery process we developed a caspase cleavage prediction method using the recently published curated MerCASBA database of experimentally determined caspase substrates and a Random Forest classification method. On both internal and external test sets, the ranking of predicted cleavage positions is superior to all previously developed prediction methods. The in silico predicted caspase cleavage positions in human proteins are available from a relational database: CaspDB. Our database provides information about potential cleavage sites in a verified set of all human proteins collected in Uniprot and their orthologs, allowing for tracing of cleavage motif conservation. It also provides information about the positions of disease-annotated single nucleotide polymorphisms, and posttranslational modifications that may modulate the caspase cleaving efficiency. PMID:25330111

  5. An Efficient Piecewise Linear Model for Predicting Activity of Caspase-3 Inhibitors

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    Alireza Foroumadi

    2012-09-01

    Full Text Available Background and purpose of the study Multimodal distribution of descriptors makes it more difficult to fit a single global model to model the entire data set in quantitative structure activity relationship (QSAR studies.Methods:The linear (Multiple linear regression; MLR, non-linear (Artificial neural network; ANN, and an approach based on "Extended Classifier System in Function approximation" (XCSF were applied herein to model the biological activity of 658 caspase-3 inhibitors. Results:Various kinds of molecular descriptors were calculated to represent the molecular structures of the compounds. The original data set was partitioned into the training and test sets by the K-means classification method. Prediction error on the test data set indicated that the XCSF as a local model estimates caspase-3 inhibition activity, better than the global models such as MLR and ANN. The atom-centered fragment type CR2X2, electronegativity, polarizability, and atomic radius and also the lipophilicity of the molecule, were the main independent factors contributing to the caspase-3 inhibition activity. Conclusions:The results of this study may be exploited for further design of novel caspase-3 inhibitors.

  6. An efficient piecewise linear model for predicting activity of caspase-3 inhibitors

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    Firoozpour Loghman

    2012-09-01

    Full Text Available Abstract Background and purpose of the study Multimodal distribution of descriptors makes it more difficult to fit a single global model to model the entire data set in quantitative structure activity relationship (QSAR studies. Methods The linear (Multiple linear regression; MLR, non-linear (Artificial neural network; ANN, and an approach based on “Extended Classifier System in Function approximation” (XCSF were applied herein to model the biological activity of 658 caspase-3 inhibitors. Results Various kinds of molecular descriptors were calculated to represent the molecular structures of the compounds. The original data set was partitioned into the training and test sets by the K-means classification method. Prediction error on the test data set indicated that the XCSF as a local model estimates caspase-3 inhibition activity, better than the global models such as MLR and ANN. The atom-centered fragment type CR2X2, electronegativity, polarizability, and atomic radius and also the lipophilicity of the molecule, were the main independent factors contributing to the caspase-3 inhibition activity. Conclusions The results of this study may be exploited for further design of novel caspase-3 inhibitors.

  7. Blood peptidome-degradome profile of breast cancer.

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    Yufeng Shen

    Full Text Available BACKGROUND: Cancer invasion and metastasis are closely associated with activities within the degradome; however, little is known about whether these activities can be detected in the blood of cancer patients. METHODOLOGY AND PRINCIPAL FINDINGS: The peptidome-degradome profiles of pooled blood plasma sampled from 15 breast cancer patients (BCP and age, race, and menopausal status matched control healthy persons (HP were globally characterized using advanced comprehensive separations combined with tandem Fourier transform mass spectrometry and new data analysis approaches that facilitated top-down peptidomic analysis. The BCP pool displayed 71 degradome protein substrates that encompassed 839 distinct peptidome peptides. In contrast, the HP 50 degradome substrates found encompassed 425 peptides. We find that the ratios of the peptidome peptide relative abundances can vary as much as >4000 fold between BCP and HP. The experimental results also show differential degradation of substrates in the BCP sample in their functional domains, including the proteolytic and inhibitory sites of the plasmin-antiplasmin and thrombin-antithrombin systems, the main chains of the extracellular matrix protection proteins, the excessive degradation of innate immune system key convertases and membrane attack complex components, as well as several other cancer suppressor proteins. CONCLUSIONS: Degradomics-peptidomics profiling of blood plasma is highly sensitive to changes not evidenced by conventional bottom-up proteomics and potentially provides unique signatures of possible diagnostic utility.

  8. Caspase cleavage sites in the human proteome: CaspDB, a database of predicted substrates

    OpenAIRE

    Cieplak, Piotr

    2015-01-01

    Caspases are enzymes belonging to a conserved family of cysteine-dependent aspartic-specific proteases that are involved in vital cellular processes and play a prominent role in apoptosis and inflammation. Determining all relevant protein substrates of caspases remains a challenging task. Over 1500 caspase substrates have been discovered in the human proteome according to published data and new substrates are discovered on a daily basis. To aid the discovery process we devel...

  9. Low levels of Caspase-3 predict favourable response to 5FU-based chemotherapy in advanced colorectal cancer: Caspase-3 inhibition as a therapeutic approach.

    Science.gov (United States)

    Flanagan, L; Meyer, M; Fay, J; Curry, S; Bacon, O; Duessmann, H; John, K; Boland, K C; McNamara, D A; Kay, E W; Bantel, H; Schulze-Bergkamen, H; Prehn, J H M

    2016-01-01

    Colorectal cancer (CRC) is one of the most common cancers in the Western world. 5-Fluorouracil (5FU)-based chemotherapy (CT) remains the mainstay treatment of CRC in the advanced setting, and activates executioner caspases in target cells. Executioner caspases are key proteins involved in cell disassembly during apoptosis. Activation of executioner caspases also has a role in tissue regeneration and repopulation by stimulating signal transduction and cell proliferation in neighbouring, non-apoptotic cells as reported recently. Tissue microarrays (TMAs) consisting of tumour tissue from 93 stage II and III colon cancer patients were analysed by immunohistochemistry. Surprisingly, patients with low levels of active Caspase-3 had an increased disease-free survival time. This was particularly pronounced in patients who received 5FU-based adjuvant CT. In line with this observation, lower serum levels of active Caspase-3 were found in patients with metastasised CRC who revealed stable disease or tumour regression compared with those with disease progression. The role of Caspase-3 in treatment responses was explored further in primary human tumour explant cultures from fresh patient tumour tissue. Exposure of explant cultures to 5FU-based CT increased the percentage of cells positive for active Caspase-3 and Terminal Deoxynucleotidyl Transferase dUTP Nick end Labelling (TUNEL), but also the expression of regeneration and proliferation markers β-Catenin and Ki-67, as well as cyclooxygenase-2 (COX-2). Of note, selective inhibition of Caspase-3 with Ac-DNLD-CHO, a selective, reversible inhibitor of Caspase-3, significantly reduced the expression of proliferation markers as well as COX-2. Inhibition of COX-2 with aspirin or celecoxib did not affect Caspase-3 levels but also reduced Ki-67 and β-Catenin levels, suggesting that Caspase-3 acted via COX-2 to stimulate cell proliferation and tissue regeneration. This indicates that low levels of active Caspase-3 may represent a

  10. An efficient piecewise linear model for predicting activity of caspase-3 inhibitors

    OpenAIRE

    Alireza Foroumadi; Eslam Pourbasheer; Sholeh Dehghani; Khadijeh Sadatnezhad; Loghman Firoozpour; Abbas Shafiee; Massoud Amanlou

    2012-01-01

    Abstract Background and purpose of the study Multimodal distribution of descriptors makes it more difficult to fit a single global model to model the entire data set in quantitative structure activity relationship (QSAR) studies. Methods The linear (Multiple linear regression; MLR), non-linear (Artificial neural network; ANN), and an approach based on “Extended Classifier System in Function approximation” (XCSF) were applied herein to model the biological activity of 658 caspase-3 inhibitors....

  11. Substrate-driven mapping of the degradome by comparison of sequence logos.

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    Julian E Fuchs

    Full Text Available Sequence logos are frequently used to illustrate substrate preferences and specificity of proteases. Here, we employed the compiled substrates of the MEROPS database to introduce a novel metric for comparison of protease substrate preferences. The constructed similarity matrix of 62 proteases can be used to intuitively visualize similarities in protease substrate readout via principal component analysis and construction of protease specificity trees. Since our new metric is solely based on substrate data, we can engraft the protease tree including proteolytic enzymes of different evolutionary origin. Thereby, our analyses confirm pronounced overlaps in substrate recognition not only between proteases closely related on sequence basis but also between proteolytic enzymes of different evolutionary origin and catalytic type. To illustrate the applicability of our approach we analyze the distribution of targets of small molecules from the ChEMBL database in our substrate-based protease specificity trees. We observe a striking clustering of annotated targets in tree branches even though these grouped targets do not necessarily share similarity on protein sequence level. This highlights the value and applicability of knowledge acquired from peptide substrates in drug design of small molecules, e.g., for the prediction of off-target effects or drug repurposing. Consequently, our similarity metric allows to map the degradome and its associated drug target network via comparison of known substrate peptides. The substrate-driven view of protein-protein interfaces is not limited to the field of proteases but can be applied to any target class where a sufficient amount of known substrate data is available.

  12. Profiling human protein degradome delineates cellular responses to proteasomal inhibition and reveals a feedback mechanism in regulating proteasome homeostasis

    OpenAIRE

    Yu, Tao; Tao, Yonghui; Yang, Meiqiang; Chen, Peng; Gao, XiaoBo; Zhang, Yanbo; Zhang,Tao; Chen, Zi; Hou, Jian; Zhang, Yan; Ruan, Kangcheng; Wang, Hongyan; Hu, Ronggui

    2014-01-01

    Global change in protein turnover (protein degradome) constitutes a central part of cellular responses to intrinsic or extrinsic stimuli. However, profiling protein degradome remains technically challenging. Recently, inhibition of the proteasome, e.g., by using bortezomib (BTZ), has emerged as a major chemotherapeutic strategy for treating multiple myeloma and other human malignancies, but systematic understanding of the mechanisms for BTZ drug action and tumor drug resistance is yet to be a...

  13. Caspase Protocols in Mice

    OpenAIRE

    Kaushal, Varsha; Herzog, Christian; Haun, Randy S.; Kaushal, Gur P.

    2014-01-01

    Members of the caspase family of proteases are evolutionarily conserved cysteine proteases that play a crucial role as the central executioners of the apoptotic pathway. Since the discovery of caspases, many methods have been developed to detect their activation and are widely used in basic and clinical studies. In a mouse tissue, caspase activation can be monitored by cleavage of caspase-specific synthetic substrates and by detecting cleaved caspase by western blot analysis of the tissue ext...

  14. Compressed images for affinity prediction-2 (CIFAP-2): an improved machine learning methodology on protein-ligand interactions based on a study on caspase 3 inhibitors.

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    Erdas, Ozlem; Andac, Cenk A; Gurkan-Alp, A Selen; Alpaslan, Ferda Nur; Buyukbingol, Erdem

    2015-01-01

    The aim of this study is to propose an improved computational methodology, which is called Compressed Images for Affinity Prediction-2 (CIFAP-2) to predict binding affinities of structurally related protein-ligand complexes. CIFAP-2 method is established based on a protein-ligand model from which computational affinity information is obtained by utilizing 2D electrostatic potential images determined for the binding site of protein-ligand complexes. The quality of the prediction of the CIFAP-2 algorithm was tested using partial least squares regression (PLSR) as well as support vector regression (SVR) and adaptive neuro-fuzzy ınference system (ANFIS), which are highly promising prediction methods in drug design. CIFAP-2 was applied on a protein-ligand complex system involving Caspase 3 (CASP3) and its 35 inhibitors possessing a common isatin sulfonamide pharmacophore. As a result, PLSR affinity prediction for the CASP3-ligand complexes gave rise to the most consistent information with reported empirical binding affinities (pIC(50)) of the CASP3 inhibitors. PMID:25578823

  15. Transcriptome and Degradome of microRNAs and Their Targets in Response to Drought Stress in the Plants of a Diploid and Its Autotetraploid Paulownia australis

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    Niu, Suyan; Wang, Yuanlong; Zhao, Zhenli; Deng, Minjie; Cao, Lin; Yang, Lu; Fan, Guoqiang

    2016-01-01

    MicroRNAs (miRNAs) are small, non-coding RNAs that play vital roles in plant growth, development, and stress response. Increasing numbers of studies aimed at discovering miRNAs and analyzing their functions in plants are being reported. In this study, we investigated the effect of drought stress on the expression of miRNAs and their targets in plants of a diploid and derived autotetraploid Paulownia australis. Four small RNA (sRNA) libraries and four degradome libraries were constructed from diploid and autotetraploid P. australis plants treated with either 75% or 25% relative soil water content. A total of 33 conserved and 104 novel miRNAs (processing precision value > 0.1) were identified, and 125 target genes were identified for 36 of the miRNAs by using the degradome sequencing. Among the identified miRNAs, 54 and 68 were differentially expressed in diploid and autotetraploid plants under drought stress (25% relative soil water content), respectively. The expressions of miRNAs and target genes were also validated by quantitative real-time PCR. The results showed that the relative expression trends of the randomly selected miRNAs were similar to the trends predicted by Illumina sequencing. And the correlations between miRNAs and their target genes were also analyzed. Furthermore, the functional analysis showed that most of these miRNAs and target genes were associated with plant development and environmental stress response. This study provided molecular evidence for the possible involvement of certain miRNAs in the drought response and/or tolerance in P. australis, and certain level of differential expression between diploid and autotetraploid plants. PMID:27388154

  16. Transcriptome and Degradome of microRNAs and Their Targets in Response to Drought Stress in the Plants of a Diploid and Its Autotetraploid Paulownia australis.

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    Suyan Niu

    Full Text Available MicroRNAs (miRNAs are small, non-coding RNAs that play vital roles in plant growth, development, and stress response. Increasing numbers of studies aimed at discovering miRNAs and analyzing their functions in plants are being reported. In this study, we investigated the effect of drought stress on the expression of miRNAs and their targets in plants of a diploid and derived autotetraploid Paulownia australis. Four small RNA (sRNA libraries and four degradome libraries were constructed from diploid and autotetraploid P. australis plants treated with either 75% or 25% relative soil water content. A total of 33 conserved and 104 novel miRNAs (processing precision value > 0.1 were identified, and 125 target genes were identified for 36 of the miRNAs by using the degradome sequencing. Among the identified miRNAs, 54 and 68 were differentially expressed in diploid and autotetraploid plants under drought stress (25% relative soil water content, respectively. The expressions of miRNAs and target genes were also validated by quantitative real-time PCR. The results showed that the relative expression trends of the randomly selected miRNAs were similar to the trends predicted by Illumina sequencing. And the correlations between miRNAs and their target genes were also analyzed. Furthermore, the functional analysis showed that most of these miRNAs and target genes were associated with plant development and environmental stress response. This study provided molecular evidence for the possible involvement of certain miRNAs in the drought response and/or tolerance in P. australis, and certain level of differential expression between diploid and autotetraploid plants.

  17. Caspases: An apoptosis mediator

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    Tapan Kumar Palai

    2015-03-01

    Full Text Available The process of programmed cell death, or apoptosis, is generally characterized by distinct morphological characteristics and energy - dependent biochemical mechanisms. Apoptosis is a widely conserved phenomenon helping many processes, including normal cell turnover, proper development and functioning of the immune system, hormone dependent atrophy etc. Inappropriate apoptosis (either low level or high level leads to many developmental abnormalities like, neurodegenerative diseases, ischemic damage, autoimmune disorders and many types of cancer. To use cells for therapeutic purposes through generating cell lines, it is critical to study the cell cycle machinery and signalling pathways that controls cell death and apoptosis. Apoptotic pathways provide a fundamental protective mechanism that decreases cellular sensitivity to damaging events and allow proper developmental process in multi-cellular organisms. Major mediator of apoptosis is a family of proteins known as caspases. There are mainly fourteen types of caspases but out of them only ten caspasese have got essential role in controlling the process of apoptosis. These ten caspases have been categorized into either initiator caspases (caspase 2, 8, 9, 10 or executioner caspases (caspase 3, 6, 7. Although various types of caspases have been identified so far, the exact mechanisms of action of these groups of proteins is still to be fully understood. The aim of this review is to provide a detail overview of role of different caspases in regulating the process of apoptosis.

  18. Allosteric modulation of caspases.

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    Häcker, Hans-Georg; Sisay, Mihiret Tekeste; Gütschow, Michael

    2011-11-01

    Caspases are proteolytic enzymes mainly involved in the induction and execution phases of apoptosis. This type of programmed cell death is an essential regulatory process required to maintain the integrity and homeostasis of multicellular organisms. Inappropriate apoptosis is attributed a key role in many human diseases, including neurodegenerative disorders, ischemic damage, autoimmune diseases and cancer. Allosteric modulation of the function of a protein occurs when the regulatory trigger, such as the binding of a small effector or inhibitor molecule, takes place some distance from the protein's active site. In recent years, several caspases have been identified that possess allosteric sites and binding of small molecule to these sites resulted in the modulation of enzyme activities. Regulation of caspase activity by small molecule allosteric modulators is believed to be of great therapeutic importance. In this review we give brief highlights on recent developments in identifying and characterizing natural and synthetic allosteric inhibitors as well as activators of caspases and discuss their potential in drug discovery and protein engineering. PMID:21807025

  19. Caspase Family Proteases and Apoptosis

    Institute of Scientific and Technical Information of China (English)

    Ting-Jun FAN; Li-Hui HAN; Ri-Shan CONG; Jin LIANG

    2005-01-01

    Apoptosis, or programmed cell death, is an essential physiological process that plays a critical role in development and tissue homeostasis. The progress of apoptosis is regulated in an orderly way by a series of signal cascades under certain circumstances. The caspase-cascade system plays vital roles in the induction, transduction and amplification of intracellular apoptotic signals. Caspases, closely associated with apoptosis, are aspartate-specific cysteine proteases and members of the interleukin-1β-converting enzyme family. The activation and function of caspases, involved in the delicate caspase-cascade system, are regulated by various kinds of molecules, such as the inhibitor of apoptosis protein, Bcl-2 family proteins, calpain,and Ca2+. Based on the latest research, the members of the caspase family, caspase-cascade system and caspase-regulating molecules involved in apoptosis are reviewed.

  20. Identification and functional characterization of two executioner caspases in Crassostrea gigas.

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    Tao Qu

    Full Text Available Caspase-3 and caspase-7 are two key effector caspases that play important roles in apoptotic pathways that maintain normal tissue and organ development and homeostasis. However, little is known about the sequence, structure, activity, and function of effector caspases upon apoptosis in mollusks, especially marine bivalves. In this study, we investigated the possible roles of two executioner caspases in the regulation of apoptosis in the Pacific oyster Crassostrea gigas. A full-length caspase-3-like gene named Cgcaspase-3 was cloned from C.gigas cDNA, encoding a predicted protein containing caspase family p20 and p10 domain profiles and a conserved caspase active site motif. Phylogenetic analysis demonstrated that both Cgcaspase-3 and Cgcaspase-1 may function as effector caspases clustered in the invertebrate branch. Although the sequence identities between the two caspases was low, both enzymes possessed executioner caspase activity and were capable of inducing cell death. These results suggested that Cgcaspase-3 and Cgcaspase-1 were two effector caspases in C. gigas. We also observed that nucleus-localized Cgcaspase-3, may function as a caspase-3-like protein and cytoplasm-localized Cgcaspase-1 may function as a caspase-7-like protein. Both Cgcaspase-3 and Cgcaspase-1 mRNA expression increased after larvae settled on the substratum, suggesting that both caspases acted in several tissues or organs that degenerated after oyster larvae settlement. The highest caspase expression levels were observed in the gills indicating that both effector caspases were likely involved in immune or metabolic processes in C. gigas.

  1. Allosteric modulation of caspase 3 through mutagenesis

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    Jad Walters

    2012-06-01

    Full Text Available A mutation in the allosteric site of the caspase 3 dimer interface of Val266 to histidine abolishes activity of the enzyme, and models predict that the mutation mimics the action of small molecule allosteric inhibitors by preventing formation of the active site. Mutations were coupled to His266 at two sites in the interface, E124A and Y197C. We present results from X-ray crystallography, enzymatic activity and molecular dynamics simulations for seven proteins, consisting of single, double and triple mutants. The results demonstrate that considering allosteric inhibition of caspase 3 as a shift between discrete ‘off-state’ or ‘on-state’ conformations is insufficient. Although His266 is accommodated in the interface, the structural defects are propagated to the active site through a helix on the protein surface. A more comprehensive view of allosteric regulation of caspase 3 requires the representation of an ensemble of inactive states and shows that subtle structural changes lead to the population of the inactive ensemble.

  2. Caspase-10 triggers Bid cleavage and caspase cascade activation in FasL-induced apoptosis.

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    Milhas, Delphine; Cuvillier, Olivier; Therville, Nicole; Clavé, Patricia; Thomsen, Mogens; Levade, Thierry; Benoist, Hervé; Ségui, Bruno

    2005-05-20

    In contrast to caspase-8, controversy exists as to the ability of caspase-10 to mediate apoptosis in response to FasL. Herein, we have shown activation of caspase-10, -3, and -7 as well as B cell lymphoma-2-interacting domain (Bid) cleavage and cytochrome c release in caspase-8-deficient Jurkat (I9-2) cells treated with FasL. Apoptosis was clearly induced as illustrated by nuclear and DNA fragmentation. These events were inhibited by benzyloxycarbonyl-VAD-fluoromethyl ketone, a broad spectrum caspase inhibitor, indicating that caspases were functionally and actively involved. Benzyloxycarbonyl-AEVD-fluoromethyl ketone, a caspase-10 inhibitor, had a comparable effect. FasL-induced cell death was not completely abolished by caspase inhibitors in agreement with the existence of a cytotoxic caspase-independent pathway. In subpopulations of I9-2 cells displaying distinct caspase-10 expression levels, cell sensitivity to FasL correlated with caspase-10 expression. A robust caspase activation, Bid cleavage, and DNA fragmentation were observed in cells with high caspase-10 levels but not in those with low levels. In vitro, caspase-10, as well as caspase-8, could cleave Bid to generate active truncated Bid (p15). Altogether, our data strongly suggest that caspase-10 can serve as an initiator caspase in Fas signaling leading to Bid processing, caspase cascade activation, and apoptosis. PMID:15772077

  3. Caspase exploitation by Legionella pneumophila

    OpenAIRE

    Kathrin eKrause; Amer, Amal O.

    2016-01-01

    Legionella pneumophila remains a major health concern, especially for hospitalized patients. L. pneumophila in the environment can survive extracellular or as protozoan parasite within amoeba. After human infection it efficiently replicates in alveolar macrophages without activating inflammasome assembly and cleavage of caspase-1. In contrast murine macrophages actively recognize intracellular L. pneumophila via inflammasome components which initiate pro-inflammatory cytokine secretion, phago...

  4. Caspase Exploitation by Legionella pneumophila

    OpenAIRE

    Krause, Kathrin; Amer, Amal O.

    2016-01-01

    Legionella pneumophila remains a major health concern, especially for hospitalized patients. L. pneumophila in the environment can survive extracellular or as protozoan parasite within amoeba. After human infection it efficiently replicates in alveolar macrophages without activating inflammasome assembly and cleavage of caspase-1. In contrast murine macrophages actively recognize intracellular L. pneumophila via inflammasome components which initiate pro-inflammatory cytokine secretion, phago...

  5. Structural Features of Caspase-Activating Complexes

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    Hyun Ho Park

    2012-04-01

    Full Text Available Apoptosis, also called programmed cell death, is an orderly cellular suicide program that is critical for the development, immune regulation and homeostasis of a multi-cellular organism. Failure to control this process can lead to serious human diseases, including many types of cancer, neurodegenerative diseases, and autoimmununity. The process of apoptosis is mediated by the sequential activation of caspases, which are cysteine proteases. Initiator caspases, such as caspase-2, -8, -9, and -10, are activated by formation of caspase-activating complexes, which function as a platform to recruit caspases, providing proximity for self-activation. Well-known initiator caspase-activating complexes include (1 DISC (Death Inducing Signaling Complex, which activates caspases-8 and 10; (2 Apoptosome, which activates caspase-9; and (3 PIDDosome, which activates caspase-2. Because of the fundamental biological importance of capases, many structural and biochemical studies to understand the molecular basis of assembly mechanism of caspase-activating complexes have been performed. In this review, we summarize previous studies that have examined the structural and biochemical features of caspase-activating complexes. By analyzing the structural basis for the assembly mechanism of the caspase-activating complex, we hope to provide a comprehensive understanding of caspase activation by these important oligomeric complexes.

  6. A global comparison of the human and T. brucei degradomes gives insights about possible parasite drug targets.

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    Susan T Mashiyama

    Full Text Available We performed a genome-level computational study of sequence and structure similarity, the latter using crystal structures and models, of the proteases of Homo sapiens and the human parasite Trypanosoma brucei. Using sequence and structure similarity networks to summarize the results, we constructed global views that show visually the relative abundance and variety of proteases in the degradome landscapes of these two species, and provide insights into evolutionary relationships between proteases. The results also indicate how broadly these sequence sets are covered by three-dimensional structures. These views facilitate cross-species comparisons and offer clues for drug design from knowledge about the sequences and structures of potential drug targets and their homologs. Two protease groups ("M32" and "C51" that are very different in sequence from human proteases are examined in structural detail, illustrating the application of this global approach in mining new pathogen genomes for potential drug targets. Based on our analyses, a human ACE2 inhibitor was selected for experimental testing on one of these parasite proteases, TbM32, and was shown to inhibit it. These sequence and structure data, along with interactive versions of the protein similarity networks generated in this study, are available at http://babbittlab.ucsf.edu/resources.html.

  7. Revisiting Caspase-11 Function in Host Defense

    OpenAIRE

    Ng, Tessie M.; Monack, Denise M.

    2013-01-01

    Pro-inflammatory caspases play important roles in innate immunity. Much attention has focused on caspase-1, which acts to eliminate pathogens by obliterating their replicative niches as well as alerting the host to their presence. Emerging data now sheds light on the lesser-studied pro-inflammatory caspase-11 in the combat between host and pathogen. With new tools available, researchers are now further elucidating the mechanisms by which caspase-11 contributes to host defense. Here, we review...

  8. Integration of small RNAs, degradome and transcriptome sequencing in hyperaccumulator Sedum alfredii uncovers a complex regulatory network and provides insights into cadmium phytoremediation.

    Science.gov (United States)

    Han, Xiaojiao; Yin, Hengfu; Song, Xixi; Zhang, Yunxing; Liu, Mingying; Sang, Jiang; Jiang, Jing; Li, Jihong; Zhuo, Renying

    2016-06-01

    The hyperaccumulating ecotype of Sedum alfredii Hance is a cadmium (Cd)/zinc/lead co-hyperaccumulating species of Crassulaceae. It is a promising phytoremediation candidate accumulating substantial heavy metal ions without obvious signs of poisoning. However, few studies have focused on the regulatory roles of miRNAs and their targets in the hyperaccumulating ecotype of S. alfredii. Here, we combined analyses of the transcriptomics, sRNAs and the degradome to generate a comprehensive resource focused on identifying key regulatory miRNA-target circuits under Cd stress. A total of 87 721 unigenes and 356 miRNAs were identified by deep sequencing, and 79 miRNAs were differentially expressed under Cd stress. Furthermore, 754 target genes of 194 miRNAs were validated by degradome sequencing. A gene ontology (GO) enrichment analysis of differential miRNA targets revealed that auxin, redox-related secondary metabolism and metal transport pathways responded to Cd stress. An integrated analysis uncovered 39 pairs of miRNA targets that displayed negatively correlated expression profiles. Ten miRNA-target pairs also exhibited negative correlations according to a real-time quantitative PCR analysis. Moreover, a coexpression regulatory network was constructed based on profiles of differentially expressed genes. Two hub genes, ARF4 (auxin response factor 4) and AAP3 (amino acid permease 3), which might play central roles in the regulation of Cd-responsive genes, were uncovered. These results suggest that comprehensive analyses of the transcriptomics, sRNAs and the degradome provided a useful platform for investigating Cd hyperaccumulation in S. alfredii, and may provide new insights into the genetic engineering of phytoremediation. PMID:26801211

  9. Small-molecule caspase inhibitors

    International Nuclear Information System (INIS)

    The review considers low-molecular weight inhibitors of caspases, cysteine proteases being key contributors to apoptosis (programmed cell death). The inhibitors with aspartic acid residues or various heterocyclic systems (both synthetic and natural) are covered. Their possible mechanisms of action are discussed. Data on inhibitor structure-activity relationship studies are systematically surveyed. The interactions of the non-peptide fragments of an inhibitor with the enzymes are examined. Examples of the use of some inhibitors for apoptosis suppression are provided.

  10. Small-molecule caspase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Zhenodarova, S M [Institute for Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow region (Russian Federation)

    2010-02-28

    The review considers low-molecular weight inhibitors of caspases, cysteine proteases being key contributors to apoptosis (programmed cell death). The inhibitors with aspartic acid residues or various heterocyclic systems (both synthetic and natural) are covered. Their possible mechanisms of action are discussed. Data on inhibitor structure-activity relationship studies are systematically surveyed. The interactions of the non-peptide fragments of an inhibitor with the enzymes are examined. Examples of the use of some inhibitors for apoptosis suppression are provided.

  11. Small-molecule caspase inhibitors

    Science.gov (United States)

    Zhenodarova, S. M.

    2010-02-01

    The review considers low-molecular weight inhibitors of caspases, cysteine proteases being key contributors to apoptosis (programmed cell death). The inhibitors with aspartic acid residues or various heterocyclic systems (both synthetic and natural) are covered. Their possible mechanisms of action are discussed. Data on inhibitor structure-activity relationship studies are systematically surveyed. The interactions of the non-peptide fragments of an inhibitor with the enzymes are examined. Examples of the use of some inhibitors for apoptosis suppression are provided.

  12. Caspase-7 and dental apoptosis

    Czech Academy of Sciences Publication Activity Database

    Matalová, Eva; Tucker, A. S.; Švandová, Eva; Berghe, T.V.; Sharpe, P. T.

    Fyziologický ústav AV ČR, v. v. i.. Roč. 60, č. 4 (2011), 35P-35P ISSN 0862-8408. [Proceedings of the Czech and Slovak Physiological Societies. 09.02.2011-11.02.2011, Bratislava] R&D Projects: GA AV ČR KJB500450802; GA AV ČR IAA600450904 Institutional research plan: CEZ:AV0Z50450515 Keywords : caspase * protein * dental Subject RIV: ED - Physiology http://www.biomed.cas.cz/physiolres/2011/4_11.htm

  13. Caspases and their role in inflammation and ischemic neuronal death. Focus on caspase-12.

    Science.gov (United States)

    García de la Cadena, Selene; Massieu, Lourdes

    2016-07-01

    Caspases are cysteine proteases, which play important roles in different processes including, apoptosis and inflammation. Caspase-12, expressed in mouse and human, is classified as an inflammatory caspase. However, in humans caspase-12 gene has acquired different mutations that result in the expression of different variants. Caspase-12 is generally recognized as a negative regulator of the inflammatory response induced by infections, because it inhibits the activation of caspase-1 in inflammasome complexes, the production of the pro-inflammatory cytokines IL-1β and IL-18 and the overall response to sepsis. In contrast, caspase-4, the human paralog of caspase-12, exerts a positive modulatory action of the inflammatory response to infectious agents. The role of caspase-12 and caspase-4 in inflammation associated with cerebral ischemia, a condition that results from a transient or permanent reduction of cerebral blood flow, is still unknown. Among the mechanisms involved in ischemic brain injury, apoptosis and inflammation have important roles. Under these conditions, disturbances in the homeostasis of the endoplasmic reticulum (ER) take place, leading to ER stress, caspase activation and apoptosis. Caspase-12 up-regulation and processing has been observed after the ischemic episode but its role in apoptosis is controversial. Cleavage of caspase-4 also occurs during ER stress but its role in ischemic brain injury is unknown. Throughout this review evidence supporting a role of caspase-12 and caspase-4 on the modulation of the inflammatory response to infection and their potential contribution to ER stress-induced apoptosis, is discussed. Understanding the actions of rodent caspase-12 and human caspase-4 will help us to elucidate their role in different pathological conditions, which to date is not well understood. PMID:27142195

  14. Caspase Exploitation by Legionella pneumophila

    Science.gov (United States)

    Krause, Kathrin; Amer, Amal O.

    2016-01-01

    Legionella pneumophila remains a major health concern, especially for hospitalized patients. L. pneumophila in the environment can survive extracellular or as protozoan parasite within amoeba. After human infection it efficiently replicates in alveolar macrophages without activating inflammasome assembly and cleavage of caspase-1. In contrast murine macrophages actively recognize intracellular L. pneumophila via inflammasome components which initiate pro-inflammatory cytokine secretion, phagosomal maturation and pyroptotic cell death thereby leading to bacterial restriction. During this process flagellin-dependent and -independent signaling pathways trigger the canonical as well as the non-canonical inflammasome. This review describes the current knowledge about L. pneumophila-induced inflammasome pathways in permissive and restrictive host cells. PMID:27148204

  15. 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: EA - Cell Biology Impact factor: 2.386, year: 2014

  16. Identification of miRNAs and Their Targets in Cotton Inoculated with Verticillium dahliae by High-Throughput Sequencing and Degradome Analysis

    Directory of Open Access Journals (Sweden)

    Yujuan Zhang

    2015-06-01

    Full Text Available MicroRNAs (miRNAs are a group of endogenous small non-coding RNAs that play important roles in plant growth, development, and stress response processes. Verticillium wilt is a vascular disease in plants mainly caused by Verticillium dahliae Kleb., the soil-borne fungal pathogen. However, the role of miRNAs in the regulation of Verticillium defense responses is mostly unknown. This study aimed to identify new miRNAs and their potential targets that are involved in the regulation of Verticillium defense responses. Four small RNA libraries and two degradome libraries from mock-infected and infected roots of cotton (both Gossypium hirsutum L. and Gossypium barbadense L. were constructed for deep sequencing. A total of 140 known miRNAs and 58 novel miRNAs were identified. Among the identified miRNAs, many were differentially expressed between libraries. Degradome analysis showed that a total of 83 and 24 genes were the targets of 31 known and 14 novel miRNA families, respectively. Gene Ontology analysis indicated that many of the identified miRNA targets may function in controlling root development and the regulation of Verticillium defense responses in cotton. Our findings provide an overview of potential miRNAs involved in the regulation of Verticillium defense responses in cotton and the interactions between miRNAs and their corresponding targets. The profiling of these miRNAs lays the foundation for further understanding of the function of small RNAs in regulating plant response to fungal infection and Verticillium wilt in particular.

  17. A designed redox-controlled caspase

    Energy Technology Data Exchange (ETDEWEB)

    Witkowski, Witold A.; Hardy, Jeanne A. (UMASS, Amherst)

    2011-09-15

    Caspases are a powerful class of cysteine proteases. Introduction of activated caspases in healthy or cancerous cells results in induction of apoptotic cell death. In this study, we have designed and characterized a version of caspase-7 that can be inactivated under oxidizing extracellular conditions and then reactivated under reducing intracellular conditions. This version of caspase-7 is allosterically inactivated when two of the substrate-binding loops are locked together via an engineered disulfide. When this disulfide is reduced, the protein regains its full function. The inactive loop-locked version of caspase-7 can be readily observed by immunoblotting and mass spectrometry. The reduced and reactivated form of the enzyme observed crystallographically is the first caspase-7 structure in which the substrate-binding groove is properly ordered even in the absence of an active-site ligand. In the reactivated structure, the catalytic-dyad cysteine-histidine are positioned 3.5 {angstrom} apart in an orientation that is capable of supporting catalysis. This redox-controlled version of caspase-7 is particularly well suited for targeted cell death in concert with redox-triggered delivery vehicles.

  18. Inactivation of Effector Caspases through Nondegradative Polyubiquitylation

    DEFF Research Database (Denmark)

    Ditzel, Mark; Broemer, Meike; Tenev, Tencho;

    2008-01-01

    Ubiquitin-mediated inactivation of caspases has long been postulated to contribute to the regulation of apoptosis. However, detailed mechanisms and functional consequences of caspase ubiquitylation have not been demonstrated. Here we show that the Drosophila Inhibitor of Apoptosis 1, DIAP1, blocks...... reduces the caspase's proteolytic velocity. Disruption of drICE ubiquitylation, either by mutation of DIAP1's E3 activity or drICE's ubiquitin-acceptor lysines, abrogates DIAP1's ability to neutralize drICE and suppress apoptosis in vivo. We also show that DIAP1 rests in an "inactive" conformation that...

  19. 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.

  20. Ripoptosome Analysis by Caspase-8 Coimmunoprecipitation.

    Science.gov (United States)

    Feoktistova, Maria; Geserick, Peter; Leverkus, Martin

    2016-01-01

    The biochemical signaling of cell death pathways is executed at a number of different intracellular and/or membrane-bound high-molecular mass complexes. It is crucial to be able to detect the formation, differences in assembly, and differential composition of such complexes to understand their contribution to the execution phase of apoptotic or necroptotic cell death. We describe here the use of caspase-8 coimmunoprecipitation in the spontaneously transformed keratinocyte cell line, HaCaT, to study the formation and composition of the Ripoptosome, a complex that is based on the serine-threonine kinase receptor-interacting protein 1 (RIPK1). However, the method can be adapted for use with other antibodies and cell lines. This protocol determines whether cells form the Ripoptosome complex, which is important for both apoptosis and necroptosis execution. Caspase-8 is an indispensible Ripoptosome component; therefore, caspase-8 antibodies are used to pull down the respective complex. However, the method cannot discriminate whether this complex triggers apoptosis (through the RIPK1 → FADD → caspase-8 activation pathway), necroptosis (through the RIPK1 → RIPK3 → MLKL activation pathway) or nondeath signaling. The actual signaling output (death or nondeath signaling) depends on the stoichiometry of the respective molecules as well as on the activity of FLIP, caspase-8, or other factors. PMID:26933246

  1. Small RNA and degradome deep sequencing reveals drought-and tissue-specific micrornas and their important roles in drought-sensitive and drought-tolerant tomato genotypes.

    Science.gov (United States)

    Candar-Cakir, Bilgin; Arican, Ercan; Zhang, Baohong

    2016-08-01

    Drought stress has adverse impacts on plant production and productivity. MicroRNAs (miRNAs) are one class of noncoding RNAs regulating gene expression post-transcriptionally. In this study, we employed small RNA and degradome sequencing to systematically investigate the tissue-specific miRNAs responsible to drought stress, which are understudied in tomato. For this purpose, root and upground tissues of two different drought-responsive tomato genotypes (Lycopersicon esculentum as sensitive and L. esculentum var. cerasiforme as tolerant) were subjected to stress with 5% polyethylene glycol for 7 days. A total of 699 conserved miRNAs belonging to 578 families were determined and 688 miRNAs were significantly differentially expressed between different treatments, tissues and genotypes. Using degradome sequencing, 44 target genes were identified associated with 36 miRNA families. Drought-related miRNAs and their targets were enriched functionally by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Totally, 53 miRNAs targeted 23 key drought stress- and tissue development-related genes, including DRP (dehydration-responsive protein), GTs (glycosyltransferases), ERF (ethylene responsive factor), PSII (photosystem II) protein, HD-ZIP (homeodomain-leucine zipper), MYB and NAC-domain transcription factors. miR160, miR165, miR166, miR171, miR398, miR408, miR827, miR9472, miR9476 and miR9552 were the key miRNAs functioning in regulation of these genes and involving in tomato response to drought stress. Additionally, plant hormone signal transduction pathway genes were differentially regulated by miR169, miR172, miR393, miR5641, miR5658 and miR7997 in both tissues of both sensitive and tolerant genotypes. These results provide new insight into the regulatory role of miRNAs in drought response with plant hormone signal transduction and drought-tolerant tomato breeding. PMID:26857916

  2. Identification of miRNAs and Their Targets in the Liverwort Marchantia polymorpha by Integrating RNA-Seq and Degradome Analyses.

    Science.gov (United States)

    Lin, Pin-Chun; Lu, Chia-Wei; Shen, Bing-Nan; Lee, Guan-Zong; Bowman, John L; Arteaga-Vazquez, Mario A; Liu, Li-Yu Daisy; Hong, Syuan-Fei; Lo, Chu-Fang; Su, Gong-Min; Kohchi, Takayuki; Ishizaki, Kimitsune; Zachgo, Sabine; Althoff, Felix; Takenaka, Mizuki; Yamato, Katsuyuki T; Lin, Shih-Shun

    2016-02-01

    Bryophytes (liverworts, hornworts and mosses) comprise the three earliest diverging lineages of land plants (embryophytes). Marchantia polymorpha, a complex thalloid Marchantiopsida liverwort that has been developed into a model genetic system, occupies a key phylogenetic position. Therefore, M. polymorpha is useful in studies aiming to elucidate the evolution of gene regulation mechanisms in plants. In this study, we used computational, transcriptomic, small RNA and degradome analyses to characterize microRNA (miRNA)-mediated pathways of gene regulation in M. polymorpha. The data have been integrated into the open access ContigViews-miRNA platform for further reference. In addition to core components of the miRNA pathway, 129 unique miRNA sequences, 11 of which could be classified into seven miRNA families that are conserved in embryophytes (miR166a, miR390, miR529c, miR171-3p, miR408a, miR160 and miR319a), were identified. A combination of computational and degradome analyses allowed us to identify and experimentally validate 249 targets. In some cases, the target genes are orthologous to those of other embryophytes, but in other cases, the conserved miRNAs target either paralogs or members of different gene families. In addition, the newly discovered Mpo-miR11707.1 and Mpo-miR11707.2 are generated from a common precursor and target MpARGONAUTE1 (LW1759). Two other newly discovered miRNAs, Mpo-miR11687.1 and Mpo-miR11681.1, target the MADS-box transcription factors MpMADS1 and MpMADS2, respectively. Interestingly, one of the pentatricopeptide repeat (PPR) gene family members, MpPPR_66 (LW9825), the protein products of which are generally involved in various steps of RNA metabolism, has a long stem-loop transcript that can generate Mpo-miR11692.1 to autoregulate MpPPR_66 (LW9825) mRNA. This study provides a foundation for further investigations of the RNA-mediated silencing mechanism in M. polymorpha as well as of the evolution of this gene silencing pathway in

  3. Reprogramming Caspase-7 Specificity by Regio-Specific Mutations and Selection Provides Alternate Solutions for Substrate Recognition.

    Science.gov (United States)

    Hill, Maureen E; MacPherson, Derek J; Wu, Peng; Julien, Olivier; Wells, James A; Hardy, Jeanne A

    2016-06-17

    The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. Here, we report a new approach based on a caged green fluorescent protein (CA-GFP) reporter that allows for flow-cytometry-based selection in bacteria or other cell types enabling selection of intracellular protease specificity, regardless of the compositional complexity of the protease. Here, we apply this approach to introduce the specificity of caspase-6 into caspase-7, an intracellular cysteine protease important in cellular remodeling and cell death. We found that substitution of substrate-contacting residues from caspase-6 into caspase-7 was ineffective, yielding an inactive enzyme, whereas saturation mutagenesis at these positions and selection by directed evolution produced active caspases. The process produced a number of nonobvious mutations that enabled conversion of the caspase-7 specificity to match caspase-6. The structures of the evolved-specificity caspase-7 (esCasp-7) revealed alternate binding modes for the substrate, including reorganization of an active site loop. Profiling the entire human proteome of esCasp-7 by N-terminomics demonstrated that the global specificity toward natural protein substrates is remarkably similar to that of caspase-6. Because the esCasp-7 maintained the core of caspase-7, we were able to identify a caspase-6 substrate, lamin C, that we predict relies on an exosite for substrate recognition. These reprogrammed proteases may be the first tool built with the express intent of distinguishing exosite dependent or independent substrates. This approach to specificity reprogramming should also be generalizable across a wide range of proteases. PMID:27032039

  4. Sox11 Reduces Caspase-6 Cleavage and Activity.

    Directory of Open Access Journals (Sweden)

    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. Identification of miRNAs and Their Target Genes Associated with Sweet Corn Seed Vigor by Combined Small RNA and Degradome Sequencing.

    Science.gov (United States)

    Gong, Shumin; Ding, Yanfei; Huang, Shanxia; Zhu, Cheng

    2015-06-10

    High seed vigor is significant for agriculture. Low seed vigor of sweet corn hindered the popularization of sweet corn (Zea mays L. saccharata Sturt). To better understand the involvement and regulatory mechanism of miRNAs with seed vigor, small RNA libraries from seeds non-artificially aged and artificially aged for 2 days were generated by small RNA sequencing. A total of 27 differentially expressed miRNAs were discovered, of which 10 were further confirmed by real-time quantitative polymerase chain reaction. Furthermore, targets of miRNAs were identified by degradome sequencing. A total of 1142 targets that were potentially cleaved by 131 miRNAs were identified. Gene ontology (GO) annotations of target transcripts indicated that 26 target genes cleaved by 9 differentially expressed miRNAs might play roles in the regulation of seed vigor, such as peroxidase superfamily protein targeted by PC-5p-213179_17 playing a role in the oxidation-reduction process and response to oxidative stress. These findings provide valuable information to understand the involvement of miRNAs with seed vigor. PMID:25997082

  6. Skin Barrier Defects Caused by Keratinocyte-Specific Deletion of ADAM17 or EGFR Are Based on Highly Similar Proteome and Degradome Alterations.

    Science.gov (United States)

    Tholen, Stefan; Wolf, Cristina; Mayer, Bettina; Knopf, Julia D; Löffek, Stefanie; Qian, Yawen; Kizhakkedathu, Jayachandran N; Biniossek, Martin L; Franzke, Claus-Werner; Schilling, Oliver

    2016-05-01

    Keratinocyte-specific deletion of ADAM17 in mice impairs terminal differentiation of keratinocytes leading to severe epidermal barrier defects. Mice deficient for ADAM17 in keratinocytes phenocopy mice with a keratinocyte-specific deletion of epidermal growth factor receptor (EGFR), which highlights the role of ADAM17 as a "ligand sheddase" of EGFR ligands. In this study, we aim for the first proteomic/degradomic approach to characterize the disruption of the ADAM17-EGFR signaling axis and its consequences for epidermal barrier formation. Proteomic profiling of the epidermal proteome of mice deficient for either ADAM17 or EGFR in keratinocytes at postnatal days 3 and 10 revealed highly similar protein alterations for ADAM17 and EGFR deficiency. These include massive proteome alterations of structural and regulatory components important for barrier formation such as transglutaminases, involucrin, filaggrin, and filaggrin-2. Cleavage site analysis using terminal amine isotopic labeling of substrates revealed increased proteolytic processing of S100 fused-type proteins including filaggrin-2. Alterations in proteolytic processing are supported by altered abundance of numerous proteases upon keratinocyte-specific Adam17 or Egfr deletion, among them kallikreins, cathepsins, and their inhibitors. This study highlights the essential role of proteolytic processing for maintenance of a functional epidermal barrier. Furthermore, it suggests that most defects in formation of the postnatal epidermal barrier upon keratinocyte-specific ADAM17 deletion are mediated via EGFR. PMID:27089454

  7. Coxsackievirus B3-induced apoptosis and Caspase-3

    Institute of Scientific and Technical Information of China (English)

    JIAN PING YUAN; WEI ZHAO; HONG TAO WANG; KAI YU WU; TAO LI; XIAO KUI GUO; SHAN QING TONG

    2003-01-01

    Cell death can be classified into two categories: apoptosis and necrosis. Apoptotic pathway can beeither caspase-dependent or caspase-independent. Caspase-independent cytopathic effect (CPE) has beendescribed. In order to evaluate the pattern of HeLa cell death induced by Coxsackievirus B3 (CVB3)and whether apoptosis involves caspase activation, we co-cultivated HeLa cells with CVB3 and detectedthe cytopathic changes, the alteration of mRNA and protein expression of caspase-3 gene plus caspase-3activity, as well as analyzing DNA fragmentation before and after caspase-3 activity inhibition. Accordingto the results, we propose that CVB3 may induce apoptosis and necrosis in HeLa cells, the latter appearingmuch earlier. Caspase-3 is activated at the levels of both transcription and translation, and procaspase-3 isproteolytically cleaved, thus leading to the continuous increasing of both caspase-3 precursor protein and itssubunit. However, besides CPE, apoptosis induced by CVB3 is not a direct consequence of the activationof caspase-3, or caspase-3 is not the only effector molecule in apoptotic cell death, for caspase-3 inhibitorcan not decrease DNA fragmentation. Some other biochemical mechanisms may participate in the process,whose role weakens the effect of inhibiting caspase-3 activity.

  8. Molluscan death effector domain (DED)-containing caspase-8 gene from disk abalone (Haliotis discus discus): molecular characterization and expression analysis.

    Science.gov (United States)

    Lee, Youngdeuk; De Zoysa, Mahanama; Whang, Ilson; Lee, Sukkyoung; Kim, Yucheol; Oh, Chulhong; Choi, Cheol Young; Yeo, Sang-Yeob; Lee, Jehee

    2011-02-01

    The caspase family represents aspartate-specific cysteine proteases that play key roles in apoptosis and immune signaling. In this study, we cloned the first death effector domain (DED)-containing molluscan caspase-8 gene from disk abalone (Haliotis discus discus), which is named as hdCaspase-8. The full-length hdCaspase was 2855 bp, with a 1908 bp open reading frame encoding 636 amino acids. The hdCaspase-8 had 72 kDa predicted molecular mass with an estimated isoelectric point (PI) of 6.0. The hdCaspase-8 amino acid sequence contained the characteristic feature of an N-terminal two DED, a C-terminal catalytic domain and the caspase family cysteine active site ⁵¹³KPKLFFLQACQG⁵²⁴. Phylogenetic analysis results showed that hdCaspase-8 is more similar to the invertebrate Tubifex tubifex (sludge worm) caspase-8. Real-time RT-PCR results showed that hdCaspase-8 constitutively and ubiquitously expressed in all tested tissue of unchallenged disk abalone. The basal expression level of hdCaspase-8 in gill tissue was higher than all other tested tissues. The hdCaspase-8 mRNA expression in gill and hemocytes was significantly up-regulated by exposure to bacteria (Vibrio alginolyticus, Vibrio parahemolyticus and Listeria monocytogenes) and VHSV (viral hemorrhagic septicemia virus), as compared to control animals. These results suggest that hdCaspase-8 may be involved in immune response reactions in disk abalone. PMID:21130887

  9. Proteasomal regulation of caspase-8 in cancer cell apoptosis

    OpenAIRE

    Fiandalo, Michael V.; Schwarze, Steven R.; Kyprianou, Natasha

    2013-01-01

    Previous studies demonstrated that proteasome inhibition sensitizes TRAIL resistant prostate cancer cells to TRAIL-mediated apoptosis via stabilization of the active p18 subunit of caspase-8. The present study investigated the impact of proteasome inhibition on caspase-8 stability, ubiquitination, trafficking, and activation in cancer cells. Using caspase-8 deficient neuroblastoma (NB7) cells for reconstituting non-cleavable mutant forms of caspase-8, we demonstrated that the non-cleavable fo...

  10. Allopurinol’s effect on caspase-3 and caspase-8 activity in hypoxic-ischemic newborn rats

    OpenAIRE

    Kenan Özcan; Mehmet Satar; Necmiye Canacankatan; Erdal Taşkın; Kenan Dağlıoğlu

    2013-01-01

    Aim: During reperfusion period of hypoxia-ischemia, cyclooxygenase and xanthine oxidase pathways are induced. A xanthine oxidase inhibitor, allopurinol has been shown to be neuroprotective in hypoxic- ischemic encephalopathy. Caspase-8 and caspase-3 have a key role in neuronal apoptosis. We aimed to test repeated doses of allopurinol’s effect on caspase-3 and caspase-8 activities in newborn rats with hypoxic-ischemic encephalopathy. Material and Method: Seven days old newborn rats were taken ...

  11. Delayed activation of caspase-independent apoptosis during heart failure in transgenic mice overexpressing caspase inhibitor CrmA

    OpenAIRE

    Bae, Soochan; Siu, Parco M.; Choudhury, Sangita; Ke, Qingen; Choi, Jun H.; Koh, Young Y.; Kang, Peter M.

    2010-01-01

    Although caspase activation is generally thought to be necessary to induce apoptosis, recent evidence suggests that apoptosis can be activated in the setting of caspase inhibition. In this study, we tested the hypothesis that caspase-independent apoptotic pathways contribute to the development of heart failure in the absence of caspase activation. Acute cardiomyopathy was induced using a single dose of doxorubicin (Dox, 20 mg/kg) injected into male wild-type (WT) and transgenic (Tg) mice with...

  12. The Enigmatic Roles of Caspases in Tumor Development

    International Nuclear Information System (INIS)

    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

  13. Does Caspase-6 Have a Role in Perinatal Brain Injury?

    Science.gov (United States)

    Baburamani, Ana A.; Miyakuni, Yasuka; Vontell, Regina; Supramaniam, Veena G.; Svedin, Pernilla; Rutherford, Mary; Gressens, Pierre; Mallard, Carina; Takeda, Satoru; Thornton, Claire; Hagberg, Henrik

    2015-01-01

    Apoptotic mechanisms are centre stage for the development of injury in the immature brain, and caspases have been shown to play a pivotal role during brain development and in response to injury. The inhibition of caspases using broad-spectrum agents such as Q-VD-OPh is neuroprotective in the immature brain. Caspase-6, an effector caspase, has been widely researched in neurodevelopmental disorders and found to be important following adult stroke, but its function in the neonatal brain has yet to be detailed. Furthermore, caspases may be important in microglial activation; microglia are required for optimal brain development and following injury, and their close involvement during neuronal cell death suggests that apoptotic cues such as caspase activation may be important in microglial activation. Therefore, in this study we aimed to investigate the possible apoptotic and non-apoptotic functions caspase-6 may have in the immature brain in response to hypoxia-ischaemia. We examined whether caspases are involved in microglial activation. We assessed cleaved caspase-6 expression following hypoxia-ischaemia and conducted primary microglial cultures to assess whether the broad-spectrum inhibitor Q-VD-OPh or caspase-6 gene deletion affected lipopolysaccharide (LPS)-mediated microglial activation and phenotype. We observed cleaved caspase-6 expression to be low but present in the cell body and cell processes in both a human case of white matter injury and 72 h following hypoxia-ischaemia in the rat. Gene deletion of caspase-6 did not affect the outcome of brain injury following mild (50 min) or severe (60 min) hypoxia-ischaemia. Interestingly, we did note that cleaved caspase-6 was co-localised with microglia that were not of apoptotic morphology. We observed that mRNA of a number of caspases was modulated by low-dose LPS stimulation of primary microglia. Q-VD-OPh treatment and caspase-6 gene deletion did not affect microglial activation but modified slightly the M2b

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

    International Nuclear Information System (INIS)

    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

  15. 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.

  16. Plant caspase-like proteases in plant programmed cell death

    OpenAIRE

    Xu, Qixian; Zhang, Lingrui

    2009-01-01

    Programmed cell death (PCD) is a genetically-controlled disassembly of the cell. In animal systems, the central core execution switch for apoptotic PCD is the activation of caspases (Cysteine-containing Aspartate-specific proteases). Accumulating evidence in recent years suggests the existence of caspase-like activity in plants and its functional involvement in various types of plant PCD, although no functional homologs of animal caspases were identified in plant genome. In this mini-review, ...

  17. Allopurinol’s effect on caspase-3 and caspase-8 activity in hypoxic-ischemic newborn rats

    Directory of Open Access Journals (Sweden)

    Kenan Özcan

    2013-03-01

    Full Text Available Aim: During reperfusion period of hypoxia-ischemia, cyclooxygenase and xanthine oxidase pathways are induced. A xanthine oxidase inhibitor, allopurinol has been shown to be neuroprotective in hypoxic- ischemic encephalopathy. Caspase-8 and caspase-3 have a key role in neuronal apoptosis. We aimed to test repeated doses of allopurinol’s effect on caspase-3 and caspase-8 activities in newborn rats with hypoxic-ischemic encephalopathy. Material and Method: Seven days old newborn rats were taken and there were 10 rats in each group. After Ethical Committee was approved (TIBDAM-25, rats were subjected to left carotid artery ligation and hypoxia (8% oxygen and 92% nitrogen for two and half hours. Hypoxic ischemic rats treated with 24 mg/kg allopurinol 30 minutes and 12 hours (AL48 group, and 30 minutes, 12 and 24 hours (AL72 group after hypoxic- ischemic insult. Twenty four hours after last dose, rats were decapitated. The others groups were sham and saline- treated hypoxic- ischemic (H-I group. Caspase- 3 and caspase- 8 activities were measured in both hemispheres.Results: There was no difference in caspase-3 and caspase-8 activities between right and left brain hemispheres in each group (p>0.05. Caspase-3 and caspase-8 activities were significantly lower in sham group when compared to H-I group, AL48 and AL72 groups (all of it, p=0.0001. Even though there were no difference activities of caspase- 3 and caspase- 8 between H-I group and AL48 group (p>0.05, activities of caspase- 3 and caspase- 8 in AL72 group were significantly lower than H-I group and AL48 group (respectively p= 0.0001, p=0.001. Conclusions: Decreased activities of caspase- 3 and caspase- 8 in AL72 group may suggest that totally dosage of 72 mg/kg allopurinol may be effective for reducing neuronal apoptosis in newborn rats with hypoxic- ischemic insult. (Turk Arch Ped 2013; 48: 48-52

  18. 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.

  19. 胱天蛋白酶(caspase)的前结构域%The Prodomain of Caspase

    Institute of Scientific and Technical Information of China (English)

    梁赤周; 马志章

    2001-01-01

    @@ 胱天蛋白酶(caspase)是白细胞介素-1β转化酶(interleukin-1 β enzyme,ICE)家族的总称,Caspase(cysteine aspartate-special proteases)的含义是该类蛋白酶的活性部位为极为保守的半胱氨酸(cysteine)残基(取第一个字母“c”),又特异性切割底物的天冬氨酸,用“aspase”表示,简称caspase,该酶在细胞凋亡过程中起关键作用,是目前研究的热点.现已发现的caspase有14种,它们均以无活性的酶原的形式存在,包括一个N末端前结构域(prodomain)及大、小两个亚单位.

  20. Apo cytochrome c inhibits caspases by preventing apoptosome formation

    International Nuclear Information System (INIS)

    Caspases are cysteine proteases and potent inducers of apoptosis. Their activation and activity is therefore tightly regulated. There are several mechanisms by which caspases can be activated but one key pathway involves release of holo cytochrome c from mitochondria into the cytoplasm. Cytoplasmic holo cytochrome c binds to apoptotic protease activating factor-1 (Apaf-1), driving the formation of an Apaf-1 oligomer (the apoptosome) which in turn binds and activates caspase-9. Previously we showed that the apo form of cytochrome c (lacking heme) can bind Apaf-1 and block both holo-dependent caspase activation in cell extracts and Bax-induced apoptosis in cells. Here we tested the ability of apo cytochrome c to inhibit caspase-9 activation induced by recombinant Apaf-1. Furthermore, using purified proteins and size exclusion chromatography we show that apo cytochrome c prevents holo cytochrome c-dependent apoptosome formation

  1. Proinflammatory caspase-2-mediated macrophage cell death induced by a rough attenuated Brucella suis strain.

    Science.gov (United States)

    Chen, Fang; Ding, Xicheng; Ding, Ying; Xiang, Zuoshuang; Li, Xinna; Ghosh, Debashis; Schurig, Gerhardt G; Sriranganathan, Nammalwar; Boyle, Stephen M; He, Yongqun

    2011-06-01

    Brucella spp. are intracellular bacteria that cause an infectious disease called brucellosis in humans and many domestic and wildlife animals. B. suis primarily infects pigs and is pathogenic to humans. The macrophage-Brucella interaction is critical for the establishment of a chronic Brucella infection. Our studies showed that smooth virulent B. suis strain 1330 (S1330) prevented programmed cell death of infected macrophages and rough attenuated B. suis strain VTRS1 (a vaccine candidate) induced strong macrophage cell death. To further investigate the mechanism of VTRS1-induced macrophage cell death, microarrays were used to analyze temporal transcriptional responses of murine macrophage-like J774.A1 cells infected with S1330 or VTRS1. In total 17,685 probe sets were significantly regulated based on the effects of strain, time and their interactions. A miniTUBA dynamic Bayesian network analysis predicted that VTRS1-induced macrophage cell death was mediated by a proinflammatory gene (the tumor necrosis factor alpha [TNF-α] gene), an NF-κB pathway gene (the IκB-α gene), the caspase-2 gene, and several other genes. VTRS1 induced significantly higher levels of transcription of 40 proinflammatory genes than S1330. A Mann-Whitney U test confirmed the proinflammatory response in VTRS1-infected macrophages. Increased production of TNF-α and interleukin 1β (IL-1β) were also detected in the supernatants in VTRS1-infected macrophage cell culture. Hyperphosphorylation of IκB-α was observed in macrophages infected with VTRS1 but not S1330. The important roles of TNF-α and IκB-α in VTRS1-induced macrophage cell death were further confirmed by individual inhibition studies. VTRS1-induced macrophage cell death was significantly inhibited by a caspase-2 inhibitor but not a caspase-1 inhibitor. The role of caspase-2 in regulating the programmed cell death of VTRS1-infected macrophages was confirmed in another study using caspase-2-knockout mice. In summary, VTRS1

  2. Acquired resistance to metformin in breast cancer cells triggers transcriptome reprogramming toward a degradome-related metastatic stem-like profile.

    Science.gov (United States)

    Oliveras-Ferraros, Cristina; Vazquez-Martin, Alejandro; Cuyàs, Elisabet; Corominas-Faja, Bruna; Rodríguez-Gallego, Esther; Fernández-Arroyo, Salvador; Martin-Castillo, Begoña; Joven, Jorge; Menendez, Javier A

    2014-01-01

    Therapeutic interventions based on metabolic inhibitor-based therapies are expected to be less prone to acquired resistance. However, there has not been any study assessing the possibility that the targeting of the tumor cell metabolism may result in unforeseeable resistance. We recently established a pre-clinical model of estrogen-dependent MCF-7 breast cancer cells that were chronically adapted to grow (> 10 months) in the presence of graded, millimolar concentrations of the anti-diabetic biguanide metformin, an AMPK agonist/mTOR inhibitor that has been evaluated in multiple in vitro and in vivo cancer studies and is now being tested in clinical trials. To assess what impact the phenomenon of resistance might have on the metformin-like "dirty" drugs that are able to simultaneously hit several metabolic pathways, we employed the ingenuity pathway analysis (IPA) software to functionally interpret the data from Agilent whole-human genome arrays in the context of biological processes, networks, and pathways. Our findings establish, for the first time, that a "global" targeting of metabolic reprogramming using metformin certainly imposes a great selective pressure for the emergence of new breast cancer cellular states. Intriguingly, acquired resistance to metformin appears to trigger a transcriptome reprogramming toward a metastatic stem-like profile, as many genes encoding the components of the degradome (KLK11, CTSF, FREM1, BACE-2, CASP, TMPRSS4, MMP16, HTRA1), cancer cell migration and invasion factors (TP63, WISP2, GAS3, DKK1, BCAR3, PABPC1, MUC1, SPARCL1, SEMA3B, SEMA6A), stem cell markers (DCLK1, FAK), and key pro-metastatic lipases (MAGL and Cpla2) were included in the signature. Because this convergent activation of pathways underlying tumor microenvironment interactions occurred in low-proliferative cancer cells exhibiting a notable downregulation of the G 2/M DNA damage checkpoint regulators that maintain genome stability (CCNB1, CCNB2, CDC20, CDC25C, AURKA

  3. Identification and Functional Characterization of Two Executioner Caspases in Crassostrea gigas

    OpenAIRE

    Tao Qu; Baoyu Huang; Linlin Zhang; Li Li; Fei Xu; Wen Huang; Chunyan Li; Yishuai Du; Guofan Zhang

    2014-01-01

    Caspase-3 and caspase-7 are two key effector caspases that play important roles in apoptotic pathways that maintain normal tissue and organ development and homeostasis. However, little is known about the sequence, structure, activity, and function of effector caspases upon apoptosis in mollusks, especially marine bivalves. In this study, we investigated the possible roles of two executioner caspases in the regulation of apoptosis in the Pacific oyster Crassostrea gigas. A full-length caspase-...

  4. Implication of Caspase-3 as a Common Therapeutic Target for Multineurodegenerative Disorders and Its Inhibition Using Nonpeptidyl Natural Compounds

    Directory of Open Access Journals (Sweden)

    Saif Khan

    2015-01-01

    Full Text Available Caspase-3 has been identified as a key mediator of neuronal apoptosis. The present study identifies caspase-3 as a common player involved in the regulation of multineurodegenerative disorders, namely, Alzheimer’s disease (AD, Parkinson’s disease (PD, Huntington’s disease (HD, and amyotrophic lateral sclerosis (ALS. The protein interaction network prepared using STRING database provides a strong evidence of caspase-3 interactions with the metabolic cascade of the said multineurodegenerative disorders, thus characterizing it as a potential therapeutic target for multiple neurodegenerative disorders. In silico molecular docking of selected nonpeptidyl natural compounds against caspase-3 exposed potent leads against this common therapeutic target. Rosmarinic acid and curcumin proved to be the most promising ligands (leads mimicking the inhibitory action of peptidyl inhibitors with the highest Gold fitness scores 57.38 and 53.51, respectively. These results were in close agreement with the fitness score predicted using X-score, a consensus based scoring function to calculate the binding affinity. Nonpeptidyl inhibitors of caspase-3 identified in the present study expeditiously mimic the inhibitory action of the previously identified peptidyl inhibitors. Since, nonpeptidyl inhibitors are preferred drug candidates, hence, discovery of natural compounds as nonpeptidyl inhibitors is a significant transition towards feasible drug development for neurodegenerative disorders.

  5. 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. PMID:27130972

  6. Prediction

    OpenAIRE

    Woollard, W.J.

    2006-01-01

    In this chapter we will look at the ways in which you can use ICT in the classroom to support hypothesis and prediction and how modern technology is enabling: pattern seeking, extrapolation and interpolation to meet the challenges of the information explosion of the 21st century.

  7. Mechanismus der Inhibition und Aktivierung der Caspase-aktivierten DNase

    OpenAIRE

    Kutscher, Daniel

    2011-01-01

    Der DNA Fragmentierungsfaktor DFF ist ein apoptotischer Protein Komplex, der aus den Untereinheiten CAD (Caspase aktivierte DNase) und ICAD (Inhibitor der Caspase aktivierten DNase) besteht. Da bis dato noch keine komplette Kristallstruktur des DFF Komplexes zur Verfügung steht, ist größtenteils immer noch unklar wie die beiden Untereinheiten, CAD und ICAD, auf molekularer Ebene miteinander interagieren. Der genaue Mechanismus der CAD Inhibition durch ICAD ist daher ebenso ungeklärt. In der v...

  8. The marine lipopeptide somocystinamide A triggers apoptosis via caspase 8

    OpenAIRE

    Wrasidlo, Wolf; Mielgo, Ainhoa; Torres, Vicente A; Barbero, Simone; Stoletov, Konstantin; Suyama, Takashi L.; Klemke, Richard L.; Gerwick, William H.; Carson, Dennis A.; Stupack, Dwayne G.

    2008-01-01

    Screening for novel anticancer drugs in chemical libraries isolated from marine organisms, we identified the lipopeptide somocystinamide A (ScA) as a pluripotent inhibitor of angiogenesis and tumor cell proliferation. The antiproliferative activity was largely attributable to induction of programmed cell death. Sensitivity to ScA was significantly increased among cells expressing caspase 8, whereas siRNA knockdown of caspase 8 increased survival after exposure to ScA. ScA rapidly and efficien...

  9. Discovery of Potent, Selective and Reversible Caspase-3 Inhibitors

    Institute of Scientific and Technical Information of China (English)

    Han Yongxin; John Tam; Paul Tawa; Donald W. Nicholson; Robert J. Zamboni; André Giroux; John Colucci; Christopher I. Bayly; Daniel J. Mckay; Sophie Roy; Steve Xanthoudakis; John Vaillancourt; Dita M. Rasper

    2004-01-01

    Recent studies towards understanding the molecular mechanisms of apoptosis have revealed the importance of a group of cysteinyl aspartate specific proteases, the caspases, in the programmed cell death process (Hengartner, M.O. Nature 2000, 407, 770). Caspase-3, in particular,has been characterized as the dominant effector caspase involved in the proteolytic cleavage of a variety of protein substrates including cytoskeletal proteins, kinases and DNA repair enzymes during apoptosis (Nicholson, D. W. Cell Death Differ. 1999, 6, 1028). The development of potent and selective caspase-3 inhibitors has thus emerged as an attractive therapeutic target. In the presentation,the identification of a series of potent, selective and reversible non-peptidyl caspase-3 inhibitors containing a pyrazinone core (1) will be presented. SAR optimization at R1, R2, R3 and R4 led to the discovery of inhibitors such as 2 with excellent in vitro activities (IC50 against rh-caspase-3: 5 nM; IC50 against camptothecin induced apoptotic cell death in NT2 cells: 20 nM). Compounds such as 2 also displayed excellent in vivo activities in a number of animal models of acute injuries (see: Methot, N. et al, J. Exp. Med. 2004, 119, 199; Toulmond, S. et al, British J. Pharm. 2004, 141,689; Holtzman,D.M. et al, JBC, 2002, 277, 30128), and selected examples will be discussed during the presentation.

  10. Methylene Blue Inhibits Caspases by Oxidation of the Catalytic Cysteine.

    Science.gov (United States)

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

    2015-01-01

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

  11. The calpain, caspase 12, caspase 3 cascade leading to apoptosis is altered in F508del-CFTR expressing cells.

    Directory of Open Access Journals (Sweden)

    Mathieu Kerbiriou

    Full Text Available In cystic fibrosis (CF, the most frequent mutant variant of the cystic fibrosis transmembrane conductance regulator (CFTR, F508del-CFTR protein, is misfolded and retained in the endoplasmic reticulum (ER. We previously showed that the unfolded protein response (UPR may be triggered in CF. Since prolonged UPR activation leads to apoptosis via the calcium-calpain-caspase-12-caspase-3 cascade and because apoptosis is altered in CF, our aim was to compare the ER stress-induced apoptosis pathway between wild type (Wt and F508del-CFTR expressing cells. Here we show that the calcium-calpain-caspase-12-caspase-3 cascade is altered in F508del-CFTR expressing cells. We propose that this alteration is involved in the altered apoptosis triggering observed in CF.

  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. Caspase 3 chemiluminiscence activity determination in apoptotic cells and design of Caspase 3 sensor

    Czech Academy of Sciences Publication Activity Database

    Lišková, Marcela; Klepárník, Karel; Pazdera, P.; Foret, František

    Brno : Ústav analytické chemie AV ČR, v. v. i, 2012 - (Foret, F.; Křenková, J.; Guttman, A.; Klepárník, K.; Boček, P.), s. 344-348 ISBN 978-80-904959-1-3. [CECE 2012. International Interdisciplinary Meeting on Bioanalysis /9./. Brno (CZ), 01.11.2012-02.11.2012] R&D Projects: GA ČR(CZ) GAP301/11/2055; GA ČR GAP206/11/2377; GA MŠk(CZ) EE2.3.20.0182; GA ČR GA203/08/1680 Institutional research plan: CEZ:AV0Z40310501 Keywords : Caspase 3 * apoptosis * chemiluminescence * FRET Subject RIV: CB - Analytical Chemistry, Separation http://hdl.handle.net/11104/0215649

  14. Prediction

    CERN Document Server

    Sornette, Didier

    2010-01-01

    This chapter first presents a rather personal view of some different aspects of predictability, going in crescendo from simple linear systems to high-dimensional nonlinear systems with stochastic forcing, which exhibit emergent properties such as phase transitions and regime shifts. Then, a detailed correspondence between the phenomenology of earthquakes, financial crashes and epileptic seizures is offered. The presented statistical evidence provides the substance of a general phase diagram for understanding the many facets of the spatio-temporal organization of these systems. A key insight is to organize the evidence and mechanisms in terms of two summarizing measures: (i) amplitude of disorder or heterogeneity in the system and (ii) level of coupling or interaction strength among the system's components. On the basis of the recently identified remarkable correspondence between earthquakes and seizures, we present detailed information on a class of stochastic point processes that has been found to be particu...

  15. Caspase-2 protects against oxidative stress in vivo.

    Science.gov (United States)

    Shalini, S; Puccini, J; Wilson, C H; Finnie, J; Dorstyn, L; Kumar, S

    2015-09-17

    Caspase-2 belongs to the caspase family of cysteine proteases with established roles in apoptosis. Recently, caspase-2 has been implicated in nonapoptotic functions including maintenance of genomic stability and tumor suppression. Our previous studies demonstrated that caspase-2 also regulates cellular redox status and delays the onset of several ageing-related traits. In the current study, we tested stress tolerance ability in caspase-2-deficient (Casp2(-/-)) mice by challenging both young and old mice with a low dose of the potent reactive oxygen species (ROS) generator, PQ that primarily affects lungs. In both groups of mice, PQ induced pulmonary damage. However, the lesions in caspase-2 knockout mice were consistently and reproducibly more severe than those in wild-type (WT) mice. Furthermore, serum interleukin (IL)-1β and IL-6 levels were higher in PQ-exposed aged Casp2(-/-) mice indicating increased inflammation. Interestingly, livers from Casp2(-/-) mice displayed karyomegaly, a feature commonly associated with ageing and aneuploidy. Given that Casp2(-/-) mice show impaired antioxidant defense, we tested oxidative damage in these mice. Protein oxidation significantly increased in PQ-injected old Casp2(-/-) mice. Moreover, FoxO1, SOD2 and Nrf2 expression levels were reduced and induction of superoxide dismutase (SOD) and glutathione peroxidase activity was not observed in PQ-treated Casp2(-/-) mice. Strong c-Jun amino-terminal kinase (JNK) activation was observed in Casp2(-/-) mice, indicative of increased stress. Together, our data strongly suggest that caspase-2 deficiency leads to increased cellular stress largely because these mice fail to respond to oxidative stress by upregulating their antioxidant defense mechanism. This makes the mice more vulnerable to exogenous challenges and may partly explain the shorter lifespan of Casp2(-/-) mice. PMID:25531319

  16. The miRNAs and their regulatory networks responsible for pollen abortion in Ogura-CMS Chinese cabbage revealed by high-throughput sequencing of miRNAs, degradomes and transcriptomes

    Directory of Open Access Journals (Sweden)

    Xiaochun eWei

    2015-10-01

    Full Text Available Chinese cabbage (Brassica rapa ssp. pekinensis is one of the most important vegetables in Asia and is cultivated across the world. Ogura-type cytoplasmic male sterility (Ogura-CMS has been widely used in the hybrid breeding industry for Chinese cabbage and many other cruciferous vegetables. Although, the cause of Ogura-CMS has been localized to the orf138 locus in the mitochondrial genome, however, the mechanism by which nuclear genes respond to the mutation of the mitochondrial orf138 locus is unclear. In this study, a series of whole genome small RNA, degradome and transcriptome analyses were performed on both Ogura-CMS and its maintainer Chinese cabbage buds using deep sequencing technology. A total of 289 known miRNAs derived from 69 families (including 23 new families first reported in B. rapa and 426 novel miRNAs were identified. Among these novel miRNAs, both 3-p and 5-p miRNAs were detected on the hairpin arms of 138 precursors. Ten known and 49 novel miRNAs were down-regulated, while one known and 27 novel miRNAs were up-regulated in Ogura-CMS buds compared to the fertile plants. Using degradome analysis, a total of 376 mRNAs were identified as targets of 30 known miRNA families and 100 novel miRNAs. A large fraction of the targets were annotated as reproductive development related. Our transcriptome profiling revealed that the expression of the targets was finely tuned by the miRNAs. Two novel miRNAs were identified that were specifically highly expressed in Ogura-CMS buds and sufficiently suppressed two pollen development essential genes: sucrose transporter SUC1 and H+-ATPase 6. These findings provide clues for the contribution of a potential miRNA regulatory network to bud development and pollen engenderation. This study contributes new insights to the communication between the mitochondria and chromosome and takes one step toward filling the gap in the regulatory network from the orf138 locus to pollen abortion in Ogura-CMS plants

  17. Oxidative modification of caspase-9 facilitates its activation via disulfide-mediated interaction with Apaf-1

    Institute of Scientific and Technical Information of China (English)

    Yong Zuo; Binggang Xiang; Jie Yang; Xuxu Sun; Yumei Wang; Hui Cang; Jing Yi

    2009-01-01

    Intracellular reactive oxygen species (ROS) are known to regulate apoptosis. Activation of caspase-9, the initial caspase in the mitochondrial apoptotic cascade, is closely associated with ROS, but it is unclear whether ROS regulate caspase-9 via direct oxidative modification. The present study aims to elucidate the molecular mechanisms by which ROS mediate caspase-9 activation. Our results show that the cellular oxidative state facilitates caspase-9 activation. Hydrogen peroxide treatment causes the activation of caspase-9 and apoptosis, and promotes an interaction between caspase-9 and apoptotic protease-activating factor 1 (Apaf-1) via disulfide formation. In addition, in an in vitro mitochondria-free system, the thiol-oxidant diamide promotes auto-cleavage of caspase-9 and the caspase-9/ Apaf-1 interaction by facilitating the formation of disulfide-linked complexes. Finally, a point mutation at C403 of caspase-9 impairs both H202-promoted caspase-9 activation and interaction with Apaf-1 through the abolition of disulfide formation. The association between cytochrome c and the C403S mutant is significantly weaker than that between cytochrome c and wild-type caspase-9, indicating that oxidative modification of caspase-9 contributes to apoptosome formation under oxidative stress. Taken together, oxidative modification of caspase-9 by ROS can mediate its interaction with Apaf-1, and can thus promote its auto-cleavage and activation. This mechanism may facilitate apoptosome formation and caspase-9 activation under oxidative stress.

  18. Caspase-11 Modulates Inflammation and Attenuates Toxoplasma gondii Pathogenesis.

    Science.gov (United States)

    Coutermarsh-Ott, Sheryl L; Doran, John T; Campbell, Caroline; Williams, Tere M; Lindsay, David S; Allen, Irving C

    2016-01-01

    Toxoplasma gondii is an obligate intracellular parasite that is the etiologic agent responsible for toxoplasmosis. Infection with T. gondii results in activation of nucleotide binding domain and leucine rich repeat containing receptors (NLRs). NLR activation leads to inflammasome formation, the activation of caspase-1, and the subsequent cleavage of IL-1β and IL-18. Recently, a noncanonical inflammasome has been characterized which functions through caspase-11 and appears to augment many biological functions previously considered to be dependent upon the canonical inflammasome. To better elucidate the function of this noncanonical inflammasome in toxoplasmosis, we utilized Asc (-/-) and Casp11 (-/-) mice and infected these animals with T. gondii. Our data indicates that caspase-11 modulates the innate immune response to T. gondii through a mechanism which is distinct from that currently described for the canonical inflammasome. Asc (-/-) mice demonstrated increased disease pathogenesis during the acute phase of T. gondii infection, whereas Casp11 (-/-) mice demonstrated significantly attenuated disease pathogenesis and reduced inflammation. This attenuated host response was associated with reduced local and systemic cytokine production, including diminished IL-1β. During the chronic phase of infection, caspase-11 deficiency resulted in increased neuroinflammation and tissue cyst burden in the brain. Together, our data suggest that caspase-11 functions to protect the host by enhancing inflammation during the early phase of infection in an effort to minimize disease pathogenesis during later stages of toxoplasmosis. PMID:27378827

  19. Preparation of anti-mouse caspase-12 mRNA hammerhead ribozyme and identification of its activity in vitro

    Institute of Scientific and Technical Information of China (English)

    Shan Jiang; Qing Xie; Wei Zhang; Xia-Qiu Zhou; You-Xin Jin

    2005-01-01

    AIM: To prepare and identify specific anti-mouse caspase12 hammerhead ribozymes in vitro, in order to select a more effective ribozyme against mouse caspase-12 as a potential tool to rescue cells from endoplasmic reticulum stress induced apoptosis.METHODS: Two hammerhead ribozymes directed separately against 138 and 218 site of nucleotide of mouse caspase-12 mRNA were designed by computer software,and their DNA sequences were synthesized. The synthesized ribozymes were cloned into an eukaryotic expression vector-neorpBSKU6 and embedded in U6 SnRNA context for further study. Mouse caspase-12 gene segment was cloned into PGEM-T vector under the control of T7 RNA polymerase promoter (containing gene sequence from positions nt 41 to nt 894) as target. In vitro transcription both the ribozymes and target utilize T7 promoter. The target was labeled with [α-32P]UTP, while ribozymes were not labeled. After gel purification the RNAs were dissolved in RNase free water. Ribozyme and target were incubated for 90 min at 37 ℃ in reaction buffer (40 mmol/L Tris-HCL,pH 7.5, 10 mmol/L Mg2+). Molar ratio of ribozyme vs target was 30:1. Samples were analyzed on 6% PAGE (containing 8 mol/L urea).RESULTS: Both caspase-12 and ribozyme gene sequences were successfully cloned into expression vector confirmed by sequencing. Ribozymes and caspase-12 mRNA were obtained by in vitro transcription. Cleavage experiment showed that in a physiological similar condition (37 ℃,pH 7.5), Rz138 and Rz218 both cleaved targets at predicted sites, for Rz138 the cleavage efficiency was about 100%,for Rz218 the value was 36.66%.CONCLUSION: Rz138 prepared in vitro can site specific cleave mouse caspase-12 mRNA with an excellent efficiency. It shows a potential to suppress the expression of caspase-12 in vivo, thus provided a new way to protect cells from ER stress induced apoptosis.

  20. Nitric oxide induces caspase activity in boar spermatozoa.

    Science.gov (United States)

    Moran, J M; Madejón, L; Ortega Ferrusola, C; Peña, F J

    2008-07-01

    Nitric oxide (NO) is a highly reactive free radical that plays a key role in intra- and intercellular signaling. Production of radical oxygen species and an apoptotic-like phenomenon have recently been implicated in cryodamage during sperm cryopreservation. The objective of the present study was to evaluate the effect of sodium nitroprusside (SNP), an NO donor, on boar sperm viability. Semen samples were pooled from four boars that were routinely used for artificial insemination. Flow cytometry was used to compare semen incubated with SNP to control semen. Specifically, NO production was measured using the NO indicator dye diaminofluorescein diacetate, and caspase activity was determined using the permeable pan-caspase inhibitor Z-VAD linked to FITC. SNP induced a significant increase in the percentage of sperm cells showing caspase activity, from 9.3% in control samples to 76.2% in SNP-incubated samples (Pboar sperm damage. PMID:18433854

  1. 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.

  2. Substrate-Induced Conformational Changes Occur in All Cleaved Forms of Caspase-6

    Energy Technology Data Exchange (ETDEWEB)

    S Vaidya; E Velazquez-Delgado; G Abbruzzese; J Hardy

    2011-12-31

    Caspase-6 is an apoptotic cysteine protease that also governs disease progression in Huntington's and Alzheimer's diseases. Caspase-6 is of great interest as a target for treatment of these neurodegenerative diseases; however, the molecular basis of caspase-6 function and regulation remains poorly understood. In the recently reported structure of caspase-6, the 60's and 130's helices at the base of the substrate-binding groove extend upward, in a conformation entirely different from that of any other caspase. Presently, the central question about caspase-6 structure and function is whether the extended conformation is the catalytically competent conformation or whether the extended helices must undergo a large conformational rearrangement in order to bind substrate. We have generated a series of caspase-6 cleavage variants, including a novel constitutively two-chain form, and determined crystal structures of caspase-6 with and without the intersubunit linker. This series allows evaluation of the role of the prodomain and intersubunit linker on caspase-6 structure and function before and after substrate binding. Caspase-6 is inherently more stable than closely related caspases. Cleaved caspase-6 with both the prodomain and the linker present is the most stable, indicating that these two regions act in concert to increase stability, but maintain the extended conformation in the unliganded state. Moreover, these data suggest that caspase-6 undergoes a significant conformational change upon substrate binding, adopting a structure that is more like canonical caspases.

  3. 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.

  4. Modulation of intracellular caspase machinery using organ culture approach

    Czech Academy of Sciences Publication Activity Database

    Matalová, Eva; Fleischmannová, Jana; Norek, Adam; Míšek, Ivan

    Brno : Fyziologický ústav LF MU, 2007, 8P. [Fyziologické dny /83./. Brno (CZ), 06.02.2007-08.02.2007] Institutional research plan: CEZ:AV0Z50450515 Keywords : caspase machinery Subject RIV: EB - Genetics ; Molecular Biology

  5. Caspase-7 tooth germ and hair follicle development and maintenance

    Czech Academy of Sciences Publication Activity Database

    Veselá, Barbora; Matalová, Eva

    Rome : ECDO, 2012. 257-257. [Euroconference from Death to Eternity /20./ and Training course on Concepts and Methods in Programmed Cell Death /9./. 14.09.2012-17.09.2012, Rome] R&D Projects: GA ČR(CZ) GAP502/12/1285 Institutional support: RVO:67985904 Keywords : caspase-7 * hair follicle * tooth germ Subject RIV: EA - Cell Biology

  6. Inhibition of caspases prevents ototoxic and ongoing hair cell death

    Science.gov (United States)

    Matsui, Jonathan I.; Ogilvie, Judith M.; Warchol, Mark E.

    2002-01-01

    Sensory hair cells die after acoustic trauma or ototoxic insults, but the signal transduction pathways that mediate hair cell death are not known. Here we identify several important signaling events that regulate the death of vestibular hair cells. Chick utricles were cultured in media supplemented with the ototoxic antibiotic neomycin and selected pharmacological agents that influence signaling molecules in cell death pathways. Hair cells that were treated with neomycin exhibited classically defined apoptotic morphologies such as condensed nuclei and fragmented DNA. Inhibition of protein synthesis (via treatment with cycloheximide) increased hair cell survival after treatment with neomycin, suggesting that hair cell death requires de novo protein synthesis. Finally, the inhibition of caspases promoted hair cell survival after neomycin treatment. Sensory hair cells in avian vestibular organs also undergo continual cell death and replacement throughout mature life. It is unclear whether the loss of hair cells stimulates the proliferation of supporting cells or whether the production of new cells triggers the death of hair cells. We examined the effects of caspase inhibition on spontaneous hair cell death in the chick utricle. Caspase inhibitors reduced the amount of ongoing hair cell death and ongoing supporting cell proliferation in a dose-dependent manner. In isolated sensory epithelia, however, caspase inhibitors did not affect supporting cell proliferation directly. Our data indicate that ongoing hair cell death stimulates supporting cell proliferation in the mature utricle.

  7. Programmed cell death in plants and caspase-like activities

    NARCIS (Netherlands)

    Gaussand, Gwénael Martial Daniel Jean-Marie

    2007-01-01

    The development of multicellular organisms involves an important balance between cell growth, cell division and cell death. In animals, programmed cell death (PCD) plays a key role by forming and deleting structures, controlling cell numbers and eliminating abnormal damaged cells. Caspases were foun

  8. Angelica polymorpha Maxim Induces Apoptosis of Human SH-SY5Y Neuroblastoma Cells by Regulating an Intrinsic Caspase Pathway.

    Science.gov (United States)

    Rahman, Md Ataur; Bishayee, Kausik; Huh, Sung-Oh

    2016-02-29

    Angelica polymorpha Maxim root extract (APRE) is a popular herbal medicine used for treating stomachache, abdominal pain, stomach ulcers, and rheumatism; however the effect of APRE on cancer cells has not yet been explored. Here, we examined APRE cytotoxicity seen on target neuroblastoma cells (NB) using cell viability assays, DAPI visualization of fragmented DNA, and Western blotting analysis of candidate signaling pathways involved in proliferation and apoptosis. We demonstrated that APRE reduced cell viability in NB to a greater extent than in fibroblast cells. In addition, we found that APRE could inhibit the three classes of MAPK proteins and could also down-regulate the PI3K/AKT/GSK-3β activity all being relevant for proliferation and survival. APRE could also up-regulate Bax expression and down-regulate Bcl-2 and Mcl-1. With APRE treatment, depolarization of mitochondria membrane potential and activation of caspase-3 was demonstrated in the SH-SY5Y cells. We could not found increased activity of death receptor and caspase-8 as markers of the extrinsic apoptosis pathway for the APRE treated cells. In presence of a caspase-3 siRNA and a pan-caspase inhibitor, APRE could not reduce the viability of NB cells to a significant degree. So we predicted that with APRE, the intrinsic pathway was solely responsible for inducing apoptosis as we also showed that the non-caspase autophagy pathway or ER stress-ROS mediated pathways were not involved. These findings demonstrate that an intrinsic mitochondria-mediated apoptosis pathway mediates the apoptotic effects of APRE on SH-SY5Y cells, and that APRE shows promise as a novel agent for neuroblastoma therapy. PMID:26674967

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

    International Nuclear Information System (INIS)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    International Nuclear Information System (INIS)

    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

  12. Host-Dependent Trigger of Caspases and Apoptosis by Legionella pneumophila▿ †

    OpenAIRE

    Santic, Marina; Asare, Rexford; Doric, Miljenko; Abu Kwaik, Yousef

    2007-01-01

    The Dot/Icm system of Legionella pneumophila triggers activation of caspase-3 during early stages of infection of human macrophages, but apoptosis is delayed until late stages of infection. During early stages of infection of mouse macrophages, the organism triggers rapid caspase-1-mediated cytotoxicity, which is mediated by bacterial flagellin. However, it is not known whether caspase-1 is triggered by L. pneumophila in human macrophages or whether caspase-3 is activated in permissive or non...

  13. Expression and in vitro cleavage activity of anti-caspase-7 hammerhead ribozymes

    Institute of Scientific and Technical Information of China (English)

    Wei Zhang; Qing Xie; Xia-Qiu Zhou; Shan Jiang; You-Xin Jin

    2004-01-01

    AIM: To prepare hammerhead ribozymes against mouse caspase-7 and identify their cleavage activityin vitro, in order to select a ribozyme with specific cleavage activity against mouse caspase-7 as a potential gene therapy for apoptosis-related diseases.METHODS: Anti-caspase-7 ribozymes targeting sites 333and 394 (named Rz333 and Rz394) were designed by computer software, and their DNA sequences encoding ribozymes were synthesized. Caspase-7 DNA sequence was acquired by RT-PCR. Ribozymes and caspase-7 DNA obtained byin vitro transcription were cloned into pBSKneo U6' and pGEM-T vectors, respectively. The cleavage activity of ribozymes against mouse caspase-7 was identified by cleavage experimentsin vitro.RESULTS: Rz333 and Rz394 were designed and their DNA sequences were synthesized respectively. The expression vector of caspase-7 and plasmids containing Rz333 and Rz394 were reconstructed successfully. Ribozymes and caspase-7 mRNA were expressed byin vitro transcription.In vitro cleavage experiment showed that 243-nt and 744-nt segments were produced after caspase-7 mRNA was mixed with Rz333 in equivalent, and the cleavage efficiency was 67.98%. No cleaved segment was observed when caspase-7 mRNA was mixed with Rz394.CONCLUSION: Rz333 can site-specific cleave mouse caspase-7 mRNA, and it shows a potential for gene therapy of apoptosis-related diseases by down-regulating gene expression of caspase-7.

  14. CONSTRUCTION OF ACTIVE RECOMBINANT CASPASE-3 EUKARYOTIC EXPRESSION PLA SMID AND EFFECT OF r-CASPASE-3 ON APOPTOSIS OF PANCREATIC CARCINOMA CELLS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To construct active recombinant cas pa ses-3 gene(r-caspases-3)eukaryotic expression plasmid and observe the apoptos is inducing activity of r-caspase-3 in pancreatic carcinoma cells. Methods pcDNA3.1(+)/r-caspase-3 was constructed and pan creatic carcinoma cells(PC-Ⅱ)were transfected with the pcDNA3.1(+)/r-caspases -3 by liposomes(LipofectAMINE).The expression of r-Caspase-3 mRNA in pancreat ic carcinoma cells was detected by reverse transcription process of the polymera se chain reaction(RT-PCR), and the signs of apoptosis were examined in pancreat ic carcinoma cells by the methods of the DNA electrophoresis and flow cytometry analysis(FACS).Results The sequence inserted in pBlueSKM/r-Caspase-3 p lasmid was coincident with that of the r-caspases-3. The evaluation result of pcDNA3.1(+)/r-caspases-3 through enzyme cutting was correct. A 894bp strap was observed by RT-PCR after pancreatic carcinoma cells being transfected with the pcDNA3.1(+)/r-caspases-3 by liposomes. No strap was found in control groups. A characteristic DNA ladder was observed in pancreatic carcinoma cells DNA elect r ophoresis, and transparent hypodiploid karyotype peak was found by FACS. Conclusion The plasmid of pcDNA3.1(+)/r-Caspase-3 was c onstructed successfully, the expression of r-Caspase-3 mRMA in pancreatic carc inoma cells was confirmed by RT-PCR, and pcDNA3.1(+)/r-Caspase-3 can induce a poptosis in pancreatic carcinoma cells.

  15. Zinc-mediated regulation of caspases activity: dose-dependent inhibition or activation of caspase-3 in the human Burkitt lymphoma B cells (Ramos).

    Science.gov (United States)

    Schrantz, N; Auffredou, M T; Bourgeade, M F; Besnault, L; Leca, G; Vazquez, A

    2001-02-01

    Divalent cations, including Zinc and Manganese ions, are important modulators of cell activation. We investigated the ability of these two divalent cations to modulate apoptosis in human Burkitt lymphoma B cells line (Ramos). We found that Zinc (from 10 to 50 microM) inhibited Manganese-induced caspase-3 activation and apoptosis of Ramos cells. Higher concentration of Zinc (50 to 100 microM) did not prevent Manganese-mediated apoptosis but rather increased cell death among Ramos cells. This Zinc-mediated cell death was associated with apoptotic features such as cell shrinkage, the presence of phosphatidylserine residues on the outer leaflet of the cells, chromatin condensation, DNA fragmentation and decrease of mitochondrial transmembrane potential. Zinc-mediated apoptosis was associated with caspase-9 and caspase-3 activation as revealed by the appearance of active p35 fragment of caspase-9 and p19 and p17 of caspase-3 as well as in vivo cleavage of PARP and of a cell-permeable fluorogenic caspase-3 substrate (Phiphilux-G(1)D(2)). Both Zinc-mediated apoptosis and caspase-3 activation were prevented by the cell-permeable, broad-spectrum inhibitor of caspases (zVAD-fmk) or overexpression of bcl-2. In addition, we show that Zinc-induced loss of transmembrane mitochondrial potential is a caspase-independent event, since it is not modified by the presence of zVAD-fmk, which is inhibited by overexpression of bcl-2. These results indicate that depending on its concentration, Zinc can exert opposite effects on caspase-3 activation and apoptosis in human B lymphoma cells: concentrations below 50 microM inhibit caspase-3 activation and apoptosis whereas higher concentrations of Zinc activate a death pathway associated with apoptotic-like features and caspase-3 activation. PMID:11313717

  16. 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)

    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

  17. NP24 induces apoptosis dependent on caspase-like activity in Saccharomyces cerevisiae.

    Science.gov (United States)

    Higuchi, Naoki; Ito, Yasuhiro; Kato, Jun; Ogihara, Jun; Kasumi, Takafumi

    2016-06-01

    Tomato NP24 is a homolog of osmotin, a PR-5 protein from tobacco that can initiate apoptosis in yeast via PHO36 in the plasma membrane. We cloned and sequenced NP24 from tomato cv. Momotaro. Based on phylogenetic analysis, NP24 from Momotaro belonged to the Solanaceae clade. The amino acid sequence was identical to that of cv. Ailsa Craig including signal peptide, but the residues predicted to interact with the adiponectin receptor, ADIPOR, were slightly different from osmotin. Recombinant NP24 (rNP24) was expressed in a reductase-deficient mutant of Escherichia coli as host cell, and purified from cell extract by affinity chromatography. Purified rNP24 significantly inhibited growth of Saccharomyces cerevisiae wild-type spheroplasts. In contrast, growth of PHO36 deletion mutant (ΔIzh2) spheroplasts was not inhibited. Moreover, rNP24 induced significant activity of reactive oxygen species, caspase-like activity, and also nuclear fragmentation in wild-type spheroplast cells. These results demonstrated that rNP24 from Momotaro greatly influenced cell viability due to triggering apoptosis through PHO36. Notably, apoptosis induced by NP24 was caspase-like protease dependent. PMID:26589784

  18. 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 ostatní: GA ČR(CZ) GAP502/12/1285 Institutional support: RVO:68081715 ; RVO:67985904 Keywords : apoptosis * bioluminescence * caspase-3 Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.161, year: 2013

  19. Molar tooth development in caspase-3 deficient mice

    Czech Academy of Sciences Publication Activity Database

    Matalová, Eva; Sharpe, P. T.; Lakhani, S. A.; Roth, K. A.; Flavell, R. A.; Šetková, Jana; Míšek, Ivan; Tucker, A. S.

    2006-01-01

    Roč. 50, 5 (2006), s. 491-497. ISSN 0214-6282 R&D Projects: GA AV ČR KJB500450503; GA MŠk OC B23.001 Grant ostatní: European Molecular Biology Organization ASTF195.00-05; NIH NS41962 Institutional research plan: CEZ:AV0Z50450515 Keywords : tooth development * dental apoptosis * caspase-3 mutant Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.577, year: 2006

  20. Inactivation of the Human Vitamin D Receptor by Caspase-3

    OpenAIRE

    Malloy, Peter J.; Feldman, David

    2008-01-01

    Calcitriol actions are mediated by the vitamin D receptor (VDR), a nuclear transcription factor of the steroid-retinoid-thyroid nuclear receptor gene superfamily. Calcitriol inhibits the growth of many cells including cancer cells by inducing cell cycle arrest. In some cancer cell lines, calcitriol also induces apoptosis. In the LNCaP prostate cancer cell line, induction of apoptosis and caspase-3/7 activities by staurosporine (STS) abolished [3H]1,25-dihydroxy vitamin D3 binding and VDR prot...

  1. Tumor promotion by caspase-resistant retinoblastoma protein

    Science.gov (United States)

    Borges, Helena L.; Bird, Jeff; Wasson, Katherine; Cardiff, Robert D.; Varki, Nissi; Eckmann, Lars; Wang, Jean Y. J.

    2005-01-01

    The retinoblastoma (RB) protein regulates cell proliferation and cell death. RB is cleaved by caspase during apoptosis. A mutation of the caspase-cleavage site in the RB C terminus has been made in the mouse Rb-1 locus; the resulting Rb-MI mice are resistant to endotoxin-induced apoptosis in the intestine. The Rb-MI mice do not exhibit increased tumor incidence, because the MI mutation does not disrupt the Rb tumor suppressor function. In this study, we show that Rb-MI can promote the formation of colonic adenomas in the p53-null genetic background. Consistent with this tumor phenotype, Rb-MI reduces colorectal epithelial apoptosis and ulceration caused by dextran sulfate sodium. By contrast, Rb-MI does not affect the lymphoma phenotype of p53-null mice, in keeping with its inability to protect thymocytes and splenocytes from apoptosis. The Rb-MI protein is expressed and phosphorylated in the tumors, thereby inactivating its growth suppression function. These results suggest that RB tumor suppressor function, i.e., inhibition of proliferation, is inactivated by phosphorylation, whereas RB tumor promoting function, i.e., inhibition of apoptosis, is inactivated by caspase cleavage. PMID:16227443

  2. Caspase 6 has a protective role in SOD1(G93A) transgenic mice.

    Science.gov (United States)

    Hogg, Marion C; Mitchem, Mollie R; König, Hans-Georg; Prehn, Jochen H M

    2016-06-01

    In amyotrophic lateral sclerosis (ALS), it has been suggested that the process of neurodegeneration starts at the neuromuscular junction and is propagated back along axons towards motor neurons. Caspase-dependent pathways are well established as a cause of motor neuron death, and recent work in other disease models indicated a role for caspase 6 in axonal degeneration. Therefore we hypothesised that caspase 6 may be involved in motor neuron death in ALS. To investigate the role of caspase 6 in ALS we profiled protein levels of caspase-6 throughout disease progression in the ALS mouse model SOD1(G93A); this did not reveal differences in caspase 6 levels during disease. To investigate the role of caspase 6 further we generated a colony with SOD1(G93A) transgenic mice lacking caspase 6. Analysis of the transgenic SOD1(G93A); Casp6(-/-) revealed an exacerbated phenotype with motor dysfunction occurring earlier and a significantly shortened lifespan when compared to transgenic SOD1(G93A); Casp6(+/+) mice. Immunofluorescence analysis of the neuromuscular junction revealed no obvious difference between caspase 6(+/+) and caspase 6(-/-) in non-transgenic mice, while the SOD1(G93A) transgenic mice showed severe degeneration compared to non-transgenic mice in both genotypes. Our data indicate that caspase-6 does not exacerbate ALS pathogenesis, but may have a protective role. PMID:26976329

  3. Role of Caspase and MMPs in Amniochorionic during PROM

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Objective To study the role of cysteine aspartic acid-specific protease-3 (caspase-3),matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of matrix metallo proteinase2 (TIMP-2) in human amniochorionic membranes during premature rupture of human fetal membranes (PROM).Methods Amniochorionic membranes were collected from the following groups of women: women with spontaneous PROM (n=8), women with normal labor in term after vaginal delivery(n=8) and women undergoing elective repeat cesarean section (C-section) before the onset of labor and who had no complications of pregnancy (n=8). Caspase-3 peptides were studies with use of immunohistochemistry. Messenger ribonucleic acid (mRNA) expression for MMP-2 and its specific inhibitors TIMP-2was studied with use of reverse transcriptase-polymerase chain reaction (RT-PCR).Results 1) The expressions of Caspase-3 peptides were 62.86 ± 3.83% in PROM group, 42.33 ±2.99% in vaginal delivery group, and 20.97 ± 2.94% in C- section group. There were statistically significant changes among the three groups (P<0.05).Immunohistochemistry demonstrated the presence of Caspase-3 in the amniotic epithelial cells and chorionic cytotrophoblast cells. 2) The expressions of MMP-2 were 84. 92 ±3.68% in PROM group, 32.65 ± 2.34% in vaginal delivery group, and 30.65 ±2.77% in C-section group. There were statistically significant changes between PROM and C-section group (P<0.05). 3) The expressions of TIMP-2 were 42. 01 ± 12.17% in PROM group, 73.01 ± 14.82% in vaginal delivery group, and 88.47 ± 6.51% in C- section group. There were statistically significant changes among the three groups (P<0.05).Conclusion Caspase-3 gene expressed more in PROM than in comparative group,which caused human fetal membranes cell apoptosis increased.The expression MMP-2increased and TIMP-2 dropped in PROM, which can increase the ECM decomposing.Cell apoptosis increased and extra cellular matrix degradation dropped, which may cause weakening of the

  4. Development of cell death-based method for the selectivity screening of caspase-1 inhibitors

    DEFF Research Database (Denmark)

    Chopra, Puneet; Gupta, Shashank; Dastidar, Sunanda G; Ray, Abhijit

    2009-01-01

    used for the selectivity screening of multiple caspases in a biologically relevant context in a single assay. In this study, we have developed an assay in which DNA fragmentation, a hallmark of apoptosis, of Jurkat cell line was examined post induction with etoposide in the presence or absence of...... belonging to caspase-1 family (1, 4 and 5) are not present in the Jurkat cells or might not be involved in the etoposide-induced DNA fragmentation. Since the inhibition of caspases 3, 8 and 9 is accompanied by the down regulation of the activity of a cascade of caspases (caspases 2, 6, 7, 9 and 10......Caspase-1 selective inhibitors are novel therapeutic agents for inflammatory diseases. Selectivity assays for caspases can be initiated with purified enzyme, making these assays very costly and time consuming. Therefore, there is a need to develop a fast and reliable cell-based assay, which can be...

  5. Mechanism of mitochondrial respiratory control in caspase-3 induced positive feed back loop in apoptosis

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Caspase-3 plays a central role in the execution of apoptosis. Besides many substrates of caspase-3, mitochondria seem to be one of the candidate targets in the apoptotic process. We evaluated the effects of caspase-3 on the isolated mitochondria in detail, and especially focused on the mechanism involved in mitochondrial functions, which were not fully assessed till now. Our results showed that recombinant caspase-3 induced the increase of superoxide production, the dissipation of mitochondrial membrane potential and rate increasing of mitochondrial state 4 respiration. Caspases inhibitor, z-VAD-fmk can inhibit these effects of caspase-3 on mitochondria. Bcl-xL and cyclosporin A were also shown to be able to inhibit these changes. These results suggested a possible mechanism in caspase-3 induced disruption of mitochondrial membrane barrier which formed a positive feedback loop in apoptosis.

  6. Parthenolide protects human lens epithelial cells from oxidative stress-induced apoptosis via inhibition of activation of caspase-3 and caspase-9

    Institute of Scientific and Technical Information of China (English)

    Hangping Yao; Xiajing Tang; Xueting Shao; Lei Feng; Nanping Wu; Ke Yao

    2007-01-01

    The apoptosis of lens epithelial cells has been proposed as the common basis of cataract formation, with oxidative stress as the major cause. This study was performed to investigate the protective effect of the herbal constituent parthenolide against oxidative stress-induced apoptosis of human lens epithelial (HLE) cells and the possible molecular mechanisms involved. HLE cells (SRA01-04) were incubated with 50 μM H2O2 in the absence or presence of different doses of parthenolide (10, 20 and 50μM). To study apoptosis, the cells were assessed by morphologic examination and Annexin V-propidium iodide double staining flow cytometry; to investigate the underlying molecular mechanisms, the expression of caspase-3 and caspase-9 were assayed by Western blot and quantitative RT-PCR, and the activities of caspase-3 and caspase-9 were measured by a Chemicon caspase colorimetric activity assay kit. Stimulated with H2O2 for 18h, a high fraction of HLE cells underwent apoptosis, while in the presence of parthenolide of different concentrations, dose-dependent blocking of HLE cell apoptosis was observed. The expression of caspase-3 and caspase-9 induced by H2O2 in HLE cells was significantly reduced by parthenolide both at the protein and mRNA levels, and the activation of caspase-3 and caspase-9 was also suppressed by parthenolide in a dose-dependent manner. In conclusion, parthenolide prevents HLE cells from oxidative stress-induced apoptosis through inhibition of the activation of caspase-3 and caspase-9, suggesting a potential protective effect against cataract formation.

  7. 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

  8. EMT phenotype is induced by increased Src kinase activity via Src-mediated caspase-8 phosphorylation.

    Science.gov (United States)

    Zhao, Yang; Li, XiaoJun; Sun, XiangFei; Zhang, YunFeng; Ren, Hong

    2012-01-01

    Caspase-8 governs multiple cell responses to the microenvironmental cues. However, its integration of "death-life" signalings remains elusive. In our study, the role of caspase-8-Src is well-established as a promoter for migration or metastasis in Casp8(+)Src(+) A549/H226 cells in vivo and in vitro. In particular for nude mice models, mice implanted with Casp8(+)Src(+) A459/H226 cells remarkably increased spontaneous tumor metastatic burden with a significant survival disadvantage. Additionally, we detect that Src-mediated caspase-8 phosphorylation stimulates Src phosphorylation at Tyr-416 via the linkage of Src SH2 domain with phosph-Tyr-380 site of caspase-8. In turn, activated Src can efficiently induce epithelial-mesenchymal transition (EMT) phenotypic features to promote tumor cells metastasis. Surprisingly, RXDLL motif deletion in the DEDa of caspase-8 attenuates tumor cell migration or metastasis via impairing the recruitment of caspase-8 into the cellular periphery where activated Src is subject to caspase-8 phosphorylation. Together, a simple model is that the peripherization of caspase-8 is well-poised to facilitate Src-mediated caspase-8 phosphrylation at Tyr-380, then binding of phospho-Tyr380 of caspase-8 to Src SH2 domain may maintain Src in an active conformation to induce EMT phenotype, a key step toward cancer metastasis. PMID:22508042

  9. A caspase active site probe reveals high fractional inhibition needed to block DNA fragmentation.

    Science.gov (United States)

    Méthot, Nathalie; Vaillancourt, John P; Huang, JingQi; Colucci, John; Han, Yongxin; Ménard, Stéphane; Zamboni, Robert; Toulmond, Sylvie; Nicholson, Donald W; Roy, Sophie

    2004-07-01

    Apoptotic markers consist of either caspase substrate cleavage products or phenotypic changes that manifest themselves as a consequence of caspase-mediated substrate cleavage. We have shown recently that pharmacological inhibitors of caspase activity prevent the appearance of two such apoptotic manifestations, alphaII-spectrin cleavage and DNA fragmentation, but that blockade of the latter required a significantly higher concentration of inhibitor. We investigated this phenomenon through the use of a novel radiolabeled caspase inhibitor, [(125)I]M808, which acts as a caspase active site probe. [(125)I]M808 bound to active caspases irreversibly and with high sensitivity in apoptotic cell extracts, in tissue extracts from several commonly used animal models of cellular injury, and in living cells. Moreover, [(125)I]M808 detected active caspases in septic mice when injected intravenously. Using this caspase probe, an active site occupancy assay was developed and used to measure the fractional inhibition required to block apoptosis-induced DNA fragmentation. In thymocytes, occupancy of up to 40% of caspase active sites had no effect on DNA fragmentation, whereas inhibition of half of the DNA cleaving activity required between 65 and 75% of active site occupancy. These results suggest that a high and persistent fractional inhibition will be required for successful caspase inhibition-based therapies. PMID:15067000

  10. 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.                   

  11. Caspase-dependent retinal ganglion cell apoptosis in the rat model of acute diabetes

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Background Neural apoptosis is generally believed to be mediated by two distinct pathways, caspase-dependant and caspase-independent pathways. This study investigated the apoptotic pathways involved in retinal ganglion ceils in acute diabetes in rats. Methods Diabetes was induced in male Wistar rats by a peritoneal injection of streptozotocin (STZ). Expression and localization of caspase-3 and apoptosis-inducing factor (AIF) proteins in the retina of diabetic rats was examined by Western blotting and immunohistochemistry analyses. Terminal transferase dUTP nick end labeling (TUNEL) assay and immunofluorescent staining specific for caspase-3 and AIF were applied to analyze for apoptosis of retinal ganglion cells. In addition, a caspase-3 inhibitor DEVD-CHO was injected intravitreally to further determine the apoptotic pathways of retinal ganglion cells triggered in acute diabetes. Results Two weeks after induction of diabetes, a significant increase in caspase-3 protein expression and localization occurred in the nerve fiber layer, ganglion cell layer, and inner plexiform layer of the retina. Four weeks after the onset of diabetes, the increase in caspase-3 expression was profound eight weeks postinduction of diabetes (P<0.05). Meanwhile, no AIF protein expression was detected in this study. In addition, intravitreal administration of the caspase-3 inhibitor DEVD-CHO reduced apoptosis of retinal ganglion cells by its direct inhibitory action on caspase-3. Conclusion Caspase-dependent apoptotic pathways may be the main stimulant of STZ-induced retinal ganglion cell apoptosis in acute diabetes.

  12. Ecstasy-Induced Caspase Expression Alters Following Ginger Treatment

    OpenAIRE

    Asl, Sara Soleimani; Pourheydar, Bagher; Dabaghian, Fataneh; Nezhadi, Akram; ROOINTAN, AMIR; Mehdizadeh, Mehdi

    2013-01-01

    Introduction Exposure to 3-4, methylenedioxymethamphetamine (MDMA) leads to cell death. Herein, we studied the protective effects of ginger on MDMA- induced apoptosis. Methods 15 Sprague dawley male rats were administrated with 0, 10 mg/kg MDMA, or MDMA along with 100mg/kg ginger, IP for 7 days. Brains were removed to study the caspase 3, 8, and 9 expressions in the hippocampus by RT-PCR. Data was analyzed by SPSS 16 software using the one-way ANOVA test. Results MDMA treatment resulted in a ...

  13. 人难治性颞叶癫痫神经细胞凋亡与 Caspase 3,4的表达%Apoptosis and expression of Caspase 3 and Caspase 4 in neurocytes of refractory human temporal lobe epilepsy

    Institute of Scientific and Technical Information of China (English)

    林若庭; 蔡若蔚; 张鹏飞; 林元相

    2016-01-01

    目的:观察人难治性颞叶癫痫(TLE)颞叶组织神经细胞凋亡及半胱氨酸天冬氨酸蛋白酶(Caspase )3和Caspase4的表达情况。方法免疫组化S-P染色法检测1993年1月至2008年5月福建医科大学病理学系与福建医科大学附属第一医院神经外科手术切除的30例人难治性颞叶癫痫组( TLE组)与10例脑外伤组(对照组)颞叶组织中神经细胞Caspase 3与Caspase 4的表达并分析其表达差异, TUNEL 法检测两组神经细胞的凋亡情况。结果 TLE 组颞叶组织神经细胞存在Caspase 3和Caspase 4的阳性表达且表达明显高于对照组( Caspase 3:0.69±0.10比0.26±0.05,t=12.905,P<0.01;Caspase 4:0.62±0.10比0.24±0.05,t=11.880,P<0.01),TLE组颞叶组织神经细胞凋亡指数(AI)较对照组显著增加(12.6±3.1比2.5±1.9,t=9.664,P<0.01)。结论人难治性TLE颞叶组织可能存在Caspase 3,Caspase 4转导的神经细胞凋亡。%Objective To study apoptosis and expression of Caspase 3 and Caspase 4 in temporal lobe neurocytes of refractory human temporal lobe epilepsy ( TLE).Methods The temporal tissue samples were obtained from 30 cases of refractory TLE ( TLE group) and 10 cases of brain trauma ( contrast group) between January 1993 and May 2008. The surgical specimens were paraffin-embedded samples from Department of Pathology of Fujian Medical University and Department of Neurosurgery, the First Affiliated Hospital of Fujian Medical University. S-P staining of immunohistochemistry was used to detect the expression of Caspase 3 and Caspase 4 in temporal lobe neurocytes of TLE group and contrast group.Their expression was then analyzed.The terminal deoxynucleoitidyl transferase-mediated dUTP nick end labeling ( TUNEL) staining was performed to visualize and analyze the neurocytes′apoptosis of two groups.Results The expression of Caspase 3 and Caspase 4 in neurocytes of TLE

  14. p53 increases caspase-6 expression and activation in muscle tissue expressing mutant huntingtin

    DEFF Research Database (Denmark)

    Ehrnhoefer, Dagmar E; Skotte, Niels H; Ladha, Safia;

    2014-01-01

    as in muscle tissues from two different HD mouse models. p53, a transcriptional activator of caspase-6, is upregulated in neuronal cells and tissues expressing mutant huntingtin. Activation of p53 leads to a dramatic increase in levels of caspase-6 mRNA, caspase-6 activity and cleavage of lamin A....... Using mouse embryonic fibroblasts (MEFs) from YAC128 mice, we show that this increase in caspase-6 activity can be mitigated by pifithrin-α (pifα), an inhibitor of p53 transcriptional activity, but not through the inhibition of p53's mitochondrial pro-apoptotic function. Remarkably, the p53-mediated...... increase in caspase-6 expression and activation is exacerbated in cells and tissues of both neuronal and peripheral origin expressing mutant huntingtin (Htt). These findings suggest that the presence of the mutant Htt protein enhances p53 activity and lowers the apoptotic threshold, which activates caspase...

  15. Design and Synthesis of a Novel Peptidomimetic Inhibitor of Caspase-3

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Caspases, a family of cysteine proteases, comprise of highly homologous enzymes that play an important role in apoptotic cell death. Caspase-3 shows key functions in apoptosis, mediating apoptotic cascade from the intrinsic and extrinsic activation pathways. Therefore, caspase-3 is an attractive target for therapeutic intervention. For instance,inhibitors of caspase-3 have been described as promising cardioprotectants, neuroprotectants and antiarthritic agents.A novel peptidomimetic inhibitor of caspase-3, has been designed, which still has the properties of a reversible inhibitor, while the P1 site at the C-terminal remains, and only L-amino acid has been replaced by D-amino acid. Also presented here is the synthesis of the inhibitor and its inhibitory activity against caspase-3, which was tested by the fluorescent activity assay.

  16. Ekspresi Bcl-2 dan Caspase-3 Pascapaparan Hipoksia Hipobarik Intermiten

    Directory of Open Access Journals (Sweden)

    Achmad Hidayat

    2011-12-01

    Full Text Available Intermittent hypobaric hypoxia often suffered by cabin crew due to the fact that they are breathing lower pressured air inside the plane cabin. Human body will adapt by binding more oxygen and reducing hypoxia effect. Mitochondria function will be irritated by hypoxia which affect, outer mithochondrial membrane permeability due to decrease of Bcl-2 protein. Later on if hypoxia continues mitochondrial membrane will leaked cytocrome-c will released and apoptotic pathway will occur. The purpose of this study was to analyze Bcl-2 protein as antiapoptosis and caspase-3 as apoptosis indicator of intermittent hypobaric hypoxia exposure. Experimental study >was subjected to Spraque Dawley male mice during January–April 2010 by exposing them to several intermittent hypobaric hypoxias (one to four treatment in an interval of one week. Protein expression on mice heart cell were detected by immunohistochemistry in the Department of Pathology Anatomy Padjadjaran University-RS Dr. Hasan Sadikin Bandung and western blot methods in Department Biomolecullar Indonesia University Jakarta. Bcl-2 protein expressions increased according with the frequency of intermittent hypobaric hypoxia exposures while a reverse trend was found for caspase-3 protein expressions (rs=-0.448, p=0.013. From the study it can be concluded that apoptosis will be decreased as a result of intermittent hypobaric hypoxia exposures, which occurred from natural adaptation mechanism indicated by decrease of cell apoptosis and cardio protective effect will be emerged.

  17. TNF-alpha-induced mitochondrial alterations in human T cells requires FADD and caspase-8 activation but not RIP and caspase-3 activation.

    Science.gov (United States)

    Shakibaei, Mehdi; Sung, Bokyung; Sethi, Gautam; Aggarwal, Bharat B

    2010-09-15

    Although much is known about how TNF-alpha induces apoptosis in the presence of inhibitors of protein synthesis, little is known about how it induces apoptosis without these inhibitors. In this report we investigated temporal sequence of events induced by TNF-alpha in the absence of protein synthesis. Regardless of whether we measured the effects by plasma membrane phosphotidylserine accumulation, by DNA strand breaks, or activation of caspases, significant changes were observed only between 12-24 h of TNF-alpha treatment. One of the earliest changes observed after TNF-alpha treatment was mitochondrial swelling at 10 min; followed by cytochrome c and Smac release at 10-30 min, and then heterochromatin clumping occurred at 60 min. While genetic deletion of receptor-interaction protein (RIP) had no effect on TNF-alpha-induced mitochondrial damage, deletion of Fas-associated death domain (FADD) abolished the TNF-induced mitochondrial swelling. Since pan-caspase inhibitor z-VAD-fmk abolished the TNF-alpha-induced mitochondrial changes, z-DEVD-fmk, an inhibitor of caspase-3 had no effect, suggesting that TNF-alpha-induced mitochondrial changes or cytochrome c and Smac release requires caspase-8 but not caspase-3 activation. Overall, our results indicated that mitochondrial changes are early events in TNF-alpha-induced apoptosis and that these mitochondrial changes require recruitment of FADD and caspase-8 activation, but not caspase-3 activation or RIP recruitment. PMID:20136500

  18. Differential Activity of Caspase-3 Regulates Susceptibility of Lung and Breast Tumor Cell Lines to Paclitaxel

    OpenAIRE

    Odonkor, Charles Amoatey; Achilefu, Samuel

    2008-01-01

    Recent development of tumor resistance to paclitaxel presents a major problem to cancer treatment. An unsettled controversy in the cancer chemotherapy field, however, is whether caspases play a prominent role in paclitaxel-induced death in tumors. Previous studies suggest that cleavage of caspase-3 is not instrumental for the execution of death in tumors treated with paclitaxel, while other reports indicate that caspase-dependent pathways may be critical for paclitaxel cytotoxicity. In this s...

  19. Pharmacophore Modeling and Docking Studies on Some Nonpeptide-Based Caspase-3 Inhibitors

    OpenAIRE

    Simant Sharma; Arijit Basu; Agrawal, R K

    2013-01-01

    Neurodegenerative disorders are major consequences of excessive apoptosis caused by a proteolytic enzyme known as caspase-3. Therefore, caspase-3 inhibition has become a validated therapeutic approach for neurodegenerative disorders. We performed pharmacophore modeling on some synthetic derivatives of caspase-3 inhibitors (pyrrolo[3,4-c]quinoline-1,3-diones) using PHASE 3.0. This resulted in the common pharmacophore hypothesis AAHRR.6 which might be responsible for the biological activity: tw...

  20. Coordinated host responses during pyroptosis: caspase-1-dependent lysosome exocytosis and inflammatory cytokine maturation

    OpenAIRE

    Bergsbaken, Tessa; Fink, Susan L.; den Hartigh, Andreas B.; Loomis, Wendy P.; Cookson, Brad T.

    2011-01-01

    Activation of caspase-1 leads to pyroptosis, a program of cell death characterized by cell lysis and inflammatory cytokine release. Caspase-1 activation triggered by multiple NLRs (NLRC4, NLRP1b, or NLRP3) leads to loss of lysosomes via their fusion with the cell surface, or lysosome exocytosis. Active caspase-1 increased cellular membrane permeability and intracellular calcium levels, which facilitated lysosome exocytosis and release of host antimicrobial factors and microbial products. Lyso...

  1. Estrous sheep serum enables in vitro capacitation of ram spermatozoa while preventing caspase activation.

    Science.gov (United States)

    Del Olmo, E; García-Álvarez, O; Maroto-Morales, A; Ramón, M; Jiménez-Rabadán, P; Iniesta-Cuerda, M; Anel-Lopez, L; Martinez-Pastor, F; Soler, A J; Garde, J J; Fernández-Santos, M R

    2016-01-15

    Estrous sheep serum (ESS) is considered the most efficient agent for in vitro capacitation of ram spermatozoa. We have explored the relationship between caspase activation and capacitation in ram. Semen samples from 17 rams were cryopreserved. In vivo fertility was evaluated after intrauterine artificial insemination. Samples were submitted to four treatments: control, ESS (10%), caspase inhibitor (Z-VAD-FMK), and estrous ewe serum plus caspase inhibitor (I + E). Sperm samples were incubated for 30 minutes at 38.5 °C and 5% CO2 and analyzed with flow cytometry for mitochondrial membrane potential (MitoTracker deep red), sperm viability and apoptosis-like changes (YO-PRO-1/propidium iodide), acrosomal status (peanut agglutinin-fluorescein isothiocyanate), membrane fluidity (merocyanine 540), and caspase activity (Vybrant FAM kits for polycaspases, caspase-8, and caspases 3-7). Estrous sheep serum induced changes compatible with capacitation, doubling the proportion of viable spermatozoa with increased merocyanine 540 and increasing YO-PRO-1(+) and acrosome-reacted spermatozoa (P < 0.05). Incubation increased the proportion of spermatozoa with activated caspases (P < 0.05), which was abolished by the treatments. We detected a simultaneous decrease in the proportion of the viable and caspase(-) spermatozoa after the incubation, which was prevented by the presence of estrous ewe serum (P < 0.05). The analysis of caspases 3/7 and 8 resulted in less marked differences. Fertility was positively related to viability and inactivated caspases and negatively to viable-capacitated spermatozoa and active caspases. In vitro induction of capacitation in thawed ram spermatozoa by using ESS suggests a downregulation in apoptotic pathways. However, males with the lowest fertility showed parameters similar to high-fertility males, suggesting that other factors were involved apart from capacitation and/or caspase activation. PMID:26474680

  2. Expression of second mitochondria-derived activator of caspases, X-linked inhibitor of apoptosis protein, and caspase-3 in pituitary adenomas

    Institute of Scientific and Technical Information of China (English)

    Dong Li; Gang Huo; Liang Wang; Qinglin Feng; Maoyuan Tang

    2011-01-01

    Studies concerning correlations between pituitary adenomas and cell apoptosis have mainly focused on upstream apoptosis signaling, but seldom on downstream mediators. In the present study, second mitochondria-derived activator of caspases (Smac), X-linked inhibitor of apoptosis protein (XIAP), and caspase-3 protein were qualitatively analyzed using imrnunohistochemistry, and quantified by western blot. Smac, XIAP, and caspase-3 mRNA expressions were detected by reverse transcription-PCR. Results showed that XIAP protein and mRNA expressions were greater in the invasive pituitary adenoma group compared with the noninvasive pituitary adenoma group. However, Smac and caspase-3 protein and mRNA expressions were lower in the invasive pituitary adenoma group compared with the noninvasive pituitary adenoma group. In the invasive pituitary adenomas, Smac expression was positively correlated with caspase-3 protein and mRNA expression (Protein: r=0.55, P<0.01;mRNA: r=0.50, P<0.01). Smac and caspase-3 expressions were negatively correlated with XIAP protein and mRNA expression (Protein: r=-0.56, -0.64, P<0.01;mRNA:r=-0.69,-0.67,P<0.01). However, no significant differences in correlation among Srnac, XIAP, and caspase-3 were detectable in noninvasive pituitary adenomas. These data indicated that high expression of XIAP and low expression of Smac and caspase-3 suppressed cell apoptosis and led to enhanced invasiveness of pituitary adenomas. Thus, Smac, XIAP, and caspase-3 may be useful markers in determining the invasive behavior of pituitary adenomas.

  3. Regulation of NF-κB signaling by caspases and MALT1 paracaspase

    Institute of Scientific and Technical Information of China (English)

    Jens Staal; Tine Bekaert; Rudi Beyaert

    2011-01-01

    Caspases are intracellular proteases that are best known for their function in apoptosis signaling.It has become evident that many caspases also function in other signaling pathways that propagate cell proliferation and inflammation,but studies on the inflammatory function of caspases have mainly been limited to caspase-1-mediated cytokine processing.Emerging evidence,however,indicates an important contribution of caspases as mediators or regulators of nuclear factor-κB(NF-κB)signaling,which plays a key role in inflammation and immunity.Much still needs to be learned about the mechanisms that govern the activation and regulation of NF-κB by caspases,and this review provides an update of this area.Whereas apoptosis signaling is dependent on the catalytic activity of caspases,they mainly act as scaffolding platforms for other signaling proteins in the case of NF-κB signaling.Caspase proteolytic activity,however,counteracts the pro-survival function of NF-κB by cleaving specific signaling molecules.A striking exception is the paracaspase mucosa-associated lymphoid tissue 1(MALT1),whose adaptor and proteolytic activity are both needed to initiate a full blown NF-κB response in antigen-stimulated lymphocytes.Understanding the role of caspases and MALT1 in the regulation of NF-κB signaling is of high interest for therapeutic immunomodulation.

  4. GSH-dependent regulation of Fas-mediated caspase-8 activation by acrolein.

    OpenAIRE

    Hristova, Milena; Heuvelmans, Sjanneke; van der Vliet, Albert

    2007-01-01

    Activation of the cysteine protease caspase-8 by the death receptor Fas (CD95/APO-1) in B lymphoblastoid SKW6.4 cells or Jurkat T cells is associated with GSH depletion. Conversely, GSH depletion by the aldehyde acrolein (3–30 μM) was associated with inhibition of Fas-induced caspase-8 activation, although GSH depletion by buthionine sulfoximine (BSO) did not affect caspase-8 activation. In contrast to BSO, acrolein caused a loss of caspase-8 cysteine content in association with direct alkyla...

  5. 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.

  6. Molecular basis of caspase-1 polymerization and its inhibition by a new capping mechanism.

    Science.gov (United States)

    Lu, Alvin; Li, Yang; Schmidt, Florian I; Yin, Qian; Chen, Shuobing; Fu, Tian-Min; Tong, Alexander B; Ploegh, Hidde L; Mao, Youdong; Wu, Hao

    2016-05-01

    Inflammasomes are cytosolic caspase-1-activation complexes that sense intrinsic and extrinsic danger signals, and trigger inflammatory responses and pyroptotic cell death. Homotypic interactions among Pyrin domains and caspase recruitment domains (CARDs) in inflammasome-complex components mediate oligomerization into filamentous assemblies. Several cytosolic proteins consisting of only interaction domains exert inhibitory effects on inflammasome assembly. In this study, we determined the structure of the human caspase-1 CARD domain (caspase-1(CARD)) filament by cryo-electron microscopy and investigated the biophysical properties of two caspase-1-like CARD-only proteins: human inhibitor of CARD (INCA or CARD17) and ICEBERG (CARD18). Our results reveal that INCA caps caspase-1 filaments, thereby exerting potent inhibition with low-nanomolar Ki on caspase-1(CARD) polymerization in vitro and inflammasome activation in cells. Whereas caspase-1(CARD) uses six complementary surfaces of three types for filament assembly, INCA is defective in two of the six interfaces and thus terminates the caspase-1 filament. PMID:27043298

  7. 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.

  8. Expression of Livin, Survivin and Caspase-3 in human brain astrocytoma%Livin、Survivin和Caspase-3在星形细胞瘤中的表达

    Institute of Scientific and Technical Information of China (English)

    赵树鹏; 靳彩玲; 赵新利; 周文科

    2012-01-01

    目的 探讨Livin、Survivin和Caspase-3在星形细胞瘤中的表达.方法 采用免疫组织化学法检测50例星形细胞瘤标本及10例正常脑组织中Livin、Survivin和Caspase-3的表达,分析Livin、Survivin的表达与Caspase-3表达的关系.结果 Caspase-3在正常脑组织中的阳性表达率显著高于在星形细胞瘤(P<0.05),且在Ⅰ~Ⅱ级星形细胞瘤中的阳性表达率高于Ⅲ~Ⅳ级(P<0.05).Livin和Survivin在星形细胞瘤中的阳性表达率显著高于正常脑组织(P<0.05),且在Ⅲ~Ⅳ级星形细胞瘤中的阳性表达率明显高于Ⅰ~Ⅱ级(P<0.05).星形细胞瘤中Livin和Survivin 的表达与Caspase-3蛋白的表达均呈负相关(r分别为-0.520和-0.360,P<0.05).结论 Livin、Survivin和Caspase-3的表达可能与星形细胞瘤的发生及发展有关.%Objective To explore the expression of Livin, Survivin and Caspase-3 in human brain astrocytoma. Methods The expressions of Livin, Survivin and Caspase-3 in fifty astrocytomas and ten normal brain tissues were detected with immunohistochemical method,and the relationships of Caspase-3 with Livin,Survivin were analyzed. Results The positive rate of Caspase-3 in normal tissue was significantly higher than that in human brain astrocytoma (P <0. 05) ,it was higher in gradeⅠand II than in grade Ⅲ and Ⅳ(P <0. 05) . The positive rates of Livin and Survivin in human brain astrocytoma were significantly higher than those in normal brain tissue(P <0. 05) ,and they were higher in grade Ⅲ and IV than in grade I andII (P <0. 05) . The expressions of Livin and Survivin were negatively correlated with those of Caspase-3 in human brain astro-cytoma( r = - 0.520,r = - 0.360 ,P < 0.05). Conclusion Livin,Survivin and Caspase-3 may play an important role in incidence and development of human brain astrocytoma.

  9. Caspase-3 serves as an intracellular immune receptor specific for lipopolysaccharide in oyster Crassostrea gigas.

    Science.gov (United States)

    Xu, Jiachao; Jiang, Shuai; Li, Yiqun; Li, Meijia; Cheng, Qi; Zhao, Depeng; Yang, Bin; Jia, Zhihao; Wang, Lingling; Song, Linsheng

    2016-08-01

    Apoptosis is a form of programmed cell death process controlled by a family of cysteine proteases called caspases, which plays a crucial role in the immune system homeostasis. The apoptosis and the detailed regulation mechanism have been well studied in vertebrate, but the information in lower animals, especially invertebrates, is still very limited. In the present study, Caspase-3 in the Pacific oyster Crassostrea gigas (designated CgCaspase-3) was enriched by lipopolysaccharide (LPS) affinity chromatography and further identified by MALDI-TOF/TOF-mass spectrometry. The binding activity of CgCaspase-3 to LPS was confirmed by enzyme-linked immunosorbent assay, and surface plasmon resonance analysis revealed its high binding specificity and moderate binding affinity (KD = 1.08 × 10(-6) M) to LPS. The recombinant CgCaspase-3 exhibited high proteolytic activity to substrate Ac-DEVD-pNA and relatively weak activity to substrate Ac-DMQD-pNA and Ac-VDQQD-pNA. The binding of CgCaspase-3 to LPS significantly inhibited its proteolytic activity toward AC-DEVD-pNA in vitro. The over-expression of CgCaspase-3 leaded to the phosphatidylserine exposure on the external plasma membrane and the cleavage of poly (ADP-ribose) polymerase, which reduced cell viability, and finally induced cell apoptosis. But the cell apoptosis mediated by CgCaspase-3 in vivo was significantly inhibited by the treatment of LPS. These results collectively indicated that CgCaspase-3 could serve as an intracellular LPS receptor, and the interaction of LPS with CgCaspase-3 specifically inhibited the cell apoptosis induced by CgCaspase-3. PMID:26993662

  10. Inhibition of caspase-mediated apoptosis by peroxynitrite in traumatic brain injury.

    Science.gov (United States)

    Lau, Anthony; Arundine, Mark; Sun, Hong-Shuo; Jones, Michael; Tymianski, Michael

    2006-11-01

    In traumatic brain injury (TBI), neurons surviving the primary insult may succumb through poorly understood secondary mechanisms. In vitro, cortical neurons exposed to stretch injury exhibited enhanced vulnerability to NMDA, apoptotic-like DNA fragmentation, peroxynitrite (PN) formation, and cytoplasmic cytochrome c accumulation. Surprisingly, caspase-3 activity was undetectable by both immunoblotting and fluorogenic activity assays. Therefore, we hypothesized that PN directly inhibits caspases in these neurons. Consistent with this, stretch injury in cultured neurons elicited tyrosine nitration of procaspase-3, but not caspase-9 or Apaf-1, suggesting a direct interaction of PN with caspase-3. In an ex vivo system, PN inhibited the activity of caspase-3, and this inhibition was reversible with the addition of the sulfhydryl reducing agent dithiothreitol, indicating that PN inhibits caspases by cysteinyl oxidation. Moreover, in cultures, the PN donor 3-morpholinosydnonimine (SIN-1) blocked staurosporine-induced caspase-3 activation and its downstream effects including PARP-1 [poly-(ADP-ribose) polymerase-1] cleavage and phosphotidylserine inversion, suggesting that peroxynitrite can inhibit caspase-3-mediated apoptosis. To examine these mechanisms in vivo, rats were exposed to a lateral fluid percussion injury (FPI). FPI caused increased neuronal protein nitration that colocalized with TUNEL staining, indicating that PN was associated with neurodegeneration. Caspase-3 activity was inhibited in brain lysates harvested after FPI and was restored by adding dithiothreitol. Our data show that caspase-mediated apoptosis is inhibited in neurons subjected to stretch in vitro and to TBI in vivo, mostly because of cysteinyl oxidation of caspase-3 by PN. However, this is insufficient to prevent cell death, indicating that the TBI therapy may, at a minimum, require a combination of both anti-apoptotic and anti-oxidant strategies. PMID:17093075

  11. Phylogenomics of caspase-activated DNA fragmentation factor

    International Nuclear Information System (INIS)

    The degradation of nuclear DNA by DNA fragmentation factor (DFF) is a key step in apoptosis of mammalian cells. Using comparative genomics, we have here determined the evolutionary history of the genes encoding the two DFF subunits, DFFA (also known as ICAD) and DFFB (CAD). Orthologs of DFFA and DFFB were identified in Nematostella vectensis, a representative of the primitive metazoan clade cnidarians, and in various vertebrates and insects, but not in representatives of urochordates, echinoderms, and nematodes. The domains mediating the interaction of DFFA and DFFB, a caspase cleavage site in DFFA, and the amino acid residues critical for endonuclease activity of DFFB were conserved in Nematostella. These findings suggest that DFF has been a part of the primordial apoptosis system of the eumetazoan common ancestor and that the ancient cell death machinery has degenerated in several evolutionary lineages, including the one leading to the prototypical apoptosis model, Caenorhabditis elegans

  12. 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.

    Directory of Open Access Journals (Sweden)

    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

  13. Tissue Crowding Induces Caspase-Dependent Competition for Space.

    Science.gov (United States)

    Levayer, Romain; Dupont, Carole; Moreno, Eduardo

    2016-03-01

    Regulation of tissue size requires fine tuning at the single-cell level of proliferation rate, cell volume, and cell death. Whereas the adjustment of proliferation and growth has been widely studied [1-5], the contribution of cell death and its adjustment to tissue-scale parameters have been so far much less explored. Recently, it was shown that epithelial cells could be eliminated by live-cell delamination in response to an increase of cell density [6]. Cell delamination was supposed to occur independently of caspase activation and was suggested to be based on a gradual and spontaneous disappearance of junctions in the delaminating cells [6]. Studying the elimination of cells in the midline region of the Drosophila pupal notum, we found that, contrary to what was suggested before, Caspase 3 activation precedes and is required for cell delamination. Yet, using particle image velocimetry, genetics, and laser-induced perturbations, we confirmed [6] that local tissue crowding is necessary and sufficient to drive cell elimination and that cell elimination is independent of known fitness-dependent competition pathways [7-9]. Accordingly, activation of the oncogene Ras in clones was sufficient to compress the neighboring tissue and eliminate cells up to several cell diameters away from the clones. Mechanical stress has been previously proposed to contribute to cell competition [10, 11]. These results provide the first experimental evidences that crowding-induced death could be an alternative mode of super-competition, namely mechanical super-competition, independent of known fitness markers [7-9], that could promote tumor growth. PMID:26898471

  14. P53-mediated cell cycle arrest and apoptosis through a caspase-3-independent, but caspase-9-dependent pathway in oridonin-treated MCF-7 human breast cancer cells

    Institute of Scientific and Technical Information of China (English)

    Qiao CUI; Jing-hua YU; Jin-nan WU; Shin-ichi TASHIRO; Satoshi ONODERA; Mutsuhiko MINAMI; Takashi IKEJIMA

    2007-01-01

    Aim: To study the caspase-3-independent mechanisms in oridonin-induced MCF-7 human breast cancer cell apoptosis in vitro. Methods: The viability of oridonin-treated MCF-7 cells was measured by MTT (thiazole blue) assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleoso-mal DNA fragmentation was assayed by agarose gel electrophoresis. The apoptotic ratio was determined by lactate dehydrogenase assay. Cell cycle alternation and mitochondrial membrane potential were measured by flow cytometric analysis. Bax, Bcl-2, caspase-3, caspase-9, heat shock protein (Hsp)90, p53, p-p53, p21, Poly (ADP-ribose) polymerase (PARP), and the inhibitor of caspase-activated Dnase (ICAD) protein expressions were detected by Western blot analysis. Results: Oridonin inhibited cell growth in a time- and dose-dependent manner. Cell cycle was altered through the upregulation of p53 and p21 protein expressions. Pan-caspase inhibitor Z-VAD-fmk and calpain inhibitor Ⅱ both decreased cell death ratio. Nucleosomal DNA fragmentation and the downregulation of △ψmit were detected in oridonin-induced MCF-7 cell apoptosis, which was involved in a postmitochondrial caspase-9-dependent pathway. Decreased Bcl-2 and Hsp90 expression levels and increased Bax and p21 expression levels were positively correlated with elevated levels of phosphorylated p53 phosphorylation. Moreover, PARP was partially cleaved by calpain rather than by capase-3. Conclusion: DNA damage provoked alternations in the mitochondrial and caspase-9 pathways as well as p53-mediated cell cycle arrest, but was not related to caspase-3 activity in oridonin-induced MCF-7 cells.

  15. 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.

  16. Caspase-3和bax在视网膜母细胞瘤中的表达%Expression of caspase-3 and bax gene protein in retinoblastoma

    Institute of Scientific and Technical Information of China (English)

    孙红; 惠延年; 王立勤; 马吉献

    2003-01-01

    目的: 观察凋亡及凋亡调控基因caspase-3/bax在视网膜母细胞瘤(retinoblastoma, RB)中的表达及与凋亡的相关性. 方法: 收集35例RB标本,对其分别进行caspase-3和bax免疫组织化学染色,观察表达情况及染色强度. 结果: Caspase-3及bax在未分化型(n=15)分别有较好的表达(11/12例),caspase-3及bax在分化型(n=20)中也有较好的表达(17/18例). 正常视网膜组织中无caspase-3及bax的表达. 结论: 凋亡在RB中是存在的,caspase-3及bax在RB的发生发展中起重要作用.

  17. Caspase-1 and 3 Inhibiting Drimane Sesquiterpenoids from the Extremophilic Fungus, Penicillium solitum

    OpenAIRE

    Stierle, Donald B.; Stierle, Andrea A.; Girtsman, Teri; McIntyre, Kyle; Nichols, Jesse

    2012-01-01

    Two new drimane sesquiterpene lactones and one new tricarboxylic acid derivative were isolated from the Berkeley Pit extremophilic fungus Penicillium solitum. The structures of these compounds were deduced by spectroscopic analysis. Berkedrimanes A and B inhibited the signal transduction enzymes caspase-1 and caspase-3 and mitigated the production of interleukin 1-β in the induced THP-1 (promonocytic leukemia cell line) assay.

  18. Two-Photon Enzymatic Probes Visualizing Sub-cellular/Deep-brain Caspase Activities in Neurodegenerative Models.

    Science.gov (United States)

    Qian, Linghui; Zhang, Cheng-Wu; Mao, Yanli; Li, Lin; Gao, Nengyue; Lim, Kah-Leong; Xu, Qing-Hua; Yao, Shao Q

    2016-01-01

    Caspases work as a double-edged sword in maintaining cell homeostasis. Highly regulated caspase activities are essential during animal development, but dysregulation might lead to different diseases, e.g. extreme caspase activation is known to promote neurodegeneration. At present, visualization of caspase activation has mostly remained at the cellular level, in part due to a lack of cell-permeable imaging probes capable of direct, real-time investigations of endogenous caspase activities in deep tissues. Herein, we report a suite of two-photon, small molecule/peptide probes which enable sensitive and dynamic imaging of individual caspase activities in neurodegenerative models under physiological conditions. With no apparent toxicity and the ability of imaging endogenous caspases both in different subcellular organelles of mammalian cells and in brain tissues, these probes serve as complementary tools to conventional histological analysis. They should facilitate future explorations of caspases at molecular, cellular and organism levels and inspire development of novel two-photon probes against other enzymes. PMID:27210613

  19. Caspase-1 and IL-1β processing in a teleost fish.

    Directory of Open Access Journals (Sweden)

    Marta I R Reis

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

  20. Caspase-2 promotes obesity, the metabolic syndrome and nonalcoholic fatty liver disease.

    Science.gov (United States)

    Machado, M V; Michelotti, G A; Jewell, M L; Pereira, T A; Xie, G; Premont, R T; Diehl, A M

    2016-01-01

    Obesity and its resulting metabolic disturbances are major health threats. In response to energy surplus, overtaxed adipocytes release fatty acids and pro-inflammatory factors into the circulation, promoting organ fat accumulation (including nonalcoholic fatty liver disease), insulin resistance and the metabolic syndrome. Recently, caspase-2 was linked to lipoapoptosis, so we hypothesized that caspase-2 might be a critical determinant of metabolic syndrome pathogenesis. Caspase-2-deficient and wild-type mice were fed a Western diet (high-fat diet, enriched with saturated fatty acids and 0.2% cholesterol, supplemented with fructose and glucose in the drinking water) for 16 weeks. Metabolic and hepatic outcomes were evaluated. In vitro studies assessed the role of caspase-2 in adipose tissue proliferative properties and susceptibility for lipoapoptosis. Caspase-2-deficient mice fed a Western diet were protected from abdominal fat deposition, diabetes mellitus, dyslipidemia and hepatic steatosis. Adipose tissue in caspase-2-deficient mice was more proliferative, upregulated mitochondrial uncoupling proteins consistent with browning, and was resistant to cell hypertrophy and cell death. The liver was protected from steatohepatitis through a decrease in circulating fatty acids and more efficient hepatic fat metabolism, and from fibrosis as a consequence of reduced fibrogenic stimuli from fewer lipotoxic hepatocytes. Caspase-2 deficiency protected mice from diet-induced obesity, metabolic syndrome and nonalcoholic fatty liver disease. Further studies are necessary to assess caspase-2 as a therapeutic target for those conditions. PMID:26890135

  1. Chemotherapy increases caspase-cleaved cytokeratin 18 in the serum of breast cancer patients

    International Nuclear Information System (INIS)

    Apoptosis is thought to be induced by chemotherapy in cancer patients. Therefore, the measurement of its amplitude may be a useful tool to predict the effectiveness of cancer treatment sooner than conventional methods do. In the study presented, apoptosis was assessed with an ELISA-based assay in which caspase-cleaved cytokeratin 18 (M30-antigen), a novel specific biomarker of apoptosis, is measured. Thirty seven patients with malignant (nonmetastatic and metastatic) breast cancer, 35 patients with benign breast disease, and 34 healthy subjects were studied. Cancer patients received neoadjuvant chemotherapy consisting of either fluorouracil, epirubicin, and cyclophosphamide (FEC) or epirubicin plus docetaxel (ED). Apoptosis was detected before chemotherapy, 24 and 48 h after chemotherapy in the malignant group. It was found that the baseline apoptosis level in either malignant but nonmetastatic group or benign group was not statistically different from that in the control group (p>0.05). However, it was statistically significantly higher in the metastatic group than that in the control group (p<0.05). Following the drug application, M30-antigen levels significantly increased at 24 h (p<0.05). The baseline M30-antigen levels increased about 3-times in patients showing tumor regression. M30-antigen level is increased after chemotherapy and its measurement may help clinicians to predict the effectiveness of chemotherapy sooner in breast cancer cases although confirmative larger trials are needed

  2. 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

    are not well suited to specifically assess caspase-6 activity in the presence of other, confounding protease activities, as often encountered in cell and tissue samples. Here we report the development of a method that overcomes this limitation by using a protein substrate, lamin A, which is highly...... specific for caspase-6 cleavage at amino acid 230. Using a neo-epitope antibody against cleaved lamin A, we developed an electrochemiluminescence-based ELISA assay that is suitable to specifically detect and quantify caspase-6 activity in highly apoptotic cell extracts. The method is more sensitive than......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 methods...

  3. Deficiency of caspase 3 in tumor xenograft impairs therapeutic effect of measles virus Edmoston strain.

    Science.gov (United States)

    Wang, Biao; Yan, Xu; Guo, Qingguo; Li, Yan; Zhang, Haiyan; Xie, Ji Sheng; Meng, Xin

    2015-06-30

    The oncolytic measles virus Edmonston (MV-Edm) strain shows considerable oncolytic activity against a variety of human tumors. In this study, we report MV-Edm is able to trigger apoptosis pathways in infected tumor cells and elucidate the roles of cellular apoptosis in the whole oncolytic process. We also show that activated caspase 3, a key executioner of apoptosis, plays key roles in the oncolytic virotherapy. Activated caspase 3 can accelerate viral replication in cervical cancer cells and enhance the killing effects of the virus. Deficiency of caspase 3 either in tumor cells or in tumor xenograft significantly desensitized tumor to oncolysis with MV-Edm. In the infected cells, caspase 3 regulates interferon α release, which can inhibit viral replication in neighboring tumor cells. We propose that caspase-3 activation enhances the oncolytic effects of MV-Edm, thus inhibiting tumor growth in mice. PMID:25909216

  4. Retinal Cell Death Caused by Sodium Iodate Involves Multiple Caspase-Dependent and Caspase-Independent Cell-Death Pathways

    Directory of Open Access Journals (Sweden)

    Jasmin Balmer

    2015-07-01

    Full Text Available Herein, we have investigated retinal cell-death pathways in response to the retina toxin sodium iodate (NaIO3 both in vivo and in vitro. C57/BL6 mice were treated with a single intravenous injection of NaIO3 (35 mg/kg. Morphological changes in the retina post NaIO3 injection in comparison to untreated controls were assessed using electron microscopy. Cell death was determined by TdT-mediated dUTP-biotin nick end labeling (TUNEL staining. The activation of caspases and calpain was measured using immunohistochemistry. Additionally, cytotoxicity and apoptosis in retinal pigment epithelial (RPE cells, primary retinal cells, and the cone photoreceptor (PRC cell line 661W were assessed in vitro after NaIO3 treatment using the ApoToxGlo™ assay. The 7-AAD/Annexin-V staining was performed and necrostatin (Nec-1 was administered to the NaIO3-treated cells to confirm the results. In vivo, degenerating RPE cells displayed a rounded shape and retracted microvilli, whereas PRCs featured apoptotic nuclei. Caspase and calpain activity was significantly upregulated in retinal sections and protein samples from NaIO3-treated animals. In vitro, NaIO3 induced necrosis in RPE cells and apoptosis in PRCs. Furthermore, Nec-1 significantly decreased NaIO3-induced RPE cell death, but had no rescue effect on treated PRCs. In summary, several different cell-death pathways are activated in retinal cells as a result of NaIO3.

  5. Expression of Survivin, CyclinD1, p21WAF1, Caspase-3 in Cervical Cancer and Its Relation with Prognosis

    Institute of Scientific and Technical Information of China (English)

    LU Shi; ZHANG Baohua; WANG Zehua

    2005-01-01

    The implications of Survivin, CyclinD1, p21WAF1, Caspase-3 in the development, progression and prognosis in cervical cancer were investigated. By using immunohistochemical SP method, the expression of Survivin, CyclinD1, p21WAF1 , Caspase-3 was detected in 41 cases of cervical cancer, 17 cases of cervical intraepithelial neoplasia (CIN) and 10 cases of normal tissues, and their relation with pathological grade, clinical stage, metastasis and survival time was analyzed.The results showed that the positive expression rate of Survivin, CyclinD1 in cervical cancer was significantly higher than in CIN group and normal control group (P<0.05). The median survival time in the patients with cervical cancer positive for Survivin and CyclinD1 was significantly shorter than in those with negative expression (P<0.05). The expression of both Survivin and CyclinD1 was not related with tumor grade, clinical stage and metastasis (P>0. 05). The positive expression rate of p21WAF1 , Caspase-3 in cervical ca rcer was significantly lower than in CIN group and normal control group (P<0.05), and had a close relation with tumor grade (P<0.05). The expression of Survivin in cervical cancer in cervical cancer was negatively associated with that of Caspase-3 (P<0.01), but positively with that of CyclinD1 (P<0.01). Cox Multivariate analysis revealed that Survivin was the independent prognostic indicator influencing the survival time of the patients with cervical cancer (P<0.05). It was suggested that the high expression of Survivin or CyclinD1, and low expression of p21WAF1 or Caspase-3 was closely correlated with the development of cervical cancer. Survivin and CyclinD1 could be used as a useful indicator to predict the prognosis of cervical cancer.

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

    Directory of Open Access Journals (Sweden)

    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.

  7. Testosterone undecanoate and depo medroxyprogesterone acetate induced azoospermia through increased expression of spermatogenic cell caspase 3

    Directory of Open Access Journals (Sweden)

    Nukman Moeloek

    2008-09-01

    Full Text Available The administration of a combination of testosterone undecanoate (TU, a long-acting androgen and depo-medroxyprogesterone acetate (DMPA were investigated in term of suppression of rat sperm concentration in vivo to azoospermia through increasing activity of spermatogenic cell caspase 3. Adult Sprague Dawley rats received TU and DMPA of 2.5 mg and 1.25 mg, respectively, a regimen known to rapidly reduce intra testicular testosterone and to produce azoospermia within 12 weeks. Caspase 3 positive sperm cells increased compared with control levels during 6 weeks post-injection and increased further through 60 weeks. Immunohistochemistry for caspase 3 revealed that spermatocytes represented the predominant caspase 3 positive germ cells. Modest immunoreactivity for caspase-3 was localized to nuclear region of the germ cells of control and treated testes. Immunohistochemistry study revealed significantly increased caspase-3 expression in nuclei of germ cells during administration of TU+DMPA to rats. Additionally, the caspase 3 content was significantly increased in germ cells during rats were administered TU+DMPA (453.90±84.88 cells/200 seminiferous tubules and caspase 3 significant increase in immunoreactivity was localized to the nuclei of spermatogonia, spermatocytes, and spermatids. Taken together, these results indicated that azoospermia due to reduced intratesticular testosterone concentration was caspase-3 activation dependent and suggested that the increase in active caspase-3 in the nucleus may be involved in the induction of decreased sperm production. (Med J Indones 2008; 17: 149-56Keywords: TU, DMPA, sperm concentration, germ cells

  8. 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.

  9. 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

  10. When Beauty Is Skin Deep: Regulation of the Wound Response by Caspase-8, RIPK3, and the Inflammasome.

    Science.gov (United States)

    Vince, James E

    2015-08-01

    Caspase-8 downregulation is observed in the epidermis of wounded skin, whereas permanent epidermal caspase-8 deletion causes chronic skin inflammation, suggesting that caspase-8 is a critical regulator of skin homeostasis and, possibly, the wound response. In this issue, Lee et al. document how epidermal caspase-8 deletion, or cutaneous wounding, results in increased NF-κB activation to drive keratinocyte caspase-1 expression and subsequent secretion of the pro-inflammatory cytokines, IL-1β and IL-1α. Consequently, loss of NF-κB activity, caspase-1, or the IL-1 receptor delays wound healing. Previous studies have documented how chronic skin inflammation in caspase-8-deficient mice is rescued by RIPK3 co-deletion. Therefore, targeting caspase-1, IL-1, or RIPK3 itself may benefit treatment of chronic inflammatory skin diseases, or where an inappropriate inflammatory response proves detrimental to wound healing, such as in type 2 diabetes. PMID:26174535

  11. Differential Efficacy of Caspase Inhibitors on Apoptosis Markers during Sepsis in Rats and Implication for Fractional Inhibition Requirements for Therapeutics

    OpenAIRE

    Méthot, Nathalie; Huang, JingQi; Coulombe, Nathalie; Vaillancourt, John P.; Rasper, Dita; Tam, John; Han, Yongxin; Colucci, John; Zamboni, Robert; Xanthoudakis, Steven; Toulmond, Sylvie; Nicholson, Donald W; Roy, Sophie

    2004-01-01

    A rodent model of sepsis was used to establish the relationship between caspase inhibition and inhibition of apoptotic cell death in vivo. In this model, thymocyte cell death was blocked by Bcl-2 transgene, indicating that apoptosis was predominantly dependent on the mitochondrial pathway that culminates in caspase-3 activation. Caspase inhibitors, including the selective caspase-3 inhibitor M867, were able to block apoptotic manifestations both in vitro and in vivo but with strikingly differ...

  12. Some natural flavonoids are competitive inhibitors of Caspase-1, −3 and −7 despite their cellular toxicity

    OpenAIRE

    White, J. Brandon; Beckford, Jeremy; Yadegarynia, Sina; Ngo, Nhi; Lialiutska, Tetiana; d’Alarcao, Marc

    2012-01-01

    A common feature of both apoptosis and inflammation is the activation of caspases. Caspases are aspartate-directed cysteine proteases that have numerous cellular targets. It has been discovered that several flavonoids are inhibitors of caspases. Flavonoids are members of a family of polyphenolic compounds from plants that have many biological properties, one of which is the ability to induce cell death. Some flavonoids are selective inhibitors of particular caspases. Since some of the inhibit...

  13. DMPD: A role for caspases in the differentiation of erythroid cells and macrophages. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17905508 A role for caspases in the differentiation of erythroid cells and macropha...;90(2):416-22. Epub 2007 Sep 2. (.png) (.svg) (.html) (.csml) Show A role for caspases in the differentiatio...n of erythroid cells and macrophages. PubmedID 17905508 Title A role for caspases

  14. Bicaudal is a conserved substrate for Drosophila and mammalian caspases and is essential for cell survival.

    Directory of Open Access Journals (Sweden)

    Emma M Creagh

    Full Text Available Members of the caspase family of cysteine proteases coordinate cell death through restricted proteolysis of diverse protein substrates and play a conserved role in apoptosis from nematodes to man. However, while numerous substrates for the mammalian cell death-associated caspases have now been described, few caspase substrates have been identified in other organisms. Here, we have utilized a proteomics-based approach to identify proteins that are cleaved by caspases during apoptosis in Drosophila D-Mel2 cells, a subline of the Schneider S2 cell line. This approach identified multiple novel substrates for the fly caspases and revealed that bicaudal/betaNAC is a conserved substrate for Drosophila and mammalian caspases. RNAi-mediated silencing of bicaudal expression in Drosophila D-Mel2 cells resulted in a block to proliferation, followed by spontaneous apoptosis. Similarly, silencing of expression of the mammalian bicaudal homologue, betaNAC, in HeLa, HEK293T, MCF-7 and MRC5 cells also resulted in spontaneous apoptosis. These data suggest that bicaudal/betaNAC is essential for cell survival and is a conserved target of caspases from flies to man.

  15. Bicaudal is a conserved substrate for Drosophila and mammalian caspases and is essential for cell survival.

    LENUS (Irish Health Repository)

    Creagh, Emma M

    2009-01-01

    Members of the caspase family of cysteine proteases coordinate cell death through restricted proteolysis of diverse protein substrates and play a conserved role in apoptosis from nematodes to man. However, while numerous substrates for the mammalian cell death-associated caspases have now been described, few caspase substrates have been identified in other organisms. Here, we have utilized a proteomics-based approach to identify proteins that are cleaved by caspases during apoptosis in Drosophila D-Mel2 cells, a subline of the Schneider S2 cell line. This approach identified multiple novel substrates for the fly caspases and revealed that bicaudal\\/betaNAC is a conserved substrate for Drosophila and mammalian caspases. RNAi-mediated silencing of bicaudal expression in Drosophila D-Mel2 cells resulted in a block to proliferation, followed by spontaneous apoptosis. Similarly, silencing of expression of the mammalian bicaudal homologue, betaNAC, in HeLa, HEK293T, MCF-7 and MRC5 cells also resulted in spontaneous apoptosis. These data suggest that bicaudal\\/betaNAC is essential for cell survival and is a conserved target of caspases from flies to man.

  16. Effects of manipulation of the caspase system on myofibrillar protein degradation in vitro.

    Science.gov (United States)

    Kemp, C M; Wheeler, T L

    2011-10-01

    Apoptosis via the intrinsic caspase 9 pathway can be induced by oxidative stressors hydrogen peroxide (H₂O₂) and N-(4 hydroxyphenol) rentinamide (fenretinide), a synthetic retinoid. Accelerated muscle atrophy and proteolysis in muscle-wasting conditions have been linked to oxidative stress and activated protease systems. Therefore, the hypothesis of this study was that proteolysis of myofibrillar proteins could be manipulated through the induction or inhibition of the caspase system. After slaughter, LM and supraspinatus muscles from callipyge (n = 5) and normal (n = 3) lambs were excised, finely diced, and incubated with treatment buffers containing oxidative stressors fenretinide or H₂O₂, recombinant caspase 3, caspase-specific inhibitor N-acetyl-Asp-Glu-Val-Asp-CHO (DEVD), or control solution. Muscle samples were incubated for 1, 2, 7, and 21 d at 4°C. Activation of the initiator caspase, caspase 9, and myofibrillar protein degradation was determined by SDS-PAGE and Western blotting. Results showed that fenretinide, H₂O₂, and recombinant caspase 3 increased (P tenderization. However, these stimulated changes were not sufficient to overcome the lack of proteolysis that is characteristic of muscle from callipyge lambs. PMID:21622882

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

    International Nuclear Information System (INIS)

    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. A potent reporter applicable to the monitoring of caspase-3-dependent proteolytic cleavage.

    Science.gov (United States)

    Park, Kyoungsook; Kang, Hyo-Jin; Ahn, Junhyoung; Yi, So Yeon; Han, Sang Hee; Park, Hye-Jung; Chung, Sang J; Chung, Bong Hyun; Kim, Moonil

    2008-11-01

    In this study, we developed a chimeric caspase-3 substrate (GST:DEVD:EGFP) comprised of glutathione-S transferase (GST) and enhanced green fluorescent protein (EGFP) with a specialized linker peptide harboring the caspase-3 cleavage sequence, DEVD. Using this reporter, we assessed the proteolytic cleavage of the artificial caspase-3 substrate for caspase-3. The common feature of this approach is that the presence of the DEVD sequence between GST and EGFP allows for caspase-3-dependent cleavage after the Asp (D) residue, resulting in the elimination of EGFP from the GST:DEVD:EGFP reporter. To the best of our knowledge, this study reports the first application employing a chimeric protein substrate, with the similar accuracy level compared to the conventional methods such as fluorometric assays. As a result, using this GST:DEVD:EGFP reporter, caspase-3 activation based on proteolytic properties could be monitored via a variety of bioanalytical techniques such as immunoblot analysis, glutathione-agarose bead assay, and on-chip visualization, providing both technical and economical advantages over the extensively utilized fluorogenic peptide assay. Our results convincingly showed that this versatile reporter (GST:DEVD:EGFP) constitutes a useful system for the monitoring of caspase-3 activation, potentially enabling the monitoring of the proteolytic activities of different intra-cellular proteases via the substitution of the cleavage sequence within the same schematic construct. PMID:18775457

  19. A ubiquitin ligase complex regulates caspase activation during sperm differentiation in Drosophila.

    Directory of Open Access Journals (Sweden)

    Eli Arama

    2007-10-01

    Full Text Available In both insects and mammals, spermatids eliminate their bulk cytoplasm as they undergo terminal differentiation. In Drosophila, this process of dramatic cellular remodeling requires apoptotic proteins, including caspases. To gain further insight into the regulation of caspases, we screened a large collection of sterile male flies for mutants that block effector caspase activation at the onset of spermatid individualization. Here, we describe the identification and characterization of a testis-specific, Cullin-3-dependent ubiquitin ligase complex that is required for caspase activation in spermatids. Mutations in either a testis-specific isoform of Cullin-3 (Cul3(Testis, the small RING protein Roc1b, or a Drosophila orthologue of the mammalian BTB-Kelch protein Klhl10 all reduce or eliminate effector caspase activation in spermatids. Importantly, all three genes encode proteins that can physically interact to form a ubiquitin ligase complex. Roc1b binds to the catalytic core of Cullin-3, and Klhl10 binds specifically to a unique testis-specific N-terminal Cullin-3 (TeNC domain of Cul3(Testis that is required for activation of effector caspase in spermatids. Finally, the BIR domain region of the giant inhibitor of apoptosis-like protein dBruce is sufficient to bind to Klhl10, which is consistent with the idea that dBruce is a substrate for the Cullin-3-based E3-ligase complex. These findings reveal a novel role of Cullin-based ubiquitin ligases in caspase regulation.

  20. Identification of Caspase-6 as a New Regulator of Alternatively Activated Macrophages.

    Science.gov (United States)

    Yao, Yongfang; Shi, Qian; Chen, Bing; Wang, Qingsong; Li, Xinda; Li, Long; Huang, Yahong; Ji, Jianguo; Shen, Pingping

    2016-08-12

    Alternatively activated macrophages (AAMs) play essential roles in the promotion of tissue remodeling, vasculogenesis, and tumor progression; however, the detailed mechanisms underlying the activation of AAMs remain largely unknown. Here, by using quantitative proteomic analysis, we identified 62 proteins that were up-regulated in IL-4-induced macrophages. Among these, Caspase-6 was increased significantly. Caspase-6 is important in the apoptotic signaling pathway; however, its role in non-apoptosis is also reported. Here, we first examined the non-apoptotic role of Caspase-6 in the alternative activation of macrophages after administration of IL-4, 4T1 tumor conditional medium, or co-culture with 4T1 cells. Both treatments promoted alternative activation of RAW264.7 cells and primary macrophages, whereas disruption of caspase-6 expression and activity could markedly suppress the biomarker levels of AAMs. Overexpression of Caspase-6 could significantly promote the activation of AAMs. Importantly, we further present evidence that caspase-6 could regulate breast cancer cell invasion by modulating MMP-2 and MMP-9 expression in 4T1 tumor-associated macrophages, as ablation of protein levels or activity of caspase-6 suppressed tumor cell invasion in vitro In conclusion, the observed results markedly expanded our views of the dynamic changes in protein composition during alternative activation of macrophages, and they revealed a critical new role of caspase-6 in regulating this cellular biological process, which suggested that caspase-6 might be a key nod molecule to regulate immunological steady-state and be a therapeutic candidate for tumor immunotherapy. PMID:27325699

  1. Radiolabeled isatin binding to caspase-3 activation induced by anti-Fas antibody

    International Nuclear Information System (INIS)

    Introduction: Noninvasive imaging methods that can distinguish apoptosis from necrosis may be useful in furthering our understanding of diseases characterized by apoptotic dysregulation as well as aiding drug development targeting apoptotic pathways. We evaluated the ability of radiolabeled isatins to quantify caspase-3 activity induced by the activation of the extrinsic apoptotic pathway by the anti-Fas antibody in mice. Methods: The behavior of three different radiolabeled isatins ([18F]WC-II-89, [18F]WC-IV-3 and [11C]WC-98) was characterized in mice with and without anti-Fas antibody treatment by microPET imaging and biodistribution studies. The activity of [18F]WC-II-89 was also compared with [99mTc]mebrofenin. The effect of pan-caspase inhibition with quinolyl-valyl-O-methylaspartyl-[2,6-difluorophenoxy]-methyl ketone (Q-VD-OPh) on [18F]WC-II-89 uptake was studied. Caspase-3 activity was confirmed by a fluorometric enzyme assay. Results: All three tracers behaved similarly in microPET and biodistribution studies. Increased retention of all tracers was observed in the livers of treated animals and several other organs, all of which demonstrated increased caspase-3 enzyme activity; however, impaired hepatobiliary excretion made attribution of these findings to caspase-3 activity difficult. The isatin [18F]WC-II-89 was retained at statistically significantly higher levels in the organs after anti-Fas antibody treatment while [99mTc]mebrofenin activity cleared, suggesting specific binding to activated caspase-3, but the magnitude of increased binding was still relatively low. Caspase inhibition with Q-VD-OPh partially blocked [18F]WC-II-89 retention but completely blocked caspase-3 enzyme activity in the liver. Conclusions: The radiolabeled isatins appear to bind specifically to caspase-3 in vivo, but their sensitivity is limited. Further optimization is required for these tracers to be useful for clinical applications.

  2. Caspase cleavage of GFAP produces an assembly-compromised proteolytic fragment that promotes filament aggregation

    Directory of Open Access Journals (Sweden)

    Mei‑Hsuan Chen

    2013-11-01

    Full Text Available IF (intermediate filament proteins can be cleaved by caspases to generate proapoptotic fragments as shown for desmin. These fragments can also cause filament aggregation. The hypothesis is that disease-causing mutations in IF proteins and their subsequent characteristic histopathological aggregates could involve caspases. GFAP (glial fibrillary acidic protein, a closely related IF protein expressed mainly in astrocytes, is also a putative caspase substrate. Mutations in GFAP cause AxD (Alexander disease. The overexpression of wild-type or mutant GFAP promotes cytoplasmic aggregate formation, with caspase activation and GFAP proteolysis. In this study, we report that GFAP is cleaved specifically by caspase 6 at VELD225 in its L12 linker domain in vitro. Caspase cleavage of GFAP at Asp225 produces two major cleavage products. While the C-GFAP (C-terminal GFAP is unable to assemble into filaments, the N-GFAP (N-terminal GFAP forms filamentous structures that are variable in width and prone to aggregation. The effect of N-GFAP is dominant, thus affecting normal filament assembly in a way that promotes filament aggregation. Transient transfection of N-GFAP into a human astrocytoma cell line induces the formation of cytoplasmic aggregates, which also disrupt the endogenous GFAP networks. In addition, we generated a neo-epitope antibody that recognizes caspase-cleaved but not the intact GFAP. Using this antibody, we demonstrate the presence of the caspase-generated GFAP fragment in transfected cells expressing a disease-causing mutant GFAP and in two mouse models of AxD. These findings suggest that caspase-mediated GFAP proteolysis may be a common event in the context of both the GFAP mutation and excess.

  3. A synthetic lethal screen identifies FAT1 as an antagonist of caspase-8 in extrinsic apoptosis

    OpenAIRE

    Kranz, Dominique; Boutros, Michael

    2014-01-01

    The extrinsic apoptosis pathway is initiated by binding of death ligands to death receptors resulting in the formation of the death-inducing signaling complex (DISC). Activation of procaspase-8 within the DISC and its release from the signaling complex is required for processing executor caspases and commiting cell death. Here, we report that the atypical cadherin FAT1 interacts with caspase-8 preventing the association of caspase-8 with the DISC. We identified FAT1 in a genome-wide siRNA scr...

  4. T-cell receptor downregulation by ceramide-induced caspase activation and cleavage of the zeta chain

    DEFF Research Database (Denmark)

    Menné, C; Lauritsen, Jens Peter Holst; Dietrich, J;

    2001-01-01

    gamma L-based motif-dependent and the tyrosine kinase-dependent pathways. This pathway is dependent on ceramide-induced activation of caspases and correlate with caspase-mediated cleavage of the zeta chain. Thus, a 10--15% downregulation of the TCR was induced following the treatment of the T cells with...... ceramide for 4 h. A close correlation between TCR downregulation, caspase activation, and cleavage of the zeta chain was found. Furthermore, the caspase inhibitors abolished the cleavage of the zeta chain and TCR downregulation in parallel with the inhibition of the caspase activity....

  5. Caspases in plants: metacaspase gene family in plant stress responses.

    Science.gov (United States)

    Fagundes, David; Bohn, Bianca; Cabreira, Caroline; Leipelt, Fábio; Dias, Nathalia; Bodanese-Zanettini, Maria H; Cagliari, Alexandro

    2015-11-01

    Programmed cell death (PCD) is an ordered cell suicide that removes unwanted or damaged cells, playing a role in defense to environmental stresses and pathogen invasion. PCD is component of the life cycle of plants, occurring throughout development from embryogenesis to the death. Metacaspases are cysteine proteases present in plants, fungi, and protists. In certain plant-pathogen interactions, the PCD seems to be mediated by metacaspases. We adopted a comparative genomic approach to identify genes coding for the metacaspases in Viridiplantae. We observed that the metacaspase was divided into types I and II, based on their protein structure. The type I has a metacaspase domain at the C-terminus region, presenting or not a zinc finger motif in the N-terminus region and a prodomain rich in proline. Metacaspase type II does not feature the prodomain and the zinc finger, but has a linker between caspase-like catalytic domains of 20 kDa (p20) and 10 kDa (p10). A high conservation was observed in the zinc finger domain (type I proteins) and in p20 and p10 subunits (types I and II proteins). The phylogeny showed that the metacaspases are divided into three principal groups: type I with and without zinc finger domain and type II metacaspases. The algae and moss are presented as outgroup, suggesting that these three classes of metacaspases originated in the early stages of Viridiplantae, being the absence of the zinc finger domain the ancient condition. The study of metacaspase can clarify their assignment and involvement in plant PCD mechanisms. PMID:26277721

  6. MSX2 overexpression inhibits gemcitabine-induced caspase-3 activity in pancreatic cancer cells

    Institute of Scientific and Technical Information of China (English)

    Shin Hamada; Kennichi Satoh; Kenji Kimura; Atsushi Kanno; Atsushi Masamune; Tooru Shimosegawa

    2005-01-01

    AIM: To evaluate the effect of MSX2 on gemcitabineinduced caspase-3 activation in pancreatic cancer cell line Panc-1.METHODS: Using V5-tagged MSX2 expression vector,stable transfectant of MSX2 was generated from Panc-1cells (Px14 cells). Cell viability under gemcitabine administration was determined by MTT assay relative to control cell line (empty-vector transfected Panc-1 cells;P-3EV cells). Hoechst staining was used for the detection of apoptotic cell. Activation of caspase-3 was assessed using Western blotting analysis and direct measurement of caspase-3 specific activities.RESULTS: MSX2 overexpression in Panc-1 cells resulted in decreased gemcitabine-induced caspase-3 activation and increased cell viability under gemcitabine treatment in Px14 cells.CONCLUSION: MSX2 exerts repressive effects on gemcitabine-induced apoptotic pathway. This novel apoptosis-regulating function of MSX2 may provide a new therapeutic target for pancreatic cancer.

  7. Expression of caspase-3 gene in apoptotic HL-60 cell and different human tumor cell lines

    International Nuclear Information System (INIS)

    Objective: To research the expression of caspase-3 gene in the apoptotic and the control HL-60 cells and in the different human tumor cell lines. Methods: Caspase-3 mRNA in the control and γ-radiation-induced apoptotic HL-60 cells, and in the 6 types of human tumor cell lines, was analysed by Northern blot. Results: The caspase-3 gene transcript was more highly expressed in leukemia cells HL-60, CEM, K562 and neuroblastoma SH-SY5Y than in cervical adenocarcinoma HeLa and breast carcinoma MCF7, and more highly in the radiation-induced apoptotic HL-60 than in the control HL-60 cells. Conclusion: The high level of expression of caspase-3 may aid the efforts to understand the tumor cell sensitivity to radiation, apoptosis and its inherent ability to survive

  8. Terazosin Treatment Induces Caspase-3 Expression in the Rat Ventral Prostate

    Science.gov (United States)

    Papadopoulos, Georgios; Vlachodimitropoulos, Dimitrios; Kyroudi, Aspasia; Kouloukoussa, Mirsini; Perrea, Despina; Mitropoulos, Dionisios

    2013-01-01

    Background Quinazoline-based alpha1-adrenergic receptor antagonists may not act solely on smooth muscle contractility. We evaluated the in vivo effect of terazosin on the expression of caspase-3 in the rat ventral prostate. Methods Fifteen Wistar rats were treated with terazosin (1.2 mg/kg body weight, given orally every second day) for 120 days. Another 15 control animals received the same amount of distilled water. The expression of caspase-3 was assessed immunohistochemically in formalin-fixed, paraffin-embedded tissue sections. Results Terazosin treatment did not affect prostate weight and histomorphology. In controls caspase-3 was expressed weakly and sporadically. In contrast, strong and weak expression was evident in 67% and 33% of the terazosin-treated specimens, respectively. Conclusions These findings implicate the induction of caspase-3 expression by terazosin as a potential molecular mechanism of its apoptotic action on prostate cells. PMID:23518907

  9. Differential efficacy of caspase inhibitors on apoptosis markers during sepsis in rats and implication for fractional inhibition requirements for therapeutics.

    Science.gov (United States)

    Méthot, Nathalie; Huang, JingQi; Coulombe, Nathalie; Vaillancourt, John P; Rasper, Dita; Tam, John; Han, Yongxin; Colucci, John; Zamboni, Robert; Xanthoudakis, Steven; Toulmond, Sylvie; Nicholson, Donald W; Roy, Sophie

    2004-01-19

    A rodent model of sepsis was used to establish the relationship between caspase inhibition and inhibition of apoptotic cell death in vivo. In this model, thymocyte cell death was blocked by Bcl-2 transgene, indicating that apoptosis was predominantly dependent on the mitochondrial pathway that culminates in caspase-3 activation. Caspase inhibitors, including the selective caspase-3 inhibitor M867, were able to block apoptotic manifestations both in vitro and in vivo but with strikingly different efficacy for different cell death markers. Inhibition of DNA fragmentation required substantially higher levels of caspase-3 attenuation than that required for blockade of other apoptotic events such as spectrin proteolysis and phosphatidylserine externalization. These data indicate a direct relationship between caspase inhibition and some apoptotic manifestations but that small quantities of uninhibited caspase-3 suffice to initiate genomic DNA breakdown, presumably through the escape of catalytic quantities of caspase-activated DNase. These findings suggest that putative caspase-independent apoptosis may be overestimated in some systems since blockade of spectrin proteolysis and other cell death markers does not accurately reflect the high degrees of caspase-3 inhibition needed to prevent DNA fragmentation. Furthermore, this requirement presents substantial therapeutic challenges owing to the need for persistent and complete caspase blockade. PMID:14718517

  10. Regional differences in the temporal expression of nonapoptotic caspase-3-positive Bergmann glial cells in the developing rat cerebellum

    Directory of Open Access Journals (Sweden)

    VelvetLee Finckbone

    2009-05-01

    Full Text Available Although caspases have been intimately linked to apoptotic events, some of the pro-apoptotic caspases also may regulate differentiation. We previously demonstrated that active caspase-3 is expressed and has an apparent non-apoptotic function during the development of cerebellar Bergmann glia. The current study seeks to further correlate active/cleaved caspase-3 expression with the developmental phenotype of Bergmann glia by examining regional differences in the temporal pattern of expression of cleaved caspase-3 immunoreactivity in lobules of the cerebellar vermis. In general, we found that the expression pattern of cleaved caspase-3 corresponds to the reported developmental temporal profile of the lobes and that its levels peak at 15 days and declines thereafter. Compared to intermediate or late maturing lobules, early maturing lobules had higher levels of active caspase-3 at earlier postnatal times. This period of postnatal development is precisely the time during which Bergmann glia initiate differentiation.

  11. Opposing roles for caspase and calpain death proteases in L-glutamate-induced oxidative neurotoxicity

    International Nuclear Information System (INIS)

    Oxidative glutamate toxicity in HT22 murine hippocampal cells is a model for neuronal death by oxidative stress. We have investigated the role of proteases in HT22 cell oxidative glutamate toxicity. L-glutamate-induced toxicity was characterized by cell and nuclear shrinkage and chromatin condensation, yet occurred in the absence of either DNA fragmentation or mitochondrial cytochrome c release. Pretreatment with the selective caspase inhibitors either benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (pan-caspase), N-acetyl-Leu-Glu-His-Asp-aldehyde (caspase 9) or N-acetyl-Ile-Glu-Thr-Asp-aldehyde (caspase 8), significantly increased L-glutamate-induced cell death with a corresponding increase in observed nuclear shrinkage and chromatin condensation. This enhancement of glutamate toxicity correlated with an increase in L-glutamate-dependent production of reactive oxygen species (ROS) as a result of caspase inhibition. Pretreating the cells with N-acetyl-L-cysteine prevented ROS production, cell shrinkage and cell death from L-glutamate as well as that associated with the presence of the pan-caspase inhibitor. In contrast, the caspase-3/-7 inhibitor N-acetyl-Asp-Glu-Val-Asp aldehyde was without significant effect. However, pretreating the cells with the calpain inhibitor N-acetyl-Leu-Leu-Nle-CHO, but not the cathepsin B inhibitor CA-074, prevented cell death. The cytotoxic role of calpains was confirmed further by: 1) cytotoxic dependency on intracellular Ca2+ increase, 2) increased cleavage of the calpain substrate Suc-Leu-Leu-Val-Tyr-AMC and 3) immunoblot detection of the calpain-selective 145 kDa α-fodrin cleavage fragment. We conclude that oxidative L-glutamate toxicity in HT22 cells is mediated via calpain activation, whereas inhibition of caspases-8 and -9 may exacerbate L-glutamate-induced oxidative neuronal damage through increased oxidative stress

  12. Contribution of the Caspase Gene Sequence Diversification to the Specifically Antiviral Defense in Invertebrate

    OpenAIRE

    Bin Zhi; Lei Wang; Guangyi Wang; Xiaobo Zhang

    2011-01-01

    Vertebrates achieve adaptive immunity of all sorts against pathogens through the diversification of antibodies. However the mechanism of invertebrates' innate immune defense against various pathogens remains largely unknown. Our study used shrimp and white spot syndrome virus (WSSV) to show that PjCaspase, a caspase gene of shrimp that is crucial in apoptosis, possessed gene sequence diversity. At present, the role of gene sequence diversity in immunity has not been characterized. To address ...

  13. Testosterone undecanoate and depo medroxyprogesterone acetate induced azoospermia through increased expression of spermatogenic cell caspase 3

    OpenAIRE

    Nukman Moeloek; Asmarinah Asmarinah; Nurjati C. Siregar; Syafruddin Ilyas

    2008-01-01

    The administration of a combination of testosterone undecanoate (TU, a long-acting androgen) and depo-medroxyprogesterone acetate (DMPA) were investigated in term of suppression of rat sperm concentration in vivo to azoospermia through increasing activity of spermatogenic cell caspase 3. Adult Sprague Dawley rats received TU and DMPA of 2.5 mg and 1.25 mg, respectively, a regimen known to rapidly reduce intra testicular testosterone and to produce azoospermia within 12 weeks. Caspase 3 positi...

  14. Dietary and behavioral interventions protect against age related activation of caspase cascades in the canine brain.

    Science.gov (United States)

    Snigdha, Shikha; Berchtold, Nicole; Astarita, Giuseppe; Saing, Tommy; Piomelli, Daniele; Cotman, Carl W

    2011-01-01

    Lifestyle interventions such as diet, exercise, and cognitive training represent a quietly emerging revolution in the modern approach to counteracting age-related declines in brain health. Previous studies in our laboratory have shown that long-term dietary supplementation with antioxidants and mitochondrial cofactors (AOX) or behavioral enrichment with social, cognitive, and exercise components (ENR), can effectively improve cognitive performance and reduce brain pathology of aged canines, including oxidative damage and Aβ accumulation. In this study, we build on and extend our previous findings by investigating if the interventions reduce caspase activation and ceramide accumulation in the aged frontal cortex, since caspase activation and ceramide accumulation are common convergence points for oxidative damage and Aβ, among other factors associated with the aged and AD brain. Aged beagles were placed into one of four treatment groups: CON--control environment/control diet, AOX--control environment/antioxidant diet, ENR--enriched environment/control diet, AOX/ENR--enriched environment/antioxidant diet for 2.8 years. Following behavioral testing, brains were removed and frontal cortices were analyzed to monitor levels of active caspase 3, active caspase 9 and their respective cleavage products such as tau and semaphorin7a, and ceramides. Our results show that levels of activated caspase-3 were reduced by ENR and AOX interventions with the largest reduction occurring with combined AOX/ENR group. Further, reductions in caspase-3 correlated with reduced errors in a reversal learning task, which depends on frontal cortex function. In addition, animals treated with an AOX arm showed reduced numbers of cells expressing active caspase 9 or its cleavage product semaphorin 7A, while ENR (but not AOX) reduced ceramide levels. Overall, these data demonstrate that lifestyle interventions curtail activation of pro-degenerative pathways to improve cellular health and are the

  15. Overexpression of Caspase-1 in adenocarcinoma of pancreas and chronic pancreatitis

    Institute of Scientific and Technical Information of China (English)

    Yin-Mo Yang; Marco Ramadani; Yan-Ting Huang

    2003-01-01

    AIM: To identify the expression of Caspase-l(interleukin1.β converting enzyme) and its role in adenoma of the pancreas and chronic pancreatitis.METHODS: The expression of Caspase-1 was assessed in 42 pancreatic cancer tissue samples, 38 chronic pancreatitis specimens, and 9 normal pancreatic tissues by immunohistochemistry and Western blot analysis.RESULTS: Overexpression of Caspase-1 was observed in both disorders, but there were differences in the expression patterns in distinct morphologic compartments. Pancreatic cancer tissues showed a clear cytoplasmatic overexpression of Caspase-1 in tumor cells of 71% of the tumors, whereas normal pancreatic tissues showed only occasional immunoreactivity. In chronic pancreatitis, overexpression of Caspase-1 was found in atrophic acinar cells (89 %),hyperplastic ducts (87 %), and dedifferentiating acinar cells (84 %). Although in atrophic cells a clear nuclear expression was found, hyperplastic ducts and dedifferentiating acinar cells showed dear cytoplasmic expression. Western blot analysis revealed a marked expression of the 45 kDa precursor of Caspase-1 in pancreatic cancer and chronic pancreatitis (80 %and 86 %, respectively). Clear bands at 30 kDa, which suggested the p10-p20 heterodimer of active Caspase-1, were found in 60 % of the cancer tissue and 14 % of the pancreatitis tissue specimens, but not in normal pancreatic tissues.CONCLUSION: Overexpression of Caspase-1 is a frequent event in pancreatic disorders and its differential expression patterns may reflect two functions of the protease. One is its participation in the apoptotic pathway in atrophic acinar cells and tumor-surrounding pancreatitis tissue, the other is its possible role in proliferative processes in pancreatic cancer cells and hyperplastic duct cells and dedifferentiating acinar cells in chronic pancreatitis.

  16. Dietary and behavioral interventions protect against age related activation of caspase cascades in the canine brain.

    Directory of Open Access Journals (Sweden)

    Shikha Snigdha

    Full Text Available Lifestyle interventions such as diet, exercise, and cognitive training represent a quietly emerging revolution in the modern approach to counteracting age-related declines in brain health. Previous studies in our laboratory have shown that long-term dietary supplementation with antioxidants and mitochondrial cofactors (AOX or behavioral enrichment with social, cognitive, and exercise components (ENR, can effectively improve cognitive performance and reduce brain pathology of aged canines, including oxidative damage and Aβ accumulation. In this study, we build on and extend our previous findings by investigating if the interventions reduce caspase activation and ceramide accumulation in the aged frontal cortex, since caspase activation and ceramide accumulation are common convergence points for oxidative damage and Aβ, among other factors associated with the aged and AD brain. Aged beagles were placed into one of four treatment groups: CON--control environment/control diet, AOX--control environment/antioxidant diet, ENR--enriched environment/control diet, AOX/ENR--enriched environment/antioxidant diet for 2.8 years. Following behavioral testing, brains were removed and frontal cortices were analyzed to monitor levels of active caspase 3, active caspase 9 and their respective cleavage products such as tau and semaphorin7a, and ceramides. Our results show that levels of activated caspase-3 were reduced by ENR and AOX interventions with the largest reduction occurring with combined AOX/ENR group. Further, reductions in caspase-3 correlated with reduced errors in a reversal learning task, which depends on frontal cortex function. In addition, animals treated with an AOX arm showed reduced numbers of cells expressing active caspase 9 or its cleavage product semaphorin 7A, while ENR (but not AOX reduced ceramide levels. Overall, these data demonstrate that lifestyle interventions curtail activation of pro-degenerative pathways to improve cellular

  17. Caspase-8 Binding to Cardiolipin in Giant Unilamellar Vesicles Provides a Functional Docking Platform for Bid

    DEFF Research Database (Denmark)

    Jalmar, Olivier; Franc¸ois-Moutal, Liberty; García-Sáez, Ana-Jesus;

    2013-01-01

    activating platform for caspase-8 at the mitochondrial membrane surface. Destabilisation of this platform alters receptor-mediated apoptosis in diseases such as Barth Syndrome, which is characterised by the presence of immature cardiolipin which does not allow caspase-8 binding. We used a simplified in vitro...... shed light on the active functional role of cardiolipin, bridging the gap between death receptors and mitochondria. © 2013 Jalmar et al....

  18. The Drosophila effector caspase Dcp-1 regulates mitochondrial dynamics and autophagic flux via SesB

    OpenAIRE

    DeVorkin, Lindsay; Go, Nancy Erro; Hou, Ying-Chen Claire; Moradian, Annie; Morin, Gregg B.; Gorski, Sharon M.

    2014-01-01

    Increasing evidence reveals that a subset of proteins participates in both the autophagy and apoptosis pathways, and this intersection is important in normal physiological contexts and in pathological settings. In this paper, we show that the Drosophila effector caspase, Drosophila caspase 1 (Dcp-1), localizes within mitochondria and regulates mitochondrial morphology and autophagic flux. Loss of Dcp-1 led to mitochondrial elongation, increased levels of the mitochondrial adenine nucleotide t...

  19. IMMUNOHISTOCHEMICAL ANALYSIS OF CASPASE-3 ACTIVITY IN LIVER BIOPSIES OF PATIENTS WITH MONO AND MIXED INFECTIONS

    Directory of Open Access Journals (Sweden)

    I. I. Tokin

    2015-01-01

    Full Text Available Objective: to study the activity of proapoptotic signal protein caspase-3 for determination of peculiarities of apoptosis regulation under liver chronic diseases.Subjects and methods. The immunohistochemical analysis of caspase-3 activity in 5 liver biopsies of the patients with mono infection of chronic hepatitis B and 5 liver biopsies of the patients with mixed infection of tuberculosis, chronic hepatitis C and human immunodeficiency virus was fulfilled. Morphological and morphometric analysis of serial microphotographs was performed using an image analysis system (microscope Leica DM 2500, digital camera Leica DFC320 R2 and a computer.Results. The activity of caspase-3 as dark brown granularity was revealed in all tis-sue components of liver (hepatocytes, epithelium of bile ducts, endotheliocytes, Kupffer cells of sinusoids, in compositions of lymphohistiocyte infiltrations. The maximal activity was discovered in hepatocytes nuclei. The expression of caspase-3 was significantly higher in liver biopsies of the patients with mixed infection. It is typical that the immunoreactive hepatocytes had not any morphological marks of apoptosis.Conclusion. The caspase-3 expression of proapoptotic signal protein caspase-3 may serve as an early marker of liver damage including the possibilities of apoptosis development.

  20. 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.

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

    International Nuclear Information System (INIS)

    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.

  2. Doxorubicin/heparin composite nanoparticles for caspase-activated prodrug chemotherapy.

    Science.gov (United States)

    Khaliq, Nisar Ul; Sandra, Febrina Carolina; Park, Dal Yong; Lee, Jae Young; Oh, Keun Sang; Kim, Dongkyu; Byun, Youngro; Kim, In-San; Kwon, Ick Chan; Kim, Sang Yoon; Yuk, Soon Hong

    2016-09-01

    Caspase-activated prodrug chemotherapy is introduced and demonstrated using the composite nanoparticles (NPs), which deliver doxorubicin (DOX) and DEVD-S-DOX together to the tumor tissue. DEVD-S-DOX, DOX linked to a peptide moiety (DEVD), is a prodrug that is cleaved into free DOX by caspase-3 upon apoptosis. DEVD-S-DOX has no therapeutic efficacy, but it changes into free DOX with the expression of caspase-3. With the accumulation of the composite NPs in the tumor tissue by the enhanced permeation and retention (EPR) effect, a small exposure of DOX in the tumor cells initiated apoptosis in a localized area of the tumor tissue, which induced caspase-3 activation. Cleavage of DEVD-S-DOX into free DOX by caspase-3 continued with repetitive activation of caspase-3 and cleavage of DEVD-S-DOX at the tumor site. The composite NPs were characterized with transmittance electron microscopy (TEM) and particle size analyzer. We then evaluated the nanoparticle drug release, therapeutic efficacy, and in vivo biodistribution for tumor targeting using a non-invasive live animal imaging technology and the quantification of DOX with high performance liquid chromatography. DOX-induced apoptosis-targeted chemotherapy (DIATC) was verified by in vitro/in vivo DEVD-S-DOX response to free DOX and cellular uptake behavior of the composite NPs with flow cytometry analysis. Significant antitumor efficacy with minimal cardiotoxicity was also observed, which supported DIATC for improved chemotherapy. PMID:27286189

  3. Avian leukosis virus subgroup J triggers caspase-1-mediated inflammatory response in chick livers.

    Science.gov (United States)

    Liu, Xue-Lan; Shan, Wen-Jie; Jia, Li-Juan; Yang, Xu; Zhang, Jin-Jing; Wu, Ya-Rong; Xu, Fa-Zhi; Li, Jin-Nian

    2016-04-01

    Many pathogens trigger caspase-1-mediated innate immune responses. Avian leukosis virus subgroup J (ALV-J) causes serious immunosuppression and diverse tumors in chicks. The caspase-1 inflammasome mechanism of response to ALV-J invading remains unclear. Here we investigated the expression of caspase-1, the inflammasome adaptor NLRP3, IL-1β and IL-18 in response to ALV-J infection in the liver of chick. We found caspase-1 mRNA expression was elevated at 5dpi and peaked at 7dpi in ALV-J infected animals. Corresponding to this, the expressions of NLRP3 and proinflammatory cytokines IL-1β and IL-18 were significantly increased at 5 or 7dpi. In addition, caspase-1 protein expression and inflammatory cell infiltration were induced after virus infection. These results indicated that ALV-J infection could trigger the caspase-1- mediated inflammatory response in chicks. Thus, an understanding of the inflammatory responses can provide a better insight into the pathogenicity of ALV-J and a possible anti-virus target for ALV-J infection. PMID:26811903

  4. Role of caspase-3 in neuronal apoptosis after experimental intracerebral hemorrhage in rats

    International Nuclear Information System (INIS)

    Objective: To investigate the relationship between the expression of caspase-3 and neuronal apoptosis after experimental intracerebral hemorrhage (ICH) in rats. Methods: ICH rat models were prepared with stereotactic infusion of autologous blood into caudate nucleus. TUNEL method of staining was used to detect apoptotic cells and immunohistochemistry technic was applied to detect caspase-3 expression in cerebral tissue of the sacrificed animals at 6h, 12h, 24h, 48h, 72h, 1w, 2w after the intracerebral blood infusion (n=5 in each group). Five rats underwent sham operation with intracerebral infusion of normal saline. Results: At 6h, TONEL positive cells could be demonstrated with peak at 72h and could still be shown at 2w. Apoptotic cells were not seen in the sham-operation group. Caspase-3 expression was significant at 6h with peak at 24h and was significantly higher than those in the sham group (P<0.05). Expression of caspase-3 was positively correlated with the number of TUNEL positive cells (r=0.547, P<0.01). Conclusion: The cellular (neuronal) apoptosis after experimental intracerebral hemorrhage was closely related to the expression of caspase-3, suggesting possible beneficial effect of selective caspase-3 inhibitors. (authors)

  5. Effect of Bcl-2 and caspase-3 on calcium distribution in apoptosis of HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Apoptosis manifests in two major execution programs downstream of the death signal: the caspase pathway and organelle dysfunction. An important antiapoptosis factor, Bcl-2 protein, contributes in caspase pathway of apoptosis. Calcium, an important intracellular signal element in cells, is also observed to have changes during apoptosis, which maybe affected by Bcl2 protein. We have previously reported that in Harringtonine (HT) induced apoptosis of HL-60 cells, there's a change of intracellular calcium distribution, moving from cytoplast especially Golgi's apparatus to nucleus and accumulating there with the highest concentration. We report here that caspase-3 becomes activated in HT-induced apoptosis of HL-60 cells, which can be inhibited by overexpression of Bcl-2 protein. No sign of apoptosis or intracellular calcium movement from Golgi's apparatus to nucleus in HL-60 cells overexpressing Bcl-2 or treated with Ac-DEVD-CHO, a specific inhibitor of caspase-3. The results indicate that activated caspase-3 can promote the movement of intracellular calcium from Golgi's apparatus to nucleus, and the process is inhibited by Ac-DEVD-CHO (inhibitor of caspas-3), and that Bcl-2 can inhibit the movement and accumulation of intracellular calcium in nucleus through its inhibition on caspase3. Calcium relocalization in apoptosis seems to be irreversible, which is different from the intracellular calcium changes caused by growth factor.

  6. Activation of caspase-3 and its correlation with shear force in bovine skeletal muscles during postmortem conditioning.

    Science.gov (United States)

    Cao, J-X; Ou, C-R; Zou, Y-F; Ye, K-P; Zhang, Q-Q; Khan, M A; Pan, D-D; Zhou, G

    2013-09-01

    The study was aimed at exploring the mechanism of tenderization by establishing a correlation between caspase-3 activity and shear force, verifying the activation occurring by analyzing active caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP) fragments, and understanding the pathways involved in activation of caspase-3 by evaluating its correlation with caspase-8 and -9 activities in LM, semitendinosus (STN), and psoas minor (PM) muscles. The results indicated that shear force decreased at 48 h in PM (P < 0.01), LM (P < 0.01), and STN (P < 0.05). We detected p22, p23, p20, and p18 caspase fragments as well as distinctive PARP fragments of 24 kDa by caspase-3 and 36 kDa by µ-calpain. Caspase-3 activity correlated with shear force negatively at 24 and 48 h in STN (P < 0.01 at 24 h; P < 0.01 at 48 h), PM (P < 0.001 at 24 h; P < 0.01 at 48 h), and LM muscles (P < 0.05 at 24 h; P < 0.01 at 48 h). The greatest activities of caspase-8 (P < 0.001 in PM and STN; P < 0.01 in LM) and caspase-9 (P < 0.001 in muscles) appeared at 4 h whereas that of caspase-3 was at 24 h (P < 0.001 in muscles). Caspase-9 activity correlated positively with caspase-3 at 4, 24, and 48 h in STN (P < 0.01 at 4 h; P < 0.05 at 24 h; P < 0.001 at 48 h) and at 4 and 96 h in PM (P < 0.001 at 4 h; P < 0.05 at 96 h) and LM muscles (P < 0.001 at 4 h; P < 0.001 at 96 h). The caspase-8 activity correlated with caspase-3 at 4, 48, and 96 h in STN (P < 0.05 at 4 h; P < 0.001 at 48 h; P < 0.05 at 96 h), at 4 and 24 h in PM (P < 0.001 at 4 h; P < 0.05 at 24 h), and at 4 and 96 h in LM (P < 0.001 at 4 h; P < 0.01 at 96 h). We concluded that caspase-3 was associated with the decline of shear force; the activation of caspase-3 was mediated by caspases -8 and -9 in muscles. However, more detailed studies are needed to define the precise mechanism for the cleavage of pro-caspases -8 and -9 during conditioning. PMID:23893998

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

    International Nuclear Information System (INIS)

    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. 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.

  9. 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.

  10. Bax translocation mediated mitochondrial apoptosis and caspase dependent photosensitizing effect of Ficus religiosa on cancer cells.

    Science.gov (United States)

    Haneef, Jazir; Parvathy, Muraleedharan; M, Parvathy; Thankayyan R, Santhosh Kumar; Sithul, Hima; Sreeharshan, Sreeja

    2012-01-01

    The main aim of the present work was to investigate the potential effect of acetone extract of Ficus religosa leaf (FAE) in multiple apoptosis signalling in human breast cancer cells. FAE treatment significantly induced dose and time dependent, irreversible inhibition of breast cancer cell growth with moderate toxicity to normal breast epithelial cells. This observation was validated using Sulforhodamine B assay. Cell cycle analysis by Flow cytometry showed cell cycle arrest in G1 phase and induction of sub-G0 peak. FAE induced chromatin condensation and displayed an increase in apoptotic population in Annexin V-FITC/PI (Fluorescein isothiocyanate/Propidium iodide) double staining. FAE stimulated the loss of mitochondrial membrane potential in multiple breast cancer cell lines when compared to normal diploid cells. To understand the role of Bax in FAE induced apoptosis, we employed a sensitive cell based platform of MCF-7 cells expressing Bax-EGFP. Bax translocation to mitochondria was accompanied by the disruption of mitochondrial membrane potential and marked elevation in LEHDase activity (Caspase 9). Consistent with this data, FAE induced Caspase activation as evidenced by ratio change in FRET Caspase sensor expressing MCF-7 cell line and cleavage of prominent Caspases and PARP. Interestingly, FAE accelerated cell death in a mitochondrial dependent manner in continuous live cell imaging mode indicating its possible photosensitizing effect. Intracellular generation of reactive oxygen species (ROS) by FAE played a critical role in mediating apoptotic cell death and photosensitizing activity. FAE induced dose and time dependent inhibition of cancer cell growth which was associated with Bax translocation and mitochondria mediated apoptosis with the activation of Caspase 9 dependent Caspase cascade. FAE also possessed strong photosensitizing effect on cancer cell line that was mediated through rapid mitochondrial transmembrane potential loss and partial Caspase

  11. Bax translocation mediated mitochondrial apoptosis and caspase dependent photosensitizing effect of Ficus religiosa on cancer cells.

    Directory of Open Access Journals (Sweden)

    Jazir Haneef

    Full Text Available The main aim of the present work was to investigate the potential effect of acetone extract of Ficus religosa leaf (FAE in multiple apoptosis signalling in human breast cancer cells. FAE treatment significantly induced dose and time dependent, irreversible inhibition of breast cancer cell growth with moderate toxicity to normal breast epithelial cells. This observation was validated using Sulforhodamine B assay. Cell cycle analysis by Flow cytometry showed cell cycle arrest in G1 phase and induction of sub-G0 peak. FAE induced chromatin condensation and displayed an increase in apoptotic population in Annexin V-FITC/PI (Fluorescein isothiocyanate/Propidium iodide double staining. FAE stimulated the loss of mitochondrial membrane potential in multiple breast cancer cell lines when compared to normal diploid cells. To understand the role of Bax in FAE induced apoptosis, we employed a sensitive cell based platform of MCF-7 cells expressing Bax-EGFP. Bax translocation to mitochondria was accompanied by the disruption of mitochondrial membrane potential and marked elevation in LEHDase activity (Caspase 9. Consistent with this data, FAE induced Caspase activation as evidenced by ratio change in FRET Caspase sensor expressing MCF-7 cell line and cleavage of prominent Caspases and PARP. Interestingly, FAE accelerated cell death in a mitochondrial dependent manner in continuous live cell imaging mode indicating its possible photosensitizing effect. Intracellular generation of reactive oxygen species (ROS by FAE played a critical role in mediating apoptotic cell death and photosensitizing activity. FAE induced dose and time dependent inhibition of cancer cell growth which was associated with Bax translocation and mitochondria mediated apoptosis with the activation of Caspase 9 dependent Caspase cascade. FAE also possessed strong photosensitizing effect on cancer cell line that was mediated through rapid mitochondrial transmembrane potential loss and

  12. Functional and biochemical characterization of the baculovirus caspase inhibitor MaviP35.

    Science.gov (United States)

    Brand, I L; Green, M M; Civciristov, S; Pantaki-Eimany, D; George, C; Gort, T R; Huang, N; Clem, R J; Hawkins, C J

    2011-01-01

    Many viruses express proteins which prevent the host cell death that their infection would otherwise provoke. Some insect viruses suppress host apoptosis through the expression of caspase inhibitors belonging to the P35 superfamily. Although a number of P35 relatives have been identified, Autographa californica (Ac) P35 and Spodoptera littoralis (Spli) P49 have been the most extensively characterized. AcP35 was found to inhibit caspases via a suicide substrate mechanism: the caspase cleaves AcP35 within its 'reactive site loop' then becomes trapped, irreversibly bound to the cleaved inhibitor. The Maruca vitrata multiple nucleopolyhedrovirus encodes a P35 family member (MaviP35) that exhibits 81% identity to AcP35. We found that this relative shared with AcP35 the ability to inhibit mammalian and insect cell death. Caspase-mediated cleavage within the MaviP35 reactive site loop occurred at a sequence distinct from that in AcP35, and the inhibitory profiles of the two P35 relatives differed. MaviP35 potently inhibited human caspases 2 and 3, DCP-1, DRICE and CED-3 in vitro, but (in contrast to AcP35) only weakly suppressed the proteolytic activity of the initiator human caspases 8, 9 and 10. Although MaviP35 inhibited the AcP35-resistant caspase DRONC in yeast, and was sensitive to cleavage by DRONC in vitro, MaviP35 failed to inhibit the proteolytic activity of bacterially produced DRONC in vitro. PMID:22170098

  13. Ofloxacin induces apoptosis in microencapsulated juvenile rabbit chondrocytes by caspase-8-dependent mitochondrial pathway

    International Nuclear Information System (INIS)

    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 β1 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

  14. Cytoprotective effect of selective small-molecule caspase inhibitors against staurosporine-induced apoptosis

    Directory of Open Access Journals (Sweden)

    Wu J

    2014-05-01

    Full Text Available Jianghong Wu, Yuren Wang, Shuguang Liang, Haiching Ma Reaction Biology Corp, Malvern, PA, USA Abstract: Caspases are currently known as the central executioners of the apoptotic pathways. Inhibition of apoptosis and promotion of normal cell survival by caspase inhibitors would be a tremendous benefit for reducing the side effects of cancer therapy and for control of neurodegenerative disorders such as Parkinson's, Alzheimer's, and Huntington's diseases. The objective of this study was to discover small-molecule caspase inhibitors with which to achieve cytoprotective effect. We completed the high-throughput screening of Bionet's 37,500-compound library (Key Organics Limited, Camelford, Cornwall, UK against caspase-1, -3, and -9 and successfully identified 43 initial hit compounds. The 43 hit compounds were further tested for cytoprotective activity against staurosporine-induced cell death in NIH3T3 cells. Nineteen compounds were found to have significant cytoprotective effects in cell viability assays. One of the compounds, RBC1023, was demonstrated to protect NIH3T3 cells from staurosporine-induced caspase-3 cleavage and activation. RBC1023 was also shown to protect against staurosporine-induced impairment of mitochondrial membrane potential. DNA microarray analysis demonstrated that staurosporine treatment induced broad global gene expression alterations, and RBC1023 co-treatment significantly restored these changes, especially of the genes that are related to cell growth and survival signaling such as Egr1, Cdc25c, cdkn3, Rhob, Nek2, and Taok1. Collectively, RBC1023 protects NIH3T3 cells against staurosporine-induced apoptosis via inhibiting caspase activity, restoring mitochondrial membrane potential, and possibly upregulating some cell survival-related gene expressions and pathways. Keywords: cell death, caspase inhibition, mitochondria, RBC1023

  15. Caspase-1 as a central regulator of high fat diet-induced non-alcoholic steatohepatitis.

    Directory of Open Access Journals (Sweden)

    Laura J Dixon

    Full Text Available Nonalcoholic steatohepatitis (NASH is associated with caspase activation. However, a role for pro-inflammatory caspases or inflammasomes has not been explored in diet-induced liver injury. Our aims were to examine the role of caspase-1 in high fat-induced NASH. C57BL/6 wild-type and caspase 1-knockout (Casp1(-/- mice were placed on a 12-week high fat diet. Wild-type mice on the high fat diet increased hepatic expression of pro-caspase-1 and IL-1β. Both wild-type and Casp1(-/- mice on the high fat diet gained more weight than mice on a control diet. Hepatic steatosis and TG levels were increased in wild-type mice on high fat diet, but were attenuated in the absence of caspase-1. Plasma cholesterol and free fatty acids were elevated in wild-type, but not Casp1(-/- mice, on high fat diet. ALT levels were elevated in both wild-type and Casp1(-/- mice on high fat diet compared to control. Hepatic mRNA expression for genes associated with lipogenesis was lower in Casp1(-/- mice on high fat diet compared to wild-type mice on high fat diet, while genes associated with fatty acid oxidation were not affected by diet or genotype. Hepatic Tnfα and Mcp-1 mRNA expression was increased in wild-type mice on high fat diet, but not in Casp1(-/- mice on high fat diet. αSMA positive cells, Sirius red staining, and Col1α1 mRNA were increased in wild-type mice on high fat diet compared to control. Deficiency of caspase-1 prevented those increases. In summary, the absence of caspase-1 ameliorates the injurious effects of high fat diet-induced obesity on the liver. Specifically, mice deficient in caspase-1 are protected from high fat-induced hepatic steatosis, inflammation and early fibrogenesis. These data point to the inflammasome as an important therapeutic target for NASH.

  16. Characterization and expression analysis of a caspase-2 in an invertebrate echinoderm sea cumber Apostichopus japonicus.

    Science.gov (United States)

    Ye, Shigen; Gao, Yang; Wang, Shengnan; Li, Qiang; Li, Ruijun; Li, Hua

    2016-01-01

    Caspase-2 is the most evolutionarily conserved member of the caspase family which mediates the programmed cell death and plays crucial roles in key cellular processes. In this study, a caspase-2 homolog was identified and functionally characterized in sea cucumber Apostichopus japonicus, which we named AjCASP. The full-length cDNA consists of 2100 bp with an ORF encoding a protein of 378 amino acids. The deduced amino acid sequence shows that AjCASP consists of a conserved CARD-CASP2 domain and a CASs domain containing two active residues, two proteolytic cleavage residues, a substrate pocket and a dimer interface as well. In addition, a p20 large subunit with a characteristic five-peptide motif (QACRG) and a p10 small subunit in C-terminal were identified in CASs domain. Above data demonstrated that AjCASP is similar to CED-3 (the caspase-2 homolog of nematode Caenorhabditis elegans), which is further confirmed by phylogenetic tree analysis. AjCASP was ubiquitously expressed in sea cucumber and the obviously higher expression level was observed in coelomocyte, respiratory tree and intestine. Real-time PCR analyses further demonstrated that AjCASP was significantly induced by LPS. Taken together, these results strongly suggest that AjCASP is a caspase-2 homolog and it may be involved in invertebrate immune response, especially in eliminating and degrading invading pathogens. PMID:26687532

  17. Caspase-like proteins: Acanthamoeba castellanii metacaspase and Dictyostelium discoideum paracaspase, what are their functions?

    Indian Academy of Sciences (India)

    Entsar Saheb; Wendy Trzyna; John Bush

    2014-12-01

    Caspases are cysteine proteases that are important regulators of programmed cell death in animals. Two novel relatives to members of the caspase families metacaspases and paracaspase have been discovered. Metacaspase type-1 was identified in Acanthamoeba castellanii, an opportunistic protozoan parasite that causes severe diseases in humans. Paracaspase was found in the non-pathogenic protozoan Dictyostelium discoideum. Since their discovery in Acanthamoeba and Dictyostelium, metacaspases and paracaspases have remained poorly characterized. At present we do not have sufficient data about the molecular function of these caspase-like proteins or their role, if any, in programmed cell death. How these caspase proteins function at the molecular level is an important area of study that will provide insight into their potential for treatment therapies against Acanthamoeba infection and other similar parasitic protozoan. Additionally, finding the molecular functions of these caspase-like proteins will provide information concerning their role in more complex organisms.The aim of this article was to review recent discoveries about metacaspases and paracaspases as regulators of apoptotic and non-apoptotic processes.

  18. Caspase-Mediated Apoptosis in Sensory Neurons of Cultured Dorsal Root Ganglia in Adult Mouse

    Directory of Open Access Journals (Sweden)

    Hamid Reza Momeni

    2013-01-01

    Full Text Available Objective: Sensory neurons in dorsal root ganglia (DRG undergo apoptosis after peripheral nerve injury. The aim of this study was to investigate sensory neuron death and the mechanism involved in the death of these neurons in cultured DRG.Materials and Methods: In this experimental study, L5 DRG from adult mouse were dissected and incubated in culture medium for 24, 48, 72 and 96 hours. Freshly dissected and cultured DRG were then fixed and sectioned using a cryostat. Morphological and biochemical features of apoptosis were investigated using fluorescent staining (Propidium iodide and Hoechst 33342 and the terminal Deoxynucleotide transferase dUTP nick end labeling (TUNEL method respectively. To study the role of caspases, general caspase inhibitor (Z-VAD.fmk, 100 μM and immunohistochemistry for activated caspase-3 were used.Results: After 24, 48, 72 and 96 hours in culture, sensory neurons not only displayed morphological features of apoptosis but also they appeared TUNEL positive. The application of Z-VAD.fmk inhibited apoptosis in these neurons over the same time period. In addition, intense activated caspase-3 immunoreactivity was found both in the cytoplasm and the nuclei of these neurons after 24 and 48 hours.Conclusion: Results of the present study show caspase-dependent apoptosis in the sensory neurons of cultured DRG from adult mouse.

  19. Caspase cleavage of cytochrome c1 disrupts mitochondrial function and enhances cytochrome c release

    Institute of Scientific and Technical Information of China (English)

    Yushan Zhu; Min Li; Xiaohui Wang; Haijing Jin; Shusen Liu; Jianxin Xu; Quan Chen

    2012-01-01

    Mitochondrial catastrophe can be the cause or consequence of apoptosis and is associated with a number of pathophysiological conditions.The exact relationship between mitochondrial catastrophe and caspase activation is not completely understood.Here we addressed the underlying mechanism,explaining how activated caspase could feedback to attack mitochondria to amplify further cytochrome e (cyto.c) release.We discovered that cytochrome c1 (cyto.c1) in the bc1 complex of the mitochondrial respiration chain was a novel substrate of caspase 3 (casp.3).We found that cyto.c1 was cleaved at the site of D106,which is critical for binding with cyto.c,following apoptotic stresses or targeted expression of casp.3 into tbe mitochondrial intermembrane space.We demonstrated that this cleavage was closely linked with further cyto.c release and mitochondrial catastrophe.These mitochondrial events could be effectively blocked by expressing non-cleavable cyto.c1 (D106A) or by caspase inhibitor z-VAD-fmk.Our results demonstrate that the cleavage of cyto.c1 represents a critical step for the feedback amplification of cyto.c release by caspases and subsequent mitochondrial catastrophe.

  20. Cross-talk between calpain and caspase proteolytic systems during neuronal apoptosis.

    Science.gov (United States)

    Neumar, Robert W; Xu, Y Anne; Gada, Hemal; Guttmann, Rodney P; Siman, Robert

    2003-04-18

    Cross-talk between calpain and caspase proteolytic systems has complicated efforts to determine their distinct roles in apoptotic cell death. This study examined the effect of overexpressing calpastatin, the specific endogenous calpain inhibitor, on the activity of the two proteolytic systems following an apoptotic stimulus. Human SH-SY5Y neuroblastoma cells were stably transfected with full-length human calpastatin cDNA resulting in 20-fold overexpression based on Western blot and 5-fold greater calpain inhibitory activity in cell extracts. Wild type and calpastatin overexpressing (CST1) cells were neuronally differentiated and apoptosis-induced with staurosporine (0.1-1.0 microm). Calpastatin overexpression decreased calpain activation, increased caspase-3-like activity, and accelerated the appearance of apoptotic nuclear morphology. Following 0.1-0.2 microm staurosporine, plasma membrane integrity based on calcein-acetoxymethyl fluorescence was significantly greater at 24 h in differentiated CST1 compared with differentiated wild type cells. However, this protective effect was lost at higher staurosporine doses (0.5-1.0 microm), which resulted in pronounced caspase-mediated degradation of the overexpressed calpastatin. These results suggest a dual role for calpains during neuronal apoptosis. In the early execution phase, calpain down-regulates caspase-3-like activity and slows progression of apoptotic nuclear morphology. Subsequent calpain activity, facilitated by caspase-mediated degradation of calpastatin, contributes to plasma membrane disruption and secondary necrosis. PMID:12576481

  1. Isoquinoline-1,3,4-trione Derivatives Inactivate Caspase-3 by Generation of Reactive Oxygen Species*S⃞

    OpenAIRE

    Du, Jun-qing; Wu, Jian; Zhang, Hua-jie; Zhang, Ya-Hui; Qiu, Bei-Ying; Wu, Fang; Chen, Yi-Hua; Li, Jing-Ya; Nan, Fa-Jun; Ding, Jian-Ping; Li, Jia

    2008-01-01

    Caspase-3 is an attractive therapeutic target for treatment of diseases involving disregulated apoptosis. We report here the mechanism of caspase-3 inactivation by isoquinoline-1,3,4-trione derivatives. Kinetic analysis indicates the compounds can irreversibly inactivate caspase-3 in a 1,4-dithiothreitol (DTT)- and oxygen-dependent manner, implying that a redox cycle might take place in the inactivation process. Reactive oxygen species detection experiments using a che...

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

    OpenAIRE

    Bloemberg, Darin; Quadrilatero, Joe

    2016-01-01

    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, calpa...

  3. Rhodamine 110-linked amino acids and peptides as substrates to measure caspase activity upon apoptosis induction in intact cells

    OpenAIRE

    Hug, H; Los, Marek Jan; Hirt, W; Debatin, K. M.

    1999-01-01

    Caspases (cysteine aspartate-specific proteases) are a structurally related group of cysteine proteases that cleave peptide bonds following specific recognition sequences. They play a central role in activating apoptosis of vertebrate cells. To measure apoptosis induced by various stimuli and at an early apoptotic stage, caspases are an ideal target. This is especially the case when apoptotic cells have to be analyzed ex vivo before phagocytes remove them. A new and sensitive caspase assay is...

  4. Effects of inhibitors on the synergistic interaction between calpain and caspase-3 during post-mortem aging of chicken meat.

    Science.gov (United States)

    Chen, Lin; Feng, Xian Chao; Zhang, Wan Gang; Xu, Xing Lian; Zhou, Guang Hong

    2012-08-29

    Calpain has been considered to be the most important protease involved in tenderization during the conversion of muscle into meat. However, recent evidence suggests the possible involvement of the key apoptosis protease, caspase, on post-mortem tenderization. This study used inhibitors of calpain and caspase-3 to treat chicken muscle immediately after slaughter and followed the changes in caspase-3 and calpain activities together with their expression during 5 days of aging. Addition of calpain inhibitors to the system resulted in significantly higher caspase-3 activities (p tenderizing process during the conversion of muscle tissue into meat. PMID:22720745

  5. The Caspase-8 Homolog Dredd Cleaves Imd and Relish but Is Not Inhibited by p35*

    Science.gov (United States)

    Kim, Chan-Hee; Paik, Donggi; Rus, Florentina; Silverman, Neal

    2014-01-01

    In Drosophila, the Imd pathway is activated by diaminopimelic acid-type peptidoglycan and triggers the humoral innate immune response, including the robust induction of antimicrobial peptide gene expression. Imd and Relish, two essential components of this pathway, are both endoproteolytically cleaved upon immune stimulation. Genetic analyses have shown that these cleavage events are dependent on the caspase-8 like Dredd, suggesting that Imd and Relish are direct substrates of Dredd. Among the seven Drosophila caspases, we find that Dredd uniquely promotes Imd and Relish processing, and purified recombinant Dredd cleaves Imd and Relish in vitro. In addition, interdomain cleavage of Dredd is not required for Imd or Relish processing and is not observed during immune stimulation. Baculovirus p35, a suicide substrate of executioner caspases, is not cleaved by purified Dredd in vitro. Consistent with this biochemistry but contrary to earlier reports, p35 does not interfere with Imd signaling in S2* cells or in vivo. PMID:24891502

  6. Evasion and interference: intracellular pathogens modulate caspase-dependent inflammatory responses.

    Science.gov (United States)

    Stewart, Mary K; Cookson, Brad T

    2016-06-01

    Pathogens have evolved to complete the virulence cycle of colonization, replication and dissemination in intimate association with a complex network of extracellular and intracellular surveillance systems that guard tissue spaces. In this Review, we discuss the strategies used by bacteria and viruses to evade or inhibit intracellular detection that is coupled to pro-inflammatory caspase-dependent protective responses. Such strategies include alterations of lipopolysaccharide (LPS) structures, the regulated expression of components of type III secretion systems, and the utilization of proteins that inhibit inflammasome formation, the enzymatic activity of caspases and cytokine signalling. Inflammation is crucial in response to exposure to pathogens, but is potentially damaging and thus tightly regulated. The threshold for the activation of pro-inflammatory caspases is determined by the immediate stimulus in the context of previous signals. Pathogen, genetic and situational factors modulate this threshold, which determines the ability of the host to resist infection while minimizing harm. PMID:27174147

  7. Correlation between neuronal injury and Caspase-3 after focal ischemia in human hippocampus

    Institute of Scientific and Technical Information of China (English)

    戚基萍; 吴爱萍; 王德生; 王立峰; 李淑霞; 徐凤琳

    2004-01-01

    Background Cerebral ischemia is a significant clinical problem, and cerebral ischemia usually causes neuron injury such as apoptosis in various brain areas, including hippocampus. Cysteinyl aspartate-specific protease (Caspases) are fundamental factors of apoptotic mechanism. Caspase-3 inhibitors show effect in attenuating brain injury after ischemia. But all the results were from animal models in research laboratories. This study aimed at investigating the correlation between the change of ischemic neuronal injury and Caspase-3 post-ischemia in human hippocampus. Methods We selected and systematized 48 post-mortem specimens from 48 patients, who died of cerebral infarction. Morphological change was firstly analyzed by observing hematoxyline/eosin-staining hippocampal sections. The expression of Caspase-3 was investigated using the methods of in situ hybridization and immunohistochemistry. Terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick-end labeling (TUNEL) method was used to clarify the involvement of Caspase-3 in neuron death. The loss of MAP 2 (MAP-2) was applied to judging the damaged area and degree of neuronal injury caused by ischemia.Results In the CA1 sector of hippocampus, Caspase-3 immunostaining modestly increased at 8 hours [8.05/high-power field (hpf)], dramatically increased at 24 hours (24.85/hpf), decreased somewhat after 72 hours. Caspase-3 mRNA was detectable at 4 hours (6.75/hpf), reached a maximum at 16 hours (17.60/hpf), faded at 72 hours. TUNEL-positive cells were detectable at 24 hours (10.76/hpf), markedly increased at 48-72 hours. The loss of MAP-2 was obviously detected at 4 hours, progressed significantly between 24 and 72 hours; MAP-2 immunoreactivity was barely detectable at 72 hours. Before 72 hours, the Caspase-3 evolution was related with the upregulation of TUNEL and the loss of MAP-2. The positive correlation between Caspase-3 mRNA and TUNEL was significant at the 0.05 level (correlation

  8. TNF-α-Induced Mitochondrial Alterations in Human T Cells Requires FADD and Caspase-8 Activation but Not RIP and Caspase-3 Activation

    OpenAIRE

    Shakibaei, Mehdi; Sung, Bokyung; Sethi, Gautam; Aggarwal, Bharat B.

    2010-01-01

    Although much is known about how TNF-α induces apoptosis in the presence of inhibitors of protein synthesis, little is known about how it induces apoptosis without these inhibitors. In this report we investigated temporal sequence of events induced by TNF-α in the absence of protein synthesis. Regardless of whether we measured the effects by plasma membrane phosphotidylserine accumulation, by DNA strand breaks, or activation of caspases, significant changes were observed only between 12–24 h ...

  9. FE65 binds Teashirt, inhibiting expression of the primate-specific caspase-4.

    Directory of Open Access Journals (Sweden)

    Yuji Kajiwara

    Full Text Available The Alzheimer disease (AD amyloid protein precursor (APP can bind the FE65 adaptor protein and this complex can regulate gene expression. We carried out yeast two-hybrid studies with a PTB domain of FE65, focusing on those genes that might be involved in nuclear signaling, and identified and validated Teashirt proteins as FE65 interacting proteins in neurons. Using reporter systems, we observed that FE65 could simultaneously recruit SET, a component of the inhibitor of acetyl transferase, and Teashirt, which in turn recruited histone deacetylases, to produce a powerful gene-silencing complex. We screened stable cell lines with a macroarray focusing on AD-related genes and identified CASP4, encoding caspase-4, as a target of this silencing complex. Chromatin immunoprecipitation showed a direct interaction of FE65 and Teashirt3 with the promoter region of CASP4. Expression studies in postmortem samples demonstrated decreasing expression of Teashirt and increasing expression of caspase-4 with progressive cognitive decline. Importantly, there were significant increases in caspase-4 expression associated with even the earliest neuritic plaque changes in AD. We evaluated a case-control cohort and observed evidence for a genetic association between the Teashirt genes TSHZ1 and TSHZ3 and AD, with the TSHZ3 SNP genotype correlating with expression of Teashirt3. The results were consistent with a model in which reduced expression of Teashirt3, mediated by genetic or other causes, increases caspase-4 expression, leading to progression of AD. Thus the cell biological, gene expression and genetic data support a role for Teashirt/caspase-4 in AD biology. As caspase-4 shows evidence of being a primate-specific gene, current models of AD and other neurodegenerative conditions may be incomplete because of the absence of this gene in the murine genome.

  10. 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.

  11. Cytoprotective effect of selective small-molecule caspase inhibitors against staurosporine-induced apoptosis.

    Science.gov (United States)

    Wu, Jianghong; Wang, Yuren; Liang, Shuguang; Ma, Haiching

    2014-01-01

    Caspases are currently known as the central executioners of the apoptotic pathways. Inhibition of apoptosis and promotion of normal cell survival by caspase inhibitors would be a tremendous benefit for reducing the side effects of cancer therapy and for control of neurodegenerative disorders such as Parkinson's, Alzheimer's, and Huntington's diseases. The objective of this study was to discover small-molecule caspase inhibitors with which to achieve cytoprotective effect. We completed the high-throughput screening of Bionet's 37,500-compound library (Key Organics Limited, Camelford, Cornwall, UK) against caspase-1, -3, and -9 and successfully identified 43 initial hit compounds. The 43 hit compounds were further tested for cytoprotective activity against staurosporine-induced cell death in NIH3T3 cells. Nineteen compounds were found to have significant cytoprotective effects in cell viability assays. One of the compounds, RBC1023, was demonstrated to protect NIH3T3 cells from staurosporine-induced caspase-3 cleavage and activation. RBC1023 was also shown to protect against staurosporine-induced impairment of mitochondrial membrane potential. DNA microarray analysis demonstrated that staurosporine treatment induced broad global gene expression alterations, and RBC1023 co-treatment significantly restored these changes, especially of the genes that are related to cell growth and survival signaling such as Egr1, Cdc25c, cdkn3, Rhob, Nek2, and Taok1. Collectively, RBC1023 protects NIH3T3 cells against staurosporine-induced apoptosis via inhibiting caspase activity, restoring mitochondrial membrane potential, and possibly upregulating some cell survival-related gene expressions and pathways. PMID:24920883

  12. Peripheral neuropathy in the Twitcher mouse involves the activation of axonal caspase 3

    Directory of Open Access Journals (Sweden)

    Ernesto R Bongarzone

    2011-10-01

    Full Text Available Infantile Krabbe disease results in the accumulation of lipid-raft-associated galactosylsphingosine (psychosine, demyelination, neurodegeneration and premature death. Recently, axonopathy has been depicted as a contributing factor in the progression of neurodegeneration in the Twitcher mouse, a bona fide mouse model of Krabbe disease. Analysis of the temporal-expression profile of MBP (myelin basic protein isoforms showed unexpected increases of the 14, 17 and 18.5 kDa isoforms in the sciatic nerve of 1-week-old Twitcher mice, suggesting an abnormal regulation of the myelination process during early postnatal life in this mutant. Our studies showed an elevated activation of the pro-apoptotic protease caspase 3 in sciatic nerves of 15- and 30-day-old Twitcher mice, in parallel with increasing demyelination. Interestingly, while active caspase 3 was clearly contained in peripheral axons at all ages, we found no evidence of caspase accumulation in the soma of corresponding mutant spinal cord motor neurons. Furthermore, active caspase 3 was found not only in unmyelinated axons, but also in myelinated axons of the mutant sciatic nerve. These results suggest that axonal caspase activation occurs before demyelination and following a dying-back pattern. Finally, we showed that psychosine was sufficient to activate caspase 3 in motor neuronal cells in vitro in the absence of myelinating glia. Taken together, these findings indicate that degenerating mechanisms actively and specifically mediate axonal dysfunction in Krabbe disease and support the idea that psychosine is a pathogenic sphingolipid sufficient to cause axonal defects independently of demyelination.

  13. Adenoviral vector mediated-expression of caspase-3 siRNA on apoptosis induced by lipopolysaccharide

    Institute of Scientific and Technical Information of China (English)

    Wu Feixiang; Yu Weifeng; Yuan Yang; Miao Xuerong; Xu Xuewu; Huang Shengdong; Sun Yuming

    2009-01-01

    Objective: To construct the recombinant adenovirus expressing small RNA of rats caspase-3 and observe the down-regulation effect of caspase-3 in neurons induced by lipopolysaccharide(LPS) in vitro. Methods: pShuttleH1-siCas3 containing Oligo DNA of the targeting sequences and pEGFPC1-Cas3 containing caspase-3 and EGFP sequences were constructed respectively, pShuttleH1-siCas3 and pEGFPC1-Cas3 were co-transfected to the 293 cells by liposomes to determine interfering efficacy by flow eytometry, pShuttleH1-siCas3 was linearized and transformed into E. coli BJ5183 cells containing backbone plasmid pAdEasy-1. The recombinant plasmid was transfected into 293 cells to package the adenovirus Ad-siCas3. The titers of adenovirus were determined by the specific 50% tissue culture infection dosage method. After virus infected the cultured hippocampus neurons, LPS-induced apoptosis and caspase-3 mRNA expression were observed. Results: It was identified that the sequence of target gene was correctly inserted into the genome of virus. The expression of green fluorescence protein was reduced by pShuttleH1-siCas3 in 293 cells. The titer of recombinant adenovirus was 1.06×1010 pfu/ml. After virus infection, caspase-3 mRNA was greatly reduced and neurons apoptosis was suppressed. Conclusion: The recombinant adenovirus expressing rats caspase-3 siRNA were successfully constructed, which may probably be further used in pain therapy by its anti-apoptosis effect.

  14. Laquinimod decreases Bax expression and reduces caspase-6 activation in neurons.

    Science.gov (United States)

    Ehrnhoefer, Dagmar E; Caron, Nicholas S; Deng, Yu; Qiu, Xiaofan; Tsang, Michelle; Hayden, Michael R

    2016-09-01

    Laquinimod is an immunomodulatory compound that has shown neuroprotective benefits in clinical trials for multiple sclerosis. Laquinimod ameliorates both white and gray matter damage in human patients, and prevents axonal degeneration in animal models of multiple sclerosis. Axonal damage and white matter loss are a common feature shared between different neurodegenerative diseases. Caspase-6 activation plays an important role in axonal degeneration on the molecular level. Increased activity of caspase-6 has been demonstrated in brain tissue from presymptomatic Huntington disease mutation carriers, and it is an early marker of axonal dysfunction. Since laquinimod is currently undergoing a clinical trial in Huntington disease (LEGATO-HD, clinicaltrials.gov ID: NCT02215616), we set out to evaluate its impact on neuronal caspase-6 activation. We find that laquinimod ameliorates DNA-damage induced activation of caspase-6 in primary neuronal cultures. This is an indirect effect that is not mediated by direct inhibition of the enzyme. The investigation of potential caspase-6 activating mechanisms revealed that laquinimod reduces the expression of Bax, a pro-apoptotic molecule that causes mitochondrial cytochrome c release and caspase activation. Bax expression is furthermore increased in striatal tissues from the YAC128 mouse model of HD in an age-dependent manner. Our results demonstrate that laquinimod can directly downregulate neuronal apoptosis pathways relevant for axonal degeneration in addition to its known effects on astrocytes and microglia in the CNS. It targets a pathway that is relevant for the pathogenesis of HD, supporting the hypothesis that laquinimod may provide clinical benefit. PMID:27296315

  15. Ginsenoside Rg1 Attenuates Isoflurane-induced Caspase-3 Activation via Inhibiting Mitochondrial Dysfunction

    Institute of Scientific and Technical Information of China (English)

    MIAO Hui Hui; ZHEN Yu; DING Guan Nan; HONG Fang Xiao; XIE Zhong Cong; TIAN Ming

    2015-01-01

    Objective The inhalation anesthetic isoflurane has been shown to induce mitochondrial dysfunction and caspase activation, which may lead to learning and memory impairment. Ginsenoside Rg1 is reported to be neuroprotective. We therefore set out to determine whether ginsenoside Rg1 can attenuate isoflurane-induced caspase activation via inhibiting mitochondrial dysfunction. Methods We investigated the effects of ginsenoside Rg1 at concentrations of 12.5, 25, and 50 µmol/L and pretreatment times of 12 h and 24 h on isoflurane-induced caspase-3 activation in H4 naïve and stably transfected H4 human neuroglioma cells that express full-length human amyloid precursor protein (APP) (H4-APP cells). For mitochondrial dysfunction, we assessed mitochondrial permeability transition pore (mPTP) and adenosine-5’-triphosphate (ATP) levels. We employed Western blot analysis, chemiluminescence, and flowcytometry. Results Here we show that pretreatment with 50 µmol/L ginsenoside Rg1 for 12 h attenuated isoflurane-induced caspase-3 activation and mitochondrial dysfunction in H4-APP cells, while pretreatment with 25 and 50 µmol/L ginsenoside Rg1 for 24 h attenuated isoflurane-induced caspase-3 activation and mitochondrial dysfunction in both H4 naïve and H4-APP cells. Conclusion These data suggest that ginsenoside Rg1 may ameliorate isoflurane-induced caspase-3 activation by inhibiting mitochondrial dysfunction. Pending further studies, these findings might recommend the use of ginsenoside Rg1 in preventing and treating isoflurane-induced neurotoxicity.

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

    Science.gov (United States)

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

    2012-02-01

    The outcome of producing apoptotic defects in cancer cells is the primary obstacle that limits the therapeutic efficacy of anticancer agents, and hence the development of novel agents targeting novel non-canonical cell death pathways has become an imperative mission for clinical research. Fisetin (3,3',4',7-tetrahydroxyflavone) is a naturally occurring flavonoid commonly found in fruits and vegetables. In this study, we investigated the potential anticancer effects of fisetin on breast cancer cells. The result showed fisetin induced higher cytotoxicity in human breast cancer MCF-7 than in MDA-MB-231 cells otherwise it did not exert any detectable cytotoxicity in non-tumorigenic MCF-10A cells. We found fisetin can trigger a novel form of atypical apoptosis in caspase-3-deficient MCF-7 cells, which was characterized by several apoptotic features, including plasma membrane rupture, mitochondrial depolarization, activation of caspase-7, -8 and -9, and PARP cleavage; however, neither DNA fragmentation and phosphotidylserine (PS) externalization was observed. Although p53 was also activated by fisetin, the fisetin-induced apoptosis was not rescued by the p53 inhibitor pifithrin-α. In contrast, the fisetin-induced apoptosis was abrogated by pan-caspase inhibitor z-VAD-fmk. Furthermore, inhibition of autophagy by fisetin was shown as additional route to prompt anticancer activity in MCF-7 cells. These data allow us to propose that fisetin appears as a new potential anticancer agent which can be applied to develop a clinical protocol of human breast cancers. PMID:21922137

  17. The level of cytokines and expression of caspase genes in rheumatoid arthritis.

    Science.gov (United States)

    Malysheva, I E; Topchieva, L V; Barysheva, O Y; Kurbatova, I V; Vasykova, O A; Vezikova, N N; Marusenko, I M; Nemova, N N

    2016-05-01

    The level of TNFα and IL6 in the blood plasma of patients with rheumatoid arthritis (RA) who received antiinflammatory therapy with methotrexate (MT) was significantly lower than in the patients without MT treatment. The level of caspase 6 and 9 gene transcripts in peripheral blood lymphocytes in patients with rheumatoid arthritic diagnosed for the first time and in patients with MT treatment were not significantly different. At the same time, the level of caspase 3 mRNA expression was significantly higher in the cells of the RA patients with MT therapy compared to the patients without MT therapy. PMID:27417728

  18. TNF-α Induces Caspase-1 Activation Independently of Simultaneously Induced NLRP3 in 3T3-L1 Cells.

    Science.gov (United States)

    Furuoka, Mana; Ozaki, Kei-Ichi; Sadatomi, Daichi; Mamiya, Sayaka; Yonezawa, Tomo; Tanimura, Susumu; Takeda, Kohsuke

    2016-12-01

    The intracellular cysteine protease caspase-1 is critically involved in obesity-induced inflammation in adipose tissue. A substantial body of evidence from immune cells, such as macrophages, has shown that caspase-1 activation depends largely on a protein complex, called the NLRP3 inflammasome, which consists of the NOD-like receptor (NLR) family protein NLRP3, the adaptor protein ASC, and caspase-1 itself. However, it is not fully understood how caspase-1 activation is regulated within adipocytes upon inflammatory stimuli. In this study, we show that TNF-α-induced activation of caspase-1 is accompanied by robust induction of NLRP3 in 3T3-L1 adipocytes but that caspase-1 activation may not depend on the NLRP3 inflammasome. Treatment of 3T3-L1 cells with TNF-α induced mRNA expression and activation of caspase-1. Although the basal expression of NLRP3 and ASC was undetectable in unstimulated cells, TNF-α strongly induced NLRP3 expression but did not induce ASC expression. Interestingly, inhibitors of the ERK MAP kinase pathway strongly suppressed NLRP3 expression but did not suppress the expression and activation of caspase-1 induced by TNF-α, suggesting that NLRP3 is dispensable for TNF-α-induced caspase-1 activation. Moreover, we did not detect the basal and TNF-α-induced expression of other NLR proteins (NLRP1a, NLRP1b, and NLRC4), which do not necessarily require ASC for caspase-1 activation. These results suggest that TNF-α induces caspase-1 activation in an inflammasome-independent manner in 3T3-L1 cells and that the ERK-dependent expression of NLRP3 may play a role independently of its canonical role as a component of inflammasomes. J. Cell. Physiol. 231: 2761-2767, 2016. © 2016 Wiley Periodicals, Inc. PMID:26989816

  19. Toxoplasma gondii inhibits cytochrome c-induced caspase activation in its host cell by interference with holo-apoptosome assembly

    Directory of Open Access Journals (Sweden)

    Kristin Graumann

    2015-05-01

    Full Text Available Inhibition of programmed cell death pathways of mammalian cells often facilitates the sustained survival of intracellular microorganisms. The apicomplexan parasite Toxoplasma gondii is a master regulator of host cell apoptotic pathways. Here, we have characterized a novel anti-apoptotic activity of T. gondii. Using a cell-free cytosolic extract model, we show that T. gondii interferes with the activities of caspase 9 and caspase 3/7 which have been induced by exogenous cytochrome c and dATP. Proteolytic cleavage of caspases 9 and 3 is also diminished suggesting inhibition of holo-apoptosome function. Parasite infection of Jurkat T cells and subsequent triggering of apoptosome formation by exogenous cytochrome c in vitro and in vivo indicated that T. gondii also interferes with caspase activation in infected cells. Importantly, parasite inhibition of cytochrome c-induced caspase activation considerably contributes to the overall anti-apoptotic activity of T. gondii as observed in staurosporine-treated cells. Co-immunoprecipitation showed that T. gondii abolishes binding of caspase 9 to Apaf-1 whereas the interaction of cytochrome c with Apaf-1 remains unchanged. Finally, T. gondii lysate mimics the effect of viable parasites and prevents holo-apoptosome functionality in a reconstituted in vitro system comprising recombinant Apaf-1 and caspase 9. Beside inhibition of cytochrome c release from host cell mitochondria, T. gondii thus also targets the holo-apoptosome assembly as a second mean to efficiently inhibit the caspase-dependent intrinsic cell death pathway.

  20. Chalcone-Induced Apoptosis through Caspase-Dependent Intrinsic Pathways in Human Hepatocellular Carcinoma Cells

    Science.gov (United States)

    Ramirez-Tagle, Rodrigo; Escobar, Carlos A.; Romero, Valentina; Montorfano, Ignacio; Armisén, Ricardo; Borgna, Vincenzo; Jeldes, Emanuel; Pizarro, Luis; Simon, Felipe; Echeverria, Cesar

    2016-01-01

    Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed cancers worldwide. Chemoprevention of HCC can be achieved through the use of natural or synthetic compounds that reverse, suppress or prevent the development of cancer progression. In this study, we investigated the antiproliferative effects and the mechanism of action of two compounds, 2,3,4′-trimethoxy-2′-hydroxy-chalcone (CH1) and 3′-bromo-3,4-dimethoxy-chalcone (CH2), over human hepatoma cells (HepG2 and Huh-7) and cultured mouse hepatocytes (HepM). Cytotoxic effects were observed over the HepG2 and Huh-7, and no effects were observed over the HepM. For HepG2 cells, treated separately with each chalcone, typical apoptotic laddering and nuclear condensation were observed. Additionally, the caspases and Bcl-2 family proteins activation by using Western blotting and immunocytochemistry were studied. Caspase-8 was not activated, but caspase-3 and -9 were both activated by chalcones in HepG2 cells. Chalcones also induced reactive oxygen species (ROS) accumulation after 4, 8 and 24 h of treatment in HepG2 cells. These results suggest that apoptosis in HepG2 was induced through: (i) a caspase-dependent intrinsic pathway; and (ii) by alterations in the cellular levels of Bcl-2 family proteins, and also, that the chalcone moiety could be a potent candidate as novel anticancer agents acting on human hepatomas. PMID:26907262

  1. IAPs: from caspase inhibitors to modulators of NF-kappaB, inflammation and cancer

    DEFF Research Database (Denmark)

    Gyrd-Hansen, Mads; Meier, Pascal

    2010-01-01

    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...

  2. Cordycepin, a Natural Antineoplastic Agent, Induces Apoptosis of Breast Cancer Cells via Caspase-dependent Pathways.

    Science.gov (United States)

    Wang, Di; Zhang, Yongfeng; Lu, Jiahui; Wang, Yang; Wang, Junyue; Meng, Qingfan; Lee, Robert J; Wang, Di; Teng, Lesheng

    2016-01-01

    Cordycepin, a major compound separated from Cordyceps sinensis, is known as a potential novel candidate for cancer therapy. Breast cancer, the most typical cancer diagnosed among women, remains a global health problem. In this study, the anti-breast cancer property of cordycepin and its underlying mechanisms was investigated. The direct effects of cordycepin on breast cancer cells both in in vitro and in vivo experiments were evaluated. Cordycepin exerted cytotoxicity in MCF-7 and MDA-MB-231 cells confirmed by reduced cell viability, inhibition of cell proliferation, enhanced lactate dehydrogenase release and reactive oxygen species accumulation, induced mitochondrial dysfunction and nuclear apoptosis in human breast cancer cells. Cordycepin increased the activation of pro-apoptotic proteins, including caspase-8, caspase-9, caspase-3 and Bax, and suppressed the expression of the anti-apoptotic protein, B-cell lymphoma 2 (Bcl-2). The inhibition on MCF-7-xenografted tumor growth in nude mice further confirmed cordycepin's anti-breast cancer effect. These aforementioned results reveal that cordycepin induces apoptosis in human breast cancer cells via caspase-dependent pathways. The data shed light on the possibility of cordycepin being a safe agent for breast cancer treatment. PMID:26996021

  3. 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: 3.197, year: 2014

  4. Involvement of JNK and Caspase Activation in Hoiamide A-Induced Neurotoxicity in Neocortical Neurons

    Directory of Open Access Journals (Sweden)

    Zhengyu Cao

    2015-02-01

    Full Text Available The frequent occurrence of Moorea producens (formerly Lyngbya majuscula blooms has been associated with adverse effects on human health. Hoiamide A is a structurally unique cyclic depsipeptide isolated from an assemblage of the marine cyanobacteria M. producens and Phormidium gracile. We examined the influence of hoiamide A on neurite outgrowth in neocortical neurons and found that it suppressed neurite outgrowth with an IC50 value of 4.89 nM. Further study demonstrated that hoiamide A stimulated lactic acid dehydrogenase (LDH efflux, nuclear condensation and caspase-3 activity with EC50 values of 3.66, 2.55 and 4.33 nM, respectively. These data indicated that hoiamide A triggered a unique neuronal death profile that involves both necrotic and apoptotic mechanisms. The similar potencies and similar time-response relationships between LDH efflux and caspase-3 activation/nuclear condensation suggested that both necrosis and apoptosis may derive from interaction with a common molecular target. The broad-spectrum caspase inhibitor, Z-VAD-FMK completely inhibited hoiamide A-induced neurotoxicity. Additionally, hoiamide A stimulated JNK phosphorylation, and a JNK inhibitor attenuated hoiamide A-induced neurotoxicity. Collectively, these data demonstrate that hoiamide A-induced neuronal death requires both JNK and caspase signaling pathways. The potent neurotoxicity and unique neuronal cell death profile of hoiamide A represents a novel neurotoxic chemotype from marine cyanobacteria.

  5. Caspases and osteogenic markers—in vitro screening of inhibition impact

    Czech Academy of Sciences Publication Activity Database

    Adamová, Eva; Janečková, Eva; Klepárník, Karel; Matalová, Eva

    2016-01-01

    Roč. 52, č. 2 (2016), s. 144-148. ISSN 1071-2690 R&D Projects: GA ČR(CZ) GA14-37368G; GA ČR(CZ) GA14-28254S Institutional support: RVO:68081715 ; RVO:67985904 Keywords : osteogenesis * caspases * gene expression Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 1.145, year: 2014

  6. Interaction of CSR1 with XIAP reverses inhibition of caspases and accelerates cell death.

    Science.gov (United States)

    Zheng, Zhong-Liang; Tan, Lang-Zhu; Yu, Yan P; Michalopoulos, George; Luo, Jian-Hua

    2012-08-01

    Cellular Stress Response 1 (CSR1) is a tumor suppressor gene that is located at 8p21, a region that is frequently deleted in prostate cancer as well as a variety of human malignancies. Previous studies have indicated that the expression of CSR1 induces cell death. In this study, we found that CSR1 interacts with X-linked Inhibitor of Apoptosis Protein (XIAP), using yeast two-hybrid screening analyses. XIAP overexpression has been found in many human cancers, and forced expression of XIAP blocks apoptosis. Both in vitro and in vivo analyses indicated that the C-terminus of CSR1 binds XIAP with high affinity. Through a series of in vitro recombinant protein-binding analyses, the XIAP-binding motif in CSR1 was determined to include amino acids 513 to 572. Targeted knock-down of XIAP enhanced CSR1-induced cell death, while overexpression of XIAP antagonized CSR1 activity. The binding of CSR1 with XIAP enhanced caspase-9 and caspase-3 protease activities, and CSR1-induced cell death was dramatically reduced on expression of a mutant CSR1 that does not bind XIAP. However, a XIAP mutant that does not interact with caspase-9 had no impact on CSR1-induced cell death. These results suggest that cell death is induced when CSR1 binds XIAP, preventing the interaction of XIAP with caspases. Thus, this study may have elucidated a novel mechanism by which tumor suppressors induce cell death. PMID:22683311

  7. Caspase-7 participates in differentiation of cells forming dental hard tissues

    Czech Academy of Sciences Publication Activity Database

    Matalová, Eva; Lesot, H.; Švandová, Eva; Vanden Berghe, T.; Sharpe, P. T.; Healy, C.; Vandenabeele, P.; Tucker, A. S.

    2013-01-01

    Roč. 55, č. 5 (2013), s. 615-621. ISSN 0012-1592 R&D Projects: GA ČR GAP304/11/1418 Grant ostatní: GA ČR(CZ) GAP502/12/1285 Institutional support: RVO:67985904 Keywords : ameloblast * apoptosis * caspase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.178, year: 2013

  8. 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.

  9. Evidence that endoplasmic reticulum (ER) stress and caspase-4 activation occur in human neutrophils

    International Nuclear Information System (INIS)

    Apoptosis can result from activation of three major pathways: the extrinsic, the intrinsic, and the most recently identified endoplasmic reticulum (ER) stress-mediated pathway. While the two former pathways are known to be operational in human polymorphonuclear neutrophils (PMNs), the existence of the ER stress-mediated pathway, generally involving caspase-4, has never been reported in these cells. Recently, we have documented that arsenic trioxide (ATO) induced apoptosis in human PMNs by a mechanism that needs to be further investigated. In this study, using immunofluorescence and electron microscopy, we present evidence of ER alterations in PMNs activated by the ER stress inducer arsenic trioxide (ATO). Several key players of the unfolded protein response, including GRP78, GADD153, ATF6, XBP1 and eIF2α are expressed and activated in PMNs treated with ATO or other ER stress inducers. Although caspase-4 is expressed and activated in neutrophils, treatment with a caspase-4 inhibitor did not attenuate the pro-apoptotic effect of ATO at a concentration that reverses caspase-4 processing and activation. Our results demonstrate for the first time that the ER stress-mediated apoptotic pathway operates in human neutrophils.

  10. Non-apoptotic role for caspase-7 in hair follicles and the surrounding tissue

    Czech Academy of Sciences Publication Activity Database

    Veselá, Barbora; Švandová, Eva; Vanden Berghe, T.; Tucker, A. S.; Vandenabeele, P.; Matalová, Eva

    2015-01-01

    Roč. 46, 4-5 (2015), s. 443-455. ISSN 1567-2379 R&D Projects: GA ČR GB14-37368G Institutional support: RVO:67985904 Keywords : caspase-7 * development * hair follicles Subject RIV: EA - Cell Biology Impact factor: 1.815, year: 2014

  11. Effect of glycyrrhizic flavone on the expression of caspase -3 and caspase - 12 in photoaging skin of mice%甘草黄酮对小鼠光老化皮肤中caspase-3、caspase-12表达的影响

    Institute of Scientific and Technical Information of China (English)

    宁舒鹏; 杨桂兰; 王佳媚; 白景瑞; 龙朝钦; 罗洋

    2012-01-01

    目的:研究皮肤外用甘草黄酮对紫外线照射引起的小鼠光老化皮肤的影响.方法:BALB/C小鼠50只随机分成5组:模型组(A)、基质组(B)、甘草黄酮组(C)、薇诺娜组(D)、正常对照组(E),模拟UVB长期照射,造成皮肤光老化小鼠模型.B、C、D组照射同时分别外用基质乳膏、甘草黄酮乳膏、薇诺娜防晒霜( SPF30,PA+++).组织切片HE染色观察皮肤结构改变;以ELISA法测定皮肤组织caspasse -3、caspase - 12含量.结果:A、B组小鼠皮肤组织caspasse -3、caspase - 12含量明显增加,病理切片呈现明显光老化状态;C、D组capasse -3、caspase - 12含量与E组差异无统计学意义(P>0.05).结论:甘草黄酮乳膏对紫外线照射引起的小鼠皮肤光老化具有保护作用,其机制可能为甘草黄酮通过抑制caspase - 12活化,进而阻断caspase-3的激活,抑制细胞的凋亡.%Objective: To investigate the effect of glycyrrhizic flavone cream on the photoaging skin of mice induced by ultraviolet radiation B ( UVB). Methods: Fifty BALB / C mice were randomly divided into 5 groups (n = 5 in each group) , photoaging model group (A), matrix group (B), glycyrrhizic flavone group (C) , Winona group (D), normal control group (E). The mouse model of photoaging skin was established by long - term UVB radiation. Matrix cream, glycyrrhizic flavone cream, and Winona cream (SPF30, PA + + + ) were used respectively on the back of mice in groups of B, C and D before UVB radiation. Histological sections were stained with HE to observe the structural changes of skin. Caspasse - 3 and caspase - 12 levels were detected by ELISA. Results; Expression of caspasae -3 and caspase - 12 in skin tissue of A and B group were significantly upregulated and obvious skin photoage was seen. The differences of cspasse - 3 and caspase -12 expression between C, D and E group were not statistically significant (P >0. 05). Conclusion; The glycyrrhizic flavone cream could protect skin from

  12. Study on HepG-2 apoptosis induced by saponins isolated from Asparagus and the effects on the activities of caspase-3,8,9

    Institute of Scientific and Technical Information of China (English)

    JI Yu-bin; XU He; JI Chen-feng

    2008-01-01

    Objective To study the effect of saponins of asparagus on apoptosis and the variations of caspaseS, caspase-9 and caspase-3 activity in the process of asparagus induced apoptosis in HepG-2, to investigate the apoptosis mechanism further. Methods Asparagus on apoptosis effects on tumor cells cultured-HepG-2 with different concentrations at different time, IC50 value was measured by MTT assay, the apoptosis rate was determined by FCM with AnnexinV/PI staining, their apoptotic morphology were observed by electron microscopy and Colorimetric method was used to measure caspase-8,9 and caspase-3 activities. Results Experiments of antitumour in vivo showed that saponins of asparagus can inhibit the growth of tumor cell of HepG-2 in evidence, IC50 was 101.15 mg·L-1. Cultured for 72 h, the apoptosis rate had positive increased with concentrations. Apoptotic morphology was observed by electron microscopy. The activities of caspase-8, easpase-9 and caspase-3 had positive increased with concentrations. And have significant difference compared with negative control group(P<0.01). The activities of caspase-8 were high at 24 h, but the activities of caspase-9 and caspase-3 is high at 48 h. Conclusions Aaponins of asparagus can inhibit the growth of tumor cell of HepG2, and the underlying mechanism might be related to up regulation of caspase-8, 9 activity which subsequently transforms caspase-3 into its active form.

  13. Vitamin C Attenuates Isoflurane-Induced Caspase-3 Activation and Cognitive Impairment.

    Science.gov (United States)

    Cheng, Baiqi; Zhang, Yiying; Wang, Arthur; Dong, Yuanlin; Xie, Zhongcong

    2015-12-01

    Anesthetic isoflurane has been reported to induce caspase-3 activation. The underlying mechanism(s) and targeted intervention(s), however, remain largely to be determined. Vitamin C (VitC) inhibits oxidative stress and apoptosis. We therefore employed VitC to further determine the up-stream mechanisms and the down-stream consequences of the isoflurane-induced caspase-3 activation. H4 human neuroglioma cells overexpressed human amyloid precursor protein (H4-APP cells) and rat neuroblastoma cells were treated either with (1) 2% isoflurane or (2) with the control condition, plus saline or 400 μM VitC for 3 or 6 h. Western blot analysis and fluorescence assay were utilized at the end of the experiments to determine caspase-3 activation, levels of reactive oxygen species and ATP, and mitochondrial function. The interaction of isoflurane (1.4% for 2 h) and VitC (100 mg/kg) on cognitive function in mice was also assessed in the fear conditioning system. Here, we show for the first time that the VitC treatment attenuated the isoflurane-induced caspase-3 activation. Moreover, VitC mitigated the isoflurane-induced increases in the levels of reactive oxygen species, opening of mitochondrial permeability transition pore, reduction in mitochondrial membrane potential, and the reduction in ATP levels in the cells. Finally, VitC ameliorated the isoflurane-induced cognitive impairment in the mice. Pending confirmation from future studies, these results suggested that VitC attenuated the isoflurane-induced caspase-3 activation and cognitive impairment by inhibiting the isoflurane-induced oxidative stress, mitochondrial dysfunction, and reduction in ATP levels. These findings would promote further research into the underlying mechanisms and targeted interventions of anesthesia neurotoxicity. PMID:25367886

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

    Science.gov (United States)

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

    2014-02-01

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

  15. β-Amyloid induces nuclear protease-mediated lamin fragmentation independent of caspase activation.

    Science.gov (United States)

    Ramasamy, Vijay Sankar; Islam, Md Imamul; Haque, Md Aminul; Shin, Song Yub; Park, Il-Seon

    2016-06-01

    β-Amyloid (Aβ), a hallmark peptide of Alzheimer's disease, induces both caspase-dependent apoptosis and non-apoptotic cell death. In this study, we examined caspase-independent non-apoptotic cell death preceding caspase activation in Aβ42-treated cells. We first determined the optimal treatment conditions for inducing cell death without caspase activation and selected a double-treatment method involving the incubation of cells with Aβ42 for 4 and 6h (4+6h sample). We observed that levels of lamin A (LA) and lamin B (LB) were reduced in the 4+6h samples. This reduction was decreased by treatment with suc-AAPF-CMK, an inhibitor of nuclear scaffold (NS) protease, but not by treatment with z-VAD-FMK, a pan-caspase inhibitor. In addition, suc-AAPF-CMK decreased the changes in nuclear morphology observed in cells in the 4+6h samples, which were different from nuclear fragmentation observed in STS-treated cells. Furthermore, suc-AAPF-CMK inhibited cell death in the 4+6h samples. LA and LB fragmentation occurred in the isolated nuclei and was also inhibited by suc-AAPF-CMK. Together, these data indicated that the fragmentation of LA and LB in the Aβ42-treated cells was induced by an NS protease, whose identity is not clearly determined yet. A correlation between Aβ42 toxicity and the lamin fragmentation by NS protease suggests that inhibition of the protease could be an effective method for controlling the pathological process of AD. PMID:26876308

  16. Macrophage specific caspase-1/11 deficiency protects against cholesterol crystallization and hepatic inflammation in hyperlipidemic mice.

    Directory of Open Access Journals (Sweden)

    Tim Hendrikx

    Full Text Available While non-alcoholic steatohepatitis (NASH is characterized by hepatic steatosis combined with inflammation, the mechanisms triggering hepatic inflammation are unknown. In Ldlr(-/- mice, we have previously shown that lysosomal cholesterol accumulation in Kupffer cells (KCs correlates with hepatic inflammation and cholesterol crystallization. Previously, cholesterol crystals have been shown to induce the activation of inflammasomes. Inflammasomes are protein complexes that induce the processing and release of pro-inflammatory cytokines IL-1b and IL-18 via caspase-1 activation. Whereas caspase-1 activation is independent of caspase-11 in the canonical pathway of inflammasome activation, caspase-11 was found to trigger caspase-1-dependent IL-1b and IL-18 in response to non-canonical inflammasome activators. So far, it has not been investigated whether inflammasome activation stimulates the formation of cholesterol crystals. We hypothesized that inflammasome activation in KCs stimulates cholesterol crystallization, thereby leading to hepatic inflammation.Ldlr (-/- mice were transplanted (tp with wild-type (Wt or caspase-1/11(-/- (dKO bone marrow and fed either regular chow or a high-fat, high-cholesterol (HFC diet for 12 weeks. In vitro, bone marrow derived macrophages (BMDM from wt or caspase-1/11(-/- mice were incubated with oxLDL for 24h and autophagy was assessed.In line with our hypothesis, caspase-1/11(-/--tp mice had less severe hepatic inflammation than Wt-tp animals, as evident from liver histology and gene expression analysis in isolated KCs. Mechanistically, KCs from caspase-1/11(-/--tp mice showed less cholesterol crystals, enhanced cholesterol efflux and increased autophagy. In wt BMDM, oxLDL incubation led to disturbed autophagy activity whereas BMDM from caspase-1/11(-/- mice had normal autophagy activity.Altogether, these data suggest a vicious cycle whereby disturbed autophagy and decreased cholesterol efflux leads to newly formed

  17. A novel synthetic route of the caspase 3 sensor for determination of caspase 3 based on Förster resonance energy transfer

    Czech Academy of Sciences Publication Activity Database

    Lišková, Marcela; Klepárník, Karel; Pazdera, P.; Foret, František

    Baltimore, 2012. L-146. [ITP 2012. International Symposium, Exhibit & Workshops on Electro- and Liquid Phase-separation Techniques /19./. 30.09.2012-03.10.2012, Baltimore] R&D Projects: GA ČR(CZ) GAP301/11/2055; GA ČR(CZ) GBP206/12/G014; GA ČR GAP206/11/2377; GA MŠk(CZ) CZ.1.07/2.3.00/20.0182; GA TA ČR(CZ) TA 02010672 Institutional support: RVO:68081715 Keywords : Caspase 3 * apoptpsis * FRET Subject RIV: CB - Analytical Chemistry, Separation

  18. c-FLIP及Caspase-8表达在乳腺癌组织中的表达及临床意义%Expression of c-FLIP and Caspase-8 in Breast Cancer and Their Clinical Significance

    Institute of Scientific and Technical Information of China (English)

    潘峥; 陈军; 覃冠德; 覃思繁

    2012-01-01

    Objective To investigate the expression of c-FLIP and Caspase-8 in breast cancer and their correlation with metastasis and recurrence. Methods Immunohistochemisty was employed to determine the expression of c-FLIP and Caspase-8 in 80 breast carcinomas and paired adjacent breast tissues. Results c-FLIP was overexpressed in breast cancers compared with paired adjacent breast tissues. The expression of c-FLIP was significantly correlated with TNM stages, histological grade, axillary lymph node metastasis, the recurrence of tumor and the express of C-erbB-2. The expression of Caspase-8 was significantly lower in breast cancers compared with paired adjacent breast tissues. The expression of Caspase-8 was significantly correlated with histological grade, tumor size. There was a significantly negative relationship of c-FLIP with Caspase-8. Conclusion The overexpres-sion of c-FLIP and the inactivation of Caspase-8 might enhance the tumour cell proliferation and malignance in breast cancer.%目的 探讨凋亡相关蛋白c-FLIP和Caspase-8在人乳腺癌组织中的表达及相关性,分析其与乳腺癌转移复发的关系.方法 应用免疫组织化学法检测80例乳腺癌组织和相应的癌旁组织中c-FLIP和Caspase-8的表达情况.结果 80例乳腺癌组织中c-FLIP表达的阳性率显著高于相应的癌旁组织(P<0.05),c-FLIP的表达与TNM分期、组织学分级、腋窝淋巴结转移、术后复发及C-erbB-2表达密切相关(P<0.05);Caspase-8表达的阳性率显著低于相应的癌旁组织(P<0.05),Caspase-8的表达与组织学分级、肿瘤大小密切相关(P<0.05).c-FLIP表达和Caspase-8表达有显著负相关性(γ=-0.380,P=0.001).结论 c-FLIP的高表达及Caspase-8表达缺失可能在乳腺癌发生发展中起重要作用.

  19. In vitro degradation of bovine myofibrils is caused by μ-calpain, not caspase-3.

    Science.gov (United States)

    Mohrhauser, D A; Underwood, K R; Weaver, A D

    2011-03-01

    Tenderness is a key palatability trait influencing perception of consumers of meat quality and is influenced by a multitude of factors, including postmortem proteolysis. A fundamental understanding of this biological mechanism regulating tenderness is necessary to decrease variability and increase consumer satisfaction. However, reports regarding the enzyme systems involved in postmortem tenderization are conflicting. Therefore, the objective of this study was to determine if caspase-3 is responsible for the degradation of myofibrillar proteins during aging. Bovine semitendinosus muscles were removed from 2 carcasses. Muscle from the left side of each carcass was excised 20 min postmortem and utilized for in vitro analysis of protein degradation. Muscle strips were dissected from the semitendinosus, restrained to maintain length, and placed in a neutral buffer containing protease inhibitors. Upon rigor completion, myofibrils were isolated from each strip and sarcomere length was determined. Samples with similar sarcomere lengths were selected to minimize the effect of sarcomere length on proteolysis. Myofibrils were then incubated at 22°C with μ-calpain, caspase-3, or μ-calpain + caspase-3 for 0.25, 1, 3, 24, 48, or 72 h at optimum pH for enzyme activity. The semitendinosus from the right side of each carcass was excised 1 d postmortem, cut into 2.54-cm steaks, vacuum-packaged, and allowed to age for 2, 4, 7, or 10 d to evaluate normal protein degradation during beef aging. Proteolysis of troponin T, α-actinin, and desmin was monitored using SDS-PAGE and Western blotting techniques, whereas proteolysis of titin and nebulin was monitored using SDS-vertical agarose gel electrophoresis and Western blotting. Analysis of Western blots revealed no change in abundance of intact troponin T, desmin, titin, or nebulin over time in myofibrils incubated with caspase-3. However, abundance of these proteins subjected to digestion with μ-calpain and μ-calpain + caspase-3

  20. Uterine Endoplasmic Reticulum Stress and Its Unfolded Protein Response May Regulate Caspase 3 Activation in the Pregnant Mouse Uterus

    Science.gov (United States)

    Suresh, Arvind; Subedi, Kalpana; Kyathanahalli, Chandrashekara; Jeyasuria, Pancharatnam; Condon, Jennifer C.

    2013-01-01

    We have previously proposed that uterine caspase-3 may modulate uterine contractility in a gestationally regulated fashion. The objective of this study was to determine the mechanism by which uterine caspase-3 is activated and consequently controlled in the pregnant uterus across gestation. Utilizing the mouse uterus as our gestational model we examined the intrinsic and extrinsic apoptotic signaling pathways and the endoplasmic reticulum stress response as potential activators of uterine caspase-3 at the transcriptional and translational level. Our study revealed robust activation of the uterine myocyte endoplasmic reticulum stress response and its adaptive unfolded protein response during pregnancy coinciding respectively with increased uterine caspase-3 activity and its withdrawal to term. In contrast the intrinsic and extrinsic apoptotic signaling pathways remained inactive across gestation. We speculate that physiological stimuli experienced by the pregnant uterus likely potentiates the uterine myocyte endoplasmic reticulum stress response resulting in elevated caspase-3 activation, which is isolated to the pregnant mouse myometrium. However as term approaches, activation of an elevated adaptive unfolded protein response acts to limit the endoplasmic reticulum stress response inhibiting caspase-3 resulting in its decline towards term. We speculate that these events have the capacity to regulate gestational length in a caspase-3 dependent manner. PMID:24058658

  1. Uterine endoplasmic reticulum stress and its unfolded protein response may regulate caspase 3 activation in the pregnant mouse uterus.

    Directory of Open Access Journals (Sweden)

    Arvind Suresh

    Full Text Available We have previously proposed that uterine caspase-3 may modulate uterine contractility in a gestationally regulated fashion. The objective of this study was to determine the mechanism by which uterine caspase-3 is activated and consequently controlled in the pregnant uterus across gestation. Utilizing the mouse uterus as our gestational model we examined the intrinsic and extrinsic apoptotic signaling pathways and the endoplasmic reticulum stress response as potential activators of uterine caspase-3 at the transcriptional and translational level. Our study revealed robust activation of the uterine myocyte endoplasmic reticulum stress response and its adaptive unfolded protein response during pregnancy coinciding respectively with increased uterine caspase-3 activity and its withdrawal to term. In contrast the intrinsic and extrinsic apoptotic signaling pathways remained inactive across gestation. We speculate that physiological stimuli experienced by the pregnant uterus likely potentiates the uterine myocyte endoplasmic reticulum stress response resulting in elevated caspase-3 activation, which is isolated to the pregnant mouse myometrium. However as term approaches, activation of an elevated adaptive unfolded protein response acts to limit the endoplasmic reticulum stress response inhibiting caspase-3 resulting in its decline towards term. We speculate that these events have the capacity to regulate gestational length in a caspase-3 dependent manner.

  2. 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

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

    International Nuclear Information System (INIS)

    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 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: ► During acetaminophen overdose caspase-3 can be activated in fed mice of certain outbred strains. ► Hepatic ATP levels are not the determining factor for caspase activity. ► Caspase-3 activity does not result in increased hepatocellular apoptotic cell death. ► Neutrophil recruitment during

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

    Science.gov (United States)

    Bloemberg, Darin; Quadrilatero, Joe

    2016-06-01

    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. PMID:27104214

  5. Purification, crystallization and preliminary crystallographic characterization of the caspase-recruitment domain of human Nod1

    International Nuclear Information System (INIS)

    The caspase-recruitment domain of the cytosolic pathogen receptor Nod1 was crystallized. X-ray diffraction data were collected to 1.9 Å resolution. The caspase-recruitment domain (CARD) is known to play an important role in apoptosis and inflammation as an essential protein–protein interaction domain. The CARD of the cytosolic pathogen receptor Nod1 was overexpressed in Escherichia coli and purified by affinity chromatography and gel filtration. The purified CARD was crystallized at 277 K using the microseeding method. X-ray diffraction data were collected to 1.9 Å resolution. The crystals belong to space group P31 or P32, with unit-cell parameters a = b = 79.1, c = 80.9 Å. Preliminary analysis indicates that there is one dimeric CARD molecule in the asymmetric unit

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

    Directory of Open Access Journals (Sweden)

    Liang Pang

    2015-05-01

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

  7. Resveratrol and clofarabine induces a preferential apoptosis-activating effect on malignant mesothelioma cells by Mcl-1 down-regulation and caspase-3 activation.

    Science.gov (United States)

    Lee, Yoon-Jin; Lee, Yong-Jin; Lee, Sang-Han

    2015-03-01

    We previously demonstrated that resveratrol and clofarabine elicited a marked cytotoxicity on malignant mesothelioma (MM) MSTO-211H cells but not on the corresponding normal mesothelial MeT-5A cells. Little is known of the possible molecules that could be used to predict preferential chemosensitivity on MSTO-211H cells. Resveratrol and clofarabine induced down-regulation of Mcl-1 protein level in MSTO-211H cells. Treatment of cells with cycloheximide in the presence of proteasome inhibitor MG132 suggested that Mcl-1 protein levels were regulated at the post-translational step. The siRNA-based knockdown of Mcl-1 in MSTO-211H cells triggered more growth-inhibiting and apoptosis-inducing effects with the resultant cleavages of procaspase-3 and its substrate PARP, increased caspase-3/7 activity, and increased percentage of apoptotic propensities. However, the majority of the observed changes were not shown in MeT-5A cells. Collectively, these studies indicate that the preferential activation of caspase cascade in malignant cells might have important applications as a therapeutic target for MM. PMID:24924397

  8. Molecular cloning, immunohistochemical localization, characterization and expression analysis of caspase-9 from the purse red common carp (Cyprinus carpio) exposed to cadmium

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Dian; Xu, Zhen’e [Medical College of Nanchang University, Nanchang 330006 (China); Institute of Immunotherapy, Nanchang University, Nanchang 330006 (China); Zhang, Xiaoyan [Medical College of Nanchang University, Nanchang 330006 (China); Wang, Hongmei [Medical College of Nanchang University, Nanchang 330006 (China); Institute of Immunotherapy, Nanchang University, Nanchang 330006 (China); Wang, Yannan [Medical College of Nanchang University, Nanchang 330006 (China); Min, Weiping, E-mail: weiping.min@gmail.com [Medical College of Nanchang University, Nanchang 330006 (China); Institute of Immunotherapy, Nanchang University, Nanchang 330006 (China); Jiangxi Academy of Medical Sciences, Nanchang 330006 (China)

    2013-10-15

    Highlights: •The cDNA of caspase-9 in common carp was cloned. •The evolutionary conservation including caspase recruitment domain, large and small subunits was clarified. •The mRNA level of caspase-9 cannot be used as a major marker at an earlier point in the apoptotic cascade. •Caspase-9 cleavage form was detected. •Immunopositive staining was limited to the cytoplasm of renal tubular epithelial cells. -- Abstract: Caspase-9, the essential initiator caspase is believed to play a central role in mitochondria-mediated apoptosis signaling. In this study, we isolated the caspase-9 gene from common carp, one of the most important industrial aquatic animals in China using rapid amplification of cDNA ends (RACE). The deduced amino acid sequence of caspase-9, composed of 436 amino acids, showed approximately 47.6% identity and 64.7% similarity to human caspase-9. It also possessed a conserved caspase-associated recruitment domain (CARD), a large subunit and a small subunit. Phylogenetic analysis clearly demonstrated that caspase-9 formed a clade with cyprinid fish caspase-9. Real-time quantitative PCR analysis revealed that caspase-9 transcripts were not significantly increased in kidney after exposure to cadmium (Cd). Whereas caspase-9 cleaved fragments were detected using Western blot analysis with the same Cd treatment condition. Furthermore, the result of immunohistochemical detection showed immunoreactivities were predominantly limited to the cytoplasm of renal tubular epithelial cells and no remarkable changes of immunopositive staining were observed after Cd treatment. Accordingly, the results signify that caspase-9 may play an essential role in Cd induced apoptosis.

  9. Molecular cloning, immunohistochemical localization, characterization and expression analysis of caspase-9 from the purse red common carp (Cyprinus carpio) exposed to cadmium

    International Nuclear Information System (INIS)

    Highlights: •The cDNA of caspase-9 in common carp was cloned. •The evolutionary conservation including caspase recruitment domain, large and small subunits was clarified. •The mRNA level of caspase-9 cannot be used as a major marker at an earlier point in the apoptotic cascade. •Caspase-9 cleavage form was detected. •Immunopositive staining was limited to the cytoplasm of renal tubular epithelial cells. -- Abstract: Caspase-9, the essential initiator caspase is believed to play a central role in mitochondria-mediated apoptosis signaling. In this study, we isolated the caspase-9 gene from common carp, one of the most important industrial aquatic animals in China using rapid amplification of cDNA ends (RACE). The deduced amino acid sequence of caspase-9, composed of 436 amino acids, showed approximately 47.6% identity and 64.7% similarity to human caspase-9. It also possessed a conserved caspase-associated recruitment domain (CARD), a large subunit and a small subunit. Phylogenetic analysis clearly demonstrated that caspase-9 formed a clade with cyprinid fish caspase-9. Real-time quantitative PCR analysis revealed that caspase-9 transcripts were not significantly increased in kidney after exposure to cadmium (Cd). Whereas caspase-9 cleaved fragments were detected using Western blot analysis with the same Cd treatment condition. Furthermore, the result of immunohistochemical detection showed immunoreactivities were predominantly limited to the cytoplasm of renal tubular epithelial cells and no remarkable changes of immunopositive staining were observed after Cd treatment. Accordingly, the results signify that caspase-9 may play an essential role in Cd induced apoptosis

  10. Cytosolic caspases mediate mislocalised SOD2 depletion in an in vitro model of chronic prion infection

    Directory of Open Access Journals (Sweden)

    Layla Sinclair

    2013-07-01

    Oxidative stress as a contributor to neuronal death during prion infection is supported by the fact that various oxidative damage markers accumulate in the brain during the course of this disease. The normal cellular substrate of the causative agent, the prion protein, is also linked with protective functions against oxidative stress. Our previous work has found that, in chronic prion infection, an apoptotic subpopulation of cells exhibit oxidative stress and the accumulation of oxidised lipid and protein aggregates with caspase recruitment. Given the likely failure of antioxidant defence mechanisms within apoptotic prion-infected cells, we aimed to investigate the role of the crucial antioxidant pathway components, superoxide dismutases (SOD 1 and 2, in an in vitro model of chronic prion infection. Increased total SOD activity, attributable to SOD1, was found in the overall population coincident with a decrease in SOD2 protein levels. When apoptotic cells were separated from the total population, the induction of SOD activity in the infected apoptotic cells was lost, with activity reduced back to levels seen in mock-infected control cells. In addition, mitochondrial superoxide production was increased and mitochondrial numbers decreased in the infected apoptotic subpopulation. Furthermore, a pan-caspase probe colocalised with SOD2 outside of mitochondria within cytosolic aggregates in infected cells and inhibition of caspase activity was able to restore cellular levels of SOD2 in the whole unseparated infected population to those of mock-infected control cells. Our results suggest that prion propagation exacerbates an apoptotic pathway whereby mitochondrial dysfunction follows mislocalisation of SOD2 to cytosolic caspases, permitting its degradation. Eventually, cellular capacity to maintain oxidative homeostasis is overwhelmed, thus resulting in cell death.

  11. Quantification of active caspases in stem cells: single cell analysis at femtogram level

    Czech Academy of Sciences Publication Activity Database

    Adamová, Eva; Lišková, Marcela; Klepárník, Karel; Hampl, Aleš; Matalová, Eva

    Leipzig: Fraunhofer Institute for Cell Therapy and Immunology, 2013. s. 358. [World Conference on Regenerative Medicine 2013 /WCRM 2013/. 23.10.2013-25.10.2013, Leipzig] R&D Projects: GA ČR GAP206/11/2377; GA ČR GAP304/11/1418 Institutional support: RVO:68081715 ; RVO:67985904 ; RVO:68378041 Keywords : caspases * cell analysis * cancer cell Subject RIV: CB - Analytical Chemistry, Separation; EA - Cell Biology (UZFG-Y); EB - Genetics ; Molecular Biology (UEM-P)

  12. Primary enamel knot cell death in Apaf-1 and caspase-9 deficient mice

    Czech Academy of Sciences Publication Activity Database

    Šetková, Jana; Matalová, Eva; Sharpe, P. T.; Míšek, Ivan; Tucker, A. S.

    2007-01-01

    Roč. 52, 1 (2007), s. 15-19. ISSN 0003-9969 R&D Projects: GA AV ČR KJB500450503; GA ČR GA304/04/0101; GA MŠk OC B23.001 Institutional research plan: CEZ:AV0Z50450515 Keywords : dental apoptosis * apoptosome * apaf-1 knockout * caspase-9 knockout Subject RIV: FF - HEENT, Dentistry Impact factor: 1.554, year: 2007

  13. Cancer gene therapy with iCaspase-9 transcriptionally targeted to tumor endothelial cells

    OpenAIRE

    Song, Wenying; Dong, Zhihong; Jin, Taocong; Mantellini, Maria G.; Núñez, Gabriel; Jacques E Nör

    2008-01-01

    Antiangiogenic therapies have shown varying results partly because each tumor type secretes a distinct panel of angiogenic factors to sustain its own microvascular network. In addition, recent evidence demonstrated that tumors develop resistance to antiangiogenic therapy by turning on alternate angiogenic pathways when one pathway is therapeutically inhibited. Here, we test the hypothesis that expression of a caspase-based artificial death switch in tumor-associated endothelial cells will dis...

  14. Prokaryotic Caspase Homologs: Phylogenetic Patterns and Functional Characteristics Reveal Considerable Diversity

    Science.gov (United States)

    Asplund-Samuelsson, Johannes; Bergman, Birgitta; Larsson, John

    2012-01-01

    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. PMID:23185476

  15. Macrophage Activation Redirects Yersinia-Infected Host Cell Death from Apoptosis to Caspase-1-Dependent Pyroptosis

    OpenAIRE

    Bergsbaken, Tessa; Cookson, Brad T.

    2007-01-01

    Infection of macrophages by Yersinia species results in YopJ-dependent apoptosis, and naïve macrophages are highly susceptible to this form of cell death. Previous studies have demonstrated that macrophages activated with lipopolysaccharide (LPS) prior to infection are resistant to YopJ-dependent cell death; we found this simultaneously renders macrophages susceptible to killing by YopJ− Yersinia pseudotuberculosis (Yptb). YopJ− Yptb-induced macrophage death was dependent on caspase-1 activat...

  16. Annexin A1 processing is associated with caspase-dependent apoptosis in BZR cells.

    Science.gov (United States)

    Debret, R; El Btaouri, H; Duca, L; Rahman, I; Radke, S; Haye, B; Sallenave, J M; Antonicelli, F

    2003-07-10

    Annexins are widely distributed and have been described in lung as well as in other cells and tissues. Annexin I (ANX AI) is a member of the calcium-dependent phospholipid binding protein family. Besides its anti-inflammatory function, ANX AI has been involved in several mechanisms such as the Erk repression pathway or apoptosis. To investigate the role of ANX AI on apoptosis in broncho-alveolar cells, we have constructed a plasmid containing the ANX AI full length cDNA. Transfected BZR cells displayed a higher level of both forms of ANX AI (37 and 33 kDa) as well as a decrease in cell viability (two-fold versus cells transfected with an empty vector). In order to analyse the endogenous ANX AI processing during stimulus-induced apoptosis, BZR cells were treated with a commonly used inducer, i.e. C2 ceramides. In these conditions, microscopic analysis revealed chromatin condensation in dying cells and the Bcl-2, Bcl-x(L)/Bax mRNA balance was altered. Caspase-3 is one of the key executioners of apoptosis, being responsible for the cleavage of many proteins such as the nuclear enzyme poly(ADP-ribose) polymerase (PARP). We demonstrate that caspase-3 was activated after 4 h treatment in the presence of ceramide leading to the cleavage of PARP. Dose-response experiments revealed that cell morphology and viability modifications following ceramide treatment were accompanied by an increase in endogenous ANX AI processing. Interestingly, in both ceramide and transfection experiments, the ANX AI cleaved form was enhanced whereas pre-treatment with the caspase inhibitor Z-VAD-fmk abolished ANX AI cleavage. In conclusion, this study demonstrates a complex regulatory role of caspase-dependent apoptosis where ANX AI is processed at the N-terminal region which could give susceptibility to apoptosis upon ceramide treatment. PMID:12832039

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

    OpenAIRE

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

    2011-01-01

    Adaptive immune responses are induced in liver after major stresses such as hemorrhagic shock (HS) and trauma. There is emerging evidence that the inflammasome, the multiprotein platform that induces caspase-1 activation and promotes interleukin (IL)-1β and IL-18 processing, is activated in response to cellular oxidative stress, such as after hypoxia, ischemia and HS. Additionally, damage-associated molecular patterns, such as those released after injury, have been shown to activate the infla...

  18. 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

  19. Tripeptidyl Peptidase II Promotes Maturation of Caspase-1 in Shigella flexneri-Induced Macrophage Apoptosis

    OpenAIRE

    Hilbi, Hubert; Puro, Robyn J.; Zychlinsky, Arturo

    2000-01-01

    The invasive enteropathogenic bacterium Shigella flexneri activates apoptosis in macrophages. Shigella-induced apoptosis requires caspase-1. We demonstrate here that tripeptidyl peptidase II (TPPII), a cytoplasmic, high-molecular-weight protease, participates in the apoptotic pathway triggered by Shigella. The TPPII inhibitor Ala-Ala-Phe-chloromethylketone (AAF-cmk) and clasto-lactacystin β-lactone (lactacystin), an inhibitor of both TPPII and the proteasome, protected macrophages from Shigel...

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

    OpenAIRE

    Liu, Guangyi; Tao WANG; WANG, TINGING; Song, Jinming; Zhou, Zhen

    2013-01-01

    Neuron apoptosis is known to mediate a change of ethology following cerebral ischemia-reperfusion injury in rats. Additionally, Bcl-2, Bax and caspase-3 proteins may exert a significant effect on neuron injury. The aim of this study was to investigate the role, mechanism of action and clinical significance of these proteins in neuron apoptosis and functional impairment following cerebral ischemia-reperfusion injury in rats. Sixty male healthy adult Wistar rats were randomly assigned into cont...

  1. DIETARY PHYTOCHEMICALS INDUCE p53- AND CASPASE-INDEPENDENT CELL DEATH IN HUMAN NEUROBLASTOMA CELLS

    OpenAIRE

    Sukumari-Ramesh, Sangeetha; Bentley, J Nicole; Laird, Melissa D.; Singh, Nagendra; Vender, John R.; Dhandapani, Krishnan M.

    2011-01-01

    Neuroblastoma (NB) is the most prevalent pediatric solid tumor and a leading cause of cancer-related death in children. In the present study, a novel cytotoxic role for the dietary compounds, curcumin, andrographolide, wedelolactone, dibenzoylmethane, and tanshinone IIA was identified in human S-type NB cells, SK-N-AS and SK-N-BE(2). Mechanistically, cell death appeared apoptotic by flow cytometry; however, these effects proceeded independently from both caspase-3 and p53 activation, as asses...

  2. Experimental Cerebral Malaria Develops Independently of Caspase Recruitment Domain-Containing Protein 9 Signaling

    OpenAIRE

    Julius Clemence R Hafalla; Burgold, Jan; Dorhoi, Anca; Gross, Olaf; Ruland, Jürgen; Stefan H. E. Kaufmann; Matuschewski, Kai

    2012-01-01

    The outcome of infection depends on multiple layers of immune regulation, with innate immunity playing a decisive role in shaping protection or pathogenic sequelae of acquired immunity. The contribution of pattern recognition receptors and adaptor molecules in immunity to malaria remains poorly understood. Here, we interrogate the role of the caspase recruitment domain-containing protein 9 (CARD9) signaling pathway in the development of experimental cerebral malaria (ECM) using the murine Pla...

  3. Effects of Berberine on the Expression of Caspase-3 Gene in Human Cervical Cancer Hela Cell%小檗碱对宫颈癌Hela细胞Caspase-3基因表达的影响

    Institute of Scientific and Technical Information of China (English)

    张丽萍; 张志军; 程萍; 肖劲松

    2010-01-01

    日的:研究小檗碱(berberlne)对人宫颈癌Hela细胞体外增殖、凋亡及凋亡相关基因Caspase-3表达的影响.方法:WIT法测定不同浓度(0~40 μmol/L)berberine干预Hela细胞后的凋亡率;RT-PCR检测Caspase-3 mRNA表达水平.结果:berberine呈剂量-时间依赖方式抑制Hela细胞的生长(P<0.01);且随着berberine浓度增高,细胞凋亡增加,Caspase-3 mRNA表达上调(P<0.001).结论:berberine抑制人宫颈癌Hela细胞生长、诱导凋亡的机制可能与上调Caspase-3 mRNA表达有关.

  4. Caspase-3在大鼠中枢神经系统的表达研究%Study on the expression of caspase-3 in central nervous system of rat

    Institute of Scientific and Technical Information of China (English)

    陈雪梅; 杜显刚; 官鹏; 谭志巍; 王亚琴

    2005-01-01

    目的研究caspase-3在大鼠中枢神经系统的表达及其意义.方法用western-blot方法对出生1天和3个月SD大鼠的大脑皮质、中脑和小脑组织的caspase-3进行半定量测定.结果出生1天大鼠脑的caspase-3表达较高,出生3个月大鼠脑的caspase-3表达较低.结论caspase-3在中枢神经系统的发育成熟过程中对神经元的凋亡起着关键性作用.

  5. Effcts of Vitamin C on A549 Cell Proliferation, Apoptosis and Expressions of Caspase, Survivin

    Directory of Open Access Journals (Sweden)

    Lanzhen HUANG

    2010-02-01

    Full Text Available Background and objective It was proven that Vitamin C could inhibit the growth of many types of tumors as an antioxidant. The aim of this study is to explore role of Vitamin C in proliferation and apoptosis of lung carcinoma cell line A549 and the underlying mechanism. Methods A549 cells were cultured in vitro and incubated with Vitamin C. The cell viability was measured by growth curve and clonogentic assay. Flow cytometry was used to analyze cell cycle and detect apoptosis. The levels of expression of Caspase-3 mRNA and Survivin mRNA were detected by RT-PCR. Results Vitamin C of 400 μg/mL, 4 mg/mL significantly inhibited the growth of A549 cell lines (P=0.024, P=0.015, respectively. Flow cytometry showed that the cells major stagnation stayed in the G0/G1 and S phase and the apoptotic rate increased with time prolonged. Vitamin C signifiantly up-ragulated the expression of Caspase-3 mRNA, but had no effect on Survivin mRNA. Conclusion Vitamin C can inhibit the proliferation of A549, block A549 cells in G0/G1 and S phase, and induce apoptosis of A549 cells. Apotosis occurred by up-ragulated the expressionof Caspase-3.

  6. Relationship between triterpenoid anticancer drug resistance, autophagy, and caspase-1 in adult T-cell leukemia

    Science.gov (United States)

    Nakanishi, Tsukasa; Morita, Kentaro; Tsukada, Junichi; Kanazawa, Tamotsu

    2016-01-01

    We previously reported that the inflammasome inhibitor cucurbitacin D (CuD) induces apoptosis in human leukemia cell lines. Here, we investigated the effects of CuD and a B-cell lymphoma extra-large (Bcl-xL) inhibitor on autophagy in peripheral blood lymphocytes (PBL) isolated from adult T-cell leukemia (ATL) patients. CuD induced PBL cell death in patients but not in healthy donors. This effect was not significantly inhibited by treatment with rapamycin or 3-methyladenine (3-MA). The Bcl-xL inhibitor Z36 induced death in primary cells from ATL patients including that induced by CuD treatment, effects that were partly inhibited by 3-MA. Similarly, cell death induced by the steroid prednisolone was enhanced in the presence of Z36. A western blot analysis revealed that Z36 also promoted CuD-induced poly(ADP ribose) polymerase cleavage. Interestingly, the effects of CuD and Z36 were attenuated in primary ATL patient cells obtained upon recurrence after umbilical cord blood transplantation, as compared to those obtained before chemotherapy. Furthermore, cells from this patient expressed a high level of caspase-1, and treatment with caspase-1 inhibitor-enhanced CuD-induced cell death. Taken together, these results suggest that rescue from resistance to steroid drugs can enhance chemotherapy, and that caspase-1 is a good marker for drug resistance in ATL patients. PMID:27190722

  7. Agarol, an ergosterol derivative from Agaricus blazei, induces caspase-independent apoptosis in human cancer cells.

    Science.gov (United States)

    Shimizu, Takamitsu; Kawai, Junya; Ouchi, Kenji; Kikuchi, Haruhisa; Osima, Yoshiteru; Hidemi, Rikiishi

    2016-04-01

    Agaricus blazei (A. blazei) is a mushroom with many biological effects and active ingredients. We purified a tumoricidal substance from A. blazei, an ergosterol derivative, and named it 'Agarol'. Cytotoxic effects of Agarol were determined by the MTT assay using A549, MKN45, HSC-3, and HSC-4 human carcinoma cell lines treated with Agarol. Apoptosis was detected by flow cytometry analysis. Reactive oxygen species (ROS) levels and mitochondria membrane potential (∆ψm) were also determined by flow cytometry. Western blot analysis was used to quantify the expression of apoptosis-related proteins. Agarol predominantly induced apoptosis in two p53-wild cell lines (A549 and MKN45) compared to the other p53-mutant cell lines (HSC-3 and HSC-4). Further mechanistic studies revealed that induction of apoptosis is associated with increased generation of ROS, reduced ∆ψm, release of apoptosis-inducing factor (AIF) from the mitochondria to the cytosol, upregulation of Bax, and downregulation of Bcl-2. Caspase-3 activities did not increase, and z-VAD-fmk, a caspase inhibitor, did not inhibit the Agarol-induced apoptosis. These findings indicate that Agarol induces caspase-independent apoptosis in human carcinoma cells through a mitochondrial pathway. The in vivo anticancer activity of Agarol was confirmed in a xenograft murine model. This study suggests a molecular mechanism by which Agarol induces apoptosis in human carcinoma cells and indicates the potential use of Agarol as an anticancer agent. PMID:26893131

  8. BGP-15 inhibits caspase-independent programmed cell death in acetaminophen-induced liver injury

    International Nuclear Information System (INIS)

    It has been recently shown that acute acetaminophen toxicity results in endoplasmic reticulum redox stress and an increase in cells with apoptotic phenotype in liver. Since activation of effector caspases was absent, the relevance of caspase-independent mechanisms in acetaminophen-induced programmed cell death was investigated. BGP-15, a drug with known protective actions in conditions involving redox imbalance, has been co-administered with a single sublethal dose of acetaminophen. Proapoptotic events and outcome of the injury were investigated. ER redox alterations and early ER-stress-related signaling events induced by acetaminophen, such as ER glutathione depletion, phosphorylation of eIF2α and JNK and induction of the transcription factor GADD153, were not counteracted by co-treatment with BGP-15. However, BGP-15 prevented AIF mitochondria-to-nucleus translocation and mitochondrial depolarization. BGP-15 co-treatment attenuated the rate of acetaminophen-induced cell death as assessed by apoptotic index and enzyme serum release. These results reaffirm that acute acetaminophen toxicity involves oxidative stress-induced caspase-independent cell death. In addition, pharmacological inhibition of AIF translocation may effectively protect against or at least delay acetaminophen-induced programmed cell death.

  9. Albumin-binding caspase-cleavable prodrug that is selectively activated in radiation exposed local tumor.

    Science.gov (United States)

    Chung, Seung Woo; Choi, Jeong Uk; Lee, Beom Seok; Byun, Julia; Jeon, Ok-Cheol; Kim, Seong Who; Kim, In-San; Kim, Sang Yoon; Byun, Youngro

    2016-07-01

    Existence of the genomically and epigenomically diverse subclones in a tumor severely limits the therapeutic efficacy of targeted agents. To overcome such a limitation, we prepared a novel targeted prodrug, EMC-DEVD-S-DOX, which comprises two important features: radiation-induced apoptosis targeting and albumin-binding properties. In particular, the prodrug binds circulating albumin after intravenous administration and then activated by caspase-3, which is upregulated from apoptotic cells that responded to radiotherapy. The prodrug was designed to bind circulating albumin to extend half-life and facilitate tumor accumulation in order to increase the possibility of contacting caspase-3, which is only transiently upregulated during apoptosis. Our results showed that EMC-DEVD-S-DOX had a prolonged half-life with enhanced tumor accumulation, which clearly benefited the therapeutic effect of the prodrug. Also, agreeing with the in vitro studies that showed ignorable cytotoxic effect in the absence of caspase-3, the prodrug was effective only when combined with radiotherapy without any noticeable systemic toxicity in vivo. Due to the highly selective action of EMC-DEVD-S-DOX independent to the complex genomic profiles of tumor, the prodrug would overcome the limitation of current targeted therapy and potentiate radiotherapy in the clinical oncology. PMID:27085176

  10. Pharmacophore Modeling and Docking Studies on Some Nonpeptide-Based Caspase-3 Inhibitors

    Directory of Open Access Journals (Sweden)

    Simant Sharma

    2013-01-01

    Full Text Available Neurodegenerative disorders are major consequences of excessive apoptosis caused by a proteolytic enzyme known as caspase-3. Therefore, caspase-3 inhibition has become a validated therapeutic approach for neurodegenerative disorders. We performed pharmacophore modeling on some synthetic derivatives of caspase-3 inhibitors (pyrrolo[3,4-c]quinoline-1,3-diones using PHASE 3.0. This resulted in the common pharmacophore hypothesis AAHRR.6 which might be responsible for the biological activity: two aromatic rings (R mainly in the quinoline nucleus, one hydrophobic (H group (CH3, and two acceptor (A groups (–C=O. After identifying a valid hypothesis, we also developed an atom-based 3D-QSAR model applying the PLS algorithm. The developed model was statistically robust (q2=0.53; pred_r2=0.80. Additionally, we have performed molecular docking studies, cross-validated our results, and gained a deeper insight into its molecular recognition process. Our developed model may serve as a query tool for future virtual screening and drug designing for this particular target.

  11. Caspase dependent apoptotic inhibition of melanoma and lung cancer cells by tropical Rubus extracts.

    Science.gov (United States)

    George, Blassan Plackal Adimuriyil; Abrahamse, Heidi; Hemmaragala, Nanjundaswamy M

    2016-05-01

    Rubus fairholmianus Gard. inhibits human melanoma (A375) and lung cancer (A549) cell growth by the caspase dependent apoptotic pathway. Herbal products have a long history of clinical use and acceptance. They are freely available natural compounds that can be safely used to prevent various ailments. The plants and plant derived products became the basis of traditional medicine system throughout the world for thousands of years. The effects of R. fairholmianus root acetone extract (RFRA) on the proliferation of A375 and A549 cells was examined in this study. RFRA led to a decrease in cell viability, proliferation and an increase in cytotoxicity in a dose dependent manner when compared with control and normal skin fibroblast cells (WS1). The morphology of treated cells supported apoptotic cell death. Annexin V/propidium iodide staining indicated that RFRA induced apoptosis in A375 and A549 cells and the percentages of early and late apoptotic populations significantly increased. Moreover, the apoptotic inducing ability of RFRA when analysing effector caspase 3/7 activity, indicated a marked increase in treated cells. In summary, we have shown the anticancer effects of RFRA in A375 and A549 cancer cells via induction of caspase dependent apoptosis in vitro. The extract is more effective against melanoma; which may suggest the usefulness of RFRA-based anticancer therapies. PMID:27133056

  12. STEREOLOGIC ESTIMATION OF KI-67, CASPASE 3 AND GSTP1 POSITIVE CELLS IN PROSTATE LESIONS

    Directory of Open Access Journals (Sweden)

    Luis Santamaría

    2011-05-01

    Full Text Available Cell proliferation, caspase 3 and pi-form of glutathione S transferase (GSTP1 were evaluated in prostate carcinoma (PCA, proliferative inflammatory atrophy (PIA and prostate intraepithelial neoplasia (PIN. Forty biopsies were classified as: without morphological lesions (controls: CTR, PIA, PIN and PCA. Ki67, caspase3 and GSTP1 were immunostained. The following estimates were performed: Numerical densities of Ki67+ cells (NVEPKi67, of all epithelial cells (NVEPtotal and of GSTP1+ cells (NVEPGSTP1; labelling index for Ki67 (LIKi67; volume fraction to caspase 3 positive tissue (VVcaspase 3 and of GSTP1 positive tissue (VVGSTP1. ANOVA was performed to compare the groups. NVEPtotal and NVEPKi67 were increased in PIA. LIKi67 was only increased in PCA. VVcaspase 3 was decreased in PIN and PCA. VVEGSTP1 was decreased in PCA. In our results PIA lacks the characteristics of a premalignant lesion. The result may be explained by the use of unbiased quantitative methods, the inadequate definition of PIA and the scarce inflammation observed in the samples with PIA included in this study.

  13. The Drosophila effector caspase Dcp-1 regulates mitochondrial dynamics and autophagic flux via SesB.

    Science.gov (United States)

    DeVorkin, Lindsay; Go, Nancy Erro; Hou, Ying-Chen Claire; Moradian, Annie; Morin, Gregg B; Gorski, Sharon M

    2014-05-26

    Increasing evidence reveals that a subset of proteins participates in both the autophagy and apoptosis pathways, and this intersection is important in normal physiological contexts and in pathological settings. In this paper, we show that the Drosophila effector caspase, Drosophila caspase 1 (Dcp-1), localizes within mitochondria and regulates mitochondrial morphology and autophagic flux. Loss of Dcp-1 led to mitochondrial elongation, increased levels of the mitochondrial adenine nucleotide translocase stress-sensitive B (SesB), increased adenosine triphosphate (ATP), and a reduction in autophagic flux. Moreover, we find that SesB suppresses autophagic flux during midoogenesis, identifying a novel negative regulator of autophagy. Reduced SesB activity or depletion of ATP by oligomycin A could rescue the autophagic defect in Dcp-1 loss-of-function flies, demonstrating that Dcp-1 promotes autophagy by negatively regulating SesB and ATP levels. Furthermore, we find that pro-Dcp-1 interacts with SesB in a nonproteolytic manner to regulate its stability. These data reveal a new mitochondrial-associated molecular link between nonapoptotic caspase function and autophagy regulation in vivo. PMID:24862573

  14. Serum levels of caspase-cleaved cytokeratin-18 and mortality are associated in severe septic patients: pilot study.

    Directory of Open Access Journals (Sweden)

    Leonardo Lorente

    Full Text Available Apoptosis is increased in sepsis. Cytokeratin 18 (CK-18, a protein of the intermediate filament group present in most epithelial and parenchymal cells, is cleaved by the action of caspases and released into the blood as caspase-cleaved CK (CCCK-18 during apoptosis. Circulating levels of CCCK-18 have scarcely been explored in septic patients. In one study with 101 severe septic patients, the authors reported higher serum CCCK-18 levels in non-survivors than in survivors; however, the sample size was too small to demonstrate an association between serum CCCK-18 levels and early mortality and whether they could be used as a biomarker to predict outcomes in septic patients. Thus, these were the objectives of this study with a large series of patients.We performed a prospective, multicenter, observational study in six Spanish Intensive Care Units with 224 severe septic patients. Blood samples were collected at the time that severe sepsis was diagnosed to determine serum levels of CCCK-18, tumor necrosis factor (TNF-alpha, interleukin (IL-6 and IL-10. The end point was 30-day mortality.Non-surviving patients (n = 80 showed higher serum CCCK-18 levels (P391 u/L were associated with 30-day survival (Odds ratio = 2.687; 95% confidence interval = 1.449-4.983; P = 0.002, controlling for SOFA score, serum lactic acid levels and age. Kaplan-Meier survival analysis showed that the risk of death in septic patients with serum CCCK-18 levels >391 u/L was higher than in patients with lower values (Hazard Ratio = 3.1; 95% CI = 1.96-4.84; P<0.001. Serum CCCK-18 levels were positively associated with serum levels of IL-6 and lactic acid, and with SOFA and APACHE scores.The major novel finding of our study, the largest cohort of septic patients providing data on circulating CCCK-18 levels, was that serum CCCK-18 levels are associated with mortality in severe septic patients.

  15. Evaluation of Bcl-2, Bcl-x and Cleaved Caspase-3 in Malignant Peripheral Nerve Sheath Tumors and Neurofibromas

    Directory of Open Access Journals (Sweden)

    KARIN S. CUNHA

    2013-11-01

    Full Text Available AIMS: To study the expression of Bcl-2, Bcl-x, as well the presence of cleaved caspase-3 in neurofibromas and malignant peripheral nerve sheath tumors. The expression of Bcl-2 and Bcl-x and the presence of cleaved caspase 3 were compared to clinicopathological features of malignant peripheral nerve sheath tumors and their impact on survival rates were also investigated. MATERIALS AND METHODS: The evaluation of Bcl-2, Bcl-x and cleaved caspase-3 was performed by immunohistochemistry using tissue microarrays in 28 malignant peripheral nerve sheath tumors and 38 neurofibromas. Immunoquantification was performed by computerized digital image analysis. CONCLUSIONS: Apoptosis is altered in neurofibromas and mainly in malignant peripheral nerve sheath tumors. High levels of cleaved caspase-3 are more common in tumors with more aggressive histological features and it is associated with lower disease free survival of patients with malignant peripheral nerve sheath tumors.

  16. A facile method to prepare large quantities of active caspase-3 overexpressed by auto-induction in the C41(DE3) strain.

    Science.gov (United States)

    Hwang, Dohyeon; Kim, Sang Ah; Yang, Eun Gyeong; Song, Hyun Kyu; Chung, Hak Suk

    2016-10-01

    Since human Caspase-3, a member of the cysteine protease family, plays important roles not only in the apoptosis pathway as an executioner protein, but also in neurological disorders as a critical factor, biomedical researchers have been interested in the development of modulators of caspase-3 activity. Such studies require large quantities of purified active caspase-3. So far, purification of soluble caspase-3 from full-length human caspase-3 in Escherichia coli (E. coli) yields only several mg from a liter of culture media. Therefore, a number of alternative strategies to purify active caspase-3 have been described in the literature, including refolding and protein engineering. In this study, we systematically study the effects of host E. coli strains and growth conditions on purifications of active caspase-3 from full-length human caspase-3. Using a combination of conditions that include use of the C41(DE3) strain, low-temperature expression, and auto-induction that induces caspase-3 expression depending on metabolic state of the individual host cell, we are able to obtain 14-17 mg caspase-3 per liter of culture, an amount that is about 7 times larger than published results. This optimized expression and purification method for caspase-3 can be easily scaled up to facilitate the demand for active enzyme. PMID:27320415

  17. Fluorescence-quenching-based homogeneous caspase-3 activity assay using photon upconversion

    Energy Technology Data Exchange (ETDEWEB)

    Vuojola, Johanna, E-mail: johanna.vuojola@utu.fi [Department of Biotechnology, University of Turku, Tykistoekatu 6A, FI-20520 Turku (Finland); Riuttamaeki, Terhi; Kulta, Essi; Arppe, Riikka; Soukka, Tero [Department of Biotechnology, University of Turku, Tykistoekatu 6A, FI-20520 Turku (Finland)

    2012-05-06

    Highlights: Black-Right-Pointing-Pointer We demonstrate the use of photon upconversion in a caspase-3 activity assay. Black-Right-Pointing-Pointer The separation-free assay uses an internally quenched substrate peptide. Black-Right-Pointing-Pointer UCPs enable simple instrumentation and total elimination of autofluorescence. Black-Right-Pointing-Pointer A sensitive assay with high signal-to-background ratios was achieved. Black-Right-Pointing-Pointer Suitable for high-throughput screening through miniaturization and white plates. - Abstract: Caspase proteases are key mediators in apoptosis and thus of great interest in pharmaceutical industry. Enzyme-activity assays are commonly employed in the screening of protease inhibitors that are potential drug candidates. Conventional homogeneous fluorescence-based assays are susceptible to autofluorescence originating from biological material. This background autofluorescence can be eliminated by using upconverting phosphors (UCPs) that emit visible light upon excitation at near-infrared. In the assay energy was transferred from a UCP-donor to a conventional fluorophore acceptor that resided at one end of a caspase-3-specific substrate peptide. Attached to the other end was a quencher molecule that was used to attenuate the acceptor emission through intramolecular energy transfer in an intact peptide. In non-inhibitory conditions the enzyme reaction separated the fluorophore from the quencher and the emission of the fluorophore was recovered. The method was applied for the detection and characterization of a known caspase-3 inhibitor Z-DEVD-FMK, and the assay gave IC{sub 50} values of approximately 13 nM for this inhibitor. We have demonstrated the applicability of UCPs on a fluorescence-quenching-based homogeneous enzyme-activity assay for the detection of caspase-3 inhibitors. The use of near-infrared excitable UCPs enables inexpensive instrumentation and total elimination of autofluorescence, while the use of an

  18. Continuous monitoring of caspase-3 activation induced by propofol in developing mouse brain.

    Science.gov (United States)

    Konno, Ayumi; Nishimura, Akiko; Nakamura, Shiro; Mochizuki, Ayako; Yamada, Atsushi; Kamijo, Ryutaro; Inoue, Tomio; Iijima, Takehiko

    2016-06-01

    The neurotoxicity of anesthetics on the developing brain has drawn the attention of anesthesiologists. Several studies have shown that apoptosis is enhanced by exposure to anesthesia during brain development. Although apoptosis is a physiological developmental step occurring before the maturation of neural networks and the integration of brain function, pathological damage also involves apoptosis. Previous studies have shown that prolonged exposure to anesthetics causes apoptosis. Exactly when the apoptotic cascade starts in the brain remains uncertain. If it starts during the early stage of anesthesia, even short-term anesthesia could harm the brain. Therefore, apoptogenesis should be continuously monitored to elucidate when the apoptotic cascade is triggered by anesthesia. Here, we describe the development of a continuous monitoring system to detect caspase-3 activation using an in vivo model. Brain slices from postnatal days 0-4 SCAT3 transgenic mice with a heterozygous genotype (n=20) were used for the monitoring of caspase-3 cleavage. SCAT3 is a fusion protein of ECFP and Venus connected by a caspase-3 cleavable peptide, DEVD. A specimen from the hippocampal CA1 sector was mounted on a confocal laser microscope and was continuously superfused with artificial cerebrospinal fluid, propofol (2,6-diisopropylphenol, 1μM or 10μM), and dimethyl sulfoxide. Images were obtained every hour for five hours. A pixel analysis of the ECFP/Venus ratio images was performed using a histogram showing the number of pixels with each ratio. In the histogram of the ECFP/Venus ratio, an area with a ratio>1 indicated the number of pixels from caspase-3-activated CA1 neurons. We observed a shift in the histogram toward the right over time, indicating caspase-3 activation. This right-ward shift dramatically changed at five hours in the propofol 1μM and 10μM groups and was obviously different from that in the control group. Thus, real-time fluorescence energy transfer (FRET) imaging

  19. Caspase 3 involves in neuroplasticity, microglial activation and neurogenesis in the mice hippocampus after intracerebral injection of kainic acid

    OpenAIRE

    Tzeng, Tsai-Teng; Tsay, Huey-Jen; Chang, Luping; Hsu, Chia-Lin; Lai, Tzu-Hsuan; Huang, Fong-Lee; Shiao, Young-Ji

    2013-01-01

    Background The roles of caspase 3 on the kainic acid-mediated neurodegeneration, dendritic plasticity alteration, neurogenesis, microglial activation and gliosis are not fully understood. Here, we investigate hippocampal changes using a mouse model that receive a single kainic acid-intracerebral ventricle injection. The effects of caspase 3 inhibition on these changes were detected during a period of 1 to 7 days post kainic acid injection. Result Neurodegeneration was assessed by Fluoro-Jade ...

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

    Science.gov (United States)

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

    2016-05-27

    The BH3-only protein Bid is known as a critical mediator of the mitochondrial pathway of apoptosis following death receptor activation. However, since full-length Bid possesses potent apoptotic activity, the role of a caspase-mediated Bid cleavage is not established in vivo In addition, due to the fact that multiple caspases cleave Bid at the same site in vitro, the identity of the Bid-cleaving caspase during death receptor signaling remains uncertain. Moreover, as Bid maintains its overall structure following its cleavage by caspase 8, it remains unclear how Bid is activated upon cleavage. Here, Bid-deficient (Bid KO) colon cancer cells were generated by gene editing, and were reconstituted with wild-type or mutants of Bid. While the loss of Bid blocked apoptosis following treatment by TNF-related apoptosis inducing ligand (TRAIL), this blockade was relieved by re-introduction of the wild-type Bid. In contrast, the caspase-resistant mutant Bid(D60E) and a BH3 defective mutant Bid(G94E) failed to restore TRAIL-induced apoptosis. By generating Bid/Bax/Bak-deficient (TKO) cells, we demonstrated that Bid is primarily cleaved by caspase 8, not by effector caspases, to give rise to truncated Bid (tBid) upon TRAIL treatment. Importantly, despite the presence of an intact BH3 domain, a tBid mutant lacking the mitochondrial targeting helices (α6 and α7) showed diminished apoptotic activity. Together, these results for the first time establish that cleavage by caspase 8 and the subsequent association with the outer mitochondrial membrane are two critical events that activate Bid during death receptor-mediated apoptosis. PMID:27053107

  1. The caspase 3-dependent apoptotic effect of pycnogenol in human oral squamous cell carcinoma HSC-3 cells

    OpenAIRE

    Yang, In-Hyoung; Shin, Ji-Ae; Kim, Lee-Han; Kwon, Ki Han; Cho, Sung-Dae

    2015-01-01

    In the present study, the apoptotic effect of pycnogenol and its molecular mechanism in human oral squamous cell carcinoma HSC-3 cells were investigated. Pycnogenol significantly inhibited the viability of HSC-3 cells and suppressed neoplastic cell transformation in HSC-3 cells and TPA-treated JB6 cells. It caused caspase-dependent apoptosis evidenced by the increase in cleaved poly (ADP-ribose) polymerase and caspase 3 in a dose-dependent manner. Pycnogenol increased Bak protein by enhancing...

  2. Uterine Endoplasmic Reticulum Stress and Its Unfolded Protein Response May Regulate Caspase 3 Activation in the Pregnant Mouse Uterus

    OpenAIRE

    Suresh, Arvind; Subedi, Kalpana; Kyathanahalli, Chandrashekara; Jeyasuria, Pancharatnam; Condon, Jennifer C.

    2013-01-01

    We have previously proposed that uterine caspase-3 may modulate uterine contractility in a gestationally regulated fashion. The objective of this study was to determine the mechanism by which uterine caspase-3 is activated and consequently controlled in the pregnant uterus across gestation. Utilizing the mouse uterus as our gestational model we examined the intrinsic and extrinsic apoptotic signaling pathways and the endoplasmic reticulum stress response as potential activators of uterine cas...

  3. Genetically Encoded FRET-Sensor Based on Terbium Chelate and Red Fluorescent Protein for Detection of Caspase-3 Activity

    OpenAIRE

    Goryashchenko, Alexander S.; Maria G. Khrenova; Bochkova, Anna A.; Ivashina, Tatiana V.; Vinokurov, Leonid M.; Alexander P. Savitsky

    2015-01-01

    This article describes the genetically encoded caspase-3 FRET-sensor based on the terbium-binding peptide, cleavable linker with caspase-3 recognition site, and red fluorescent protein TagRFP. The engineered construction performs two induction-resonance energy transfer processes: from tryptophan of the terbium-binding peptide to Tb3+ and from sensitized Tb3+ to acceptor—the chromophore of TagRFP. Long-lived terbium-sensitized emission (microseconds), pulse excitation source, and time-resolved...

  4. 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.

  5. Prolonged mitotic arrest induces a caspase-dependent DNA damage response at telomeres that determines cell survival.

    Science.gov (United States)

    Hain, Karolina O; Colin, Didier J; Rastogi, Shubhra; Allan, Lindsey A; Clarke, Paul R

    2016-01-01

    A delay in the completion of metaphase induces a stress response that inhibits further cell proliferation or induces apoptosis. This response is thought to protect against genomic instability and is important for the effects of anti-mitotic cancer drugs. Here, we show that mitotic arrest induces a caspase-dependent DNA damage response (DDR) at telomeres in non-apoptotic cells. This pathway is under the control of Mcl-1 and other Bcl-2 family proteins and requires caspase-9, caspase-3/7 and the endonuclease CAD/DFF40. The gradual caspase-dependent loss of the shelterin complex protein TRF2 from telomeres promotes a DDR that involves DNA-dependent protein kinase (DNA-PK). Suppression of mitotic telomere damage by enhanced expression of TRF2, or the inhibition of either caspase-3/7 or DNA-PK during mitotic arrest, promotes subsequent cell survival. Thus, we demonstrate that mitotic stress is characterised by the sub-apoptotic activation of a classical caspase pathway, which promotes telomere deprotection, activates DNA damage signalling, and determines cell fate in response to a prolonged delay in mitosis. PMID:27230693

  6. Protective effect of novel substituted nicotine hydrazide analogues against hypoxic brain injury in neonatal rats via inhibition of caspase.

    Science.gov (United States)

    Deng, Chang-Bo; Li, Juan; Li, Lu-Yi; Sun, Feng-Jie

    2016-07-01

    In hypoxic-ischemic injury of the brain of neonates, the level of caspase-3 was found to be aberrantly activated. Its overexpression leads to the alteration of cytoskeleton protein fodrin and loss of DNA repair enzyme which ultimately results in neurological impairment and disability. Concerning this, the present study was intended to develop novel nicotine hydrazide analogues as caspase inhibitors via efficient synthetic route. These compounds were subsequently tested for inhibitory activity against caspase-3 and -7 where they exhibit highly potent activity against caspase-3 revealing compound 5k as most potent inhibitor (IC50=19.4±2.5μM). In Western blot analysis, 5k considerably inhibits the overexpression of caspase-3. The aryl nicotinate of compound 5k, as indicated by molecular docking was found to engage His121 and critical enzyme thiols, i.e., Cys163 of caspase-3 for its potent activity. Moreover, histopathological examination of brain tissues and hippocampus neurons showed that compound 5k considerably improves the brain injury and exert neuroprotective effects in hypoxic-ischemic (HI). In brain homogenate, 5k significantly improves the activity of MDA, SOD, GSH-Px, CAT and T-AOC to exert its beneficial effect against oxidative stress induced by HI injury. PMID:27216999

  7. Isolation and characterization of a Solanum tuberosum subtilisin-like protein with caspase-3 activity (StSBTc-3).

    Science.gov (United States)

    Fernández, María Belén; Daleo, Gustavo Raúl; Guevara, María Gabriela

    2015-01-01

    Plant proteases with caspase-like enzymatic activity have been widely studied during the last decade. Previously, we have reported the presence and induction of caspase-3 like activity in the apoplast of potato leaves during Solanum tuberosum- Phytophthora infestans interaction. In this work we have purified and identified a potato extracellular protease with caspase-3 like enzymatic activity from potato leaves infected with P. infestans. Results obtained from the size exclusion chromatography show that the isolated protease is a monomeric enzyme with an estimated molecular weight of 70 kDa approximately. Purified protease was analyzed by MALDI-TOF MS, showing a 100% of sequence identity with the deduced amino acid sequence of a putative subtilisin-like protease from S. tuberosum (Solgenomics protein ID: PGSC0003DMP400018521). For this reason the isolated protease was named as StSBTc-3. This report constitutes the first evidence of isolation and identification of a plant subtilisin-like protease with caspase-3 like enzymatic activity. In order to elucidate the possible function of StSBTc-3 during plant pathogen interaction, we demonstrate that like animal caspase-3, StSBTc-3 is able to produce in vitro cytoplasm shrinkage in plant cells and to induce plant cell death. This result suggest that, StSBTc-3 could exert a caspase executer function during potato- P. infestans interaction, resulting in the restriction of the pathogen spread during plant-pathogen interaction. PMID:25486023

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

    International Nuclear Information System (INIS)

    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

  9. 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.

  10. Caspase-3 Contributes to ZO-1 and Cl-5 Tight-Junction Disruption in Rapid Anoxic Neurovascular Unit Damage

    Science.gov (United States)

    de Curtis, Marco; Kuhlmann, Christoph R. W.; Luhmann, Heiko J.

    2011-01-01

    Background Tight-junction (TJ) protein degradation is a decisive step in hypoxic blood-brain barrier (BBB) breakdown in stroke. In this study we elucidated the impact of acute cerebral ischemia on TJ protein arrangement and the role of the apoptotic effector protease caspase-3 in this context. Methodology/Principal Findings We used an in vitro model of the neurovascular unit and the guinea pig whole brain preparation to analyze with immunohistochemical methods the BBB properties and neurovascular integrity. In both methodological approaches we observed rapid TJ protein disruptions after 30 min of oxygen and glucose deprivation or middle cerebral artery occlusion, which were accompanied by strong caspase-3 activation in brain endothelial cells (BEC). Surprisingly only few DNA-fragmentations were detected with TUNEL stainings in BEC. Z-DEVD-fmk, an irreversible caspase-3 inhibitor, partly blocked TJ disruptions and was protective on trans-endothelial electrical resistance. Conclusions/Significance Our data provide evidence that caspase-3 is rapidly activated during acute cerebral ischemia predominantly without triggering DNA-fragmentation in BEC. Further we detected fast TJ protein disruptions which could be partly blocked by caspase-3 inhibition with Z-DEVD-fmk. We suggest that the basis for clinically relevant BBB breakdown in form of TJ disruptions is initiated within minutes during ischemia and that caspase-3 contributes to this process. PMID:21364989

  11. Correlation of the expression of survivin and caspase-3 proteins in juvenile laryngeal papilloma%儿童喉乳头状瘤组织中Survivin及Caspase-3蛋白表达及相关性研究

    Institute of Scientific and Technical Information of China (English)

    汪武; 周远大; 何海霞

    2009-01-01

    目的:研究儿童喉乳头状瘤中survivin及caspase-3蛋白的表达及其相互关系.方法:应用免疫组化方法对儿童喉乳头状瘤组织、儿童声带小结组织及正常的喉黏膜组织中survivin及caspase-3的表达进行检测并进行相关性分析.结果:42例儿童喉乳头状瘤组织中survivin阳性表达率为57.14%, 明显高于儿童声带小结组(P<0.01)和正常黏膜组(P<0.01);42例喉乳头状瘤组织中caspase-3阳性表达率为26.19%, 显著高于儿童声带小结组和正常喉黏膜组 (P<0.01);Spearman相关性分析显示, 儿童喉乳头状瘤中survivin与caspase-3的表达呈显著负相关(r=-0.682, P<0.01).结论:Survivin的高表达和caspase-3的低表达可能在儿童喉乳头状瘤发生发展过程中起着重要作用.%AIM: To investigate correlation between the expression of survivin and caspase-3 proteins in juvenile laryngeal papilloma. METHODS: The expression of survivin and caspase-3 proteins were detected with immunohistochemial method in 43 cases of juvenile laryngeal papilloma, 25 vocal nodules and 25 normal laryngeal mucosa. RESULTS: The positive rates of survivin protein in juvenile laryngeal papilloma were 57.14% and higher than that in voeal nodules (P<0.01)and the normal laryngeal moeusa (P<0.01). And the Caspase-3 protein positive rate was 26.19% in juvenile laryngeal papilloma and lower than that in voeal nodules and the normal laryngeal mucosa (P<0.01).There was a significant negative correlation between the expression of survivin and caspase-3 in juvenile laryngeal papilloma. CONCLUSION: The abnormal expression of survivin and caspase-3 may play important role in the pathogenesis of juvenile laryngeal papilloma.

  12. Study on Caspase-3 activity on trichloroethylene-induced human keratinocyte apoptosis%三氯乙烯诱导人角质形成细胞凋亡中Caspase-3活力的研究

    Institute of Scientific and Technical Information of China (English)

    汪立杰; 叶良平; 沈彤; 朱启星

    2009-01-01

    目的 观察三氯乙烯(TCE)诱导离体培养的人角质形成细胞(KC)Caspase-3活力变化及细胞凋亡情况,探讨TCE诱导KC凋亡的可能信号通路.方法 以不同浓度(0.125、0.250、0.500、1.000、2.000 mmol/L)TCE对离体分离培养的KC分别染毒至4、8、12、24 h;Caspase-3抑制剂(Z-DEVD-FMK)预处理组,先用100 μmol/L Z-DEVD-FMK预处理细胞1 h,然后再用2.000 mmol/L TCE染毒12 h.用分光光度法检测细胞Caspase-3活力变化,借助Annexin-V/PI双染和流式细胞仪检测细胞凋亡情况.结果 与空白对照相比,TCE染毒4 h,各TCE剂量组Caspase-3活力无明显变化(P>0.05);染毒8 h,1.000 mmol/LTCE组Caspase-3活力和2.000 mmol/LTCE组Caspase-3活力,与对照组相比差异有显著性(P0.01).结论 在TCE诱导离体培养的KC凋亡中,Caspase-3的活化可能发挥了重要的作用.

  13. Caspase-3在roscovitine诱发PC12细胞凋亡中发挥重要作用%Caspase-3 plays a required role in PC12 cell apoptotic death induced by roscovitine

    Institute of Scientific and Technical Information of China (English)

    高建新; 周玉琴; 张茹华; 马雪莲; 刘克敬

    2005-01-01

    我们已证实周期蛋白激酶(cyclin-dependent kinases)cdk2、cdc2和cdk5抑制剂roscovitine诱导PC12细胞凋亡.本实验应用caspase-3免疫细胞化学与hoechst 33342荧光化学双标、MTT比色法细胞活性测定和Western blot方法,研究了caspase-3在roscovitine所致PC12细胞凋亡中的作用.结果显示,roscovitine(50μmol/L)处理PC12细胞12 h,细胞核染色质凝缩及核碎片形成,同时胞浆中出现caspase-3阳性标志,caspase-3阳性细胞占细胞总数的42%.非特异性caspases抑制剂Z-VAD-FMK(50 μmol/L)和caspase-3特异性抑制剂Z-DEVD-FMK(100 μmol/L)可部分降低roscovitine所致的细胞死亡,使细胞存活率分别由29.03%(roscovitine)增至58.06%(Z-VAD-FMK+roscovitine)和45.16%(Z-DEVD-FMK+roscovitine);用单克隆non-erythroid α-spectrin抗体检测roscovitine处理组细胞匀浆提取液,表明caspase-3裂解的特异性spectrin 120 kDa蛋白产物较对照组显著增加.提示细胞凋亡成分caspases参与roscovitine所致的细胞凋亡,其中caspase-3发挥重要作用.

  14. 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)

    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

  15. Negative regulation of caspase 3-cleaved PAK2 activity by protein phosphatase 1

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The p21-activated kinase 2 (PAK2) is activated by binding of small G proteins, Cdc42 and Rac, or through proteolytic cleavage by caspases or caspase-like proteases. Activation by both small G protein and caspase requires autophosphorylation at Thr-402 of PAK2. Although activation of PAK2 has been investigated for nearly a decade, the mechanism of PAK2 downregulation is unclear. In this study, we have applied the kinetic theory of substrate reaction during modification of enzyme activity to study the regulation mechanism of PAK2 activity by the catalytic subunit of protein phosphatase 1 (PP1α). On the basis of the kinetic equation of the substrate reaction during the reversible phosphorylation of PAK2, all microscopic kinetic constants for the free enzyme and enzyme-substrate(s) complexes have been determined. The results indicate that (1) PP1α can act directly on phosphorylated Thr-402 in the activation loop of PAK2 and down-regulate its kinase activity; (2) binding of the exogenous protein/peptide substrates at the active site of PAK2 decreases both the rates of PAK2 autoactivation and inactivation. The present method provides a novel approach for studying reversible phosphorylation reactions. The advantage of this method is not only its usefulness in study of substrate effects on enzyme modification but also its convenience in study of modification reaction directly involved in regulation of enzyme activity. This initial study should provide a foundation for future structural and mechanistic work of protein kinases and phosphatases.

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

    Directory of Open Access Journals (Sweden)

    R.L. Figueira

    2016-01-01

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

  17. Thymoquinone inhibits autophagy and induces cathepsin-mediated, caspase-independent cell death in glioblastoma cells.

    Directory of Open Access Journals (Sweden)

    Ira O Racoma

    Full Text Available Glioblastoma is the most aggressive and common type of malignant brain tumor in humans, with a median survival of 15 months. There is a great need for more therapies for the treatment of glioblastoma. Naturally occurring phytochemicals have received much scientific attention because many exhibit potent tumor killing action. Thymoquinone (TQ is the bioactive compound of the Nigella sativa seed oil. TQ has anti-oxidant, anti-inflammatory and anti-neoplastic actions with selective cytotoxicity for human cancer cells compared to normal cells. Here, we show that TQ selectively inhibits the clonogenicity of glioblastoma cells as compared to normal human astrocytes. Also, glioblastoma cell proliferation could be impaired by chloroquine, an autophagy inhibitor, suggesting that glioblastoma cells may be dependent on the autophagic pathway for survival. Exposure to TQ caused an increase in the recruitment and accumulation of the microtubule-associated protein light chain 3-II (LC3-II. TQ also caused an accumulation of the LC3-associated protein p62, confirming the inhibition of autophagy. Furthermore, the levels of Beclin-1 protein expression were unchanged, indicating that TQ interferes with a later stage of autophagy. Finally, treatment with TQ induces lysosome membrane permeabilization, as determined by a specific loss of red acridine orange staining. Lysosome membrane permeabilization resulted in a leakage of cathepsin B into the cytosol, which mediates caspase-independent cell death that can be prevented by pre-treatment with a cathepsin B inhibitor. TQ induced apoptosis, as determined by an increase in PI and Annexin V positive cells. However, apoptosis appears to be caspase-independent due to failure of the caspase inhibitor z-VAD-FMK to prevent cell death and absence of the typical apoptosis related signature DNA fragmentation. Inhibition of autophagy is an exciting and emerging strategy in cancer therapy. In this vein, our results describe a

  18. Negative regulation of caspase 3-cleaved PAK2 activity by protein phosphatase 1

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The p21-activated kinase 2 (PAK2) is activated by binding of small G proteins, Cdc42 and Rac, or through proteolytic cleavage by caspases or caspase-like proteases. Activation by both small G protein and caspase requires autophosphorylation at Thr-402 of PAK2. Although activation of PAK2 has been investigated for nearly a decade, the mechanism of PAK2 downregulation is unclear. In this study, we have applied the kinetic theory of substrate reaction during modification of enzyme activity to study the regulation mechanism of PAK2 activity by the catalytic subunit of protein phosphatase 1 (PP1α). On the basis of the kinetic equation of the substrate reaction during the reversible phosphorylation of PAK2, all microscopic kinetic constants for the free enzyme and enzyme-substrate(s) complexes have been determined. The results indicate that (1) PP1α can act directly on phosphorylated Thr-402 in the acti-vation loop of PAK2 and down-regulate its kinase activity; (2) binding of the exogenous protein/peptide substrates at the active site of PAK2 decreases both the rates of PAK2 autoactivation and inactivation. The present method provides a novel approach for studying reversible phosphorylation reactions. The advantage of this method is not only its usefulness in study of substrate effects on enzyme modifica-tion but also its convenience in study of modification reaction directly involved in regulation of enzyme activity. This initial study should provide a foundation for future structural and mechanistic work of protein kinases and phosphatases.

  19. Molecular cloning and characterization of four caspases members in Apostichopus japonicus.

    Science.gov (United States)

    Shao, Yina; Li, Chenghua; Zhang, Weiwei; Duan, Xuemei; Li, Ye; Jin, Chunhua; Xiong, Jinbo; Qiu, Qiongfen

    2016-08-01

    The caspase family representing aspartate-specific cysteine proteases have been demonstrated to possess key roles in apoptosis and immune response. We previously demonstrated that LPS challenged Apostichopus japonicus coelomocyte could significantly induced apoptosis in vitro. However, apoptosis related molecules were scarcely investigated in this economic species. In the present work, we cloned and characterized four members caspase family from A. japonicus (designated as Ajcaspase-2, Ajcaspase-3, Ajcaspase-6, and Ajcaspase-8, respectively) by RACE. Multiple sequence alignment and structural analysis revealed that all Ajcaspases contained the conservative CASC domain at C terminal, in which some unique features for each Ajcaspase made them different from each other. These specific domains together with phylogenetic analysis supported that all these four identified proteins belonged to novel members of apoptotic signaling pathway in sea cucumber. Tissue distribution analysis revealed that four Ajcaspase genes were constitutively expressed in all examined tissues. The expression of Ajcaspase-2 was tightly correlated with that of Ajcaspase-8 in each detected tissues. Ajcaspase-3 and Ajcaspase-6 transcripts were both highly expressed in immune tissue of coelomocytes. Furthermore, the Vibrio splendidus challenged sea cucumber coelomocytes could significantly up-regulate the mRNA expressions of four genes. The expression levels of Ajcaspase-2 and Ajcaspase-8 were relative earlier than those of Ajcaspase-6 and Ajcaspase-3, respectively, which could be inferred that Ajcapase-2 might directly modulate Ajcaspase-6, and Ajcaspase-8 initiate the expression of Ajcaspase-3. The induce expressions differed among each Ajcaspase depending upon their roles such as initiator or effector caspase. All our results demonstrated that four Ajcaspases present diversified functions in apoptotic cascade signaling pathway of sea cucumber under immune response. PMID:27245866

  20. Caspase-8 regulates TNF-α-induced epithelial necroptosis and terminal ileitis.

    Science.gov (United States)

    Günther, Claudia; Martini, Eva; Wittkopf, Nadine; Amann, Kerstin; Weigmann, Benno; Neumann, Helmut; Waldner, Maximilian J; Hedrick, Stephen M; Tenzer, Stefan; Neurath, Markus F; Becker, Christoph

    2011-09-15

    Dysfunction of the intestinal epithelium is believed to result in the excessive translocation of commensal bacteria into the bowel wall that drives chronic mucosal inflammation in Crohn's disease, an incurable inflammatory bowel disease in humans characterized by inflammation of the terminal ileum. In healthy individuals, the intestinal epithelium maintains a physical barrier, established by the tight contact of cells. Moreover, specialized epithelial cells such as Paneth cells and goblet cells provide innate immune defence functions by secreting mucus and antimicrobial peptides, which hamper access and survival of bacteria adjacent to the epithelium. Epithelial cell death is a hallmark of intestinal inflammation and has been discussed as a possible pathogenic mechanism driving Crohn's disease in humans. However, the regulation of epithelial cell death and its role in intestinal homeostasis remain poorly understood. Here we demonstrate a critical role for caspase-8 in regulating necroptosis of intestinal epithelial cells (IECs) and terminal ileitis. Mice with a conditional deletion of caspase-8 in the intestinal epithelium (Casp8(ΔIEC)) spontaneously developed inflammatory lesions in the terminal ileum and were highly susceptible to colitis. Casp8(ΔIEC) mice lacked Paneth cells and showed reduced numbers of goblet cells, indicating dysregulated antimicrobial immune cell functions of the intestinal epithelium. Casp8(ΔIEC) mice showed increased cell death in the Paneth cell area of small intestinal crypts. Epithelial cell death was induced by tumour necrosis factor (TNF)-α, was associated with increased expression of receptor-interacting protein 3 (Rip3; also known as Ripk3) and could be inhibited on blockade of necroptosis. Lastly, we identified high levels of RIP3 in human Paneth cells and increased necroptosis in the terminal ileum of patients with Crohn's disease, suggesting a potential role of necroptosis in the pathogenesis of this disease. Together, our

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

    Czech Academy of Sciences Publication Activity Database

    Lišková, Marcela; Křenková, Jana; Klepárník, Karel; Pazdera, P.; Foret, František

    Brno: Ústav analytické chemie AV ČR, v. v. i, 2015 - (Foret, F.; Křenková, J.; Drobníková, I.; Klepárník, K.), s. 137-140 ISBN 978-80-904959-3-7. [CECE 2015. International Interdisciplinary Meeting on Bioanalysis /12./. Brno (CZ), 21.09.2015-23.09.2015] R&D Projects: GA ČR(CZ) GA14-28254S Institutional support: RVO:68081715 Keywords : caspases * click chemistry * cell analysis Subject RIV: CB - Analytical Chemistry, Separation http://www.ce-ce.org/CECE2015/CECE%202015%20proceedings_full.pdf

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

    Czech Academy of Sciences Publication Activity Database

    Lišková, Marcela; Křenková, Jana; Klepárník, Karel; Pazdera, P.; Foret, František

    Brno : Ústav analytické chemie AV ČR, v. v. i, 2015 - (Foret, F.; Křenková, J.; Drobníková, I.; Klepárník, K.), s. 137-140 ISBN 978-80-904959-3-7. [CECE 2015. International Interdisciplinary Meeting on Bioanalysis /12./. Brno (CZ), 21.09.2015-23.09.2015] R&D Projects: GA ČR(CZ) GA14-28254S Institutional support: RVO:68081715 Keywords : caspases * click chemistry * cell analysis Subject RIV: CB - Analytical Chemistry, Separation http://www.ce-ce.org/CECE2015/CECE%202015%20proceedings_full.pdf

  3. Changes in the expression of caspase-12 after acute myocardial infarction in rats

    OpenAIRE

    LI, RUI-JUN; HE, KUN-LUN; Xin Li; Liu, Chun-Lei; Wang, Li-li

    2011-01-01

    Objective To observe the expression of caspase-12 in rat acute myocardial infarction model,and explore the role of endoplasmic reticulum stress in cardiomyocyte apoptosis after myocardial infarction.Methods A total of 80 male Wistar rats were randomly divided into sham operation group(S group) and myocardial infarction group(MI group).Animals in each group were then randomly reassigned into 1h,6h,12h and 24h subgroups according to the observation time(10 rats for each subgroup).Models of myoc...

  4. Imaging of caspase-3 activation by a novel FRET probe composed of CFP and DsRed

    Science.gov (United States)

    Lin, Juquiang; Zhang, Zhihong; Liu, Bifeng; Luo, Qingming

    2006-01-01

    Caspases-3 is a kind of cysteine proteases and plays an important role in cell apoptosis. It has been reported that caspase-3 activation can be real-time detected in living cells by fluorescence resonance energy transfer (FRET) between an enhanced cyan fluorescent protein and enhanced yellow fluorescent protein. However, the large spectral overlap between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) emission and the highly sensitivity to pH of YFP restricted their detecting sensitivity and reliability. CFP and red fluorescent protein (DsRed) possess superb wavelength separation of donor and acceptor emission spectra and DsRed was insensitive to pH, so the FRET probe composed of CFP and DsRed would be more suitable for imaging caspase-3 activation than the FRET probe composed of CFP and YFP. We constructed a vector that encoded CRS (caspase-3 recognition site) fused with CFP and DsRed (CFP-CRS-DsRed). In CFP-CRS-DsRed expressing tumor cells, FRET from CFP to DsRed could be detected. In the Clinical applications of cancer chemotherapy, cisplatin is one of the most broadly used drugs. It was already confirmed that caspase-3 was activated in HeLa cell treated by cisplatin. When the cells were stimulated with cisplatin, we found that the FRET efficient was remarkably decreased and then disappeared. It indicated that actived caspase-3 cleaved the CFP-CRS-DsRed fusion protein at CRS site. Thus, the FRET probe of CFP-CRS-DsRed could sensitively and reliably monitor caspase-3 activation in living cell. This probe will be highly useful for rapid-screening potential drugs that may target the apoptotic process and for imaging tumors in vivo.

  5. Silencing of Pokemon enhances caspase-dependent apoptosis via fas- and mitochondria-mediated pathways in hepatocellular carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Yu-Qin Zhang

    Full Text Available The role of Pokemon (POK erythroid myeloid ontogenic actor, a recently identified POK transcription factor with proto-oncogenic activity, in hepatocellular carcinogenesis has only been assessed by a few studies. Our previous study revealed that Pokemon is overexpressed in hepatocellular carcinomas (HCC and promotes HCC cell proliferation and migration via an AKT- and ERK- dependent manner. In the present study, we used the TUNEL assay and FACS analysis to demonstrate that oxaliplatin induced apoptosis was significantly increased in cells with silenced Pokemon. Western blots showed that p53 expression and phosphorylation were significantly increased in Pokemon defective cells, thereby initiating the mitochondria-mediated and death receptor-mediated apoptotic pathways. In the mitochondria-mediated pathway, expression of pro-apoptotic Bcl-2 family members (including Bad, Bid, Bim and Puma as well as AIF was increased and decreasing the mitochondrial membrane potential resulted in cytochrome C released from mitochondrial in HepG2 si-Pokemon cells. In addition, upon oxaliplatin treatment of Pokemon-silenced cells, the FAS receptor, FADD and their downstream targets caspase-10 and caspase-8 were activated, causing increased release of caspase-8 active fragments p18 and p10. Increased activated caspase-8-mediated cleavage and activation of downstream effector caspases such as caspase-9 and caspase-3 was observed in HepG2 si-Pokemon cells as compared to control. Therefore, Pokemon might serve as an important mediator of crosstalk between intrinsic and extrinsic apoptotic pathways in HCC cells. Moreover, our findings suggest that Pokemon could be an attractive therapeutic target gene for human cancer therapy.

  6. Silencing of Pokemon enhances caspase-dependent apoptosis via fas- and mitochondria-mediated pathways in hepatocellular carcinoma cells.

    Science.gov (United States)

    Zhang, Yu-Qin; Xiao, Chuan-Xing; Lin, Bi-Yun; Shi, Ying; Liu, Yun-Peng; Liu, Jing-Jing; Guleng, Bayasi; Ren, Jian-Lin

    2013-01-01

    The role of Pokemon (POK erythroid myeloid ontogenic actor), a recently identified POK transcription factor with proto-oncogenic activity, in hepatocellular carcinogenesis has only been assessed by a few studies. Our previous study revealed that Pokemon is overexpressed in hepatocellular carcinomas (HCC) and promotes HCC cell proliferation and migration via an AKT- and ERK- dependent manner. In the present study, we used the TUNEL assay and FACS analysis to demonstrate that oxaliplatin induced apoptosis was significantly increased in cells with silenced Pokemon. Western blots showed that p53 expression and phosphorylation were significantly increased in Pokemon defective cells, thereby initiating the mitochondria-mediated and death receptor-mediated apoptotic pathways. In the mitochondria-mediated pathway, expression of pro-apoptotic Bcl-2 family members (including Bad, Bid, Bim and Puma) as well as AIF was increased and decreasing the mitochondrial membrane potential resulted in cytochrome C released from mitochondrial in HepG2 si-Pokemon cells. In addition, upon oxaliplatin treatment of Pokemon-silenced cells, the FAS receptor, FADD and their downstream targets caspase-10 and caspase-8 were activated, causing increased release of caspase-8 active fragments p18 and p10. Increased activated caspase-8-mediated cleavage and activation of downstream effector caspases such as caspase-9 and caspase-3 was observed in HepG2 si-Pokemon cells as compared to control. Therefore, Pokemon might serve as an important mediator of crosstalk between intrinsic and extrinsic apoptotic pathways in HCC cells. Moreover, our findings suggest that Pokemon could be an attractive therapeutic target gene for human cancer therapy. PMID:23874836

  7. Cleavage of Armadillo/beta-catenin by the caspase DrICE in Drosophila apoptotic epithelial cells

    Directory of Open Access Journals (Sweden)

    Kessler Thomas

    2009-02-01

    Full Text Available Abstract Background During apoptosis cells become profoundly restructured through concerted cleavage of cellular proteins by caspases. In epithelial tissues, apoptotic cells loose their apical/basal polarity and are extruded from the epithelium. We used the Drosophila embryo as a system to investigate the regulation of components of the zonula adherens during apoptosis. Since Armadillo/beta-catenin (Arm is a major regulator of cadherin-mediated adhesion, we analyzed the mechanisms of Arm proteolysis in apoptosis. Results We define early and late apoptotic stages and find that early in apoptosis Dα-catenin remains relatively stable, while Arm and DE-cadherin protein levels are strongly reduced. Arm is cleaved by caspases in embryo extracts and we provide evidence that the caspase-3 homolog drICE cleaves Arm in vitro and in vivo. Cleavage by drICE creates a stable protein fragment that remains associated with the plasma membrane early in apoptosis. To further understand the role of caspase-mediated cleavage of Arm, we examined potential caspase cleavage sites and found that drICE cleaves Arm at a unique DQVD motif in the N-terminal domain of the protein. Mutation of the drICE cleavage site in Arm results in a protein that is not cleaved in vitro and in vivo. Furthermore we provide evidence that cleavage of Arm plays a role in the removal of DE-cadherin from the plasma membrane during apoptosis. Conclusion This study defines the specificity of caspase cleavage of Arm in Drosophila apoptotic cells. Our data suggest that N-terminal truncation of Arm by caspases is evolutionarily conserved and thus might provide a principal mechanism involved in the disassembly of adherens junctions during apoptosis.

  8. Caspase-1 is involved in the genesis of inflammatory hypernociception by contributing to peripheral IL-1β maturation

    Directory of Open Access Journals (Sweden)

    Zamboni Dario S

    2010-10-01

    Full Text Available Abstract Background Caspase-1 is a cysteine protease responsible for the processing and secretion of IL-1β and IL-18, which are closely related to the induction of inflammation. However, limited evidence addresses the participation of caspase-1 in inflammatory pain. Here, we investigated the role of caspase-1 in inflammatory hypernociception (a decrease in the nociceptive threshold using caspase-1 deficient mice (casp1-/-. Results Mechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. The production of cytokines, PGE2 and neutrophil migration were evaluated by ELISA, radioimmunoassay and myeloperoxidase activity, respectively. The interleukin (IL-1β and cyclooxygenase (COX-2 protein expression were evaluated by western blotting. The mechanical hypernociception induced by intraplantar injection of carrageenin, tumour necrosis factor (TNFα and CXCL1/KC was reduced in casp1-/- mice compared with WT mice. However, the hypernociception induced by IL-1β and PGE2 did not differ in WT and casp1-/- mice. Carrageenin-induced TNF-α and CXCL1/KC production and neutrophil recruitment in the paws of WT mice were not different from casp1-/- mice, while the maturation of IL-1β was reduced in casp1-/- mice. Furthermore, carrageenin induced an increase in the expression of COX-2 and PGE2 production in the paw of WT mice, but was reduced in casp1-/- mice. Conclusion These results suggest that caspase-1 plays a critical role in the cascade of events involved in the genesis of inflammatory hypernociception by promoting IL-1β maturation. Because caspase-1 is involved in the induction of COX-2 expression and PGE2 production, our data support the assertion that caspase-1 is a key target to control inflammatory pain.

  9. Wild-type and mutant p53 mediate cisplatin resistance through interaction and inhibition of active caspase-9.

    Science.gov (United States)

    Chee, Jacqueline L Y; Saidin, Suzan; Lane, David P; Leong, Sai Mun; Noll, Jacqueline E; Neilsen, Paul M; Phua, Yi Ting; Gabra, Hani; Lim, Tit Meng

    2013-01-15

    The p53 gene has been implicated in many cancers due to its frequent mutations as well as mutations in other genes whose proteins directly affect p53's functions. In addition, high expression of p53 [wild-type (WT) or mutant] has been found in the cytoplasm of many tumor cells, and studies have associated these observations with more aggressive tumors and poor prognosis. Cytoplasmic mis-localization of p53 subsequently reduced its transcriptional activity and this loss-of-function (LOF) was used to explain the lack of response to chemotherapeutic agents. However, this hypothesis seemed inadequate in explaining the apparent selection for tumor cells with high levels of p53 protein, a phenomenon that suggests a gain-of-function (GOF) of these mis-localized p53 proteins. In this study, we explored whether the direct involvement of p53 in the apoptotic response is via regulation of the caspase pathway in the cytoplasm. We demonstrate that p53, when present at high levels in the cytoplasm, has an inhibitory effect on caspase-9. Concurrently, knockdown of endogenous p53 caused an increase in the activity of caspase-9. p53 was found to interact with the p35 fragment of caspase-9, and this interaction inhibits the caspase-9 activity. In a p53-null background, the high-level expression of both exogenous WT and mutant p53 increased the resistance of these cells to cisplatin, and the data showed a correlation between high p53 expression and caspase-9 inhibition. These results suggest the inhibition of caspase-9 as a potential mechanism in evading apoptosis in tumors with high-level p53 expression that is cytoplasmically localized. PMID:23255126

  10. Compound K, a metabolite of ginseng saponin, induces apoptosis via caspase-8-dependent pathway in HL-60 human leukemia cells

    International Nuclear Information System (INIS)

    Compound K [20-O-β-(D-glucopyranosyl)-20(S)-protopanaxadiol], a metabolite of the protopanaxadiol-type saponins of Panax ginseng C.A. Meyer, has been reported to possess anti-tumor properties to inhibit angiogenesis and to induce tumor apoptosis. In the present study, we investigated the effect of Compound K on apoptosis and explored the underlying mechanisms involved in HL-60 human leukemia cells. We examined the effect of Compound K on the viabilities of various cancer cell lines using MTT assays. DAPI assay, Annexin V and PI double staining, Western blot assay and immunoprecipitation were used to determine the effect of Compound K on the induction of apoptosis. Compound K was found to inhibit the viability of HL-60 cells in a dose- and time-dependent manner with an IC50 of 14 μM. Moreover, this cell death had typical features of apoptosis, that is, DNA fragmentation, DNA ladder formation, and the externalization of Annexin V targeted phosphatidylserine residues in HL-60 cells. In addition, compound-K induced a series of intracellular events associated with both the mitochondrial- and death receptor-dependent apoptotic pathways, namely, (1) the activation of caspases-3, -8, and -9; (2) the loss of mitochondrial membrane potential; (3) the release of cytochrome c and Smac/DIABLO to the cytosol; (4) the translocation of Bid and Bax to mitochondria; and (5) the downregulations of Bcl-2 and Bcl-xL. Furthermore, a caspase-8 inhibitor completely abolished caspase-3 activation, Bid cleavage, and subsequent DNA fragmentation by Compound K. Interestingly, the activation of caspase-3 and -8 and DNA fragmentation were significantly prevented in the presence of cycloheximide, suggesting that Compound K-induced apoptosis is dependent on de novo protein synthesis. The results indicate that caspase-8 plays a key role in Compound K-stimulated apoptosis via the activation of caspase-3 directly or indirectly through Bid cleavage, cytochrome c release, and caspase-9 activation

  11. 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.

  12. Increased expression of stefin B in the nucleus of T98G astrocytoma cells delays caspase activation

    Directory of Open Access Journals (Sweden)

    Tao eSun

    2012-09-01

    Full Text Available Stefin B (cystatin B is an endogenous inhibitor of cysteine proteinases localized in the nucleus and the cytosol. Loss-of-function mutations in the stefin B gene (CSTB gene were reported in patients with Unverricht-Lundborg disease (EPM1. Our previous results showed that thymocytes isolated from stefin B-deficient mice are more sensitive to apoptosis induced by the protein kinase C inhibitor staurosporin (STS than the wild-type control cells. We have also shown that the increased expression of stefin B in the nucleus of T98G astrocytoma cells delayed cell cycle progression through the S phase. In the present study we examined if the nuclear or cytosolic functions of stefin B are responsible for the accelerated induction of apoptosis observed in the cells from stefin B-deficient mice. We have shown that the overexpression of stefin B in the nucleus, but not in the cytosol of astrocytoma T98G cells, delayed caspase-3 and-7 activation. Pretreatment of cells with the pan-caspase inhibitor z-Val-Ala-Asp(OMe-fluoromethylketone completely inhibited caspase activation, while treatment with the inhibitor of calpains- and papain-like cathepsins (2S,3S-trans-epoxysuccinyl-leucylamido-3-methyl-butane ethyl ester did not prevent caspase activation. We concluded that the delay of caspase activation in T98G cells overexpressing stefin B in the nucleus is independent of cathepsin inhibition.

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

    Science.gov (United States)

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

    2009-07-01

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

  14. Effects of camptothecin, etoposide and Ca2+ on caspase-3 activity and myofibrillar disruption of chicken during postmortem ageing.

    Science.gov (United States)

    Chen, Lin; Feng, Xian Chao; Lu, Feng; Xu, Xing Lian; Zhou, Guang Hong; Li, Qing Yun; Guo, Xiang Ying

    2011-03-01

    Recently, a novel consideration has focused on the potential relationship of apoptosis and the protease caspases and the underlying mechanism for meat postmortem tenderization. In this study, apoptosis inducers, camptothecin and etoposide as well as Ca(2+) were used to treat chicken muscle immediately after slaughter and follow the changes in caspase-3 activities and changes in the myofibrillar structures during 7 days of ageing. All three treatments resulted in significantly higher caspase-3 activities during storage (p<0.05), with the natural substrates, whereas Western blotting analysis of the α-spectrin cleavage product, 120 kDa peptide (SBDP 120), showed that Ca(2+) was more effective than either camptothecin or etopside, and all were most active up to day 3 (p<0.01). According to SDS-PAGE, each treatment enhanced the accumulation of the 30 kD Troponin-T degradation product, especially during the first 3 days (p<0.05), and this was supported by the degradation of myofibrils observed by electron microscopy (TEM). TEM images showed the treatments resulted in enlargement of the I-bands and shrinkage of A-bands; however Z-lines were only slightly affected, even at day 7. The findings revealed that the three apoptosis inducers could increase myofibrillar dissociation and proteolysis during the first 3 days of chicken meat ageing. Because of the high activity of caspase-3 during the early postmortem period, it is possible that caspase-3 contributes to the conversion of muscle into meat. PMID:21055882

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

    Directory of Open Access Journals (Sweden)

    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.

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

    International Nuclear Information System (INIS)

    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 (H2O2) 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.

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

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    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.

  18. Involvement of mitochondria in acetaminophen-induced apoptosis and hepatic injury Roles of cytochrome c, Bax, Bid, and caspases

    International Nuclear Information System (INIS)

    The role of apoptosis in acetaminophen (AAP)-induced hepatic injury was investigated. Six hours after AAP administration to BALB/c mice, a significant loss of hepatic mitochondrial cytochrome c was observed that was similar in extent to the loss observed after in vivo activation of CD95 by antibody treatment. AAP-induced loss of mitochondrial cytochrome c coincided with the appearance in the cytosol of a fragment corresponding to truncated Bid (tBid). At the same time, tBid became detectable in the mitochondrial fraction, and concomitantly, Bax was found translocated to mitochondria. However, AAP failed to activate the execution caspases 3 and 7 as evidenced by a lack of procaspase processing and the absence of an increase in caspase-3-like activity. In contrast, the administration of the pan-inhibitor of caspases, benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (but not its analogue benzyloxycarbonyl-Phe-Ala-fluoromethylketone) prevented the development of liver injury by AAP and the appearance of apoptotic parenchymal cells. This correlated with the inhibition of the processing of Bid to tBid. The caspase inhibitor failed to prevent both the redistribution of Bax to the mitochondria and the loss of cytochrome c. In conclusion, apoptosis is an important causal event in the initiation of the hepatic injury inflicted by AAP. However, as suggested by the lack of activation of the main execution caspases, apoptosis is not properly executed and degenerates into necrosis

  19. The effector caspases drICE and dcp-1 have partially overlapping functions in the apoptotic pathway in Drosophila.

    Science.gov (United States)

    Xu, D; Wang, Y; Willecke, R; Chen, Z; Ding, T; Bergmann, A

    2006-10-01

    Caspases are essential components of the apoptotic machinery in both vertebrates and invertebrates. Here, we report the isolation of a mutant allele of the Drosophila effector caspase drICE as a strong suppressor of hid- (head involution defective-) induced apoptosis. This mutant was used to determine the apoptotic role of drICE. Our data are consistent with an important function of drICE for developmental and irradiation-induced cell death. Epistatic analysis suggests that drICE acts genetically downstream of Drosophila inhibitor of apoptosis protein 1 (Diap1). However, although cell death is significantly reduced in drICE mutants in all assays, it is not completely blocked. A double-mutant analysis between drICE and death caspase-1 (dcp-1), another effector caspase, reveals that some cells (type I) strictly require drICE for apoptosis, whereas other cells (type II) require either drICE or dcp-1. Thus, these data demonstrate a barely appreciated complexity in the apoptotic pathway, and are consistent with current models about effector caspase regulation in both vertebrates and invertebrates. PMID:16645642

  20. BRCA1 Zinc RING Finger Domain Disruption Alters Caspase Response in Ovarian Surface Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Kruk Patricia A

    2002-07-01

    Full Text Available Abstract Background The frequently occurring 185delAG mutation occurs in the amino-terminal zinc RING domain of the breast and ovarian cancer susceptibility gene, BRCA1. We sought to determine differential cell viability and apoptotic response of human ovarian surface epithelial cells with and without the 185delAG mutation. Results BRCA1wt and BRCA1+ cells were treated with staurosporine. Cell proliferation assays showed BRCA1wt cells grew to a greater extent compared to BRCA1+ cells. Trypan blue exclusion assays confirmed this observation. Western immunoblot analysis revealed that caspase 3 levels were higher after staurosporine treatment in BRCA1+ cells than in wild type cells, while full length DNA Fragmentation Factor 45 levels were lower in BRCA1+ cells. While there was no significant difference in levels of excision repair cross complementing protein1 (ERCC1 with BRCA1 status, BRCA1+ cells demonstrated cleavage of polyribose ADP polymerase (PARP before wild type cells. Conclusions Disruption of the BRCA1 RING domain caused altered cell viability and caspase-dependent apoptotic response after chemotoxic stress.

  1. Lacidipine Attenuates Apoptosis via a Caspase-3 Dependent Pathway in Human Kidney Cells

    Directory of Open Access Journals (Sweden)

    Aiqi Zhang

    2013-10-01

    Full Text Available Background: Acute kidney injury (AKI is common in hospitalised patients and has a poor prognosis. Therefore, new therapeutic strategies are anticipated. Lacidipine, a novel third-generation dihydropyridine calcium channel blocker, has been demonstrated effective for hypertension. However, its potential effect on renal injury remains unknown. In the present study, an in vitro model of renal ischemia reperfusion (I/R injury was used to investigate the protective effect and underlying mechanisms of lacidipine on human kidney cell (HKC apoptosis. Methods: HKCs were subjected to adenosine triphosphate (ATP depletion and recovery (0.01 µM AA, depletion for 2 h and recovery for 30 min, with or without lacidipine (1 µM and 10 µM, 24 h, then cell viability and apoptosis were determined using the cell counting kit-8 (CCK-8 assay and Annexin V flow cytometry. The expression of Bcl-2, Bax, and cytochrome c (cyt c was examined by western blot. Results: Antimycin A (AA was found to induce apoptosis of HKCs. The proportion of early apoptosis and activity of caspase-3 peaked at 30 min after ATP depletion and recovery and were attenuated by lacidipine. The expression of cyt c and Bax was decreased, while that of Bcl-2 was increased significantly in lacidipine treated group. Conclusion: We conclude that lacidipine protects HKCs against apoptosis induced by ATP depletion and recovery by regulating the caspase-3 pathway.

  2. Comparative genomics reveals conservation of filaggrin and loss of caspase-14 in dolphins.

    Science.gov (United States)

    Strasser, Bettina; Mlitz, Veronika; Fischer, Heinz; Tschachler, Erwin; Eckhart, Leopold

    2015-05-01

    The expression of filaggrin and its stepwise proteolytic degradation are critical events in the terminal differentiation of epidermal keratinocytes and in the formation of the skin barrier to the environment. Here, we investigated whether the evolutionary transition from a terrestrial to a fully aquatic lifestyle of cetaceans, that is dolphins and whales, has been associated with changes in genes encoding filaggrin and proteins involved in the processing of filaggrin. We used comparative genomics, PCRs and re-sequencing of gene segments to screen for the presence and integrity of genes coding for filaggrin and proteases implicated in the maturation of (pro)filaggrin. Filaggrin has been conserved in dolphins (bottlenose dolphin, orca and baiji) but has been lost in whales (sperm whale and minke whale). All other S100 fused-type genes have been lost in cetaceans. Among filaggrin-processing proteases, aspartic peptidase retroviral-like 1 (ASPRV1), also known as saspase, has been conserved, whereas caspase-14 has been lost in all cetaceans investigated. In conclusion, our results suggest that filaggrin is dispensable for the acquisition of fully aquatic lifestyles of whales, whereas it appears to confer an evolutionary advantage to dolphins. The discordant evolution of filaggrin, saspase and caspase-14 in cetaceans indicates that the biological roles of these proteins are not strictly interdependent. PMID:25739514

  3. 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.

  4. Propofol inhibits caspase-3 in astroglial cells: role of heme oxygenase-1.

    Science.gov (United States)

    Acquaviva, Rosaria; Campisi, Agata; Raciti, Giuseppina; Avola, Roberto; Barcellona, Maria Luisa; Vanella, Luca; Li Volti, Giovanni

    2005-04-01

    Several lines of evidence have extensively demonstrated that peroxynitrite plays a pivotal role in Central Nervous System (CNS) injuries. The present study was aimed at elucidating the molecular mechanism by which propofol attenuates peroxynitrite-mediated injury in the brain. Primary cultured astroglial cells were incubated for 18 h with a known peroxynitrite donor (SIN-1,3 mM) in the presence or absence of propofol (40 microM, 80 microM and 160 microM). The protective effects of propofol were evaluated by MTT cytotoxicity assay, LDH release, and caspase-3 activation by Western blot analysis. Appropriate propofol concentrations (ranging from 40 microM to 160 microM) significantly increased HO-1 expression and attenuated SIN-1-mediated cytotoxicity and caspase-3 activation. The protective effects of propofol were mitigated by the addition of tin-mesoporphirin (SnMP), a potent inhibitor of HO activity. The addition of a specific synthetic inhibitor of NF-kappaB abolished propofol-mediated HO-1 induction, suggesting a possible role for this nuclear transcriptional factor in our experimental conditions. These findings indicate that propofol attenuates peroxynitrite-mediated apoptosis in astroglial cells, a property that may be relevant in both physiological and pathological processes in the CNS. PMID:16181106

  5. Aspirin inhibits growth of ovarian cancer by upregulating caspase-3 and downregulating bcl-2

    Science.gov (United States)

    LI, LIN; MAO, XIAOGANG; QIN, XIAOMIN; ZHOU, MIN; XING, HUI; DONG, FAN; JIANG, XIAOYUAN; ZHUANG, WENHUI

    2016-01-01

    The aim of the present study was to investigate the effect and mechanism of different concentrations of aspirin in inhibiting the ovarian cancer of p53N236S gene knock-in mice. In total, 28 male p53S mice, with an age range of 4–6 weeks and weight of 20–25 g were selected. The animals were transplanted with SKOV3 cells to establish subdermal human ovarian cancer. The mice were randomly divided into different groups according to the aspirin concentrations (mmol/l) used, i.e., 0, 1, 2 and 3. Subsequently, intraperitoneal injection was performed once every two days for 3 weeks. The tumor volume, lifetime, tumor cell proliferation inhibition rates, caspase-3 protein and bcl-2 protein expression of the four groups were analyzed and compared. Following aspirin treatment for 1, 2 and 3 weeks, the tumor volume of the 3 mmol/l aspirin group was significantly smaller than the other groups (Povarian cancer of p53S rats due to its upregulation of the expression of caspase-3 protein and downregulation of the expression of bcl-2 protein. PMID:27347106

  6. NSD1 mitigates caspase-1 activation by listeriolysin O in macrophages.

    Directory of Open Access Journals (Sweden)

    Olivia S Sakhon

    Full Text Available Mammals and plants share pathogen-sensing systems named nod-like receptors (NLRs. Some NLRs form the inflammasome, a protein scaffold that regulates the secretion of interleukin (IL-1β and IL-18 by cleaving catalytically inactive substrates into mature cytokines. Here, we show an immune conservation between plant and mammalian NLRs and demonstrate that the murine nuclear receptor binding SET domain protein 1 (NSD1, a protein that bears similarity to the NLR regulator enhanced downy mildew 2 (EDM2 in Arabidopsis, diminishes caspase-1 activity during extracellular stimulation with Listeria monocytogenes listeriolysin O (LLO. EDM2 is known to regulate plant developmental processes, whereas NSD1 is associated with developmental disorders. We observed that NSD1 neither affects nuclear factor (NF-κB signaling nor regulates NLRP3 inflammasome gene expression at the chromatin, transcriptional or translational level during LLO stimulation of macrophages. Silencing of Nsd1 followed by LLO stimulation led to increased caspase-1 activation, enhanced post-translational maturation of IL-1β and IL-18 and elevated pyroptosis, a form of cell death associated with inflammation. Furthermore, treatment of macrophages with LLO(W492A, which lacks hemolytic activity due to a tryptophan to alanine substitution in the undecapeptide motif, indicates the importance of functional LLO for NSD1 regulation of the NLRP3 inflammasome. Taken together, our results indicate that NLR signaling in plants may be used for gene discovery in mammals.

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

    Science.gov (United States)

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

    2016-01-01

    Neonatal asphyxia can cause irreversible injury of multiple organs resulting in hypoxic-ischemic encephalopathy and necrotizing enterocolitis (NEC). This injury is dependent on time, severity, and gestational age, once the preterm babies need ventilator support. Our aim was to assess the different brain and intestinal effects of ischemia and reperfusion in neonate rats after birth anoxia and mechanical ventilation. Preterm and term neonates were divided into 8 subgroups (n=12/group): 1) preterm control (PTC), 2) preterm ventilated (PTV), 3) preterm asphyxiated (PTA), 4) preterm asphyxiated and ventilated (PTAV), 5) term control (TC), 6) term ventilated (TV), 7) term asphyxiated (TA), and 8) term asphyxiated and ventilated (TAV). We measured body, brain, and intestine weights and respective ratios [(BW), (BrW), (IW), (BrW/BW) and (IW/BW)]. Histology analysis and damage grading were performed in the brain (cortex/hippocampus) and intestine (jejunum/ileum) tissues, as well as immunohistochemistry analysis for caspase-3 and intestinal fatty acid-binding protein (I-FABP). IW was lower in the TA than in the other terms (Pmechanical ventilation, varied with gestational age, with increased expression of caspase-3 and I-FABP biomarkers. PMID:27356106

  8. 脐血中 Caspase7蛋白在羊水量异常产妇中的表达%Expression of Caspase7 in umbilical serum of pregnant women with abnormal amniotic fluid

    Institute of Scientific and Technical Information of China (English)

    李小琴; 白彩云

    2016-01-01

    Objective To explore the difference in expression of Caspase 7 in umbilical serum between pregnant women with abnormal amniotic fluid and normal pregnant women .Methods ELISA was performed to determine the concentration of Caspase 7 protein in umbilical serum of 30 cases of oligohydramnios , 30 cases of polyhydramnios and 30 normal cases .Results Caspase7 was positively expressed in umbilical serum of three groups.The expression of Caspase7 was higher in the oligohydramnios group (160.2 ±25.8 ng/mL) than in the normal group (142.9 ±18.4 ng/mL), and there was significant difference (t=2.99,P<0.05).The expression of Caspase7 was lower in the polyhydramnios group (119.2 ±21.5 ng/mL) than in the normal group (142.9 ±18.4 ng/mL) with significant difference (t=4.59, P<0.05 ) .Conclusion The expression of Caspase 7 in umbilical serum of pregnant women of oligohydramnios and polyhydramnios is abnormal, which indicates that it might be correlated with abnormal amniotic fluid .%目的:探讨Caspase7蛋白在羊水量异常和正常的产妇血清中的表达差异。方法采用酶联免疫吸附法( ELISA)分别检测30例羊水过少、30例羊水过多和30例羊水量正常产妇脐带血中Caspase7蛋白表达水平的情况,并加以分析。结果Caspase7蛋白在3组产妇血清中均有阳性表达。羊水过少组血清中Caspase7蛋白平均浓度(160.2±25.8ng/mL)比对照组高(142.9±18.4ng/mL),差异有统计学意义(t=2.99,P<0.05)。羊水过多组血清中Caspase7蛋白平均浓度(119.2±21.5ng/mL)比对照组低(142.9±18.4ng/mL),差异也有统计学意义(t=4.59,P<0.05)。结论羊水过少或过多产妇血清中Caspase7蛋白表达异常,提示其可能与羊水量异常的发生有关。

  9. B cell receptor cross-linking triggers a caspase-8-dependent apoptotic pathway that is independent of the death effector domain of Fas-associated death domain protein.

    Science.gov (United States)

    Besnault, L; Schrantz, N; Auffredou, M T; Leca, G; Bourgeade, M F; Vazquez, A

    2001-07-15

    We have previously reported that B cell receptors, depending on the degree to which they are cross-linked, can promote apoptosis in various human B cell types. In this study, we show that B cell receptors can trigger two apoptotic pathways according to cross-linking and that these pathways control mitochondrial activation in human Burkitt's lymphoma cells. Whereas soluble anti-mu Ab triggers caspase-independent mitochondrial activation, cross-linked anti-mu Ab induces an apoptotic response associated with a caspase-dependent loss of mitochondrial transmembrane potential. This B cell receptor-mediated caspase-dependent mitochondrial activation is associated with caspase-8 activation. We show here that caspase-8 inhibitors strongly decrease cross-linking-dependent B cell receptor-mediated apoptosis in Burkitt's lymphoma BL41 cells. These inhibitors act upstream from the mitochondria as they prevented the loss of mitochondrial membrane potential observed in B cell receptor-treated BL41 cells. Caspase-8 activation in these cells was also evident from the detection of cleaved fragments of caspase-8 and the cleavage of specific substrates, including Bid. Our data show that cross-linked B cell receptors induced an apoptotic pathway involving sequential caspase-8 activation, loss of mitochondrial membrane potential, and the activation of caspase-9 and caspase-3. Cells expressing a dominant negative mutant of Fas-associated death domain protein were sensitive to cross-linked B cell receptor-induced caspase-8 activation and apoptosis; therefore, this caspase-8 activation was independent of the death effector domain of Fas-associated death domain protein. PMID:11441077

  10. Influence of 5-azacytidine on expression level of XIAP and caspase-9 in HL-60 cells%5-氮杂胞苷对HL-60细胞中XIAP和Caspase-9表达水平的影响

    Institute of Scientific and Technical Information of China (English)

    任金海; 林凤茹; 郭晓楠; 张静楠; 王颖; 乔淑凯; 张丽红; 李琳

    2010-01-01

    目的 探讨5-氮杂胞苷(5-aza-CR)对HL-60细胞中XIAP和Caspase-9表达水平的影响以及XIAP与Caspase-9表达的相关性,初步了解XIAP抗凋亡的途径.方法 培养HL-60细胞,用不同浓度5-aza-CR干预,流式细胞术检测干预前后XIAP和Caspase-9的表达水平.结果 5-aza-CR干预HL-60细胞后早期凋亡率在5-aza-CR各浓度组均随干预时间的延长而增加,其中10.0μmol/L组干预4h后凋亡率达(34.26±3.15)%;XIAP表达水平在5-aza-CR各浓度组均随干预时间的延长而下降,10.0μmol/L组干预4h后降至76.22±2.84;Caspase-9表达水平在5-aza-CR各浓度组均随干预时间的延长而升高,10.0μmol/L组干预4h后升至75.45±4.64;XIAP和Caspase-9表达水平呈负相关.结论 HL-60细胞中XIAP抗凋亡途径可能与抑制Caspase-9表达相关.

  11. 瘢痕疙瘩组织中Livin、Smac、Caspase-3的表达及其相关性%Expression of Livin, Smac and Caspase-3 in keloids and their correlation

    Institute of Scientific and Technical Information of China (English)

    张远贵; 段冬; 李攀登; 陈润芳; 李之华; 高新宇; 吴炜

    2016-01-01

    BACKGROUND:Currently, there is no effective treatment for keloids that often recur. Its pathogenesis is stil entirely unclear, and fibroblast proliferation and apoptosis have become a research hotspot. OBJECTIVE:To investigate the expression of Livin, Smac and Caspase-3 in keloids and to analyze their relationship so as to preliminarily explore the significance of Livin, Smac and Caspase-3 in the pathogenesis of keloids. METHODS:RT-PCR and immunohistochemical methods were used to detect the mRNA and protein expressions of Livin, Smac and Caspase-3 in keloids (n=20) and normal skin tissues (n=20). RESULTS AND CONCLUSION:Compared with the normal skin tissue, the mRNA and protein positive expressions of Livin were significantly higher in keloids (P   目的:分析瘢痕疙瘩组织中Livin、Smac、Caspase-3的表达的相关性,初步探讨3种因子在瘢痕疙瘩组织发病机制中的作用。  方法:采用RT-PCR和免疫组织化学分别检测Livin、Smac、Caspase-3的mRNA和蛋白在20例瘢痕疙瘩组织和20例正常皮肤组织中的表达,并进行相关性分析  结果与结论:瘢痕疙瘩组织中Livin的mRNA表达水平和蛋白的阳性表达率显著高于正常皮肤组织(P <0.05),瘢痕疙瘩组织中Smac和Caspase-3的mRNA表达水平和蛋白的阳性表达率低于正常皮肤组织(P<0.05)。瘢痕疙瘩组织中Livin和Smac、Caspase-3蛋白的表达呈负关联。结果提示Livin基因高表达可能抑制了Smac、Caspase-3基因表达从而导致了瘢痕疙瘩成纤维细胞增殖和凋亡的失衡,最终导致了瘢痕疙瘩的形成。

  12. Allitridi induces apoptosis by affecting Bcl-2 expression and caspase-3 activity in human gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Hong LAN; You-yong LU

    2004-01-01

    AIM: To investigate the mechanism of allitridi-induced apoptosis in human gastric cancer cell line BGC823.METHODS: Growth inhibition by allitridi was analyzed using cell growth curve and MTT assay. Apoptotic cells were detected using staining with Hoechst 33342, and confirmed by flow cytometric analysis and DNA fragmentation analysis. The protein expression affected by allitridi was determined using Western blot. The activity of caspase-3 was measured using a fluorescence assay. RESULTS: Allitridi induced apoptosis, and then inhibited cells proliferation in human gastric cancer cell line BGC823. The protein level of Bcl-2 was decreased dramatically,while Bax and p53 were not significantly affected by allitridi. The expression and activity of caspase-3 started to increase after allitridi treatment for 72 h. CONCLUSION: Allitridi induced apoptosis through down-regulation of Bcl-2, and increased caspase-3 expression and its activity.

  13. Caspase-8 activity is part of the BeWo trophoblast cell defense mechanisms against Trypanosoma cruzi infection.

    Science.gov (United States)

    Carrillo, Ileana; Droguett, Daniel; Castillo, Christian; Liempi, Ana; Muñoz, Lorena; Maya, Juan Diego; Galanti, Norbel; Kemmerling, Ulrike

    2016-09-01

    Congenital Chagas disease is caused by the protozoan parasite Trypanosoma cruzi that must cross the placental barrier during transmission. The trophoblast constitutes the first tissue in contact with the maternal-blood circulating parasite. Importantly, the congenital transmission rates are low, suggesting the presence of local placental defense mechanisms. Cellular proliferation and differentiation as well as apoptotic cell death are induced by the parasite and constitute part of the epithelial turnover of the trophoblast, which has been suggested to be part of those placental defenses. On the other hand, caspase-8 is an essential molecule in the modulation of trophoblast turnover by apoptosis and by epithelial differentiation. As an approach to study whether T. cruzi induced trophoblast turnover and infection is mediated by caspase-8, we infected BeWo cells (a trophoblastic cell line) with the parasite and determined in the infected cells the expression and enzymatic activity of caspase-8, DNA synthesis (as proliferation marker), β-human chorionic gonadotropin (β-hCG) (as differentiation marker) and activity of Caspase-3 (as apoptotic death marker). Parasite load in BeWo cells was measured by DNA quantification using qPCR and cell counting. Our results show that T. cruzi induces caspase-8 activity and that its inhibition increases trophoblast cells infection while decreases parasite induced cellular differentiation and apoptotic cell death, but not cellular proliferation. Thus, caspase-8 activity is part of the BeWo trophoblast cell defense mechanisms against T. cruzi infection. Together with our previous results, we suggest that the trophoblast turnover is part of local placental anti-parasite mechanisms. PMID:27328973

  14. CD40L induces multidrug resistance to apoptosis in breast carcinoma and lymphoma cells through caspase independent and dependent pathways

    Directory of Open Access Journals (Sweden)

    Blay Jean-Yves

    2006-03-01

    Full Text Available Abstract Background CD40L was found to reduce doxorubicin-induced apoptosis in non Hodgkin's lymphoma cell lines through caspase-3 dependent mechanism. Whether this represents a general mechanism for other tumor types is unknown. Methods The resistance induced by CD40L against apoptosis induced by a panel of cytotoxic chemotherapeutic drugs in non Hodgkin's lymphoma and breast carcinoma cell lines was investigated. Results Doxorubicin, cisplatyl, etoposide, vinblastin and paclitaxel increased apoptosis in a dose-dependent manner in breast carcinoma as well as in non Hodgkin's lymphoma cell lines. Co-culture with irradiated L cells expressing CD40L significantly reduced the percentage of apoptotic cells in breast carcinoma and non Hodgkin's lymphoma cell lines treated with these drugs. In breast carcinoma cell lines, these 5 drugs induced an inconsistent increase of caspase-3/7 activity, while in non Hodgkin's lymphoma cell lines all 5 drugs increased caspase-3/7 activity up to 28-fold above baseline. Co-culture with CD40L L cells reduced (-39% to -89% the activation of caspase-3/7 induced by these agents in all 5 non Hodgkin's lymphoma cell lines, but in none of the 2 breast carcinoma cell lines. Co culture with CD40L L cells also blocked the apoptosis induced by exogenous ceramides in breast carcinoma and non Hodgkin's lymphoma cell lines through a caspase-3-like, 8-like and 9-like dependent pathways. Conclusion These results indicate that CD40L expressed on adjacent non tumoral cells induces multidrug resistance to cytotoxic agents and ceramides in both breast carcinoma and non Hodgkin's lymphoma cell lines, albeit through a caspase independent and dependent pathway respectively.

  15. Effect of Dexamethasone on Nitric Oxide Synthase and Caspase-3 Gene Expressions in Endotoxemia in Neonate Rat Brain

    Institute of Scientific and Technical Information of China (English)

    HUA WANG; YU-BIN WU; XIU-HUA DU

    2005-01-01

    Objective To investigate the gene and protein expressions of three isoforms of nitric oxide synthase (NOS) and gene expression of Caspase-3, and effect of dexamethasone on them in neonatal rats with lipopolysaccharide (LPS)-induced endotoxemic brain damage. Methods Expressions of the three isoforms of NOS and caspase-3 mRNA in the brain were investigated by RT-PCR in postnatal 7-day wistar rats with acute endotoxemia by intraperitoneal administration of LPS. Regional distributions of NOSs were examined by immunohistochemical technique. Results nNOS and Caspase-3 mRNA were obviously detected. eNOS mRNA was faintly expressed, but iNOS mRNA was undetectable in the control rat brain. The expressions of NOS mRNA of three isoforms were weak 2 h after LPS (5 mg/mg) delivery, peaked at 6 h, and thereafter, reduced gradually up to 24 h. The expression intensity was in the order of nNOS> iNOS> eNOS. Widespread nNOS, scattered eNOS distribution and negative iNOS were identified in the control rat brain and all isoforms of NOS could be induced by LPS which reached the apex at 24 h in the order of nNOS> iNOS> eNOS as detected by immunostaining. Although Caspase-3 mRNA could be found in all groups, DNA fragmentation was only seen at 6 h and 24 h. The expressions of NOS and Caspase-3 mRNA were inhibited in the rat brain when dexamethasone was administrated. Conclusion LPS-induced NO production induces apoptosis of neurons through mechanism involving the Caspase-3 activation, which may play an important role in the pathogenesis of brain damage during endotoxemia, and neuro-protective effects of dexamethasone may be partially realized by inhibiting the expression of NOS mRNA.

  16. Neuroprotective effects of M826, a reversible caspase-3 inhibitor, in the rat malonate model of Huntington's disease

    OpenAIRE

    Toulmond, Sylvie; Tang, Keith; Bureau, Yves; Ashdown, Helen; Degen, Sarah; O'Donnell, Ruth; Tam, John; Han, Yongxin; Colucci, John; Giroux, André; Zhu, Yanxia; Boucher, Mathieu; Pikounis, Bill; Xanthoudakis, Steven; Roy, Sophie

    2004-01-01

    Caspases, key enzymes in the apoptosis pathway, have been detected in the brain of HD patients and in animal models of the disease. In the present study, we investigated the neuroprotective properties of a new, reversible, caspase-3-specific inhibitor, M826 (3-({(2S)-2-[5-tert-butyl-3-{[(4-methyl-1,2,5-oxadiazol-3-yl)methyl]amino}-2-oxopyrazin-1(2H)-yl]butanoyl}amino)-5-[hexyl(methyl)amino]-4-oxopentanoic acid), in a rat malonate model of HD.Pharmacokinetic and autoradiography studies after i...

  17. 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

  18. Sphingosine kinase expression regulates apoptosis and caspase activation in PC12 cells.

    Science.gov (United States)

    Edsall, L C; Cuvillier, O; Twitty, S; Spiegel, S; Milstien, S

    2001-03-01

    Sphingosine-1-phosphate (SPP), a bioactive sphingolipid metabolite, suppresses apoptosis of many types of cells, including rat pheochromocytoma PC12 cells. Elucidating the molecular mechanism of action of SPP is complicated by many factors, including uptake and metabolism, as well as activation of specific G-protein-coupled SPP receptors, known as the endothelial differentiation gene-1 (EDG-1) family. In this study, we overexpressed type 1 sphingosine kinase (SPHK1), the enzyme that converts sphingosine to SPP, in order to examine more directly the role of intracellularly generated SPP in neuronal survival. Enforced expression of SPHK1 in PC12 cells resulted in significant increases in kinase activity, with corresponding increases in intracellular SPP levels and concomitant decreases in both sphingosine and ceramide, and marked suppression of apoptosis induced by trophic factor withdrawal or by C(2)-ceramide. NGF, which protects PC12 cells from serum withdrawal-induced apoptosis, also stimulated SPHK1 activity. Surprisingly, overexpression of SPHK1 had no effect on activation of two known NGF-stimulated survival pathways, extracellular signal regulated kinase ERK 1/2 and Akt. However, trophic withdrawal-induced activation of the stress activated protein kinase, c-Jun amino terminal kinase (SAPK/JNK), and activation of the executionary caspases 2, 3 and 7, were markedly suppressed. Moreover, this abrogation of caspase activation, which was prevented by the SPHK inhibitor N,N-dimethylsphingosine, was not affected by pertussis toxin treatment, indicating that the cytoprotective effect was likely not mediated by binding of SPP to cell surface G(i)-coupled SPP receptors. In agreement, there was no detectable release of SPP into the culture medium, even after substantially increasing cellular SPP levels by NGF or sphingosine treatment. In contrast to PC12 cells, C6 astroglioma cells secreted SPP, suggesting that SPP might be one of a multitude of known neurotrophic

  19. Characterization of ERK Docking Domain Inhibitors that Induce Apoptosis by Targeting Rsk-1 and Caspase-9

    International Nuclear Information System (INIS)

    The extracellular signal-regulated kinase-1 and 2 (ERK1/2) proteins play an important role in cancer cell proliferation and survival. ERK1/2 proteins also are important for normal cell functions. Thus, anti-cancer therapies that block all ERK1/2 signaling may result in undesirable toxicity to normal cells. As an alternative, we have used computational and biological approaches to identify low-molecular weight compounds that have the potential to interact with unique ERK1/2 docking sites and selectively inhibit interactions with substrates involved in promoting cell proliferation. Colony formation and water soluble tetrazolium salt (WST) assays were used to determine the effects of test compounds on cell proliferation. Changes in phosphorylation and protein expression in response to test compound treatment were examined by immunoblotting and in vitro kinase assays. Apoptosis was determined with immunoblotting and caspase activity assays. In silico modeling was used to identify compounds that were structurally similar to a previously identified parent compound, called 76. From this screen, several compounds, termed 76.2, 76.3, and 76.4 sharing a common thiazolidinedione core with an aminoethyl side group, inhibited proliferation and induced apoptosis of HeLa cells. However, the active compounds were less effective in inhibiting proliferation or inducing apoptosis in non-transformed epithelial cells. Induction of HeLa cell apoptosis appeared to be through intrinsic mechanisms involving caspase-9 activation and decreased phosphorylation of the pro-apoptotic Bad protein. Cell-based and in vitro kinase assays indicated that compounds 76.3 and 76.4 directly inhibited ERK-mediated phosphorylation of caspase-9 and the p90Rsk-1 kinase, which phosphorylates and inhibits Bad, more effectively than the parent compound 76. Further examination of the test compound's mechanism of action showed little effects on related MAP kinases or other cell survival proteins. These

  20. Characterization of ERK Docking Domain Inhibitors that Induce Apoptosis by Targeting Rsk-1 and Caspase-9

    Directory of Open Access Journals (Sweden)

    MacKerell Alexander D

    2011-01-01

    Full Text Available Abstract Background The extracellular signal-regulated kinase-1 and 2 (ERK1/2 proteins play an important role in cancer cell proliferation and survival. ERK1/2 proteins also are important for normal cell functions. Thus, anti-cancer therapies that block all ERK1/2 signaling may result in undesirable toxicity to normal cells. As an alternative, we have used computational and biological approaches to identify low-molecular weight compounds that have the potential to interact with unique ERK1/2 docking sites and selectively inhibit interactions with substrates involved in promoting cell proliferation. Methods Colony formation and water soluble tetrazolium salt (WST assays were used to determine the effects of test compounds on cell proliferation. Changes in phosphorylation and protein expression in response to test compound treatment were examined by immunoblotting and in vitro kinase assays. Apoptosis was determined with immunoblotting and caspase activity assays. Results In silico modeling was used to identify compounds that were structurally similar to a previously identified parent compound, called 76. From this screen, several compounds, termed 76.2, 76.3, and 76.4 sharing a common thiazolidinedione core with an aminoethyl side group, inhibited proliferation and induced apoptosis of HeLa cells. However, the active compounds were less effective in inhibiting proliferation or inducing apoptosis in non-transformed epithelial cells. Induction of HeLa cell apoptosis appeared to be through intrinsic mechanisms involving caspase-9 activation and decreased phosphorylation of the pro-apoptotic Bad protein. Cell-based and in vitro kinase assays indicated that compounds 76.3 and 76.4 directly inhibited ERK-mediated phosphorylation of caspase-9 and the p90Rsk-1 kinase, which phosphorylates and inhibits Bad, more effectively than the parent compound 76. Further examination of the test compound's mechanism of action showed little effects on related MAP

  1. Inactivation of cellular caspases by peptide-derived tryptophan and tyrosine peroxides

    DEFF Research Database (Denmark)

    Hampton, Mark B; Morgan, Philip E; Davies, Michael Jonathan

    show here that Trp- and Tyr-derived peptide peroxides, generated by singlet oxygen, inhibit caspase activity in the lysates of apoptotic Jurkat cells. N-Ac-Trp-OMe peroxide was the most effective inhibitor, and was 30-fold more effective than H(2)O(2) under identical conditions. As such, protein......Peroxides generated on peptides and proteins within cells, as a result of radical attack or reaction with singlet oxygen, are longer-lived than H(2)O(2) due to their poor removal by protective enzymes. These peroxides readily oxidize cysteine residues and can inactivate thiol-dependent enzymes. We...... peroxides could modulate the progression of apoptosis in cells in which they are generated....

  2. Regulation of Caspase-3 and Bcl-2 Expression in Dalton's Lymphoma Ascites Cells by Abrin

    Directory of Open Access Journals (Sweden)

    V. Ramnath

    2009-01-01

    Full Text Available The role of abrin, a toxic lectin isolated from seeds of Abrus precatorius Linn in inducing apoptosis in murine Dalton's Lymphoma Ascites (DLA cells was evaluated. Abrin when incubated at the concentration of 10 ng per million DLA cells could bring about cell death as typical morphological changes with apoptosis. However, necrotic cell death dominated when a higher dose of abrin was used. DNA samples, isolated from DLA cells treated with abrin showed fragmentation. Abrin brought about induction of apoptosis by stimulating the expression of pro-apoptotic Caspase-3, at the same time blocking the expression of Bcl-2, which is an anti apoptotic gene. However, the expression of tumor suppressor gene p53 has not been observed in control and abrin-treated DLA cells. Results suggested that abrin effectively induced apoptotic changes in the tumor cells that led to cellular death.

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

    Science.gov (United States)

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

    2016-03-18

    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. PMID:26876577

  4. Caspase recruitment domain-containing protein 9 signaling in innate immunity and inflammation.

    Science.gov (United States)

    Roth, Susanne; Ruland, Jürgen

    2013-06-01

    Caspase recruitment domain-containing protein (Card)9 is a nonredundant adapter protein that functions in the innate immune system in the assembly of multifunctional signaling complexes. Together with B cell lymphoma (Bcl)10 and the paracaspase, mucosa-associated lymphoid tissue lymphoma translocation protein (Malt)1, Card9 links spleen-tyrosine kinase (Syk)-coupled C-type lectin receptors to inflammatory responses. Card9 signaling also responds to intracellular danger sensors, such as retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLRs) and nucleotide-oligomerization domain (Nod)2. Card9 complexes are engaged upon fungal, bacterial, or viral recognition, and they are essential for host protection. Moreover, Card9 polymorphisms are commonly associated with human inflammatory diseases. Here, we discuss the molecular regulation and the physiological functions of Card9 in host defense and immune homeostasis, and provide a framework for the therapeutic targeting of Card9 signaling in immune-mediated diseases. PMID:23523010

  5. A simple device for quantitation of Caspase 3 in individual apoptotic embryonic cells

    Czech Academy of Sciences Publication Activity Database

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

    Brno: Ústav analytické chemie AV ČR, v. v. i, 2012 - (Foret, F.; Křenková, J.; Guttman, A.; Klepárník, K.; Boček, P.), s. 354-356 ISBN 978-80-904959-1-3. [CECE 2012. International Interdisciplinary Meeting on Bioanalysis /9./. Brno (CZ), 01.11.2012-02.11.2012] R&D Projects: GA ČR(CZ) GAP301/11/2055; GA ČR GAP206/11/2377; GA ČR(CZ) GPP206/11/P002 Institutional support: RVO:68081715 ; RVO:67985904 Keywords : single cell * chemiluminiscence * Caspase 3 Subject RIV: CB - Analytical Chemistry, Separation http://hdl.handle.net/11104/0215496

  6. MDMA诱导大鼠神经元凋亡及凋亡相关因子caspase-3的表达%Neuron apoptosis induced by 3,4-methylenedioxy methamphetamine and the expression of caspase-3

    Institute of Scientific and Technical Information of China (English)

    王雪; 李静; 祝三平

    2007-01-01

    目的 探讨3,4 -亚甲基二氧基甲基苯丙胺(MDMA)对实验大鼠神经元凋亡的诱导及凋亡相关因子半胱氨酸天冬氨酸特异性蛋白酶-3(caspase-3)的表达.方法 将20只Wistar雄性大鼠随机均分为1组对照组(A)、3组MDMA实验组(B、C、D).B组予MDMA(20 mg · kg-1, ip, single),C组予MDMA(20 mg · kg-1, 8 am,8 pm,ip×2 d),D组予MDMA (20 mg · kg-1 ,8 am,8 pm,ip×4 d);A组给予等体积生理盐水.采用TUNEL法检测神经元凋亡,免疫组织化学方法检测Caspase-3的表达.结果 给予MDMA后,大鼠各相关脑区有凋亡细胞形成,Caspase-3有不同程度的表达.结论 MDMA可导致神经元的凋亡,并诱导凋亡相关因子Caspase-3的表达.

  7. 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.

  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. GsdmD p30 elicited by caspase-11 during pyroptosis forms pores in membranes.

    Science.gov (United States)

    Aglietti, Robin A; Estevez, Alberto; Gupta, Aaron; Ramirez, Monica Gonzalez; Liu, Peter S; Kayagaki, Nobuhiko; Ciferri, Claudio; Dixit, Vishva M; Dueber, Erin C

    2016-07-12

    Gasdermin-D (GsdmD) is a critical mediator of innate immune defense because its cleavage by the inflammatory caspases 1, 4, 5, and 11 yields an N-terminal p30 fragment that induces pyroptosis, a death program important for the elimination of intracellular bacteria. Precisely how GsdmD p30 triggers pyroptosis has not been established. Here we show that human GsdmD p30 forms functional pores within membranes. When liberated from the corresponding C-terminal GsdmD p20 fragment in the presence of liposomes, GsdmD p30 localized to the lipid bilayer, whereas p20 remained in the aqueous environment. Within liposomes, p30 existed as higher-order oligomers and formed ring-like structures that were visualized by negative stain electron microscopy. These structures appeared within minutes of GsdmD cleavage and released Ca(2+) from preloaded liposomes. Consistent with GsdmD p30 favoring association with membranes, p30 was only detected in the membrane-containing fraction of immortalized macrophages after caspase-11 activation by lipopolysaccharide. We found that the mouse I105N/human I104N mutation, which has been shown to prevent macrophage pyroptosis, attenuated both cell killing by p30 in a 293T transient overexpression system and membrane permeabilization in vitro, suggesting that the mutants are actually hypomorphs, but must be above certain concentration to exhibit activity. Collectively, our data suggest that GsdmD p30 kills cells by forming pores that compromise the integrity of the cell membrane. PMID:27339137

  10. Caspase Mediated Synergistic Effect of Boswellia serrata Extract in Combination with Doxorubicin against Human Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Mohammad Ahmed Khan

    2014-01-01

    Full Text Available The study investigated the growth-inhibiting and apoptosis mediating effects of B. serrata extract as monotherapy and combination therapy with DOX against hepatocellular carcinoma cell lines. Boswellic acid rich fraction of B. serrata extract was prepared. MTT assay on HepG2 and Hep3B cells was carried out using B. serrata alone and in combination with DOX. Further, caspase-3 activity, TNF-α level, and IL-6 level were estimated. Isobolographic analysis was carried out to evaluate the effect of combination therapy. Additionally, protective effect of B. serrata extract on DOX induced hepatic toxicity was also evaluated in Wistar rats. B. serrata extract inhibited growth of HepG2 (IC50 value of 21.21±0.92 μg/mL as well as HepG2 (IC50 value of 18.65±0.71 μg/mL. DOX inhibited growth in HepG2 and Hep3B cells with an IC50 of 1.06±0.04 μg/mL and 1.92±0.09 μg/mL. Isobolographic analysis showed combination index (CI of DOX and B. serrata extract of 0.53±0.03 to 0.79±0.02 suggesting synergistic behavior against the two cell lines. B. serrata extract also caused dose dependent increase in caspase-3 activity, TNF-α level, and IL-6 level which was higher (P<0.001 with DOX (1 μM and B. serrata extract (20 μg/mL combination. B. serrata extract also protected Wistar rats against DOX induced hepatic toxicity.

  11. Iodinated contrast media induce neutrophil apoptosis through a mitochondrial and caspase mediated pathway.

    LENUS (Irish Health Repository)

    Fanning, N F

    2012-02-03

    Iodinated contrast media (ICM) can induce apoptosis (programmed cell death) in renal, myocardial and endothelial cells. Following intravascular injection, circulating immune cells are exposed to high concentrations of ICM. As neutrophils constitutively undergo apoptosis we hypothesized that ICM may adversely affect neutrophil survival. Our aim was to investigate the effect of ICM on neutrophil apoptosis. Neutrophils were isolated from healthy subjects and cultured in vitro with ionic (diatrizoate and ioxaglate) and non-ionic (iohexol and iotrolan) ICM. The effect of ICM on neutrophil apoptosis in both unstimulated and lipopolysaccharide-stimulated neutrophils was determined by annexin V flow cytometry. The influence of physicochemical properties of the different ICM on apoptosis of neutrophils was also studied. We further investigated the effects of ICM on key intracellular signal pathways, including p38 mitogen-activated protein kinase (MAPK) by Western blotting, and mitochondrial depolarization and caspase activity by flow cytometry. Isoiodine concentrations (20 mg ml(-1)) of ionic (diatrizoate 69.6+\\/-2.9%; ioxaglate 58.9+\\/-2.0%) and non-ionic (iohexol 57.3+\\/-2.9%; iotrolan 57.1+\\/-2.6%) ICM significantly induced neutrophil apoptosis over control levels (47.7+\\/-1.4%). The apoptotic effect of ICM was influenced by their chemical structure, with ionic ICM having a more significant (p<0.01) apoptotic effect than non-ionic ICM (p<0.05). Furthermore, ICM reversed the anti-apoptotic effect of lipopolysaccharide (1000 ng ml(-1)) treated neutrophils to control levels (23.0+\\/-3.5% to 61.2+\\/-5.3%; n=4; p<0.05). These agents induce apoptosis through a p38 MAPK independent pathway that results in mitochondrial depolarization, and is dependent on caspase activation. As neutrophils play a central role in host response to infection and injury, ICM, through induction of neutrophil apoptosis, could have a significant deleterious effect on host immune defence and

  12. 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.

  13. 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.

  14. Structure of the caspase-recruitment domain from a zebrafish guanylate-binding protein

    International Nuclear Information System (INIS)

    The crystal structure of the first zebrafish caspase-recruitment domain at 1.47 Å resolution illustrates a six-helix bundle fold similar to that of the human NLRP1 CARD. The caspase-recruitment domain (CARD) mediates homotypic protein–protein interactions that assemble large oligomeric signaling complexes such as the inflammasomes during innate immune responses. Structural studies of the mammalian CARDs demonstrate that their six-helix bundle folds belong to the death-domain superfamily, whereas such studies have not been reported for other organisms. Here, the zebrafish interferon-induced guanylate-binding protein 1 (zIGBP1) was identified that contains an N-terminal GTPase domain and a helical domain typical of the mammalian guanylate-binding proteins, followed by a FIIND domain and a C-terminal CARD similar to the mammalian inflammasome proteins NLRP1 and CARD8. The structure of the zIGBP1 CARD as a fusion with maltose-binding protein was determined at 1.47 Å resolution. This revealed a six-helix bundle fold similar to the NLRP1 CARD structure with the bent α1 helix typical of all known CARD structures. The zIGBP1 CARD surface contains a positively charged patch near its α1 and α4 helices and a negatively charged patch near its α2, α3 and α5 helices, which may mediate its interaction with partner domains. Further studies using binding assays and other analyses will be required in order to address the physiological function(s) of this zebrafish protein

  15. Mitochondrial permeabilization without caspase activation mediates the increase of basal apoptosis in cells lacking Nrf2.

    Science.gov (United States)

    Ariza, Julia; González-Reyes, José A; Jódar, Laura; Díaz-Ruiz, Alberto; de Cabo, Rafael; Villalba, José Manuel

    2016-06-01

    Nuclear factor E2-related factor-2 (Nrf2) is a cap'n'collar/basic leucine zipper (b-ZIP) transcription factor which acts as sensor of oxidative and electrophilic stress. Low levels of Nrf2 predispose cells to chemical carcinogenesis but a dark side of Nrf2 function also exists because its unrestrained activation may allow the survival of potentially dangerous damaged cells. Since Nrf2 inhibition may be of therapeutic interest in cancer, and a decrease of Nrf2 activity may be related with degenerative changes associated with aging, it is important to investigate how the lack of Nrf2 function activates molecular mechanisms mediating cell death. Murine Embryonic Fibroblasts (MEFs) bearing a Nrf2 deletion (Nrf2KO) displayed diminished cellular growth rate and shortened lifespan compared with wild-type MEFs. Basal rates of DNA fragmentation and histone H2A.X phosphorylation were higher in Nrf2KO MEFs, although steady-state levels of reactive oxygen species were not significantly increased. Enhanced rates of apoptotic DNA fragmentation were confirmed in liver and lung tissues from Nrf2KO mice. Apoptosis in Nrf2KO MEFs was associated with a decrease of Bcl-2 but not Bax levels, and with the release of the mitochondrial pro-apoptotic factors cytochrome c and AIF. Procaspase-9 and Apaf-1 were also increased in Nrf2KO MEFs but caspase-3 was not activated. Inhibition of XIAP increased death in Nrf2KO but not in wild-type MEFs. Mitochondrial ultrastructure was also altered in Nrf2KO MEFs. Our results support that Nrf2 deletion produces mitochondrial dysfunction associated with mitochondrial permeabilization, increasing basal apoptosis through a caspase-independent and AIF-dependent pathway. PMID:27016073

  16. Nascent histamine induces α-synuclein and caspase-3 on human cells

    International Nuclear Information System (INIS)

    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

  17. Deltamethrin-induced oxidative stress and mitochondrial caspase-dependent signaling pathways in murine splenocytes.

    Science.gov (United States)

    Kumar, Anoop; Sasmal, D; Bhaskar, Amand; Mukhopadhyay, Kunal; Thakur, Aman; Sharma, Neelima

    2016-07-01

    Deltamethrin (DLM) is a well-known pyrethroid insecticide used extensively in pest control. Exposure to DLM has been demonstrated to cause apoptosis in various cells. However, the immunotoxic effects of DLM on mammalian system and its mechanism is still an open question to be explored. To explore these effects, this study has been designed to first observe the interactions of DLM to immune cell receptors and its effects on the immune system. The docking score revealed that DLM has strong binding affinity toward the CD45 and CD28 receptors. In vitro study revealed that DLM induces apoptosis in murine splenocytes in a concentration-dependent manner. The earliest markers of apoptosis such as enhanced reactive oxygen species and caspase 3 activation are evident as early as 1 h by 25 and 50 µM DLM. Western blot analysis demonstrated that p38 MAP kinase and Bax expression is increased in a concentration-dependent manner, whereas Bcl 2 expression is significantly reduced after 3 h of DLM treatment. Glutathione depletion has been also observed at 3 and 6 h by 25 and 50 µM concentration of DLM. Flow cytometry results imply that the fraction of hypodiploid cells has gradually increased with all the concentrations of DLM at 18 h. N-acetyl cysteine effectively reduces the percentage of apoptotic cells, which is increased by DLM. In contrast, buthionine sulfoxamine causes an elevation in the percentage of apoptotic cells. Phenotyping data imply the effect of DLM toxicity in murine splenocytes. In brief, the study demonstrates that DLM causes apoptosis through its interaction with CD45 and CD28 receptors, leading to oxidative stress and activation of the mitochondrial caspase-dependent pathways which ultimately affects the immune functions. This study provides mechanistic information by which DLM causes toxicity in murine splenocytes. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 808-819, 2016. PMID:25534813

  18. Caspase-3 expression in spinal tissue of retinoic acid induce spiua bifida fetal rat%维甲酸诱导脊柱裂胎鼠脊髓组织中Caspase-3表达情况

    Institute of Scientific and Technical Information of China (English)

    马英桓; 袁正伟

    2012-01-01

    Objective To explore caspase-3 expression in spinal tissue of retinoic acid induced spina bifida fetal rat. Methods Pregnant Wister rats with 10 days were used. Retinoic acid dissolved in olive oil (40mg /ml) were stomach fed for preparing the rat model of spina bifida malformations 135mg / kg). Control group only received olive oil. The animals were divided into 4 groups: pregnancy of 12 days, 15 days, 17 days and 20 days. Immunohistochemical method was used to detect and compare caspase-3 expression in different groups. Results The expression of caspase-3 increased at the day 15 after pregnancy, and maintained until day 20 in the spinal tissue of modeled fetal rat, which presented significant difference compared to that of control groups at the same pregnant time. At day 15, day 17 and day 20 of pregnancy, the number of caspase-3 positive cells was more in model animals than the control. Conclusions Retinoic acid induced spina bifida fetal rat demonstrates the increased caspase-3 expression in spinal tissue of fetal rats.%目的 本文旨在探讨维甲酸诱导脊柱裂胎鼠脊髓组织Caspase-3表达情况.方法 选取孕10d Wistar大鼠,实验组用溶有维甲酸(40mg/ml)的橄榄油,以135mg/kg经胃管注入给药制作脊柱裂畸形大鼠模型;对照组选取孕10 d Wistar大鼠给等量橄榄油.将实验组及对照组按照孕12、15、17和20 d分为4组.应用免疫组织化学方法比较分析Caspase-3在对照组、畸形组胎鼠脊髓组织细胞中的分布和表达情况.结果 脊柱裂大鼠脊髓神经组织中Caspase-3在15d开始增多,一直持续到20 d胚胎大鼠.其增高情况明显高于同一时间点对照组大鼠.胚胎15、17和20 d显性脊柱裂畸形鼠脊髓组织Caspase-3阳性细胞数多于对照组,荧光强度高于对照组.结论 维甲酸诱导的脊柱裂胎鼠Caspase-3表达明显高于正常发育胎鼠.

  19. 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.

  20. TAF15 and the leukemia-associated fusion protein TAF15-CIZ/NMP4 are cleaved by caspases-3 and -7

    International Nuclear Information System (INIS)

    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 106DQPD/Y110 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.

  1. Elevated Levels of Uterine Anti-Apoptotic Signaling May Activate NFKB and Potentially Confer Resistance to Caspase 3-Mediated Apoptotic Cell Death During Pregnancy in Mice1

    Science.gov (United States)

    Jeyasuria, Pancharatnam; Subedi, Kalpana; Suresh, Arvind; Condon, Jennifer C.

    2011-01-01

    Preserving the uterus in a state of relative quiescence is vital to the maintenance of a successful pregnancy. Elevated cytoplasmic levels of uterine caspase 3 during pregnancy have been proposed as a potential regulator of uterine quiescence through direct targeting and disabling of the uterine contractile architecture. However, despite highly elevated levels of uterine caspase 3 during pregnancy, there is minimal evidence of apoptosis. This current study defines the mechanism whereby the pregnant uterine myocyte may harness the tocolytic activity of active caspases while avoiding apoptotic cell death. Using the pregnant mouse model, we have analyzed the uterus for changes in pro- and antiapoptotic signaling patterns associated with the advancing stages of pregnancy. Briefly, we have found that members of the IAP family, such as SURVIVIN and XIAP, and the Bcl2 family members, such as MCL1, are elevated in the uterine myocyte during late gestation. The IAP family members are the only endogenous inhibitors of active caspase 3, and MCL1 limits activation of caspase 3 by suppressing proapoptotic signaling. Elevated XIAP levels partner with SURVIVIN, resulting in increased levels of the antiapoptotic MCL1 via NFKB activation; these together have the potential to limit both the activity and level of active caspase 3 in the pregnant uterus as term approaches. We propose that modification of these antiapoptotic signaling partners allows the pregnant uterus to escape the apoptotic action of elevated active caspase 3 levels but also functions to limit the levels of active uterine caspase 3 near term. PMID:21566000

  2. IGF-I activates caspases 3/7, 8 and 9 but does not induce cell death in colorectal cancer cells

    International Nuclear Information System (INIS)

    Colorectal cancer is the third most common cancer in the western world. Chemotherapy is often ineffective to treat the advanced colorectal cancers due to the chemo-resistance. A major contributor to chemo-resistance is tumour-derived inhibition or avoidance of apoptosis. Insulin-like growth factor I (IGF-I) has been known to play a prominent role in colorectal cancer development and progression. The role of IGF-I in cancer cell apoptosis is not completely understood. Using three colorectal cancer cell lines and one muscle cell line, associations between IGF-I and activities of caspase 3/7, 8 and 9 have been examined; the role of insulin-like growth factor I receptor (IGF-IR) in the caspase activation has been investigated. The results show that exogenous IGF-I significantly increases activity of caspases 3/7, 8 and 9 in all cell lines used; blocking IGF-I receptor reduce IGF-I-induced caspase activation. Further studies demonstrate that IGF-I induced caspase activation does not result in cell death. This is the first report to show that while IGF-I activates caspases 3/7, 8 and 9 it does not cause colorectal cancer cell death. The study suggests that caspase activation is not synonymous with apoptosis and that activation of caspases may not necessarily induce cell death

  3. Caspase-dependent inhibition of store-operated Ca{sup 2+} entry into apoptosis-committed Jurkat cells

    Energy Technology Data Exchange (ETDEWEB)

    Onopiuk, Marta; Wierzbicka, Katarzyna; Brutkowski, Wojciech; Szczepanowska, Joanna [Department of Biochemistry, The Nencki Institute of Experimental Biology, Warsaw (Poland); Zablocki, Krzysztof, E-mail: k.zablocki@nencki.gov.pl [Department of Biochemistry, The Nencki Institute of Experimental Biology, Warsaw (Poland)

    2010-08-20

    Activation of T-cells triggers store-operated Ca{sup 2+} entry, which begins a signaling cascade leading to induction of appropriate gene expression and eventually lymphocyte proliferation and differentiation. The simultaneous enhancement of Fas ligand gene expression in activated cells allows the immune response to be limited by committing the activated cells to apoptosis. In apoptotic cells the store-operated calcium entry is significantly inhibited. It has been documented that moderate activation of Fas receptor may cause reversible inhibition of store-operated channels by ceramide released from hydrolyzed sphingomyelin. Here we show that activation of Fas receptor in T-cells results in caspase-dependent decrease of cellular STIM1 and Orai1 protein content. This effect may be responsible for the substantial inhibition of Ca{sup 2+} entry into Jurkat cells undergoing apoptosis. In turn, this inhibition might prevent overloading of cells with calcium and protect them against necrosis. -- Research highlights: {yields} Fas activation reduces STIM1 and Orai1 protein content in caspase dependent manner. {yields} Fas activation partially reduces mitochondrial potential in caspase dependent manner. {yields} Fas stimulation inhibits of store-operated Ca{sup 2+} entry in caspase dependent manner. {yields} Inhibition of Ca{sup 2+} entry in apoptotic cells may protect them from secondary necrosis.

  4. Expression of livin protein in lung cancer and its relation with the expression of pro-caspase3 protein

    Directory of Open Access Journals (Sweden)

    Hongru LI

    2008-10-01

    Full Text Available Background and objective Livin is a novel inhibitor of apoptosis protein (IAP, recent studies showed it overexpresses in a variety of carcinomas including lung cancer and contributes much to the cancerous development. The objective of this study is to explore the expression of livin in tissues of lung cancer and its relationshipwith histological types, chemotherapy, Lymph node metastasis and to study its correlation with the expression of pro-caspase3 as well. Methods Expressions of Livin and caspase3 were detected by Western blot assay in lung cancer tissues as well as in controls. Results Livin was expressed in 15 of 27 lung cancer, significantly more than those in lung para-cancerous (1/5 or benign disease lung tissues (2/12 (P 0.05. Conclusion Livin are differently expressed in different histological types of lung cancer; High levels of livin expression do not relate to chemotherapy, lymph node metastasis (P >0.05. The levels of livin tends to be positively associated with those of accordingly pro-caspase3, it is presumed that livin could bind pro-caspase3 and suppress its activation.

  5. Homocysteine thiolactone induces apoptotic DNA damage mediated by increased intracellular hydrogen peroxide and caspase 3 activation in HL-60 cells.

    Science.gov (United States)

    Huang, R F; Huang, S M; Lin, B S; Wei, J S; Liu, T Z

    2001-05-11

    The cytotoxicity of homocysteine derivatives on chromosomal damage in somatic cells is not well established. The present study used reactive homocysteine derivative of homocysteine thiolactone (Hcy) to investigate its causal effect on apoptotic DNA injury in human promyeloid HL-60 cells. Our results demonstrated that Hcy induced cell death and features of apoptosis including increased phosphotidylserine exposure on the membrane surface, increased apoptotic cells with hypoploid DNA contents, and internucleosomal DNA fragmentation, all of which occurred in a time- and concentration-dependent manner. Hcy treatment also significantly increased intracellular reactive oxygen species H2O2, which coincided with the elimination of caspase 3 proenzyme levels and increased caspase 3 activity at the time of the appearance of apoptotic DNA fragmentation. Preincubation of Hcy-treated HL-60 cells with catalase completely scavenged intracellular H2O2, thus inhibiting caspase 3 activity and protecting cells from apoptotic DNA damage. In contrast, superoxide dismutase failed to inhibit Hcy-induced DNA damage. Taken together, these results demonstrate that Hcy exerted its genotoxic effects on HL-60 cells through an apoptotic pathway, which is mediated by the activation of caspase 3 activity induced by an increase in intracellular hydrogen peroxide. PMID:11432446

  6. 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

  7. Nuclear substructure reorganization during late stageerythropoiesis is selective and does not involve caspase cleavage ofmajor nuclear substructural proteins

    Energy Technology Data Exchange (ETDEWEB)

    Krauss, Sharon Wald; Lo, Annie J.; Short, Sarah A.; Koury, MarkJ.; Mohandas, Narla; Chasis, Joel Anne

    2005-04-06

    Enucleation, a rare feature of mammalian differentiation, occurs in three cell types: erythroblasts, lens epithelium and keratinocytes. Previous investigations suggest that caspase activation functions in lens epithelial and keratinocyte enucleation, as well as in early erythropoiesis encompassing BFU-E differentiation to proerythroblast. To determine whether caspase activation contributes to later erythropoiesis and whether nuclear substructures other than chromatin reorganize, we analyzed distributions of nuclear subcompartment proteins and assayed for caspase-induced cleavage of subcompartmental target proteins in mouse erythroblasts. We found that patterns of lamin B in the filamentous network interacting with both the nuclear envelope and DNA, nuclear matrix protein NuMA, and splicing factors Sm and SC35 persisted during nuclear condensation, consistent with effective transcription of genes expressed late in differentiation. Thus nuclear reorganization prior to enucleation is selective, allowing maintenance of critical transcriptional processes independent of extensive chromosomal reorganization. Consistent with these data, we found no evidence for caspase-induced cleavage of major nuclear subcompartment proteins during late erythropoiesis, in contrast to what has been observed in early erythropoiesis and in lens epithelial and keratinocyte differentiation. These findings imply that nuclear condensation and extrusion during terminal erythroid differentiation involve novel mechanisms that do not entail major activation of apoptotic machinery.

  8. The effect of caspase-3 inhibition on interdigital tissue regression in explant cultures of developing mouse limbs

    Czech Academy of Sciences Publication Activity Database

    Kudělová, Judita; Tucker, A.S.; Dubská, L.; Chlastáková, Ivana; Doubek, J.; Matalová, Eva

    2012-01-01

    Roč. 16, č. 4 (2012), s. 295-301. ISSN 1976-8354 R&D Projects: GA AV ČR IAA600450904; GA ČR GA203/08/1680 Institutional research plan: CEZ:AV0Z50450515 Keywords : caspases * cell death * digitalization Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.639, year: 2012

  9. Compound K, a metabolite of ginseng saponin, induces apoptosis via caspase-8-dependent pathway in HL-60 human leukemia cells

    Directory of Open Access Journals (Sweden)

    Choi Jung-Hye

    2009-12-01

    Full Text Available Abstract Background Compound K [20-O-β-(D-glucopyranosyl-20(S-protopanaxadiol], a metabolite of the protopanaxadiol-type saponins of Panax ginseng C.A. Meyer, has been reported to possess anti-tumor properties to inhibit angiogenesis and to induce tumor apoptosis. In the present study, we investigated the effect of Compound K on apoptosis and explored the underlying mechanisms involved in HL-60 human leukemia cells. Methods We examined the effect of Compound K on the viabilities of various cancer cell lines using MTT assays. DAPI assay, Annexin V and PI double staining, Western blot assay and immunoprecipitation were used to determine the effect of Compound K on the induction of apoptosis. Results Compound K was found to inhibit the viability of HL-60 cells in a dose- and time-dependent manner with an IC50 of 14 μM. Moreover, this cell death had typical features of apoptosis, that is, DNA fragmentation, DNA ladder formation, and the externalization of Annexin V targeted phosphatidylserine residues in HL-60 cells. In addition, compound-K induced a series of intracellular events associated with both the mitochondrial- and death receptor-dependent apoptotic pathways, namely, (1 the activation of caspases-3, -8, and -9; (2 the loss of mitochondrial membrane potential; (3 the release of cytochrome c and Smac/DIABLO to the cytosol; (4 the translocation of Bid and Bax to mitochondria; and (5 the downregulations of Bcl-2 and Bcl-xL. Furthermore, a caspase-8 inhibitor completely abolished caspase-3 activation, Bid cleavage, and subsequent DNA fragmentation by Compound K. Interestingly, the activation of caspase-3 and -8 and DNA fragmentation were significantly prevented in the presence of cycloheximide, suggesting that Compound K-induced apoptosis is dependent on de novo protein synthesis. Conclusions The results indicate that caspase-8 plays a key role in Compound K-stimulated apoptosis via the activation of caspase-3 directly or indirectly through

  10. Moringa oleifera Gold Nanoparticles Modulate Oncogenes, Tumor Suppressor Genes, and Caspase-9 Splice Variants in A549 Cells.

    Science.gov (United States)

    Tiloke, Charlette; Phulukdaree, Alisa; Anand, Krishnan; Gengan, Robert M; Chuturgoon, Anil A

    2016-10-01

    Gold nanoparticles (AuNP's) facilitate cancer cell recognition and can be manufactured by green synthesis using nutrient rich medicinal plants such as Moringa oleifera (MO). Targeting dysregulated oncogenes and tumor suppressor genes is crucial for cancer therapeutics. We investigated the antiproliferative effects of AuNP synthesized from MO aqueous leaf extracts (MLAuNP ) in A549 lung and SNO oesophageal cancer cells. A one-pot green synthesis technique was used to synthesise MLAuNP . A549, SNO cancer cells and normal peripheral blood mononuclear cells (PBMCs) were exposed to MLAuNP and CAuNP to evaluate cytotoxicity (MTT assay); apoptosis was measured by phosphatidylserine (PS) externalization, mitochondrial depolarization (ΔΨm) (flow cytometry), caspase-3/7, -9 activity, and ATP levels (luminometry). The mRNA expression of c-myc, p53, Skp2, Fbw7α, and caspase-9 splice variants was determined using qPCR, while relative protein expression of c-myc, p53, SRp30a, Bax, Bcl-2, Smac/DIABLO, Hsp70, and PARP-1 were determined by Western blotting. MLAuNP and CAuNP were not cytotoxic to PBMCs, whilst its pro-apoptotic properties were confirmed in A549 and SNO cells. MLAuNP significantly increased caspase activity in SNO cells while MLAuNP significantly increased PS externalization, ΔΨm, caspase-9, caspase-3/7 activities, and decreased ATP levels in A549 cells. Also, p53 mRNA and protein levels, SRp30a (P = 0.428), Bax, Smac/DIABLO and PARP-1 24 kDa fragment levels were significantly increased. Conversely, MLAuNP significantly decreased Bcl-2, Hsp70, Skp2, Fbw7α, c-myc mRNA, and protein levels and activated alternate splicing with caspase-9a splice variant being significantly increased. MLAuNP possesses antiproliferative properties and induced apoptosis in A549 cells by activating alternate splicing of caspase-9. J. Cell. Biochem. 117: 2302-2314, 2016. © 2016 Wiley Periodicals, Inc. PMID:26923760

  11. Caspase dependence of the death of neonatal retinal ganglion cells induced by axon damage and induction of autophagy as a survival mechanism

    Directory of Open Access Journals (Sweden)

    C. Sternberg

    2010-10-01

    Full Text Available We examined the degeneration of post-mitotic ganglion cells in ex-vivo neonatal retinal explants following axon damage. Ultrastructural features of both apoptosis and autophagy were detected. Degenerating cells reacted with antibodies specific for activated caspase-3 or -9, consistent with the presence of caspase activity. Furthermore, peptidic inhibitors of caspase-9, -6 or -3 prevented cell death (100 µM Ac-LEDH-CHO, 50 µM Ac-VEID-CHO and 10 µM Z-DEVD-fmk, respectively. Interestingly, inhibition of autophagy by 7-10 mM 3-methyl-adenine increased the rate of cell death. Immunohistochemistry data, caspase activation and caspase inhibition data suggest that axotomy of neonatal retinal ganglion cells triggers the intrinsic apoptotic pathway, which, in turn, is counteracted by a pro-survival autophagic response, demonstrated by electron microscopy profiles and pharmacological autophagy inhibitor.

  12. Aloe emodin induces G2/M cell cycle arrest and apoptosis via activation of caspase-6 in human colon cancer cells.

    Science.gov (United States)

    Suboj, Priya; Babykutty, Suboj; Srinivas, Priya; Gopala, Srinivas

    2012-01-01

    Aloe emodin (AE), a natural anthraquinone, is reported to have antiproliferative activity in various cancer cell lines. In this study, we analyzed the molecular mechanisms involved in the growth-inhibitory activity of this hydroxyanthraquinone in colon cancer cell, WiDr. In our observation AE inhibited cell proliferation by arresting the cell cycle at the G2/M phase and inhibiting cyclin B1. AE appreciably induced cell death specifically through the induction of apoptosis and by activating caspases 9/6. Apoptotic execution was found to be solely dependent on caspase-6 rather than caspase-3 or caspase-7. This is the first study indicating that the AE induces apoptosis specifically through the activation of caspase-6. PMID:22343391

  13. Neuroprotective effects of M826, a reversible caspase-3 inhibitor, in the rat malonate model of Huntington's disease.

    Science.gov (United States)

    Toulmond, Sylvie; Tang, Keith; Bureau, Yves; Ashdown, Helen; Degen, Sarah; O'Donnell, Ruth; Tam, John; Han, Yongxin; Colucci, John; Giroux, André; Zhu, Yanxia; Boucher, Mathieu; Pikounis, Bill; Xanthoudakis, Steven; Roy, Sophie; Rigby, Michael; Zamboni, Robert; Robertson, George S; Ng, Gordon Y K; Nicholson, Donald W; Flückiger, Jean-Pierre

    2004-02-01

    1. Caspases, key enzymes in the apoptosis pathway, have been detected in the brain of HD patients and in animal models of the disease. In the present study, we investigated the neuroprotective properties of a new, reversible, caspase-3-specific inhibitor, M826 (3-([(2S)-2-[5-tert-butyl-3-[[(4-methyl-1,2,5-oxadiazol-3-yl)methyl]amino]-2-oxopyrazin-1(2H)-yl]butanoyl]amino)-5-[hexyl(methyl)amino]-4-oxopentanoic acid), in a rat malonate model of HD. 2. Pharmacokinetic and autoradiography studies after intrastriatal (i.str.) injection of 1.5 nmol of M826 or its tritiated analogue [(3)H]M826 indicated that the compound diffused within the entire striatum. The elimination half-life (T(1/2)) of M826 in the rat striatum was 3 h. 3. I.str. injection of 1.5 nmol of M826 10 min after malonate infusion induced a significant reduction (66%) in the number of neurones expressing active caspase-3 in the ipsilateral striatum. 4. Inhibition of active caspase-3 translated into a significant but moderate reduction (39%) of the lesion volume, and of cell death (24%), 24 h after injury. The efficacy of M826 at inhibiting cell death was comparable to that of the noncompetitive NMDA receptor antagonist MK801. 5. These data provide in vivo proof-of-concept of the neuroprotective effects of reversible caspase-3 inhibitors in a model of malonate-induced striatal injury in the adult rat. PMID:14744804

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

    International Nuclear Information System (INIS)

    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.

  15. Osthole prevents anti-Fas antibody-induced hepatitis in mice by affecting the caspase-3-mediated apoptotic pathway.

    Science.gov (United States)

    Okamoto, Toshihiro; Kawasaki, Toru; Hino, Okio

    2003-02-15

    Fas (Apo-1/CD95) ligand, which is a type II membrane protein, is a major inducer of apoptosis. Osthole is a coumarin derivative present in medicinal plants. The effect of osthole on hepatitis induced by anti-Fas antibody in mice was studied. Pretreatment of mice with osthole (10, 50, and 100 mg/kg, i.p.) prevented the elevation of plasma alanine aminotransferase (ALT) caused by anti-Fas antibody (175 microg/kg, i.v.). Administration of osthole to mice even at a dose of 10 mg/kg significantly inhibited of anti-Fas antibody-induced elevation of plasma ALT. Capase-3 is a cysteine protease, and treatment of mice with anti-Fas antibody caused an elevation of caspase-3 activity at 3.5 and 6 hr. Pretreatment of mice with osthole (100 mg/kg, i.p.) inhibited the elevation of caspase-3 activity caused by anti-Fas antibody. However, the addition of osthole (up to 10(-4)M) to a liver cytosol fraction isolated from mice treated with anti-Fas antibody did not inhibit caspase-3 activity in vitro. Thus, treatment of mice with osthole inhibited caspase-3 activity by an effect upstream of caspase-3 activation. The livers of mice treated with anti-Fas antibody contained apoptotic and dead cells; osthole attenuated the development of this apoptosis and cell death. The present results show that osthole prevented anti-Fas antibody-induced hepatitis by inhibiting the Fas-mediated apoptotic pathway. PMID:12566097

  16. Effect of Caspase Inhibitor Ac-DEVD-CHO on Apoptosis of Vascular Smooth Muscle Cells Induced by Artesunate

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    Jingwen Zhang

    2014-05-01

    Full Text Available Numerous studies have shown that the proliferation and apoptosis of vascular smooth muscle cells play a key role in restenosis. Artesunate is a triterpenoid with a peroxide structure and its antimalarial, antitumor, and antiangiogenetic activities can inhibit the proliferation and apoptosis of multifarious cells. Apoptosis is caused by the activation of a series of intracellular proteolytic enzymes, among which caspase-dependent apoptosis was the earliest to be recognized. The purpose of this article is to study the effects of caspase-3 inhibitor Ac-DEVD-CHO on proliferation and apoptosis of vascular smooth muscle cells induced by Artesunate and to explore the mechanism of Artesunate-induced apoptosis of vascular smooth muscle cells. By using the method based on methyl thiazolyl tetrazolium to observe the effects of Artesunate on the growth and proliferation of vascular smooth muscle cells; observing the change in cell shape before and after Artesunate administration by transmission electron microscopy; detecting the changes in cell cycle and apoptosis rates before and after drug administration by flow cytometry; detecting the activity of caspase-3 in the caspase apoptosis pathway by the Western Blot method, we found that Artesunate inhibits the growth and proliferation of vascular smooth muscle cells in a dose- and time-dependent manner within the concentration range of 7.5–120 μg/mL, and the inhibition rate of Artesunate can be as high as 89.49 % at a concentration of 120 μg/mL after acting for 72 hours; vascular smooth muscle cells show a typical apoptosis peak due to the effects of higher concentration of Artesunate. Compared with the control group, the higher-concentration group shows major variability, Ac-DEVD-CHO, however, can significantly decrease this induction; it has been detected by Western Blot that Artesunate can induce caspase-3 activity dramatically in vascular smooth muscle cells, but this activation may be remarkably

  17. Unveiling interactions among mitochondria, caspase-like proteases, and the actin cytoskeleton during plant programmed cell death (PCD.

    Directory of Open Access Journals (Sweden)

    Christina E N Lord

    Full Text Available Aponogeton madagascariensis produces perforations over its leaf surface via programmed cell death (PCD. PCD begins between longitudinal and transverse veins at the center of spaces regarded as areoles, and continues outward, stopping several cells from these veins. The gradient of PCD that exists within a single areole of leaves in an early stage of development was used as a model to investigate cellular dynamics during PCD. Mitochondria have interactions with a family of proteases known as caspases, and the actin cytoskeleton during metazoan PCD; less is known regarding these interactions during plant PCD. This study employed the actin stain Alexa Fluor 488 phalloidin, the actin depolymerizer Latrunculin B (Lat B, a synthetic caspase peptide substrate and corresponding specific inhibitors, as well as the mitochondrial pore inhibitor cyclosporine A (CsA to analyze the role of these cellular constituents during PCD. Results depicted that YVADase (caspase-1 activity is higher during the very early stages of perforation formation, followed by the bundling and subsequent breakdown of actin. Actin depolymerization using Lat B caused no change in YVADase activity. In vivo inhibition of YVADase activity prevented PCD and actin breakdown, therefore substantiating actin as a likely substrate for caspase-like proteases (CLPs. The mitochondrial pore inhibitor CsA significantly decreased YVADase activity, and prevented both PCD and actin breakdown; therefore suggesting the mitochondria as a possible trigger for CLPs during PCD in the lace plant. To our knowledge, this is the first in vivo study using either caspase-1 inhibitor (Ac-YVAD-CMK or CsA, following which the actin cytoskeleton was examined. Overall, our findings suggest the mitochondria as a possible upstream activator of YVADase activity and implicate these proteases as potential initiators of actin breakdown during perforation formation via PCD in the lace plant.

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

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    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.

  19. Effects of nerve growth factor on the expression of caspase-12 of nerve cells in cerebral ischemia/reperfusion area

    Institute of Scientific and Technical Information of China (English)

    Jiping Yang; Huaijun Liu; Ying Li; Yan Liu; Haiqing Yang

    2006-01-01

    BACKGROUND: Researches suggest that cascade reaction of cysteine protease mediated by caspase-12 can cause apoptosis after cerebral ischemia/reperfusion injury;however, nerve growth factor (NGF) can reduce apoptosis through inhibiting activation of that reaction.OBJECTTVE: To observe the effect of NGF on the expression of caspase-12 in brain tissue of rabbits with cerebral ischemia/reperfusion injury, and elucidate the protective mechanism of NGF on neural apoptosis induced by cerebral ischemia/reperfusion injury.DESIGN: Randomized controlled animal study.SETTING: Department of Image, Second Hospital, Hebei Medical University.MATERIALS: A total of 26 healthy New Zealand rabbits, of clean grade, aged 4.5-5 months, weighing (2.6±0.2) kg, were selected in this study. Reagents: NGF (Xiamen Beida Zhilu Biotechnology Co., Ltd.);caspase-12 (Santa Cruz Biotechnology Company, USA, clone number: SC-12395); caspase-3 (Santa Cruz Biotechnology Company, USA, clone number: SC-7272); biotin-antibody Ⅱ and ABC compound (Wuhan Boster Company); in situ end-labeling (ISEL, Beijing Zhongshan Company).METHODS: The experiment was carried out in the Laboratories of Nerve Molecule Image Science and Neurology of the Second Hospital of Hebei Medical University from May to August 2005. ① All animals were randomly divided into three groups. Ischemia/reperfusion (I/R) group (n=10): Left middle cerebral artery (MCA) was blocked for 2 hours and then blooded for 2 hours in order to establish focal cerebral ischemia/reperfusion models. Sham operation group (n=6): Cork was inserted with 3 cm in depth, and then pulled to common carotid artery. Other procedures were as the same as those in ischemia/reperfusion group.Treatment group (n=10): After modeling, 400 AU (16 μg/L) NGF was inserted into cerebral infarction focus sham operation group and at 3 days after reperfusion in other two groups. In addition, contents of caspase-12 and caspase-3 were measured with immunohistochemical technique; mean

  20. Induction of Caspase 8 by 5-Azacytidine renders NB cells sensitive to TRAIL%5氮杂胞苷诱导NB细胞Caspase 8表达及对TRAIL敏感性的研究

    Institute of Scientific and Technical Information of China (English)

    佟海侠; 张锦华; 张继红; 陆春伟

    2005-01-01

    目的:探讨5氮杂胞苷(5-Azacytidine)对肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor related apoptosis inducing ligand,TRAIL)诱导神经母细胞瘤(neuroblastoma,NB)细胞株SH-SY5Y凋亡的影响及其发生机制.方法:应用RT-PCR方法检测5氮杂胞苷作用前后SY5Y细胞Caspase 8的表达;应用四甲基偶氮唑蓝(MTT)比色法及流式细胞仪(FCM)检测TRAIL、5氮杂胞苷+TRAIL、5氮杂胞苷+Caspase 8抑制剂+TRAIL对SY5Y细胞生长及凋亡的影响.结果:SY5Y细胞不表达Caspase 8,5氮杂胞苷作用1、3和5d的SY5Y细胞Caspase 8表达逐渐增加;SY5Y细胞对TRAIL不敏感,经5氮杂胞苷诱导表达Caspase 8的 SY5Y 细胞对TRAIL敏感,并呈现一定的时间和剂量依赖性.联合用药组细胞凋亡率分别为(28.6±4.0)%、(38.5±4.2)%和(42.3±6.2)%,与单独用TRAIL组[(9.5±2.0)%、(10.7±1.8)%和(11.2±1.5)%]比较,差异有统计学意义,F值分别为41.131、45.014和49.405,P值均为0.000;Caspase 8抑制剂组细胞凋亡率分别为(11.8±2.0)%、(13.5±2.9)%和(15.1±3.4)%,与相应浓度的5氮杂胞苷+TRAIL组相比,细胞凋亡率明显降低,F值分别为41.131、45.014和49.405,P值均为0.000,说明Caspase 8抑制剂可明显降低TRAIL对表达Caspase 8的SY5Y细胞的杀伤作用.结论:5氮杂胞苷能增强SY5Y细胞对TRAIL的敏感性,其发生机制可能与Caspase 8表达上调有关.

  1. 5氮杂胞苷联合干扰素-γ上调神经母细胞瘤细胞Caspase 8表达的研究%5-Azacytidine and IFN-gamma cooperate to upregulate Caspase-8 expression in neuroblastoma cells

    Institute of Scientific and Technical Information of China (English)

    佟海侠; 陆春伟; 王秋实; 王立维

    2011-01-01

    Objective : To investigate if induction of Caspase 8 by demethylation agent 5 - Azacytidine( 5AZA ) and/or γ - interferon ( IFNγ ) renders neuroblastoma ( NB ) cells sensitive to tumor necrosis factor related apoptosis inducing ligand( TRAIL ). Methods: Caspase 8 expression was monitored by Western blot analysis. The effects of 5AZA,IFNγ, TRAIL, 5AZA and/or IFNγ +TRAIL, 5AZA and/or IFNγ + Caspase 8 inhibitor + TRAIL on the growth and apoptosis of NB cells were detected with flow cytometry. The relative Caspase 8 activity was measured with colorimetric assay. Results : Caspase 8 was undetectable in SH - SY5Y( SY5Y ) cells which were resistant to TRAIL , but an increased expression of Caspase 8 was found after treatment with 5AZA and/or IFNγ. The level of Caspase 8 protein in IFNγ +5AZA group was higher than that in single treatment group. Moreover, TRAIL combined with 5AZA and/or IFNγ induced apoptosis in SY5Y cells. The relative Caspase 8 activity of SY5Y cells in SAZA and/or IFNγ + TRAIL group was significantly higher than those of control group, 5AZA group, IFNγ group, TRAIL group and Caspase 8 inhibitor group. Conclusion :5 - AZA and IFNγ could cooperate to upregulate Caspase - 8 expression in NB cells. TRAIL induced apoptosis in NB cells expressing Caspase 8 with an increase of relative Caspase 8 activity.%目的:应用去甲基药5氮杂胞苷(5AZA)和/或干扰素(IFNγ)诱导神经母细胞瘤(neuroblastoma,NB)细胞Caspase 8的表达,并观察是否可以恢复NB细胞对肿瘤坏死因子相关凋亡诱导配体(TRAIL)的敏感性.方法:应用Western blot方法检测5AZA和/或IFNγ作用前后SY5Y细胞Caspase 8蛋白的表达;应用流式细胞术检测5AZA 、IFNγ、TRAIL、5AZA 和/或IFNγ+TRAIL、5AZA和/或IFNγ+Caspase 8抑制剂zIETD-FMK+TRAIL对NB细胞生长及凋亡的影响;应用比色法测定Caspase 8相对活性.结果:对TRAIL耐药的SY5Y细胞不表达Caspase 8,经5AZA 和/或IFNγ处理后Caspase 8 蛋白

  2. SRSF1 (SRp30a) regulates the alternative splicing of caspase 9 via a novel intronic splicing enhancer affecting the chemotherapeutic sensitivity of non-small cell lung cancer cells

    OpenAIRE

    Shultz, Jacqueline C.; Rachel W Goehe; Murudkar, Charuta S.; Wijesinghe, Dayanjan S.; Mayton, Eric K.; Massiello, Autumn; Hawkins, Amy J.; Mukerjee, Prabhat; Pinkerman, Ryan L.; Park, Margaret A; Chalfant, Charles E.

    2011-01-01

    Increasing evidence points to the functional importance of alternative splice variations in cancer pathophysiology with the alternative pre-mRNA processing of caspase 9 as one example. In this study, we delve into the underlying molecular mechanisms that regulate the alternative splicing of caspase 9. Specifically, the pre-mRNA sequence of caspase 9 was analyzed for RNA cis-elements known to interact with SRSF1, a required enhancer for caspase 9 RNA splicing. This analysis revealed thirteen p...

  3. Ceramide induces release of mitochondrial proapop-totic proteins in caspase-dependent and -independent manner in HT-29 cells

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    In this study, the release of mitochondrial proapoptotic intermembrane space proteins induced by ex-ogenous C2-ceramide in human colon carcinoma (HT-29) cell line was investigated. HT-29 cells were treated with 12.5, 25 and 50 μmol/L C2-ceramide in vitro. Flow cytometer was used to detect the mito-chondrial membrane potential (ΔΨm). Subcellular fractions were extracted by Mitochondrial/Cytosol Fractionation Kit after C2-ceramide treatment for 24 h. SDS-PAGE was used to determine the level of cytochrome c (Cyt c), high temperature requirement A2 (HtrA2) and second mitochondrial-derived ac-tivator of caspases (Smac) released from mitochondria, the expression of X-linked inhibitor of apop-tosis protein (XIAP) and caspase-3 for 24 h. The results showed that ΔΨm began to decrease from 6 h after 25 and 50 μmol/L C2-ceramide treatment (P<0.05) and cyclosporin A (CsA) could inhibit the col-lapse of ΔΨm through regulating mitochondrial membrane permeability transition pore. There was no effect of C2-ceramide on the expression of Cyt c, HtrA2 and Smac in the total levels. 12.5, 25 and 50 μmol/L C2-ceramide could induce Cyt c, HtrA2 and Smac to release from mitochondria to cytosol and down-regulate the expression of XIAP (P<0.05). Also there was expression of cleaved caspase-3 with C2-ceramide treatment. After the treatment with caspase inhibitor, C2-ceramide still induced the release of Cyt c and HtrA2, but Smac did not. Therefore, C2-ceramide could induce apoptosis of HT-29 cells through the mitochondria pathway. The release of Cyt c, HtrA2 and Smac from mitochondria did not occur via the same mechanism, the release of Cyt c and HtrA2 was caspase-independent and the re-lease of Smac was caspase-dependent.

  4. Ceramide induces release of mitochondrial proapoptotic proteins in caspase-dependent and -independent manner in HT-29 cells

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    In this study, the release of mitochondrial proapoptotic intermembrane space proteins induced by exogenous C2-ceramide in human colon carcinoma (HT-29) cell line was investigated. HT-29 cells were treated with 12.5, 25 and 50 μmol/L C2-ceramide in vitro. Flow cytometer was used to detect the mitochondrial membrane potential (△Ψm). Subcellular fractions were extracted by Mitochondrial/Cytosol Fractionation Kit after C2-ceramide treatment for 24 h. SDS-PAGE was used to determine the level of cytochrome c (Cyt c), high temperature requirement A2 (HtrA2) and second mitochondrial-derived activator of caspases (Smac) released from mitochondria, the expression of X-linked inhibitor of apoptosis protein (XIAP) and caspase-3 for 24 h. The results showed that △Ψm began to decrease from 6 h after 25 and 50 μmol/L C2-ceramide treatment (P<0.05) and cyclosporin A (CsA) could inhibit the collapse of △Ψm through regulating mitochondrial membrane permeability transition pore. There was no effect of C2-ceramide on the expression of Cyt c, HtrA2 and Smac in the total levels. 12.5, 25 and 50 μmol/L C2-ceramide could induce Cyt c, HtrA2 and Smac to release from mitochondria to cytosol and down-regulate the expression of XIAP (P<0.05). Also there was expression of cleaved caspase-3 with C2-ceramide treatment. After the treatment with caspase inhibitor, C2-ceramide still induced the release of Cyt c and HtrA2, but Smac did not. Therefore, C2-ceramide could induce apoptosis of HT-29 cells through the mitochondria pathway. The release of Cyt c, HtrA2 and Smac from mitochondria did not occur via the same mechanism, the release of Cyt c and HtrA2 was caspase-independent and the release of Smac was caspase-dependent.

  5. Oridonin induces apoptosis of HeLa cells via altering expres sion of Bcl-2/Bax and activating caspase-3/ICAD pathway

    Institute of Scientific and Technical Information of China (English)

    Chun-ling ZHANG; Li-jun WU; Shin-ichi TASHIRO; Satoshi ONODERA; Takashi IKEJIMA

    2004-01-01

    AIM: To study the mechanisms by which oridonin inhibited HeLa cell growth in vitro. METHODS: Viability of oridonin-induced HeLa cells was measured by MTT assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis.Caspase activity was assayed using fiuorometric protease assay. ICAD, Bcl-2, and Bax proteins expression were detected by Western blot analysis. RESULTS: Oridonin induced oligonucleosomal fragmentation of DNA and increased caspase-3 activity, on the other hand, reduced the expression of inhibitor of caspase-3-activated DNase (ICAD), a caspase-3 substrate, at 12 h in HeLa cells. Oridonin-induced DNA fragmentation, caspase-3 activation and down-regulation of ICAD expression were effectively inhibited by a caspase-3 inhibitor, z-DEVD-fmk (z-AspGlu-Val-Asp-fmk). However, pretreatment with an inhibitor of poly (ADP-ribose) polymerase (PARP), 3, 4-dihydro5-[4-(1-piperidinyl)butoxy]-1 (2H)-isoquinolinone (DPQ), did not suppress oridonin-induced HeLa cell death. In addition, oridonin-induced apoptosis was associated with an increase in the expression of the apoptosis inducer Bax, and a significant reduction in expression of the apoptosis suppressor Bcl-2 in mitochondria. CONCLUSION:Oridonin induces HeLa cells apoptosis by altering balance of Bcl-2 and Bax protein expression and activation of caspase-3/ICAD pathway.

  6. Harmol induces apoptosis by caspase-8 activation independently of Fas/Fas ligand interaction in human lung carcinoma H596 cells.

    Science.gov (United States)

    Abe, Akihisa; Yamada, Hiroyuki

    2009-06-01

    The beta-carboline alkaloids are naturally existing plant substances. It is known that these alkaloids have a wide spectrum of neuropharmacological, psychopharmacological, and antitumor effects. Therefore, they have been traditionally used in oriental medicine for the treatment of various diseases including cancers and malaria. In this study, harmol and harmalol, which are beta-carboline alkaloids, were examined for their antitumor effect on human lung carcinoma cell lines, and structure-activity relationship was also investigated. H596, H226, and A549 cells were treated with harmol and harmalol, respectively. Apoptosis was induced by harmol only in H596 cells. In contrast, harmalol had negligible cytotoxicity in three cell lines. Harmol induced caspase-3, caspase-8, and caspase-9 activities and caspase-3 activities accompanied by cleavage of poly-(ADP-ribose)-polymerase. Furthermore, harmol treatment decreased the native Bid protein, and induced the release of cytochrome c from mitochondria to cytosol. The apoptosis induced by harmol was completely inhibited by caspase-8 inhibitor and partially inhibited by caspase-9 inhibitor. The antagonistic antibody ZB4 blocked Fas ligand-induced apoptosis, but had no effect on harmol-induced apoptosis. Harmol had no significant effect on the expression of Fas. In conclusion, our results showed that the harmol could cause apoptosis-inducing effects in human lung H596 cells through caspase-8-dependent pathway but independent of Fas/Fas ligand interaction. PMID:19318910

  7. Sphingosine 1-phosphate inhibits activation of caspases that cleave poly(ADP-ribose) polymerase and lamins during Fas- and ceramide-mediated apoptosis in Jurkat T lymphocytes.

    Science.gov (United States)

    Cuvillier, O; Rosenthal, D S; Smulson, M E; Spiegel, S

    1998-01-30

    Ceramide, a sphingolipid generated by the hydrolysis of membrane-associated sphingomyelin, appears to play a role as a gauge of apoptosis. A further metabolite of ceramide, sphingosine 1-phosphate (SPP), prevents ceramide-mediated apoptosis, and it has been suggested that the balance between intracellular ceramide and SPP levels may determine the cell fate (Cuvillier, O., Pirianov, G, Kleuser, B., Vanek, P. G., Coso, O. A., Gutkind, J. S., and Spiegel, S. (1996) Nature 381, 800-803). Here, we investigated the role of SPP and the protein kinase C activator, phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), in the caspase cascade leading to the proteolysis of poly(ADP-ribose) polymerase (PARP) and lamins. In Jurkat T cells, Fas ligation or addition of exogenous C2-ceramide induced activations of caspase-3/CPP32 and caspase-7/Mch3 followed by PARP cleavage, effects that can be blocked either by SPP or TPA. Furthermore, both SPP and TPA inhibit the activation of caspase-6/Mch2 and subsequent lamin B cleavage. Ceramide, in contrast to Fas ligation, did not induce activation of caspase-8/FLICE and neither SPP nor TPA were able to prevent this activation. Thus, SPP, likely generated via protein kinase C-mediated activation of sphingosine kinase, suppresses the apoptotic pathway downstream of FLICE but upstream of the executioner caspases, caspase-3, -6, and -7. PMID:9446602

  8. PLGA-carbon nanotube conjugates for intercellular delivery of caspase-3 into osteosarcoma cells.

    Directory of Open Access Journals (Sweden)

    Qingsu Cheng

    Full Text Available Cancer has arisen to be of the most prominent health care issues across the world in recent years. Doctors have used physiological intervention as well as chemical and radioactive therapeutics to treat cancer thus far. As an alternative to current methods, gene delivery systems with high efficiency, specificity, and safety that can reduce side effects such as necrosis of tissue are under development. Although viral vectors are highly efficient, concerns have arisen from the fact that viral vectors are sourced from lethal diseases. With this in mind, rod shaped nano-materials such as carbon nanotubes (CNTs have become an attractive option for drug delivery due to the enhanced permeability and retention effect in tumors as well as the ability to penetrate the cell membrane. Here, we successfully engineered poly (lactic-co-glycolic (PLGA functionalized CNTs to reduce toxicity concerns, provide attachment sites for pro-apoptotic protein caspase-3 (CP3, and tune the temporal release profile of CP3 within bone cancer cells. Our results showed that CP3 was able to attach to functionalized CNTs, forming CNT-PLGA-CP3 conjugates. We show this conjugate can efficiently transduce cells at dosages as low as 0.05 μg/ml and suppress cell proliferation up to a week with no further treatments. These results are essential to showing the capabilities of PLGA functionalized CNTs as a non-viral vector gene delivery technique to tune cell fate.

  9. Dual role of the caspase enzymes in satellite cells from aged and young subjects.

    Science.gov (United States)

    Fulle, S; Sancilio, S; Mancinelli, R; Gatta, V; Di Pietro, R

    2013-01-01

    Satellite cell (SC) proliferation and differentiation have critical roles in skeletal muscle recovery after injury and adaptation in response to hypertrophic stimuli. Normal ageing hinders SC proliferation and differentiation, and is associated with increased expression of a number of pro-apoptotic factors in skeletal muscle. In light of previous studies that have demonstrated age-related altered expression of genes involved in SC antioxidant and repair activity, this investigation was aimed at evaluating the incidence of apoptotic features in human SCs. Primary cells were obtained from vastus lateralis of nine young (27.3±2.0 years old) and nine old (71.1±1.8 years old) subjects, and cultured in complete medium for analyses at 4, 24, 48, and 72 h. Apoptosis was assessed using AnnexinV/propidium iodide staining, the terminal deoxynucleotidyl transferase dUTP nick-end labelling technique, RT-PCR, DNA microarrays, flow cytometry, and immunofluorescence analysis. There was an increased rate of apoptotic cells in aged subjects at all of the experimental time points, with no direct correlation between AnnexinV-positive cells and caspase-8 activity. On the other hand, CASP2, CASP6, CASP7, and CASP9 and a number of cell death genes were upregulated in the aged SCs. Altogether, our data show age-related enhanced susceptibility of human SCs to apoptosis, which might be responsible for their reduced response to muscle damage. PMID:24336075

  10. Equine torovirus (BEV induces caspase-mediated apoptosis in infected cells.

    Directory of Open Access Journals (Sweden)

    Ana M Maestre

    Full Text Available Toroviruses are gastroenteritis causing agents that infect different animal species and humans. To date, very little is known about how toroviruses cause disease. Here, we describe for the first time that the prototype member of this genus, the equine torovirus Berne virus (BEV, induces apoptosis in infected cells at late times postinfection. Observation of BEV infected cells by electron microscopy revealed that by 24 hours postinfection some cells exhibited morphological characteristics of apoptotic cells. Based on this finding, we analyzed several apoptotic markers, and observed protein synthesis inhibition, rRNA and DNA degradation, nuclear fragmentation, caspase-mediated cleavage of PARP and eIF4GI, and PKR and eIF2α phosphorylation, all these processes taking place after peak virus production. We also determined that both cell death receptor and mitochondrial pathways are involved in the apoptosis process induced by BEV. BEV-induced apoptosis at late times postinfection, once viral progeny are produced, could facilitate viral dissemination in vivo and contribute to viral pathogenesis.

  11. Automatic morphological subtyping reveals new roles of caspases in mitochondrial dynamics.

    Directory of Open Access Journals (Sweden)

    Jyh-Ying Peng

    2011-10-01

    Full Text Available Morphological dynamics of mitochondria is associated with key cellular processes related to aging and neuronal degenerative diseases, but the lack of standard quantification of mitochondrial morphology impedes systematic investigation. This paper presents an automated system for the quantification and classification of mitochondrial morphology. We discovered six morphological subtypes of mitochondria for objective quantification of mitochondrial morphology. These six subtypes are small globules, swollen globules, straight tubules, twisted tubules, branched tubules and loops. The subtyping was derived by applying consensus clustering to a huge collection of more than 200 thousand mitochondrial images extracted from 1422 micrographs of Chinese hamster ovary (CHO cells treated with different drugs, and was validated by evidence of functional similarity reported in the literature. Quantitative statistics of subtype compositions in cells is useful for correlating drug response and mitochondrial dynamics. Combining the quantitative results with our biochemical studies about the effects of squamocin on CHO cells reveals new roles of Caspases in the regulatory mechanisms of mitochondrial dynamics. This system is not only of value to the mitochondrial field, but also applicable to the investigation of other subcellular organelle morphology.

  12. Sam68 is cleaved by caspases under apoptotic cell death induced by ionizing radiation

    International Nuclear Information System (INIS)

    The RNA-binding protein Sam68, a mitotic substrate of tyrosine kinases, has been reported to participate in the cell cycle, apoptosis, and signaling. In particular, overexpression of Sam68 protein is known to suppress cell growth and cell cycle progression in NIH3T3 cells. Although Sam68 is involved in many cellular activities, the function of Sam68, especially in response to apoptotic stimulation, is not well understood. In this study, we found that Sam68 protein is cleaved in immune cells undergoing apoptosis induced by γ-radiation. Moreover, we found that Sam68 cleavage was induced by apoptotic stimuli containing γ-radiation in a caspase-dependent manner. In particular, we showed that activated casepase-3, 7, 8 and 9 can directly cleave Sam68 protein through in vitro protease cleavage assay. Finally, we found that the knockdown of Sam68 attenuated γ-radiation-induced cell death and growth suppression. Conclusively, the cleavage of Sam68 is a new indicator for the cell damaging effects of ionizing radiation. (author)

  13. Reduced expression of PNUTS leads to activation of Rb-phosphatase and caspase-mediated apoptosis.

    Science.gov (United States)

    De Leon, Gabriel; Sherry, Tara C; Krucher, Nancy A

    2008-06-01

    There is abundant evidence that Retinoblastoma (Rb) activity is important in the control of cell proliferation and apoptosis. Reversible phosphorylation of the Rb protein that is carried out by cyclin dependent kinases and Protein phosphatase 1 (PP1) regulates its functions. A PP1 interacting protein, PNUTS (Phosphatase Nuclear Targeting Subunit) is proposed to be a regulator of Rb phosphorylation. In this study, PNUTS knockdown in MCF7, SKA and HCT116 cancer cells causes a reduction in viability due to increased apoptosis. However, normal cells (MCF10A breast and CCD-18Co colon) do not exhibit reduced viability when PNUTS expression is diminished. PNUTS knockdown has no effect in Rb-null Saos-2 cells. However, when Rb is stably expressed in Saos-2 cells, PNUTS knockdown reduces cell number. Knockdown of PNUTS in p53-/- HCT116 cells indicates that p53 is dispensable for the induction of apoptosis. Loss of PNUTS expression results in increased Rb-phosphatase activity and Rb dephosphorylation. E2F1 dissociates from Rb in cells depleted of PNUTS and the resulting apoptosis is dependent on caspase-8. These results indicate that Rb phosphorylation state can be manipulated by targeting Rb phosphatase activity and suggest that PNUTS may be a potential target for therapeutic pro-apoptotic strategies. PMID:18360108

  14. Proton pump inhibitors induce a caspase-independent antitumor effect against human multiple myeloma.

    Science.gov (United States)

    Canitano, Andrea; Iessi, Elisabetta; Spugnini, Enrico Pierluigi; Federici, Cristina; Fais, Stefano

    2016-07-01

    Multiple Myeloma (MM) is the second most common hematological malignancy and is responsive to a limited number of drugs. Unfortunately, to date, despite the introduction of novel drugs, no relevant increase in survival rates has been obtained. Proton pump inhibitors (PPIs) have been shown to have significant antitumor action as single agents as well as in combination with chemotherapy. This study investigates the potential anti-tumor effectiveness of two PPIs, Lansoprazole and Omeprazole, against human MM cells. We found that Lansoprazole exerts straightforward efficacy against myeloma cells, even at suboptimal concentrations (50 µM), while Omeprazole has limited cytotoxic action. The Lansoprazole anti-MM effect was mostly mediated by a caspase-independent apoptotic-like cytotoxicity, with only a secondary anti-proliferative action. This study provides clear evidence supporting the use of Lansoprazole in the strive against MM with an efficacy proven much higher than current therapeutical approaches and without reported side effects. It is however conceivable that, consistent with the results obtained in other human tumors, Lansoprazole may well be combined with existing anti-myeloma therapies with the aim to improve the low level of efficacy of the current strategies. PMID:27084522

  15. Smac Mimetic Bypasses Apoptosis Resistance in FADD- or Caspase-8-Deficient Cells by Priming for Tumor Necrosis Factor α-Induced Necroptosis

    Directory of Open Access Journals (Sweden)

    Bram Laukens

    2011-10-01

    Full Text Available Searching for new strategies to bypass apoptosis resistance, we investigated the potential of the Smac mimetic BV6 in Jurkat leukemia cells deficient in key molecules of the death receptor pathway. Here, we demonstrate for the first time that Smac mimetic primes apoptosis-resistant, FADD- or caspase-8-deficient leukemia cells for TNFα-induced necroptosis in a synergistic manner. In contrast to TNFα, Smac mimetic significantly enhances CD95-induced apoptosis in wild-type but not in FADD-deficient cells. Interestingly, Smac mimetic- and TNFα-mediated cell death occurs without characteristic features of apoptosis (i.e., caspase activation, DNA fragmentation in FADD-deficient cells. By comparison, Smac mimetic and TNFα trigger activation of caspase-8, -9, and -3 and DNA fragmentation in wild-type cells. Consistently, the caspase inhibitor zVAD.fmk fails to block Smac mimetic- and TNFα-triggered cell death in FADD- or caspase-8-deficient cells, while it confers protection in wild-type cells. By comparison, necrostatin-1, an RIP1 kinase inhibitor, abolishes Smac mimetic- and TNFα-induced cell death in FADD- or caspase-8-deficient. Thus, Smac mimetic enhances TNFα-induced cell death in leukemia cells via two distinct pathways in a context-dependent manner: it primes apoptosis-resistant cells lacking FADD or caspase-8 to TNFα-induced, RIP1-dependent and caspase-independent necroptosis, whereas it sensitizes apoptosis-proficient cells to TNFα-mediated, caspase-dependent apoptosis. These findings have important implications for the therapeutic exploitation of necroptosis as an alternative cell death program to overcome apoptosis resistance.

  16. Caspase-3、DIAPH-3蛋白在浸润性乳腺癌中的作用研究%Role of caspase-3 and DIAPH3 in invasive breast cancer

    Institute of Scientific and Technical Information of China (English)

    张玲玲; 张亚男; 耿翠芝; 丁妍; 杨会钗; 马力; 刘月平

    2016-01-01

    目的:探讨浸润性乳腺癌组织中Caspase-3、DIAPH-3蛋白的表达与临床病理特征及预后的关系。方法采用免疫组织化学EliVision法检测Caspase-3、DIAPH-3在浸润性乳腺癌(=240)及癌旁组织(=44)中的表达情况,并分析其与临床病理特征及预后的关系。结果 Caspase-3、DIAPH-3在浸润性乳腺癌组织中表达的阳性率(36.7%和40.8%)低于癌旁组织(81.8%和86.7%),<0.05。Caspase-3的阳性表达率随淋巴结转移数的增多、肿瘤长径的增加、临床分期的提高、脉管瘤栓的产生及P53的阳性表达的增强而降低。DIAPH-3的表达随淋巴结转移数的增加、脉管瘤栓的产生及临床分期的提高而降低。Kaplan- Meier单因素生存分析显示Caspase-3和DIAPH-3蛋白阳性组患者的5年生存率(95.5%、95.8%),高于阴性组(85.5%、84.5%)。COX多因素生存分析显示DIAPH-3阴性表达与患者不良预后正相关(O?=1.687,95%CI:1.032,2.758)。结论浸润性乳腺癌组织中Caspase-3、DIAPH-3蛋白表达均低于癌旁正常乳腺组织,两者均与乳腺癌的进展及预后密切相关,但两者之间无关联;DIAPH-3的低表达提示患者预后不良,可能是预测乳腺癌患者预后的独立因素之一。%Objective To study the possible association of the expressions of caspase-3 and diaphanous related formin 3 (DIAPH3) protein with clinicopathological features and prognosis in invasive breast cancer. Methods The expressions of caspase-3 and DIAPH3 protein in invasive breast cancer tissues from 240 cases and para-cancerous tissues from 44 cases were detected by immunohistochemical EliVision method, and then their relationships with the clinicopathological features and prognosis were analyzed. Results The positive-expression rates of caspase-3 and DIAPH3 protein in the invasive breast cancer tissues (36.7% and 40.8%respectively) were significantly lower than those in the para-cancerous tissues

  17. The caspase 3-dependent apoptotic effect of pycnogenol in human oral squamous cell carcinoma HSC-3 cells.

    Science.gov (United States)

    Yang, In-Hyoung; Shin, Ji-Ae; Kim, Lee-Han; Kwon, Ki Han; Cho, Sung-Dae

    2016-01-01

    In the present study, the apoptotic effect of pycnogenol and its molecular mechanism in human oral squamous cell carcinoma HSC-3 cells were investigated. Pycnogenol significantly inhibited the viability of HSC-3 cells and suppressed neoplastic cell transformation in HSC-3 cells and TPA-treated JB6 cells. It caused caspase-dependent apoptosis evidenced by the increase in cleaved poly (ADP-ribose) polymerase and caspase 3 in a dose-dependent manner. Pycnogenol increased Bak protein by enhancing its protein stability whereas other Bcl-2 family members were not altered. In addition, the treatment with pycnogenol led to the production of reactive oxygen species and N-acetyl-l-cysteine almost blocked pycnogenol-induced reactive oxygen species generation. Taken together, these findings suggest that pycnogenol may be a potential candidate for the chemoprevention or chemotherapy of human oral cancer. PMID:26798196

  18. The caspase proteolytic system in callipyge and normal lambs in longissimus, semimembranosus, and infraspinatus muscles during postmortem storage.

    Science.gov (United States)

    Kemp, C M; King, D A; Shackelford, S D; Wheeler, T L; Koohmaraie, M

    2009-09-01

    The objective of this experiment was to determine whether the caspase proteolytic system has a role in postmortem tenderization. Six ewes and 6 wethers that were noncarriers and 6 ewes and 6 wethers that were expressing the callipyge gene were used for this study. Caspase activities were determined in LM at 7 different time points during the postmortem storage period: 0 h, 4 h, 8 h, 24 h, 2 d, 7 d, and 21 d and in semimembranosus (SM) and infraspinatus (IS) muscles at 0 h, 8 h, 24 h, and 7 d from callipyge and noncallipyge (normal) lambs. Calpastatin activity was determined at 0 h, 2 d, 7 d, and 21 d and slice shear force measured at 2, 7, and 21 d in the LM. Calpastatin activity and slice shear force were greater in LM from callipyge lambs than normal lambs at each time point (P tenderization, but they may have some role through calpastatin degradation. PMID:19420232

  19. Cancer during pregnancy alters the activity of rat placenta and enhances the expression of cleaved PARP, cytochrome-c and caspase 3

    International Nuclear Information System (INIS)

    The presence of cancer makes it difficult to predict the progress of pregnancy and can be deleterious to the maternal-foetal relationship. Apoptosis may affect a range of placental functions and result in the retardation of foetal growth. In this work, we investigated the placental alterations produced by tumour growth and the effects on the expression of apoptotic factors in placental tissue. Adult female Wistar rats (90 days old, n = 54) were allocated to control (C), tumour-bearing (W), or ascitic fluid-injected (A) groups and were killed on the 16th, 19th or 21st day of pregnancy. Placental tissues were analysed using biochemical and histochemical assays. The placental protein content and glutathione-S-transferase activity were decreased in groups W and A. Histochemical analysis showed an increase in the number of cells with cleaved PARP, caspase 3 and cytochrome-c in groups W and A, indicating that the tumour growth clearly damaged placental tissue and affected the levels of apoptotic factors. These results were confirmed by western blotting. Since trophoblastic cells are responsible for maintaining a normal placental function, the uncontrolled death of these cells in response to tumour cell growth or substances derived from ascitic fluid could have a negative impact on foetal development. Further knowledge of these events may help to preserve the foetus and placenta during development

  20. The structure of the CARD8 caspase-recruitment domain suggests its association with the FIIND domain and procaspases through adjacent surfaces.

    Science.gov (United States)

    Jin, Tengchuan; Huang, Mo; Smith, Patrick; Jiang, Jiansheng; Xiao, T Sam

    2013-05-01

    CARD8 plays crucial roles in regulating apoptotic and inflammatory signaling pathways through the association of its caspase-recruitment domain (CARD) with those of procaspase-9 and procaspase-1. The CARD8 CARD has also been predicted to form an intramolecular complex with its FIIND domain. Here, the first crystal structure of the CARD8 CARD is reported; it adopts a six-helix bundle fold with a unique conformation of the α6 helix that is described here for the first time. The surface of the CARD8 CARD displays a prominent acidic patch at its α2, α3 and α5 helices that may interact with the procaspase-9 CARD, whereas an adjacent charged surface at its α3 and α4 helices may associate with the CARD8 FIIND domain without interfering with the CARD-CARD interaction. This suggests that the function of CARD8 may be regulated by both intramolecular and intermolecular interactions involving electrostatic attractions. PMID:23695559

  1. Single cell analysis of caspase-3 in apoptotic and non-apoptotic cells during mouse limb development

    Czech Academy of Sciences Publication Activity Database

    Adamová, Eva; Klepárník, Karel; Matalová, E.

    2014-01-01

    Roč. 3, - (2014), PP58. ISSN 2052-1219. [European Calcified Tissue Society Congress /41./. 17.05.2014-20.05.2014, Praha] R&D Projects: GA ČR GAP206/11/2377; GA ČR(CZ) GA14-28254S Institutional support: RVO:68081715 Keywords : single cell analysis * caspase-3 * mouse limb development Subject RIV: CB - Analytical Chemistry, Separation

  2. 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

  3. Cleavage of caspases-1, -3, -6, -8 and -9 substrates by proteases in skeletal muscles from mice undergoing cancer cachexia

    OpenAIRE

    Belizário, J E; Lorite, M. J.; Tisdale, M J

    2001-01-01

    A prominent feature of several type of cancer is cachexia. This syndrome causes a marked loss of lean body mass and muscle wasting, and appears to be mediated by cytokines and tumour products. There are several proteases and proteolytic pathways that could be responsible for the protein breakdown. In the present study, we investigated whether caspases are involved in the proteolytic process of skeletal muscle catabolism observed in a murine model of cancer cachexia (MAC16), in comparison with...

  4. BARF1 gene silencing triggers caspase-dependent mitochondrial apoptosis in Epstein-Barr virus-positive malignant cells

    Indian Academy of Sciences (India)

    Taznim Begam Mohd Mohidin; Ching Ching Ng

    2015-03-01

    Epstein-Barr virus (EBV)-encoded BARF1 (BamH1-A Rightward Frame-1) is expressed in EBV-positive malignancies such as nasopharyngeal carcinoma, EBV-associated gastric cancer, B-cell lymphoma and nasal NK/T-cell lymphoma, and has been shown to have an important role in oncogenesis. However, the mechanism by which BARF1 elicits its biological effects is unclear. We investigated the effects of BARF1 silencing on cell proliferation and apoptosis in EBV-positive malignant cells. We observed that BARF1 silencing significantly inhibits cell proliferation and induces apoptosis-mediated cell death by collapsing the mitochondrial membrane potential in AG876 and Hone-Akata cells. BARF1 knockdown up-regulates the expression of pro-apoptotic proteins and down-regulates the expression of anti-apoptotic proteins. In BARF1-down-regulated cells, the Bcl-2/BAX ratio is decreased. The caspase inhibitor z-VAD-fmk was found to rescue siBARF1-induced apoptosis in these cells. Immunoblot analysis showed significant increased levels of cleaved caspase 3 and caspase 9. We observed a significant increase in cytochrome c level as well as the formation of apoptosome complex in BARF1-silenced cells. In conclusion, siRNA-mediated BARF1 down-regulation induces caspase-dependent apoptosis via the mitochondrial pathway through modulation of Bcl-2/BAX ratio in AG876 and Hone-Akata cells. Targeting BARF1 using siRNA has the potential to be developed as a novel therapeutic strategy in the treatment of EBV-associated malignancies.

  5. Hsp90 is involved in apoptosis of Candida albicans by regulating the calcineurin-caspase apoptotic pathway.

    Science.gov (United States)

    Dai, BaoDi; Wang, Yan; Li, DeDong; Xu, Yi; Liang, RongMei; Zhao, LanXue; Cao, YongBing; Jia, JianHui; Jiang, YuanYing

    2012-01-01

    Candida albicans is the most common human fungal pathogen. Recent evidence has revealed the occurrence of apoptosis in C. albicans that is inducible by environmental stresses such as hydrogen peroxide, acetic acid, and amphotericin B. Apoptosis is regulated by the calcineurin-caspase pathway in C. albicans, and calcineurin is under the control of Hsp90 in echinocandin resistance. However, the role of Hsp90 in apoptosis of C. albicans remains unclear. In this study, we investigated the role of Hsp90 in apoptosis of C. albicans by using an Hsp90-compromised strain tetO-HSP90/hsp90 and found that upon apoptotic stimuli, including hydrogen peroxide, acetic acid or amphotericin B treatment, less apoptosis occurred, less ROS was produced, and more cells survived in the Hsp90-compromised strain compared with the Hsp90/Hsp90 wild-type strain. In addition, Hsp90-compromised cells were defective in up-regulating caspase-encoding gene CaMCA1 expression and activating caspase activity upon the apoptotic stimuli. Investigations on the relationship between Hsp90 and calcineurin revealed that activation of calcineurin could up-regulate apoptosis but could not further down-regulate apoptosis in Hsp90-compromised cells, indicating that calcineurin was downstream of Hsp90. Hsp90 inhibitor geldanamycin (GdA) could further decrease the apoptosis in calcineurin-pathway-defect strains, indicating that compromising Hsp90 function had a stronger effect than compromising calcineurin function on apoptosis. Collectively, this study demonstrated that compromised Hsp90 reduced apoptosis in C. albicans, partially through downregulating the calcineurin-caspase pathway. PMID:23028789

  6. Hsp90 is involved in apoptosis of Candida albicans by regulating the calcineurin-caspase apoptotic pathway.

    Directory of Open Access Journals (Sweden)

    BaoDi Dai

    Full Text Available Candida albicans is the most common human fungal pathogen. Recent evidence has revealed the occurrence of apoptosis in C. albicans that is inducible by environmental stresses such as hydrogen peroxide, acetic acid, and amphotericin B. Apoptosis is regulated by the calcineurin-caspase pathway in C. albicans, and calcineurin is under the control of Hsp90 in echinocandin resistance. However, the role of Hsp90 in apoptosis of C. albicans remains unclear. In this study, we investigated the role of Hsp90 in apoptosis of C. albicans by using an Hsp90-compromised strain tetO-HSP90/hsp90 and found that upon apoptotic stimuli, including hydrogen peroxide, acetic acid or amphotericin B treatment, less apoptosis occurred, less ROS was produced, and more cells survived in the Hsp90-compromised strain compared with the Hsp90/Hsp90 wild-type strain. In addition, Hsp90-compromised cells were defective in up-regulating caspase-encoding gene CaMCA1 expression and activating caspase activity upon the apoptotic stimuli. Investigations on the relationship between Hsp90 and calcineurin revealed that activation of calcineurin could up-regulate apoptosis but could not further down-regulate apoptosis in Hsp90-compromised cells, indicating that calcineurin was downstream of Hsp90. Hsp90 inhibitor geldanamycin (GdA could further decrease the apoptosis in calcineurin-pathway-defect strains, indicating that compromising Hsp90 function had a stronger effect than compromising calcineurin function on apoptosis. Collectively, this study demonstrated that compromised Hsp90 reduced apoptosis in C. albicans, partially through downregulating the calcineurin-caspase pathway.

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

    International Nuclear Information System (INIS)

    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

  8. Mitochondrial calcium uniporter silencing potentiates caspase-independent cell death in MDA-MB-231 breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Curry, Merril C.; Peters, Amelia A. [School of Pharmacy, The University of Queensland, Brisbane, Queensland 4072 (Australia); Kenny, Paraic A. [Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Roberts-Thomson, Sarah J. [School of Pharmacy, The University of Queensland, Brisbane, Queensland 4072 (Australia); Monteith, Gregory R., E-mail: gregm@uq.edu.au [School of Pharmacy, The University of Queensland, Brisbane, Queensland 4072 (Australia)

    2013-05-10

    Highlights: •Some clinical breast cancers are associated with MCU overexpression. •MCU silencing did not alter cell death initiated with the Bcl-2 inhibitor ABT-263. •MCU silencing potentiated caspase-independent cell death initiated by ionomycin. •MCU silencing promoted ionomycin-mediated cell death without changes in bulk Ca{sup 2+}. -- Abstract: The mitochondrial calcium uniporter (MCU) transports free ionic Ca{sup 2+} into the mitochondrial matrix. We assessed MCU expression in clinical breast cancer samples using microarray analysis and the consequences of MCU silencing in a breast cancer cell line. Our results indicate that estrogen receptor negative and basal-like breast cancers are characterized by elevated levels of MCU. Silencing of MCU expression in the basal-like MDA-MB-231 breast cancer cell line produced no change in proliferation or cell viability. However, distinct consequences of MCU silencing were seen on cell death pathways. Caspase-dependent cell death initiated by the Bcl-2 inhibitor ABT-263 was not altered by MCU silencing; whereas caspase-independent cell death induced by the calcium ionophore ionomycin was potentiated by MCU silencing. Measurement of cytosolic Ca{sup 2+} levels showed that the promotion of ionomycin-induced cell death by MCU silencing occurs independently of changes in bulk cytosolic Ca{sup 2+} levels. This study demonstrates that MCU overexpression is a feature of some breast cancers and that MCU overexpression may offer a survival advantage against some cell death pathways. MCU inhibitors may be a strategy to increase the effectiveness of therapies that act through the induction of caspase-independent cell death pathways in estrogen receptor negative and basal-like breast cancers.

  9. Effect of protein kinase C alpha, caspase-3, and survivin on apoptosis of oral cancer cells induced by staurosporine

    Institute of Scientific and Technical Information of China (English)

    Yu-xia ZHANG; Shi-bin YU; Jing-ping OU-YANG; Dong XIA; Min WANG; Jin-rong LI

    2005-01-01

    Aim: To elucidate inhibition of protein kinase C α (PKC α) activity by staurosporine on apoptosis of oral cancer cell line tongue squamous cell carcinoma (TSCCa)cells and to clarify the role of survivin and caspase-3 in mediating apoptosis.Methods: TSCCa cell viability was measured by MTT assay after 100 nmol/L staurosporine treatment. Apoptotic cells were identified by using phase contrast microscopy, acridine orange/ethidium bromide staining, and flow cytometry. Level of PKC α and its subcellular location were investigated using Western blot analysis.Expression of survivin and caspase-3 were evaluated using immunocytochemistry.Results: Staurosporine significantly inhibited the cell viability of TSCCa cells in a dose- and time-dependent manner. Marked cell accumulation in G2/M phase was observed after 100 nmol/L staurosporine exposure for 6 h and 12 h. In addition,the percentage of apoptosis increased in a time-dependent manner, from 2.9% in control cultures to approximately 27.4% at 100 nmol/L staurosporine treatment for24 h. Staurosporine displayed difference in inhibitory efficacy between cytosolic and membrance-derived PKC α. The content of PKCα in membrane versus cytosol decreased quickly, from 0.45 in ethanol-treated control cultures to 0.18 after staurosporine exposure for 24 h (P<0.01). After treatment withstaurosporine, a time-dependent reduction of survivin and an activation of caspase-3 were observed in TSCCa cells. Conclusion: Staurosporine inhibited cell viability and promoted apoptosis in TSCCa cells. Inhibition of PKCα activity might be a potential mechanism for staurosporine to induce apoptosis in this cell line. The cleavage of survivin and activation of caspase-3 signaling pathway might contribute to PKC α inhibition-induced apoptosis.

  10. Caspase-cleaved cytokeratin 18 fragment (M30) as marker of postoperative residual tumor load in colon cancer patients

    OpenAIRE

    Ausch, C.; Buxhofer-Ausch, V; Olszewski, U.; Hinterberger, W.; Ogris, E.; Schiessel, R.; Hamilton, G.

    2009-01-01

    Abstract Background Soluble cytokeratin 18 (CK18; M65) and a caspase-cleaved fragment of CK18 (M30) have been used as biomarkers, corresponding to tumor cell death and apoptosis respectively. Methods In the present study, M30 was quantified for the first time in serum samples of colon cancer patients pre- and postoperatively as well as during chemotherapy. Minimal residual disease (MRD) was assessed preoperatively by detection of pan-cytokeratin ant...

  11. Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis

    OpenAIRE

    Hou, Ying-Chen Claire; Chittaranjan, Suganthi; Barbosa, Sharon González; McCall, Kimberly; Gorski, Sharon M.

    2008-01-01

    A complex relationship exists between autophagy and apoptosis, but the regulatory mechanisms underlying their interactions are largely unknown. We conducted a systematic study of Drosophila melanogaster cell death–related genes to determine their requirement in the regulation of starvation-induced autophagy. We discovered that six cell death genes—death caspase-1 (Dcp-1), hid, Bruce, Buffy, debcl, and p53—as well as Ras–Raf–mitogen activated protein kinase signaling pathway components had a r...

  12. Preclinical studies identify non-apoptotic low-level caspase-3 as therapeutic target in pemphigus vulgaris.

    Directory of Open Access Journals (Sweden)

    Camille Luyet

    Full Text Available The majority of pemphigus vulgaris (PV patients suffer from a live-threatening loss of intercellular adhesion between keratinocytes (acantholysis. The disease is caused by auto-antibodies that bind to desmosomal cadherins desmoglein (Dsg 3 or Dsg3 and Dsg1 in mucous membranes and skin. A currently unresolved controversy in PV is whether apoptosis is involved in the pathogenic process. The objective of this study was to perform preclinical studies to investigate apoptotic pathway activation in PV pathogenesis with the goal to assess its potential for clinical therapy. For this purpose, we investigated mouse and human skin keratinocyte cultures treated with PV antibodies (the experimental Dsg3 monospecific antibody AK23 or PV patients IgG, PV mouse models (passive transfer of AK23 or PVIgG into adult and neonatal mice as well as PV patients' biopsies (n=6. A combination of TUNEL assay, analyses of membrane integrity, early apoptotic markers such as cleaved poly-ADP-ribose polymerase (PARP and the collapse of actin cytoskeleton failed to provide evidence for apoptosis in PV pathogenesis. However, the in vitro and in vivo PV models, allowing to monitor progression of lesion formation, revealed an early, transient and low-level caspase-3 activation. Pharmacological inhibition confirmed the functional implication of caspase-3 in major events in PV such as shedding of Dsg3, keratin retraction, proliferation including c-Myc induction, p38MAPK activation and acantholysis. Together, these data identify low-level caspase-3 activation downstream of disrupted Dsg3 trans- or cis-adhesion as a major event in PV pathogenesis that is non-synonymous with apoptosis and represents, unlike apoptotic components, a promising target for clinical therapy. At a broader level, these results posit that an impairment of adhesive functions in concert with low-level, non-lethal caspase-3 activation can evoke profound cellular changes which may be of relevance for other

  13. Apoptosis in Heart Failure: Release of Cytochrome c from Mitochondria and Activation of Caspase-3 in Human Cardiomyopathy

    Science.gov (United States)

    Narula, Jagat; Pandey, Pramod; Arbustini, Eloisa; Haider, Nezam; Narula, Navneet; Kolodgie, Frank D.; dal Bello, Barbara; Semigran, Marc J.; Bielsa-Masdeu, Anna; Dec, G. William; Israels, Sara; Ballester, Manel; Virmani, Renu; Saxena, Satya; Kharbanda, Surender

    1999-07-01

    Apoptosis has been shown to contribute to loss of cardiomyocytes in cardiomyopathy, progressive decline in left ventricular function, and congestive heart failure. Because the molecular mechanisms involved in apoptosis of cardiocytes are not completely understood, we studied the biochemical and ultrastructural characteristics of upstream regulators of apoptosis in hearts explanted from patients undergoing transplantation. Sixteen explanted hearts from patients undergoing heart transplantation were studied by electron microscopy or immunoblotting to detect release of mitochondrial cytochrome c and activation of caspase-3. The hearts explanted from five victims of motor vehicle accidents or myocardial ventricular tissues from three donor hearts were used as controls. Evidence of apoptosis was observed only in endstage cardiomyopathy. There was significant accumulation of cytochrome c in the cytosol, over myofibrils, and near intercalated discs of cardiomyocytes in failing hearts. The release of mitochondrial cytochrome c was associated with activation of caspase-3 and cleavage of its substrate protein kinase C δ but not poly(ADP-ribose) polymerase. By contrast, there was no apparent accumulation of cytosolic cytochrome c or caspase-3 activation in the hearts used as controls. The present study provides in vivo evidence of cytochrome c-dependent activation of cysteine proteases in human cardiomyopathy. Activation of proteases supports the phenomenon of apoptosis in myopathic process. Because loss of myocytes contributes to myocardial dysfunction and is a predictor of adverse outcomes in the patients with congestive heart failure, the present demonstration of an activated apoptotic cascade in cardiomyopathy could provide the basis for novel interventional strategies.

  14. Pfaffosidic Fraction from Hebanthe paniculata Induces Cell Cycle Arrest and Caspase-3-Induced Apoptosis in HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Tereza Cristina da Silva

    2015-01-01

    Full Text Available Hebanthe paniculata roots (formerly Pfaffia paniculata and popularly known as Brazilian ginseng show antineoplastic, chemopreventive, and antiproliferative properties. Functional properties of these roots and their extracts are usually attributed to the pfaffosidic fraction, which is composed mainly by pfaffosides A–F. However, the therapeutic potential of this fraction in cancer cells is not yet entirely understood. This study aimed to analyze the antitumoral effects of the purified pfaffosidic fraction or saponinic fraction on the human hepatocellular carcinoma HepG2 cell line. Cellular viability, proliferation, and apoptosis were evaluated, respectively, by MTT assay, BrdU incorporation, activated caspase-3 immunocytochemistry, and DNA fragmentation assay. Cell cycle was analyzed by flow cytometry and the cell cycle-related proteins were analyzed by quantitative PCR and Western blot. The cells exposed to pfaffosidic fraction had reduced viability and cellular growth, induced G2/M at 48 h or S at 72 h arrest, and increased sub-G1 cell population via cyclin E downregulation, p27KIP1 overexpression, and caspase-3-induced apoptosis, without affecting the DNA integrity. Antitumoral effects of pfaffosidic fraction from H. paniculata in HepG2 cells originated by multimechanisms of action might be associated with cell cycle arrest in the S phase, by CDK2 and cyclin E downregulation and p27KIP1 overexpression, besides induction of apoptosis through caspase-3 activation.

  15. 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.

  16. Ciglitazone induces caspase-independent apoptosis via p38-dependent AIF nuclear translocation in renal epithelial cells

    International Nuclear Information System (INIS)

    Peroxisome proliferator-activated receptor γ (PPARγ) agonists have been reported to induce apoptosis in a variety of cell types including renal proximal epithelial cells. However, the underlying mechanism of cell death induced by PPARγ agonists has not been clearly defined in renal proximal tubular cells. This study was therefore undertaken to determine the mechanism by which ciglitazone, a synthetic PPARγ agonist, induces apoptosis in opossum kidney (OK) cells, an established renal epithelial cell line. Ciglitazone treatment induced apoptotic cell death in a dose- and time-dependent manner. Ciglitazone caused a transient activation of ERK and sustained activation of p38 MAP kinase. Ciglitazone-mediated cell death was attenuated by the p38 inhibitor SB203580 and transfection of dominant-negative form of p38, but not by the MEK inhibitor U0126, indicating that p38 MAP kinase activation is involved in the ciglitazone-induced cell death. Although ciglitazone-induced caspase-3 activation, the ciglitazone-mediated cell death was not affected by the caspase-3 inhibitor DEVD-CHO. Ciglitazone-induced mitochondrial membrane depolarization and apoptosis-inducing factor (AIF) nuclear translocation and these effects were prevented by the p38 inhibitor. These results suggest that ciglitazone induces caspase-independent apoptosis through p38 MAP kinase-dependent AIF nuclear translocation in OK renal epithelial cells

  17. Ursolic Acid Triggers Apoptosis in Human Osteosarcoma Cells via Caspase Activation and the ERK1/2 MAPK Pathway.

    Science.gov (United States)

    Wu, Chia-Chieh; Cheng, Chun-Hsiang; Lee, Yi-Hui; Chang, Ing-Lin; Chen, Hsin-Yao; Hsieh, Chen-Pu; Chueh, Pin-Ju

    2016-06-01

    Ursolic acid (UA), a naturally occurring pentacyclic triterpene acid found in many medicinal herbs and edible plants, has been shown to trigger apoptosis in several lines of tumor cells in vitro. We found that treatment with UA suppressed the viability of human osteosarcoma MG-63 cells and induced cell cycle arrest at sub-G1 and G2/M phases. Furthermore, exposure to UA induced intracellular oxidative stress and collapse of mitochondrial membrane permeability, resulting in the subsequent activation of apoptotic caspases 8, 9, and 3 as well as PARP cleavage, and ultimately apoptosis in MG-63 cells. Moreover, protein analysis of mitogen-activated protein kinase (MAPK)-related protein expression showed an increase in activated ERK1/2, JNK, and p38 MAPK in UA-treated MG-63 cells. In addition, UA-induced apoptosis was significantly abolished in MG-63 cells that had been pretreated with inhibitors of caspase 3, 8, and 9 and ERK1/2. Furthermore, UA-treated MG-63 cells also exhibited an enhancement in Bax/Bcl-2 ratio, whereas anti-apoptotic XIAP and survivin were down-regulated. Taken together, we provide evidence demonstrating that UA mediates caspase-dependent and ERK1/2 MAPK-associated apoptosis in osteosarcoma MG-63 cells. PMID:27171502

  18. 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]. PMID:26246284

  19. Fatal Hepatitis Mediated by TNFα Requires Caspase-8 and Involves the BH3-only Proteins Bid and Bim

    Science.gov (United States)

    Kaufmann, Thomas; Jost, Philipp J; Pellegrini, Marc; Puthalakath, Hamsa; Gugasyan, Raffi; Gerondakis, Steve; Cretney, Erika; Smyth, Mark J; Silke, John; Hakem, Razq; Bouillet, Philippe; Mak, Tak W; Dixit, Vishva M; Strasser, Andreas

    2009-01-01

    SUMMARY Apoptotic death of hepatocytes, a feature and contributing factor of many chronic and acute liver diseases, can be a consequence of over-activation of the immune system. Injection with lipopolysaccharide (LPS) plus the transcriptional inhibitor D(+)-galactosamine (GalN) or mitogenic T cell activation cause fatal hepatocyte apoptosis in mice. In both settings hepatocyte killing is mediated by TNFα/TNF-R signaling but the effector mechanisms remain unclear. Our analysis of gene-targeted mice showed that caspase-8 is essential for hepatocyte killing in both settings. Loss of Bid, the pro-apoptotic BH3-only protein activated by caspase-8, and essential for Fas ligand-induced hepatocyte killing, resulted only in a minor reduction of liver damage. However, combined loss of Bid and another BH3-only protein, Bim, activated by JNK, protected mice from LPS+GalN-induced hepatitis. These observations identify caspase-8 and the BH3-only proteins Bid and Bim as potential therapeutic targets for treatment of inflammatory liver diseases. PMID:19119023

  20. Butylphthalide Suppresses Neuronal Cells Apoptosis and Inhibits JNK-Caspase3 Signaling Pathway After Brain Ischemia /Reperfusion in Rats.

    Science.gov (United States)

    Wen, Xiang-Ru; Tang, Man; Qi, Da-Shi; Huang, Xiao-Jing; Liu, Hong-Zhi; Zhang, Fang; Wu, Jian; Wang, Yi-Wen; Zhang, Xun-Bao; Guo, Ji-Qiang; Wang, Shu-Ling; Liu, Yong; Wang, Yu-Lan; Song, Yuan-Jian

    2016-10-01

    Although Butylphthalide (BP) has protective effects that reduce ischemia-induced brain damage and neuronal cell death, little is known about the precise mechanisms occurring during cerebral ischemia/reperfusion (I/R). Therefore, the aim of this study was to investigate the neuroprotective mechanisms of BP against ischemic brain injury induced by cerebral I/R through inhibition of the c-Jun N-terminal kinase (JNK)-Caspase3 signaling pathway. BP in distilled non-genetically modified Soybean oil was administered intragastrically three times a day at a dosage of 15 mg/(kg day) beginning at 20 min after I/R in Sprague-Dawley rats. Immunohistochemical staining and Western blotting were performed to examine the expression of related proteins, and TUNEL-staining was used to detect the percentage of neuronal apoptosis in the hippocampal CA1 region. The results showed that BP could significantly protect neurons against cerebral I/R-induced damage. Furthermore, the expression of p-JNK, p-Bcl2, p-c-Jun, FasL, and cleaved-caspase3 was also decreased in the rats treated with BP. In summary, our results imply that BP could remarkably improve the survival of CA1 pyramidal neurons in I/R-induced brain injury and inhibit the JNK-Caspase3 signaling pathway. PMID:27015680

  1. The correlation between MIB-1, AgNOR, and caspase-3 apoptosis with chemoradiotherapy response in cervical cancer

    International Nuclear Information System (INIS)

    Chemoradiotherapy is one of treatments for the locally advanced cervical cancer given by concurrent radiotherapy combined with chemotherapy in the same time. Chemoradiotherapy response is influenced by biological factor i.e. cell kinetic that consists of cell proliferation and death. In this research, the correlation between AgNOR, MIB-1 cell proliferation biomarker and the expression of apoptotic caspase-3 with chemoradiotherapy response of cervical cancer has been studied. Twenty one microscopic tissue samples were taken from cervical cancer biopsies before radiotherapy. The tissue samples were stained with AgNOR, whereas MIB-1 and apoptosis caspase-3 in the tissue samples were detected by immunochemistry technique. After the completion of chemoradiotherapy treatment, the clinical response was observed by pelvic control method. The result of this research show that there is no correlation between AgNOR, MIB-1 value with apoptosis (p>0.05) before chemoradiotherapy. Cell proliferation observed by AgNOR and MIB-1 before chemoradiotherapy indicate no correlation with chemoradiotherapy response, however the apoptotic expression shows positive correlation with chemoradiotherapy response. The index of caspase-3 apoptosis obtained from this research can be used for considering the chemoradiotherapy schedule for the cervical cancer patient. (author)

  2. Caspase-1 Dependent IL-1β Secretion and Antigen-Specific T-Cell Activation by the Novel Adjuvant, PCEP

    Directory of Open Access Journals (Sweden)

    Sunita Awate

    2014-06-01

    Full Text Available The potent adjuvant activity of the novel adjuvant, poly[di(sodiumcarboxylatoethylphenoxyphosphazene] (PCEP, with various antigens has been reported previously. However, very little is known about its mechanisms of action. We have recently reported that intramuscular injection of PCEP induces NLRP3, an inflammasome receptor gene, and inflammatory cytokines, including IL-1β and IL-18, in mouse muscle tissue. Caspase-1 is required for the processing of pro-forms of IL-1β and IL-18 into mature forms and is a critical constituent of the NLRP3 inflammasome. Hence, in the present study, we investigated the role of caspase-1 in the secretion of IL-1β and IL-18 in PCEP-stimulated splenic dendritic cells (DCs. Caspase inhibitor YVAD-fmk-treated splenic DCs showed significantly reduced IL-1β and IL-18 secretion in response to PCEP stimulation. Further, PCEP had no effect on the expression of MHC class II or co-stimulatory molecules, CD86 and CD40, suggesting that PCEP does not induce DC maturation. However, PCEP directly activated B-cells to induce significant production of IgM. In addition, PCEP+ovalbumin (OVA immunized mice showed significantly increased production of antigen-specific IFN-γ by CD4+ and CD8+ T-cells. We conclude that PCEP activates innate immunity, leading to increased antigen-specific T-cell responses.

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

    International Nuclear Information System (INIS)

    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

  4. Dasatinib promotes paclitaxel-induced necroptosis in lung adenocarcinoma with phosphorylated caspase-8 by c-Src.

    Science.gov (United States)

    Diao, Yan; Ma, Xiaobin; Min, WeiLi; Lin, Shuai; Kang, HuaFeng; Dai, ZhiJun; Wang, Xijing; Zhao, Yang

    2016-08-28

    Cisplatin and paclitaxel are considered to be the backbone of chemotherapy in lung adenocarcinoma. These agents show pleiotropic effects on cell death. However, the precise mechanisms remain unclear. The present study reported that phosphorylated caspase-8 at tyrosine 380 (p-Casp8) was characterized as a biomarker of chemoresistance to TP regimen (cisplatin and paclitaxel) in patients with resectable lung adenocarcinoma with significantly poorer 5-year disease-free survival (DFS) and overall survival (OS). Cisplatin killed lung adenocarcinoma cells regardless of c-Src-induced caspase-8 phosphorylation at tyrosine 380. Subsequently, we identified a novel mechanism by which paclitaxel induced necroptosis in lung adenocarcinoma cells that was dependent upon p-Casp8, receptor-interacting protein kinase 1 (RIPK1), and RIPK3. Moreover, dasatinib, a c-Src inhibitor, dephosphorylated caspase-8 to facilitate necroptosis, rather than apoptosis, in paclitaxel-treated p-Casp8-expressing lung adenocarcinoma cells. The data from our study revealed previously unrecognized roles of p-Casp8 as a positive effector in the initiation of necroptosis and as a negative effector in the repression of the interaction between RIPK1 and RIPK3. Moreover, these outcomes supported the need for further clinical studies with the goal of evaluating the efficacy of dasatinib plus paclitaxel in the treatment of lung adenocarcinoma. PMID:27195913

  5. Inhibition of Corydalis decumbens Alkaloids on Hydrogen Peroxideinduced Apoptosis of PC12 Cells through Down-regulating Caspase-3 Expression

    Institute of Scientific and Technical Information of China (English)

    YAN Ren-jie; YANG Yi-fang; LUO Yong-ming; WU Chun-zhen

    2011-01-01

    Objective To extract alkaloids from Corydalis decumbens (AsCD) by supercritical CO2 fluid extraction (SFE) and to evaluate protective effects of AsCD against hydrogen peroxide (H2O2)-induced apoptosis in rat PC12 cells.Methods AsCD were extracted by SFE and oxidative damage PC12 cells model was induced by H2O2.The survival rate of the cells was determined by MTT assay; Lactate dehydrogenase release was determined by ultraviolet spectrophotometry; Flow cytometry was used to detect apoptosis; Caspase-3 mRNA and protein were determined by real-time PCR and Western blotting assay,respectively.Results AsCD remarkably reduced the cytotoxicity,prevented membrane damage,and inhibited cell apoptosis.AsCD inhibited Caspase-3 mRNA and protein expression induced by H2O2 in PC12 cells.Conclusion AsCD possess protective effects against H2O2-induced apoptosis in PC12 cells,and the mechanism of AsCD responsible to the inhibition of apoptosis is possibly attributed to thedown-regulating Caspase-3 expression.AsCD might be useful in the treatment of oxidative stress-related neurodegenerative diseases.

  6. Induction of Manduca sexta Larvae Caspases Expression in Midgut Cells by Bacillus thuringiensis Cry1Ab Toxin

    Directory of Open Access Journals (Sweden)

    Helena Porta

    2011-01-01

    Full Text Available Bacillus thuringiensis produces crystal toxins known as Cry that are highly selective against important agricultural and human health-related insect pests. Cry proteins are pore-forming toxins that interact with specific receptors in the midgut cell membrane of susceptible larvae making pores that cause osmotic shock, leading finally to insect death. In the case of pore-forming toxins that are specific to mammalian cells, death responses at low doses may induce apoptosis or pyroptosis, depending on the cell type. The death mechanism induced by Cry toxins in insect midgut cells is poorly understood. Here, we analyze the caspases expression by RT-PCR analysis, showing that the initial response of Manduca sexta midgut cells after low dose of Cry1Ab toxin administration involves a fast and transient accumulation of caspase-1 mRNA, suggesting that pyroptosis was activated by Cry1Ab toxin as an initial response but was repressed later. In contrast, caspase-3 mRNA requires a longer period of time of toxin exposure to be activated but presents a sustained activation, suggesting that apoptosis may be a cell death mechanism induced also at low dose of toxin.

  7. Induction of caspase 8 and reactive oxygen species by ruthenium-derived anticancer compounds with improved water solubility and cytotoxicity.

    Science.gov (United States)

    Vidimar, Vania; Meng, Xiangjun; Klajner, Marcelina; Licona, Cynthia; Fetzer, Ludivine; Harlepp, Sébastien; Hébraud, Pascal; Sidhoum, Marjorie; Sirlin, Claude; Loeffler, Jean-Philippe; Mellitzer, Georg; Sava, Gianni; Pfeffer, Michel; Gaiddon, Christian

    2012-12-01

    Organometallic compounds which contain metals, such as ruthenium or gold, have been investigated as a replacement for platinum-derived anticancer drugs. They often show good antitumor effects, but the identification of their precise mode of action or their pharmacological optimization is still challenging. We have previously described a class of ruthenium(II) compounds with interesting anticancer properties. In comparison to cisplatin, these molecules have lower side effects, a reduced ability to interact with DNA, and they induce cell death in absence of p53 through CHOP/DDIT3. We have now optimized these molecules by improving their cytotoxicity and their water solubility. In this article, we demonstrate that by changing the ligands around the ruthenium we modify the ability of the compounds to interact with DNA. We show that these optimized molecules reduce tumor growth in different mouse models and retain their ability to induce CHOP/DDIT3. However, they are more potent inducers of cancer cell death and trigger the production of reactive oxygen species and the activation of caspase 8. More importantly, we show that blocking reactive oxygen species production or caspase 8 activity reduces significantly the activity of the compounds. Altogether our data suggest that water-soluble ruthenium(II)-derived compounds represent an interesting class of molecules that, depending on their structures, can target several pro-apoptotic signaling pathways leading to reactive oxygen species production and caspase 8 activation. PMID:22964219

  8. Mice lacking caspase-2 are protected from behavioral changes, but not pathology, in the YAC128 model of Huntington disease

    Directory of Open Access Journals (Sweden)

    Bissada Nagat

    2011-08-01

    Full Text Available Abstract Background Huntington Disease (HD is a neurodegenerative disorder in which caspase activation and cleavage of substrates, including the huntingtin protein, has been invoked as a pathological mechanism. Specific changes in caspase-2 (casp2 activity have been suggested to contribute to the pathogenesis of HD, however unique casp2 cleavage substrates have remained elusive. We thus utilized mice completely lacking casp2 (casp2-/- to examine the role played by casp2 in the progression of HD. This 'substrate agnostic' approach allows us to query the effect of casp2 on HD progression without pre-defining proteolytic substrates of interest. Results YAC128 HD model mice lacking casp2 show protection from well-validated motor and cognitive features of HD, including performance on rotarod, swimming T-maze, pre-pulse inhibition, spontaneous alternation and locomotor tasks. However, the specific pathological features of the YAC128 mice including striatal volume loss and testicular degeneration are unaltered in mice lacking casp2. The application of high-resolution magnetic resonance imaging (MRI techniques validates specific neuropathology in the YAC128 mice that is not altered by ablation of casp2. Conclusions The rescue of behavioral phenotypes in the absence of pathological improvement suggests that different pathways may be operative in the dysfunction of neural circuitry in HD leading to behavioral changes compared to the processes leading to cell death and volume loss. Inhibition of caspase-2 activity may be associated with symptomatic improvement in HD.

  9. Cell apoptosis in perihematomal brain regions and expression of Caspase-3 protein in patients with hypertensive intracerebral hemorrhage

    Institute of Scientific and Technical Information of China (English)

    Xinqing Zhang; Xiaoliang Yin; Kun Zhang; Zhimin Zhang; Hui Cai; Honglan Xu

    2006-01-01

    BACKGROUND: In patients with intracerebral hemorrhage (ICH), besides the space-occupying effect of hematoma, hematomal component also causes the pathological changes of perihematomal region, including the death of neurons and glial cells, vasogenic brain edema, the destruction of blood brain barrier and so on, which are the important factors to influence the prognosis of patients. Therefore, it is necessary to perform fur ther investigation and study on the pathological characteristics of injury and death of brain nerve cells. OBJECTIVE: To observe the pathological changes of apoptosis and Caspase-3 expression in perihe matomal brain regions in patients with hypertensive ICH (HICH) in different stages of onset, and analyze their relationship. DESIGN: Case-control observation. SETTING: Departments of Neurosurgery and Pathology of Beijing Chuiyangliu Hospital. PARTICIPANTS: Totally 19 patients with HICH, including 12 male, 7 female, aged (58.3±12.8) ranging from 49 to 78 years, whose mean volume of hemorrhage was (48.6±16.4) mL, were involved . All the cases conformed to the diagnostic criteria of intracerebral hemorrhage formulated in the 4th National Cerebrovascular Dis eases Conference and were confirmed by skull CT scanning. Informed consents of operation and specimens were obtained from the patients and relatives.METHODS; ①Patients with HICH who had undergone surgical evacuation of an intracerebral hematoma by traverse temporal lobe approach in the Department of Neurosurgery, Beijing Chuiyangliu Hospital from Jan uary 2004 to July 2005 were involved. Nineteen specimens of brain tissue from perihematomal region of HICH patients in different phases served as patient group. Five specimens were obtained from distant regions of patients in the super-early phase as the control group. According to the time from onset to operation, the 19 cases were divided into 3 groups: 6 cases in super-early phase(onset < 8 hours), 8 cases in early phase (onset about 8 to 24

  10. Quercetin induces apoptosis by activating caspase-3 and regulating Bcl-2 and cyclooxygenase-2 pathways in human HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    Guomin Niu; Songmei Yin; Shuangfeng Xie; Yiqing Li; Danian Nie; Liping Ma; Xiuju Wang; Yudan Wu

    2011-01-01

    Quercetin is one of the naturally occurring dietary flavo-nol compounds. It is present abundantly in plants and has chemopreventive and anticancer effects. To investigate its anticancer mechanism, we examined the activity of quercetin against acute leukemia cell line, HL-60. Our results showed that quercetin inhibited cell proliferation and induced apoptosis in a time- and dose-dependent manner. Furthermore, quercetin down-regulated the expression of anti-apoptosis protein Bcl-2 and up-regulated the expression of pro-apoptosis protein Bax. Caspase-3 was also activated by quercetin, which started a caspase-3-depended mitochodrial pathway to induce apoptosis. It was also found that quercetin inhibited the expression of the cycloocygenase-2 (Cox-2) mRNA and Cox-2 protein. Taken together, these findings suggested that quercetin induces apoptosis in a caspase-3-dependent pathway by inhibiting Cox-2 expression and regulates the expression of downstream apoptotic components, including Bcl-2 and Bax. Quercetin can be a potent and promising medicine which might be safely used in leukemia therapy.

  11. Climate prediction and predictability

    Science.gov (United States)

    Allen, Myles

    2010-05-01

    Climate prediction is generally accepted to be one of the grand challenges of the Geophysical Sciences. What is less widely acknowledged is that fundamental issues have yet to be resolved concerning the nature of the challenge, even after decades of research in this area. How do we verify or falsify a probabilistic forecast of a singular event such as anthropogenic warming over the 21st century? How do we determine the information content of a climate forecast? What does it mean for a modelling system to be "good enough" to forecast a particular variable? How will we know when models and forecasting systems are "good enough" to provide detailed forecasts of weather at specific locations or, for example, the risks associated with global geo-engineering schemes. This talk will provide an overview of these questions in the light of recent developments in multi-decade climate forecasting, drawing on concepts from information theory, machine learning and statistics. I will draw extensively but not exclusively from the experience of the climateprediction.net project, running multiple versions of climate models on personal computers.

  12. Expressão citofotométrica quantitativa da CASPASE-3 e do CD-34 no carcinoma ductal infiltrante de mama Cytophotometric expressions of CASPASE-3 and CD-34 in breast cancer

    Directory of Open Access Journals (Sweden)

    Fábio Santana dos Passos

    2009-10-01

    Full Text Available OBJETIVO: Descrever, correlacionar e comparar a expressão dos marcadores tumorais CD-34 (angiogênese e caspase-3 (apoptose em carcinoma ductal invasor de mama. MÉTODOS: Foram utilizados 22 casos de adenocarcinoma infiltrante de mama provenientes de blocos de parafina e, após preparo específico para imunoistoquímica, 15 apresentaram leitura satisfatória e foram avaliados pelo sistema de fotocitometria de imagem SAMBA 4000® e software IMMUNO®. Os parâmetros analisados foram o índice de marcagem e densidade óptica. RESULTADOS: Para o CD-34 não houve normalidade dos dados na análise do índice de marcagem, com obtenção de P=0,019, havendo normalidade para a análise da densidade óptica, com P=0,199. Para a caspase-3 houve normalidade de dados para o índice marcagem com P=0,306 e para a densidade óptica com P=0,114; não houve diferença estatística significativa entre eles em relação à média do índice de marcagem (P=0,872 e da densidade óptica (P=0,816, quando analisados os parâmetros que definem a expressão dos marcadores; existiu tendência à associação entre a densidade óptica e o índice de marcagem do marcador tumoral caspase-3, com P=0,025. Não foi observada tendência à associação quando comparados densidade óptica e índice de marcagem do marcador tumoral CD-34; índice de marcagem do marcador tumoral caspase-3 e índice de marcagem do marcador tumoral CD-34; e densidade óptica da caspase-3 com a do CD-34. CONCLUSÃO: Dos 22 casos incluídos foi possível verificar a expressão do marcador CD-34 em 18 lâminas e da caspase-3 em 22 lâminas; Para o CD-34 não houve normalidade dos dados na análise do índice de marcagem, havendo sim normalidade para a análise da densidade óptica. Para a caspase-3 houve normalidade de dados tanto para o índice de marcagem como para a densidade óptica. Existe tendência à associação entre a densidade óptica e o índice de marcagem da caspase-3. Não foi observada

  13. Astragalus saponins induce apoptosis in human gastric adenocarcinoma cells via a caspase 3-dependent pathway

    Institute of Scientific and Technical Information of China (English)

    JOSHUA K S Ko; Kathy K W Auyeung

    2008-01-01

    Objective Many Asian countriea including China, Japan and Korea have very high incidence of gastric cancer, in which about 42 % cases occur in mainland China. The precise targets and underlying mechanisms are not well understood. Our previous study revealed that Astragalus saponins (AST) showed promising effects on the suppression of the growth of HT-29 human colon cancer cells and tumor xenograft by inhibiting cell proliferation and promoting apoptosis. In the present study, we investigated the anti-carcinogenic effects of AST in AGS human gastric adenocarcinoma cells and attempted to elucidate the underlying mechanisms. Methods Growth inhibition of AGS cells was determined by using the MTT viability test. Involvement of different members of the apoptotic cascade and other growth-related factors was explored by assessment of their protein expression using Western blot analysis. Distribution of cells in different phases of the cell cycle was assessed by flow eytometry. Results Our data indicate that AST induced growth-inhibition and apoptosis in AGS cells by activating caspase 3 with subsequent poly (ADP-ribose) polymerase (PARP) cleavage. Cell cycle arrest at the G2/M phase had been observed in AST-treated AGS cells. The anti-proliferative effect of AST was associated with modulation of eydin B1 and p21. We then demonstrate that AST could downregulate the expression of VEGF, of which interaction with its receptors is important for angiogenesis during tumor formation. Conclusions Our findings suggest that AST is an effective agent in gastric cancer treatment by inducing cell cycle arrest and apoptosis, of which anti-angiogenesis could be an alternative mode of action.

  14. Megakaryocytes regulate expression of Pyk2 isoforms and caspase-mediated cleavage of actin in osteoblasts.

    Science.gov (United States)

    Kacena, Melissa A; Eleniste, Pierre P; Cheng, Ying-Hua; Huang, Su; Shivanna, Mahesh; Meijome, Tomas E; Mayo, Lindsey D; Bruzzaniti, Angela

    2012-05-18

    The proliferation and differentiation of osteoblast (OB) precursors are essential for elaborating the bone-forming activity of mature OBs. However, the mechanisms regulating OB proliferation and function are largely unknown. We reported that OB proliferation is enhanced by megakaryocytes (MKs) via a process that is regulated in part by integrin signaling. The tyrosine kinase Pyk2 has been shown to regulate cell proliferation and survival in a variety of cells. Pyk2 is also activated by integrin signaling and regulates actin remodeling in bone-resorbing osteoclasts. In this study, we examined the role of Pyk2 and actin in the MK-mediated increase in OB proliferation. Calvarial OBs were cultured in the presence of MKs for various times, and Pyk2 signaling cascades in OBs were examined by Western blotting, subcellular fractionation, and microscopy. We found that MKs regulate the temporal expression of Pyk2 and its subcellular localization. We also found that MKs regulate the expression of two alternatively spliced isoforms of Pyk2 in OBs, which may regulate OB differentiation and proliferation. MKs also induced cytoskeletal reorganization in OBs, which was associated with the caspase-mediated cleavage of actin, an increase in focal adhesions, and the formation of apical membrane ruffles. Moreover, BrdU incorporation in MK-stimulated OBs was blocked by the actin-polymerizing agent, jasplakinolide. Collectively, our studies reveal that Pyk2 and actin play an important role in MK-regulated signaling cascades that control OB proliferation and may be important for therapeutic interventions aimed at increasing bone formation in metabolic diseases of the skeleton. PMID:22447931

  15. Megakaryocytes Regulate Expression of Pyk2 Isoforms and Caspase-mediated Cleavage of Actin in Osteoblasts*

    Science.gov (United States)

    Kacena, Melissa A.; Eleniste, Pierre P.; Cheng, Ying-Hua; Huang, Su; Shivanna, Mahesh; Meijome, Tomas E.; Mayo, Lindsey D.; Bruzzaniti, Angela

    2012-01-01

    The proliferation and differentiation of osteoblast (OB) precursors are essential for elaborating the bone-forming activity of mature OBs. However, the mechanisms regulating OB proliferation and function are largely unknown. We reported that OB proliferation is enhanced by megakaryocytes (MKs) via a process that is regulated in part by integrin signaling. The tyrosine kinase Pyk2 has been shown to regulate cell proliferation and survival in a variety of cells. Pyk2 is also activated by integrin signaling and regulates actin remodeling in bone-resorbing osteoclasts. In this study, we examined the role of Pyk2 and actin in the MK-mediated increase in OB proliferation. Calvarial OBs were cultured in the presence of MKs for various times, and Pyk2 signaling cascades in OBs were examined by Western blotting, subcellular fractionation, and microscopy. We found that MKs regulate the temporal expression of Pyk2 and its subcellular localization. We also found that MKs regulate the expression of two alternatively spliced isoforms of Pyk2 in OBs, which may regulate OB differentiation and proliferation. MKs also induced cytoskeletal reorganization in OBs, which was associated with the caspase-mediated cleavage of actin, an increase in focal adhesions, and the formation of apical membrane ruffles. Moreover, BrdU incorporation in MK-stimulated OBs was blocked by the actin-polymerizing agent, jasplakinolide. Collectively, our studies reveal that Pyk2 and actin play an important role in MK-regulated signaling cascades that control OB proliferation and may be important for therapeutic interventions aimed at increasing bone formation in metabolic diseases of the skeleton. PMID:22447931

  16. Radiation-Inducible Caspase-8 Gene Therapy for Malignant Brain Tumors

    International Nuclear Information System (INIS)

    Purpose: Patients with malignant gliomas have a poor prognosis. To explore a novel and more effective approach for the treatment of patients with malignant gliomas, we designed a strategy that combines caspase-8 (CSP8) gene therapy and radiation treatment (RT). In addition, the specificity of the combined therapy was investigated to decrease the unpleasant effects experienced by the surrounding normal tissue. Methods and Materials: We constructed the plasmid pEGR-green fluorescence protein that included the radiation-inducible early growth response gene-1 (Egr-1) promoter and evaluated its characteristics. The pEGR-CSP8 was constructed and included the Egr-1 promoter and CSP8 complementary DNA. Assays that evaluated the apoptosis inducibility and cytotoxicity caused by CSP8 gene therapy combined with RT were performed using U251 and U87 glioma cells. The pEGR-CSP8 was transfected into the subcutaneous U251 glioma cells of nude mice by means of in vivo electroporation. The in vivo effects of CSP8 gene therapy combined with RT were evaluated. Results: The Egr-1 promoter yielded a better response with fractionated RT than with single-dose RT. In the assay of apoptosis inducibility and cytotoxicity, pEGR-CSP8 showed response for RT. The pEGR-CSP8 combined with RT is capable of inducing cell death effectively. In mice treated with pEGR-CSP8 and RT, apoptotic cells were detected in pathologic sections, and a significant difference was observed in tumor volumes. Conclusions: Our results indicate that radiation-inducible gene therapy may have great potential because this can be spatially or temporally controlled by exogenous RT and is safe and specific

  17. Oridonin induces apoptosis in gastric cancer through Apaf-1, cytochrome c and caspase-3 signaling pathway

    Institute of Scientific and Technical Information of China (English)

    Ke-Wang Sun; Ying-Yu Ma; Tian-Pei Guan; Ying-Jie Xia; Chang-Ming Shao; Le-Gao Chen; Ya-Jun Ren

    2012-01-01

    AIM:To investigate the effect and mechanism of oridonin on the gastric cancer cell line HGC-27 in vitro.METHODS:The inhibitory effect of oridonin on HGC-27 cells was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay.After treatment with 10 μg/mL oridonin for 24 h and 48 h,the cells were stained with acridine orange/ethidium bromide.The morphologic changes were observed under an inverted fluorescence microscope.DNA fragmentation (a hallmark of apoptosis) and lactate dehydrogenase activity were examined using DNA ladder assay and lactate dehydrogenase-release assay.After treated with oridonin (0,1.25,2.5,5 and 10 μg/mL),HGC-27cells were collected for anexin V-phycoerythrin and 7-amino-actinomycin D double staining and tested by flow cytometric analysis,and oridonin-induced apoptosis in HGC-27 cells was detected.After treatment with oridonin for 24 h,the effects of oridonin on expression of Apaf-1,Bcl-2,Bax,caspase-3 and cytochrome c were also analyzed using reverse-transcript polymerase chain reaction (RT-PCR) and Western blotting.RESULTS:Oridonin significantly inhibited the proliferation of HGC-27 cells in a dose-and time-dependent manner.The inhibition rates of HGC-27 treated with four different concentrations of oridonin for 24 h (1.25,2.5,5 and 10 μg/mL) were 1.78% ± 0.36%,4.96% ±1.59%,10.35% ± 2.76% and 41.6% ± 4.29%,respectively,which showed a significant difference (P < 0.05).The inhibition rates of HGC-27 treated with oridonin at the four concentrations for 48 h were 14.77% ± 4.21%,21.57% ± 3.75%,30.31% ± 4.91% and 61.19% ±5.81%,with a significant difference (P < 0.05).The inhibition rates of HGC-27 treated with oridonin for 72 h at the four concentrations were 25.77% ± 4.85%,31.86% ± 3.86%,48.30% ± 4.16% and 81.80% ± 6.72%,with a significant difference (P < 0.05).Cells treated with oridonin showed typical apoptotic features with acridine orange

  18. Assessment of calpain and caspase systems activities during ageing of two bovine muscles by degradation patterns of αII spectrin and PARP-1.

    Science.gov (United States)

    Saccà, Elena; Pizzutti, Nicoletta; Corazzin, Mirco; Lippe, Giovanna; Piasentier, Edi

    2016-03-01

    The activities of calpain and caspase systems during ageing in Longissimus lumborum (LL) and Infraspinatus (IS) muscles of Italian Simmental young bulls (Bos taurus) were assessed. Samples from 10 animals were collected within 20 min of exsanguination (T0), after 48 h (T1) and 7 days (T2) post mortem. Calpain and caspase activity were evaluated based on the formation of αII spectrin cleavage products of 145 kDa (SBDP145) and 120 kDa (SBDP120), respectively. Caspase activity was also assessed by the presence of poly (adenosine diphosphate-ribose) polymerase-1 (PARP-1) cleavage product. At T0, LL showed higher levels of SBDP145 than IS (P spectrin nor PARP-1 cleavage products were found. LL and IS showed different proteolysis after slaughter that was influenced more by calpain than caspase activity, which was detectable only in the early post mortem period. PMID:26950517

  19. Cadmium-induced apoptosis and necrosis in human osteoblasts: role of caspases and mitogen-activated protein kinases pathways.

    Science.gov (United States)

    Brama, M; Politi, L; Santini, P; Migliaccio, S; Scandurra, R

    2012-02-01

    Cadmium is a widespread environmental pollutant which induces severe toxic alterations, including osteomalacia and osteoporosis, likely by estrogen receptor-dependent mechanisms. Indeed, cadmium has been described to act as an endocrine disruptor and its toxicity is exerted both in vivo and in vitro through induction of apoptosis and/or necrosis by not fully clarified intracellular mechanism(s) of action. Aim of the present study was to further investigate the molecular mechanism by which cadmium might alter homeostasis of estrogen target cells, such as osteoblast homeostasis, inducing cell apoptosis and/or necrosis. Human osteoblastic cells (hFOB 1.19) in culture were used as an in vitro model to characterize the intracellular mechanisms induced by this heavy metal. Cells were incubated in the presence/ absence of 10-50 μM cadmium chloride at different times and DNA fragmentation and activation of procaspases- 8 and -3 were induced upon CdCl(2) treatment triggering apoptotic and necrotic pathways. Addition of caspase-8 and -3 inhibitors (Z-IETD-FMK and Z-DQMD-FMK) partially blocked these effects. No activation of procaspase-9 was observed. To determine the role of mitogen-activated protein kinases (MAPK) in these events, we investigated c-jun N-terminal kinase (JNK), p38 and extracellular signal-regulated protein kinase (ERK1/2) phosphorylation which were activated by 10 μM CdCl(2). Chemical inhibitors of JNK, p38, and ERK1/2, SP600125, SB202190, and PD98059, significantly reduced the phosphorylation of the kinases and blunted apoptosis. In contrast, caspase inhibitors did not reduce the cadmium-induced MAPK phosphorylation, suggesting an independent activation of these pathways. In conclusion, at least 2 pathways appear activated by cadmium in osteoblasts: a direct induction of caspase-8 followed by activation of caspase-3 and an indirect induction by phosphorylation of ERK1/2, p38, and JNK MAPK triggering activation of caspase-8 and -3. PMID:21697648

  20. 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

    Full Text Available Multi-walled carbon nanotubes (MWCNTs represent a human health risk as mice exposed by inhalation display pulmonary fibrosis. Production of IL-1β via inflammasome activation is a mechanism of MWCNT-induced acute inflammation and has been implicated in chronic fibrogenesis. Mice sensitized to allergens have elevated T-helper 2 (Th2 cytokines, IL-4 and IL-13, and are susceptible to MWCNT-induced airway fibrosis. We postulated that Th2 cytokines would modulate MWCNT-induced inflammasome activation and IL-1β release in vitro and in vivo during allergic inflammation.THP-1 macrophages were primed with LPS, exposed to MWCNTs and/or IL-4 or IL-13 for 24 hours, and analyzed for indicators of inflammasome activation. C57BL6 mice were sensitized to house dust mite (HDM allergen and MWCNTs were delivered to the lungs by oropharyngeal aspiration. Mice were euthanized 1 or 21 days post-MWCNT exposure and evaluated for lung inflammasome components and allergic inflammatory responses.Priming of THP-1 macrophages with LPS increased pro-IL-1β and subsequent exposure to MWCNTs induced IL-1β secretion. IL-4 or IL-13 decreased MWCNT-induced IL-1β secretion by THP-1 cells and reduced pro-caspase-1 but not pro-IL-1β. Treatment of THP-1 cells with STAT6 inhibitors, either Leflunomide or JAK I inhibitor, blocked suppression of caspase activity by IL-4 and IL-13. In vivo, MWCNTs alone caused neutrophilic infiltration into the lungs of mice 1 day post-exposure and increased IL-1β in bronchoalveolar lavage fluid (BALF and pro-caspase-1 immuno-staining in macrophages and airway epithelium. HDM sensitization alone caused eosinophilic inflammation with increased IL-13. MWCNT exposure after HDM sensitization increased total cell numbers in BALF, but decreased numbers of neutrophils and IL-1β in BALF as well as reduced pro-caspase-1 in lung tissue. Despite reduced IL-1β mice exposed to MWCNTs after HDM developed more severe airway fibrosis by 21 days and had increased

  1. 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

    Full Text Available Ying-Hui Chen,1,2,* Jo-Yu Wang,3,* Bo-Syong Pan,3,4 Yi-Fen Mu,3 Meng-Shao Lai,3,4 Edmund Cheung So,5 Thian-Sze Wong,6 Bu-Miin Huang3,4 1Department of Anesthesia, Chi-Mei Medical Center, Liouying, 2Department of Nursing, Min-Hwei College of Health Care Management, 3Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, 4The Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, 5Department of Anesthesia, An Nan Hospital, China Medical University, Tainan, Taiwan; 6Department of Surgery, University of Hong Kong Medical Center, Faculty of Medicine, The University of Hong Kong, Hong Kong *Authors contributed equally to this work Purpose: The present study aims to investigate whether the combination treatment of cordycepin (an extracted pure compound from Cordyceps sinensis and cisplatin (a platinum-based chemotherapy drug has better apoptotic effect in head and neck squamous cell carcinoma (HNSCC. Methods: The apoptotic influences of cordycepin and/or cisplatin treatments to human OC3, OEC-M1, and FaDu HNSCC cells were investigated by morphological observations, viability assay, flow cytometry assay, and Western blotting methods. Results: Data showed that the cell death phenomenon increased as the dosage of cordycepin or cisplatin increased, and it appeared more in cordycepin plus cisplatin cotreatment among three cell lines. Cell survival rates significantly decreased as the dosage of cordycepin or cisplatin increased, and the better apoptotic effects were observed in cotreatment. Cell cycle analysis further demonstrated that percentages of subG1 cells in cordycepin or cisplatin treatments significantly increased, suggesting that cells underwent apoptosis, and cordycepin plus cisplatin induced many more subG1 cells. Furthermore, cordycepin or cisplatin induced caspase-8, caspase-9, caspase-3, and poly adenosine diphosphate-ribose polymerase protein cleavages, and stimulated c

  2. Application of a FRET probe for Caspase-3 activation in living HeLa cells by sequentially treated cisplatin and TRAIL

    Science.gov (United States)

    Lin, Juqiang; Zhang, Zhihong; Yi, Qiushi; Zeng, Shaoqun; Luo, Qingming

    2006-02-01

    Caspase-3 is a kind of cysteine proteases that plays an important role in cell apoptosis. We have constructed a FRET (fluorescence resonance energy transfer) probe fused with ECFP (enhanced cyan fluorescence protein) and DsRed (Discosoma red fluorescent protein) with a linker containing a caspase-3 cleavage sequence (CCS, DEVD).It could be observed much change in fluorescence emission ratio when the probe was cleaved by caspase-3. Therefore, application of this probe we can real-time detected the activation of caspase-3. It was already confirmed that caspase-3 was activated in HeLa cells treated by cisplatin or TRAIL (Tumor necrosis factor (TNF)-related apoptosis-inducing ligand). In the present study, we detected the activation of caspase-3 during cisplatin or TRAIL induced apoptosis in living HeLa cells, and also observed the activation of caspase-3 caused by both cisplatin and TRAIL combined treatment. Our results demonstrated a synergistic effect between cisplatin and TRAIL. Cisplatin is one of the most broadly used drugs in the Clinical applications of cancer chemotherapy, and TRAIL, which belongs to the TNF family proteins, can selectively induce apoptosis in many transformed cells but not in normal cells. Therefore, TRAIL is a very valuably prospective utility as its potential tumor-specific cancer therapeutic. Most of anticancer drugs can induce apoptosis which mediated by the activation of caspase pathway. We can select the best synergistic effect group by our FRET probe. This finding would be useful in the design of treatment modalities for patients.

  3. Synergistic anticancer activity of dietary tea polyphenols and bleomycin hydrochloride in human cervical cancer cell: Caspase-dependent and independent apoptotic pathways.

    Science.gov (United States)

    Alshatwi, Ali A; Periasamy, Vaiyapuri Subbarayan; Athinarayanan, Jegan; Elango, Ramesh

    2016-03-01

    Bleomycin is a chemotherapeutic agent that is frequently used in the treatment of various cancers. Bleomycin causes serious adverse effects via antioxidant defense abnormalities against reactive oxygen species (ROS). However, the current cervical cancer monodrug therapy strategy has failed to produce the expected outcomes; hence, combinational therapies are gaining great interest. Tea polyphenols are also effective antioxidative and chemo-preventive agents. However, the combined effect of tea polyphenol (TPP) and bleomycin (BLM) against cervical cancer remains unknown. In this study, we focused on the potential of TPP on BLM anticancer activity against cervical cancer cells. Cervical cancer cells (SiHa) were treated with various concentrations of TPP, BLM and TPP combined with BLM (TPP-BLM), and their effects on cell growth, intracellular reactive oxygen species, poly-caspase activity, early apoptosis and the expression of caspase-3, caspase-8 and caspase-9, Bcl-2 and p53 were assessed. The MTT assay revealed that the SiHa cells were less sensitive to growth inhibition by TPP treatment compared with both BLM and the combination therapy. Nuclear staining indicated that exposure to TPP-BLM increased the percentage of apoptotic nuclei compared with a mono-agent treatment. Caspase activation assay demonstrated that proportion of early and late apoptotic/secondary necrotic cells was higher in the cells treated with the combination therapy than in those treated with either TPP or BLM alone. The TPP-BLM treatment synergistically induced apoptosis through caspase-3, caspase-8 and caspase-9 activation, Bcl-2 upregulation and p53 overexpression. This study suggests that TPP-BLM may be used as an efficient antioxidant-based combination therapy for cervical cancer. PMID:26800624

  4. Elevated Levels of Uterine Anti-Apoptotic Signaling May Activate NFKB and Potentially Confer Resistance to Caspase 3-Mediated Apoptotic Cell Death During Pregnancy in Mice1

    OpenAIRE

    Jeyasuria, Pancharatnam; Subedi, Kalpana; Suresh, Arvind; Condon, Jennifer C.

    2011-01-01

    Preserving the uterus in a state of relative quiescence is vital to the maintenance of a successful pregnancy. Elevated cytoplasmic levels of uterine caspase 3 during pregnancy have been proposed as a potential regulator of uterine quiescence through direct targeting and disabling of the uterine contractile architecture. However, despite highly elevated levels of uterine caspase 3 during pregnancy, there is minimal evidence of apoptosis. This current study defines the mechanism whereby the pr...

  5. Immunohistochemical study of cell proliferation, Bcl-2, p53, and caspase-3 expression on preneoplastic changes induced by cadmium and zinc chloride in the ventral rat prostate.

    OpenAIRE

    Arriazu, Riánsares; José M Pozuelo; Henriques-Gil, Nuno; Perucho, Teresa; Martín, Rocío; Rodríguez, Rosario; Santamaría, Luis

    2006-01-01

    KEYWORDS CLASSIFICATION: Animals;Apoptosis;biosynthesis;Biology;chemically induced;Cadmium;Cadmium Chloride;Carcinogens;Caspase 3;Caspases;Cell Proliferation;Chlorides;Immunohistochemistry;metabolism;Male;mechanisms of carcinogenesis;pathology;pharmacology;Precancerous Conditions;Proliferating Cell Nuclear Antigen;Prostate;Prostatic Intraepithelial Neoplasia;Prostatic Neoplasms;Proteins;Proto-Oncogene Proteins;Proto-Oncogene Proteins c-bcl-2;Rats;Rats,Sprague-Dawley;Research;Spain;toxicity;Ti...

  6. Involvement of mitochondria and caspase pathways in N-demethyl-clarithromycin-induced apoptosis in human cervical cancer HeLa cell

    Institute of Scientific and Technical Information of China (English)

    Ai-min QIAO; Takashi IKEJIMA; Shini-chi TASHIRO; Satoshi ONODERA; Wei-ge ZHANG; Ying-liang WU

    2006-01-01

    Aim: To study the mechanisms by which N-demethyl-clarithromycin (NDC) induces human cervical cancer HeLa cell apoptosis in vitro. Methods: The viability of N-demethyl-clarithromycin-induced HeLa cells was measured by MTT assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. Measurement of mitochondrial transmembrane potential was analyzed by a FACScan flowcytometer. Caspase-3, poly-(ADP-ribose) polymerase (PARP), caspase-activated DNase (ICAD), Bcl-2, Bax, p53, and SIRT1 protein expression and the release of cytochrome c were detected by Western blot analysis. Results: N-demethyl-clarithromycin, an anti-inflammatory substance, inhibited HeLa cell growth in a dose- and time-dependent manner.N-demethyl-clarithromycin induced HeLa cell death through the apoptotic pathways. The pan-caspase inhibitor (z-VAD-fmk), caspase-3 inhibitor (z-DEVD-fmk) and the caspase-9 inhibitor (z-LEHD-fmk) partially enhanced cell viability induced by N-demethyl-clarithromycin, but the caspase-8 inhibitor (z-IETD-fmk) had almost no effect. Caspase-3 was activated then followed by the degradation of caspase-3 substrates, the inhibitor of ICAD and PARP. Simultaneously, mitochondrial transmembrane potential was markedly reduced and the release of cytochrome c in the cytosol was increased.N-demethyl-clarithromycin upregulated the expression ratio of mitochondrial Bax/Bcl-2, and significantly increased the expression of the p53 protein. It also downregulated anti-apoptotic protein SIRT1 expression. Conclusion: N-demethyl-clarithromycin induced apoptosis in HeLa cells via the mitochondrial pathway.

  7. Estudo citofotométrico da expressão dos marcadores tumorais Caspase-3 e Ki-67 no adenocarcinoma gástrico Cytophotometric study of the expression of tumoral markers Caspase-3 and Ki-67 in gastric adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Pedro Manuel Gonzales Cuellar

    2007-06-01

    Full Text Available RACIONAL: A carcinogênese gástrica é processo complexo e depende de fatores genéticos, ambientais e infecciosos. Nos últimos anos, houve grandes avanços nos campos da genética e da biologia molecular, sobre o desenvolvimento dos tumores. Os marcadores tumorais são substâncias ausentes nos tecidos normais e que podem ser identificadas em tecidos com câncer. Através de procedimentos imunoistoquímicos eles podem ser estudados. OBJETIVOS: Descrever a expressão citofotométrica do marcador tumoral Ki-67 analisando a densidade óptica e o índice de marcagem no adenocarcinoma de estômago. Descrever a expressão citofotométrica do marcador tumoral Caspase-3 analisando a densidade óptica e o índice de marcagem no adenocarcinoma de estômago. Comparar o índice de marcagem e densidade óptica dos marcadores tumorais Ki-67 e Caspase-3 no adenocarcinoma de estômago. MÉTODO: Foram selecionados, inicialmente, 58 blocos com espécime de adenocarcinoma gástrico coletados nos Serviços de Anátomo-Patologia do Hospital do Gama - Brasília (DF e Hospital Dom Orione - Araguaina (TO, e analizados no Laboratório de Citologia e Histopalogia Ltda - CITOLAB, Curitiba (PR. Foram aproveitados 31 blocos para o estudo histológico e imunoistoquímico realizado pelo sistema de análise computarizado SAMBA 4000. RESULTADOS: Das 31 lâminas estudas, 15 (48% foram marcadas pelo marcador Ki-67, 22 (71% foram marcadas pelo marcador Caspase-3 e 14 (45% marcaram com os dois marcadores. CONCLUSÕES: A expressão citofométrica do marcador Ki-67 foi observada em 15 lâminas da amostra estudada e apresentaram média do índice de marcagem de 36,85%, enquanto a densidade óptica apresentou média de 29,33 pixels. A expressão citofotométrica do marcador Caspase-3 foi observada em 22 lâminas da amostra estudada e apresentaram média do índice de marcagem de 87,71% e 60,74 pixels de média para a densidade óptica. Na comparação do índice de marcagem dos

  8. 活化 Caspase 9在牛磺酸保护神经细胞中的保护作用%Taurine protects neuronal cells by suppressing Caspase 9 activation

    Institute of Scientific and Technical Information of China (English)

    刘阳; 王李瑶; 张庆华; 夏鹤春; 孙涛

    2014-01-01

    目的:神经系统疾病与神经细胞的凋亡密切相关。文中旨在探讨牛磺酸通过活化Caspase 9对海马神经元细胞凋亡的抑制作用,进而探讨牛磺酸对神经系统的保护作用及其机制。方法海马神经元细胞分为4组:对照组、损伤凋亡组、牛磺酸低剂量保护组、牛磺酸高剂量保护组。监测各组细胞生长状态,MTT监测各组细胞凋亡状态,免疫荧光及蛋白印迹法测定Caspase 9在各组中的表达水平。结果与对照组比较,损伤凋亡组海马神经元细胞生长不良,MTT实验示损失凋亡组细胞活力(A值为0.102±0.025)明显低于对照组(A值为0.643±0.013),低、高剂量干预组细胞活力(A值分别为0.504±0.072、0.452±0.029)明显提高(P<0.05);免疫荧光测定示损伤凋亡组Caspase 9活化明显增高(A值为61386.8±10083.6),对照组(A值为4502.2±2518.1)及牛磺酸低、高剂量保护组(A值分别为20077.4±4187.5和13976.2±7044.1)活化较低(P<0.05);蛋白印迹法示损伤凋亡组Caspase 9表达(A值为1.23)较对照组(A值为0.17)及低、高剂量保护组(A值分别为0.21和0.19),明显升高(P<0.05)。结论牛磺酸可抑制Caspase 9的活化,对神经细胞有较好的保护作用。%Objective Neurological diseases are closely associated with the apoptosis of neuronal cells .This article aims to study the inhibitory effect of taurine on the apoptosis of hippocampal neurons by activating Caspase 9 as well as its protective effect on the nervous system and its mechanisms . Methods Mouse hippocampal neuronal cells were randomly divided into four groups:control, injury and apoptosis, low-dose taurine protection, and high-dose taurine protection.The proliferation of the neuronalcells was observed, their apoptosis examined by MTT colorimetric assay, and the expression of Caspase 9 in different groups

  9. 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.

  10. Down-modulation of heat shock protein 70 and up-modulation of Caspase-3 during schisandrin B-induced apoptosis in human hepatoma SMMC-7721 cells

    Institute of Scientific and Technical Information of China (English)

    Yi-Feng Wu; Ming-Fu Cao; Yan-Ping Gao; Fei Chen; Tao Wang; Edward P. Zumbika; Kai-Xian Qian

    2004-01-01

    AIM: To investigate the effect of schisandrin B (Sch B) on proliferation and apoptosis of human hepatoma SMMC-7721 cells in vitro and regulation of Hsp70 and Caspases-3, 7, 9 expression by Sch B.METHODS: Human hepatoma cell line SMMC-7721 was cultured and treated with Sch B at various concentrations.Growth suppression was detected with MTT colorimetric assay. Cell apoptosis was confirmed by DNA ladder detection and flow cytometric analysis. The expression of Hsp70,Caspases-3, 7, 9 were analyzed by Western blot analysis.RESULTS: Sch B inhibited the growth of hepatoma SMMC-7721 cells in a dose-dependent manner, leading to a 50% decrease in cell number (LC50) value of 23.50 mg/L. Treatment with Sch B resulted in degradation of chromosomal DNA into small internucleosomal fragments, evidenced by the formation of a 180-200 bp DNA ladder on agarose gels.FCM analysis showed the peak areas of subdiploid at the increased concentration of Sch B. The results of Western bolt analysis showed that Hsp70 was down-regulated and Caspase-3 was up-regulated, while the activity of Caspases-7,-9 had no significant change.CONCLUSION: Sch B is able to inhibit the proliferation of human hepatoma SMMC-7721 cells and induce apoptosis,which goes through Caspase-3-dependent and Caspase-9-independent pathway accompanied with the down-regulation of Hsp70 protein expression at an early event.

  11. Cordycepin induces apoptosis of C6 glioma cells through the adenosine 2A receptor-p53-caspase-7-PARP pathway.

    Science.gov (United States)

    Chen, Ying; Yang, Shih-Hung; Hueng, Dueng-Yuan; Syu, Jhih-Pu; Liao, Chih-Chen; Wu, Ya-Chieh

    2014-06-01

    Cordycepin, 3'-deoxyadenosine from Cordyceps sinensis, has been shown to exert anti-tumor effects in several cancer cell lines. This study investigated the effect of cordycepin on a rat glioma cell line. Cordycepin caused apoptosis in C6 glioma cells in a time- and concentration-dependent manner, but did not affect the survival of primary cultured rat astrocytes. Cordycepin increased the total protein levels of p53 and phosphorylated p53 in the C6 cells. Levels of cleaved caspase-7 and poly (ADP-ribose) polymerase (PARP), but not cleaved caspase-3, were also increased after cordycepin treatment. Specific inhibitors for p53 and caspases abrogated cordycepin-induced caspase-7 and PARP cleavage, and prevented cordycepin-induced apoptosis. Moreover, siRNA knockdown of p53 blocked cordycepin-induced cleavage of caspase-7 and PARP. Both adenosine 2A receptor (A2AR) antagonist and small interference RNA (siRNA) knockdown of A2AR blocked cordycepin-induced apoptosis, p53 activation, and caspase-7 and PARP cleavage. These may provide a new strategy of cordycepin for glioma therapy in the future. PMID:24704558

  12. Mechanism of 2',3'-dimethoxyflavanone-induced apoptosis in breast cancer stem cells: role of ubiquitination of caspase-8 and LC3.

    Science.gov (United States)

    Tran, Thao Anh; Ahn, Kwang Seok; Song, Yeon Woo; Moon, Jeong Yong; Cho, Moonjae; Lim, Yoongho; Cho, Somi Kim

    2014-11-15

    Accumulating evidence has displayed that targeting cancer stem cells (CSCs) is a very promising way for anti-cancer therapies. 2',3'-Dimethoxyflavanone (2',3'-DMF) showed the most potent toxicity of a group of 42 flavonoids tested in MCF-7-SC breast cancer stem cells. 2',3'-DMF triggered intrinsic and extrinsic apoptosis by stimulating the cleavage of PARP and the activation of caspase-9, -8, and -3. Interestingly, 2',3'-DMF induces a dramatic increase in the conversion of LC3, a well-known marker for autophagy. However, acidic vesicular organelles (AVOs), one of the autophagic flux markers were not detected. Co-treatment with chloroquine, the lysosomal inhibitor that blocks autophagic degradation did not show any change in the degree of LC3 conversion, implying that LC3 could play a role in the non-autophagic cell death of MCF-7-SC. We found that 2',3'-DMF induces the ubiquitination of caspase-8, this resulted in an interaction between caspase-8 and LC3, which led to the aggregation and activation of caspase-8. Co-treating cells with 2',3'-DMF and 3-methyladenine, an inhibitor of LC3 lipidation, reduced the activation of caspase-8. These findings provide novel insights into the anti-cancer effects of 2',3'-DMF in breast cancer stem cells by revealing that it induced apoptosis in accompany with the activation of caspase-8 mediated by LC3 conversion. PMID:25132643

  13. Crocus sativus L. (Saffron Stigma Aqueous Extract Induces Apoptosis in Alveolar Human Lung Cancer Cells through Caspase-Dependent Pathways Activation

    Directory of Open Access Journals (Sweden)

    Saeed Samarghandian

    2013-01-01

    Full Text Available Worldwide, lung cancer is the most common form of cancer. Saffron has been used in folk medicine for centuries. We investigated the potential of saffron to induce cytotoxic and apoptotic effects in lung cancer cells (A549. We also examined the caspase-dependent pathways activation of saffron-induced apoptosis against the A549 cells. A549 cells were incubated with different concentrations of saffron extract; then cell morphological changes, cell viability, and apoptosis were determined by the normal invertmicroscope, MTT assay, Annexin V and propidium iodide, and flow cytometric analysis, respectively. Activated caspases were detected by treatment of saffron in lung cancer cells using fluorescein-labeled inhibitors of polycaspases. The proliferation of the A549 cells were decreased after treatment with saffron in a dose- and time-dependent manner. The percentage of apoptotic cells increased with saffron concentrations. Saffron induced morphological changes, decreased percentage of viable cells, and induced apoptosis. Saffron could induce apoptosis in the A549 cells and activate caspase pathways. The levels of caspases involved in saffron-induced apoptosis in the A549 cells indicating caspase-dependent pathway were induced by saffron. The anticancer activity of the aqueous extract of saffron could be attributed partly to its inhibition of the cell proliferation and induction of apoptosis in cancer cells through caspase-dependent pathways activation.

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

    Science.gov (United States)

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

    2013-10-01

    We describe here a simple fluorometric assay for the highly sensitive detection of caspase-3 activities on the basis of the inner-filter effect of gold nanoparticles (AuNPs) on CdTe quantum dots (QDs). The method takes advantage of the high molar absorptivity of the plasmon band of gold nanoparticles as well as the large absorption band shift from 520 to 680 nm upon nanoparticle aggregation. When labeled with a peptide possessing the caspase-3 cleavage sequence (DEVD), the monodispersed Au-Ps (peptide-modified AuNPs) exhibited a tendency to aggregate when exposed to caspase-3, which induced the absorption band transition from 520 to 680 nm and turned on the fluorescence of the CdTe QDs for caspase-3 sensing. Under optimum conditions, a high sensitivity towards caspase-3 was achieved with a detection limit as low as 18 pM, which was much lower than the corresponding assays based on absorbance or other approaches. Overall, we demonstrated a facile and sensitive approach for caspase-3 detection, and we expected that this method could be potentially generalized to design more fluorescent assays for sensing other bioactive entities. PMID:24015837

  15. 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. PMID:23675438

  16. IntApop: a web service for predicting apoptotic protein interactions in humans.

    Science.gov (United States)

    Zhou, Nan; Zhang, Jinchun; Feng, Ling; Lu, Bangmin; Wang, Zijie; Sun, Rong; Wu, Chuanfang; Bao, Jinku

    2013-12-01

    Apoptosis, a type of cell death, is necessary for maintaining tissue homeostasis and removing malignant cells. Interrupted apoptosis process contributes to carcinogenesis, developmental defects, autoimmune diseases and neurological disorders. Due to the complexity of the process, the molecular dynamics and relative interactions of individual proteins responsible for the activation or inhibition of apoptosis should be researched systematically. In this study, we integrate known protein interactions from databases DIP, IntAct, MINT, HPRD and BioGRID by Naïve Bayes classifier. The receiver operation characteristic (ROC) curve with the area under the ROC curve (AUC) of 0.797 indicates it has a good performance in prediction. Then, we predict the global human apoptotic protein interactions network. Within it, we not only identify the already known interactions of caspases (caspase-8/-10, caspase-9, caspase-3/-6/-7) and Bcl-2 family, but also reveal that Bid can interact with casein kinases (CSK21/22/2B, KC1A, KC1E); both of B2LA1 and B2CL2 can interact with Bid, Bax and Bak; caspase-8 interacts with autophagic proteins (MLP3B, MLP3A and LRRk2). Consequently, we make an initial step to develop the web service IntApop that provides an appropriate platform for apoptosis researchers, systems biologists and translational clinician scientists to predict apoptotic protein interactions in human. In addition, the interaction network can be visualized online, making it a widely applicable systems biology tool for apoptosis and cancer researchers. PMID:24120734

  17. miR-155 targets Caspase-3 mRNA in activated macrophages.

    Science.gov (United States)

    De Santis, Rebecca; Liepelt, Anke; Mossanen, Jana C; Dueck, Anne; Simons, Nadine; Mohs, Antje; Trautwein, Christian; Meister, Gunter; Marx, Gernot; Ostareck-Lederer, Antje; Ostareck, Dirk H

    2016-01-01

    To secure the functionality of activated macrophages in the innate immune response, efficient life span control is required. Recognition of bacterial lipopolysaccharides (LPS) by toll-like receptor 4 (TLR4) induces downstream signaling pathways, which merge to induce the expression of cytokine genes and anti-apoptotic genes. MicroRNAs (miRNAs) have emerged as important inflammatory response modulators, but information about their functional impact on apoptosis is scarce. To identify miRNAs differentially expressed in response to LPS, cDNA libraries from untreated and LPS-activated murine macrophages were analyzed by deep sequencing and regulated miRNA expression was verified by Northern blotting and qPCR. Employing TargetScan(TM) we identified CASPASE-3 (CASP-3) mRNA that encodes a key player in apoptosis as potential target of LPS-induced miR-155. LPS-dependent primary macrophage activation revealed TLR4-mediated enhancement of miR-155 expression and CASP-3 mRNA reduction. Endogenous CASP-3 and cleaved CASP-3 protein declined in LPS-activated macrophages. Accumulation of miR-155 and CASP-3 mRNA in miRNA-induced silencing complexes (miRISC) was demonstrated by ARGONAUTE 2 (AGO2) immunoprecipitation. Importantly, specific antagomir transfection effectively reduced mature miR-155 and resulted in significantly elevated CASP-3 mRNA levels in activated macrophages. In vitro translation assays demonstrated that the target site in the CASP-3 mRNA 3'UTR mediates miR-155-dependent Luciferase reporter mRNA destabilization. Strikingly, Annexin V staining of macrophages transfected with antagomir-155 and stimulated with LPS prior to staurosporine (SSP) treatment implied that LPS-induced miR-155 prevents apoptosis through CASP-3 mRNA down-regulation. In conclusion, we report that miR-155-mediated CASP-3 mRNA destabilization in LPS-activated RAW 264.7 macrophages suppresses apoptosis, as a prerequisite to maintain their crucial function in inflammation. PMID:26574931

  18. The cyclin-dependent kinase inhibitor roscovitine and the nucleoside analog sangivamycin induce apoptosis in caspase-3 deficient breast cancer cells independent of caspase mediated P-glycoprotein cleavage: Implications for therapy of drug resistant breast cancers

    OpenAIRE

    Cappellini, Alessandra; Chiarini, Francesca; Ognibene, Andrea; McCubrey, James A; Martelli, Alberto M.

    2009-01-01

    Resistance to multiple chemotherapeutic agents is a common clinical problem which can arise during cancer treatment. Drug resistance often involves overexpression of the multidrug resistance MDR1 gene, encoding P-glycoprotein (P-gp), a 170-kDa glycoprotein belonging to the ATP-binding cassette superfamily of membrane transporters. We have recently demonstrated apoptosis-induced, caspase-3-dependent P-gp cleavage in human T-lymphoblastoid CEM-R VBL100 cells. However, P-gp contain many aspartat...

  19. Nerve cells apoptosis and Fas and Caspase-8 expression among chronic alcoholic intoxication rats%慢性酒精中毒大鼠神经细胞凋亡及Fas、Caspase8蛋白的表达

    Institute of Scientific and Technical Information of China (English)

    段玉香; 张永利; 王彦

    2012-01-01

    目的 研究慢性酒精中毒大鼠神经细胞凋亡及Fas、Caspase8蛋白表达情况.方法 逐步增加饮水中酒精浓度,建立慢性酒精中毒大鼠动物模型,HE染色光镜下观察大鼠脑组织病理变化,Tunnel法检测大鼠脑细胞凋亡情况,免疫组化法检测Fas、Caspase8蛋白表达情况.结果 酒精组大脑皮质、海马、小脑等部位均有神经细胞数目缺失及细胞变性和损伤,酒精组各脑区凋亡细胞数和Fas、Casepse8蛋白表达阳性细胞数均高于对照组,两组比较差异有统计学意义(P<0.05).结论 慢性酒精中毒大鼠脑存在明显神经细胞凋亡,伴有Fas、Caspase-8蛋白表达增加,提示细胞死亡受体途径在慢性酒精中毒大鼠脑损伤病理过程中发挥作用.%Objective To understand nerve cells apoptosis and Fas and Casepse -8 protein expression status a-mong chronic alcoholic intoxication rats. Method Established chronic alcoholic intoxication rat model by increasing alcohol percentage of drinking gradually. Pathological changes were observed by light microscope, cells apoptosis and Fas and Casepse -8 protein expression were examined by Tunnel and Tmmunohistochemical method. Results In alcohol group, there were nerve cells missing, degeneration and damage in cerebral cortex, seahorse and cerebellum. The cells number of apoptosis and the positive cell of expression Fas and caspase -8 were obviously higher than control group, there was statistical significance between the 2 groups (P < 0.01) . Condusions There were obviously nerve cells apoptosis as well as the expression of Fas and Casepse -8 increased in the rats, which indicated that Fas and Casepse -8 protein attribute to nerve apoptosis and involved in the pathogenesis of chronic alcoholism.

  20. 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

    Benzene is a well-known environmental pollutant that can induce hematotoxicity, aplastic anemia, acute myelogenous leukemia, and lymphoma. However, although benzene metabolites are known to induce oxidative stress and disrupt the cell cycle, the mechanism underlying lympho/leukemogenicity is not fully understood. Caspase-4 (alias caspase-11) and -12 are inflammatory caspases implicated in inflammation and endoplasmic reticulum stress-induced apoptosis. The objectives of this study were to investigate the altered expression of caspase-4 and -12 in mouse bone marrow after benzene exposure and to determine whether their alterations are associated with benzene-induced bone marrow toxicity, especially cellular apoptosis. In addition, we evaluated whether the p53 gene is involved in regulating the mechanism, using both wild-type (WT) mice and mice lacking the p53 gene. For this study, 8-week-old C57BL/6 mice [WT and p53 knockout (KO)] were administered a benzene solution (150 mg/kg diluted in corn oil) via oral gavage once daily, 5 days/week, for 1 or 2 weeks. Blood and bone marrow cells were collected and cell counts were measured using a Coulter counter. Total mRNA and protein extracts were prepared from the harvested bone marrow cells. Then qRT-PCR and Western blotting were performed to detect changes in the caspases at the mRNA and protein level, respectively. A DNA fragmentation assay and Annexin-V staining were carried out on the bone marrow cells to detect apoptosis. Results indicated that when compared to the control, leukocyte number and bone marrow cellularity decreased significantly in WT mice. The expression of caspase-4 and -12 mRNA increased significantly after 12 days of benzene treatment in the bone marrow cells of benzene-exposed p53KO mice. However, apoptosis detection assays indicated no evidence of apoptosis in p53KO or WT mice. In addition, no changes of other apoptosis-related caspases, such as caspase-3 and -9, were found in WT or p53KO mice at the

  1. 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

  2. Expression of a naturally occurring angiotensin AT(1) receptor cleavage fragment elicits caspase-activation and apoptosis.

    Science.gov (United States)

    Cook, Julia L; Singh, Akannsha; DeHaro, Dawn; Alam, Jawed; Re, Richard N

    2011-11-01

    Several transmembrane receptors are documented to accumulate in nuclei, some as holoreceptors and others as cleaved receptor products. Our prior studies indicate that a population of the 7-transmembrane angiotensin type-1 receptor (AT(1)R) is cleaved in a ligand-augmented manner after which the cytoplasmic, carboxy-terminal cleavage fragment (CF) traffics to the nucleus. In the present report, we determine the precise cleavage site within the AT(1)R by mass spectrometry and Edman sequencing. Cleavage occurs between Leu(305) and Gly(306) at the junction of the seventh transmembrane domain and the intracellular cytoplasmic carboxy-terminal domain. To evaluate the function of the CF distinct from the holoreceptor, we generated a construct encoding the CF as an in-frame yellow fluorescent protein fusion. The CF accumulates in nuclei and induces apoptosis in CHO-K1 cells, rat aortic smooth muscle cells (RASMCs), MCF-7 human breast adenocarcinoma cells, and H9c2 rat cardiomyoblasts. All cell types show nuclear fragmentation and disintegration, as well as evidence for phosphotidylserine displacement in the plasma membrane and activated caspases. RASMCs specifically showed a 5.2-fold increase (P < 0.001) in CF-induced active caspases compared with control and a 7.2-fold increase (P < 0.001) in cleaved caspase-3 (Asp174). Poly(ADP-ribose)polymerase was upregulated 4.8-fold (P < 0.001) in CF expressing cardiomyoblasts and colocalized with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). CF expression also induces DNA laddering, the gold-standard for apoptosis in all cell types studied. CF-induced apoptosis, therefore, appears to be a general phenomenon as it is observed in multiple cell types including smooth muscle cells and cardiomyoblasts. PMID:21813711

  3. Discovery of novel second mitochondria-derived activator of caspase mimetics as selective inhibitor of apoptosis protein inhibitors.

    Science.gov (United States)

    Wang, Jin; Li, Wei

    2014-05-01

    Inhibitor of apoptosis (IAP) proteins are widely considered as promising cancer drug targets, especially for drug-resistant tumors. Mimicking the IAP-binding motif of second mitochondria-derived activator of caspases (SMAC) is a rational strategy to design potential IAP inhibitors. In this report, we used the bioactive conformation of AVPI tetrapeptide in the N terminus of SMAC as a template and performed a shape-based virtual screening against a drug-like compound library to identify novel IAP inhibitors. Top hits were subsequently docked to available IAP crystal structures as a secondary screening followed by validation using in vitro biologic assays. Four novel hit compounds were identified to potently inhibit cell growth in two human melanoma (A375 and M14) and two human prostate (PC-3 and DU145) cancer cell lines. The best compound, UC-112 [5-((benzyloxy)methyl)-7-(pyrrolidin-1-ylmethyl)quinolin-8-ol], has IC50 values ranging from 0.7 to 3.4 µM. UC-112 also potently inhibits the growth of P-glycoprotein (P-gp)-overexpressed multidrug-resistant cancer cells, strongly activates caspase-3/7 and caspase-9 activities, and selectively downregulates survivin level at a concentration as low as 1 µM. Coincubation of UC-112 with a known proteasome inhibitor Z-Leu-Leu-Leu-CHO (MG-132) rescued survivin inhibition, consistent with the anticipated mechanism of action for UC-112. As a single agent, UC-112 strongly inhibits tumor growth and reduces both X chromosome-linked IAP and survivin levels in an A375 human melanoma xenograft model in vivo. Overall, our study identified novel scaffolds, especially UC-112, as new platforms on which potent and selective IAP antagonists can be developed. PMID:24623800

  4. Docetaxel induces Bcl-2- and pro-apoptotic caspase-independent death of human prostate cancer DU145 cells.

    Science.gov (United States)

    Ogura, Takeharu; Tanaka, Yoshiyuki; Tamaki, Hiroki; Harada, Mamoru

    2016-06-01

    Docetaxel is a useful chemotherapeutic agent for the first-line treatment of hormone-refractory prostate cancer. Abnormal expression of Bcl-2 is commonly found in cancer cells, which increases their anti-apoptotic potency and chemoresistance. We investigated the effects of Bcl-2 expression status on the susceptibility of DU145 cells, an androgen-independent human prostate cancer cell line, to docetaxel and other anticancer agents. A panel of Bcl-2-expressing DU145 cell lines was established. Bcl-2 expression levels were unrelated to the susceptibility of DU145 cells to docetaxel. The sensitivity of DU145 cells to cisplatin fluctuated, and the sensitivity to tumor necrosis factor (TNF)-α was decreased by Bcl-2 overexpression. In a xenograft mouse model, overexpression of Bcl-2 drastically decreased the sensitivity of DU145 cells to cisplatin and TNF-α; however, there was no change in the response to docetaxel. Fluorescent microscopy revealed that Bcl-2-overexpression had no effect on the docetaxel-induced death of DU145 cells, but significantly decreased DU145 cell death induced by cisplatin or TNF-α. Interestingly, docetaxel hardly induced caspase-3/7 activation in control or Bcl-2-overexpressing DU145 cells, but did at a low level in LNCaP cells, another prostate cancer cell line. Moreover, in contrast to LNCaP cells, the reduced viabilities of docetaxel-treated control and Bcl-2-overexpressing DU145 cells were not restored by the addition of either a Bid inhibitor or a panel of pro-apoptotic caspase inhibitors. These findings indicate that the antitumor effects of docetaxel on DU145 cells are independent of both Bcl-2 and pro-apoptotic caspases. PMID:27082738

  5. Acetaminophen induces a caspase-dependent and Bcl-XL sensitive apoptosis in human hepatoma cells and lymphocytes.

    Science.gov (United States)

    Boulares, A Hamid; Zoltoski, Anna J; Stoica, Bogdan A; Cuvillier, Olivier; Smulson, Mark E

    2002-01-01

    Acetaminophen is a widely used analgesic and antipyretic drug that exhibits toxicity at high doses to the liver and kidneys. This toxicity has been attributed to cytochrome P-450-generated metabolites which covalently modify target proteins. Recently, acetaminophen, in its unmetabolized form, has been shown to affect a variety of cells and tissues, for instance, testicular and lymphoid tissues and lymphocyte cell lines. The effects on cell viability of acetaminophen at a concentration comparable to that achieved in plasma during acetaminophen toxicity have now been examined with a hepatoma cell line SK-Hep1, primary human peripheral blood lymphocytes and human Jurkat T cells. Acetaminophen reduced cell viability in a time-dependent manner. Staining of cells with annexin-V also revealed that acetaminophen induced, after 8 hr of treatment, a loss of the asymmetry of membrane phospholipids, which is an early event associated with apoptosis. Acetaminophen triggered the release of cytochrome c from mitochondria into the cytosol, activation of caspase-3, 8, and 9, cleavage of poly(ADP-ribose) polymerase, and degradation of lamin B1 and DNA. Whereas cleavage of DNA into internucleosomal fragments was apparent in acetaminophen treated SK-Hep1 and primary lymphocytes, DNA was only degraded to 50-kb fragments in treated Jurkat cells. Overexpression of the antiapoptotic protein Bcl-XL prevented these various apoptotic events induced by acetaminophen in Jurkat cells. Caspase-8 activation was a postmictochondrial event and occurred in a Fas-independent manner. These results demonstrate that acetaminophen induces caspases-dependent apoptosis with mitochondria as a primary target. These results also reiterate the potential role of apoptosis in acetaminophen hepatic and extrahepatic toxicity. PMID:12005112

  6. 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

  7. Activity identification of chimeric anti-caspase-3 mRNA hammerhead ribozyme in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    XU; Renhuan; (

    2001-01-01

    [1]Andrew, F., Gerard, E., A license to kill, Cell, 1996, 85: 781-784.[2]Thornberry, N. A., Lazebnik, Y., Caspases: Enemies within, Science, 1998, 281: 1312-1316.[3]Kijima, H., Ishida, H., Ohkawa, T. et al., Therapeutic application of ribozymes, Pharmacol. Ther., 1995, 68: 247-264.[4]Phylactou, L. A., Kilpatrick, M. W., Wood, M. J., Ribozymes as therapeutic tools for genetic disease, Hum. Mol. Genet., 1998, 7(10): 1649-1653.[5]Bettrand, E., Pictet, R ., Grange, T., Can heamerhead ribozymes be efficient tools inactivate gene function? Nucleic Acids Res., 1994, 22: 293-300.[6]Lieber, A., Strauss, M., Selection of efficient cleavage sites in target RNAs by using a ribozyme expression library, Mol. Cell Biol., 1995, 15: 540-551.[7]Xu, R. H., Zhou, X. Q., Xie, Q. et al., Preparation and identification of hammerhead ribozyme in vitro against rat caspase-3 mRNA fragment, Chin. J. Hepatol., 2000,8: 361-363.[8]Liu, J., Jin, Y. X., Wang, D. B., A novel vector for abundant expression of antisense RNA, triplex-forming RNA and ribozyme in vivo, High Technology Letters, 2000, 6: 84-88.[9]Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., New York: Cold Spring Harbor Laboratory Press, 1989.[10]Porter, A. G., J?nicke, R. U., Emerging roles of caspase-3 in apoptosis, Cell Death Differ, 1999, 6: 99-104.[11]Cryns, V., Yuan, J., Proteases to die for, Genes Dev., 1998, 12: 1551-1570.[12]Narendra, K. V., Anikumar, R. K., Fritz, E., Recent developments in the hammerhead ribozyme field, Nucleic Acids Research, 1998, 26: 5237-5242.

  8. Effect of perindopril on endoplasmic reticulum stress-related protein GRP78 and caspase-12 in rats with myocardial infarction

    Directory of Open Access Journals (Sweden)

    Yun-yun HE

    2014-03-01

    Full Text Available Objective  To investigate whether the effect of perindopril on ameliorating heart function is associated with inhibiting endoplasmic reticulum stress (ERS signaling pathway. Methods  Myocardial infarction (MI models in male SD rats were reproduced by ligation of the left anterior descending coronary artery (LAD. Rats that survived post-MI within 24h were randomly divided into model group (n=8 and perindopril group (n=8. Eight sham-operated rats were used as controls which underwent the same surgical procedure except that the suture around the coronary artery was not tied. The rats in peridopril group were treated with peridopril in a dose of 2mg/(kg.d for 4 weeks. The rats in model group and sham group received an equal amount of saline. At the end of 4 weeks, transthoracic echocardiogram was performed to measure the ventricular morphology and heart function, including left ventricular end systolic diameter (LVESD and end diastolic diameter (LVEDD, as well as left ventricular ejection fraction (LVEF and fraction shortening (FS. The expression levels of GRP78 and caspase-12 protein were assessed with immunohistochemistry or Western blotting. Results  Compared with the sham-operated group, the LVEDD and LVESD significantly increased after 4 weeks of MI, while the EF and FS decreased in model group. Perindopril significantly inhibited ventricular hypertrophy and improved heart function (P<0.05. Western blotting or immunohistochemistry showed that the important endoplasmic reticulum stress markers, GRP78 and caspase-12 significantly increased in perindopril treated rats after MI, showing that perindopril significantly attenuated these changes. Conclusion Perindopril may improve the heart function, which may be partly related to a decrease in the expression level of GRP78 and caspase-12, and by blocking ERS signaling pathway. DOI: 10.11855/j.issn.0577-7402.2014.01.01

  9. Induction of Apoptosis by Green Synthesized Gold Nanoparticles Through Activation of Caspase-3 and 9 in Human Cervical Cancer Cells

    Science.gov (United States)

    Baharara, Javad; Ramezani, Tayebe; Divsalar, Adeleh; Mousavi, Marzieh; Seyedarabi, Arefeh

    2016-01-01

    Background: Gold Nanoparticles (GNPs) are used in imaging and molecular diagnostic applications. As the development of a novel approach in the green synthesis of metal nanoparticles is of great importance and a necessity, a simple and safe method for the synthesis of GNPs using plant extracts of Zataria multiflora leaves was applied in this study and the results on GNPs’ anticancer activity against HeLa cells were reported. Methods: The GNPs were characterized by UV-visible spectroscopy, FTIR, TEM, DLS and Zeta-potential measurements. In addition, the cellular up-take of nanoparticles was investigated using Dark Field Microscopy (DFM). Induction of apoptosis by high dose of GNPs in HeLa cells was assessed by MTT assay, Acridin orange, DAPI staining, Annexin V/PI double-labeling flow cytometry and caspase activity assay. Results: UV-visible spectroscopy results showed a surface plasmon resonance band for GNPs at 530 nm. FTIR results demonstrated an interaction between plant extract and nanoparticles. TEM images revealed different shapes for GNPs and DLS results indicated that the GNPs range in size from 10 to 42 nm. The Zeta potential values of the synthesized GNPs were between 30 to 50 Mev, indicating the formation of stable particles. As evidenced by MTT assay, GNPs inhibit proliferation of HeLa cells in dose-dependent GNPs and cytotoxicity of GNPs in Bone Marrow Mesenchymal Stem Cell (BMSCs) was lower than cancerous cells. At nontoxic concentrations, the cellular up-take of the nanoparticles took place. Acridin orange and DAPI staining showed morphological changes in the cell’s nucleus due to apoptosis. Finally, caspase activity assay demonstrated HeLa cell’s apoptosis through caspase activation. Conclusion: The results showed that GNPs have the ability to induce apoptosis in HeLa cells. PMID:27141266

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

    International Nuclear Information System (INIS)

    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 G2/M arrest and appearance of a distinctive SubG1 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

  11. Abrin P2 suppresses proliferation and induces apoptosis of colon cancer cells via mitochondrial membrane depolarization and caspase activation.

    Science.gov (United States)

    Yu, Ying; Yang, Runmei; Zhao, Xiuyun; Qin, Dandan; Liu, Zhaoyang; Liu, Fang; Song, Xin; Li, Liqin; Feng, Renqing; Gao, Nannan

    2016-05-01

    To explore the cytotoxic mechanism of abrin P2 on human colon cancer HCT-8 cells, abrin P2 was isolated from the seed of Abrus precatorius L. It was found that abrin P2 exhibited cytotoxicity toward 12 different human cancer cell lines. Our results demonstrated that abrin P2 suppressed the proliferation of human colon cancer cells (HCT-8 cells) and induced cell cycle arrest at the S and G2/M phases. The mechanism by which abrin P2 inhibited cell proliferation was via the down-regulation of cyclin B1, proliferating cell nuclear antigen and Ki67, as well as the up-regulation of P21. In addition, abrin P2 induced a dose- and time-dependent increase in the rate of HCT-8 cell apoptosis. Treatment with both Z-VAD-FMK, a broad-spectrum caspase inhibitor, and abrin P2 demonstrated that abrin P2 induced HCT-8 cell apoptosis via the activation of caspases. Together, our results revealed that abrin P2-induced apoptosis in HCT-8 cells was associated with the activation of caspases-3/-8/-9, the reduction in the Bcl-2/Bax ratio, the loss of mitochondrial membrane potential, and the increase in cytochrome c release. We further showed that abrin P2 administration effectively suppressed the growth of colon cancer xenografts in nude mice. This is the first report that abrin P2 effectively inhibits colon cancer cell growth in vivo and in vitro by suppressing proliferation and inducing apoptosis. PMID:27055473

  12. Phycocyanin for protecting brain ischemia-reperfusion injury and its effect on the expression of Caspase-3 mRNA

    Institute of Scientific and Technical Information of China (English)

    Xuewei Yang; Yunliang Guo; Hongbing Chen

    2006-01-01

    BACKGROUND: Phycocyanin can anti-oxidize and clear free radial. Whether its protective effect on brain is related to Caspase-3, the promoter and operator of apoptosis, is highly concerned.OBJECTIVE: To observe phycocyanin for protecting nerve function and reducing the size of cerebral infarction of rats with brain ischemia-reperfusion and its effect on the expression of Caspase-3 mRNA.DESIGN: A randomized controlled experiment.SETrING: Institute of Cerebrovascular Disease, Affiliated Hospital of Medical College of Qingdao University.MATERIALS: Totally 84 adult healthy female Wistar rats, weighing 210 to 250 g, of clean grade, were provided by the Animal Experimental Center of Shandong University. Phycocyanin (Institute of Oceanography of Chinese Academy of Sciences) was used.METHODS: This experiment was carried out in the Key Laboratory for Prevention and Treatment of Brain Diseases during May to December 2005. ① The rats were randomized into sham-operation group (n=4),control group (n=40) and phycocyanin-treated group (n=40). Middle cerebral artery occlusion/reperfusion (MACO/R) models were created on the rats of control and phycocyanin-treated groups with suture-occluded method by inserting a thread into left side external-internal carotid artery. In the sham-operation group, inserting suture was omitted. After ischemia for 1 hour and reperfusion for 2 hours, suspension of phycocyanin was intragastrically administrated into the rats of the phycocyanin-treated group at 100 mg/kg , and the same volume of normal saline was isochronously administrated into the rats of control group as the same. ② Six rats were chosen respectively from the control group and phycocyanin-treated group, then neurologic impairment degrees of rats were evaluated according to Bederson's grading. ③ Six rats were chosen respectively from the control and phycocyanin-treated groups. The isolated brain tissue was stained with triphenyltetrazolium chloride, and then the size of cerebral

  13. Dynamics of caspase-3 activation and inhibition in embryonic micromasses evaluated by a photon-counting chemiluminiscence approach

    Czech Academy of Sciences Publication Activity Database

    Chlastáková, Ivana; Lišková, Marcela; Kudělová, Judita; Dubská, L.; Klepárník, Karel; Matalová, Eva

    2012-01-01

    Roč. 48, č. 9 (2012), s. 545-549. ISSN 1071-2690 R&D Projects: GA ČR(CZ) GAP206/11/2377; GA MŠk(CZ) EE2.3.20.0182 Grant ostatní: GA ČR(CZ) GAP502/12/1285 Institutional research plan: CEZ:AV0Z50450515; CEZ:AV0Z40310501 Keywords : Caspases * apoptpsis * chemiluminiscence * camptothecin Subject RIV: CB - Analytical Chemistry, Separation; ED - Physiology (UZFG-Y) Impact factor: 1.289, year: 2012

  14. A new class of fluorinated 5-pyrrolidinyl-sulfonyl isatin caspase inhibitors for PET imaging of apoptosis

    International Nuclear Information System (INIS)

    Thirteen compounds in a new class of fluorinated 5-pyrrolidinyl-sulfonyl isatin derivatives were synthesised that have potent and selective inhibitory activity against effector caspases-3 and -7. With in vivo animal PET imaging studies of cerebral ischemia being planned, N-benzylation with selected para-substituted benzylic halides allowed systematic variation of lipophilicity (logP 1.94-3.31) without decreasing inhibition potency (IC50). From this series the p-methoxybenzyl analogue was selected for initial 'proof-of-concept' [18F]- fluoride radiolabelling which proceeded in good yield and purity with no need for a protection/de-protection strategy. (authors)

  15. 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 Un