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Sample records for caspase degradome prediction

  1. Uncovering leaf rust responsive miRNAs in wheat (Triticum aestivum L.) using high-throughput sequencing and prediction of their targets through degradome analysis.

    Science.gov (United States)

    Kumar, Dhananjay; Dutta, Summi; Singh, Dharmendra; Prabhu, Kumble Vinod; Kumar, Manish; Mukhopadhyay, Kunal

    2017-01-01

    Deep sequencing identified 497 conserved and 559 novel miRNAs in wheat, while degradome analysis revealed 701 targets genes. QRT-PCR demonstrated differential expression of miRNAs during stages of leaf rust progression. Bread wheat (Triticum aestivum L.) is an important cereal food crop feeding 30 % of the world population. Major threat to wheat production is the rust epidemics. This study was targeted towards identification and functional characterizations of micro(mi)RNAs and their target genes in wheat in response to leaf rust ingression. High-throughput sequencing was used for transcriptome-wide identification of miRNAs and their expression profiling in retort to leaf rust using mock and pathogen-inoculated resistant and susceptible near-isogenic wheat plants. A total of 1056 mature miRNAs were identified, of which 497 miRNAs were conserved and 559 miRNAs were novel. The pathogen-inoculated resistant plants manifested more miRNAs compared with the pathogen infected susceptible plants. The miRNA counts increased in susceptible isoline due to leaf rust, conversely, the counts decreased in the resistant isoline in response to pathogenesis illustrating precise spatial tuning of miRNAs during compatible and incompatible interaction. Stem-loop quantitative real-time PCR was used to profile 10 highly differentially expressed miRNAs obtained from high-throughput sequencing data. The spatio-temporal profiling validated the differential expression of miRNAs between the isolines as well as in retort to pathogen infection. Degradome analysis provided 701 predicted target genes associated with defense response, signal transduction, development, metabolism, and transcriptional regulation. The obtained results indicate that wheat isolines employ diverse arrays of miRNAs that modulate their target genes during compatible and incompatible interaction. Our findings contribute to increase knowledge on roles of microRNA in wheat-leaf rust interactions and could help in rust

  2. Genome-wide identification of vegetative phase transition-associated microRNAs and target predictions using degradome sequencing in Malus hupehensis.

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    Xing, Libo; Zhang, Dong; Li, Youmei; Zhao, Caiping; Zhang, Songwen; Shen, Yawen; An, Na; Han, Mingyu

    2014-12-17

    A long juvenile period between germination and flowering is a common characteristic among fruit trees, including Malus hupehensis (Pamp.) Rehd., which is an apple rootstock widely used in China. microRNAs (miRNAs) play an important role in the regulation of phase transition and reproductive growth processes. M. hupehensis RNA libraries, one adult and one juvenile phase, were constructed using tree leaves and underwent high-throughput sequencing. We identified 42 known miRNA families and 172 novel miRNAs. We also identified 127 targets for 25 known miRNA families and 168 targets for 35 unique novel miRNAs using degradome sequencing. The identified miRNA targets were categorized into 58 biological processes, and the 123 targets of known miRNAs were associated with phase transition processes. The KEGG analysis revealed that these targets were involved in starch and sucrose metabolism, and plant hormone signal transduction. Expression profiling of miRNAs and their targets indicated multiple regulatory functions in the phase transition. The higher expression level of mdm-miR156 and lower expression level of mdm-miR172 in the juvenile phase leaves implied that these two small miRNAs regulated the phase transition. mdm-miR160 and miRNA393, which regulate genes involved in auxin signal transduction, could also be involved in controlling this process. The identification of known and novel miRNAs and their targets provides new information on this regulatory process in M. hupehensis, which will contribute to the understanding of miRNA functions during growth, phase transition and reproduction in woody fruit trees. The combination of sRNA and degradome sequencing can be used to better illustrate the profiling of hormone-regulated miRNAs and miRNA targets involving complex regulatory networks, which will contribute to the understanding of miRNA functions during growth, phase transition and reproductive growth in perennial woody fruit trees.

  3. Novel Bioinformatics-Based Approach for Proteomic Biomarkers Prediction of Calpain-2 & Caspase-3 Protease Fragmentation: Application to βII-Spectrin Protein

    Science.gov (United States)

    El-Assaad, Atlal; Dawy, Zaher; Nemer, Georges; Kobeissy, Firas

    2017-01-01

    The crucial biological role of proteases has been visible with the development of degradomics discipline involved in the determination of the proteases/substrates resulting in breakdown-products (BDPs) that can be utilized as putative biomarkers associated with different biological-clinical significance. In the field of cancer biology, matrix metalloproteinases (MMPs) have shown to result in MMPs-generated protein BDPs that are indicative of malignant growth in cancer, while in the field of neural injury, calpain-2 and caspase-3 proteases generate BDPs fragments that are indicative of different neural cell death mechanisms in different injury scenarios. Advanced proteomic techniques have shown a remarkable progress in identifying these BDPs experimentally. In this work, we present a bioinformatics-based prediction method that identifies protease-associated BDPs with high precision and efficiency. The method utilizes state-of-the-art sequence matching and alignment algorithms. It starts by locating consensus sequence occurrences and their variants in any set of protein substrates, generating all fragments resulting from cleavage. The complexity exists in space O(mn) as well as in O(Nmn) time, where N, m, and n are the number of protein sequences, length of the consensus sequence, and length per protein sequence, respectively. Finally, the proposed methodology is validated against βII-spectrin protein, a brain injury validated biomarker.

  4. Prognostic value of inhibitors of apoptosis proteins (IAPs) and caspases in prostate cancer: caspase-3 forms and XIAP predict biochemical progression after radical prostatectomy

    International Nuclear Information System (INIS)

    Rodríguez-Berriguete, Gonzalo; Torrealba, Norelia; Ortega, Miguel Angel; Martínez-Onsurbe, Pilar; Olmedilla, Gabriel; Paniagua, Ricardo; Guil-Cid, Manuel; Fraile, Benito; Royuela, Mar

    2015-01-01

    The expression status of apoptotic regulators, such as caspases and inhibitors of apoptosis proteins (IAPs), could reflect the aggressiveness of tumors and, therefore, could be useful as prognostic markers. We explored the associations between tumor expression of caspases and IAPs and clinicopathological features of prostate cancer – clinical and pathological T stage, Gleason score, preoperative serum PSA levels, perineural invasion, lymph node involvement, surgical margin status and overall survival – and evaluated its capability to predict biochemical progression after radical prostatectomy. Protein expression of caspases (procaspase-8, cleaved caspase-8, procaspase-3, cleaved caspase-3, caspase-7 and procaspase-9) and IAPs (cIAP1/2, cIAP2, NAIP, Survivin and XIAP) was analyzed by immunohistochemistry in radical prostatectomy samples from 84 prostate cancer patients. Spearman’s test, Kaplan-Meier curves, and univariate and multivariate Cox proportional hazard regression analysis were performed. cIAP1/2, cIAP2, Survivin, procaspase-8, cleaved caspase-8, procaspase-3 and caspase-7 expression correlated with at least one clinicopathological feature of the disease. Patients negative for XIAP, procaspase-3 or cleaved caspase-3 had a significantly worse prognosis. Of note, XIAP, procaspase-3 and cleaved caspase-3 were predictors of biochemical progression independent of Gleason score and pathological T stage. Our results indicate that alterations in the expression of IAPs and caspases contribute to the malignant behavior of prostate tumors and suggest that tumor expression of XIAP, procaspase-3 and cleaved caspase-3 may help to identify prostate cancer patients at risk of progression

  5. Targeted degradomics in protein terminomics and protease substrate discovery

    DEFF Research Database (Denmark)

    Savickas, Simonas; auf dem Keller, Ulrich

    2017-01-01

    extensive degradomics target lists that now can be tested with help of selected and parallel reaction monitoring (S/PRM) in complex biological systems, where proteases act in physiological environments. In this minireview, we describe the general principles of targeted degradomics, outline the generic...

  6. Dissecting Degradomes: Analysis of Protease-Coding Genes.

    Science.gov (United States)

    Álvarez-Eguiluz, Ángel; Díaz-Navarro, Ander; Puente, Xose S

    2018-01-01

    Proteases constitute up to 3% of all protein-coding genes in a vertebrate genome and participate in numerous physiological and pathological processes. The characterization of the degradome of one organism, the set of all genes encoding proteolytic enzymes, and the comparison to the degradome of other species have proved useful to identify genetic differences that are helpful to elucidate the molecular basis of diverse biological processes, the different susceptibility to disease, and the evolution of the structure and function of proteases. Here we describe the main procedures involved in the characterization of the degradome of an organism for which its genome sequence is available.

  7. Tudor staphylococcal nuclease is an evolutionarily conserved component of the programmed cell death degradome.

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    Sundström, Jens F; Vaculova, Alena; Smertenko, Andrei P; Savenkov, Eugene I; Golovko, Anna; Minina, Elena; Tiwari, Budhi S; Rodriguez-Nieto, Salvador; Zamyatnin, Andrey A; Välineva, Tuuli; Saarikettu, Juha; Frilander, Mikko J; Suarez, Maria F; Zavialov, Anton; Ståhl, Ulf; Hussey, Patrick J; Silvennoinen, Olli; Sundberg, Eva; Zhivotovsky, Boris; Bozhkov, Peter V

    2009-11-01

    Programmed cell death (PCD) is executed by proteases, which cleave diverse proteins thus modulating their biochemical and cellular functions. Proteases of the caspase family and hundreds of caspase substrates constitute a major part of the PCD degradome in animals. Plants lack close homologues of caspases, but instead possess an ancestral family of cysteine proteases, metacaspases. Although metacaspases are essential for PCD, their natural substrates remain unknown. Here we show that metacaspase mcII-Pa cleaves a phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease), during both developmental and stress-induced PCD. TSN knockdown leads to activation of ectopic cell death during reproduction, impairing plant fertility. Surprisingly, human TSN (also known as p100 or SND1), a multifunctional regulator of gene expression, is cleaved by caspase-3 during apoptosis. This cleavage impairs the ability of TSN to activate mRNA splicing, inhibits its ribonuclease activity and is important for the execution of apoptosis. Our results establish TSN as the first biological substrate of metacaspase and demonstrate that despite the divergence of plants and animals from a common ancestor about one billion years ago and their use of distinct PCD pathways, both have retained a common mechanism to compromise cell viability through the cleavage of the same substrate, TSN.

  8. 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.

  9. 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.

  10. Combined Small RNA and Degradome Sequencing Reveals Novel MiRNAs and Their Targets in the High-Yield Mutant Wheat Strain Yunong 3114.

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    Feng Chen

    Full Text Available Wheat is one of the main food sources worldwide; large amount studies have been conducted to improve wheat production. MicroRNAs (miRNAs with about 20-30 nucleotide are a class of regulatory small RNAs (sRNAs, which could regulate gene expression through sequence-specific base pairing with target mRNAs, playing important roles in plant growth. An ideal plant architecture (IPA is crucial to enhance yield in bread wheat. In this study, the high-yield wheat strain Yunong 3114 was EMS-mutagenesis from the wild-type strain Yunong 201, exhibiting a preferable plant structure compared with the wild-type strain. We constructed small RNA and degradome libraries from Yunong 201 and Yunong 3114, and performed small RNA sequencing of these libraries in order identify miRNAs and their targets related to IPA in wheat. Totally, we identified 488 known and 837 novel miRNAs from Yunong 3114 and 391 known and 533 novel miRNAs from Yunong 201. The number of miRNAs in the mutant increased. A total of 37 known and 432 putative novel miRNAs were specifically expressed in the mutant strain; furthermore, 23 known and 159 putative novel miRNAs were specifically expressed in the wild-type strain. A total of 150 known and 100 novel miRNAs were differentially expressed between mutant and wild-type strains. Among these differentially expressed novel miRNAs, 4 and 8 predict novel miRNAs were evidenced by degradome sequencing and showed up-regulated and down-regulated expressions in the mutant strain Yunong 3114, respectively. Targeted gene annotation and previous results indicated that this set of miRNAs is related to plant structure. Our results further suggested that miRNAs may be necessary to obtain an optimal wheat structure.

  11. 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.

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

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

    2002-01-01

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

  13. Genome-wide identification of the Phaseolus vulgaris sRNAome using small RNA and degradome sequencing.

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    Formey, Damien; Iñiguez, Luis Pedro; Peláez, Pablo; Li, Yong-Fang; Sunkar, Ramanjulu; Sánchez, Federico; Reyes, José Luis; Hernández, Georgina

    2015-06-02

    MiRNAs and phasiRNAs are negative regulators of gene expression. These small RNAs have been extensively studied in plant model species but only 10 mature microRNAs are present in miRBase version 21, the most used miRNA database, and no phasiRNAs have been identified for the model legume Phaseolus vulgaris. Thanks to the recent availability of the first version of the common bean genome, degradome data and small RNA libraries, we are able to present here a catalog of the microRNAs and phasiRNAs for this organism and, particularly, we suggest new protagonists in the symbiotic nodulation events. We identified a set of 185 mature miRNAs, including 121 previously unpublished sequences, encoded by 307 precursors and distributed in 98 families. Degradome data allowed us to identify a total of 181 targets for these miRNAs. We reveal two regulatory networks involving conserved miRNAs: those known to play crucial roles in the establishment of nodules, and novel miRNAs present only in common bean, suggesting a specific role for these sequences. In addition, we identified 125 loci that potentially produce phased small RNAs, with 47 of them having all the characteristics of being triggered by a total of 31 miRNAs, including 14 new miRNAs identified in this study. We provide here a set of new small RNAs that contribute to the broader knowledge of the sRNAome of Phaseolus vulgaris. Thanks to the identification of the miRNA targets from degradome analysis and the construction of regulatory networks between the mature microRNAs, we present here the probable functional regulation associated with the sRNAome and, particularly, in N2-fixing symbiotic nodules.

  14. Caspases : more than just killers?

    OpenAIRE

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

    2001-01-01

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

  15. Caspases: more than just killers?

    Science.gov (United States)

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

    2001-01-01

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

  16. The caspase-activated DNase

    DEFF Research Database (Denmark)

    Larsen, Brian D; Sørensen, Claus S

    2017-01-01

    CAD-induced DNA breaks. Furthermore, an apparent consequence of CAD activity is also emerging, as a potential source of oncogenic mutations. This review will discuss the mechanisms underlying CAD-induced DNA breaks and highlight how CAD activity promotes diverse cell fates....... and integral to this balance. Importantly, the view of apoptotic signal transduction has expanded over the previous decades. Subapoptotic caspase signaling has surfaced as mechanism that can promote the adoption of a range of cellular fates. An emerging mechanism of subapoptotic caspase signaling...... is the activation of the caspase-activated DNase (CAD) through controlled cleavage of the inhibitor of CAD (ICAD). CAD-induced DNA breaks incite a DNA damage response, frequently invoking p53 signaling, that transduces a change in cell fate. Cell differentiation and senescence are fates demonstrated to arise from...

  17. Genome-wide characterization of rice black streaked dwarf virus-responsive microRNAs in rice leaves and roots by small RNA and degradome sequencing.

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    Sun, Zongtao; He, Yuqing; Li, Junmin; Wang, Xu; Chen, Jianping

    2015-04-01

    MicroRNAs (miRNAs) are small, non-coding RNAs which typically function by guiding cleavage of target mRNAs. They play important roles in development, abiotic stress and responses to pathogens. Four small RNA libraries and four degradome libraries were constructed from the leaves and roots of healthy rice and plants infected with Rice black streaked dwarf virus (RBSDV). Analysis of the deep sequencing results showed that the expression patterns of 14 miRNAs in leaves and 16 miRNAs in roots changed significantly in response to RBSDV infection. Some responses were similar in roots and leaves, but many miRNAs responded differently in different tissues. The results were confirmed for selected miRNAs by quantitative real-time PCR. By using degradome sequencing, a total of 104 target transcripts for 17 conserved and 16 non-conserved miRNAs were shown to be responsive to RBSDV infection. Fifteen novel miRNAs were also identified by small RNA and degradome sequencing. The results provide new insights into the regulatory networks of miRNAs and their targets in different plant tissues in response to virus infection. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  18. Comparison of miRNAs and Their Targets in Seed Development between Two Maize Inbred Lines by High-Throughput Sequencing and Degradome Analysis.

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    Feng-Yao Wu

    Full Text Available MicroRNAs (miRNAs play an important role in plant growth, development, and response to environment. For identifying and comparing miRNAs and their targets in seed development between two maize inbred lines (i.e. PH6WC and PH4CV, two sRNAs and two degradome libraries were constructed. Through high-throughput sequencing and miRNA identification, 55 conserved and 24 novel unique miRNA sequences were identified in two sRNA libraries; moreover, through degradome sequencing and analysis, 137 target transcripts corresponding to 38 unique miRNA sequences were identified in two degradome libraries. Subsequently, 16 significantly differentially expressed miRNA sequences were verified by qRT-PCR, in which 9 verified sequences obviously target 30 transcripts mainly involved with regulation in flowering and development in embryo. Therefore, the results suggested that some miRNAs (e.g. miR156, miR171, miR396 and miR444 related reproductive development might differentially express in seed development between the PH6WC and PH4CV maize inbred lines in this present study.

  19. 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.

  20. Pharmacological caspase inhibitors: research towards therapeutic perspectives.

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    Kudelova, J; Fleischmannova, J; Adamova, E; Matalova, E

    2015-08-01

    Caspases are key molecules of apoptosis and the inflammatory response. Up-regulation of the caspase cascade contributes to human pathologies such as neurodegenerative and immune disorders. Thus, blocking the excessive apoptosis by pharmacological inhibitors seems promising for therapeutic interventions in such diseases. Caspase inhibitors, both natural and artificial, have been used as research tools and have helped to define the role of the individual caspases in apoptosis and in non-apoptotic processes. Moreover, some caspase inhibitors have demonstrated their therapeutic efficiency in the reduction of cell death and inflammation in animal models of human diseases. However, no drug based on caspase inhibition has been approved on the market until now. Thus, the development of therapeutic approaches that specifically target caspases remains a great challenge and is now the focus of intense biological and clinical interest. Here, we provide a brief review of recent knowledge about pharmacological caspase inhibitors with special focus on their proposed clinical applications.

  1. Identification of miRNAs and their targets through high-throughput sequencing and degradome analysis in male and female Asparagus officinalis.

    Science.gov (United States)

    Chen, Jingli; Zheng, Yi; Qin, Li; Wang, Yan; Chen, Lifei; He, Yanjun; Fei, Zhangjun; Lu, Gang

    2016-04-12

    MicroRNAs (miRNAs), a class of non-coding small RNAs (sRNAs), regulate various biological processes. Although miRNAs have been identified and characterized in several plant species, miRNAs in Asparagus officinalis have not been reported. As a dioecious plant with homomorphic sex chromosomes, asparagus is regarded as an important model system for studying mechanisms of plant sex determination. Two independent sRNA libraries from male and female asparagus plants were sequenced with Illumina sequencing, thereby generating 4.13 and 5.88 million final clean reads, respectively. Both libraries predominantly contained 24-nt sRNAs, followed by 21-nt sRNAs. Further analysis identified 154 conserved miRNAs, which belong to 26 families, and 39 novel miRNA candidates seemed to be specific to asparagus. Comparative profiling revealed that 63 miRNAs exhibited significant differential expression between male and female plants, which was confirmed by real-time quantitative PCR analysis. Among them, 37 miRNAs were significantly up-regulated in the female library, whereas the others were preferentially expressed in the male library. Furthermore, 40 target mRNAs representing 44 conserved and seven novel miRNAs were identified in asparagus through high-throughput degradome sequencing. Functional annotation showed that these target mRNAs were involved in a wide range of developmental and metabolic processes. We identified a large set of conserved and specific miRNAs and compared their expression levels between male and female asparagus plants. Several asparagus miRNAs, which belong to the miR159, miR167, and miR172 families involved in reproductive organ development, were differentially expressed between male and female plants, as well as during flower development. Consistently, several predicted targets of asparagus miRNAs were associated with floral organ development. These findings suggest the potential roles of miRNAs in sex determination and reproductive developmental processes in

  2. 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.

  3. Inflammasomes and inflammatory caspases in skin inflammation.

    Science.gov (United States)

    Iversen, Lars; Johansen, Claus

    2008-11-01

    The inflammatory caspases comprise a subclass of caspases associated with immune responses. Caspase-1 was the first identified member of this class, which also includes caspase-4, -5, -11 and -12. Caspase-1 was identified as the IL-1beta-converting enzyme and, more recently, it has also been shown to activate IL-18 and IL-33. Activation of the inflammatory caspases occurs upon assembly of multiprotein complexes, termed inflammasomes. The inflammasomes and inflammatory caspases are part of the innate immune system, which constitutes the first line of defense that detects pathogens, such as nonself antigens, bacterial and viral components, and other danger signals, and orchestrates the immune response. Inflammasomes and inflammatory caspases have also been suggested to bridge the innate immune responses to the adaptive immune system. More recently, the expression and role of inflammasomes and inflammatory caspases have been studied in both human and rodent skin, and findings have indicated a possible key role of these regulators of the immune system in the pathogenesis of inflammatory skin diseases. This article will review some of the most recent findings, identifying inflammasomes and inflammatory caspases as potential inducers and regulators of skin inflammation in contact hypersensitivity and psoriasis.

  4. Active caspase-3 expression levels as bioindicator of individual radiosensitivity

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    NEYLIANE F.G. DOS SANTOS

    Full Text Available ABSTRACT Several molecules and events involved in cell response to radiation-induced damage have been investigated towards a personalized radiotherapy. Considering the importance of active caspase-3 in the proteolytic cascade that ensures radiation-induced apoptosis execution, this research was designed to evaluate the expression levels of this protein as a bioindicator of individual radiosensitivity. Peripheral blood samples of 10 healthy individuals were gamma-irradiated (cobalt-60 source with 1, 2 and 4 Gy (control: non-irradiated samples, and active caspase-3 expression levels were measured in lymphocytes, by flow cytometry, ex vivo and after different times of in vitro incubation (24, 48 and 72 hours. Short-term incubation of 24 h was the most adequate condition to evidence correlations between dose radiation and active caspase-3 expression. For each radiation dose, it was observed a significant inter-individual variation in active caspase-3 expression intensity, suggesting that this parameter may be suitable for evidence individual radiosensitivity. The methodology presented and discussed in this work may help to predict healthy tissues response to radiation exposure toward the better patient outcome.

  5. Combined small RNA and degradome sequencing to identify miRNAs and their targets in response to drought in foxtail millet.

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    Wang, Yongqiang; Li, Lin; Tang, Sha; Liu, Jianguang; Zhang, Hanshuang; Zhi, Hui; Jia, Guanqing; Diao, Xianmin

    2016-04-12

    Foxtail millet (Setaria italica) is a diploid C4 panicoid species. Because of its prominent drought resistance, small genome size, self-pollination, and short life cycle, foxtail millet has become an ideal model system for studying drought tolerance of crops. MicroRNAs (miRNAs) are endogenous, small RNAs that play important regulatory roles in the development and stress response in plants. In this study, we applied Illumina sequencing to systematically investigate the drought-responsive miRNAs derived from S. italica inbred An04-4783 seedlings grown under control and drought conditions. Degradome sequencing was applied to confirm the targets of these miRNAs at a global level. A total of 81 known miRNAs belonging to 28 families were identified, among which 14 miRNAs were upregulated and four were downregulated in response to drought. In addition, 72 potential novel miRNAs were identified, three of which were differentially expressed under drought conditions. Degradome sequencing analysis showed that 56 and 26 genes were identified as targets of known and novel miRNAs, respectively. Our analysis revealed post-transcriptional remodeling of cell development, transcription factors, ABA signaling, and cellar homeostasis in S.italica in response to drought. This preliminary characterization provided useful information for further studies on the regulatory networks of drought-responsive miRNAs in foxtail millet.

  6. Pharmacological caspase inhibitors: Research towards therapeutic perspectives

    Czech Academy of Sciences Publication Activity Database

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

    2015-01-01

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

  7. Pharmacological caspase inhibitors: Research towards therapeutic perspectives

    Czech Academy of Sciences Publication Activity Database

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

    2015-01-01

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

  8. Caspase-12 and the inflammatory response to Yersinia pestis.

    NARCIS (Netherlands)

    Ferwerda, B.; McCall, M.B.B.; Vries, M.C. de; Hopman, J.C.W.; Maiga, B.; Dolo, A.; Doumbo, O.; Daou, M.; Jong, D.J. de; Joosten, L.A.B.; Tissingh, R.A.; Reubsaet, F.A.; Sauerwein, R.W.; Meer, J.W.M. van der; Ven, A.J.A.M. van der; Netea, M.G.

    2009-01-01

    BACKGROUND: Caspase-12 functions as an antiinflammatory enzyme inhibiting caspase-1 and the NOD2/RIP2 pathways. Due to increased susceptibility to sepsis in individuals with functional caspase-12, an early-stop mutation leading to the loss of caspase-12 has replaced the ancient genotype in Eurasia

  9. Identification of the degradome of Isp-1, a major intracellular serine protease of Bacillus subtilis, by two-dimensional gel electrophoresis and matrix- assisted laser desorption/ionization-time of flight analysis.

    Science.gov (United States)

    Lee, Ah Young; Goo Park, Sung; Kho, Chang Won; Young Park, Sun; Cho, Sayeon; Lee, Sang Chul; Lee, Do Hee; Myung, Pyung Keun; Park, Byoung Chul

    2004-11-01

    Intracellular serine protease-1 (Isp-1) is a major intracellular serine protease of Bacillus subtilis, whose functions still remain largely unknown. Furthermore, physiological substrates are yet to be determined. To identify Isp-1 substrates, we digested extract obtained from an Isp-1 deficient Bacillus mutant with purified Isp-1 and examined eliminated or decreased spots by two-dimensional gel and matrix-assisted laser desorption/ionization-time of flight analyses. Proteins degraded by Isp-1, termed the Isp-1 degradome, are involved in a variety of cellular functions such as DNA packing, genetic competence, and protein secretion. From the degradome we selected ClpC and EF-Tu as putative Isp-1 substrates and studied their in vitro degradation. ClpC and EF-Tu contain putative cleavage sites for Isp-1. N-terminal sequencing of in vitro proteolytic fragments of ClpC and EF-Tu revealed that these sites are indeed recognized and cleaved by Isp-1. Moreover, the cellular levels of ClpC and EF-Tu were dramatically reduced at the late stationary phase, where the expression level of Isp-1 was greatly increased. These results suggest that the regulated proteolysis of ClpC by Isp-1 plays an important role in the stationary phase adaptive response. This degradomic approach could provide a powerful tool for finding physiological substrates of many proteolytic enzymes whose functions remain to be determined.

  10. Genome-Wide Analysis of Gene and microRNA Expression in Diploid and Autotetraploid Paulownia fortunei (Seem Hemsl. under Drought Stress by Transcriptome, microRNA, and Degradome Sequencing

    Directory of Open Access Journals (Sweden)

    Zhenli Zhao

    2018-02-01

    Full Text Available Drought is a common and recurring climatic condition in many parts of the world, and it can have disastrous impacts on plant growth and development. Many genes involved in the drought response of plants have been identified. Transcriptome, microRNA (miRNA, and degradome analyses are rapid ways of identifying drought-responsive genes. The reference genome sequence of Paulownia fortunei (Seem Hemsl. is now available, which makes it easier to explore gene expression, transcriptional regulation, and post-transcriptional in this species. In this study, four transcriptome, small RNA, and degradome libraries were sequenced by Illumina sequencing, respectively. A total of 258 genes and 11 miRNAs were identified for drought-responsive genes and miRNAs in P. fortunei. Degradome sequencing detected 28 miRNA target genes that were cleaved by members of nine conserved miRNA families and 12 novel miRNAs. The results here will contribute toward enriching our understanding of the response of Paulownia fortunei trees to drought stress and may provide new direction for further experimental studies related the development of molecular markers, the genetic map construction, and other genomic research projects in Paulownia.

  11. 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.

  12. Krebs Cycle Moonlights in Caspase Regulation

    OpenAIRE

    Minis, Adi; Steller, Hermann

    2016-01-01

    In this issue of Developmental Cell, Aram et al. (2016) identify a mechanism that uses a Krebs cycle protein to control local activation of a ubiquitin ligase complex at the mitochondrial outer membrane for temporally and spatially restricted caspase activation during Drosophila sperm differentiation.

  13. Krebs Cycle Moonlights in Caspase Regulation.

    Science.gov (United States)

    Minis, Adi; Steller, Hermann

    2016-04-04

    In this issue of Developmental Cell, Aram et al. (2016) identify a mechanism that uses a Krebs cycle protein to control local activation of a ubiquitin ligase complex at the mitochondrial outer membrane for temporally and spatially restricted caspase activation during Drosophila sperm differentiation. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Conformational similarity in the activation of caspase-3 and -7 revealed by the unliganded and inhibited structures of caspase-7

    Energy Technology Data Exchange (ETDEWEB)

    Agniswamy, Johnson; Fang, Bin; Weber, Irene T.; (GSU)

    2009-09-08

    Caspase-mediated apoptosis has important roles in normal cell differentiation and aging and in many diseases including cancer, neuromuscular disorders and neurodegenerative diseases. Therefore, modulation of caspase activity and conformational states is of therapeutic importance. We report crystal structures of a new unliganded conformation of caspase-7 and the inhibited caspase-7 with the tetrapeptide Ac-YVAD-Cho. Different conformational states and mechanisms for substrate recognition have been proposed based on unliganded structures of the redundant apoptotic executioner caspase-3 and -7. The current study shows that the executioner caspase-3 and -7 have similar conformations for the unliganded active site as well as the inhibitor-bound active site. The new unliganded caspase-7 structure exhibits the tyrosine flipping mechanism in which the Tyr230 has rotated to block entry to the S2 binding site similar to the active site conformation of unliganded caspase-3. The inhibited structure of caspase-7/YVAD shows that the P4 Tyr binds the S4 region specific to polar residues at the expense of a main chain hydrogen bond between the P4 amide and carbonyl oxygen of caspase-7 Gln 276, which is similar to the caspase-3 complex. This new knowledge of the structures and conformational states of unliganded and inhibited caspases will be important for the design of drugs to modulate caspase activity and apoptosis.

  15. Plasma concentration of Caspase-8 is associated with short sleep duration and the risk of incident diabetes mellitus.

    Science.gov (United States)

    Svensson, Thomas; Svensson, Akiko Kishi; Kitlinski, Mariusz; Almgren, Peter; Engström, Gunnar; Nilsson, Jan; Orho-Melander, Marju; Nilsson, Peter M; Melander, Olle

    2018-02-01

    The biological mechanism for the association between sleep duration and incident diabetes mellitus (DM) is unclear. Sleep duration and Caspase-8, a marker of apoptotic activity, have both been implicated in beta cell function. To investigate the associations between sleep duration and plasma Caspase-8, and incident DM, respectively. Prospective cohort study. The Malmö Diet and Cancer (MDC) Study is a population-based, prospective study run in the city of Malmö, Sweden. 4023 individuals from the MDC Study aged 45-68 years at baseline without a history of prevalent DM, and with information on habitual sleep duration. Incident DM. Mean follow-up time was 17.8 years. Sleep duration was the only behavioural variable significantly associated with plasma Caspase-8. Plasma Caspase-8 was significantly associated with incident DM per standard deviation of its transformed continuous form (hazard ratio [HR]= 1.24, 95% confidence interval [CI] 1.13-1.36), and when dichotomized into high (quartile 4) (HR=1.44, 95%CI: 1.19-1.74) compared to low (quartiles 1-3) concentrations. Caspase-8 interacted with sleep duration; compared to 7-8 hours of sleep and low plasma Caspase-8, individuals with high plasma Caspase-8 and sleep duration <6 hours (HR=3.54, 95%CI: 2.12-5.90), 6-7 hours (HR=1.81, 95%CI: 1.24-2.65), and 8-9 hours (HR=1.54, 95%CI: 1.09-2.18) were at significantly increased risks of incident DM. Sleep duration is associated with plasma Caspase-8. Caspase-8 independently predicts DM years before disease onset and modifies the effect of sleep duration on incident DM. Future studies should investigate if change of sleep duration modifies plasma concentrations of Caspase-8. Copyright © 2018 Endocrine Society

  16. Camalexin induces apoptosis in T-leukemia Jurkat cells by increased concentration of reactive oxygen species and activation of caspase-8 and caspase-9.

    Science.gov (United States)

    Mezencev, Roman; Updegrove, Taylor; Kutschy, Peter; Repovská, Mária; McDonald, John F

    2011-07-01

    Camalexin, a major indole phytoalexin of Arabidopsis thaliana, accumulates in various cruciferous plants in response to environmental stress and reportedly displays antimicrobial activities against various plant pathogens. However, its cytotoxicity against eukaryotic cells and potential as a prospective drug for human diseases has been examined only in a limited context. Our data demonstrate the time- and concentration-dependent cytotoxicity of camalexin on human T-leukemia Jurkat cells in the micromolar range, and the lower potency of cytotoxic effects on human lymphoblasts and primary fibroblasts. Cytotoxicity of camalexin is enhanced by the glutathione-depleting agent buthionine sulfoximine and completely blocked by pan-caspase inhibitor Z-VAD-FMK. Treatment of Jurkat cells with camalexin resulted in activation of caspase-8, caspase-9, caspases-3/7, and apoptosis that was detected by the presence of a sub-G1 population of cells, externalization of phosphatidyl serine and decreased mitochondrial membrane potential. Staining with 2',7'-dichlorodihydrofluorescein diacetate and dihydroethidium bromide displayed increased concentration of reactive oxygen species (ROS) early in camalexin-treated Jurkat cells, prior to the onset of apoptosis, while staining with MitoSOX(™) dye identified mitochondria as a source of increased ROS. Our data suggest that this phytochemical, which has a wide range of predicted pharmacological activities, induces apoptosis in Jurkat leukemia cells through increased ROS followed by dissipation of mitochondrial membrane potential and execution of caspase-9- and caspase-8-initiated apoptosis. This is, to the best of our knowledge, the first report on antileukemic activity and mode of action of this unique indole phytoalexin.

  17. 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. Copyright © 2010 Elsevier Ltd. All rights reserved.

  18. Caspase-12 and the inflammatory response to Yersinia pestis.

    Science.gov (United States)

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

    2009-09-01

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

  19. The impact of caspase-12 on susceptibility to candidemia.

    NARCIS (Netherlands)

    Rosentul, D.C.; Plantinga, T.S.; Scott, W.K.; Alexander, B.D.; Geer, N.M. van de; Perfect, J.R.; Kullberg, B.J.; Johnson, M.D.; Netea, M.G.

    2012-01-01

    Candida is one of the leading causes of sepsis, and an effective host immune response to Candida critically depends on the cytokines IL-1beta and IL-18, which need caspase-1 cleavage to become bioactive. Caspase-12 has been suggested to inhibit caspase-1 activation and has been implicated as a

  20. Comparative Analysis of Fruit Ripening-Related miRNAs and Their Targets in Blueberry Using Small RNA and Degradome Sequencing.

    Science.gov (United States)

    Hou, Yanming; Zhai, Lulu; Li, Xuyan; Xue, Yu; Wang, Jingjing; Yang, Pengjie; Cao, Chunmei; Li, Hongxue; Cui, Yuhai; Bian, Shaomin

    2017-12-19

    MicroRNAs (miRNAs) play vital roles in the regulation of fruit development and ripening. Blueberry is an important small berry fruit crop with economical and nutritional value. However, nothing is known about the miRNAs and their targets involved in blueberry fruit ripening. In this study, using high-throughput sequencing of small RNAs, 84 known miRNAs belonging to 28 families and 16 novel miRNAs were identified in white fruit (WF) and blue fruit (BF) libraries, which represent fruit ripening onset and in progress, respectively. Among them, 41 miRNAs were shown to be differentially expressed during fruit maturation, and 16 miRNAs representing 16 families were further chosen to validate the sRNA sequencing data by stem-loop qRT-PCR. Meanwhile, 178 targets were identified for 41 known and 7 novel miRNAs in WF and BF libraries using degradome sequencing, and targets of miR160 were validated using RLM-RACE (RNA Ligase-Mediated (RLM)-Rapid Amplification of cDNA Ends) approach. Moreover, the expression patterns of 6 miRNAs and their targets were examined during fruit development and ripening. Finally, integrative analysis of miRNAs and their targets revealed a complex miRNA-mRNA regulatory network involving a wide variety of biological processes. The findings will facilitate future investigations of the miRNA-mediated mechanisms that regulate fruit development and ripening in blueberry.

  1. In vivo assessment of protease dynamics in cutaneous wound healing by degradomics analysis of porcine wound exudates.

    Science.gov (United States)

    Sabino, Fabio; Hermes, Olivia; Egli, Fabian E; Kockmann, Tobias; Schlage, Pascal; Croizat, Pierre; Kizhakkedathu, Jayachandran N; Smola, Hans; auf dem Keller, Ulrich

    2015-02-01

    Proteases control complex tissue responses by modulating inflammation, cell proliferation and migration, and matrix remodeling. All these processes are orchestrated in cutaneous wound healing to restore the skin's barrier function upon injury. Altered protease activity has been implicated in the pathogenesis of healing impairments, and proteases are important targets in diagnosis and therapy of this pathology. Global assessment of proteolysis at critical turning points after injury will define crucial events in acute healing that might be disturbed in healing disorders. As optimal biospecimens, wound exudates contain an ideal proteome to detect extracellular proteolytic events, are noninvasively accessible, and can be collected at multiple time points along the healing process from the same wound in the clinics. In this study, we applied multiplexed Terminal Amine Isotopic Labeling of Substrates (TAILS) to globally assess proteolysis in early phases of cutaneous wound healing. By quantitative analysis of proteins and protein N termini in wound fluids from a clinically relevant pig wound model, we identified more than 650 proteins and discerned major healing phases through distinctive abundance clustering of markers of inflammation, granulation tissue formation, and re-epithelialization. TAILS revealed a high degree of proteolysis at all time points after injury by detecting almost 1300 N-terminal peptides in ∼450 proteins. Quantitative positional proteomics mapped pivotal interdependent processing events in the blood coagulation and complement cascades, temporally discerned clotting and fibrinolysis during the healing process, and detected processing of complement C3 at distinct time points after wounding and by different proteases. Exploiting data on primary cleavage specificities, we related candidate proteases to cleavage events and revealed processing of the integrin adapter protein kindlin-3 by caspase-3, generating new hypotheses for protease

  2. Transcriptome-Wide Analysis of Botrytis elliptica Responsive microRNAs and Their Targets in Lilium Regale Wilson by High-Throughput Sequencing and Degradome Analysis

    Directory of Open Access Journals (Sweden)

    Xue Gao

    2017-05-01

    Full Text Available MicroRNAs, as master regulators of gene expression, have been widely identified and play crucial roles in plant-pathogen interactions. A fatal pathogen, Botrytis elliptica, causes the serious folia disease of lily, which reduces production because of the high susceptibility of most cultivated species. However, the miRNAs related to Botrytis infection of lily, and the miRNA-mediated gene regulatory networks providing resistance to B. elliptica in lily remain largely unexplored. To systematically dissect B. elliptica-responsive miRNAs and their target genes, three small RNA libraries were constructed from the leaves of Lilium regale, a promising Chinese wild Lilium species, which had been subjected to mock B. elliptica treatment or B. elliptica infection for 6 and 24 h. By high-throughput sequencing, 71 known miRNAs belonging to 47 conserved families and 24 novel miRNA were identified, of which 18 miRNAs were downreguleted and 13 were upregulated in response to B. elliptica. Moreover, based on the lily mRNA transcriptome, 22 targets for 9 known and 1 novel miRNAs were identified by the degradome sequencing approach. Most target genes for elliptica-responsive miRNAs were involved in metabolic processes, few encoding different transcription factors, including ELONGATION FACTOR 1 ALPHA (EF1a and TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR 2 (TCP2. Furthermore, the expression patterns of a set of elliptica-responsive miRNAs and their targets were validated by quantitative real-time PCR. This study represents the first transcriptome-based analysis of miRNAs responsive to B. elliptica and their targets in lily. The results reveal the possible regulatory roles of miRNAs and their targets in B. elliptica interaction, which will extend our understanding of the mechanisms of this disease in lily.

  3. Fibroblast activation protein-α, a stromal cell surface protease, shapes key features of cancer associated fibroblasts through proteome and degradome alterations.

    Science.gov (United States)

    Koczorowska, M M; Tholen, S; Bucher, F; Lutz, L; Kizhakkedathu, J N; De Wever, O; Wellner, U F; Biniossek, M L; Stahl, A; Lassmann, S; Schilling, O

    2016-01-01

    Cancer associated fibroblasts (CAFs) constitute an abundant stromal component of most solid tumors. Fibroblast activation protein (FAP) α is a cell surface protease that is expressed by CAFs. We corroborate this expression profile by immunohistochemical analysis of colorectal cancer specimens. To better understand the tumor-contextual role of FAPα, we investigate how FAPα shapes functional and proteomic features of CAFs using loss- and gain-of function cellular model systems. FAPα activity has a strong impact on the secreted CAF proteome ("secretome"), including reduced levels of anti-angiogenic factors, elevated levels of transforming growth factor (TGF) β, and an impact on matrix processing enzymes. Functionally, FAPα mildly induces sprout formation by human umbilical vein endothelial cells. Moreover, loss of FAPα leads to a more epithelial cellular phenotype and this effect was rescued by exogenous application of TGFβ. In collagen contraction assays, FAPα induced a more contractile cellular phenotype. To characterize the proteolytic profile of FAPα, we investigated its specificity with proteome-derived peptide libraries and corroborated its preference for cleavage carboxy-terminal to proline residues. By "terminal amine labeling of substrates" (TAILS) we explored FAPα-dependent cleavage events. Although FAPα acts predominantly as an amino-dipeptidase, putative FAPα cleavage sites in collagens are present throughout the entire protein length. In contrast, putative FAPα cleavage sites in non-collagenous proteins cluster at the amino-terminus. The degradomic study highlights cell-contextual proteolysis by FAPα with distinct positional profiles. Generally, our findings link FAPα to key aspects of CAF biology and attribute an important role in tumor-stroma interaction to FAPα. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  4. 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.

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

    Directory of Open Access Journals (Sweden)

    Sebastian Horn

    2017-04-01

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

  6. Caspases -2, -3, -6, and -9, but not caspase-1, are activated in sepsis-induced thymocyte apoptosis.

    Science.gov (United States)

    Tinsley, K W; Cheng, S L; Buchman, T G; Chang, K C; Hui, J J; Swanson, P E; Karl, I E; Hotchkiss, R S

    2000-01-01

    Sepsis induces extensive lymphocyte cell death that may contribute to immune depression and morbidity/mortality in the disorder. bcl-2 is a member of a new class of oncogenes that prevents cell death from an array of noxious stimuli. Transgenic mice that overexpress BCL-2 in T lymphocytes are resistant to sepsis-induced T cell apoptosis, and mortality was decreased in sepsis. The purpose of this study was to identify key initiator and executioner "caspases" involved in sepsis-induced lymphocyte apoptosis and to determine if BCL-2 acts prior to caspase activation. Thymi were removed 5-22 h post-cecal ligation and puncture (CLP) or sham surgery. Apoptosis was evaluated in thymocytes by annexin-V FITC labeling and flow cytometry. Caspase-1 activity was determined by western blot analysis of the procaspase protein and p20 subunit of the activated caspase; activities of caspases -2, -6, and -9 were determined by colorimetric assays using specific substrates conjugated to a color reporter molecule. Caspase-3 activity was determined both by western blot and by a fluorogenic assay in which a fluorescent compound was generated. Thymocytes from CLP mice had markedly increased apoptosis and activation of caspases -2, -3, -6, and -9 in comparison with thymocytes of sham-operated mice. Caspase-1 was not activated. BCL-2 prevented sepsis-induced thymocyte apoptosis and inhibited activation of all caspases. We conclude that sepsis causes activation of multiple caspases and that BCL-2 acts upstream as an inhibitor of caspase activation. The pattern of caspase activation suggests a mitochondrial mediated pathway.

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

    Science.gov (United States)

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

    2003-11-14

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

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

    Science.gov (United States)

    Vagner, Tatyana; Mouravlev, Alexandre; Young, Deborah

    2015-01-01

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

  9. 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.

  10. Caspase-like proteins: Acanthamoeba castellanii metacaspase and ...

    Indian Academy of Sciences (India)

    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 ...

  11. Activation of caspase-9 is required for UV-induced apoptosis of human keratinocytes.

    Science.gov (United States)

    Sitailo, Leonid A; Tibudan, Shalini S; Denning, Mitchell F

    2002-05-31

    UV radiation from the sun activates both the membrane death receptor and the intrinsic or mitochondrial apoptotic signaling pathways in epidermal keratinocytes, triggering apoptosis and affording protection against skin cancer formation. We have investigated the involvement of caspase-9 in the UV death effector pathway in human keratinocytes, since this is the initiating caspase in the mitochondrial pathway required for UV-induced apoptosis in some, but not all, cell types. UV radiation triggered activation of caspase-3, caspase-9, and caspase-8 with similar kinetics, although the rank order of activation was caspase-3 > caspase-9 > caspase-8. Inhibition of caspase-9 with either the peptide inhibitor benzyloxycarbonyl-Leu-Glu(OCH(3))-His-Asp(OCH(3))-fluoromethyl ketone, or expression of a catalytically inactive caspase-9 by retroviral transduction, protected normal keratinocytes from UV-induced apoptosis. HaCaT keratinocytes harboring mutant p53 alleles were also protected from UV-induced apoptosis by the dominant negative caspase-9. The dominant negative caspase-9 blocked UV-induced activation of caspase-3, caspase-9, and caspase-8, and also protected cells from the loss of mitochondrial membrane potential. In contrast, the dominant negative caspase-9 did not protect from anti-Fas-induced apoptosis or caspase activation. These results identify caspase-9 as the critical upstream caspase initiating apoptosis by UV radiation in human keratinocytes, the relevant cell type for this important environmental carcinogen.

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

    Science.gov (United States)

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

    2012-01-01

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

  13. Tau and Caspase 3 as Targets for Neuroprotection

    Directory of Open Access Journals (Sweden)

    Anat Idan-Feldman

    2012-01-01

    Full Text Available The peptide drug candidate NAP (davunetide has demonstrated protective effects in various in vivo and in vitro models of neurodegeneration. NAP was shown to reduce tau hyperphosphorylation as well as to prevent caspase-3 activation and cytochrome-3 release from mitochondria, both characteristic of apoptotic cell death. Recent studies suggest that caspases may play a role in tau pathology. The purpose of this study was to evaluate the effect of NAP on tau hyperphosphorylation and caspase activity in the same biological system. Our experimental setup used primary neuronal cultures subjected to oxygen-glucose deprivation (OGD, with and without NAP or caspase inhibitor. Cell viability was assessed by measuring mitochondrial activity (MTS assay, and immunoblots were used for analyzing protein level. It was shown that apoptosis was responsible for all cell death occurring following ischemia, and NAP treatment showed a concentration-dependent protection from cell death. Ischemia caused an increase in the levels of active caspase-3 and hyperphosphorylated tau, both of which were prevented by either NAP or caspase-inhibitor treatment. Our data suggest that, in this model system, caspase activation may be an upstream event to tau hyperphosphorylation, although additional studies will be required to fully elucidate the cascade of events.

  14. Intrinsic-mediated caspase activation is essential for cardiomyocyte hypertrophy

    Science.gov (United States)

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

    2013-01-01

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

  15. 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.

  16. Prospective clinical biomarkers of caspase-mediated apoptosis associated with neuronal and neurovascular damage following stroke and other severe brain injuries: Implications for chronic neurodegeneration

    Directory of Open Access Journals (Sweden)

    Olena Y Glushakova

    2017-01-01

    Full Text Available Acute brain injuries, including ischemic and hemorrhagic stroke, as well as traumatic brain injury (TBI, are major worldwide health concerns with very limited options for effective diagnosis and treatment. Stroke and TBI pose an increased risk for the development of chronic neurodegenerative diseases, notably chronic traumatic encephalopathy, Alzheimer's disease, and Parkinson's disease. The existence of premorbid neurodegenerative diseases can exacerbate the severity and prognosis of acute brain injuries. Apoptosis involving caspase-3 is one of the most common mechanisms involved in the etiopathology of both acute and chronic neurological and neurodegenerative diseases, suggesting a relationship between these disorders. Over the past two decades, several clinical biomarkers of apoptosis have been identified in cerebrospinal fluid and peripheral blood following ischemic stroke, intracerebral and subarachnoid hemorrhage, and TBI. These biomarkers include selected caspases, notably caspase-3 and its specific cleavage products such as caspase-cleaved cytokeratin-18, caspase-cleaved tau, and a caspase-specific 120 kDa αII-spectrin breakdown product. The levels of these biomarkers might be a valuable tool for the identification of pathological pathways such as apoptosis and inflammation involved in injury progression, assessment of injury severity, and prediction of clinical outcomes. This review focuses on clinical studies involving biomarkers of caspase-3-mediated pathways, following stroke and TBI. The review further examines their prospective diagnostic utility, as well as clinical utility for improved personalized treatment of stroke and TBI patients and the development of prophylactic treatment chronic neurodegenerative disease.

  17. Non-steroidal Anti-inflammatory Drugs Are Caspase Inhibitors.

    Science.gov (United States)

    Smith, Christina E; Soti, Subada; Jones, Torey A; Nakagawa, Akihisa; Xue, Ding; Yin, Hang

    2017-03-16

    Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most commonly used drugs in the world. While the role of NSAIDs as cyclooxygenase (COX) inhibitors is well established, other targets may contribute to anti-inflammation. Here we report caspases as a new pharmacological target for NSAID family drugs such as ibuprofen, naproxen, and ketorolac at physiologic concentrations both in vitro and in vivo. We characterize caspase activity in both in vitro and in cell culture, and combine computational modeling and biophysical analysis to determine the mechanism of action. We observe that inhibition of caspase catalysis reduces cell death and the generation of pro-inflammatory cytokines. Further, NSAID inhibition of caspases is COX independent, representing a new anti-inflammatory mechanism. This finding expands upon existing NSAID anti-inflammatory behaviors, with implications for patient safety and next-generation drug design. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. 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

    2015-09-24

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

  19. Molecular cloning, characterization and expression analysis of caspase-3 from the oriental river prawn, Macrobrachium nipponense when exposed to acute hypoxia and reoxygenation.

    Science.gov (United States)

    Sun, Shengming; Xuan, Fujun; Fu, Hongtuo; Zhu, Jian; Ge, Xianping; Wu, Xugan

    2017-03-01

    Caspases are present in the cytosol as inactive proenzymes but become activated when apoptosis is initiated, playing an essential role at various stages of the process. In this study, a caspase-3 (Mncaspase-3c) was cloned from gill of the oriental river prawn Macrobrachium nipponense by reverse-transcription polymerase chain reaction and rapid amplification of cDNA ends, and its properties were characterized. The 1730-bp cDNA contained an open reading frame of 1566 bp, a 123-bp 5'-untranslated region (UTR), and a 41-bp 3'-UTR containing a poly(A) tail. The molecular mass of the deduced amino acid (aa) sequence (521 aa) was 56.3 kDa with an estimated pI of 5.01. The MnCaspase-3c sequence contained a predicted caspase family p20 domain and a caspase family p10 domain at positions 236-367 and 378-468 respectively. Recombinant MnCaspase-3c protein was expressed in Escherichia coli and purified. In vitro activity assays indicated that the recombinant MnCaspase-3c hydrolyzed the substrate Ac-DEVD-pNA, suggesting a physiological role as a caspase-3. Caspase-3c gene transcripts were distributed in all M. nipponense tissues tested by quantitative RT-PCR, being especially abundant in hemocytes. Comet assays in gill tissues showed an obvious time-dependent response to hypoxia. Furthermore, Mncaspase-3c, at both the mRNA and protein levels, was demonstrated to participate in the apoptotic process in gill after stimulation by acute hypoxia. Overall, these results indicate that hypoxia triggers apoptosis in shrimp gill tissues. Copyright © 2017. Published by Elsevier Ltd.

  20. Suture compression induced midpalatal suture chondrocyte apoptosis with increased caspase-3, caspase-9, Bad, Bak, Bax and Bid expression.

    Science.gov (United States)

    Lan, Tingting; Zhao, Hanchi; Xiang, Bilu; Wang, Jun; Liu, Yang

    2017-07-22

    Previous studies found bone resorption and chondrocytes loss in mouse models of mid-palatal suture when given continuous compressive force, although chondrocytes response remained unknown. Herein, we design this study to determine how continuous compression force induces chondrocytes apoptosis. Thirty C57BL/6 male mice (aged 6 weeks) were randomly assigned into controls (not ligated to a spring), blank controls (ligated with no compression) and the compression group (ligated with 20-g compression). After 4 d, palatal tissues were sampled and stained by TB and safranin-O. Tunel staining measured the percentage of apoptotic chondrocytes, and immunohistochemistry was performed to label apoptosis-associated proteins (e.g., Bcl-2, Bcl-xl, Bax, Bak, Bid, Bad, caspase-3, caspase-8 and caspase-9). Intergroup comparison was made by the rank sum test, and P compression group was significantly decreased, while the control group remained largely unaltered. Tunel staining showed that apoptotic cell numbers in the mid-palatal suture were significantly higher than the control group. Immunohistochemistry showed that mice in the compression group had significantly increased expression of caspase-3, caspase-9, Bad, Bak, Bax and Bid; However, caspase-8 remained unaltered. No expression of Bcl-2 and Bcl-xl was detected. Continuous compression force induces chondrocytes apoptosis in the mid-palatal suture. This process might be associated with the mitochondrial pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    Pearce, John A.

    2013-02-01

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

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

    NARCIS (Netherlands)

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

    2011-01-01

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

  3. 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.

  4. Caspase-12 ablation preserves muscle function in the mdx mouse

    Science.gov (United States)

    Moorwood, Catherine; Barton, Elisabeth R.

    2014-01-01

    Duchenne muscular dystrophy (DMD) is a devastating muscle wasting disease caused by mutations in dystrophin. Several downstream consequences of dystrophin deficiency are triggers of endoplasmic reticulum (ER) stress, including loss of calcium homeostasis, hypoxia and oxidative stress. During ER stress, misfolded proteins accumulate in the ER lumen and the unfolded protein response (UPR) is triggered, leading to adaptation or apoptosis. We hypothesized that ER stress is heightened in dystrophic muscles and contributes to the pathology of DMD. We observed increases in the ER stress markers BiP and cleaved caspase-4 in DMD patient biopsies, compared with controls, and an increase in multiple UPR pathways in muscles of the dystrophin-deficient mdx mouse. We then crossed mdx mice with mice null for caspase-12, the murine equivalent of human caspase-4, which are resistant to ER stress. We found that deleting caspase-12 preserved mdx muscle function, resulting in a 75% recovery of both specific force generation and resistance to eccentric contractions. The compensatory hypertrophy normally found in mdx muscles was normalized in the absence of caspase-12; this was found to be due to decreased fibre sizes, and not to a fibre type shift or a decrease in fibrosis. Fibre central nucleation was not significantly altered in the absence of caspase-12, but muscle fibre degeneration found in the mdx mouse was reduced almost to wild-type levels. In conclusion, we have identified heightened ER stress and abnormal UPR signalling as novel contributors to the dystrophic phenotype. Caspase-4 is therefore a potential therapeutic target for DMD. PMID:24879640

  5. Developing a powerful In Silico tool for the discovery of novel caspase-3 substrates: a preliminary screening of the human proteome

    Directory of Open Access Journals (Sweden)

    Ayyash Muneef

    2012-01-01

    Full Text Available Abstract Background Caspases are a family of cysteinyl proteases that regulate apoptosis and other biological processes. Caspase-3 is considered the central executioner member of this family with a wide range of substrates. Identification of caspase-3 cellular targets is crucial to gain further insights into the cellular mechanisms that have been implicated in various diseases including: cancer, neurodegenerative, and immunodeficiency diseases. To date, over 200 caspase-3 substrates have been identified experimentally. However, many are still awaiting discovery. Results Here, we describe a powerful bioinformatics tool that can predict the presence of caspase-3 cleavage sites in a given protein sequence using a Position-Specific Scoring Matrix (PSSM approach. The present tool, which we call CAT3, was built using 227 confirmed caspase-3 substrates that were carefully extracted from the literature. Assessing prediction accuracy using 10 fold cross validation, our method shows AUC (area under the ROC curve of 0.94, sensitivity of 88.83%, and specificity of 89.50%. The ability of CAT3 in predicting the precise cleavage site was demonstrated in comparison to existing state-of-the-art tools. In contrast to other tools which were trained on cleavage sites of various caspases as well as other similar proteases, CAT3 showed a significant decrease in the false positive rate. This cost effective and powerful feature makes CAT3 an ideal tool for high-throughput screening to identify novel caspase-3 substrates. The developed tool, CAT3, was used to screen 13,066 human proteins with assigned gene ontology terms. The analyses revealed the presence of many potential caspase-3 substrates that are not yet described. The majority of these proteins are involved in signal transduction, regulation of cell adhesion, cytoskeleton organization, integrity of the nucleus, and development of nerve cells. Conclusions CAT3 is a powerful tool that is a clear improvement over

  6. Blockade of processing/activation of caspase-3 by hypoxia

    International Nuclear Information System (INIS)

    Han, Sang Hee; Kim, Moonil; Park, Kyoungsook; Kim, Tae-Hyoung; Seol, Dai-Wu

    2008-01-01

    Tumor hypoxia, which is caused by the rapid proliferation of tumor cells and aberrant vasculature in tumors, results in inadequate supplies of oxygen and nutrients to tumor cells. Paradoxically, these unfavorable growth conditions benefit tumor cell survival, although the mechanism is poorly understood. We have demonstrated for the first time that hypoxia inhibits TRAIL-induced apoptosis by blocking translocation of Bax from cytosol to the mitochondria in tumor cells. However, it is largely unknown how hypoxia-inhibited Bax translocation attenuates TRAIL-induced apoptosis. Here, we demonstrate that despite its inhibitory activity in TRAIL-induced apoptosis, hypoxia does not affect TRAIL-triggered proximal apoptotic signaling events, including caspase-8 activation and Bid cleavage. Instead, hypoxia inhibited processing of caspase-3, leading to incomplete activation of the caspase. Importantly, hypoxia-blocked translocation of Bax to the mitochondria significantly inhibited releasing the mitochondrial factors, such as cytochrome c and Smac/DIABLO, to the cytosol in response to TRAIL. It is well-known that complete processing/activation of caspase-3 requires Smac/DIABLO released from mitochondria. Therefore, our data indicate that an engagement of the apoptotic mitochondrial events leading to caspase-3 activation is blocked by hypoxia. Our data shed new light on understanding of the apoptotic signal transduction and targets regulated by tumor hypoxia

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

  8. A Dimeric Smac/Diablo Peptide Directly Relieves Caspase-3 Inhibition by XIAP: Dynamic and Cooperative Regulation of XIAP by Smac/Diablo

    OpenAIRE

    Gao, Zhonghua; Tian, Yuan; Wang, Junru; Yin, Qian; Wu, Hao; Li, Yue-Ming; Jiang, Xuejun

    2007-01-01

    Caspase activation, the executing event of apoptosis, is under deliberate regulation. IAP proteins inhibit caspase activity whereas Smac/Diablo antagonizes IAP. XIAP, a ubiquitous IAP, can inhibit both caspase-9, the initiator caspase of the mitochondrial apoptotic pathway, and the downstream effector caspases, caspase-3 and caspase-7. Smac neutralizes XIAP inhibition of caspase-9 by competing for binding of the BIR3 domain of XIAP with caspase-9, whereas how Smac liberates effector caspases ...

  9. 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.

  10. 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

  11. Ethyl Alcohol Extract of Hizikia fusiforme Induces Caspase ...

    African Journals Online (AJOL)

    Ethyl Alcohol Extract of Hizikia fusiforme Induces Caspase-dependent Apoptosis in Human Leukemia U937 Cells by Generation of Reactive Oxygen Species. C-H Kang, S-H Kang, S-H Boo, S-Y Park, D-O Moon, G-Y Kim ...

  12. 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.

  13. 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

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

    Science.gov (United States)

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

    2016-11-15

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

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

    Directory of Open Access Journals (Sweden)

    Eva Svandova

    2018-03-01

    Full Text Available Caspases are well known proteases in the context of inflammation and apoptosis. Recently, novel roles of pro-apoptotic caspases have been reported, including findings related to the development of hard tissues. To further investigate these emerging functions of pro-apoptotic caspases, the in vivo localisation of key pro-apoptotic caspases (-3,-6,-7,-8, and -9 was assessed, concentrating on the development of two neighbouring hard tissues, cells participating in odontogenesis (represented by the first mouse molar and intramembranous osteogenesis (mandibular/alveolar bone. The expression of the different caspases within the developing tissues was correlated with the apoptotic status of the cells, to produce a picture of whether different caspases have potentially distinct, or overlapping non-apoptotic functions. The in vivo investigation was additionally supported by examination of caspases in an osteoblast-like cell line in vitro. Caspases-3,-7, and -9 were activated in apoptotic cells of the primary enamel knot of the first molar; however, caspase-7 and -8 activation was also associated with the non-apoptotic enamel epithelium at the same stage and later with differentiating/differentiated odontoblasts and ameloblasts. In the adjacent bone, active caspases-7 and -8 were present abundantly in the prenatal period, while the appearance of caspases-3,-6, and -9 was marginal. Perinatally, caspases-3 and -7 were evident in some osteoclasts and osteoblastic cells, and caspase-8 was abundant mostly in osteoclasts. In addition, postnatal activation of caspases-7 and -8 was retained in osteocytes. The results provide a comprehensive temporo-spatial pattern of pro-apoptotic caspase activation, and demonstrate both unique and overlapping activation in non-apoptotic cells during development of the molar tooth and mandibular/alveolar bone. The importance of caspases in osteogenic pathways is highlighted by caspase inhibition in osteoblast-like cells, which

  16. Tyrosine Phosphorylation of Caspase-8 Abrogates Its Apoptotic Activity and Promotes Activation of c-Src

    Science.gov (United States)

    Tsang, Jennifer LY; Jia, Song Hui; Parodo, Jean; Plant, Pamela; Lodyga, Monika; Charbonney, Emmanuel; Szaszi, Katalin; Kapus, Andras; Marshall, John C.

    2016-01-01

    Src family tyrosine kinases (SFKs) phosphorylate caspase-8A at tyrosine (Y) 397 resulting in suppression of apoptosis. In addition, the phosphorylation of caspase-8A at other sites including Y465 has been implicated in the regulation of caspase-8 activity. However, the functional consequences of these modifications on caspase-8 processing/activity have not been elucidated. Moreover, various Src substrates are known to act as potent Src regulators, but no such role has been explored for caspase-8. We asked whether the newly identified caspase-8 phosphorylation sites might regulate caspase-8 activation and conversely, whether caspase-8 phosphorylation might affect Src activity. Here we show that Src phosphorylates caspase-8A at multiple tyrosine sites; of these, we have focused on Y397 within the linker region and Y465 within the p12 subunit of caspase-8A. We show that phosphomimetic mutation of caspase-8A at Y465 prevents its cleavage and the subsequent activation of caspase-3 and suppresses apoptosis. Furthermore, simultaneous phosphomimetic mutation of caspase-8A at Y397 and Y465 promotes the phosphorylation of c-Src at Y416 and increases c-Src activity. Finally, we demonstrate that caspase-8 activity prevents its own tyrosine phosphorylation by Src. Together these data reveal that dual phosphorylation converts caspase-8 from a pro-apoptotic to a pro-survival mediator. Specifically, tyrosine phosphorylation by Src renders caspase-8 uncleavable and thereby inactive, and at the same time converts it to a Src activator. This novel dynamic interplay between Src and caspase-8 likely acts as a potent signal-integrating switch directing the cell towards apoptosis or survival. PMID:27101103

  17. Caspase-1 cleavage of transcription factor GATA4 and regulation of cardiac cell fate.

    Science.gov (United States)

    Aries, A; Whitcomb, J; Shao, W; Komati, H; Saleh, M; Nemer, M

    2014-12-11

    Caspase-1 or interleukin-1β (IL-1β) converting enzyme is a pro-inflammatory member of the caspase family. An IL-1β-independent role for caspase-1 in cardiomyocyte cell death and heart failure has emerged but the mechanisms underlying these effects are incompletely understood. Here, we report that transcription factor GATA4, a key regulator of cardiomyocyte survival and adaptive stress response is an in vivo and in vitro substrate for caspase-1. Caspase-1 mediated cleavage of GATA4 generates a truncated protein that retains the ability to bind DNA but lacks transcriptional activation domains and acts as a dominant negative regulator of GATA4. We show that caspase-1 is rapidly activated in cardiomyocyte nuclei treated with the cell death inducing drug Doxorubicin. We also find that inhibition of caspase-1 alone is as effective as complete caspase inhibition at rescuing GATA4 degradation and myocyte cell death. Caspase-1 inhibition of GATA4 transcriptional activity is rescued by HSP70, which binds directly to GATA4 and masks the caspase recognition motif. The data identify a caspase-1 nuclear substrate and suggest a direct role for caspase-1 in transcriptional regulation. This mechanism may underlie the inflammation-independent action of caspase-1 in other organs.

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

    Directory of Open Access Journals (Sweden)

    Fengmei Song

    2018-04-01

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

  19. Comparative Analysis of miRNAs and Their Target Transcripts between a Spontaneous Late-Ripening Sweet Orange Mutant and Its Wild-Type Using Small RNA and Degradome Sequencing.

    Science.gov (United States)

    Wu, Juxun; Zheng, Saisai; Feng, Guizhi; Yi, Hualin

    2016-01-01

    Fruit ripening in citrus is not well-understood at the molecular level. Knowledge of the regulatory mechanism of citrus fruit ripening at the post-transcriptional level in particular is lacking. Here, we comparatively analyzed the miRNAs and their target genes in a spontaneous late-ripening mutant, "Fengwan" sweet orange (MT) ( Citrus sinensis L. Osbeck), and its wild-type counterpart ("Fengjie 72-1," WT). Using high-throughput sequencing of small RNAs and RNA degradome tags, we identified 107 known and 21 novel miRNAs, as well as 225 target genes. A total of 24 miRNAs (16 known miRNAs and 8 novel miRNAs) were shown to be differentially expressed between MT and WT. The expression pattern of several key miRNAs and their target genes during citrus fruit development and ripening stages was examined. Csi-miR156k, csi-miR159, and csi-miR166d suppressed specific transcription factors ( GAMYBs, SPLs , and ATHBs ) that are supposed to be important regulators involved in citrus fruit development and ripening. In the present study, miRNA-mediated silencing of target genes was found under complicated and sensitive regulation in citrus fruit. The identification of miRNAs and their target genes provide new clues for future investigation of mechanisms that regulate citrus fruit ripening.

  20. piPipes: a set of pipelines for piRNA and transposon analysis via small RNA-seq, RNA-seq, degradome- and CAGE-seq, ChIP-seq and genomic DNA sequencing.

    Science.gov (United States)

    Han, Bo W; Wang, Wei; Zamore, Phillip D; Weng, Zhiping

    2015-02-15

    PIWI-interacting RNAs (piRNAs), 23-36 nt small silencing RNAs, repress transposon expression in the metazoan germ line, thereby protecting the genome. Although high-throughput sequencing has made it possible to examine the genome and transcriptome at unprecedented resolution, extracting useful information from gigabytes of sequencing data still requires substantial computational skills. Additionally, researchers may analyze and interpret the same data differently, generating results that are difficult to reconcile. To address these issues, we developed a coordinated set of pipelines, 'piPipes', to analyze piRNA and transposon-derived RNAs from a variety of high-throughput sequencing libraries, including small RNA, RNA, degradome or 7-methyl guanosine cap analysis of gene expression (CAGE), chromatin immunoprecipitation (ChIP) and genomic DNA-seq. piPipes can also produce figures and tables suitable for publication. By facilitating data analysis, piPipes provides an opportunity to standardize computational methods in the piRNA field. Supplementary information, including flowcharts and example figures for each pipeline, are available at Bioinformatics online. piPipes is implemented in Bash, C++, Python, Perl and R. piPipes is free, open-source software distributed under the GPLv3 license and is available at http://bowhan.github.io/piPipes/. Phillip.Zamore@umassmed.edu or Zhiping.Weng@umassmed.edu Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press.

  1. 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.

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

  3. Bile Acid-induced Apoptosis in Hepatocytes Is Caspase-6-dependent

    NARCIS (Netherlands)

    Rust, Christian; Wild, Nadine; Bernt, Carina; Vennegeerts, Timo; Wimmer, Ralf; Beuers, Ulrich

    2009-01-01

    Apoptosis induced by hydrophobic bile acids is thought to contribute to liver injury during cholestasis. Caspase-6 is an executioner caspase that also appears to have regulatory functions in hematopoetic cell lines. We aimed to elucidate the role of caspase-6 in bile acid-induced apoptosis. The

  4. Ecstasy-Induced Caspase Expression Alters Following Ginger Treatment

    Directory of Open Access Journals (Sweden)

    Sara Soleimani Asl

    2013-11-01

    Full Text Available 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 significant increase in caspase 3, 8, and 9 as compared to the sham group (p<0.001. Ginger administration however, appeared to significantly decrease the same (p<0.001. Discussion: Our findings suggest that ginger consumption may lead to the improvement of MDMA-induced neurotoxicity.

  5. Caspases in yeast apoptosis-like death: facts and artefacts

    Czech Academy of Sciences Publication Activity Database

    Váchová, Libuše; Palková, Z.

    2007-01-01

    Roč. 7, - (2007), s. 12-21 ISSN 1567-1356 R&D Projects: GA ČR GA525/05/0297; GA MŠk(CZ) LC531 Institutional research plan: CEZ:AV0Z50200510 Keywords : caspases and metacaspases * apoptosis and programmed cell death in yeast * saccharomyces cerevisiae Subject RIV: EE - Microbiology, Virology Impact factor: 2.812, year: 2007

  6. 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 - others: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

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

    Czech Academy of Sciences Publication Activity Database

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2004-01-01

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

  9. Changes in ultrastructure, protease and caspase-like activities during flower senescence in Lilium longiflorum.

    Science.gov (United States)

    Battelli, Riccardo; Lombardi, Lara; Rogers, Hilary J; Picciarelli, Piero; Lorenzi, Roberto; Ceccarelli, Nello

    2011-05-01

    The last phase of flower development is senescence during which nutrients are recycled to developing tissues. The ultimate fate of petal cells is cell death. In this study we used the ethylene-insensitive Lilium longiflorum as a model system to characterize Lily flower senescence from the physiological, biochemical and ultrastructural point of view. Lily flower senescence is highly predictable: it starts three days after flower opening, before visible signs of wilting, and ends with the complete wilting of the corolla within 10 days. The earliest events in L. longiflorum senescence include a fall in fresh and dry weight, fragmentation of nuclear DNA and cellular disruption. Mesophyll cell degradation is associated with vacuole permeabilization and rupture. Protein degradation starts later, coincident with the first visible signs of tepal senescence. A fall in total protein is accompanied by a rise in total proteases, and also by a rise of three classes of caspase-like activity with activities against YVAD, DEVD and VEID. The timing of the appearance of these caspase-like activities argues against their involvement in the regulation of the early stages of senescence, but their possible role in the regulation of the final stages of senescence and cell death is discussed. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  10. 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

    2009-01-01

    be 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...... 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...

  11. A Novel Method for Imaging Apoptosis Using a Caspase-1 Near-Infrared Fluorescent Probe

    Directory of Open Access Journals (Sweden)

    Shanta M. Messerli

    2004-03-01

    Full Text Available Here we describe a novel method for imaging apoptosis in cells using a near-infrared fluorescent (NIRF probe selective for caspase-1 (interleukin β-converting enzyme, ICE. This biocompatible, optically quenched ICE-NIRF probe incorporates a peptide substrate, which can be selectively cleaved by caspase-1, resulting in the release of fluorescence signal. The specificity of this probe for caspase-1 is supported by various lines of evidence: 1 activation by purified caspase-1, but not another caspase in vitro; 2 activation of the probe by infection of cells with a herpes simplex virus amplicon vector (HGC-ICE-IacZ expressing a catalytically active caspase-1-IacZ fusion protein; 3 inhibition of HGC-ICE-IacZ vector-induced activation of the probe by coincubation with the caspase-1 inhibitor YVAD-cmk, but not with a caspase-3 inhibitor; and 4 activation of the probe following standard methods of inducing apoptosis with staurosporine, ganciclovir, or ionizing radiation in culture. These results indicate that this novel ICE-NIRF probe can be used in monitoring endogenous and vector-expressed caspase-1 activity in cells. Furthermore, tumor implant experiments indicate that this ICE-NIRF probe can be used to detect caspase-1 activity in living animals. This novel ICE-NIRF probe should prove useful in monitoring endogenous and vector-expressed caspase-1 activity, and potentially apoptosis in cell culture and in vivo.

  12. Caspase-10 Is the Key Initiator Caspase Involved in Tributyltin-Mediated Apoptosis in Human Immune Cells

    Directory of Open Access Journals (Sweden)

    Harald F. Krug

    2012-01-01

    Full Text Available Tributyltin (TBT is one of the most toxic compounds produced by man and distributed in the environment. A multitude of toxic activities have been described, for example, immunotoxic, neurotoxic, and endocrine disruptive effects. Moreover, it has been shown for many cell types that they undergo apoptosis after treatment with TBT and the cell death of immune cells could be the molecular background of its immunotoxic effect. As low as 200 nM up to 1 μM of TBT induces all signs of apoptosis in Jurkat T cells within 1 to 24 hrs of treatment. When compared to Fas-ligand control stimulation, the same sequence of events occurs: membrane blebbing, phosphatidylserine externalisation, the activation of the “death-inducing signalling complex,” and the following sequence of cleavage processes. In genetically modified caspase-8-deficient Jurkat cells, the apoptotic effects are only slightly reduced, whereas, in FADD-negative Jurkat cells, the TBT effect is significantly diminished. We could show that caspase-10 is recruited by the TRAIL-R2 receptor and apoptosis is totally prevented when caspase-10 is specifically inhibited in all three cell lines.

  13. 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

  14. Two coffins and a funeral: early or late caspase activation determines two types of apoptosis induced by DNA damaging agents.

    Science.gov (United States)

    Oropesa-Ávila, Manuel; de la Cruz-Ojeda, Patricia; Porcuna, Jesús; Villanueva-Paz, Marina; Fernández-Vega, Alejandro; de la Mata, Mario; de Lavera, Isabel; Rivero, Juan Miguel Suarez; Luzón-Hidalgo, Raquel; Álvarez-Córdoba, Mónica; Cotán, David; Zaderenko, Ana Paula; Cordero, Mario D; Sánchez-Alcázar, José A

    2017-03-01

    Cell cytoskeleton makes profound changes during apoptosis including the organization of an Apoptotic Microtubule Network (AMN). AMN forms a cortical structure which plays an important role in preserving plasma membrane integrity during apoptosis. Here, we examined the cytoskeleton rearrangements during apoptosis induced by camptothecin (CPT), a topoisomerase I inhibitor, in human H460 and porcine LLCPK-1α cells. Using fixed and living cell imaging, we showed that CPT induced two dose- and cell cycle-dependent types of apoptosis characterized by different cytoskeleton reorganizations, time-dependent caspase activation and final apoptotic cell morphology. In the one referred as "slow" (~h) or round-shaped, apoptosis was characterized by a slow contraction of the actinomyosin ring and late caspase activation. In "slow" apoptosis the γ-tubulin complexes were not disorganized and microtubules were not depolymerized at early stages. In contrast, "fast" (~min) or irregular-shaped apoptosis was characterized by early caspase activation followed by full contraction of the actinomyosin ring. In fast apoptosis γ-tubulin complexes were disorganized and microtubules were initially depolymerized. However, after actinomyosin contraction, microtubules were reformed adopting a cortical but irregular disposition near plasma membrane. In addition to distinctive cytoskeleton reorganization kinetics, round and irregular-shaped apoptosis showed different biological properties with respect to AMN maintenance, plasma membrane integrity and phagocytes response. Our results suggest that the knowledge and modulation of the type of apoptosis promoted by genotoxic agents may be important for deciding a better therapeutic option and predicting the immune response in cancer treatment.

  15. Erythrocyte caspase-3 levels in children with chronic kidney disease.

    Science.gov (United States)

    Polak-Jonkisz, D; Purzyc, L; Szcepańska, M; Makulska, I

    2013-02-01

    In chronic kidney disease (CKD), a number of intra- and extracellular factors, e.g., uremic toxins, mechanic, oxidative or osmotic stress - induce changes (rearrangements) in the structure of cytoplasmatic membrane, while also simultaneously deregulating blood cell metabolism and, in consequence, contributing to preliminary ageing and suicidal death of red blood cells (RBCs).The aim of the reported study was an evaluation of caspase-3 and lactate dehydrogenase activities and of ATP concentrations in erythrocytes as cellular responses to CKD progress. Conservatively treated sixty (60) CKD children were enrolled into the study and divided, according to CKD progression (stage I-IV). The control group consisted of twenty-five (25) healthy children. The activity of caspase-3 (Casp-3) and lactate dehydrogenase (LDH) were spectrophotometrically assayed in haemolysed erythrocytes. Adenosine triphosphate (ATP(e)) concentrations were measured by means of a luciferin-luciferase kit. A gradual increase of LDH and ATP levels was observed in transition from CKD stage I to stage III. In Group IV, the levels of those parameters were statistically significantly lower than in the control group. The activity of Casp-3 in Group I was comparable to that in healthy children. The highest activity of Casp-3 was observed in Group III. 1. The activity of caspase-3 in RBCs of CKD children grows with progression of the disease. 2. The lower LDH activities and the ATP concentration drop below the values characteristic for the control group, as observed in stage IV of CKD, indicate a compromised energy balance. Copyright © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2017-11-15

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

  17. AlCl3 induces lymphocyte apoptosis in rats through the mitochondria-caspase dependent pathway.

    Science.gov (United States)

    Li, Miao; Song, Miao; Ren, Li-Min; Xiu, Chun-Yu; Liu, Jian-Yu; Zhu, Yan-zhu; Li, Yan-Fei

    2016-04-01

    To investigate apoptosis mechanisms in lymphocytes induced by aluminum trichloride (AlCl3) through the mitochondria-caspase dependent pathway, the spleen lymphocytes of rats were cultured with RPMI-1640 medium and exposed to AlCl3·6H2O in the final concentrations of 0 (control group, CG), 0.3 (low-dose group, LG), 0.6 (mid-dose group, MG), and 1.2 (high-dose group, HG) mmol·L(-1) for 24 h, respectively. Mitochondrial transmembrane potential (ΔΨm), cytochrome C (Cyt C) protein expression in cytoplasm, Caspase-3 and Caspase-9 activity, Bcl-2, Bax, Caspase-3 and Caspase-9 mRNA expressions, DNA ladder and lymphocytes apoptosis index were detected. The results showed that Cyt C protein expression in cytoplasm, Caspase-3 and Caspase-9 activity, Bcl-2, Bax, Caspase-3 and Caspase-9 mRNA expressions, the ratio of Bcl-2 and Bax mRNA expression, lymphocytes apoptosis index increased, while ΔΨm decreased in the AlCl3-treated groups compared with those in CG. The results indicate that AlCl3 induces lymphocyte apoptosis in rats through the mitochondria-caspase dependent pathway. © 2014 Wiley Periodicals, Inc.

  18. 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.                   

  19. Expression of Caspase Signaling Components in the Outer Membranes of Chronic Subdural Hematomas.

    Science.gov (United States)

    Osuka, Koji; Watanabe, Yasuo; Usuda, Nobuteru; Aoyama, Masahiro; Iwami, Kenichiro; Takeuchi, Mikinobu; Watabe, Takeya; Takayasu, Masakazu

    2017-11-15

    Chronic subdural hematoma (CSDH) is fundamentally treatable through surgery, although CSDH recurs in some cases. We have observed several cases of spontaneous resolution of CSDH outer membranes, including in trabecular CSDH, after trepanation surgery. In this study, we examined the expression of molecules involved in caspase signaling in CSDH outer membranes. Eight patients whose outer membranes were obtained successfully during trepanation surgery were included in this study. The expression of Fas; Fas-associated death domain (FADD); tumor necrosis factor receptor type 1-associated death domain (TRADD); receptor-interacting protein (RIP); caspases 3, 7, 8, and 9; poly-(ADP-ribose) polymerase (PARP); DNA fragmentation factor 45 (DFF45) and β-actin was examined by Western blot analysis. The expression levels of PARP, caspase-3, and cleaved caspase-3 were also examined by immunohistochemistry. Fas; FADD; TRADD; RIP; caspases 3, 7, 8, and 9; PARP, and DFF45 were detected in nearly all samples. Caspase-3 and PARP were localized in the endothelial cells of vessels and in fibroblasts in CSDH outer membranes. In addition, cleaved caspase-3 was detected in fibroblasts. We detected molecules of the caspase signaling pathway in CSDH outer membranes. In particular, cleaved caspase-3 was detected, which suggests that apoptosis may occur within these membranes. Thus, during the growth of CSDH outer membranes, the caspase signaling pathway may be restrained. Once the pathway is activated, gradual resolution of CSDH outer membranes may occur. Therefore, these molecules may be novel therapeutic targets for intractable CSDH.

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Science.gov (United States)

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

    2013-12-01

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

  2. Microparticulate Caspase-1 Regulates Gasdermin-D and Pulmonary Vascular Endothelial Cell Injury.

    Science.gov (United States)

    Mitra, Srabani; Exline, Matthew; Habyarimana, Fabien; Gavrilin, Mikhail; Baker, Paul; Masters, Seth L; Wewers, Mark D; Sarkar, Anasuya

    2018-01-24

    Lung endothelial cell apoptosis and injury occurs throughout all stages of acute lung injury (ALI/ARDS) and impacts disease progression. Caspases 1, 4 and 5 are essential for completion of the apoptotic program known as pyroptosis that also involves pro-inflammatory cytokines. Because GSDM-D mediates pyroptotic death and is essential for pore formation, we hypothesized that it may direct caspase-1 encapsulated microparticle (MP) release and mediate endothelial cell death. Our current work provides evidence that GSDM-D is released by LPS stimulated THP1 monocytic cells where it is packaged into microparticles along with active caspase-1. Furthermore, only MP released from stimulated monocytic cells that contain both cleaved GSDM-D and active caspase-1 induce endothelial cell apoptosis. MPs pretreated with caspase-1 inhibitor, YVAD, or pan-caspase inhibitor, ZVAD, do not contain cleaved GSDM-D. MPs from caspase-1KO cells are also deficient in p30 active GSDM-D, further confirming that caspase-1 regulates GSDM-D function. Although control MPs contained cleaved GSDM-D without caspase-1, these fractions were unable to induce cell death, suggesting that encapsulation of both caspase-1 and GSDM-D is essential for cell death induction. Release of microparticulate active caspase-1 was abrogated in GSDM-KO cells, although cytosolic caspase-1 activation was not impaired. Lastly, higher levels of microparticulate GSDM-D was detected in septic ARDS patient plasma when compared to healthy donors. Taken together, these findings suggest that GSDM-D regulates the release of microparticulate active caspase-1 from monocytes essential for induction of cell death and thereby may play a critical role in sepsis-induced endothelial cell injury.

  3. Caspase-2 mediates a Brucella abortus RB51-induced hybrid cell death having features of apoptosis and pyroptosis

    Directory of Open Access Journals (Sweden)

    Denise Nicole Bronner

    2013-11-01

    Full Text Available Programmed cell death (PCD can play a crucial role in tuning the immune response to microbial infection. Although PCD can occur in different forms, all are mediated by a family of proteases called caspases. Caspase-2 is the most conserved caspase; however its function in cell death is ill-defined. Previously we demonstrated that live attenuated cattle vaccine strain Brucella abortus RB51 induces caspase-2-mediated PCD of infected macrophages. However, the mechanism of caspase-2-mediated cell death pathway remained unclear. In this study, we found that caspase-2 mediated proinflammatory cell death of RB51-infected macrophages and regulated many genes in different PCD pathways. We show that the activation of proapoptotic caspases-3 and -8 was dependent upon caspase-2. Caspase-2 regulated mitochondrial cytochrome c release and TNFα production, both of which are known to activate caspase-3 and caspase-8, respectively. In addition to TNFα, RB51-induced caspase-1 and IL-1β production was also driven by caspase-2-mediated mitochondrial dysfunction. Interestingly, pore formation, a phenomenon commonly associated with caspase-1-mediated pyroptosis, occurred; however it did not contribute to RB51-induced proinflammatory cell death. Our data suggest that caspase-2 acts as an initiator caspase that mediates a novel RB51-induced hybrid cell death that simulates but differs from typical apoptosis and pyroptosis. The initiator role of the caspase-2-mediated cell death was also conserved in cellular stress-induced cell death of macrophages treated with etoposide, naphthalene, or anti-Fas. Caspase-2 also regulated caspase-3 and -8 activation, as well as cell death in macrophages treated with each of the three reagents. Taken together, our data has demonstrated that caspase-2 can play an important role in mediating a proinflammatory response and a hybrid cell death that demonstrates features of both apoptosis and pyroptosis.

  4. 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-6...

  5. Activation of two caspase cascades, caspase 8/3/6 and caspase 9/3/6, during photodynamic therapy using a novel photosensitizer, ATX-S10(Na), in normal human keratinocytes.

    Science.gov (United States)

    Takahashi, Hidetoshi; Itoh, Yasuhiro; Miyauchi, Yuki; Nakajima, Susumu; Sakata, Isao; Ishida-Yamamoto, Akemi; Iizuka, Hajime

    2003-11-01

    Photodynamic therapy (PDT) is a potent treatment for skin tumors. Although the therapeutic effect of PDT is supposed to be due to cellular cytotoxicity, the precise mechanism is still unknown. ATX-S10(Na) [13,17-bis(1-carboxypropionyl)carbamoylethyl-8-ethenyl-2-hydroxy-3-hydroxyiminoethylidene-2,7,12,18-tetramethylporphyrin sodium salt], a novel hydrophilic chlorin photosensitizer, shows good accumulation in tumors and is suitable for use in PDT. In this study, we investigated the mechanism of PDT-induced cell death using ATX-S10(Na). . Following ATX-S10(Na) treatment for 12 h, normal human keratinocytes (NHK) were irradiated using a diode laser. PDT-induced cell death and the activity of various caspases were measured. Activation of Fas antigen was also determined by immunoprecipitation analysis. The expression of Bax, cytochrome c, and apoptosis-inducing factor (AIF) was determined by Western blotting. ATX-S10(Na)-PDT had induced apoptosis of NHK by 2 h and the maximal effect was observed at 6 h following irradiation. The effect was suppressed by pretreatment of NHK with inhibitors of caspases 3, 6, 8 and 9. A caspase activity assay revealed the sequential activation of caspases 8, 3 and 6, and caspases 9, 3 and 6, respectively. Immunoprecipitation analysis indicated multimerization of Fas antigen without Fas ligand binding in ATX-S10(Na)-PDT-treated NHK. Western blotting revealed cytosolic release of cytochrome c and AIF accompanied by decreased Bax expression in the cytosol. ATX-S10(Na)-PDT induces apoptosis of NHK, and this was mediated by sequential activation of two caspase cascades, caspases 8, 3 and 6, and caspases 9, 3 and 6. This was accompanied by multimerization of Fas antigen and cytosolic release of cytochrome c and AIF.

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

    Science.gov (United States)

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

    2017-09-26

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

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

    Science.gov (United States)

    Mitra, Srabani

    2015-01-01

    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. PMID:26710067

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

    Science.gov (United States)

    Mitra, Srabani; Wewers, Mark D; Sarkar, Anasuya

    2015-01-01

    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.

  9. Dying glioma cells establish a proangiogenic microenvironment through a caspase 3 dependent mechanism

    Science.gov (United States)

    Feng, Xiao; Yu, Yang; He, Sijia; Cheng, Jin; Gong, Yanping; Zhang, Zhengxiang; Yang, Xuguang; Xu, Bing; Liu, Xinjian; Li, Chuan-Yuan; Tian, Ling; Huang, Qian

    2017-01-01

    Vascular recovery or re-angiogenesis after radiotherapy plays a significant role in tumor recurrence, whereas molecular mechanisms of this process remain elusive. In this work, we found that dying glioma cells promoted post-irradiation angiogenesis through a caspase 3 dependent mechanism. Evidence in vitro and in vivo indicated that caspase 3 inhibition undermined proangiogenic effects of dying glioma cells. Proteolytic inactivation of caspase 3 in glioma cells reduced tumorigenicity. Importantly, we identified that NF-κB/COX-2/PGE2 axis acted as downstream signaling of caspase 3, mediating proangiogenic response after irradiation. Additionally, VEGF-A, regulated by caspase 3 possibly through phosphorylated eIF4E, was recognized as another downstream factor participating in the proangiogenic response. In conclusion, these data demonstrated that caspase 3 in dying glioma cells supported the proangiogenic response after irradiation by governing NF-κB/COX-2/PGE2 axis and p-eIF4E/VEGF-A signaling. While inducing caspase 3 activation has been a generally-adopted notion in cancer therapeutics, our study counterintuitively illustrated that caspase 3 activation in dying glioma cells unfavorably supported post-irradiation angiogenesis, suggesting that radiotherapy combined with caspase 3 inhibitors may be more effective strategies due to restricted post-irradiation angiogenesis. PMID:27826040

  10. 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.

  11. The limits for detection of activated caspases of spermatozoa by western blot in human semen.

    Science.gov (United States)

    Brugnon, F; Pons-Rejraji, H; Artonne, C; Janny, L; Grizard, G

    2012-08-01

    Detection of activated caspases of spermatozoa could be helpful to evaluate male infertility. Although western blot is validated as a highly specific method to detect the proteins extracted from cells, the ability of this technique to detect activated sperm caspases in human semen may be limited. Indeed, round cells, which potentially contain some activated caspases, may be present in semen and interfere with the detection of activated sperm caspases. Moreover, it is necessary to evaluate the minimum amount of spermatozoa necessary to optimise the detection of activated caspases in semen samples. Our results showed that interference due to round cells contained in semen with activated caspase-3 requires separation of spermatozoa by density migration. This sperm preparation selects a mature sperm population that does not reflect the whole sperm population, and in infertile men with oligoasthenoteratozoospermia, the amount of spermatozoa thus selected is usually low. Moreover, the western blot technique's low detection sensitivity and the low level of caspase enzyme activity in human spermatozoa for activated caspase-3, -8 and -9 mean that large quantities of spermatozoa are needed to detect the expression of the activated caspases. These limitations prevent this method being used for routine analysis in clinical practice. © 2012 Blackwell Verlag GmbH.

  12. Small RNA sequencing and degradome analysis of developing fibers of short fiber mutants Ligon-lintles-1 (Li 1 ) and -2 (Li 2 ) revealed a role for miRNAs and their targets in cotton fiber elongation.

    Science.gov (United States)

    Naoumkina, Marina; Thyssen, Gregory N; Fang, David D; Hinchliffe, Doug J; Florane, Christopher B; Jenkins, Johnie N

    2016-05-17

    The length of cotton fiber is an important agronomic trait that directly affects the quality of yarn and fabric. Understanding the molecular basis of fiber elongation would provide a means for improvement of fiber length. Ligon-lintless-1 (Li 1 ) and -2 (Li 2 ) are monogenic and dominant mutations that result in an extreme reduction in the length of lint fiber on mature seeds. In a near-isogenic state with wild type cotton these two short fiber mutants provide an effective model system to study the mechanisms of fiber elongation. Plant miRNAs regulate many aspects of growth and development. However, the mechanism underlying the miRNA-mediated regulation of fiber development is largely unknown. Small RNA libraries constructed from developing fiber cells of the short fiber mutants Li 1 and Li 2 and their near-isogenic wild type lines were sequenced. We identified 24 conservative and 147 novel miRNA families with targets that were detected through degradome sequencing. The distribution of the target genes into functional categories revealed the largest set of genes were transcription factors. Expression profiles of 20 miRNAs were examined across a fiber developmental time course in wild type and short fiber mutations. We conducted correlation analysis between miRNA transcript abundance and the length of fiber for 11 diverse Upland cotton lines. The expression patterns of 4 miRNAs revealed significant negative correlation with fiber lengths of 11 cotton lines. Our results suggested that the mutations have changed the regulation of miRNAs expression during fiber development. Further investigations of differentially expressed miRNAs in the Li 1 and Li 2 mutants will contribute to better understanding of the regulatory mechanisms of cotton fiber development. Four miRNAs negatively correlated with fiber length are good candidates for further investigations of miRNA regulation of important genotype dependent fiber traits. Thus, our results will contribute to further studies

  13. A Type III Effector NleF from EHEC Inhibits Epithelial Inflammatory Cell Death by Targeting Caspase-4

    Directory of Open Access Journals (Sweden)

    Ting Song

    2017-01-01

    Full Text Available Enterohemorrhagic E. coli (EHEC is a highly pathogenic bacterial strain capable of inducing severe gastrointestinal disease. Here, we show that EHEC uses the T3SS effector NleF to counteract the host inflammatory response by dampening caspase-4-mediated inflammatory epithelial cell death and by preventing the production of IL-1β. The other two inflammatory caspases, caspase-1 and caspase-5, are not involved in EHEC ΔnleF-induced inflammatory cell death. We found that NleF not only interrupted the heterodimerization of caspase-4-p19 and caspase-4-p10, but also inhibited the interaction of caspase-1 and caspase-4. The last four amino acids of the NleF carboxy terminus are essential in inhibiting caspase-4-dependent inflammatory cell death.

  14. Caspase-2 mediates a Brucella abortus RB51-induced hybrid cell death having features of apoptosis and pyroptosis.

    Science.gov (United States)

    Bronner, Denise N; O'Riordan, Mary X D; He, Yongqun

    2013-01-01

    Programmed cell death (PCD) can play a crucial role in tuning the immune response to microbial infection. Although PCD can occur in different forms, all are mediated by a family of proteases called caspases. Caspase-2 is the most conserved caspase, however, its function in cell death is ill-defined. Previously we demonstrated that live attenuated cattle vaccine strain Brucella abortus RB51 induces caspase-2-mediated and caspase-1-independent PCD of infected macrophages. We also discovered that rough attenuated B. suis strain VTRS1 induces a caspase-2-mediated and caspase-1-independent proinflammatory cell death in infected macrophages, which was tentatively coined "caspase-2-mediated pyroptosis". However, the mechanism of caspase-2-mediated cell death pathway remained unclear. In this study, we found that caspase-2 mediated proinflammatory cell death of RB51-infected macrophages and regulated many genes in different PCD pathways. We show that the activation of proapoptotic caspases-3 and -8 was dependent upon caspase-2. Caspase-2 regulated mitochondrial cytochrome c release and TNFα production, both of which are known to activate caspase-3 and caspase-8, respectively. In addition to TNFα, RB51-induced caspase-1 and IL-1β production was also driven by caspase-2-mediated mitochondrial dysfunction. Interestingly, pore formation, a phenomenon commonly associated with caspase-1-mediated pyroptosis, occurred; however, unlike its role in S. typhimurium-induced pyroptosis, pore formation did not contribute to RB51-induced proinflammatory cell death. Our data suggest that caspase-2 acts as an initiator caspase that mediates a novel RB51-induced hybrid cell death that simulates but differs from typical non-proinflammatory apoptosis and caspase-1-mediated proinflammatory pyroptosis. The initiator role of the caspase-2-mediated cell death was also conserved in cellular stress-induced cell death of macrophages treated with etoposide, naphthalene, or anti-Fas. Caspase-2 also

  15. 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

  16. PQ1, a Quinoline Derivative, Induces Apoptosis in T47D Breast Cancer Cells through Activation of Caspase-8 and Caspase-9

    Science.gov (United States)

    Ding, Ying; Nguyen, Thu Annelise

    2013-01-01

    Apoptosis, a programmed cell death, is an important control mechanism of cell homeostasis. Deficiency in apoptosis is one of the key features of cancer cells, allowing cells to escape from death. Activation of apoptotic signaling pathway has been a target of anti-cancer drugs in an induction of cytotoxicity. PQ1, 6-methoxy-8-[(3-aminopropyl)amino]-4-methyl-5-(3-trifluoromethylphenyloxy)quinoline, has been reported to decrease the viability of cancer cells and attenuate xenograft tumor growth. However, the mechanism of the anti-cancer effect is still unclear. To evaluate whether the cytotoxicity of PQ1 is related to induction of apoptosis, the effect of PQ1 on apoptotic pathways was investigated in T47D breast cancer cells. PQ1-treated cells had an elevation of cleaved caspase-3 compared to controls. Studies of intrinsic apoptotic pathway showed that PQ1 can activate the intrinsic checkpoint protein caspase-9, enhance the level of pro-apoptotic protein Bax, and release cytochrome c from mitochondria to cytosol; however, PQ1 has no effect on the level of anti-apoptotic protein Bcl-2. Further studies also demonstrated that PQ1 can activate the key extrinsic player, caspase-8. Pre-treatment of T47D cells with caspase-8 or caspase-9 inhibitor suppressed the cell death induced by PQ1, while pre-treatment with caspase-3 inhibitor completely counteracted the effect of PQ1 on cell viability. This report provides evidence that PQ1 induces cytotoxicity via activation of both caspase-8 and caspase-9 in T47D breast cancer cells. PMID:23677255

  17. PQ1, a quinoline derivative, induces apoptosis in T47D breast cancer cells through activation of caspase-8 and caspase-9.

    Science.gov (United States)

    Ding, Ying; Nguyen, Thu Annelise

    2013-09-01

    Apoptosis, a programmed cell death, is an important control mechanism of cell homeostasis. Deficiency in apoptosis is one of the key features of cancer cells, allowing cells to escape from death. Activation of apoptotic signaling pathway has been a target of anti-cancer drugs in an induction of cytotoxicity. PQ1, 6-methoxy-8-[(3-aminopropyl)amino]-4-methyl-5-(3-trifluoromethylphenyloxy)quinoline, has been reported to decrease the viability of cancer cells and attenuate xenograft tumor growth. However, the mechanism of the anti-cancer effect is still unclear. To evaluate whether the cytotoxicity of PQ1 is related to induction of apoptosis, the effect of PQ1 on apoptotic pathways was investigated in T47D breast cancer cells. PQ1-treated cells had an elevation of cleaved caspase-3 compared to controls. Studies of intrinsic apoptotic pathway showed that PQ1 can activate the intrinsic checkpoint protein caspase-9, enhance the level of pro-apoptotic protein Bax, and release cytochrome c from mitochondria to cytosol; however, PQ1 has no effect on the level of anti-apoptotic protein Bcl-2. Further studies also demonstrated that PQ1 can activate the key extrinsic player, caspase-8. Pre-treatment of T47D cells with caspase-8 or caspase-9 inhibitor suppressed the cell death induced by PQ1, while pre-treatment with caspase-3 inhibitor completely counteracted the effect of PQ1 on cell viability. This report provides evidence that PQ1 induces cytotoxicity via activation of both caspase-8 and caspase-9 in T47D breast cancer cells.

  18. Overexpression of Smac promotes Cisplatin-induced apoptosis by activating caspase-3 and caspase-9 in lung cancer A549 cells.

    Science.gov (United States)

    Qin, Sida; Yang, Chengcheng; Wang, Xifang; Xu, Chongwen; Li, Shuo; Zhang, Boxiang; Ren, Hong

    2013-03-01

    Second mitochondrial-derived activator of caspase (Smac) plays crucial roles in mitochondrial apoptosis pathways and promotes chemotherapy-induced apoptosis. In this study, Smac levels were examined in various lung cancer cell lines, and the effects of overexpressed Smac in the nonsmall-cell lung cancer cell line A549 were assayed by stable transfection of Smac. Subsequently, MTT assays, cell counting, and flow cytometry were applied to show that overexpression of Smac inhibits cell viability and cell growth and enhances apoptosis after cisplatin treatment. Western blotting was performed before and after cisplatin treatment to demonstrate that drug treatment could release Smac from mitochondria into the cytosol and promote apoptosis by activating caspase-3 and caspase-9. Promotion of apoptosis by cytosolic Smac could be blocked by pretreating cells with the caspase-9 inhibitor z-LEHD-FMK. Our findings indicate that overexpressed Smac significantly inhibited A549 cell growth and promoted apoptosis following cisplatin treatment due to the release of Smac from mitochondria into the cytosol, which increased the activities of caspase-3 and caspase-9.

  19. Cutting Edge in IFN Regulation: Inflammatory Caspases Cleave cGAS.

    Science.gov (United States)

    Heidegger, Simon; Haas, Tobias; Poeck, Hendrik

    2017-03-21

    Caspases have important functions beyond their established role in driving inflammation and apoptosis. In this issue of Immunity, Wang et al. (2017) demonstrate that inflammasome-triggered caspases cleave and inactivate the DNA sensor cGAS, thus restricting the type I interferon response to cytosolic DNA. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  1. 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.

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

    Science.gov (United States)

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

    2017-10-01

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

  3. 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

    Caspase-8 is involved in death receptor-mediated apoptosis in type II cells, the proapoptotic programme of which is triggered by truncated Bid. Indeed, caspase-8 and Bid are the known intermediates of this signalling pathway. Cardiolipin has been shown to provide an anchor and an essential...... system that mimics contact sites and/or cardiolipin-enriched microdomains at the outer mitochondrial surface in which the platform consisting of caspase-8, Bid and cardiolipin was reconstituted in giant unilamellar vesicles. We analysed these vesicles by flow cytometry and confirm previous results...... that demonstrate the requirement for intact mature cardiolipin for caspase-8 activation and Bid binding and cleavage. We also used confocal microscopy to visualise the rupture of the vesicles and their revesiculation at smaller sizes due to alteration of the curvature following caspase-8 and Bid binding...

  4. The Caspase Pathway as a Possible Therapeutic Target in Experimental Pemphigus

    Directory of Open Access Journals (Sweden)

    Deyanira Pacheco-Tovar

    2011-01-01

    Full Text Available Apoptosis plays a role in pemphigus IgG-dependent acantholysis; theoretically, the blockade of the caspase pathway could prevent the blistering that is caused by pemphigus autoantibodies. Using this strategy, we attempted to block the pathogenic effect of pemphigus IgG in Balb/c mice by using the caspase inhibitor Ac-DEVD-CMK. This inhibitor was administrated before the injection of pemphigus IgG into neonatal mice. The main results of the present investigation are as follows: (1 pemphigus IgG induces intraepidermal blisters in Balb/c neonatal mice; (2 keratinocytes around the blister and acantholytic cells undergo apoptosis; (3 the caspases inhibitor Ac-DEVD-CMK prevents apoptosis; (4 the inhibition of the caspase pathway prevents blister formation. In conclusion, inhibition of the caspase pathway may be a promising therapeutic tool that can help in the treatment of pemphigus flare ups.

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

    International Nuclear Information System (INIS)

    Ulukaya, Engin; Karaagac, Esra; Ari, Ferda; Oral, Arzu Y.; Adim, Saduman B.; Tokullugil, Asuman H.; Evrensel, Türkkan

    2011-01-01

    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

  6. PP128. Placental Caspase-3 gene polymorphisms is associated with preeclampsia.

    Science.gov (United States)

    Hsu, C-D; Polavarapu, S; Parton, L

    2012-07-01

    Increased placental trophoblastic apoptosis (programmed cell death) was previously reported in pregnancies complicated by preeclampsia. Caspase-3 is one of the key executioners of apoptosis. Caspase are expressed in many tissues including human placental trophoblast and other tissues. Variations in the promoter area of the Caspase genes may modulate apoptotic signaling, contributing to an increased risk of preeclampsia To determine if gene polymorphisms of Caspase 3 proteins differ between patient with and without preeclampsia. Forty-three singleton placentas were studied. Twenty-two placentas were with preeclampsia and 21 were normotensive controls. DNA was extracted from placentas using QIAAmp DNA Minikit. Genotyping of Caspase 3 +567 was determined by real-time PCR using the Applied Biosystems Prism 7900 HT SDS machine. Chi-square and Fisher's exact tests were used for statistical analysis. There were no significant differences in maternal age, parity or race between the two groups. Preeclamptic placentas had higher frequency of wild type TT of Caspase-3 SNP (+567) as compared with normotensive controls (59% versus 28.5%). Preeclamptic placentas expressed significantly more genotype of TT of Caspase-3 SNP (+567) than normotensive patients when compared to CC (p=0.02). The alle frequencies of the Caspase SNP (+567) in preeclampstic placentas were 0.77 and 0.23 for T and C, respectively, as compared to 0.52 and 0.48, respectively, in placentas from normotensive pregnancies. Immune intolerance of maternal and placental interaction plays an important role in the pathogenesis of preeclampsia. Increased of placental apoptosis was reported in pregnancy complicated with preeclamsia. Our findings indicate placental Caspase 3 (+567) gene polymorphisms is associated with preeclampsia. Altered placental alle frequencies and caspase-3 SNP (+567) in preeclampsia further suggests preeclampsia is a trophoblastic disorder. Copyright © 2012. Published by Elsevier B.V.

  7. Overexpression of FADD and Caspase-8 inhibits proliferation and promotes apoptosis of human glioblastoma cells.

    Science.gov (United States)

    Wang, Hong-Bin; Li, Tao; Ma, Dong-Zhou; Ji, Yan-Xin; Zhi, Hua

    2017-09-01

    The study aimed at exploring the effects involved in Fas-Associated protein with Death Domain (FADD) expression and cysteine-aspartic acid specific protease-8 (Caspase-8) in relation to the proliferation and apoptosis of human glioblastoma (GBM) cells. 93 GBM tissues and 64 normal brain tissues were the central mediums used for the investigation of the study. Cultured human GBM SC189 cells were divided into separate groups including the blank negative control (NC), FADD and Caspase-8 groups. The mRNA and protein expressions of FADD and Caspase-8 in tissues and human glioblastoma (GBM) cells were detected using qRT-PCR and Western blotting techniques. Cell proliferation was tested by CCK-8. Flow cytometry was used for the measure of cell cycle and apoptosis rates. The mRNA and protein expressions of FADD and Caspase-8 in GBM tissues were less than the levels of expression displayed in normal brain tissues. Correlations between the expressions of FADD and Caspase-8 in GBM tissues were analyzed as being linked with the clinical grades of GBM patients. Patients in stage III+IV displayed lower expressions of FADD and Caspase-8 than patients in stage I+II. In comparison with the blank group, the FADD and Caspase-8 groups showed decreased proliferation rates of SHG44 cells and lower ratios of cells in the S phase and Bcl-2 expression. Greater ratios of cells in the G0/G1 stage as well as increased cell apoptosis and expressions of Caspase-8 and Bax were exhibited. The expression of FADD in the FADD group was higher than the blank group, however no significant differences in FADD expression was observed between the blank and Caspase-8 groups. The data obtained during the study demonstrated that overexpression of FADD and Caspase-8 suppresses proliferation whilst promoting the apoptosis of human GBM cells. Copyright © 2017. Published by Elsevier Masson SAS.

  8. 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.

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

    Science.gov (United States)

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

    2015-04-01

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

  11. The IAP-antagonist ARTS initiates caspase activation upstream of cytochrome C and SMAC/Diablo

    Science.gov (United States)

    Edison, N; Zuri, D; Maniv, I; Bornstein, B; Lev, T; Gottfried, Y; Kemeny, S; Garcia-Fernandez, M; Kagan, J; Larisch, S

    2012-01-01

    ARTS (Sept4_i2) is a pro-apoptotic tumor suppressor protein that functions as an antagonist of X-linked IAP (XIAP) to promote apoptosis. It is generally thought that mitochondrial outer membrane permeabilization (MOMP) occurs before activation of caspases and is required for it. Here, we show that ARTS initiates caspase activation upstream of MOMP. In living cells, ARTS is localized to the mitochondrial outer membrane. In response to apoptotic signals, ARTS translocates rapidly to the cytosol in a caspase-independent manner, where it binds XIAP and promotes caspase activation. This translocation precedes the release of cytochrome C and SMAC/Diablo, and ARTS function is required for the normal timing of MOMP. We also show that ARTS-induced caspase activation leads to cleavage of the pro-apoptotic Bcl-2 family protein Bid, known to promote MOMP. We propose that translocation of ARTS initiates a first wave of caspase activation that can promote MOMP. This leads to the subsequent release of additional mitochondrial factors, including cytochrome C and SMAC/Diablo, which then amplifies the caspase cascade and causes apoptosis. PMID:21869827

  12. 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.

  13. Axonal cleaved caspase-3 regulates axon targeting and morphogenesis in the developing auditory brainstem

    Directory of Open Access Journals (Sweden)

    Sarah E Rotschafer

    2016-10-01

    Full Text Available Caspase-3 is a cysteine protease that is most commonly associated with cell death. Recent studies have shown additional roles in mediating cell differentiation, cell proliferation, and development of cell morphology. We investigated the role of caspase-3 in the development of chick auditory brainstem nuclei during embryogenesis. Immunofluorescence from embryonic days E6-13 revealed that the temporal expression of cleaved caspase-3 follows the ascending anatomical pathway. Expression is first seen in the auditory portion of VIIIth nerve including central axonal regions projecting to nucleus magnocellularis (NM, then later in NM axons projecting to nucleus laminaris (NL, and subsequently in NL dendrites. To examine the function of cleaved caspase-3 in chick auditory brainstem development, we blocked caspase-3 cleavage in developing chick embryos with the caspase-3 inhibitor Z-DEVD-FMK from E6 to E9, then examined NM and NL morphology and NM axonal targeting on E10. NL lamination in treated embryos was disorganized and the neuropil around NL contained a significant number of glial cells normally excluded from this region. Additionally, NM axons projected into inappropriate portions of NL in Z-DEVD-FMK treated embyros. We found that the presence of misrouted axons was associated with more severe NL disorganization. The effects of axonal caspase-3 inhibition on both NL morphogenesis and NM axon targeting suggest that these developmental processes are coordinated, likely through communication between axons and their targets.

  14. Caspase-2 associates with FAN through direct interaction and overlapping functionality.

    Science.gov (United States)

    Forsberg, Jeremy; Li, Xinge; Zamaraev, Aleksey V; Panaretakis, Theocharis; Zhivotovsky, Boris; Olsson, Magnus

    2018-04-06

    Caspase-2 has been implicated in diverse cellular processes, and the identification of factors with which it interacts has steadily increased. In the present study, we report a direct interaction between caspase-2 and factor associated with neutral sphingomyelinase activation (FAN) using yeast two-hybrid screening and co-immunoprecipitation. Further, stable suppression of caspase-2 expression in HEK293T and HeLa cells enabled a systematic investigation of putative novel enzyme functionalities, especially with respect to ceramide production, cell migration, IL-6 production and vesicular homeostasis, all of which have been previously reported to be associated with FAN. Lipidomics excluded the involvement of caspase-2 in the generation of ceramide species, but caspase-2-dependent deregulation of IL-6 release, vesicular size and delayed cell relocation supported an association between caspase-2 and FAN. Collectively, these data identify a novel caspase-2-interacting factor, FAN, and expand the role for the enzyme in seemingly non-apoptotic cellular mechanisms. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. Overexpression of caspase-1 in pancreatic disorders: implications for a function besides apoptosis.

    Science.gov (United States)

    Ramadani, M; Gansauge, F; Schlosser, S; Yang, Y; Beger, H G; Gansauge, S

    2001-01-01

    The caspases are known to play a crucial role in the triggering and execution of apoptosis in a variety of cell types. We assessed the expression of caspase-1 in 42 pancreatic cancer tissue samples, 38 chronic pancreatitis specimens, and nine normal pancreatic tissues by immunohistochemistry and Western blot analysis. We found a clear overexpression of caspase-1 in both disorders, but differences in the expression patterns in distinct morphologic compartments. Pancreatic cancer tissue showed a clear cytoplasmatic overexpression of caspase-1 in tumor cells in 71% of the tumors, whereas normal pancreatic tissue showed only occasional immunoreactivity. In chronic pancreatitis an 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 clear 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, suggested to represent the p10-p20 heterodimer of active caspase-1, were found in 60% of the cancer tissue and 14% of the pancreatitis tissue specimens. Since we found a highly significant correlation between cytoplasm overexpression of caspase-1 in pancreatic cancer and overexpression of the known prognostic factors cyclin D1, epidermal growth factor, and epidermal growth factor receptor, it is plausible that caspase-1 has a yet unknown function in proliferative processes in addition to its well-known role in the apoptotic pathway.

  16. Cell-in-Cell Death Is Not Restricted by Caspase-3 Deficiency in MCF-7 Cells

    Science.gov (United States)

    Wang, Shan; He, Meifang; Li, Linmei; Liang, Zhihua; Zou, Zehong

    2016-01-01

    Purpose Cell-in-cell structures are created by one living cell entering another homotypic or heterotypic living cell, which usually leads to the death of the internalized cell, specifically through caspase-dependent cell death (emperitosis) or lysosome-dependent cell death (entosis). Although entosis has attracted great attention, its occurrence is controversial, because one cell line used in its study (MCF-7) is deficient in caspase-3. Methods We investigated this issue using MCF-7 and A431 cell lines, which often display cell-in-cell invasion, and have different levels of caspase-3 expression. Cell-in-cell death morphology, microstructures, and signaling pathways were compared in the two cell lines. Results Our results confirmed that MCF-7 cells are caspase-3 deficient with a partial deletion in the CASP-3 gene. These cells underwent cell death that lacked typical apoptotic properties after staurosporine treatment, whereas caspase-3-sufficient A431 cells displayed typical apoptosis. The presence of caspase-3 was related neither to the lysosome-dependent nor to the caspase-dependent cell-in-cell death pathway. However, the existence of caspase-3 was associated with a switch from lysosome-dependent cell-in-cell death to the apoptotic cell-in-cell death pathway during entosis. Moreover, cellular hypoxia, mitochondrial swelling, release of cytochrome C, and autophagy were observed in internalized cells during entosis. Conclusion The occurrence of caspase-independent entosis is not a cell-specific process. In addition, entosis actually represents a cellular self-repair system, functioning through autophagy, to degrade damaged mitochondria resulting from cellular hypoxia in cell-in-cell structures. However, sustained autophagy-associated signal activation, without reduction in cellular hypoxia, eventually leads to lysosome-dependent intracellular cell death. PMID:27721872

  17. Acinetobacter calcoaceticus-baumannii complex strains induce caspase-dependent and caspase-independent death of human epithelial cells.

    Science.gov (United States)

    Krzymińska, Sylwia; Frąckowiak, Hanna; Kaznowski, Adam

    2012-09-01

    We investigated interactions of human isolates of Acinetobacter calcoaceticus-baumannii complex strains with epithelial cells. The results showed that bacterial contact with the cells as well as adhesion and invasion were required for induction of cytotoxicity. The infected cells revealed hallmarks of apoptosis characterized by cell shrinking, condensed chromatin, and internucleosomal fragmentation of nuclear DNA. The highest apoptotic index was observed for 4 of 10 A. calcoaceticus and 4 of 7 A. baumannii strains. Moreover, we observed oncotic changes: cellular swelling and blebbing, noncondensed chromatin, and the absence of DNA fragmentation. The highest oncotic index was observed in cells infected with 6 A. calcoaceticus isolates. Cell-contact cytotoxicity and cell death were not inhibited by the pan-caspase inhibitor z-VAD-fmk. Induction of oncosis was correlated with increased invasive ability of the strains. We demonstrated that the mitochondria of infected cells undergo structural and functional alterations which can lead to cell death. Infected apoptotic and oncotic cells exhibited loss of mitochondrial transmembrane potential (ΔΨ(m)). Bacterial infection caused generation of nitric oxide and reactive oxygen species. This study indicated that Acinetobacter spp. induced strain-dependent distinct types of epithelial cell death that may contribute to the pathogenesis of bacterial infection.

  18. The apoptotic function analysis of p53, Apaf1, Caspase3 and Caspase7 during the spermatogenesis of the Chinese fire-bellied newt Cynops orientalis.

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    Da-Hui Wang

    Full Text Available BACKGROUND: Spontaneous and stress-induced germ cell apoptosis during spermatogenesis of multicellular organisms have been investigated broadly in mammals. Spermatogenetic process in urodele amphibians was essentially like that in mammals in spite of morphological differences; however, the mechanism of germ cell apoptosis in urodele amphibians remains unknown. The Chinese fire-belly newt, Cynops orientalis, was an excellent organism for studying germ cell apoptosis due to its sensitiveness to temperature, strong endurance of starvation, and sensitive skin to heavy metal exposure. METHODOLOGY/PRINCIPAL FINDINGS: TUNEL result showed that spontaneous germ cell apoptosis took place in normal newt, and severe stress-induced apoptosis occurred to spermatids and sperm in response to heat shock (40°C 2 h, cold exposure (4°C 12 h, cadmium exposure (Cd 36 h, and starvation stress. Quantitative reverse transcription polymerase chain reactions (qRT-PCR showed that gene expression of Caspase3 or Caspase7 was obviously elevated after stress treatment. Apaf1 was not altered at its gene expression level, and p53 was significantly decreased after various stress treatment. Caspase assay demonstrated that Caspase-3, -8, -9 enzyme activities in newt testis were significantly elevated after heat shock (40°C 2 h, cold exposure (4°C 12 h, and cadmium exposure (Cd 36 h, while Caspase3 and Caspase8 activities were increased with Caspase9 significantly decreased after starvation treatment. CONCLUSIONS/SIGNIFICANCE: Severe germ cell apoptosis triggered by heat shock, cold exposure, and cadmium exposure was Caspase3 dependent, which probably involved both extrinsic and intrinsic pathways. Apaf1 may be involved in this process without elevating its gene expression. But starvation-induced germ cell apoptosis was likely mainly through extrinsic pathway. p53 was probably not responsible for stress-induced germ cell apoptosis in newt testis. The intriguing high occurrence

  19. Cleaved caspase-3 in lung epithelium of children who died with acute respiratory distress syndrome

    NARCIS (Netherlands)

    Bem, Reinout A.; van der Loos, Chris M.; van Woensel, Job B. M.; Bos, Albert P.

    2010-01-01

    OBJECTIVE: To investigate the extent of cleaved caspase-3 immunostaining in lung epithelial cells in children with acute respiratory distress syndrome. DESIGN: Observational study in sixteen children who died with acute respiratory distress syndrome and diffuse alveolar damage. SETTING: Pediatric

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

    Science.gov (United States)

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

    2013-01-01

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

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

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    Antje Bast

    2014-03-01

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

  2. Ordering of ceramide formation and caspase-9 activation in CD95L-induced Jurkat leukemia T cell apoptosis.

    Science.gov (United States)

    Lafont, Elodie; Dupont, Romain; Andrieu-Abadie, Nathalie; Okazaki, Toshiro; Schulze-Osthoff, Klaus; Levade, Thierry; Benoist, Hervé; Ségui, Bruno

    2012-04-01

    Ceramide, a biologically active sphingolipid in cell death signaling, accumulates upon CD95L treatment, concomitantly to apoptosis induction in Jurkat leukemia T cells. Herein, we show that ceramide did not increase in caspase-8 and -10-doubly deficient Jurkat cells in response to CD95L, indicating that apical caspases are essential for CD95L-triggered ceramide formation. Jurkat cells are typically defined as type 2 cells, which require the activation of the mitochondrial pathway for efficient apoptosis induction in response to CD95L. Caspase-9-deficient Jurkat cells significantly resisted CD95L-induced apoptosis, despite ceramide accumulation. Knock-down of sphingomyelin synthase 1, which metabolizes ceramide to sphingomyelin, enhanced (i) CD95L-triggered ceramide production, (ii) cytochrome c release from the mitochondria and (iii) caspase-9 activation. Exogenous ceramide-induced caspase-3 activation and apoptosis were impaired in caspase-9-deficient Jurkat cells. Conversely, caspase-9 re-expression in caspase-9-deficient Jurkat cells restored caspase-3 activation and apoptosis upon exogenous ceramide treatment. Collectively, our data provide genetic evidence that CD95L-triggered endogenous ceramide increase in Jurkat leukemia T cells (i) is not a mere consequence of cell death and occurs mainly in a caspase-9-independent manner, (ii) is likely involved in the pro-apoptotic mitochondrial pathway leading to caspase-9 activation. Copyright © 2012 Elsevier B.V. All rights reserved.

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

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

    2003-12-01

    To identify the expression of Caspase-1(interleukin-1beta converting enzyme) and its role in adenoma of the pancreas and chronic pancreatitis. 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. 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 clear 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. 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.

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

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    Alejandro Romero

    2011-02-01

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

  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) GB14-37368G; GA ČR(CZ) GA14-28254S Institutional support: RVO:67985904 ; RVO:68081715 Keywords : osteogenesis * chondrogenesis * caspases * caspase-3 * gene expression Subject RIV: EA - Cell Biology; CB - Analytical Chemistry, Separation (UIACH-O) Impact factor: 0.897, year: 2016

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

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    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

  7. 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

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

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    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.

  9. [The expression and significance of smac, XIAP, caspase-3 in nonnasal inverted papilloma].

    Science.gov (United States)

    Yang, Lihui; Shan, Chunguang; Huang, Hongmei; Xu, Qiurong; Zhao, Ying; Meng, Yajing; Zhang, Zhihong

    2012-07-01

    To explore the expression and significance of second mitochondria derived activator of caspase (Smac), X-linked inhibitor of apoptosis protein (XIAP)and cysteine containing aspartate specific protease 3 (caspase-3) in the growth, development and carcinogenesis of the nonnasal inverted papilloma (NIP). Immunohistochemical method was used to detect the expression of Smac, XIAP, caspase-3 in 10 cases of nasal cavity mucosae (NM) and 45 cases of NIP, the group of NIP including 25 cases of NIP without dysplasia, 11 cases of NIP with dysplasia, and 9 cases of NIP with malignant transformation to squamous cell carcinoma (SCC). The intensity of the positive expression of Smac, Caspase-3 in NIP were lower than NM, the intensity of the positive expression decreased with the decreasing degree of histological differentiation. There was a significant difference between NIP without dysplasia and SCC. It was presented with a progressive tendency for the expression of XIAP in the group of NM and NIP. The lower degree of histological differentiation, the higher intensity of the positive expression. The expression between NIP without dysplasia and SCC had a significant difference. Smac negatively correlated with XIAP (r(s) = -0.323, P Smac positively correlated with caspase 3 (r(s) = 0.424, P Smac, XIAP, caspase 3 might be associated with the growth and carcinogenesis of NIP.

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

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    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.

  11. 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

    2014-01-01

    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.

  12. Caspase 3 in dying tumor cells mediates post-irradiation angiogenesis

    Science.gov (United States)

    Zhang, Zhengxiang; Yu, Yang; Cheng, Jin; Gong, Yanping; Li, Chuan-Yuan; Huang, Qian

    2015-01-01

    Cytotoxic radiotherapy unfavorably induces tumor cells to generate various proangiogenic substances, promoting post-irradiation angiogenesis (PIA), which is one of major causes of radiotherapy failure. Though several studies have reported some mechanisms behind PIA, they have not yet described the beginning proangiogenic motivator buried in the irradiated microenvironment. In this work, we revealed that dying tumor cells induced by irradiation prompted PIA via a caspase 3 dependent mechanism. Proteolytic inactivation of caspase 3 in dying tumor cells by transducing a dominant-negative version weakened proangiogenic effects in vitro and in vivo. In addition, inhibition of caspase 3 activity suppressed tumor angiogenesis and tumorigenesis in xenograft mouse model. Importantly, we identified vascular endothelial growth factor (VEGF)-A as a downstream proangiogenic factor regulated by caspase 3 possibly through Akt signaling. Collectively, these findings indicated that besides acting as a key executioner in apoptosis, caspase 3 in dying tumor cells may play a central role in driving proangiogenic response after irradiation. Thus, radiotherapy in combination with caspase 3 inhibitors may be a novel promising therapeutic strategy to reduce tumor recurrence due to restrained PIA. PMID:26431328

  13. Caffeic Acid Induced Apoptosis in MG63 Osteosarcoma Cells Through Activation of Caspases

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    Ferry Sandra

    2017-03-01

    Full Text Available Background: Caffeic acid has been reported that when it is combined with all-trans retinoic acid, it can inhibit proliferation activity of SaOS-2 or OSA-01 cells. In addition, caffeic acid merely could reduce cell viability of SaOS-2 cells. However, there is not any study in caffeic acid's possible effect to induce apoptosis in osteosarcoma cell. Materials and Methods: MG-63 cells were cultured in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum. Cells were treated with various concentrations of caffeic acid. Apoptosis were analyzed with Sub-G1 assay and activation of caspase-8, -9, and -3 were analyzed with immunoblotting. Caffeic acid-induced percentage of apoptotic cells and cleaved-8, -9, -3 were then statistically analyzed. Results: Sub-G1 results showed that caffeic acid significantly induced apoptosis in MG-63 osteosarcoma cells in concentration dependent manner. Immunoblotting results showed that caffeic acid induced cleavage of caspase-8, -9 and -3. Cleaved-caspase-8 and -9 were increased at 1-hour treatment of caffeic acid, while cleaved-caspase 3 was increased markedly at 6-hours treatment of caffeic acid. Conclusions: Caffeic acid induces apoptosis significantly in concentration dependent manner through caspase-dependent intrinsic apoptotic pathway. Keywords: caffeic acid, osteosarcoma, MG-63, apoptosis, caspase

  14. Cas Ilgly Induces Apoptosis in Glioma C6 Cells In Vitro and In Vivo through Caspase-Dependent and Caspase-Independent Mechanisms

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    Cristina Trejo-Solís

    2005-06-01

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

  15. Molecular dynamics-assisted pharmacophore modeling of caspase-3-isatin sulfonamide complex: Recognizing essential intermolecular contacts and features of sulfonamide inhibitor class for caspase-3 binding.

    Science.gov (United States)

    Kumar, Sivakumar Prasanth; Patel, Chirag N; Jha, Prakash C; Pandya, Himanshu A

    2017-12-01

    The identification of isatin sulfonamide as a potent small molecule inhibitor of caspase-3 had fuelled the synthesis and characterization of the numerous sulfonamide class of inhibitors to optimize for potency. Recent works that relied on the ligand-based approaches have successfully shown the regions of optimizations for sulfonamide scaffold. We present here molecular dynamics-based pharmacophore modeling of caspase-3-isatin sulfonamide crystal structure, to elucidate the essential non-covalent contacts and its associated pharmacophore features necessary to ensure caspase-3 optimal binding. We performed 20ns long dynamics of this crystal structure to extract global conformation states and converted into structure-based pharmacophore hypotheses which were rigorously validated using an exclusive focussed library of experimental actives and inactives of sulfonamide class by Receiver Operating Characteristic (ROC) statistic. Eighteen structure-based pharmacophore hypotheses with better sensitivity and specificity measures (>0.6) were chosen which collectively showed the role of pocket residues viz. Cys163 (S 1 sub-site; required for covalent and H bonding with Michael acceptor of inhibitors), His121 (S 1 ; π stack with bicyclic isatin moiety), Gly122 (S 1 ; H bond with carbonyl oxygen) and Tyr204 (S 2 ; π stack with phenyl group of the isatin sulfonamide molecule) as stringent binding entities for enabling caspase-3 optimal binding. The introduction of spatial pharmacophore site points obtained from dynamics-based pharmacophore models in a virtual screening strategy will be helpful to screen and optimize molecules belonging to sulfonamide class of caspase-3 inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Amplification activation loop between caspase-8 and -9 dominates artemisinin-induced apoptosis of ASTC-a-1 cells.

    Science.gov (United States)

    Xiao, Fenglian; Gao, Weijie; Wang, Xiaoping; Chen, Tongsheng

    2012-06-01

    Although caspases have been demonstrated to be involved in artemisinin (ARTE)-induced apoptosis, their exact functions are not well understood. The aim of this report is to explore the roles of caspase-8, -9 and -3 during ARTE-induced apoptosis in human lung adenocarcinoma (ASTC-a-1) cells. ARTE treatment induces a rapid generation of reactive oxygen species (ROS), and ROS-dependent apoptosis as well as the activation of caspase-8, -9 and -3 via time- and dose-dependent fashion. Of upmost importance, inhibition of caspase-8 or -9, but not caspase-3, almost completely blocks the ARTE-induced not only activation of the caspase-8, -9 and -3 but also apoptosis. In addition, the apoptotic process triggered by ARTE does not involve the Bid cleavage, tBid translocation, significant loss of mitochondrial membrane potential and cytochrome c release from mitochondria. Moreover, silencing Bax/Bak does not prevent the ATRE-induced cell death as well as the activation of caspase-8, -9 and -3. Collectively, our data firstly demonstrate that ARTE triggers a ROS-mediated positive feedback amplification activation loop between caspase-8 and -9 independent of mitochondria, which dominantly mediated the ARTE-induced apoptosis via a caspase-3-independent apoptotic pathway in ASTC-a-1 cells. Our findings imply a potential to develop new derivatives from artemisinin to effectively initiate the amplification activation loop of caspases.

  17. Apoptosis of CTLL-2 cells induced by an immunosuppressant, ISP-I, is caspase-3-like protease-independent.

    Science.gov (United States)

    Yamaji, T; Nakamura, S; Takematsu, H; Kawasaki, T; Kozutsumi, Y

    2001-04-01

    In our previous study, the sphingosine-like immunosuppressant ISP-1 was shown to induce apoptosis in the mouse cytotoxic T cell line CTLL-2. In this study, we characterized the ISP-1-induced apoptotic pathway. Although caspase-3-like protease activity increases concomitantly with ISP-1-induced apoptosis in CTLL-2 cells, the apoptosis is not inhibited by caspase-3-like protease inhibitors, i.e. DEVD-cho and z-DEVD-fmk. In contrast, sphingosine-induced apoptosis in CTLL-2 cells is caspase-3-like protease-dependent. A caspase inhibitor with broad specificity, z-VAD-fmk, protects cells from apoptosis induced by ISP-1, indicating that ISP-1-induced apoptosis is dependent on caspase(s) other than caspase-3. Overexpression of Bcl-2 or Bcl-xL suppresses the apoptosis induced by ISP-1, although sphingosine-induced apoptosis is not efficiently inhibited by Bcl-2. Finally, ISP-1-induced mitochondrial depolarization, which is thought to be a checkpoint dividing the apoptotic pathway into upstream and downstream stages, is not inhibited by DEVD-cho, but is inhibited by z-VAD-fmk. These data suggest that a pathway dependent on caspase(s) other than caspase-3 is involved upstream of mitochondrial depolarization in ISP-1-induced apoptosis.

  18. Overexpression of caspase-1 (interleukin-1beta converting enzyme) in chronic pancreatitis and its participation in apoptosis and proliferation.

    Science.gov (United States)

    Ramadani, M; Yang, Y; Gansauge, F; Gansauge, S; Beger, H G

    2001-05-01

    Caspase-1, formerly designated interleukin-1beta converting enzyme, was the first described member of a group of cysteine proteases called caspases. It is suggested that caspases play an important role in apoptosis, but recent observations could show that caspase-1 might also be involved in cellular proliferation. We investigated the expression of caspase-1 in 38 chronic pancreatitis tissues, six pancreatitis tissues from patients with pancreatic carcinoma and nine normal pancreatic tissues by immunohistochemistry. Western blot analysis was used to confirm the immunohistochemical findings. We found a clear expression of caspase-1 in chronic pancreatitis, but not in normal pancreatic tissues. Interestingly, we found expression of caspase-1 in three distinct morphologic compartments: (i) in atrophic acinar cells (31 of 35; 89%), (ii) proliferating cells of ductal origin (33 of 38; 87%), and (iii) in acinar cells redifferentiating to form tubular structures (26 of 31; 83%). These immunohistochemical findings were confirmed by Western blot analysis, which showed an expression of caspase-1 in 85% of the tissues. No correlation was found between any of the examined clinicopathologic features and the caspase-1 expression in chronic pancreatitis. In conclusion, the expression of caspase-1 is a frequent event in chronic pancreatitis and its distribution pattern may reflect two functions of this protease: on one hand its participation in the apoptotic pathway in atrophic acinar cells and, on the other hand, its role in proliferation and differentiation in proliferating duct cells.

  19. 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.

  20. 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.

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

    International Nuclear Information System (INIS)

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Banun Kusumawardani

    2016-04-01

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

  3. 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

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

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

    International Nuclear Information System (INIS)

    Sheng Zhiguo; Cao Xiaojuan; Peng Shuangqing; Wang Changyong; Li Qianqian; Wang Yimei; Liu Mifeng

    2008-01-01

    Quinolones (QNs)-induced arthropathy is an important toxic effect in immature animals leading to restriction of their therapeutic use in pediatrics. However, the exact mechanism still remains unclear. Recently, we have demonstrated that ofloxacin, a typical QN, induces apoptosis of alginate microencapsulated juvenile rabbit joint chondrocytes by disturbing the β 1 integrin functions and inactivating the ERK/MAPK signaling pathway. In this study, we extend our initial observations to further elucidate the mechanism(s) of ofloxacin-induced apoptosis by utilizing specific caspase inhibitors. Pretreatment with both caspase-9-specific inhibitor zLEHD-fmk and caspase-8 inhibitor zIETD-fmk attenuated ofloxacin-induced apoptosis and activation of caspase-3 of chondrocyte in a concentration-dependent manner, as determined by fluorescent dye staining, enzyme activity assay and immunoblotting. Furthermore, the activation of caspase-9, -8 and -3 stimulated by ofloxacin was significantly inhibited in the presence of zIETD-fmk while pretreatment with zLEHD-fmk only blocked the activation of caspase-9 and -3. Ofloxacin also stimulated a concentration-dependent translocation of cytochrome c from mitochondria into the cytosol and a decrease of mitochondrial transmembrane potential, which was completely inhibited by zIETD-fmk. In addition, ofloxacin was found to increase the level of Bax, tBid, p53 in a concentration- and time-dependent manner. Taken together, The current results indicate that the caspase-8-dependent mitochondrial pathway is primarily involved in the ofloxacin-induced apoptosis of microencapsulated juvenile rabbit joint chondrocytes

  6. Impacts of Bone Marrow Stem Cells on Caspase-3 Levels after Spinal Cord Injury in Mice.

    Science.gov (United States)

    Gashmardi, Noushin; Hosseini, Seyed Ebrahim; Mehrabani, Davood; Edalatmanesh, Mohammad Amin; Khodabandeh, Zahra

    2017-11-01

    Spinal cord injury (SCI) is a drastic disability that leads to spinal cord impairment. This study sought to determine the effects of bone marrow stem cells (BMSCs) on caspase-3 levels after acute SCI in mice. Forty-two mice were randomly divided into 3 groups: control (2 subcategories), subjected to no intervention; sham (3 subcategories), subjected to acute SCI; and experimental (2 subcategories), subjected to SCI and cell transplantation. In the experimental group, 2×10 5 BMSCs were injected intravenously 1 day after SCI. The mesenchymal property of the cells was assessed. The animals in the 3 groups were sacrificed 1, 21, and 35 days after the induction of injury and caspase-3 levels were evaluated using a caspase-3 assay kit. The obtained values were analyzed with ANOVA and Tukey tests using GraphPad and SPSS. Based on the assessments, the transplanted cells were spindle-shaped and were negative for the hematopoietic markers of CD34 and CD45 and positive for the expression of the mesenchymal marker of CD90 and osteogenic induction. The caspase-3 levels showed a significant increase in the sham and experimental groups in comparison to the control group. One day after SCI, the caspase-3 level was significantly higher in the sham group (1.157±0.117) than in the other groups (P<0.000). Twenty-one days after SCI, the caspase-3 level was significantly lower in the experimental group than in the sham group (0.4±0.095 vs. 0.793±0.076; P˂0.000). Thirty-five days following SCI, the caspase-3 level was lower in the experimental group than in the sham group (0.223±0.027 vs. 0.643±0.058; P˂0.000). We conclude that BMSC transplantation was able to downregulate the caspase-3 level after acute SCI, underscoring the role of caspase-3 as a marker for the assessment of treatment efficacy in acute SCI.

  7. Impacts of Bone Marrow Stem Cells on Caspase-3 Levels after Spinal Cord Injury in Mice

    Directory of Open Access Journals (Sweden)

    Noushin Gashmardi

    2017-11-01

    Full Text Available Spinal cord injury (SCI is a drastic disability that leads to spinal cord impairment. This study sought to determine the effects of bone marrow stem cells (BMSCs on caspase-3 levels after acute SCI in mice. Forty-two mice were randomly divided into 3 groups: control (2 subcategories, subjected to no intervention; sham (3 subcategories, subjected to acute SCI; and experimental (2 subcategories, subjected to SCI and cell transplantation. In the experimental group, 2×105 BMSCs were injected intravenously 1 day after SCI. The mesenchymal property of the cells was assessed. The animals in the 3 groups were sacrificed 1, 21, and 35 days after the induction of injury and caspase-3 levels were evaluated using a caspase-3 assay kit. The obtained values were analyzed with ANOVA and Tukey tests using GraphPad and SPSS. Based on the assessments, the transplanted cells were spindle-shaped and were negative for the hematopoietic markers of CD34 and CD45 and positive for the expression of the mesenchymal marker of CD90 and osteogenic induction. The caspase-3 levels showed a significant increase in the sham and experimental groups in comparison to the control group. One day after SCI, the caspase-3 level was significantly higher in the sham group (1.157±0.117 than in the other groups (P<0.000. Twenty-one days after SCI, the caspase-3 level was significantly lower in the experimental group than in the sham group (0.4±0.095 vs. 0.793±0.076; P˂0.000. Thirty-five days following SCI, the caspase-3 level was lower in the experimental group than in the sham group (0.223±0.027 vs. 0.643±0.058; P˂0.000. We conclude that BMSC transplantation was able to downregulate the caspase-3 level after acute SCI, underscoring the role of caspase-3 as a marker for the assessment of treatment efficacy in acute SCI.

  8. 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.

  9. Triglyceride-induced macrophage cell death is triggered by caspase-1.

    Science.gov (United States)

    Son, Sin Jee; Rhee, Ki-Jong; Lim, Jaewon; Kim, Tae Ue; Kim, Tack-Joong; Kim, Yoon Suk

    2013-01-01

    Triglyceride (TG) induces macrophage cell death which contributes to the development of atherosclerosis. We confirmed that exogenous TG accumulates in human THP-1 macrophages and causes cell death. TG treated THP-1 macrophages exhibited no change in tumor necrosis factor (TNF)-α, interleukin (IL)-18, macrophage inflammatory protein (MIP)-1α, and IL-1R1 receptor mRNA expression. However, there was a marked decrease in IL-1β mRNA expression but an increase in IL-1β protein secretion. Decreased expression of IL-1β mRNA and increased secretion of IL-1β protein was not the direct cause of cell death. Until now, TG was assumed to induce necrotic cell death in macrophages. Since caspase-1 is known to be involved in activation and secretion of IL-1β protein and pyroptotic cell death, next we determined whether caspase-1 is associated with TG-induced macrophage cell death. We found an increase in caspase-1 activity in TG-treated THP-1 macrophages and inhibition of caspase-1 activity using a specific inhibitor partially rescued cell death. These results suggest activation of the pyroptotic pathway by TG. This is the first report implicating the activation of caspase-1 and the triggering of the pyroptosis pathway in TG-induced macrophage cell death.

  10. Early apoptotic reorganization of spliceosomal proteins involves caspases, CAD and rearrangement of NuMA.

    Science.gov (United States)

    Dieker, Jürgen; Iglesias-Guimarais, Victoria; Décossas, Marion; Stevenin, James; van der Vlag, Johan; Yuste, Victor J; Muller, Sylviane

    2012-02-01

    The reorganization of nuclear structures is an important early feature of apoptosis and involves the activity of specific proteases and nucleases. Well-known is the condensation and fragmentation of chromatin; however, much less is understood about the mechanisms involved in the reorganization of structures from the interchromatin space, such as interchromatin granule clusters (IGCs). In this study, we show that the initial enlargement and rounding-up of IGCs correlate with a decrease in mRNA transcription and are caspase-independent, but involve protein phosphatases PP1/PP2A. Subsequently, multiple enlarged IGCs dissociate from chromatin and fuse into a single structure. The dissociation requires caspase activity and involves caspase-activated DNase (CAD). Apoptotic IMR-5 cells, lacking a proper processing of CAD, show multiple enlarged IGCs that remain linked with chromatin. Overexpression of CAD in IMR-5 cells results in the dissociation of IGCs from chromatin, but the fusion into a single structure remains disturbed. Nuclear matrix protein NuMA is reorganized in a caspase-dependent way around fused IGCs. In conclusion, we show here that the apoptotic rearrangement of IGCs, the nuclear matrix and chromatin are closely associated, occur in defined stages and depend on the activity of protein phosphatases, caspases and CAD. © 2011 John Wiley & Sons A/S.

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

    Science.gov (United States)

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

    2016-04-04

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

  12. Detergent sclerosants at sub-lytic concentrations induce endothelial cell apoptosis through a caspase dependent pathway.

    Science.gov (United States)

    Cooley-Andrade, Osvaldo; Cheung, Kelvin; Chew, An-Ning; Connor, David Ewan; Parsi, Kurosh

    2016-07-01

    To investigate the apoptotic effects of detergent sclerosants sodium tetradecylsulphate (STS) and polidocanol (POL) on endothelial cells at sub-lytic concentrations. Human umbilical vein endothelial cells (HUVECs) were isolated and labelled with antibodies to assess for apoptosis and examined with confocal microscopy and flow cytometry. Isolated HUVECs viability was assessed using propidium iodide staining. Early apoptosis was determined by increased phosphatidylserine exposure by lactadherin binding. Caspase 3, 8, 9 and Bax activation as well as inhibitory assays with Pan Caspase (Z-VAD-FMK) and Bax (BI-6C9) were assessed to identify apoptotic pathways. Porimin activation was used to assess cell membrane permeability. Cell lysis reached almost 100 % with STS at 0.3 % and with POL at 0.6 %. Apoptosis was seen with both STS and POL at concentrations ranging from 0.075 to 0.15 %. PS exposure increased with both STS and POL and exhibited a dose-dependent trend. Active Caspase 3, 8 and 9 but not Bax were increased in HUVECs stimulated with low concentrations of both STS and POL. Inhibitory assays demonstrated Caspase 3, 8, 9 inhibition at low concentrations (0.075 to 0.6 %) with both STS and POL. Both agents increased the activation of porimin at all concentrations. Both sclerosants induced endothelial cell (EC) apoptosis at sub-lytic concentrations through a caspase-dependant pathway. Both agents induced EC oncosis.

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

    Science.gov (United States)

    Saheb, Entsar; Trzyna, Wendy; Bush, John

    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.

  14. Caspase-8 inactivation in T cells increases necroptosis and suppresses autoimmunity in Bim−/− mice

    Science.gov (United States)

    Bohgaki, Toshiyuki; Mozo, Julien; Salmena, Leonardo; Matysiak-Zablocki, Elzbieta; Bohgaki, Miyuki; Sanchez, Otto; Strasser, Andreas

    2011-01-01

    Dysregulation of either the extrinsic or intrinsic apoptotic pathway can lead to various diseases including immune disorders and cancer. In addition to its role in the extrinsic apoptotic pathway, caspase-8 plays nonapoptotic functions and is essential for T cell homeostasis. The pro-apoptotic BH3-only Bcl-2 family member Bim is important for the intrinsic apoptotic pathway and its inactivation leads to autoimmunity that is further exacerbated by loss of function of the death receptor Fas. We report that inactivation of caspase-8 in T cells of Bim−/− mice restrained their autoimmunity and extended their life span. We show that, similar to caspase-8−/− T cells, Bim−/− T cells that also lack caspase-8 displayed elevated levels of necroptosis and that inhibition of this cell death process fully rescued the survival and proliferation of these cells. Collectively, our data demonstrate that inactivation of caspase-8 suppresses the survival and proliferative capacity of Bim−/− T cells and restrains autoimmunity in Bim−/− mice. PMID:22006951

  15. Intracellular antibody-caspase-mediated cell killing: An approach for application in cancer therapy

    Science.gov (United States)

    Tse, Eric; Rabbitts, Terence H.

    2000-10-01

    Antibodies have been expressed inside cells in an attempt to ablate the function of oncogene products. To make intracellular antibodies more generally applicable and effective in cancer therapy, we have devised a method in which programmed cell death or apoptosis can be triggered by specific antibody-antigen interaction. When intracellular antibodies are linked to caspase 3, the "executioner" in the apoptosis pathway, and bind to the target antigen, the caspase 3 moieties are self-activated and thereby induce cell killing. We have used this strategy in a model system with two pairs of intracellular antibodies and antigens. In vivo coexpression of an antibody-caspase 3 fusion with its antigenic target induced apoptosis that was specific for antibody, antigen, and active caspase 3. Moreover, the antibody-caspase 3 fusion protein was not toxic to cells in the absence of antigen. Therefore, intracellular antibody-mediated apoptosis should be useful as a specific therapeutic approach for the treatment of cancers, a situation where target cell killing is required.

  16. Hydrogen peroxide-mediated necrosis induction in HUVECs is associated with an atypical pattern of caspase-3 cleavage.

    Science.gov (United States)

    Csordas, Adam; Wick, Georg; Bernhard, David

    2006-06-10

    Oxidative stress, continuously exerted during chronic inflammation, has been implicated as a major causative agent of cellular dysfunction and cell death. In the present study, we investigated the impact of oxidative stress on the mode of cell death in HUVECs using H2O2 as a model reagent. We found that the predominant form of cell death was necrosis. Necrosis induction was accompanied by a distinct mode of caspase-3 cleavage, yielding a 29-kDa fragment. While inhibition of caspases could not prevent the generation of the 29-kDa fragment, general protease inhibitors, such as leupeptin and LLNL, proved to be effective in inhibiting the distinct processing pattern of caspase-3. These results suggest that caspases can act as substrates for non-caspase proteases in cells primed for necrosis induction. Thus, the pattern of caspase-3 cleavage might reflect the proteolytic system engaged in the cell death machinery in HUVECs.

  17. Caspase-1 contributes to Chlamydia trachomatis-induced upper urogenital tract inflammatory pathologies without affecting the course of infection.

    Science.gov (United States)

    Cheng, Wen; Shivshankar, Pooja; Li, Zhongyu; Chen, Lili; Yeh, I-Tien; Zhong, Guangming

    2008-02-01

    Chlamydia trachomatis infection induces inflammatory pathologies in the upper genital tract, potentially leading to ectopic pregnancy and infertility in the affected women. Caspase-1 is required for processing and release of the inflammatory cytokines interleukin-1beta (IL-1beta), IL-18, and possibly IL-33. In the present study, we evaluated the role of caspase-1 in chlamydial infection and pathogenesis. Although chlamydial infection induced caspase-1 activation and processing of IL-1beta, mice competent and mice deficient in caspase-1 experienced similar courses of chlamydial infection in their urogenital tracts, suggesting that Chlamydia-activated caspase-1 did not play a significant role in resolution of chlamydial infection. However, when genital tract tissue pathologies were examined, the caspase-1-deficient mice displayed much reduced inflammatory damage. The reduction in inflammation was most obvious in the fallopian tube tissue. These observations demonstrated that although caspase-1 is not required for controlling chlamydial infection, caspase-1-mediated responses can exacerbate the Chlamydia-induced inflammatory pathologies in the upper genital tract, suggesting that the host caspase-1 may be targeted for selectively attenuating chlamydial pathogenicity without affecting the host defense against chlamydial infection.

  18. Integrity of XIAP is essential for effective activity recovery of apoptosome and its downstream caspases by Smac/Diablo.

    Science.gov (United States)

    Attaran-Bandarabadi, Faezeh; Abhari, Behnaz Ahangarian; Neishabouri, Shima Hallaj; Davoodi, Jamshid

    2017-08-01

    Contribution of individual BIR domains to Smac antagonism is investigated. Ammonium citrate was used to activate caspase-9 and pro-caspase-9 (D315, D330/A). However, the presence of citrate resulted in autoproteolysis of pro-caspase-9 and its inhibition by XIAP BIR3, which was not observed for apoptosome activated pro-caspase-9 indicating abnormal behavior of pro-caspase-9 in kosmotropic citrate salt. Thus, we used Apaf-1(residues 1-591) to activate caspase-9 through the formation of mini-apoptosome instead. Inhibition of apoptosome by XIAP BIR-1-2-3 was observed to be similar to that of BIR3 indicating that the cleavage of XIAP does not affect its potency. However, BIR1-2-3 was more prone to Smac antagonism due to simultaneous interaction of two BIR domains from XIAP with two N-terminal binding sites of Smac. Therefore, despite the role in caspase-9 activation, Apaf-1 does not influence caspase-9 inhibition by XIAP. In addition, caspase-3, -7 and -9 activity recovery by Smac protein and peptide were more efficient for BIR1-2-3 than for BIR1-2. Consequently, it can be proposed that the presence of multiple BIR domains for XIAP among different species along with dimeric nature of Smac are evolutionary designed to strengthen the antagonistic activity of Smac culminating in efficient induction of cell death. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. The co-chaperone p23 is degraded by caspases and the proteasome during apoptosis

    DEFF Research Database (Denmark)

    Mollerup, Jens; Berchtold, Martin Werner

    2005-01-01

    The heat shock protein 90 co-chaperone p23 has recently been shown to be up-regulated in cancer cells and down-regulated in atheroschlerotic plaques. We found that p23 is degraded during apoptosis induced by several stimuli, including Fas and TNFa-receptor activation as well as staurosporine...... treatment. Caspase inhibition protected p23 from degradation in several cell lines. In addition, recombinant caspase-3 and 8 cleaved p23 at Asp 142 generating a degradation product of 18 kDa as seen in apoptotic cells. Truncated p23 is further degraded in a proteasome dependent process during apoptosis....... Furthermore, we found that the anti-aggregating activity of truncated p23 was reduced compared to full length p23 indicating that caspase mediated p23 degradation contributes to protein destabilisation in apoptosis....

  20. Essential roles of Caspase-3 in facilitating Myc-induced genetic instability and carcinogenesis.

    Science.gov (United States)

    Cartwright, Ian M; Liu, Xinjian; Zhou, Min; Li, Fang; Li, Chuan-Yuan

    2017-07-10

    The mechanism for Myc-induced genetic instability is not well understood. Here we show that sublethal activation of Caspase-3 plays an essential, facilitative role in Myc-induced genomic instability and oncogenic transformation. Overexpression of Myc resulted in increased numbers of chromosome aberrations and γH2AX foci in non-transformed MCF10A human mammary epithelial cells. However, such increases were almost completely eliminated in isogenic cells with CASP3 gene ablation. Furthermore, we show that endonuclease G, an apoptotic nuclease downstream of Caspase-3, is directly responsible for Myc-induced genetic instability. Genetic ablation of either CASP3 or ENDOG prevented Myc-induced oncogenic transformation of MCF10A cells. Taken together, we believe that Caspase-3 plays a critical, unexpected role in mediating Myc-induced genetic instability and transformation in mammalian cells.

  1. Regulatory effect of caspase-11 on interleukin-1β in the fungal keratitis.

    Science.gov (United States)

    Zhu, Keke; Mu, Hongmei; Pi, Baimu

    2016-11-01

    Caused by fungus, fungal keratitis is a kind of infections corneal disease with high rate of blindness, which patients are mainly farmers in developing countries. Interleukin, as important proinflammatory cytokines, involve in immune defense process against fungal infection of cornea. The expression of interleukin in the pathogenesis of fungal keratitis, especially the main source of its cells, is not clear and the cell signaling pathways which regulate the synthesis and modification of interleukin is still unknown. Caspase-11 was obtained and cultured. And the ELISA and Western-blot methods were used to explore the regulatory effect of Caspse-11 on Interleukin-1β in the fungal keratitis. neutrophils were the main cell lineage of IL-1β to take part in the innate anti-fungi immunity in the cornea; IL-1β generation induced by fungal infection might not be through the pre-excitation in the classical signal pathway; TLR4/TRIF pathway was not involved in pro-IL-1β generation; while Dectin-1/syk pathway was involved in IL-1β generation in the fungal keratitis; Caspase-l participated in the modification of IL-1β to change from the precursor into the mature body; but NLRP3 inflammasome and ASC inflammasome were not involved in IL-1β generation; Caspase-11 was involved in IL-1β generation through regulating the modified process of Caspase-l to turning from precursor into mature body. TLR4/TRIF pathway and NLRP3 inflammasome and ASC inflammasome are not involved in the pro-IL-1β generation, while Caspase-l, Caspase-11 and Dectin-1/syk pathway are involved in the IL-1β generation.

  2. Caspase-2 mediated apoptotic and necrotic murine macrophage cell death induced by rough Brucella abortus.

    Directory of Open Access Journals (Sweden)

    Fang Chen

    Full Text Available Brucella species are Gram-negative, facultative intracellular bacteria that cause zoonotic brucellosis. Survival and replication inside macrophages is critical for establishment of chronic Brucella infection. Virulent smooth B. abortus strain 2308 inhibits programmed macrophage cell death and replicates inside macrophages. Cattle B. abortus vaccine strain RB51 is an attenuated rough, lipopolysaccharide O antigen-deficient mutant derived from smooth strain 2308. B. abortus rough mutant RA1 contains a single wboA gene mutation in strain 2308. Our studies demonstrated that live RB51 and RA1, but not strain 2308 or heat-killed Brucella, induced both apoptotic and necrotic cell death in murine RAW264.7 macrophages and bone marrow derived macrophages. The same phenomenon was also observed in primary mouse peritoneal macrophages from mice immunized intraperitoneally with vaccine strain RB51 using the same dose as regularly performed in protection studies. Programmed macrophage cell death induced by RB51 and RA1 was inhibited by a caspase-2 inhibitor (Z-VDVAD-FMK. Caspase-2 enzyme activation and cleavage were observed at the early infection stage in macrophages infected with RB51 and RA1 but not strain 2308. The inhibition of macrophage cell death promoted the survival of rough Brucella cells inside macrophages. The critical role of caspase-2 in mediating rough B. abortus induced macrophage cell death was confirmed using caspase-2 specific shRNA. The mitochondrial apoptosis pathway was activated in macrophages infected with rough B. abortus as demonstrated by increase in mitochondrial membrane permeability and the release of cytochrome c to cytoplasm in macrophages infected with rough Brucella. These results demonstrate that rough B. abortus strains RB51 and RA1 induce apoptotic and necrotic murine macrophage cell death that is mediated by caspase-2. The biological relevance of Brucella O antigen and caspase-2-mediated macrophage cell death in Brucella

  3. Caspase-2 mediated apoptotic and necrotic murine macrophage cell death induced by rough Brucella abortus.

    Science.gov (United States)

    Chen, Fang; He, Yongqun

    2009-08-28

    Brucella species are Gram-negative, facultative intracellular bacteria that cause zoonotic brucellosis. Survival and replication inside macrophages is critical for establishment of chronic Brucella infection. Virulent smooth B. abortus strain 2308 inhibits programmed macrophage cell death and replicates inside macrophages. Cattle B. abortus vaccine strain RB51 is an attenuated rough, lipopolysaccharide O antigen-deficient mutant derived from smooth strain 2308. B. abortus rough mutant RA1 contains a single wboA gene mutation in strain 2308. Our studies demonstrated that live RB51 and RA1, but not strain 2308 or heat-killed Brucella, induced both apoptotic and necrotic cell death in murine RAW264.7 macrophages and bone marrow derived macrophages. The same phenomenon was also observed in primary mouse peritoneal macrophages from mice immunized intraperitoneally with vaccine strain RB51 using the same dose as regularly performed in protection studies. Programmed macrophage cell death induced by RB51 and RA1 was inhibited by a caspase-2 inhibitor (Z-VDVAD-FMK). Caspase-2 enzyme activation and cleavage were observed at the early infection stage in macrophages infected with RB51 and RA1 but not strain 2308. The inhibition of macrophage cell death promoted the survival of rough Brucella cells inside macrophages. The critical role of caspase-2 in mediating rough B. abortus induced macrophage cell death was confirmed using caspase-2 specific shRNA. The mitochondrial apoptosis pathway was activated in macrophages infected with rough B. abortus as demonstrated by increase in mitochondrial membrane permeability and the release of cytochrome c to cytoplasm in macrophages infected with rough Brucella. These results demonstrate that rough B. abortus strains RB51 and RA1 induce apoptotic and necrotic murine macrophage cell death that is mediated by caspase-2. The biological relevance of Brucella O antigen and caspase-2-mediated macrophage cell death in Brucella pathogenesis and

  4. Q-VE-OPh, a Negative Control for O-Phenoxy-Conjugated Caspase Inhibitors

    Directory of Open Access Journals (Sweden)

    Benjamin Southerland

    2010-06-01

    Full Text Available The broad-spectrum apoptosis (caspase inhibitor, Q-VD-OPh, has been shown to have no side effects and is effective at a much lower concentration than other FMK-type caspase inhibitors. However, an appropriate negative control to use with this inhibi- tor has not been available. In this study, we developed and analyzed a new compound, based on the Q-VD-OPh backbone, which acts as a cognate negative control. To create the negative control, we substituted a glutamate residue for the aspartate residue to create Q-VE-OPh, thereby retaining the identical charge and molecular properties with only the addition of an extra –CH2 group. The purity and quality were assessed by ion trap mass spectrometry and verified by nuclear magnetic resonance. We determined the effectiveness of Q-VE-OPh, in comparison to Q-VD-OPh, to prevent DNA fragmentation in human Jurkat T leukemia cells that were induced to undergo apoptosis. DNA fragmentation was analyzed by agarose gel electrophoresis for the presence of DNA laddering, the hallmark indicator of apoptosis. Our results indicate that apoptosis was potently inhibited by Q-VD-OPh. In stark contrast, Q-VE-OPh did not inhibit apoptosis at a similar dose but required at least 20 times greater concentration than Q-VD-OPh to have any inhibitory effect. Western blot analysis showed that Q-VE-OPh was similarly less effective at inhibiting the activation of the extrinsic (caspase 8 and intrinsic (caspase 9 initiator caspases. Cell proliferation and viability studies further demonstrate that Q-VE-OPh is non-toxic, even at high concentration. Our data indicate that the specificity, effectiveness, and absence of toxicity of Q-VE-OPh provides the appropriate and superior negative control for in vitro and in vivo studies when analyzing the effects of o-phenoxy caspase inhibitors.

  5. Q-VE-OPh, a Negative Control for O-Phenoxy-Conjugated Caspase Inhibitors

    Directory of Open Access Journals (Sweden)

    Benjamin Southerland

    2010-01-01

    Full Text Available The broad-spectrum apoptosis (caspase inhibitor, Q-VD-OPh, has been shown to have no side effects and is effective at a much lower concentration than other FMK-type caspase inhibitors. However, an appropriate negative control to use with this inhibitor has not been available. In this study, we developed and analyzed a new compound, based on the Q-VD-OPh backbone, which acts as a cognate negative control. To create the negative control, we substituted a glutamate residue for the aspartate residue to create Q-VE-OPh , thereby retaining the identical charge and molecular properties with only the addition of an extra -CH2 group. The purity and quality were assessed by ion trap mass spectrometry and verified by nuclear magnetic resonance. We determined the effectiveness of Q-VE-OPh, in comparison to Q-VD-OPh, to prevent DNA fragmentation in human Jurkat T leukemia cells that were induced to undergo apoptosis. DNA fragmentation was analyzed by agarose gel electrophoresis for the presence of DNA laddering, the hallmark indicator of apoptosis. Our results indicate that apoptosis was potently inhibited by Q-VD-OPh. In stark contrast, Q-VE-OPh did not inhibit apoptosis at a similar dose but required at least 20 times greater concentration than Q-VD-OPh to have any inhibitory effect. Western blot analysis showed that Q-VE-OPh was similarly less effective at inhibiting the activation of the extrinsic (caspase 8 and intrinsic (caspase 9 initiator caspases. Cell proliferation and viability studies further demonstrate that Q-VE-OPh is non-toxic, even at high concentration. Our data indicate that the specificity, effectiveness, and absence of toxicity of Q-VE-OPh provides the appropriate and superior negative control for in vitro and in vivo studies when analyzing the effects of o-phenoxy caspase inhibitors.

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

    Czech Academy of Sciences Publication Activity Database

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

    2015-01-01

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

  7. Caspase-3 cleavage of GGA3 stabilizes BACE: implications for Alzheimer's disease.

    Science.gov (United States)

    Vassar, Robert

    2007-06-07

    BACE initiates the production of beta-amyloid (Abeta), the likely cause of Alzheimer's disease (AD). In this issue of Neuron, Tesco et al. show that during apoptosis caspase-3 cleaves the adaptor protein GGA3, which is required for BACE lysosomal degradation, consequently stabilizing BACE and elevating Abeta generation.

  8. TNFα inhibits skeletal myogenesis through a PW1-dependent pathway by recruitment of caspase pathways

    Science.gov (United States)

    Coletti, Dario; Yang, Ellen; Marazzi, Giovanna; Sassoon, David

    2002-01-01

    Cachexia is associated with poor prognosis in patients with chronic disease. Tumor necrosis factor-alpha (TNFα) plays a pivotal role in mediating cachexia and has been demonstrated to inhibit skeletal muscle differentiation in vitro. It has been proposed that TNFα-mediated activation of NFκB leads to down regulation of MyoD, however the mechanisms underlying TNFα effects on skeletal muscle remain poorly understood. We report here a novel pathway by which TNFα inhibits muscle differentiation through activation of caspases in the absence of apoptosis. TNFα-mediated caspase activation and block of differentiation are dependent upon the expression of PW1, but occur independently of NFκB activation. PW1 has been implicated previously in p53-mediated cell death and can induce bax translocation to the mitochondria. We show that bax-deficient myoblasts do not activate caspases and differentiate in the presence of TNFα, highlighting a role for bax-dependent caspase activation in mediating TNFα effects. Taken together, our data reveal that TNFα inhibits myogenesis by recruiting components of apoptotic pathways through PW1. PMID:11847111

  9. Mitochondrial permeabilisation engages NF-κB dependent anti-tumour activity under caspase deficiency

    Science.gov (United States)

    Giampazolias, Evangelos; Bock, Florian; Cloix, Catherine; Cao, Kai; Roca, Alba; Lopez, Jonathan; Ichim, Gabriel; Proïcs, Emma; Rubio-Patiño, Camila; Fort, Loic; Yatim, Nader; Woodham, Emma; Orozco, Susana; Taraborrelli, Lucia; Peltzer, Nieves; Lecis, Daniele; Machesky, Laura; Walczak, Henning; Albert, Matthew L.; Milling, Simon; Oberst, Andrew; Ricci, Jean-Ehrland; Ryan, Kevin M.; Blyth, Karen; Tait, Stephen W.G.

    2017-01-01

    Apoptosis represents a key anti-cancer therapeutic effector mechanism. During apoptosis, mitochondrial outer membrane permeabilisation (MOMP) typically kills cells even in the absence of caspase activity. Caspase activity can also have a variety of unwanted consequences that include DNA-damage. We therefore investigated whether MOMP-induced caspase-independent cell death (CICD) might be a better way to kill cancer cells. We find that cells undergoing CICD display potent pro-inflammatory effects relative to apoptosis. Underlying this, MOMP was found to stimulate NF-κB activity through the down-regulation of inhibitor of apoptosis (IAP) proteins. Strikingly, engagement of CICD displays potent anti-tumorigenic effects, often promoting complete tumour regression in a manner dependent on intact immunity. Our data demonstrate that by activating NF-κB, MOMP can exert additional signalling functions besides triggering cell death. Moreover, they support a rationale for engaging caspase-independent cell death in cell-killing anti-cancer therapies. PMID:28846096

  10. 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.

  11. 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

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

    Directory of Open Access Journals (Sweden)

    Rodrigo Ramirez-Tagle

    2016-02-01

    Full Text Available 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.

  13. 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: 2.221, year: 2015

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

    Science.gov (United States)

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

    2014-01-01

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

  15. Multiple Pseudomonas species secrete exolysin-like toxins and provoke Caspase-1-dependent macrophage death.

    Science.gov (United States)

    Basso, Pauline; Wallet, Pierre; Elsen, Sylvie; Soleilhac, Emmanuelle; Henry, Thomas; Faudry, Eric; Attrée, Ina

    2017-10-01

    Pathogenic bacteria secrete protein toxins that provoke apoptosis or necrosis of eukaryotic cells. Here, we developed a live-imaging method, based on incorporation of a DNA-intercalating dye into membrane-damaged host cells, to study the kinetics of primary bone marrow-derived macrophages (BMDMs) mortality induced by opportunistic pathogen Pseudomonas aeruginosa expressing either Type III Secretion System (T3SS) toxins or the pore-forming toxin, Exolysin (ExlA). We found that ExlA promotes the activation of Caspase-1 and maturation of interleukin-1β. BMDMs deficient for Caspase-1 and Caspase-11 were resistant to ExlA-induced death. Furthermore, by using KO BMDMs, we determined that the upstream NLRP3/ASC complex leads to the Caspase-1 activation. We also demonstrated that Pseudomonas putida and Pseudomonas protegens and the Drosophila pathogen Pseudomonas entomophila, which naturally express ExlA-like toxins, are cytotoxic toward macrophages and provoke the same type of pro-inflammatory death as does ExlA + P. aeruginosa. These results demonstrate that ExlA-like toxins of two-partner secretion systems from diverse Pseudomonas species activate the NLRP3 inflammasome and provoke inflammatory pyroptotic death of macrophages. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

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

    DEFF Research Database (Denmark)

    Gyrd-Hansen, Mads; Meier, Pascal

    2010-01-01

    . The development of such inhibitors has radically changed our knowledge of the signalling processes that are regulated by IAPs. Recent studies indicate that IAPs not only regulate caspases and apoptosis, but also modulate inflammatory signalling and immunity, mitogenic kinase signalling, proliferation and mitosis...

  17. Hepatocyte caspase-8 is an essential modulator of steatohepatitis in rodents

    NARCIS (Netherlands)

    Hatting, M.; Zhao, G.; Schumacher, F.; Sellge, G.; Masaoudi, Al M.; Gaßler, N.; Boekschoten, M.V.; Müller, M.R.; Liedtke, C.; Cubero, F.J.; Trautwein, C.

    2013-01-01

    In human and murine models of nonalcoholic steatohepatitis (NASH), increased hepatocyte apoptosis is a critical mechanism contributing to inflammation and fibrogenesis. Caspase 8 (Casp8) is essential for death-receptor-mediated apoptosis activity and therefore its modulation might be critical for

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

    International Nuclear Information System (INIS)

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

    2006-01-01

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

  19. 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

    2016-02-22

    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.

  20. Effect of Turkish pollen and propolis extracts on caspase-3 activity in ...

    African Journals Online (AJOL)

    Management, High School of Health, University of Gumushane, Gumushane, 4Karadeniz Advanced Technology Research and. Application Centre ... Conclusion: Turkish pollen and propolis individually increase apoptosis and the activity of caspase-3 in. HL-60 cells. ... essential and aromatic oils, 5 % pollen and 5 %.

  1. 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.

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

    Science.gov (United States)

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

    2012-10-10

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

  3. Association of caspase-1 polymorphisms with Chagas cardiomyopathy among individuals in Santa Cruz, Bolivia.

    Science.gov (United States)

    Fu, Katherine Yih-Jia; Zamudio, Roxana; Henderson-Frost, Jo; Almuedo, Alex; Steinberg, Hannah; Clipman, Steven Joseph; Duran, Gustavo; Marcus, Rachel; Crawford, Thomas; Alyesh, Daniel; Colanzi, Rony; Flores, Jorge; Gilman, Robert Hugh; Bern, Caryn

    2017-01-01

    Trypanosoma cruzi (Tc) infection is usually acquired in childhood in endemic areas, leading to Chagas disease, which progresses to Chagas cardiomyopathy in 20-30% of infected individuals over decades. The pathogenesis of Chagas cardiomyopathy involves the host inflammatory response to T. cruzi, in which upstream caspase-1 activation prompts the cascade of inflammatory chemokines/cytokines, cardiac remodeling, and myocardial dysfunction. The aim of the present study was to examine the association of two caspase-1 single nucleotide polymorphisms (SNPs) with cardiomyopathy. We recruited infected (Tc+, n = 149) and uninfected (Tc-, n = 87) participants in a hospital in Santa Cruz, Bolivia. Cardiac status was classified (I, II, III, IV) based on Chagas cardiomyopathy-associated electrocardiogram findings and ejection fractions on echocardiogram. Genotypes were determined using Taqman probes via reverse transcription-polymerase chain reaction of peripheral blood DNA. Genotype frequencies were analyzed according to three inheritance patterns (dominant, recessive, additive) using logistic regression adjusted for age and sex. The AA allele for the caspase-1 SNP rs501192 was more frequent in Tc+ cardiomyopathy (classes II, III, IV) patients compared to those with a normal cardiac status (class I) [odds ratio (OR) = -2.18, p = 0.117]. This trend approached statistical significant considering only Tc+ patients in class I and II (OR = -2.64, p = 0.064). Caspase-1 polymorphisms may play a role in Chagas cardiomyopathy development and could serve as markers to identify individuals at higher risk for priority treatment.

  4. Caspase-6 Induces 7A6 Antigen Localization to Mitochondria During FAS-induced Apoptosis of Jurkat Cells.

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    Suita, Hiroaki; Shinomiya, Takahisa; Nagahara, Yukitoshi

    2017-04-01

    Mitochondria are central to apoptosis. However, apoptosis progression involving mitochondria is not fully understood. A factor involved in mitochondria-mediated apoptosis is 7A6 antigen. 7A6 localizes to mitochondria from the cytosol during apoptosis, which seems to involve 'effector' caspases. In this study, we investigated the precise role of effector caspases in 7A6 localization to mitochondria during apoptosis. Human T-cell lymphoma Jurkat cells were treated with an antibody against FAS. 7A6 localization was analyzed by confocal laser scanning microscopy and flow cytometry. Caspases activation was determined by western blot analysis. 7A6 localization to mitochondria during anti-FAS-induced apoptosis was significantly reduced by the caspase-6 inhibitor, N-acetyl-Val-Glu-Ile-Asp-aldehyde, but not by the caspase-3 inhibitor, N-acetyl-Asp-Asn-Leu-Asp-aldehyde, nor caspase-7/3 inhibitor, N-acetyl-Asp-Gln-Thr-Asp-aldehyde. Moreover, caspase-6 down-regulation suppressed 7A6 localization to mitochondria. Caspase-6 regulates 7A6 localization to mitochondria during anti-FAS-induced apoptosis of Jurkat cells. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  5. Independent Induction of Caspase-8 and cFLIP Expression during Colorectal Carcinogenesis in Sporadic and HNPCC Adenomas and Carcinomas

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    D. M. Heijink

    2007-01-01

    Full Text Available Background: TNF-Related Apoptosis Inducing Ligand (TRAIL is a promising agent for the induction of apoptosis in neoplastic tissues. Important determinants of TRAIL sensitivity are two intracellular proteins of the TRAIL pathway, caspase-8 and its anti-apoptotic competitor cellular Flice-Like Inhibitory Protein (cFLIP. Methods: The aim of this study was to investigate basic expression of caspase-8 and cFLIP in normal colorectal epithelium (n = 20, colorectal adenomas (n = 66 and colorectal carcinomas (n = 44 using immunohistochemistry performed on both sporadic and Hereditary Non-Polyposis Colorectal Cancer (HNPCC or Lynch syndrome-associated adenomas and carcinomas. Results: Expression of both caspase-8 and cFLIP was similar in cases with sporadic and hereditary origin. Expression of caspase-8 in colorectal adenomas and carcinomas was increased when compared to normal colon tissue (P = 0.02. Nuclear, paranuclear as well as cytoplasmic localizations of caspase-8 were detected. Immunohistochemistry revealed an upregulation of cFLIP in colorectal carcinomas in comparison to normal epithelium and colorectal adenomas (P < 0.001. A large variation in the caspase-8/cFLIP ratio was observed between the individual adenomas and carcinomas. Conclusion: Caspase-8 and cFLIP are upregulated during colorectal carcinogenesis. Upregulation of caspase-8 and/or downregulation of cFLIP may be interesting approaches to maximize TRAIL sensitivity in colorectal neoplasms.

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

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    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.

  7. The broad-spectrum caspase inhibitor Boc-Asp-CMK induces cell death in human leukaemia cells.

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    Frydrych, Ivo; Mlejnek, Petr; Dolezel, Petr; Zoumpourlis, Vassilis; Krumpochova, Petra

    2008-08-01

    Synthetic caspase inhibitors and particularly broad-spectrum caspase inhibitors can prevent cells from death or at least slow down cell death process and abrogate some apoptotic hallmarks [Kitanaka, C., Kuchino, Y., 1999. Caspase-independent programmed cell death with necrotic morphology. Cell Death and Differentiation 6, 508-515]. However, not all synthetic caspase inhibitors diminish cell death. We have found that the broad-spectrum caspase inhibitor Boc-Asp-CMK induced cell death at micromolar concentrations in human leukaemia cells. Interestingly, low concentrations of Boc-Asp-CMK induced cell death with apoptotic hallmarks. Increasing concentrations of Boc-Asp-CMK led to necrotic cell death. The switch between apoptosis and necrosis seemed to depend upon the degree of inhibition of executioner caspases, including caspase-3/7 with Boc-Asp-CMK. Interestingly, caspase-3 processing was not inhibited even for the highest concentration of Boc-Asp-CMK used. We assume, that toxic properties of Boc-Asp-CMK can be attributed to the chloromethylketone residuum in its molecule, as its analogue Boc-Asp-FMK with fluoromethylketone residuum was more than 13 times less toxic. Our results further indicated that toxicity of Boc-Asp-CMK might arise from its interference with mitochondrial metabolism.

  8. Natural activation of caspase-3 is required for the development of operant behavior in postnatal ontogenesis.

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    Kudryashova, I V; Stepanichev, M Yu; Gulyaeva, N V

    2009-01-01

    We have previously demonstrated transient increases in caspase-3 activity in the hippocampus of rat pups from age 17 days. We report here our studies on the effects of inhibition of caspase-3 during this period on the acquisition of a two-way avoidance reaction. Rat pups received intracerebroventricular doses of the caspase-3 inhibitor Z-DEVD-FMK On postnatal day 18. Control animals of the same age received the inactive peptide Z-FA-FMK or isotonic saline solution. Inhibition of caspase-3 during the period of its natural activation in the hippocampus during early ontogenesis was found to impair the development of operant behavior in rats. This was apparent as a reduction in the efficiency of learning during acquisition of active avoidance reactions and decreases in the numbers of intersignal reactions. Administration of the inhibitor had no specific action on the types of conditioned reflex activity less associated with operant learning. Thus, there were no differences between the experimental and control groups in the numbers of emotional reactions to the conditioned stimulus. The number of orientational-investigative conditioned reactions also showed no change after administration of Z-DEVD-FMK. On the background of the reduction in the efficiency of the acquisition of the conditioned active avoidance reflex, the number of incomplete acts, in contrast to other types of conditioned reactions, increased significantly after administration of Z-DEVD-FMK, which is evidence for the persistence of the ability to form associative connections between activation of the conditioned signal and the need to move to the other sector. The difficulty in these animals arose at the decision-taking stage on choosing the appropriate form of behavior. Changes in orientational-investigative behavior were not associated with inhibition of caspase-3 during the critical period of development, as the effects of Z-DEVD-FMK and Z-FA-FMK were similar.

  9. Hypoxia/reoxygenation impairs memory formation via adenosine-dependent activation of caspase 1.

    Science.gov (United States)

    Chiu, Gabriel S; Chatterjee, Diptaman; Darmody, Patrick T; Walsh, John P; Meling, Daryl D; Johnson, Rodney W; Freund, Gregory G

    2012-10-03

    After hypoxia, a critical adverse outcome is the inability to create new memories. How anterograde amnesia develops or resolves remains elusive, but a link to brain-based IL-1 is suggested due to the vital role of IL-1 in both learning and brain injury. We examined memory formation in mice exposed to acute hypoxia. After reoxygenation, memory recall recovered faster than memory formation, impacting novel object recognition and cued fear conditioning but not spatially cued Y-maze performance. The ability of mice to form new memories after hypoxia/reoxygenation was accelerated in IL-1 receptor 1 knockout (IL-1R1 KO) mice, in mice receiving IL-1 receptor antagonist (IL-1RA), and in mice given the caspase 1 inhibitor Ac-YVAD-CMK. Mechanistically, hypoxia/reoxygenation more than doubled caspase 1 activity in the brain, which was localized to the amygdala compared to the hippocampus. This reoxygenation-dependent activation of caspase 1 was prevented by broad-spectrum adenosine receptor (AR) antagonism with caffeine and by targeted A1/A2A AR antagonism with 8-cyclopentyl-1,3-dipropylxanthine plus 3,7-dimethyl-1-propargylxanthine. Additionally, perfusion of adenosine activated caspase 1 in the brain, while caffeine blocked this action by adenosine. Finally, resolution of anterograde amnesia was improved by both caffeine and by targeted A1/A2A AR antagonism. These findings indicate that amygdala-based anterograde amnesia after hypoxia/reoxygenation is sustained by IL-1β generated through adenosine-dependent activation of caspase 1 after reoxygenation.

  10. Cordycepin Induced MA-10 Mouse Leydig Tumor Cell Apoptosis through Caspase-9 Pathway

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    Chun-Yi Jen

    2011-01-01

    Full Text Available In the present study, the apoptotic effect of cordycepin on MA-10 cells, a mouse Leydig tumor cell line, was investigated. Results demonstrated that the number of rounding-up cell increased by cordycepin (10 μM to 5 mM for 24 h, and cells with plasma membrane blebbing could be observed by 100 μM cordycepin. In viability test, MA-10 cell surviving rate significantly decreased as the dosage (10 μM to 5 mM and duration (3–24 h of cordycepin treatment increased (P < 0.05. Cordycepin at 100 μM and 1 mM for 24 h treatment induced significant DNA fragmentation (P < 0.05. In addition, the percentage of G1 and G2/M phase cell significantly declined by cordycepin (100 μM and 1 mM for 24 h treatment, while the percentages of subG1 phase cell increased by 100 μM and/or 1 mM cordycepin in 6, 12 and 24 h treatments (P < 0.05, respectively, which highly suggested that cordycepin induced MA-10 cell apoptosis. In mechanism study with the treatments of caspases, c-Jun NH2 terminal kinase (JNK or reactive oxygen species (ROS inhibitors plus cordycepin for 24 h, only caspases inhibitor suppressed subG1 phase in MA-10 cells. Moreover, western blotting results showed that cordycepin induced caspase-9, -3 and -7 protein expressions, but not caspase-8, in time- and dose-dependent manners. In conclusion, cordycepin induced apoptosis in MA-10 mouse Leydig tumor cells through a caspase-9 and -3 and -7 dependent pathway.

  11. Regulation of axon arborization pattern in the developing chick ciliary ganglion: Possible involvement of caspase 3.

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    Katow, Hidetaka; Kanaya, Teppei; Ogawa, Tomohisa; Egawa, Ryo; Yawo, Hiromu

    2017-04-01

    During a certain critical period in the development of the central and peripheral nervous systems, axonal branches and synapses are massively reorganized to form mature connections. In this process, neurons search their appropriate targets, expanding and/or retracting their axons. Recent work suggested that the caspase superfamily regulates the axon morphology. Here, we tested the hypothesis that caspase 3, which is one of the major executioners in apoptotic cell death, is involved in regulating the axon arborization. The embryonic chicken ciliary ganglion was used as a model system of synapse reorganization. A dominant negative mutant of caspase-3 precursor (C3DN) was made and overexpressed in presynaptic neurons in the midbrain to interfere with the intrinsic caspase-3 activity using an in ovo electroporation method. The axon arborization pattern was 3-dimensionally and quantitatively analyzed in the ciliary ganglion. The overexpression of C3DN significantly reduced the number of branching points, the branch order and the complexity index, whereas it significantly elongated the terminal branches at E6. It also increased the internodal distance significantly at E8. But, these effects were negligible at E10 or later. During E6-8, there appeared to be a dynamic balance in the axon arborization pattern between the "targeting" mode, which is accompanied by elongation of terminal branches and the pruning of collateral branches, and the "pathfinding" mode, which is accompanied by the retraction of terminal branches and the sprouting of new collateral branches. The local and transient activation of caspase 3 could direct the balance towards the pathfinding mode. © 2017 Japanese Society of Developmental Biologists.

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

    Science.gov (United States)

    Qi, Jian Hua; Anand-Apte, Bela

    2015-04-01

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

  13. Paradoxical sleep deprivation changes testicular malondialdehyde and caspase-3 expression in male rats

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    Fitranto Arjadi

    2015-08-01

    Full Text Available BACKGROUND Sleep deprivation is a significant problem among adult men and is considered as a risk factor for several diseases. Paradoxical sleep deprivation (PSD induces Leydig cell apoptosis through elevation of corticosterone, with testicular malondialdehyde (MDA and Leydig cell caspase-3 expression as parameters. The aim of this study was to observe testicular MDA level and caspase-3 expression treated with paradoxical sleep deprivation (PSD, immobilization, and footshock stress and to determine the stress model with a significant effect in white male rats (Rattus norvegicus . METHODS This experimental randomized study of posttest only with control group design was conducted on 24 white male Wistar strain rats, randomly allocated into four treatment groups, i.e. control (K1 without any stress treatment, PSD (KII, immobilization (KIII, and footshock stress (KIV. Treatments were given for 25 days to produce chronic stress. Testicular MDA concentration was examined by the ELISA method while caspase-3 was examined by the TUNEL method. RESULTS Mean testicular MDA concentration with one-way ANOVA test showed differences in means between the groups (p=0.000 and post hoc Tukey-HSD test showed significant results between PSD stress group versus control, immobilization and footshock stress groups. One-way ANOVA test showed a significant difference in caspase-3 expression in at least two treatment groups (p=0.008 and post-hoc Tuckey-LSD test showed significant differences between controls and all stress groups. CONCLUSION Sleep deprivation is a type of stress inducing changes in testicular MDA concentration and caspase-3 expression in male rat testes.

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

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    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

  16. Phytochemical composition, antibacterial and anticancer activities of Trifolium cherleri extract on lung cancer cell line (A549 and analysis of caspase 3 and caspase 9 apoptosis genes expression

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    Amir Mirzaie

    2017-08-01

    Methods: This experimental study was performed in Islamic Azad University, from December 2016 to February 2017. At first, the phytochemical constituents of T. cherleri extract were determined using gas chromatography-mass spectrometry (GC-MS method. Subsequently, the antibacterial activity of the extract was evaluated against some gram positive and negative pathogenic bacteria included Staphylococcus aureus ATCC 25923, Streptococcus pyogenes ATCC 19615, Salmonella enteritidis ATCC 13076 and Listeria monocytogenes ATCC 35152 via minimum inhibitory concentration (MIC method. Moreover, anticancer potential of extract was examined by colorimetric MTT assay toward lung cancer (A549 cell line. Then, the evaluation of caspase 3 and 9 apoptosis gene expression was determined using Real-Time Polymerase Chain Reaction (Real-Time PCR technique. Moreover, the Real-Time PCR was performed using relative quantitative method. Results: The phytochemical analyses of T. cherleri extract showed the 20 major components and the most frequent component was belonged to hexadecanoic acid, ethyl ester (20.7% and 2-Pentadecanone, 6,10,14-trimethyl (19.9%. The extract had maximum antibacterial effects against Staphylococcus aureus and Streptococcus pyogenes. There was a dose dependent increase in the cytotoxicity effect of extract against A549 cancer cell. Moreover, the Real-Time PCR results indicated that the caspase 3 and caspase 9 gene expression was significantly up-regulated 2.57±0.27 (P<0.05, and 3.3±0.46 (P<0.05, respectively. Conclusion: The results of this study showed that the T. cherleri extract had significant anti-bacterial and anti-cancer effects and it appear that the extract has potential uses for pharmaceutical industries. Moreover, it could be considered as a promising source for novel drug compounds, but more studies are needed.

  17. EFEK PERLAKUAN LOGAM BERAT KADMIUM TERHADAP APOPTOSIS MELALUI AKTIVASI CASPASE-3 BULU BABI Deadema setosum: APLIKASI BIOMONITORING PENCEMARAN DI PERAIRAN LAUT (Effect of Cadmium Heavy Metal Treatment Toward Apoptosis Through Caspase-3 Activation of Sea

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    Dominggus Rumahlatu

    2014-05-01

    Full Text Available ABSTRAK Kadmium yang terakumulasi pada hewan dapat menyebabkan karsinogenik, mutagenik, dan teratogenik. Pada penelitian ini, dilakukan secara eksperimen nonfaktorial dalam RAL 6 level dengan 7 ulangan selama 4 minggu, untuk mengkaji efek perlakuan logam berat Cd terhadap apoptosis melalui aktivasi protein caspase-3 bulu babi Deadema setosum. Penelitian dilakukan di laboratorium Balai LIPI Ambon dalam 6 bak aquarium berukuran 100 x 60 x 70 cm3.Tiap bak perlakuan diisi 200 L air laut yang diganti satu kali setiap minggu. Konsentrasi perlakuan adalah 0, 1, 3, 6, 9, dan 12 µg/L Cd terlarut. Pada tiap bak diterapkan satu level perlakuan konsentrasi Cd, dan tiap bak itu dihuni oleh 7 individu D. setosum sebagai ulangan. Usia hewan uji sekitar 8 bulan berbobot 90 g dengan lingkar tubuh 15 cm. Hewan uji diberi pakan lamun. Pengukuran konsentrasi protein caspase-3 dilakukan pada organ hepar dengan metode Caspase Colorimetric Assay Kit, sedangkan pemeriksaan apoptosis dilakukan dengan teknik pengecatan Hematoxilen-Eosin (HE. Data penelitian terkait konsentrasi protein caspase-3 dianalisis dengan One Way Anova dan uji lanjut dihitung dengan Duncan 0,05. Hasil penelitian memperlihatkan bahwa perlakuan Cd sangat signifikan meningkatkan kadar protein caspase-3; semakin tinggi kadar Cd, kadar protein caspase-3 juga makin tinggi. Konsentrasi protein caspase-3 pada konsentrasi perlakuan 12 µg/L Cd adalah yang paling tinggi dibanding kontrol. Gambaran histologi hepar D. setosum yang mengalami apoptosis atas dasar pengecatan HE sangat sesuai dengan tiap konsentrasi protein caspase-3 yang terekam. Hasil ini menunjukkan bahwa protein caspase-3 memiliki potensi sebagai satu alternatif biomonitoring pencemaran Cd pada tingkat seluler D. setosum di perairan laut.    ABSTRACT Cadmium which accumulated in animals could cause carcinogenic, mutagenic and teratogenic. In this research, non factorial experiments conducted in Complete Random Design (CRD 6 level with 7

  18. Apoptosis induced by chlormethine and ionizing radiations in normal and tumoral lymphocytes: role of caspase-3

    International Nuclear Information System (INIS)

    Holl, V.P.

    2000-01-01

    Apoptosis can be induced by various stimuli like ionizing radiations or alkylating agents. Recent works have shown that apoptosis due to ionizing radiations can be initiated by DNA and cell membrane alterations, via radical species generation, implying the in fine activation of effector caspases, and in particular caspase-3. The main goal of this work is to clarify the role of caspase-3 in the radio-induced apoptosis mechanisms and to study the effects of apoptosis inhibition on the behaviour of the damaged cells. The effects of activation and caspase-3 activity inhibition on the progress of spontaneous, radio-induced or chlormethine-induced apoptosis have been evaluated for normal and tumoral lymphocytes. A chemical molecule, the ebselen, which can mime the action of the endogenous glutathione peroxidase, and a tetra-peptide inhibitor, AC-DEVD-CHO, selective of effector caspases, have been selected. The results indicate an inhibition by ebselen of all morphological and biochemical characteristics of chlormethine-induced apoptosis and a restoring of the cells viability. This seleno-organic compound also reduces the drop of the intra-cellular glutathione level and the loss of the trans-membrane potential (M) of the mitochondrion in the MOLT-4 tumoral cells treated with chlormethine. In parallel, the AC-DEVD-CHO effect on apoptosis induction has been tested. This inhibitor stops some chlormethine-induced criteria of apoptosis without affecting the final loss of the mitochondrial M and the cells proliferation. AC-DEVD-CHO has been also incubated just before the irradiation of the culture cells. The inhibition of the specific DEVD caspases prevents the inter-nucleosomal fragmentation of DNA and partially delays the externalization of phosphatidylserine without changing the viability of the irradiated cells. Moreover, the analysis of the AC-DEVD-CHO pre-treated irradiated cells floating on the surface shows a strong mitochondrial lactate dehydrogenase activity, which

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

    Science.gov (United States)

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

    2017-12-26

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

  20. RNA silencing of Mcl-1 enhances ABT-737-mediated apoptosis in melanoma: role for a caspase-8-dependent pathway.

    Science.gov (United States)

    Keuling, Angela M; Felton, Kathleen E A; Parker, Arabesque A M; Akbari, Majid; Andrew, Susan E; Tron, Victor A

    2009-08-17

    Malignant melanoma is resistant to almost all conventional forms of chemotherapy. Recent evidence suggests that anti-apoptotic proteins of the Bcl-2 family are overexpressed in melanoma and may contribute to melanoma's striking resistance to apoptosis. ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-xl and Bcl-w, has demonstrated efficacy in several forms of leukemia, lymphoma as well as solid tumors. However, overexpression of Mcl-1, a frequent observance in melanoma, is known to confer ABT-737 resistance. Here we report that knockdown of Mcl-1 greatly reduces cell viability in combination with ABT-737 in six different melanoma cell lines. We demonstrate that the cytotoxic effect of this combination treatment is due to apoptotic cell death involving not only caspase-9 activation but also activation of caspase-8, caspase-10 and Bid, which are normally associated with the extrinsic pathway of apoptosis. Caspase-8 (and caspase-10) activation is abrogated by inhibition of caspase-9 but not by inhibitors of the death receptor pathways. Furthermore, while caspase-8/-10 activity is required for the full induction of cell death with treatment, the death receptor pathways are not. Finally, we demonstrate that basal levels of caspase-8 and Bid correlate with treatment sensitivity. Our findings suggest that the combination of ABT-737 and Mcl-1 knockdown represents a promising, new treatment strategy for malignant melanoma. We also report a death receptor-independent role for extrinsic pathway proteins in treatment response and suggest that caspase-8 and Bid may represent potential markers of treatment sensitivity.

  1. RNA silencing of Mcl-1 enhances ABT-737-mediated apoptosis in melanoma: role for a caspase-8-dependent pathway.

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    Angela M Keuling

    Full Text Available BACKGROUND: Malignant melanoma is resistant to almost all conventional forms of chemotherapy. Recent evidence suggests that anti-apoptotic proteins of the Bcl-2 family are overexpressed in melanoma and may contribute to melanoma's striking resistance to apoptosis. ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-xl and Bcl-w, has demonstrated efficacy in several forms of leukemia, lymphoma as well as solid tumors. However, overexpression of Mcl-1, a frequent observance in melanoma, is known to confer ABT-737 resistance. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that knockdown of Mcl-1 greatly reduces cell viability in combination with ABT-737 in six different melanoma cell lines. We demonstrate that the cytotoxic effect of this combination treatment is due to apoptotic cell death involving not only caspase-9 activation but also activation of caspase-8, caspase-10 and Bid, which are normally associated with the extrinsic pathway of apoptosis. Caspase-8 (and caspase-10 activation is abrogated by inhibition of caspase-9 but not by inhibitors of the death receptor pathways. Furthermore, while caspase-8/-10 activity is required for the full induction of cell death with treatment, the death receptor pathways are not. Finally, we demonstrate that basal levels of caspase-8 and Bid correlate with treatment sensitivity. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that the combination of ABT-737 and Mcl-1 knockdown represents a promising, new treatment strategy for malignant melanoma. We also report a death receptor-independent role for extrinsic pathway proteins in treatment response and suggest that caspase-8 and Bid may represent potential markers of treatment sensitivity.

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

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

    2017-06-01

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

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

    Science.gov (United States)

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

    2013-08-01

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

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

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    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

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

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    Darin Bloemberg

    2016-06-01

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

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

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    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.

  7. Novel mechanisms for caspase inhibition protecting cardiac function with chronic pressure overload

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    Park, Misun; Vatner, Stephen F.; Yan, Lin; Gao, Shumin; Yoon, Seunghun; Lee, Grace Jung Ah; Xie, Lai-Hua; Kitsis, Richard N.; Vatner, Dorothy E.

    2013-01-01

    Myocyte apoptosis is considered a major mechanism in the pathogenesis of heart failure. Accordingly, manipulations that inhibit apoptosis are assumed to preserve cardiac function by maintaining myocyte numbers. We tested this assumption by examining the effects of caspase inhibition (CI) on cardiac structure and function in C57BL/6 mouse with pressure overload model induced by transverse aortic constriction (TAC). CI preserved left ventricular (LV) function following TAC compared with the veh...

  8. 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 - others:GA ČR(CZ) GAP502/12/1285 Program:GA Institutional support: RVO:67985904 Keywords : ameloblast * apoptosis * caspase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.178, year: 2013

  9. Sulfur Mustard Induces Apoptosis in Lung Epithelial Cells via a Caspase Amplification Loop

    Science.gov (United States)

    2010-01-01

    SM inhalation by normal breathing. Twenty-four hours after SM exposure, bronchoalveolar lavage (BAL) samples were collected to analyze cellular com...grafted onto nude mice . ol et al. (2009) recently showed that peptide inhibitors specific or caspase-8 or -9 could prevent SM-induced microvesication...ntibodies and reprobed with other antibodies to compare different proteins from he same blot as described in Rosenthal et al. (2003). .5. Bronchoalveolar

  10. The loss of functional caspase-12 in Europe is a pre-neolithic event.

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    Montserrat Hervella

    Full Text Available Caspase-12 (CASP12 modulates the susceptibility to sepsis. In humans, the "C" allele at CASP12 rs497116 has been associated with an increased risk of sepsis. Instead, the derived "T" allele encodes for an inactive caspase-12. Interestingly, Eurasians are practically fixed for the inactive variant, whereas in Sub-Saharan Africa the active variant is still common (~24%. This marked structure has been explained as a function of the selective advantage that the inactive caspase-12 confers by increasing resistance to infection. As regards to both when positive selection started acting and as to the speed with which fixation was achieved in Eurasia, estimates depend on the method and assumptions used, and can vary substantially. Using experimental evidence, we propose that, least in Eurasia, the increase in the frequency of the T allele might be related to the selective pressure exerted by the increase in zoonotic diseases transmission caused by the interplay between increased human population densities and a closer contact with animals during the Neolithic. METHODOLOG/PRINCIPAL FINDINGS: We genotyped CASP12 rs497116 in prehistoric individuals from 6 archaeological sites from the North of the Iberian Peninsula that date from Late Upper Paleolithic to Late Neolithic. DNA extraction was done from teeth lacking cavities or breakages using standard anti-contamination procedures, including processing of the samples in a positive pressure, ancient DNA-only chamber, quantitation of DNAs by qPCR, duplication, replication, genotyping of associated animals, or cloning of PCR products. Out of 50, 24 prehistoric individuals could finally be genotyped for rs497116. Only the inactive form of CASP12 was found.We demonstrate that the loss of caspase-12 in Europe predates animal domestication and that consequently CASP12 loss is unlikely to be related to the impact of zoonotic infections transmitted by livestock.

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

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    Johannes Asplund-Samuelsson

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

  12. Crocetin prevents retinal degeneration induced by oxidative and endoplasmic reticulum stresses via inhibition of caspase activity.

    Science.gov (United States)

    Yamauchi, Mika; Tsuruma, Kazuhiro; Imai, Shunsuke; Nakanishi, Tomohiro; Umigai, Naofumi; Shimazawa, Masamitsu; Hara, Hideaki

    2011-01-10

    Crocetin is a carotenoid that is the aglicone of crocin, which are found in saffron stigmas (Crocus sativus L.) and gardenia fruit (Gardenia jasminoides Ellis). In this study, we investigated the effects of crocetin on retinal damage. To examine whether crocetin affects stress pathways, we investigated intracellular oxidation induced by reactive oxygen species, expression of endoplasmic reticulum (ER) stress-related proteins, disruption of the mitochondrial membrane potential (ΔΨ(m)), and caspases activation. In vitro, we employed cultured retinal ganglion cells (RGC-5, a mouse ganglion cell-line transformed using E1A virus). Cell damage was induced by tunicamycin or hydrogen peroxide (H(2)O(2)) exposure. Crocetin at a concentration of 3μM showed the inhibitory effect of 50-60% against tunicamycin- and H(2)O(2)-induced cell death and inhibited increase in caspase-3 and -9 activity. Moreover, crocetin inhibited the enzymatic activity of caspase-9 in a cell-free system. In vivo, retinal damage in mice was induced by exposure to white light at 8000lx for 3h after dark adaptation. Photoreceptor damage was evaluated by measuring the outer nuclear layer thickness at 5days after light exposure and recording the electroretinogram (ERG). Retinal cell damage was also detected with Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining at 48h after light exposure. Crocetin at 100mg/kg, p.o. significantly inhibited photoreceptor degeneration and retinal dysfunction and halved the expression of TUNEL-positive cells. These results indicate that crocetin has protective effects against retinal damage in vitro and in vivo, suggesting that the mechanism may inhibit increase in caspase-3 and -9 activities after retinal damage. Copyright © 2010 Elsevier B.V. All rights reserved.

  13. 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

  14. Association of caspase-1 polymorphisms with Chagas cardiomyopathy among individuals in Santa Cruz, Bolivia

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    Katherine Yih-Jia Fu

    Full Text Available Abstract INTRODUCTION: Trypanosoma cruzi (Tc infection is usually acquired in childhood in endemic areas, leading to Chagas disease, which progresses to Chagas cardiomyopathy in 20-30% of infected individuals over decades. The pathogenesis of Chagas cardiomyopathy involves the host inflammatory response to T. cruzi, in which upstream caspase-1 activation prompts the cascade of inflammatory chemokines/cytokines, cardiac remodeling, and myocardial dysfunction. The aim of the present study was to examine the association of two caspase-1 single nucleotide polymorphisms (SNPs with cardiomyopathy. METHODS: We recruited infected (Tc+, n = 149 and uninfected (Tc−, n = 87 participants in a hospital in Santa Cruz, Bolivia. Cardiac status was classified (I, II, III, IV based on Chagas cardiomyopathy-associated electrocardiogram findings and ejection fractions on echocardiogram. Genotypes were determined using Taqman probes via reverse transcription-polymerase chain reaction of peripheral blood DNA. Genotype frequencies were analyzed according to three inheritance patterns (dominant, recessive, additive using logistic regression adjusted for age and sex. RESULTS: The AA allele for the caspase-1 SNP rs501192 was more frequent in Tc+ cardiomyopathy (classes II, III, IV patients compared to those with a normal cardiac status (class I [odds ratio (OR = −2.18, p = 0.117]. This trend approached statistical significant considering only Tc+ patients in class I and II (OR = −2.64, p = 0.064. CONCLUSIONS: Caspase-1 polymorphisms may play a role in Chagas cardiomyopathy development and could serve as markers to identify individuals at higher risk for priority treatment.

  15. Caspase 2 activation and ER stress drive rapid Jurkat cell apoptosis by clofibrate.

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    Fabio Penna

    Full Text Available Differently from the antiapoptotic action most commonly assigned to peroxisome proliferators (PPs, we demonstrated that some of them, clofibrate (CF in particular, display clearcut apoptogenic properties on rat hepatoma cell lines. We and others could confirm that CF as well as various other PPs can induce apoptosis in a variety of cells, including human liver, breast and lung cancer cell lines. The present study was aimed at investigating the cytotoxic action of CF on a neoplastic line of different origin, the human T leukemia Jurkat cells. We observed that CF rapidly triggers an extensive and morphologically typical apoptotic process on Jurkat cells, though not in primary T cells, which is completely prevented by the polycaspase inhibitor zVADfmk. Gene silencing studies demonstrated that CF-induced apoptosis in Jurkat cells is partially dependent on activation of caspase 2. Looking for a possible trigger of caspase 2 activation, we observed increased levels of phosphorylated eIF2α and JNK in CF-treated cells. Moreover, intracellular Ca(2+ homeostasis was perturbed. Together, these findings are suggestive for the occurrence of ER stress, an event that is known to have the potential to activate caspase 2. The present observations demonstrate that CF induces in Jurkat cells a very fast and extensive apoptosis, that involves induction of ER stress and activation of caspases 2 and 3. Since apoptosis in Jurkat cells occurs at pharmacologically relevant concentrations of CF, the present findings encourage further in depth analysis in order to work out the potential implications of CF cytotoxcity on leukemic cells.

  16. Antiproliferative activity and caspase enhancement properties of Annona muricata leaves extract against colorectal cancer cells

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    Lili Indrawati

    2016-10-01

    Full Text Available Background: The prevalence of colorectal cancer is rising in Asia including Indonesia. Annona muricata tea leaves, that is traditionally used for maintaining health, and lately being used by cancer patients. The objectives of this study is to investigate its effects in human colorectal cancer cell in vitro and ex vivo.Methods: Thirty patients with colorectal cancer (CRC were enrolled in a randomized double-blind placebo-controlled trial. They were equally divided into two groups: those treated with 300 mg A. muricata leaf extract and placebo daily for 8 weeks. Serum from supplemented CRC patients of both groups was compared for caspase 9 and caspase 8 enhancement activity. Antiproliferative effect of water extract of A. muricata leaves and its fractions were evaluated against colorectal cancer cell line (DLD-1 and COLO 205 compared with 5-fluorouracil and placebo, the dose range was 62.5-2,000 µg/mL. Method used was 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay. Data were analyzed by Mann-Whitney U test. The p value was set at 0.05.Results: Ethanol-soluble fraction of A. muricata leaves extract water extract (ESFAM leaves extract had cytotoxicity effects on DLD-1 as well as COLO 205 cell line, as shown by the lower IC50 compared to 5-fluorouracil and placebo, 20.59 μg/mL and 654.9μg/mL, respectively. Serum of subjects supplemented with extract significantly induced caspase 9 (p=0.001 activity of DLD-1 colorectal cancer cell line, but not for caspase 8 activity (p=0.372.Conclusion: The study's results suggest the cytotoxicity potential of  A. muricata  leaves extract  in in vitro and ex vivo studies.

  17. 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

  18. HSV and glycoprotein J inhibit caspase activation and apoptosis induced by granzyme B or Fas.

    Science.gov (United States)

    Jerome, K R; Chen, Z; Lang, R; Torres, M R; Hofmeister, J; Smith, S; Fox, R; Froelich, C J; Corey, L

    2001-10-01

    HSV-1 inhibits apoptosis of infected cells, presumably to ensure that the infected cell survives long enough to allow completion of viral replication. Because cytotoxic lymphocytes kill their targets via the induction of apoptosis, protection from apoptosis could constitute a mechanism of immune evasion for HSV. Several HSV genes are involved in the inhibition of apoptosis, including Us5, which encodes glycoprotein J (gJ). Viruses deleted for Us5 showed defects in inhibition of caspase activation after Fas ligation or UV irradiation. Transfected cells expressing the Us5 gene product gJ were protected from Fas- or UV-induced apoptosis, as measured by morphology, caspase activation, membrane permeability changes, or mitochondrial transmembrane potential. In contrast, caspase 3 activation in mitochondria-free cell lysates by granzyme (gr)B was inhibited equivalently by Us5 deletion and rescue viruses, suggesting that gJ is not required for HSV to inhibition this process. However, mitochondria-free lysates from transfected cells expressing Us5/gJ were protected from grB-induced caspase activation, suggesting that Us5/gJ is sufficient to inhibit this process. Transfected cells expressing Us5/gJ were also protected from death induced by incubation with purified grB and perforin. These findings suggest that HSV has a comprehensive set of immune evasion functions that antagonize both Fas ligand- and grB-mediated pathways of CTL-induced apoptosis. The understanding of HSV effects on killing by CTL effector mechanisms may shed light on the incomplete control of HSV infections by the immune system and may allow more rational approaches to the development of immune modulatory treatments for HSV infection.

  19. TNFα-induced macrophage death via caspase-dependent and independent pathways

    Science.gov (United States)

    Tran, Tri M.; Temkin, Vladislav; Shi, Bo; Pagliari, Lisa; Daniel, Soizic; Ferran, Christiane; Pope, Richard M.

    2009-01-01

    Macrophages are the principal source of TNFα, yet they are highly resistant to TNFα-mediated cell death. Previously, employing in vitro differentiated human macrophages, we showed that following the inhibition of NF-κB, TNFα-induced caspase-8 activation contributes to DNA fragmentation but is not necessary for the loss of the inner mitochondrial transmembrane potential (ΔΨm) or cell death. We here extend these observations to demonstrate that, when NF-κB is inhibited in macrophages, TNFα alters lysosomal membrane permeability (LMP). This results in the release of cathepsin B with subsequent loss of ΔΨm and caspase-8 independent cell death. Interestingly, the cytoprotective, NF-κB-dependent protein A20 was rapidly induced in macrophages treated with TNFα. Ectopic expression of A20 in macrophages preserves LMP following treatment with TNFα, and as a result, mitochondrial integrity is safeguarded and macrophages are protected from cell death. These observations demonstrate that TNFα triggers both caspase 8-dependent and -independent cell death pathways in macrophages and identify a novel mechanism by which A20 protects these cells against both pathways. PMID:19152111

  20. Sequential Cdk1 and Plk1 phosphorylation of caspase-8 triggers apoptotic cell death during mitosis.

    Science.gov (United States)

    Matthess, Yves; Raab, Monika; Knecht, Rainald; Becker, Sven; Strebhardt, Klaus

    2014-05-01

    Caspase-8 is crucial for cell death induction, especially via the death receptor pathway. The dysregulated expression or function of caspase-8 can promote tumor formation, progression and treatment resistance in different human cancers. Here, we show procaspase-8 is regulated during the cell cycle through the concerted inhibitory action of Cdk1/cyclin B1 and polo-like kinase 1 (Plk1). By phosphorylating S387 in procaspase-8 Cdk1/cyclin B1 generates a phospho-epitope for the binding of the PBD of Plk1. Subsequently, S305 in procaspase-8 is phosphorylated by Plk1 during mitosis. Using an RNAi-based strategy we could demonstrate that the extrinsic cell death is increased upon Fas-stimulation when endogenous caspase-8 is replaced by a mutant (S305A) mimicking the non-phosphorylated form. Together, our data show that sequential phosphorylation by Cdk1/cyclin B1 and Plk1 decreases the sensitivity of cells toward stimuli of the extrinsic pathway during mitosis. Thus, the clinical Plk1 inhibitor BI 2536 decreases the threshold of different cancer cell types toward Fas-induced cell death. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  1. Expression of caspase-3, p53 and Bcl-2 in generalized aggressive periodontitis

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    Özdemir B Handan

    2006-06-01

    Full Text Available Abstract Background Apoptosis, or programmed cell death is a form of physiological cell death. It is increased or decreased in the presence of infection, inflammation or tissue remodelling. Previous studies suggest that apoptosis is involved in the pathogenesis of inflammatory periodontal disease. The aim of the present study was to investigate the clinical features and known indicators of apoptosis (p53, Bcl-2, Caspase-3 in patients with generalized aggressive periodontitis (GAP Methods Eight patients with GAP, who had sites with probing depths (PD > 5 mm, and 10 periodontally-healthy persons were included in the study. Clinical examinations and PD were performed, and the plaque index and gingival index were recorded. Gingival tissues biopsies were obtained from active site of each patient and from healthy individuals. The expression of caspase-3, Bcl-2, and p53 was evaluated by immunohistochemistry Results There were no significant differences between GAP and control group with respect to levels of caspase-3 and p53 expression (P > 0.05. Contrary, the frequency of grade 3 expression of Bcl-2 was higher in GAP group than the control group. Conclusion The higher frequency of Bcl-2 expression in GAP group indicates and delayed apoptosis can lead to increasing resident inflammatory cells in periodontal tissues and resulting in progressive periodontal destruction.

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

    Science.gov (United States)

    Setkova, J; Matalova, E; Sharpe, P T; Misek, I; Tucker, A S

    2007-01-01

    During molar development, apoptosis occurs in a well-characterised pattern suggesting several roles for cell death in odontogenesis. However, molecular mechanisms of dental apoptosis are only poorly understood. In this study, Apaf-1 and caspase-9 knockouts were used to uncover the engagement of these members of the apoptotic machinery during early tooth development, concentrating primarily on their function in the apoptotic elimination of primary enamel knot cells. Molar tooth germ morphology, proliferation and apoptosis were investigated on frontal histological sections of murine heads at embryonic days (ED) 15.5, the stage when the primary enamel knot is eliminated apoptotically. In molar tooth germs of both knockouts, no apoptosis was observed according to morphological (haematoxylin-eosin) as well as biochemical criteria (TUNEL). Morphology of the mutant tooth germs, however, was not changed. Additionally, knockout mice showed no changes in proliferation compared to wild type mice. According to our findings on knockout embryos, Apaf-1 and caspase-9 are involved in apoptosis during tooth development; however, they seem dispensable and not necessary for proper tooth shaping. Compensatory or other mechanisms of cell death may act to eliminate the primary enamel knot cells in the absence of Apaf-1 and caspase-9.

  3. Endogenous α-crystallin inhibits expression of caspase-3 induced by hypoxia in retinal neurons.

    Science.gov (United States)

    Ying, Xi; Peng, Yanli; Zhang, Jiaping; Wang, Xingli; Wu, Nan; Zeng, Yuxiao; Wang, Yi

    2014-08-28

    To investigate the expression of endogenous, hypoxic stress-induced α-crystallin and caspase-3 in rat retinal neurons in vitro. Retinal neurons were cultured from Long-Evans rats. The expression of endogenous α-crystallin was analyzed by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). Furthermore, hypoxic exposure was performed in cultured cells, and the expression of endogenous α-crystallin and caspase-3 was assayed by Western blotting. Positive α-crystallin staining was observed in cultured retinal neurons, and expression of endogenous α-crystallin mRNA peaked 3-5d after inoculation (Pendogenous, hypoxic stress-induced α-crystallin expression increased gradually, peaking 6h after hypoxia. The expression was more abundant compared to the control (Pendogenous α-crystallin in retinal neurons, especially over-expression induced by hypoxic stress, results in the down regulation of caspase-3. The data suggest that endogenous α-crystallin may act as an endogenous neuroprotective factor in retinal neurons. Copyright © 2014 Elsevier Inc. All rights reserved.

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

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    Tsukasa Nakanishi

    2016-05-01

    Full Text Available 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.

  5. Nickel induces interleukin-1β secretion via the NLRP3-ASC-caspase-1 pathway.

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    Li, Xiujin; Zhong, Fei

    2014-04-01

    Exposure to nickel (Ni(2+)) can trigger allergic reactions in susceptible individuals, which is widely accepted as the major cause of allergic contact hypersensitivity (CHS) worldwide. Although Ni(2+)-induced proinflammatory responses clearly play a pivotal role in CHS, the underlying molecular mechanism has not been fully defined. Here we report that Ni(2+) activates the NLRP3-ASC-caspase-1 immune signaling pathway in antigen-presenting cells, leading to the proteolytic processing and secretion of a proinflammatory cytokine, interleukin-1β (IL-1β). The activation of this signaling axis is independent of phagolysosome-cathepsin B pathway. Instead, Ni(2+) induces mitochondrial reactive oxygen species accumulation and cation fluxes, both of which are required for activating the NLRP3-ASC-caspase-1 pathway. Together, these results identified a novel innate immune signaling pathway (NLRP3-ASC-caspase-1-IL-1β) activated by Ni(2+) and provided a mechanistic basis for optimizing the therapeutic intervention against Ni(2+)-induced allergy in patients.

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

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

    2010-02-01

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

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

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    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.

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

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

    2017-01-01

    Rabies virus is a highly neurovirulent RNA virus, which causes about 59000 deaths in humans each year. Previously, we described macrophage cytotoxicity upon infection with rabies virus. Here we examined the type of cell death and the role of specific caspases in cell death and disease development upon infection with two laboratory strains of rabies virus: Challenge Virus Standard strain-11 (CVS-11) is highly neurotropic and lethal for mice, while the attenuated Evelyn–Rotnycki–Abelseth (ERA) strain has a broader cell tropism, is non-lethal and has been used as an oral vaccine for animals. Infection of Mf4/4 macrophages with both strains led to caspase-1 activation and IL-1β and IL-18 production, as well as activation of caspases-3, -7, -8, and -9. Moreover, absence of caspase-3, but not of caspase-1 and -11 or -7, partially inhibited virus-induced cell death of bone marrow-derived macrophages. Intranasal inoculation with CVS-11 of mice deficient for either caspase-1 and -11 or -7 or both IL-1β and IL-18 led to general brain infection and lethal disease similar to wild-type mice. Deficiency of caspase-3, on the other hand, significantly delayed the onset of disease, but did not prevent final lethal outcome. Interestingly, deficiency of caspase-1/11, the key executioner of pyroptosis, aggravated disease severity caused by ERA virus, whereas wild-type mice or mice deficient for either caspase-3, -7, or both IL-1β and IL-18 presented the typical mild symptoms associated with ERA virus. In conclusion, rabies virus infection of macrophages induces caspase-1- and caspase-3-dependent cell death. In vivo caspase-1/11 and caspase-3 differently affect disease development in response to infection with the attenuated ERA strain or the virulent CVS-11 strain, respectively. Inflammatory caspases seem to control attenuated rabies virus infection, while caspase-3 aggravates virulent rabies virus infection. PMID:28280602

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

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    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.

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

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    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.

  11. Ulinastatin Alone Does Not Reduce Caspase 3-mediated Apoptosis in Protease-positive Aeromonas hydrophilia-induced Sepsis

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    Bai-Horng Su

    2007-01-01

    Conclusion: PPAH-induced sepsis has a high mortality that is related to lymphocyte apoptosis. Ulinastatin alone does not significantly reduce caspase 3-mediated lymphocyte apoptosis. [J Formos Med Assoc 2007; 106(2:97-104

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

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    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.

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

    Science.gov (United States)

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

    2013-02-01

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

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

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    Toshihiko Suzuki

    2007-08-01

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

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

    Science.gov (United States)

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

    1998-12-18

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

  16. The Value of Caspase-3 after the Application of Annona muricata Leaf Extract in COLO-205 Colorectal Cancer Cell Line

    Science.gov (United States)

    Syam, Ari Fahrial; Meilany, Sofy; Laksono, Bayu; Prabu, Oryza Gryagus; Bekti, Heri Setiyo; Indrawati, Lili; Makmun, Dadang

    2017-01-01

    Annona muricata, commonly known as soursop, contains annonacin, acetogenin, and polyphenol which are known to have chemopreventive effects on cancer. In this study, we tend to evaluate the apoptosis-inducing effect of soursop (Annona muricata) leaf extract on the colorectal cancer cell line COLO-205 through the activities of caspase-3 which is a marker of cell apoptosis. Cell cultures were incubated with soursop leaf with a concentration of 10 μg/ml and then compared with those of the incubated positive control leucovorin 10 μg/ml and placebo as a negative control. The apoptotic activity of caspase-3 was measured with ELISA. After the administration of each treatment in the colorectal cancer cell line COLO-205, the expression of caspase-3 activity was 1422 ng/ml after incubation with the extract of Annona muricata leaves, 1373 ng/ml after the administration of leucovorin, and 1297 ng/ml in the one with placebo. Annona muricata leaf extract elevated caspase-3 by 1.09 times compared to that of the pure cell line. Annona muricata leaf extract had a higher value of caspase-3 activity than leucovorin and placebo in the COLO-205 colorectal cancer cell line. These results may suggest that Annona muricata leaf extract had anticancer properties by enhancing caspase-3 activity which is a proapoptotic marker. PMID:28487731

  17. Activation of the NLRP3/caspase-1 inflammasome in human dental pulp tissue and human dental pulp fibroblasts.

    Science.gov (United States)

    Jiang, Wenkai; Lv, Haipeng; Wang, Haijing; Wang, Diya; Sun, Shukai; Jia, Qian; Wang, Peina; Song, Bing; Ni, Longxing

    2015-08-01

    The NLRP3/caspase-1 inflammasome pathway plays an important role in cellular immune defence against bacterial infection; however, its function in human dental pulp tissue and human dental pulp fibroblasts remains poorly understood. We demonstrate that NLRP3 protein expression occurs to a greater extent in pulp tissue with irreversible pulpitis than in normal pulp tissue and in tissue with reversible pulpitis. Caspase-1 is present in its active (cleaved) form only in pulp tissue with irreversible pulpitis. NLRP3 and caspase-1 are expressed in the odontoblast layers in normal human dental pulp tissue, whereas in inflamed pulp tissue, the odontoblast layers are disrupted and dental pulp cells are positive for NLRP3 and caspase-1. Additionally, we investigate the role of the NLRP3/caspase-1 inflammasome pathway in human dental pulp fibroblasts and show that ATP activates the P2X7 receptor on the cell membrane triggering K(+) efflux and inducing the gradual recruitment of the membrane pore pannexin-1. Extracellular lipopolysaccharide is able to penetrate the cytosol and activate NLRP3. Furthermore, the low intracellular K(+) concentration in the cytosol triggers reactive oxygen species generation, which also induces the NLRP3 inflammasome. Thus, the NLRP3/caspase-1 pathway has a biological role in the innate immune response mounted by human dental pulp fibroblasts.

  18. Caspase pathway of elaidic acid (9t-C18:1)-induced apoptosis in human umbilical vein endothelial cells.

    Science.gov (United States)

    Qiu, Bin; Hu, Jiang-Ning; Liu, Rong; Fan, Ya-Wei; Li, Jing; Li, Yu; Deng, Ze-Yuan

    2012-03-01

    Although TFAs (trans fatty acids) do have effects on many endothelial functions, systemic inflammation and immune disorders, only limited experimental evidence is available that TFAs participate in the pathogenesis of endothelial cell apoptosis. HUVEC (human umbilical vein endothelial cells) were grown in medium with elaidic acid (9t-C18:1) at 50, 100, 200 and 400 μmol/l for 24 h. Apoptosis was measured by flow cytometry, and caspase 3, 8 and 9 activities by colorimetric assay and their mRNA expression by qRT-PCR (quantitative real-time PCR). Results showed that 9t-C18:1 induced apoptosis of HUVEC in a dose-dependent manner. The activities and mRNA expression of caspases 8, 9 and 3 were significantly increased compared with that of the control. Z-IETD-FMK and Z-LEHD-FMK inhibited the activation of caspase 3 and apoptosis induced by 9t-C18:1. Also Z-IETD-FMK inhibited the activation of caspase 9. mRNA expressions of Bid and Smac (second mitochondria-derived activator of caspase)/DIABLO [direct IAP (inhibitor of apoptosis)-binding protein with low pI] were also significantly elevated. We conclude that 9t-C18:1 induces apoptosis of HUVEC through activating caspases 8, 9 and 3. The death receptor pathway and the mitochondrial pathway both participated in the apoptosis course induced by 9t-C18:1.

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

    Science.gov (United States)

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

    2005-01-01

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

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

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    James Elliot Scott

    2016-03-01

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

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

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    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.

  2. The Value of Caspase-3 after the Application of Annona muricata Leaf Extract in COLO-205 Colorectal Cancer Cell Line.

    Science.gov (United States)

    Abdullah, Murdani; Syam, Ari Fahrial; Meilany, Sofy; Laksono, Bayu; Prabu, Oryza Gryagus; Bekti, Heri Setiyo; Indrawati, Lili; Makmun, Dadang

    2017-01-01

    Annona muricata , commonly known as soursop, contains annonacin, acetogenin, and polyphenol which are known to have chemopreventive effects on cancer. In this study, we tend to evaluate the apoptosis-inducing effect of soursop ( Annona muricata ) leaf extract on the colorectal cancer cell line COLO-205 through the activities of caspase-3 which is a marker of cell apoptosis. Cell cultures were incubated with soursop leaf with a concentration of 10  μ g/ml and then compared with those of the incubated positive control leucovorin 10  μ g/ml and placebo as a negative control. The apoptotic activity of caspase-3 was measured with ELISA. After the administration of each treatment in the colorectal cancer cell line COLO-205, the expression of caspase-3 activity was 1422 ng/ml after incubation with the extract of Annona muricata leaves, 1373 ng/ml after the administration of leucovorin, and 1297 ng/ml in the one with placebo. Annona muricata leaf extract elevated caspase-3 by 1.09 times compared to that of the pure cell line. Annona muricata leaf extract had a higher value of caspase-3 activity than leucovorin and placebo in the COLO-205 colorectal cancer cell line. These results may suggest that Annona muricata leaf extract had anticancer properties by enhancing caspase-3 activity which is a proapoptotic marker.

  3. The Value of Caspase-3 after the Application of Annona muricata Leaf Extract in COLO-205 Colorectal Cancer Cell Line

    Directory of Open Access Journals (Sweden)

    Murdani Abdullah

    2017-01-01

    Full Text Available Annona muricata, commonly known as soursop, contains annonacin, acetogenin, and polyphenol which are known to have chemopreventive effects on cancer. In this study, we tend to evaluate the apoptosis-inducing effect of soursop (Annona muricata leaf extract on the colorectal cancer cell line COLO-205 through the activities of caspase-3 which is a marker of cell apoptosis. Cell cultures were incubated with soursop leaf with a concentration of 10 μg/ml and then compared with those of the incubated positive control leucovorin 10 μg/ml and placebo as a negative control. The apoptotic activity of caspase-3 was measured with ELISA. After the administration of each treatment in the colorectal cancer cell line COLO-205, the expression of caspase-3 activity was 1422 ng/ml after incubation with the extract of Annona muricata leaves, 1373 ng/ml after the administration of leucovorin, and 1297 ng/ml in the one with placebo. Annona muricata leaf extract elevated caspase-3 by 1.09 times compared to that of the pure cell line. Annona muricata leaf extract had a higher value of caspase-3 activity than leucovorin and placebo in the COLO-205 colorectal cancer cell line. These results may suggest that Annona muricata leaf extract had anticancer properties by enhancing caspase-3 activity which is a proapoptotic marker.

  4. Smac mimetic triggers necroptosis in pancreatic carcinoma cells when caspase activation is blocked.

    Science.gov (United States)

    Hannes, Sabine; Abhari, Behnaz Ahangarian; Fulda, Simone

    2016-09-28

    Evasion of apoptosis represents a key mechanism of treatment resistance of pancreatic cancer (PC) and contributes to the poor prognosis of this cancer type. Here, we report that induction of necroptosis is an alternative strategy to trigger programmed cell death in apoptosis-resistant PC cells. We show that the second mitochondrial activator of caspases (Smac) mimetic BV6 that antagonizes inhibitor of apoptosis (IAP) proteins induces necroptosis in PC cells in which apoptosis is blocked by the caspase inhibitor zVAD.fmk. Intriguingly, BV6 switches autocrine/paracrine production of tumor necrosis factor (TNF)α by PC cells into a death signal and also acts in concert with exogenously supplied TNFα to trigger necroptosis, when caspase activation is simultaneously blocked. BV6 stimulates TNFα production and formation of the receptor-interacting protein (RIP)1/RIP3-containing necrosome complex in PC cells. Knockdown of TNF receptor 1 (TNFR1) protects PC cells from BV6- or BV6/TNFα-mediated cell death, demonstrating that TNFα autocrine/paracrine signaling by PC cells contributes to BV6-induced necroptosis. Importantly, genetic silencing of receptor interacting protein kinase 3 (RIPK3) or mixed lineage kinase domain-like protein (MLKL) significantly rescues PC cells from BV6- or BV6/TNFα-induced cell death. Similarly, pharmacological inhibition of RIP1, RIP3 or MLKL significantly reduces BV6- or BV6/TNFα-stimulated cell death. By demonstrating that Smac mimetics can bypass resistance to apoptosis by triggering necroptosis as an alternative form of programmed cell death, our findings have important implications for the design of new treatment concepts for PC. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2012-02-01

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

  6. 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.

  7. The Role of Protamine 2 Gene Expression and Caspase 9 Activity in Male Infertility.

    Science.gov (United States)

    Zalata, Adel A; Mokhtar, Naglaa; Atwa, Amany; Khaled, Mohamed; Shaker, Olfat G

    2016-03-01

    Approximately 15% of couples are affected by infertility with the man responsible in almost half of the cases. PRMs (protamines) confer a higher order of DNA packaging in sperm than that in somatic cells. Because of the critical roles of PRMs in spermatid differentiation, aberrations in PRM expression or changes in protein structure could be causes of certain types of idiopathic human male infertility. The aim of this study was to give insight into the role of PRM2 gene expression and caspase 9 activity in the pathogenesis of male infertility. The current study included 70 men with idiopathic infertility and 64 fertile men who attended the andrology outpatient clinic at Mansoura University Hospital. Semen sample analyses were done according to WHO recommendations. The acrosome reaction of spermatozoa recovered from each sample was assessed. Samples were separated using discontinuous gradient separation. From each semen sample mature sperm were separated from immature sperm. The resulting samples were divided into 2 parts, including one to determine caspase 9 activity and the other for RNA extraction and reverse transcriptase-polymerase chain reaction of PRM2 gene expression. The polymerase chain reaction product was electrophoresed on 2% agarose gel. PRM2 gene expression was significantly decreased in immature sperm extracted from the fertile and infertile groups. Caspase 9 activity was significantly increased in immature sperm extracted from both groups. Low levels of PRM2 may be associated with morphological abnormalities, initiation of the apoptotic pathway and decreasing sperm motility. PRM2 may be an important marker to better understand the key regulatory pathway of spermatogenesis and it may act as a crucial part of fertilization. Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  8. Monomerization of cytosolic mature smac attenuates interaction with IAPs and potentiation of caspase activation.

    OpenAIRE

    Stephen P Burke; Jeffrey B Smith

    2010-01-01

    The four residues at the amino-terminus of mature Smac/DIABLO are an IAP binding motif (IBM). Upon exit from mitochondria, mature Smac interacts with inhibitor of apoptosis proteins (IAPs), abrogating caspase inhibition. We used the ubiquitin fusion model to express mature Smac in the cytosol. Transiently expressed mature Smac56-239 (called Smac56) and Smac60-239 (called Smac60), which lacks the IBM, interacted with X-linked inhibitor of apoptosis protein (XIAP). However, stable expression pr...

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

    DEFF Research Database (Denmark)

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

    2002-01-01

    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...... 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...

  10. 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.

  11. 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.

  12. 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)

    Cho, Sung-Hee; Chung, Kyung-Sook; Choi, Jung-Hye; Kim, Dong-Hyun; Lee, Kyung-Tae

    2009-01-01

    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 IC 50 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

  13. Antiapoptotic effects of caspase inhibitors on H2O2-treated lung cancer cells concerning oxidative stress and GSH.

    Science.gov (United States)

    Park, Woo Hyun

    2018-04-01

    Exogenous hydrogen peroxide (H 2 O 2 ) induces oxidative stress and apoptosis in cancer cells. This study evaluated the antiapoptotic effects of pan-caspase and caspase-3, -8, or -9 inhibitors on H 2 O 2 -treated Calu-6 and A549 lung cancer cells in relation to reactive oxygen species (ROS) and glutathione (GSH). Treatment with 50-500 μM H 2 O 2 inhibited the growth of Calu-6 and A549 cells at 24 h and induced apoptosis in these cells. All the tested caspase inhibitors significantly prevented cell death in H 2 O 2 -treated lung cancer cells. H 2 O 2 increased intracellular ROS levels, including that of O 2 ·- , at 1 and 24 h. It also increased the activity of catalase but decreased the activity of SOD. In addition, H 2 O 2 triggered GSH deletion in Calu-6 and A549 cells at 24 h. It reduced GSH levels in Calu-6 cells at 1 h but increased them at 24 h. Caspase inhibitors decreased O 2 ·- levels in H 2 O 2 -treated Calu-6 cells at 1 h and these inhibitors decreased ROS levels, including that of O 2 ·- , in H 2 O 2 -treated A549 cells at 24 h. Caspase inhibitors partially attenuated GSH depletion in H 2 O 2 -treated A549 cells and increased GSH levels in these cells at 24 h. However, the inhibitors did not affect GSH deletion and levels in Calu-6 cells at 24 h. In conclusion, H 2 O 2 induced caspase-dependent apoptosis in Calu-6 and A549 cells, which was accompanied by increases in ROS and GSH depletion. The antiapoptotic effects of caspase inhibitors were somewhat related to the suppression of H 2 O 2 -induced oxidative stress and GSH depletion.

  14. The Roles of ROS and Caspases in TRAIL-Induced Apoptosis and Necroptosis in Human Pancreatic Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Min Zhang

    Full Text Available Death signaling provided by tumor necrosis factor (TNF-related apoptosis-inducing ligand (TRAIL can induce death in cancer cells with little cytotoxicity to normal cells; this cell death has been thought to involve caspase-dependent apoptosis. Reactive oxygen species (ROS are also mediators that induce cell death, but their roles in TRAIL-induced apoptosis have not been elucidated fully. In the current study, we investigated ROS and caspases in human pancreatic cancer cells undergoing two different types of TRAIL-induced cell death, apoptosis and necroptosis. TRAIL treatment increased ROS in two TRAIL-sensitive pancreatic cancer cell lines, MiaPaCa-2 and BxPC-3, but ROS were involved in TRAIL-induced apoptosis only in MiaPaCa-2 cells. Unexpectedly, inhibition of ROS by either N-acetyl-L-cysteine (NAC, a peroxide inhibitor, or Tempol, a superoxide inhibitor, increased the annexin V-/propidium iodide (PI+ early necrotic population in TRAIL-treated cells. Additionally, both necrostatin-1, an inhibitor of receptor-interacting protein kinase 1 (RIP1, and siRNA-mediated knockdown of RIP3 decreased the annexin V-/PI+ early necrotic population after TRAIL treatment. Furthermore, an increase in early apoptosis was induced in TRAIL-treated cancer cells under inhibition of either caspase-2 or -9. Caspase-2 worked upstream of caspase-9, and no crosstalk was observed between ROS and caspase-2/-9 in TRAIL-treated cells. Together, these results indicate that ROS contribute to TRAIL-induced apoptosis in MiaPaCa-2 cells, and that ROS play an inhibitory role in TRAIL-induced necroptosis of MiaPaCa-2 and BxPC-3 cells, with caspase-2 and -9 playing regulatory roles in this process.

  15. 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.

  16. Early increase in DcR2 expression and late activation of caspases in the platelet storage lesion.

    Science.gov (United States)

    Plenchette, S; Moutet, M; Benguella, M; N'Gondara, J P; Guigner, F; Coffe, C; Corcos, L; Bettaieb, A; Solary, E

    2001-10-01

    Platelet transfusion is widely used to prevent bleeding in patients with severe thrombocytopenia. The maximal storage duration of platelet concentrates is usually 5 days, due to the platelet storage lesion that impairs their functions when stored for longer times. Some of the morphological and biochemical changes that characterize this storage lesion are reminiscent of cell death by apoptosis. The present study analyzed whether proteins involved in nucleated cell apoptosis could play a role in the platelet storage lesion. Storage of leukocyte-depleted platelets obtained by apheresis is associated with a late and limited activation of caspases, mainly caspase-3. This event correlates with an increased expression of the pro-apoptotic BH3-only protein Bim in the particulate fraction and a slight and late release of the pro-apoptotic mitochondrial protein Diablo/Smac in the cytosol. Platelets do not express the death receptors Fas, DR4 and DR5 on their plasma membrane, while the expression of the decoy receptor DcR2 increases progressively during platelet storage. Addition of low concentrations of the cryoprotector dimethylsulfoxide accelerates platelet caspase activation during storage, an effect that is partially prevented by the caspase inhibitor z-VAD-fmk. Altogether, DcR2 expression on the plasma membrane is an early event while caspase activation is a late event during platelet storage. These observations suggest that caspases are unlikely to account for the platelet storage lesion. As a consequence, addition of caspase inhibitors may not improve the quality of platelet concentrates stored in standard conditions.

  17. 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.

  18. Changes of caspase activities involved in apoptosis of a macrophage-like cell line J774.1/JA-4 treated with lipopolysaccharide (LPS) and cycloheximide.

    Science.gov (United States)

    Karahashi, H; Amano, F

    2000-02-01

    The addition of lipopolysaccharide (LPS) together with cycloheximide (CHX) induced apoptosis in a subline of a J774.1 macrophage-like cell line, JA-4, as judged by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL)-staining and poly(adenosine 5'-diphosphate (ADP)-ribose) polymerase (PARP)-cleavage. Caspase activities were examined in these macrophages in vitro using fluorogenic substrates such as acetyl-DEVD-aminomethyl coumarine (Ac-DEVD-AMC, caspase-3-like), acetyl-YVAD-aminomethyl coumarine (Ac-YVAD-AMC, caspase-1-like), acetyl-VEID-aminomethyl coumarine (Ac-VEID-AMC, caspase-6-like), and carbobenzoxy-IETD-aminofluoro coumarine (Z-IETD-AFC; caspase-8-like). Kinetic studies revealed these caspase activities with different Km and Vmax values in extracts of apoptotic macrophages. In the course of apoptosis, caspase-3-like activity increased first at 75 min, simultaneously with the appearance of TUNEL staining and prior to PARP cleavage, and then caspase-6 and 8-like activities increased at 90 and 105 min, respectively. However, caspase-1-like activity did not change throughout the experiment. Furthermore, removal of LPS and CHX by extensive washing of the cells for 60 min completely abolished the apoptosis and the subsequent release of lactate dehydrogenase (LDH) during additional incubation until 4 h after LPS addition. However, washing of the cells after 75 min or later resulted in the progress of apoptosis and LDH release, which was coordinated with the elevation of caspase-3-like activity at 60 min and that of caspase-6 or 8-like activity at 90 min, but not with that of caspase-1-like activity. These results suggest that caspase-3-like activity represents the most apical caspase among these caspases in terms of the intiation of apoptosis in macrophages treated with LPS and CHX. In the present study, we also provide evidence on the relatively low specificities of a series of caspase inhibitors other

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

    Directory of Open Access Journals (Sweden)

    Carole Minker

    2015-01-01

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

  20. Monomerization of cytosolic mature smac attenuates interaction with IAPs and potentiation of caspase activation.

    Directory of Open Access Journals (Sweden)

    Stephen P Burke

    2010-10-01

    Full Text Available The four residues at the amino-terminus of mature Smac/DIABLO are an IAP binding motif (IBM. Upon exit from mitochondria, mature Smac interacts with inhibitor of apoptosis proteins (IAPs, abrogating caspase inhibition. We used the ubiquitin fusion model to express mature Smac in the cytosol. Transiently expressed mature Smac56-239 (called Smac56 and Smac60-239 (called Smac60, which lacks the IBM, interacted with X-linked inhibitor of apoptosis protein (XIAP. However, stable expression produced wild type Smac56 that failed to homodimerize, interact with XIAP, and potentiate caspase activation. Cytosolic Smac60 retained these functions. Cytosolic Smac56 apparently becomes posttranslationally modified at the dimer interface region, which obliterated the epitope for a monoclonal antibody. Cytosolic Smacδ, which has the IBM but lacks amino acids 62-105, homodimerized and weakly interacted with XIAP, but failed to potentiate apoptosis. These findings suggest that the IBM of Smac is a recognition point for a posttranslational modification(s that blocks homodimerization and IAP interaction, and that amino acids 62-105 are required for the proapoptotic function of Smac.

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

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Kuan; Bai, Bingyang; Ta, Yajie; Zhang, Tingling; Xiao, Zikang; Wang, Peng George, E-mail: pwang@nankai.edu.cn; Zhang, Lianwen, E-mail: lianwen@nankai.edu.cn

    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. - Highlights: • Reduced NEDD4-1 correlates with increased overall O-GlcNAc level. • OGT negatively regulates NEDD4-1 stability. • O-GlcNAc regulates NEDD4-1 through caspase-mediated pathway.

  2. Caspase inhibitor IDN6556 facilitates marginal mass islet engraftment in a porcine islet autotransplant model.

    Science.gov (United States)

    McCall, Michael D; Maciver, Allison M; Kin, Tatsuya; Emamaullee, Juliet; Pawlick, Rena; Edgar, Ryan; Shapiro, A M James

    2012-07-15

    Large numbers of islets are lost in the early phase after clinical islet transplantation, through apoptosis, necrosis, or innate inflammatory injury. We previously demonstrated the efficacy of a series of caspase inhibitors in mouse models on islet engraftment through reduction in early posttransplant apoptosis. We studied IDN6556, a caspase inhibitor with a first-pass effect, in a large animal (pig) intraportal marginal mass islet autotransplant model. Total pancreatectomy and marginal mass islet autotransplantation were carried out in Yucatan miniature swine to explore the effects of IDN6556 on islet engraftment. Pigs were treated with IDN6556 at a dose of 20 mg/kg orally twice daily (n=7) or phosphate-buffered saline control (n=6) orally for 7 days, and blood glucose was monitored for 1 month. Glucose tolerance and acute insulin release were determined at 1 month. There were no differences in islet procurement, isolation, or islet functional parameters between the two groups. Pigs receiving IDN6556 had lower fasting blood glucose level after transplantation and a higher percentage (100% vs. 33.3%) showed fasting blood glucose levels less than 11 mM. This translated into an enhanced metabolic reserve and acute insulin release for pigs in the treatment group. IDN6556 led to enhanced islet engraftment in this large animal islet transplant model. Although this study has limitations including a short interval of study (1 month) and the use of unpurified islets, the results justify early clinical trials of IDN6556 in islet transplantation.

  3. 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. © 2015 The Authors. Experimental Dermatology Published by John Wiley & Sons Ltd.

  4. The role of Bax and caspase-3 in doppel-induced apoptosis of cerebellar granule cells.

    Science.gov (United States)

    Didonna, Alessandro; Sussman, Joshua; Benetti, Federico; Legname, Giuseppe

    2012-07-01

    Doppel (Dpl) protein is a paralog of the prion protein (PrP) that shares 25% sequence similarity with the C-terminus of PrP, a common N-glycosylation site and a C-terminal signal peptide for attachment of a glycosylphophatidyl inositol anchor. Whereas PrP (C) is highly expressed in the central nervous system (CNS), Dpl is detected mostly in testes and its ectopic expression in the CNS leads to ataxia as well as Purkinje and granule cell degeneration in the cerebellum. The mechanism through which Dpl induces neurotoxicity is still debated. In the present work, primary neuronal cultures derived from postnatal cerebellar granule cells of wild-type and PrP-knockout FVB mice were used in order to investigate the molecular events that occur upon exposure to Dpl. Treatment of cultured cerebellar neurons with recombinant Dpl produced apoptosis that could be prevented by PrP co-incubation. When primary neuronal cultures from Bax-deficient mice were incubated with Dpl, no apoptosis was observed, suggesting an important role of Bax in triggering neurodegeneration. Similarly, cell survival increased when recDpl-treated cells were incubated with an inhibitor of caspase-3, which mediates apoptosis in mammalian cells. Together, our findings raise the possibility that Bax and caspase-3 feature in Dpl-mediated apoptosis.

  5. Expression of death receptor 4 induces caspase-independent cell death in MMS-treated yeast.

    Science.gov (United States)

    Kang, Mi-Sun; Lee, Sung-Keun; Park, Chang-Shin; Kang, Ju-Hee; Bae, Sung-Ho; Yu, Sung-Lim

    2008-11-14

    DR4, a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, is a key element in the extrinsic pathway of TRAIL/TRAIL receptor-related apoptosis that exerts a preferential toxic effect against tumor cells. However, TRAIL and DR4 are expressed in various normal cells, and recent studies indicate that DR4 has a number of non-apoptotic functions. In this study, we evaluated the effects of human DR4 expression in yeast to determine the function of DR4 in normal cells. The expression of DR4 in yeast caused G1 arrest, which resulted in transient growth inhibition. Moreover, treatment of DR4-expressing yeast with a DNA damaging agent, MMS, elicited drastic, and sustained cell growth inhibition accompanied with massive apoptotic cell death. Further analysis revealed that cell death in the presence of DNA damage and DR4 expression was not dependent on the yeast caspase, YCA1. Taken together, these results indicate that DR4 triggers caspase-independent programmed cell death during the response of normal cells to DNA damage.

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

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    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. Caspase Cleavages of the Lymphocyte-oriented Kinase Prevent Ezrin, Radixin, and Moesin Phosphorylation during Apoptosis*

    Science.gov (United States)

    Leroy, Catherine; Belkina, Natalya V.; Long, Thavy; Deruy, Emeric; Dissous, Colette; Shaw, Stephen; Tulasne, David

    2016-01-01

    The lymphocyte-oriented kinase (LOK), also called serine threonine kinase 10 (STK10), is synthesized mainly in lymphocytes. It is involved in lymphocyte migration and polarization and can phosphorylate ezrin, radixin, and moesin (the ERM proteins). In a T lymphocyte cell line and in purified human lymphocytes, we found LOK to be cleaved by caspases during apoptosis. The first cleavage occurs at aspartic residue 332, located between the kinase domain and the coiled-coil regulation domain. This cleavage generates an N-terminal fragment, p50 N-LOK, containing the kinase domain and a C-terminal fragment, which is further cleaved during apoptosis. Although these cleavages preserve the entire kinase domain, p50 N-LOK displays no kinase activity. In apoptotic lymphocytes, caspase cleavages of LOK are concomitant with a decrease in ERM phosphorylation. When non-apoptotic lymphocytes from mice with homozygous and heterozygous LOK knockout were compared, the latter showed a higher level of ERM phosphorylation, but when apoptosis was induced, LOK−/− and LOK+/− lymphocytes showed the same low level, confirming in vivo that LOK-induced ERM phosphorylation is prevented during lymphocyte apoptosis. Our results demonstrate that cleavage of LOK during apoptosis abolishes its kinase activity, causing a decrease in ERM phosphorylation, crucial to the role of the ERM proteins in linking the plasma membrane to actin filaments. PMID:26945071

  8. Caspase Cleavages of the Lymphocyte-oriented Kinase Prevent Ezrin, Radixin, and Moesin Phosphorylation during Apoptosis.

    Science.gov (United States)

    Leroy, Catherine; Belkina, Natalya V; Long, Thavy; Deruy, Emeric; Dissous, Colette; Shaw, Stephen; Tulasne, David

    2016-05-06

    The lymphocyte-oriented kinase (LOK), also called serine threonine kinase 10 (STK10), is synthesized mainly in lymphocytes. It is involved in lymphocyte migration and polarization and can phosphorylate ezrin, radixin, and moesin (the ERM proteins). In a T lymphocyte cell line and in purified human lymphocytes, we found LOK to be cleaved by caspases during apoptosis. The first cleavage occurs at aspartic residue 332, located between the kinase domain and the coiled-coil regulation domain. This cleavage generates an N-terminal fragment, p50 N-LOK, containing the kinase domain and a C-terminal fragment, which is further cleaved during apoptosis. Although these cleavages preserve the entire kinase domain, p50 N-LOK displays no kinase activity. In apoptotic lymphocytes, caspase cleavages of LOK are concomitant with a decrease in ERM phosphorylation. When non-apoptotic lymphocytes from mice with homozygous and heterozygous LOK knockout were compared, the latter showed a higher level of ERM phosphorylation, but when apoptosis was induced, LOK(-/-) and LOK(+/-) lymphocytes showed the same low level, confirming in vivo that LOK-induced ERM phosphorylation is prevented during lymphocyte apoptosis. Our results demonstrate that cleavage of LOK during apoptosis abolishes its kinase activity, causing a decrease in ERM phosphorylation, crucial to the role of the ERM proteins in linking the plasma membrane to actin filaments. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Caspase-Dependent Apoptosis Induced by Telomere Cleavage and TRF2 Loss

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    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.

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

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    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.

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

    Science.gov (United States)

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

    2017-10-01

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

  12. The inhibition effect of P-glycoprotein on cytochrome c release and caspase-3 activation by X-ray irradiation

    International Nuclear Information System (INIS)

    Chen Jun; Yu Zhijian; Xiao Mingxing; Pu Junguo; Zhang Zhiren

    2006-01-01

    Objective: To investigate the effects of P-glycoprotein (P-gp) on cytochrome c (Cyt c) release and caspase-3 activation in drug-resistant tumor cell after X-ray irradiation. Methods: Anti-P-gp McAb UIC-2 was applied to block P-gp function. MCF-7/Adr, P-gp-overexpressed drug-resistant breast cancer cell line, was irradiated by X-rays. Flow cytometry was used to measure apoptosis, dynamic changes of mitochondrial cytochrome c release and caspase-3 activation at various time after X-ray irradiation. Results: Both the expression of cyt c and the activation of caspase-3 in P-gp-blocked group were up-regulated significantly, compared with those in control group at 6 h, 12 h, and 24 h after X-ray irradiation, respectively. Conclusion: The current study showed that inhibition of P-gp function can increase cytochrome c release and caspase-3 activation. It suggested that P-gp inhibits cytochrome c release and caspase-3 activation induced by X-ray irradiation. (authors)

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

    Science.gov (United States)

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

    2017-11-01

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

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

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    Mickael Ros

    2016-01-01

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

  15. H2O2 INDUCES APOPTOSIS OF RABBIT CHONDROCYTES VIA BOTH THE EXTRINSIC AND THE CASPASE-INDEPENDENT INTRINSIC PATHWAYS

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    CAIPING ZHUANG

    2013-07-01

    Full Text Available Osteoarthritis (OA, one of the most common joint diseases with unknown etiology, is characterized by the progressive destruction of articular cartilage and the apoptosis of chondrocytes. The purpose of this study is to elucidate the molecular mechanisms of H2O2-mediated rabbit chondrocytes apoptosis. CCK-8 assay showed that H2O2 treatment induced a remarkable reduction of cell viability, which was further verified by the remarkable phosphatidylserine externalization after H2O2 treatment for 1 h, the typical characteristics of apoptosis. H2O2 treatment induced a significant dysfunction of mitochondrial membrane potential (ΔΨm, but did not induce casapse-9 activation, indicating that H2O2 treatment induced caspase-independent intrinsic apoptosis that was further verified by the fact that silencing of AIF but not inhibiting caspase-9 potently prevented H2O2-induced apoptosis. H2O2 treatment induced a significant increase of caspase-8 and -3 activation, and inhibition of caspase-8 or -3 significantly prevented H2O2-induced apoptosis, suggesting that the extrinsic pathway played an important role. Collectively, our findings demonstrate that H2O2 induces apoptosis via both the casapse-8-mediated extrinsic and the caspase-independent intrinsic apoptosis pathways in rabbit chondrocytes.

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

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    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.

  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. Predicted Trans-Acting siRNAs in the Human Brain

    Science.gov (United States)

    Liu, Xiaoshuang; Zhang, Guangxin; Zhang, Changqing; Wang, Jin

    2015-01-01

    Endogenous small non-coding RNAs play pivotal roles in regulating gene expression in eukaryotes. Many studies have investigated the function and molecular mechanism of microRNAs in the development and disease of various organisms via mRNA repression of protein-coding genes. Recent findings indicate microRNAs might trigger the generation of trans-acting small interfering RNAs (ta-siRNAs). The interaction among different types of small RNA molecules reveals an even more complicated and elaborate pattern of RNA regulation during gene expression than previously thought. We developed a method for mining ta-siRNA sequences and evaluated the performance of our novel method using data from Arabidopsis thaliana. Additionally, using small RNA and degradome data for the human brain, we identified 155 small RNAs that satisfied ta-siRNA characteristics. The DRAXIN and ATCAY genes, which are preferentially expressed in the human brain, were predicted to be the targets of 12 potential ta-siRNAs. PMID:25654231

  19. Neuronal deletion of caspase 8 protects against brain injury in mouse models of controlled cortical impact and kainic acid-induced excitotoxicity.

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    Maryla Krajewska

    Full Text Available Acute brain injury is an important health problem. Given the critical position of caspase 8 at the crossroads of cell death pathways, we generated a new viable mouse line (Ncasp8(-/-, in which the gene encoding caspase 8 was selectively deleted in neurons by cre-lox system.Caspase 8 deletion reduced rates of neuronal cell death in primary neuronal cultures and in whole brain organotypic coronal slice cultures prepared from 4 and 8 month old mice and cultivated up to 14 days in vitro. Treatments of cultures with recombinant murine TNFα (100 ng/ml or TRAIL (250 ng/mL plus cyclohexamide significantly protected neurons against cell death induced by these apoptosis-inducing ligands. A protective role of caspase 8 deletion in vivo was also demonstrated using a controlled cortical impact (CCI model of traumatic brain injury (TBI and seizure-induced brain injury caused by kainic acid (KA. Morphometric analyses were performed using digital imaging in conjunction with image analysis algorithms. By employing virtual images of hundreds of brain sections, we were able to perform quantitative morphometry of histological and immunohistochemical staining data in an unbiased manner. In the TBI model, homozygous deletion of caspase 8 resulted in reduced lesion volumes, improved post-injury motor performance, superior learning and memory retention, decreased apoptosis, diminished proteolytic processing of caspases and caspase substrates, and less neuronal degeneration, compared to wild type, homozygous cre, and caspase 8-floxed control mice. In the KA model, Ncasp8(-/- mice demonstrated superior survival, reduced seizure severity, less apoptosis, and reduced caspase 3 processing. Uninjured aged knockout mice showed improved learning and memory, implicating a possible role for caspase 8 in cognitive decline with aging.Neuron-specific deletion of caspase 8 reduces brain damage and improves post-traumatic functional outcomes, suggesting an important role for this

  20. Attenuation of the ELAV1-like protein HuR sensitizes adenocarcinoma cells to the intrinsic apoptotic pathway by increasing the translation of caspase-2L.

    Science.gov (United States)

    Winkler, C; Doller, A; Imre, G; Badawi, A; Schmid, T; Schulz, S; Steinmeyer, N; Pfeilschifter, J; Rajalingam, K; Eberhardt, W

    2014-07-10

    Caspase-2 represents the most conserved member of the caspase family, which exhibits features of both initiator and effector caspases. Using ribonucleoprotein (RNP)-immunoprecipitation assay, we identified the proapoptotic caspase-2L encoding mRNA as a novel target of the ubiquitous RNA-binding protein HuR in DLD-1 colon carcinoma cells. Unexpectedly, crosslinking-RNP and RNA probe pull-down experiments revealed that HuR binds exclusively to the caspase-2-5' untranslated region (UTR) despite that the 3' UTR of the mRNA bears several adenylate- and uridylate-rich elements representing the prototypical HuR binding sites. By using RNAi-mediated loss-of-function approach, we observed that HuR regulates the mRNA and in turn the protein levels of caspase-2 in a negative manner. Silencing of HuR did not affect the stability of caspase-2 mRNA but resulted in an increased redistribution of caspase-2 transcripts from RNP particles to translational active polysomes implicating that HuR exerts a direct repressive effect on caspase-2 translation. Consistently, in vitro translation of a luciferase reporter gene under the control of an upstream caspase-2-5'UTR was strongly impaired after the addition of recombinant HuR, whereas translation of caspase-2 coding region without the 5'UTR is not affected by HuR confirming the functional role of the caspase-2-5'UTR. Functionally, an elevation in caspase-2 level by HuR knockdown correlated with an increased sensitivity of cells to apoptosis induced by staurosporine- and pore-forming toxins as implicated by their significant accumulation in the sub G1 phase and an increase in caspase-2, -3 and poly ADP-ribose polymerase cleavage, respectively. Importantly, HuR knockdown cells remained insensitive toward STS-induced apoptosis if cells were additionally transfected with caspase-2-specific siRNAs. Collectively, our findings support the hypothesis that HuR by acting as an endogenous inhibitor of caspase-2-driven apoptosis may essentially

  1. Methoxychlor induces atresia by altering Bcl2 factors and inducing caspase activity in mouse ovarian antral follicles in vitro

    Science.gov (United States)

    Basavarajappa, Mallikarjuna S.; Karman, Bethany N.; Wang, Wei; Gupta, Rupesh K.; Flaws, Jodi A.

    2012-01-01

    Methoxychlor (MXC) is an organochlorine pesticide widely used in many countries against various species of insects that attack crops and domestic animals. MXC reduces fertility by increasing atresia (death) of antral follicles in vivo. MXC also induces atresia of antral follicles after 96 h in vitro. The current work tested the hypothesis that MXC induces morphological atresia at early time points (24 and 48 h) by altering pro-apoptotic (Bax, Bok, Casp3, and caspase activity) and anti-apoptotic (Bcl2 and Bcl-xL) factors in the follicles. The results indicate that at 24 h, MXC increased Bcl-xL and Bax mRNA levels and increased the ratio of Bax/Bcl2. At 48–96 h, MXC induced morphological atresia. At 24–96 h, MXC increased caspase activities. These data suggest that MXC may induce atresia by altering Bcl2 factors and inducing caspase activities in antral follicles. PMID:23000595

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

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

    International Nuclear Information System (INIS)

    Voorzanger-Rousselot, Nathalie; Alberti, Laurent; Blay, Jean-Yves

    2006-01-01

    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. 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. 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. 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

  4. Caspase inhibitors affect the kinetics and dimensions of tracheary elements in xylogenic Zinnia (Zinnia elegans cell cultures

    Directory of Open Access Journals (Sweden)

    Schel Jan HN

    2010-08-01

    Full Text Available Abstract Background The xylem vascular system is composed of fused dead, hollow cells called tracheary elements (TEs that originate through trans-differentiation of root and shoot cambium cells. TEs undergo autolysis as they differentiate and mature. The final stage of the formation of TEs in plants is the death of the involved cells, a process showing some similarities to programmed cell death (PCD in animal systems. Plant proteases with functional similarity to proteases involved in mammalian apoptotic cell death (caspases are suggested as an integral part of the core mechanism of most PCD responses in plants, but participation of plant caspase-like proteases in TE PCD has not yet been documented. Results Confocal microscopic images revealed the consecutive stages of TE formation in Zinnia cells during trans-differentiation. Application of the caspase inhibitors Z-Asp-CH2-DCB, Ac-YVAD-CMK and Ac-DEVD-CHO affected the kinetics of formation and the dimensions of the TEs resulting in a significant delay of TE formation, production of larger TEs and in elimination of the 'two-wave' pattern of TE production. DNA breakdown and appearance of TUNEL-positive nuclei was observed in xylogenic cultures and this was suppressed in the presence of caspase inhibitors. Conclusions To the best of our knowledge this is the first report showing that caspase inhibitors can modulate the process of trans-differentiation in Zinnia xylogenic cell cultures. As caspase inhibitors are closely associated with cell death inhibition in a variety of plant systems, this suggests that the altered TE formation results from suppression of PCD. The findings presented here are a first step towards the use of appropriate PCD signalling modulators or related molecular genetic strategies to improve the hydraulic properties of xylem vessels in favour of the quality and shelf life of plants or plant parts.

  5. NLRP3 controls Trypanosoma cruzi infection through a caspase-1-dependent IL-1R-independent NO production.

    Science.gov (United States)

    Gonçalves, Virginia M; Matteucci, Kely C; Buzzo, Carina L; Miollo, Bruna H; Ferrante, Danny; Torrecilhas, Ana C; Rodrigues, Mauricio M; Alvarez, Jose M; Bortoluci, Karina R

    2013-01-01

    Trypanosoma cruzi (T. cruzi) is an intracellular protozoan parasite and the etiological agent of Chagas disease, a chronic infectious illness that affects millions of people worldwide. Although the role of TLR and Nod1 in the control of T. cruzi infection is well-established, the involvement of inflammasomes remains to be elucidated. Herein, we demonstrate for the first time that T. cruzi infection induces IL-1β production in an NLRP3- and caspase-1-dependent manner. Cathepsin B appears to be required for NLRP3 activation in response to infection with T. cruzi, as pharmacological inhibition of cathepsin B abrogates IL-1β secretion. NLRP3(-/-) and caspase1(-/-) mice exhibited high numbers of T. cruzi parasites, with a magnitude of peak parasitemia comparable to MyD88(-/-) and iNOS(-/-) mice (which are susceptible models for T. cruzi infection), indicating the involvement of NLRP3 inflammasome in the control of the acute phase of T. cruzi infection. Although the inflammatory cytokines IL-6 and IFN-γ were found in spleen cells from NLRP3(-/-) and caspase1(-/-) mice infected with T. cruzi, these mice exhibited severe defects in nitric oxide (NO) production and an impairment in macrophage-mediated parasite killing. Interestingly, neutralization of IL-1β and IL-18, and IL-1R genetic deficiency demonstrate that these cytokines have a minor effect on NO secretion and the capacity of macrophages to control T. cruzi infection. In contrast, inhibition of caspase-1 with z-YVAD-fmk abrogated NO production by WT and MyD88(-/-) macrophages and rendered them as susceptible to T. cruzi infection as NLRP3(-/-) and caspase-1(-/-) macrophages. Taken together, our results demonstrate a role for the NLRP3 inflammasome in the control of T. cruzi infection and identify NLRP3-mediated, caspase-1-dependent and IL-1R-independent NO production as a novel effector mechanism for these innate receptors.

  6. NLRP3 controls Trypanosoma cruzi infection through a caspase-1-dependent IL-1R-independent NO production.

    Directory of Open Access Journals (Sweden)

    Virginia M Gonçalves

    Full Text Available Trypanosoma cruzi (T. cruzi is an intracellular protozoan parasite and the etiological agent of Chagas disease, a chronic infectious illness that affects millions of people worldwide. Although the role of TLR and Nod1 in the control of T. cruzi infection is well-established, the involvement of inflammasomes remains to be elucidated. Herein, we demonstrate for the first time that T. cruzi infection induces IL-1β production in an NLRP3- and caspase-1-dependent manner. Cathepsin B appears to be required for NLRP3 activation in response to infection with T. cruzi, as pharmacological inhibition of cathepsin B abrogates IL-1β secretion. NLRP3(-/- and caspase1(-/- mice exhibited high numbers of T. cruzi parasites, with a magnitude of peak parasitemia comparable to MyD88(-/- and iNOS(-/- mice (which are susceptible models for T. cruzi infection, indicating the involvement of NLRP3 inflammasome in the control of the acute phase of T. cruzi infection. Although the inflammatory cytokines IL-6 and IFN-γ were found in spleen cells from NLRP3(-/- and caspase1(-/- mice infected with T. cruzi, these mice exhibited severe defects in nitric oxide (NO production and an impairment in macrophage-mediated parasite killing. Interestingly, neutralization of IL-1β and IL-18, and IL-1R genetic deficiency demonstrate that these cytokines have a minor effect on NO secretion and the capacity of macrophages to control T. cruzi infection. In contrast, inhibition of caspase-1 with z-YVAD-fmk abrogated NO production by WT and MyD88(-/- macrophages and rendered them as susceptible to T. cruzi infection as NLRP3(-/- and caspase-1(-/- macrophages. Taken together, our results demonstrate a role for the NLRP3 inflammasome in the control of T. cruzi infection and identify NLRP3-mediated, caspase-1-dependent and IL-1R-independent NO production as a novel effector mechanism for these innate receptors.

  7. 1800MHz Microwave Induces p53 and p53-Mediated Caspase-3 Activation Leading to Cell Apoptosis In Vitro.

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    Fuqiang Xing

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

  8. 1800MHz Microwave Induces p53 and p53-Mediated Caspase-3 Activation Leading to Cell Apoptosis In Vitro

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2017-07-01

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

  10. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

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

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    Marco A Ataide

    2014-01-01

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

  12. Malaria-Induced NLRP12/NLRP3-Dependent Caspase-1 Activation Mediates Inflammation and Hypersensitivity to Bacterial Superinfection

    Science.gov (United States)

    Ataide, Marco A.; Andrade, Warrison A.; Zamboni, Dario S.; Wang, Donghai; Souza, Maria do Carmo; Franklin, Bernardo S.; Elian, Samir; Martins, Flaviano S.; Pereira, Dhelio; Reed, George; Fitzgerald, Katherine A.; Golenbock, Douglas T.; Gazzinelli, Ricardo T.

    2014-01-01

    Cyclic paroxysm and high fever are hallmarks of malaria and are associated with high levels of pyrogenic cytokines, including IL-1β. In this report, we describe a signature for the expression of inflammasome-related genes and caspase-1 activation in malaria. Indeed, when we infected mice, Plasmodium infection was sufficient to promote MyD88-mediated caspase-1 activation, dependent on IFN-γ-priming and the expression of inflammasome components ASC, P2X7R, NLRP3 and/or NLRP12. Pro-IL-1β expression required a second stimulation with LPS and was also dependent on IFN-γ-priming and functional TNFR1. As a consequence of Plasmodium-induced caspase-1 activation, mice produced extremely high levels of IL-1β upon a second microbial stimulus, and became hypersensitive to septic shock. Therapeutic intervention with IL-1 receptor antagonist prevented bacterial-induced lethality in rodents. Similar to mice, we observed a significantly increased frequency of circulating CD14+CD16−Caspase-1+ and CD14dimCD16+Caspase-1+ monocytes in peripheral blood mononuclear cells from febrile malaria patients. These cells readily produced large amounts of IL-1β after stimulation with LPS. Furthermore, we observed the presence of inflammasome complexes in monocytes from malaria patients containing either NLRP3 or NLRP12 pyroptosomes. We conclude that NLRP12/NLRP3-dependent activation of caspase-1 is likely to be a key event in mediating systemic production of IL-1β and hypersensitivity to secondary bacterial infection during malaria. PMID:24453977

  13. Reactive Carbonyl Species Activate Caspase-3-Like Protease to Initiate Programmed Cell Death in Plants.

    Science.gov (United States)

    Biswas, Md Sanaullah; Mano, Jun'ichi

    2016-07-01

    Reactive oxygen species (ROS)-triggered programmed cell death (PCD) is a typical plant response to biotic and abiotic stressors. We have recently shown that lipid peroxide-derived reactive carbonyl species (RCS), downstream products of ROS, mediate oxidative signal to initiate PCD. Here we investigated the mechanism by which RCS initiate PCD. Tobacco Bright Yellow-2 cultured cells were treated with acrolein, one of the most potent RCS. Acrolein at 0.2 mM caused PCD in 5 h (i.e. lethal), but at 0.1 mM it did not (sublethal). Specifically, these two doses caused critically different effects on the cells. Both lethal and sublethal doses of acrolein exhausted the cellular glutathione pool in 30 min, while the lethal dose only caused a significant ascorbate decrease and ROS increase in 1-2 h. Prior to such redox changes, we found that acrolein caused significant increases in the activities of caspase-1-like protease (C1LP) and caspase-3-like protease (C3LP), the proteases which trigger PCD. The lethal dose of acrolein increased the C3LP activity 2-fold more than did the sublethal dose. In contrast, C1LP activity increments caused by the two doses were not different. Acrolein and 4-hydroxy-(E)-2-nonenal, another RCS, activated both proteases in a cell-free extract from untreated cells. H 2 O 2 at 1 mM added to the cells increased C1LP and C3LP activities and caused PCD, and the RCS scavenger carnosine suppressed their activation and PCD. However, H 2 O 2 did not activate the proteases in a cell-free extract. Thus the activation of caspase-like proteases, particularly C3LP, by RCS is an initial biochemical event in oxidative signal-stimulated PCD in plants. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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

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    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

  15. Avian encephalomyelitis virus nonstructural protein 2C induces apoptosis by activating cytochrome c/caspase-9 pathway

    International Nuclear Information System (INIS)

    Liu Jue; Wei Ting; Kwang, Jimmy

    2004-01-01

    The nonstructural protein 2C is highly conserved among picornaviruses and plays an important role in the assembly of mature virions, membrane association, and viral RNA synthesis. The investigation of other potential functions of nonstructural protein 2C from avian encephalomyelitis virus (AEV) resulted in identifying for the first time that the protein 2C is involved in apoptosis. Expression of the protein 2C on chick embryo brain (CEB) and Cos-7 cells produced TUNEL-positive cells characterized by a cleavage of cellular DNA and the formation of membrane-enclosed apoptotic bodies. Analysis of the protein 2C showed that the N-terminal domain containing 35 amino acid (aa) residues (between 46 and 80 aa) is associated with apoptotic function. Transfection of the deletion mutant lacking this 35 aa's into CEB and Cos-7 cells failed to induce apoptosis. Furthermore, the protein 2C induced apoptosis in the transfected CEB and Cos-7 cells through activation of caspase-9 rather than caspase-8 followed by activation of caspase-3 pathway. Analysis of the Western blots of caspase-3 and caspase-9 showed the characteristics of active caspase-3 and -9 in the 2C-transfected CEB and Cos-7 cells as seen in the AEV-infected CEB cells while they were in the form of procaspase-3 and procaspase-9 in the 2C mutant-transfected cells. To further elucidate the mechanism of the 2C-induced apoptosis, the 2C-transfected CEB and Cos-7 cells were fractionated into mitochondria and cytosol and subjected for Western blotting, located cytochrome c in the mitochondria as well as the cytosol fractions, while it was only sequestered in the mitochondrial fraction in the mutant 2C-transfected cells. The protein 2C was located in the mitochondria and cytosol of the transfected/infected CEB and transfected Cos-7 cells, but the mutant lost its ability to localize to the mitochondria. Altogether, the results demonstrate that the protein 2C localized to the mitochondria of the transfected cells triggered

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

    Directory of Open Access Journals (Sweden)

    Lisa Trisciuoglio

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

  17. Depletion of membrane cholesterol compromised caspase-8 imparts in autophagy induction and inhibition of cell migration in cancer cells.

    Science.gov (United States)

    Kumar, Mukesh; Irungbam, Karuna; Kataria, Meena

    2018-01-01

    Cholesterol in lipid raft plays crucial role on cancer cell survival during metastasis of cancer cells. Cancer cells are reported to enrich cholesterol in lipid raft which make them more susceptible to cell death after cholesterol depletion than normal cells. Methyl-β-cyclodextrin (MβCD), an amphipathic polysaccharide known to deplete the membrane cholesterol, induces cell death selectively in cancer cells. Present work was designed to identify the major form of programmed cell death in membrane cholesterol depleted cancer cells (MDA-MB 231 and 4T1) and its impact on migration efficiency of cancer cells. Membrane cholesterol alteration and morphological changes in 4T1 and MDA-MB 231 cancer cells by MβCD were measured by fluorescent microscopy. Cell death and cell proliferation were observed by PI, AO/EB and MTT assay respectively. Programme cell death was confirmed by flow cytometer. Caspase activation was assessed by MTT and PI after treatments with Z-VAD [OME]-FMK, mitomycin c and cycloheximide. Necroptosis, autophagy, pyroptosis and paraptosis were examined by cell proliferation assay and flow cytometry. Relative quantitation of mRNA of caspase-8, necroptosis and autophagy genes were performed. Migration efficiency of cancer cells were determined by wound healing assay. We found caspase independent cell death in cholesterol depleted MDA-MB 231 cells which was reduced by (3-MA) an autophagy inhibitor. Membrane cholesterol depletion neither induces necroptosis, paraptosis nor pyroptosis in MDA-MB 231 cells. Subsequent activation of caspase-8 after co-incubation of mitomycin c and cycloheximide separately, restored the cell viability in cholesterol depleted MDA-MB 231 cells. Down regulation of caspase-8 mRNA in cholesterol depleted cancer cells ensures that caspase-8 indirectly promotes the induction of autophagy. In another experiment we have demonstrated that membrane cholesterol depletion reduces the migration efficiency in cancer cells. Together our

  18. Deletion and aberrant CpG island methylation of Caspase 8 gene in medulloblastoma.

    Science.gov (United States)

    Gonzalez-Gomez, Pilar; Bello, M Josefa; Inda, M Mar; Alonso, M Eva; Arjona, Dolores; Amiñoso, Cinthia; Lopez-Marin, Isabel; de Campos, Jose M; Sarasa, Jose L; Castresana, Javier S; Rey, Juan A

    2004-09-01

    Aberrant methylation of promoter CpG islands in human genes is an alternative genetic inactivation mechanism that contributes to the development of human tumors. Nevertheless, few studies have analyzed methylation in medulloblastomas. We determined the frequency of aberrant CpG island methylation for Caspase 8 (CASP8) in a group of 24 medulloblastomas arising in 8 adult and 16 pediatric patients. Complete methylation of CASP8 was found in 15 tumors (62%) and one case displayed hemimethylation. Three samples amplified neither of the two primer sets for methylated or unmethylated alleles, suggesting that genomic deletion occurred in the 5' flanking region of CASP8. Our findings suggest that methylation commonly contributes to CASP8 silencing in medulloblastomas and that homozygous deletion or severe sequence changes involving the promoter region may be another mechanism leading to CASP8 inactivation in this neoplasm.

  19. Granzyme A Cleaves a Mitochondrial Complex I Protein to Initiate Caspase-Independent Cell Death

    Science.gov (United States)

    Martinvalet, Denis; Dykxhoorn, Derek M.; Ferrini, Roger; Lieberman, Judy

    2010-01-01

    SUMMARY The killer lymphocyte protease granzyme A (GzmA) triggers caspase-independent target cell death with morphological features of apoptosis. We previously showed that GzmA acts directly on mitochondria to generate reactive oxygen species (ROS) and disrupt the transmembrane potential (ΔΨm) but does not permeabilize the mitochondrial outer membrane. Mitochondrial damage is critical to GzmA-induced cell death since cells treated with superoxide scavengers are resistant to GzmA. Here we find that GzmA accesses the mitochondrial matrix to cleave the complex I protein NDUFS3, an iron-sulfur subunit of the NADH:ubiquinone oxidoreductase complex I, after Lys56 to interfere with NADH oxidation and generate superoxide anions. Target cells expressing a cleavage site mutant of NDUFS3 are resistant to GzmA-mediated cell death but remain sensitive to GzmB. PMID:18485875

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

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    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.

  1. Pharmacology of smac mimetics; chemotype differentiation based on physical association with caspase regulators and cellular transport.

    Science.gov (United States)

    Talbott, Randy L; Borzilleri, Robert M; Chaudhry, Charu; Fargnoli, Joseph; Shen, Henry; Fairchild, Craig; Barnhart, Bryan; Ortega, Marie; McDonagh, Thomas E; Vuppugalla, Ragini; Vite, Gregory D; Hunt, John T; Gottardis, Marco; Naglich, Joseph G

    2015-11-01

    Cellular levels of inhibitor of apoptosis (IAP) proteins are elevated in multiple human cancers and their activities often play a part in promoting cancer cell survival by blocking apoptotic pathways, controlling signal transduction pathways and contributing to resistance. These proteins function through interactions of their BIR (baculoviral IAP repeat) protein domains with pathway components and these interactions are endogenously antagonized by Smac/Diablo (second mitochondrial activator of caspases/direct IAP binding protein with low isoelectric point). This report describes development of synthetic smac mimetics (SM) and compares their binding, antiproliferative and anti-tumor activities. All dimeric antagonists inhibit in vitro smac tetrapeptide binding to recombinant IAP proteins, rescue IAP-bound caspase-3 activity and show anti-proliferative activity against human A875 melanoma cells. One heterodimeric SM, SM3, binds tightly to IAP proteins in vitro and slowly dissociates (greater than two hours) from these protein complexes compared to the other antagonists. In addition, in vitro SM anti-proliferation potency is influenced by ABCB1 transporter (ATP-binding cassette, sub-family B; MDR1, P-gp) activities and one antagonist, SM5, does not appear to be an ABCB1 efflux pump substrate. All dimeric smac mimetics inhibit the growth of human melanoma A875 tumors implanted in athymic mice at well-tolerated doses. One antagonist, SM4, shows broad spectrum in vivo anti-tumor activity and modulates known pharmacodynamic markers of IAP antagonism. These data taken together demonstrate the range of diverse dimeric IAP antagonist activities and supports their potential as anticancer agents. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Drosophila spaghetti and doubletime link the circadian clock and light to caspases, apoptosis and tauopathy.

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    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

  3. Cigarette smoke causes caspase-independent apoptosis of bronchial epithelial cells from asthmatic donors.

    Directory of Open Access Journals (Sweden)

    Fabio Bucchieri

    Full Text Available Epidemiologic studies have demonstrated important links between air pollution and asthma. Amongst these pollutants, environmental cigarette smoke is a risk factor both for asthma pathogenesis and exacerbation. As the barrier to the inhaled environment, the bronchial epithelium is a key structure that is exposed to cigarette smoke.Since primary bronchial epithelial cells (PBECs from asthmatic donors are more susceptible to oxidant-induced apoptosis, we hypothesized that they would be susceptible to cigarette smoke-induced cell death.PBECs from normal and asthmatic donors were exposed to cigarette smoke extract (CSE; cell survival and apoptosis were assessed by fluorescence-activated cell sorting, and protective effects of antioxidants evaluated. The mechanism of cell death was evaluated using caspase inhibitors and immunofluorescent staining for apoptosis-inducing factor (AIF.Exposure of PBEC cultures to CSE resulted in a dose-dependent increase in cell death. At 20% CSE, PBECs from asthmatic donors exhibited significantly more apoptosis than cells from non-asthmatic controls. Reduced glutathione (GSH, but not ascorbic acid (AA, protected against CSE-induced apoptosis. To investigate mechanisms of CSE-induced apoptosis, caspase-3 or -9 inhibitors were tested, but these failed to prevent apoptosis; in contrast, CSE promoted nuclear translocation of AIF from the mitochondria. GSH reduced the number of nuclear-AIF positive cells whereas AA was ineffective.Our results show that PBECs from asthmatic donors are more susceptible to CSE-induced apoptosis. This response involves AIF, which has been implicated in DNA damage and ROS-mediated cell-death. Epithelial susceptibility to CSE may contribute to the impact of environmental tobacco smoke in asthma.

  4. 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.

  5. 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

  6. 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.

  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. 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.

  9. Photosensitized methyl paraben induces apoptosis via caspase dependent pathway under ambient UVB exposure in human skin cells.

    Science.gov (United States)

    Dubey, Divya; Chopra, Deepti; Singh, Jyoti; Srivastav, Ajeet K; Kumari, Smita; Verma, Ankit; Ray, Ratan Singh

    2017-10-01

    Methyl paraben (MP), is a widely used preservative in pharmaceutical, food and cosmetic products. Its molecular mechanism under ambient ultraviolet radiation is not well understood. We investigated photosensitizing mechanism of MP under ambient UVB (0.6 mW/cm 2 ) intensity. MP showed dose dependent decrease in cell viability of human keratinocyte cell line (HaCaT) by MTT and NRU assays. Study showed 40% reduction in antimicrobial activity of UVB irradiated MP through E. coli culture. Photosensitized MP (25 μg/ml) significantly enhanced lipid peroxidation, intracellular ROS generation and disrupted mitochondrial membrane integrity. MP induced loss of lysosomal membrane integrity and endoplasmic reticulum (ER) mediated stress evident from Ca +2 release. Phototoxicity of MP showed nuclear fragmentation, phosphatidylserine translocation, 30% tail DNA and micronuclei formation. Study showed mitochondria mediated apoptosis via upregulation of Bax, Apaf-1, Cytochrome C and Caspase-3. Upregulation of Caspase-12 (2 folds) specifically showed role of ER in apoptosis. Specific caspase inhibitor, Z-VAD-FMK showed involvement of caspase cascade pathway in apoptosis. Results indicate that photosensitive MP leads to oxidative stress mediated DNA damage and apoptosis through mitochondria and ER. MP causes deleterious effects and its long term exposure to human skin may promote skin diseases. Therefore, MP should be replaced by other photosafe preservatives for humans. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2011-07-01

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

  11. Microbiota Composition, HSP70 and Caspase-3 Expression as Marker for Colorectal Cancer Patients in Aceh, Indonesia.

    Science.gov (United States)

    Yusuf, Fauzi; Ilyas, Syafruddin; Damanik, Harun Al Rasyid; Fatchiyah, Fatchiyah

    2016-10-01

    to investigate the relationship between microbiota composition with HSP70 and Caspase-3 expressions in colon tissue as an initial study to develop the candidate for early detection of colorectal cancer for Indonesian patients. this is a cross-sectional study on 32 patients undergoing colonoscopy; 16 patients of colorectal cancer (CRC) while the other 16 patients are not (colitis and internal hemorrhoid). The composition of microbiota in stool samples was examined using 16S rRNA Denaturing Gradient Gel Electrophoresis (DDGE) while expression of HSP70 was examined by immunohistochemistry and Caspase-3 by using Haematoxylin-Eosin(HE) staining to determine the morphological changes in colon tissue. analysis of PCR-DDGE shows a different composition of microbiota between patients with CRC and non-CRC. All CRC patients showed disappearance of dominant band from Bifidobacterium groups. Histological observation based on Inter Class Correlation (ICC) test from all slide showed a high scores (5.2-9.2) in CRC patients and low scores (1.7-2.4) in non-CRC patients. HSP70 expression was increased significantly in CRC patients with the highest percentage of 84%, while expression of caspase-3 decreased with the highest percentage of 21%. Statistical analysis showed that the incidence of colorectal cancer was associated with the expression of HSP 70 (pcolorectal cancer patients that show disappearance of dominant band, while expression of HSP70 increased and the Caspase-3 expression decreased significantly.

  12. Independent induction of caspase-8 and cFLIP expression during colorectal carcinogenesis in sporadic and HNPCC adenomas and carcinomas

    NARCIS (Netherlands)

    Heijink, D. M.; Kleibeuker, J. H.; Jalving, M.; Ek, W. Boersma-van; Koornstra, J. J.; Wesseling, J.; de Jong, S.

    2007-01-01

    Background: TNF-Related Apoptosis Inducing Ligand (TRAIL) is a promising agent for the induction of apoptosis in neoplastic tissues. Important determinants of TRAIL sensitivity are two intracellular proteins of the TRAIL pathway, caspase-8 and its anti-apoptotic competitor cellular Flice-Like

  13. 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

  14. 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

  15. 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.

  16. Distinct pro-apoptotic properties of Zhejiang saffron against human lung cancer via a caspase-8-9-3 cascade.

    Science.gov (United States)

    Liu, Dan-Dan; Ye, Yi-Lu; Zhang, Jing; Xu, Jia-Ni; Qian, Xiao-Dong; Zhang, Qi

    2014-01-01

    Lung cancer is the leading cause of cancer-related death worldwide. Here we investigated the antitumor effect and mechanism of Zhejiang (Huzhou and Jiande) saffron against lung cancer cell lines, A549 and H446. Using high performance liquid chromatography (HPLC), the contents of crocin I and II were determined. In vitro, MTT assay and annexin-V FITC/PI staining showed cell proliferation activity and apoptosis to be changed in a dose- and time-dependent manner. The inhibition effect of Jiande saffron was the strongest. In vivo, when mice were orally administered saffron extracts at dose of 100mg/kg/d for 28 days, xenograft tumor size was reduced, and ELISA and Western blotting analysis of caspase-3, -8 and -9 exhibited stronger expression and activity than in the control. In summary, saffron from Zhejiang has significant antitumor effects in vitro and in vivo through caspase-8-caspase-9-caspase-3 mediated cell apoptosis. It thus appears to have more potential as a therapeutic agent.

  17. Placental apoptosis in preeclampsia, intrauterine growth retardation, and HELLP syndrome: an immunohistochemical study with caspase-3 and bcl-2.

    Science.gov (United States)

    Cali, U; Cavkaytar, S; Sirvan, L; Danisman, N

    2013-01-01

    To examine the placental expression of caspase-3 and bcl-2 in pregnancies complicated by preeclampsia, IUGR, and HELLP syndrome. A prospective case-control study was conducted on 50 pregnant women between December 2006 and August 2007 at Dr. Zekai Tahir Burak Women Health Research and Education Hospital, Ankara, Turkey. Placental tissue samples were obtained from 15 pregnancies complicated by preeclampsia, 15 pregnancies with normotensive IUGR, five pregnancies with HELLP syndrome, and 15 gestational age-matched normotensive pregnancies without intrauterine infection as a control group. The placental expression of caspase-3 and bcl-2 has been investigated by immunohistochemical staining. Caspase-3 immunostaining score was significantly higher in each group when compared with the control group (p = 0.002). However there was no statistically signifant difference with bcl-2 immunostaining in each group when compared with the control group. Apoptotic marker caspase-3 is significantly increased in the villous trophoblasts of patients with preeclampsia, HELLP syndrome, and IUGR indicating increased placental apoptosis.

  18. E. adenophorum Induces Cell Cycle and Apoptosis of Renal Cells through Mitochondrial Pathway and Caspase Activation in Saanen Goat.

    Directory of Open Access Journals (Sweden)

    Yajun He

    Full Text Available The cytotoxicity effects of E. adenophorum on cell cycle and apoptosis of renal cells in Saanen goat was evaluated by TUNEL, DAPI, AO/EB staining, DNA fragmentation assay, Caspase activity, Western-blot, qRT-PCR and flow cytometry analysis. 16 saanen goats randomly divided into four groups were fed on 0%, 40%, 60% and 80% E. adenophorum diets. The Results showed that E. adenophorum induced typical apoptotic features of renal cells. E. adenophorum significantly suppressed renal cells viability, caused cell cycle activity arrest and induced typical apoptotic features in a dose-dependent manner. However, the protein levels of Fas/FasL, Bid and caspase-8 did not appear significant changes in the process of E. adenophorum-induced apoptosis. Moreover, E. adenophorum administration slightly decreased Bcl-2 expression, promoted Bax translocation to mitochondria, triggered the release of Cyt c from mitochondria into cytosol and activated caspase-9, -3, and cleaved PARP. The mitochondrial p53 translocation was significantly activated, accompanied by a significant increase in the loss of ΔΨm, Cyt c release and caspase-9 activation. Above all, these data suggest that E. adenophorum induces renal cells apoptosis via the activation of mitochondria-mediated apoptosis pathway in renal cells. These findings may provide new insights to understand the mechanisms involved in E. adenophorum-caused cytotoxicity of renal cells.

  19. Crude extract of garlic induced caspase-3 gene expression leading to apoptosis in human colon cancer cells.

    Science.gov (United States)

    Su, Chin-Cheng; Chen, Guang-Wei; Tan, Tzu-Wei; Lin, Jaung-Gung; Chung, Jing-Gung

    2006-01-01

    Garlic (Allium sativum) is a popular spice, a remedy for a variety of ailments and is also known for its medicinal uses as an antibiotic, antithrombotic and antineoplastic agent. Epidemiological and animal studies have shown that garlic consumption reduces the incidence of cancer e.g. in the stomach, colon, breast and cervix. The aim of this study was to investigate whether garlic extract has any influence on caspase-3 activity and gene expression and on the signal induction of apoptosis in vitro. As an assay system, the flow cytometry assay, Western blotting and cDNA microarray were applied in human colon cancer colo 205 cells. Our results indicated that garlic extract, when administered to the colo 205 cell cultures, reduced the percetange of viable cells, induced apoptosis, increased the levels of Bax, cytochrome c and caspase-3, but decreased the level of Bcl-2. The results also showed that raw extract of garlic decreased the mitochondrial membrane potential and increased the caspase-3 activity and gene expression. We conclude that crude extract of garlic can induce apoptosis in colo 205 cells through caspase -3 activity, by means of a mitochondrial-dependent mechanism.

  20. The viral nucleocapsid protein of transmissible gastroenteritis coronavirus (TGEV) is cleaved by caspase-6 and -7 during TGEV-induced apoptosis.

    Science.gov (United States)

    Eléouët, J F; Slee, E A; Saurini, F; Castagné, N; Poncet, D; Garrido, C; Solary, E; Martin, S J

    2000-05-01

    The transmissible gastroenteritis coronavirus (TGEV), like many other viruses, exerts much of its cytopathic effect through the induction of apoptosis of its host cell. Apoptosis is coordinated by a family of cysteine proteases, called caspases, that are activated during apoptosis and participate in dismantling the cell by cleaving key structural and regulatory proteins. We have explored the caspase activation events that are initiated upon infection of the human rectal tumor cell line HRT18 with TGEV. We show that TGEV infection results in the activation of caspase-3, -6, -7, -8, and -9 and cleavage of the caspase substrates eIF4GI, gelsolin, and alpha-fodrin. Surprisingly, the TGEV nucleoprotein (N) underwent proteolysis in parallel with the activation of caspases within the host cell. Cleavage of the N protein was inhibited by cell-permeative caspase inhibitors, suggesting that this viral structural protein is a target for host cell caspases. We show that the TGEV nucleoprotein is a substrate for both caspase-6 and -7, and using site-directed mutagenesis, we have mapped the cleavage site to VVPD(359) downward arrow. These data demonstrate that viral proteins can be targeted for destruction by the host cell death machinery.

  1. 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.

  2. The structure of the BIR3 domain of cIAP1 in complex with the N-terminal peptides of SMAC and caspase-9

    Energy Technology Data Exchange (ETDEWEB)

    Kulathila, Raviraj; Vash, Brian; Sage, David; Cornell-Kennon, Susan; Wright, Kirk; Koehn, James; Stams, Travis; Clark, Kirk; Price, Allen ((Novartis)); ((Emmanuel))

    2009-06-24

    The inhibitor of apoptosis protein (IAP) family of molecules inhibit apoptosis through the suppression of caspase activity. It is known that the XIAP protein regulates both caspase-3 and caspase-9 through direct protein-protein interactions. Specifically, the BIR3 domain of XIAP binds to caspase-9 via a 'hotspot' interaction in which the N-terminal residues of caspase-9 bind in a shallow groove on the surface of XIAP. This interaction is regulated via SMAC, the N-terminus of which binds in the same groove, thus displacing caspase-9. The mechanism of suppression of apoptosis by cIAP1 is less clear. The structure of the BIR3 domain of cIAP1 (cIAP1-BIR3) in complex with N-terminal peptides from both SMAC and caspase-9 has been determined. The binding constants of these peptides to cIAP1-BIR3 have also been determined using the surface plasmon resonance technique. The structures show that the peptides interact with cIAP1 in the same way that they interact with XIAP: both peptides bind in a similar shallow groove in the BIR3 surface, anchored at the N-terminus by a charge-stabilized hydrogen bond. The binding data show that the SMAC and caspase-9 peptides bind with comparable affinities (85 and 48 nM, respectively).

  3. Ultrafine particles of Ulmus davidiana var. japonica induce apoptosis of gastric cancer cells via activation of caspase and endoplasmic reticulum stress.

    Science.gov (United States)

    Ahn, Joungjwa; Lee, Jong Suk; Yang, Kyung Mi

    2014-06-01

    Small-sized particles are more suitable for targeted delivery and are therapeutically more effective than large-sized particles. In this study, we investigated the anticancer effects of ultrafine particles of Ulmus davidiana var. japonica (ufUJ) on human gastric cancer cell lines SNU-1, SNU-216, and SNU-484. ufUJ induced apoptosis by the proteolytic activation of caspase-9, caspase-6, and caspase-3 and cleavage of poly (ADP-ribose) polymerase. The expression levels of the endoplasmic reticulum stress-related protein BiP markedly increased after ufUJ treatment. BiP knockdown decreased ufUJ-induced cell death. ufUJ-induced apoptosis was inhibited by the caspase-3 inhibitor z-DEVD-fmk, caspase-6 inhibitor z-VEID-fmk, and caspase-9 inhibitor z-LEHD-fmk, and by siRNAs against caspases 3, 6, and 9. Gastric cancer cells did not show anchorage-independent growth in the presence of ufUJ. However, cells treated with caspase inhibitors showed an enhanced colony-forming ability. These findings may be helpful in the prevention of gastric cancer and in the development of functional foods.

  4. 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

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

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    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. © 2016 Wiley Periodicals, Inc.

  6. The cytotoxic macrolide FD-891 induces caspase-8-dependent mitochondrial release of cytochrome c and subsequent apoptosis in human leukemia Jurkat cells.

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    Inaba, Susumu; Eguchi, Tadashi; Motegi, Atsushi; Mizoue, Kazutoshi; Usui, Takeo; Nagai, Kazuo; Kataoka, Takao

    2009-09-01

    The 16-membered macrolide FD-891 exerts cytotoxicity toward several cancer cell lines. In this study, we showed that FD-891 induces apoptosis in various human cancer cell lines. Human leukemia Jurkat cells were highly sensitive to FD-891, exhibiting caspase activation and mitochondrial release of cytochrome c into the cytosol at early time points after exposure to FD-891. By contrast, Jurkat cells deficient in caspase-8 were resistant to FD-891-induced apoptosis and manifested little induction of cytochrome c release as well as caspase-9 processing. Consistent with these results, the overexpression of the Bcl-2 family member Bcl-x(L) or the caspase-8 modulator c-FLIP(L) markedly prevented FD-891-induced apoptosis. These results clearly demonstrate that FD-891 triggers caspase-8-dependent mitochondrial release of cytochrome c and subsequent apoptosis in Jurkat cells.

  7. Lack of caspase-3 attenuates immobilization-induced muscle atrophy and loss of tension generation along with mitigation of apoptosis and inflammation

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    Zhu, Shimei; Nagashima, Michio; Khan, Mahammad A.S; Yasuhara, Shingo; Kaneki, Masao; Jeevendra Martyn, J. A.

    2012-01-01

    Introduction Immobilization by casting induces disuse muscle atrophy (DMA). Methods Using wild type (WT) and caspase-3 knockout (KO) mice, we evaluated the effect of caspase-3 on muscle mass, apoptosis and inflammation during DMA. Results Caspase-3 deficiency significantly attenuated muscle mass decrease [gastrocnemius: 28 ± 1% in KO vs. 41 ± 3% in WT; soleus: 47 ± 2% in KO vs. 56 ± 2% in WT; (P immobilized versus contralateral hindlimb. Lack of caspase-3 decreased immobilization-induced increased apoptotic myonuclei (3.2-fold) and macrophage infiltration (2.2-fold) in soleus muscle and attenuated increased monocyte chemoattractant protein-1 mRNA expression (2-fold in KO vs. 18-fold in WT) in gastrocnemius. Conclusion Caspase-3 plays a key role in DMA and associated decreased tension, presumably by acting on the apoptosis and inflammation pathways. PMID:23401051

  8. 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.

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

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

    2015-01-01

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

  10. Microbiota Composition, HSP70 and Caspase-3 Expression as Marker for Colorectal Cancer Patients in Aceh, Indonesia

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    Fauzi Yusuf

    2017-02-01

    Full Text Available Aim: to investigate the relationship between microbiota composition with HSP70 and Caspase-3 expressions in colon tissue as an initial study to develop the candidate for early detection of colorectal cancer for Indonesian patients. Methods: this is a cross-sectional study on 32 patients undergoing colonoscopy; 16 patients of colorectal cancer (CRC while the other 16 patients are not (colitis and internal hemorrhoid. The composition of microbiota in stool samples was examined using 16S rRNA Denaturing Gradient Gel Electrophoresis (DDGE while expression of HSP70 was examined by immunohistochemistry and Caspase-3 by using Haematoxylin-Eosin(HE staining to determine the morphological changes in colon tissue. Results: analysis of PCR-DDGE shows a different composition of microbiota between patients with CRC and non-CRC. All CRC patients showed disappearance of dominant band from Bifidobacterium groups. Histological observation based on Inter Class Correlation (ICC test from all slide showed a high scores (5.2-9.2 in CRC patients and low scores (1.7-2.4 in non-CRC patients. HSP70 expression was increased significantly in CRC patients with the highest percentage of 84%, while expression of caspase-3 decreased with the highest percentage of 21%. Statistical analysis showed that the incidence of colorectal cancer was associated with the expression of HSP 70 (p<0.001, and Caspase 3 (p<0.001. Conclusion: bifidobacterium is an important indicator for colorectal cancer patients that show disappearance of dominant band, while expression of HSP70 increased and the Caspase-3 expression decreased significantly.

  11. p38 Activation Is Required Upstream of Potassium Current Enhancement and Caspase Cleavage in Thiol Oxidant-Induced Neuronal Apoptosis

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    McLaughlin, BethAnn; Pal, Sumon; Tran, Minhnga P.; Parsons, Andrew A.; Barone, Frank C.; Erhardt, Joseph A.; Aizenman, Elias

    2013-01-01

    Oxidant-induced neuronal apoptosis has been shown to involve potassium and zinc dysregulation, energetic dysfunction, activation of stress-related kinases, and caspase cleavage. The temporal ordering and interdependence of these events was investigated in primary neuronal cultures exposed to the sulfhydryl oxidizing agent 2,2′-dithiodipyridine (DTDP), a compound that induces the intracellular release of zinc. We previously observed that tetraethylammonium (TEA), high extracellular potassium, or cysteine protease inhibitors block apoptosis induced by DTDP. We now report that both p38 and extracellular signal-regulated kinase phosphorylation are evident in neuronal cultures within 2 hr of a brief exposure to 100 μm DTDP. However, only p38 inhibition is capable of blocking oxidant-induced toxicity. Cyclohexamide or actinomycin D does not attenuate DTDP-induced cell death, suggesting that posttranslational modification of existing targets, rather than transcriptional activation, is responsible for the deleterious effects of p38. Indeed, an early robust increase in TEA-sensitive potassium channel currents induced by DTDP is attenuated by p38 inhibition but not by caspase inhibition. Moreover, we found that activation of p38 is required for caspase 3 and 9 cleavage, suggesting that potassium currents enhancement is required for caspase activation. Finally, we observed that DTDP toxicity could be blocked with niacinamide or benzamide, inhibitors of poly (ADP-ribose) synthetase. Based on these findings, we conclude that oxidation of sulfhydryl groups on intracellular targets results in intracellular zinc release, p38 phosphorylation, enhancement of potassium currents, caspase cleavage, energetic dysfunction, and translationally independent apoptotic cell death. PMID:11331359

  12. A ROS-dependent and Caspase-3-mediated apoptosis in sheep bronchial epithelial cells in response to Mycoplasma Ovipneumoniae infections.

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    Xue, Di; Li, Yanan; Jiang, Zhongjia; Deng, Guangcun; Li, Min; Liu, Xiaoming; Wang, Yujiong

    2017-05-01

    Mycoplasma Ovipneumoniae (M. ovipneumoniae) is a primary etiological agent of enzootic pneumonia in sheep and goats. It can enter and colonize ovine respiratory epithelial cells to establish an infection, which leads a serious cell death of epithelial cells. However, the nature of the interaction between pathogen of M. ovipneumoniae and host cells in the cell injury is currently not well understood. In this study, we investigated the epithelial cell apoptosis caused by an infection of M. ovipneumoniae in sheep primary air-liquid interface (ALI) epithelial cultures. The results showed that M. ovipneumoniae could specifically bind to ciliated cells at early stage of infection. Flow cytometric analysis demonstrated that an infection of M. ovipneumoniae induced a time-dependent cell apoptotic cell death, accompanied with an increased production of extracellular nitric oxide (NO), intracellular reactive oxygen species (ROS) production and activation of caspase-3 signaling in sheep bronchial epithelial cells. The induced cell apoptosis was further confirmed by a transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assay. Interestingly, the M. ovipneumoniae-induced apoptosis and activation of caspase-3 were correlated with the production of ROS but not NO. Mechanistically, M. ovipneumoniae-induced cell apoptosis was mediated by a mechanism by increasing the expression of phosphorylation of p38 and pro-apoptotic proteins, and activating caspase-3, caspase-8 and poly ADP-ribose polymerase (PARP) cleavage. These results suggest a ROS-dependent and caspase-3-mediated cell apoptosis in sheep bronchial epithelial cells in response to M. ovipneumoniae infections. Copyright © 2017. Published by Elsevier B.V.

  13. 2-aryl benzimidazole conjugate induced apoptosis in human breast cancer MCF-7 cells through caspase independent pathway.

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    Nayak, V Lakshma; Nagesh, Narayana; Ravikumar, A; Bagul, Chandrakant; Vishnuvardhan, M V P S; Srinivasulu, Vunnam; Kamal, Ahmed

    2017-01-01

    Apoptosis is a representative form of programmed cell death, which has been assumed to be critical for cancer prevention. Thus, any agent that can induce apoptosis may be useful for cancer treatment and apoptosis induction is arguably the most potent defense against cancer promotion. In our previous studies, 2-aryl benzimidazole conjugates were synthesized and evaluated for their antiproliferative activity and one of the new molecule (2f) was considered as a potential lead. This lead molecule showed significant antiproliferative activity against human breast cancer cell line, MCF-7. The results of the present study revealed that this compound arrested the cell cycle at G2/M phase. Topoisomerase II inhibition assay and Western blot analysis suggested that this compound effectively inhibits topoisomerase II activity which leads to apoptotic cell death. Apoptosis induction in MCF-7 cells was further confirmed by loss of mitochondrial membrane potential (∆Ψm), release of cytochrome c from mitochondria, an increase in the level of apoptosis inducing factor (AIF), generation of reactive oxygen species (ROS), up regulation of proapoptotic protein Bax and down regulation of anti apoptotic protein Bcl-2. Apoptosis assay using Annexin V-FITC assay also suggested that this compound induced cell death by apoptosis. However, compound 2f induced apoptosis could not be reversed by Z-VAD-FMK (a pan-caspase inhibitor) demonstrated that the 2f induced apoptosis was caspase independent. Further, 2f treatment did not activate caspase-7 and caspase-9 activity, suggesting that this compound induced apoptosis in breast cancer cells via a caspase independent pathway. Most importantly, this compound was less toxic towards non-tumorigenic breast epithelial cells, MCF-10A. Furthermore, docking studies also support the potentiality of this molecule to bind to the DNA topoisomerase II.

  14. Caspase dependent and independent mechanisms of apoptosis across gestation in a sheep model of placental insufficiency and intrauterine growth restriction.

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    Monson, Troy; Wright, Tanner; Galan, Henry L; Reynolds, Paul R; Arroyo, Juan A

    2017-05-01

    Increased placental apoptosis is a hallmark of intrauterine growth restricted (IUGR). Several molecules have been shown to be involved in the control of apoptosis during this disease. Our objective was to determine the expression of Bcl2, Bax, phospho XIAP, AIF, caspase 3 and 9, and telomerase activity across gestation in an ovine hyperthermia-induced model of IUGR. Pregnant sheep were placed in hyperthermic (HT) conditions to induce IUGR along with age-matched controls. Placental tissues were collected at 55 (early), 95 (mid-gestation) and 130 (near-term) days of gestational age (dGA) to determine the expression of apoptotic molecules during the development of IUGR. Compared to the control placenta, IGUR pregnancies showed: significantly reduced placental Bcl2 in early gestation (55 dGA) with a significant increase observed at mid gestation (95 dGA); decreased placental pXIAP at both mid and near term gestational days (95 and 130 dGA); placental AIF increased only at 55 dGA (early gestation); active caspase 3 increased at both mid and near term gestational days (95 and 130 dGA); caspase 9 only increased at mid gestation (95 dGA) and decreased Telomerase activity near term. Placental apoptosis, mediated in part by the apoptosis related molecule, participates in the development of IUGR. Findings from this study suggest a caspase-independent apoptotic pathway during early gestation and caspase-dependent apoptosis at mid and near term gestation. The data also implicate decreased activation of XIAP as a plausible factor involved in the control of placental apoptosis during IUGR.

  15. Unveiling interactions among mitochondria, caspase-like proteases, and the actin cytoskeleton during plant programmed cell death (PCD.

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    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.

  16. Geranylated 4-Phenylcoumarins Exhibit Anticancer Effects against Human Prostate Cancer Cells through Caspase-Independent Mechanism.

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    Noor Shahirah Suparji

    Full Text Available Geranylated 4-phenylcoumarins, DMDP-1 & -2 isolated from Mesua elegans were investigated for anticancer potential against human prostate cancer cells. Treatment with DMDP-1 & -2 resulted in cell death in a time and dose dependent manner in an MTT assay on all cancer cell lines tested with the exception of lung adenocarcinoma cells. DMDP-1 showed highest cytotoxic efficacy in PC-3 cells while DMDP-2 was most potent in DU 145 cells. Flow cytometry indicated that both coumarins were successful to induce programmed cell death after 24 h treatment. Elucidation on the mode-of-action via protein arrays and western blotting demonstrated death induced without any significant expressions of caspases, Bcl-2 family proteins and cleaved PARP, thus suggesting the involvement of caspase-independent pathways. In identifying autophagy, analysis of GFP-LC3 showed increased punctate in PC-3 cells pre-treated with CQ and treated with DMDP-1. In these cells decreased expression of autophagosome protein, p62 and cathepsin B further confirmed autophagy. In contrary, the DU 145 cells pre-treated with CQ and treated with DMDP-2 has reduced GFP-LC3 punctate although the number of cells with obvious GFP-LC3 puncta was significantly increased in the inhibitor-treated cells. The increase level of p62 suggested leakage of cathepsin B into the cytosol to trigger potential downstream death mediators. This correlated with increased expression of cathepsin B and reduced expression after treatment with its inhibitor, CA074. Also auto-degradation of calpain-2 upon treatment with DMDP-1 &-2 and its inhibitor alone, calpeptin compared with the combination treatment, further confirmed involvement of calpain-2 in PC-3 and DU 145 cells. Treatment with DMDP-1 & -2 also showed up-regulation of total and phosphorylated p53 levels in a time dependent manner. Hence, DMDP-1 & -2 showed ability to activate multiple death pathways involving autophagy, lysosomal and endoplasmic reticulum death

  17. Expressão da CASPASE-3 e CD-34 no adenocarcinoma de próstata

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    Vicente Paulo da Motta

    Full Text Available OBJETIVO: 1. Avaliar em qual percentual as células tumorais se marcam com caspase-3 e CD-34; 2. quantificá-los nas células tumorais; 3. verificar correlação entre quantificação e grau de malignidade tumoral; 4. correlacioná-los entre si. MÉTODOS: Estudaram-se 38 blocos com adenocarcinoma, classificados por Gleason e marcação imunoistoquímica para caspase-3 e CD-34. As proteínas imunomarcadas foram quantificadas no software Immuno do Sistema Samba 4000 de citofotometria de imagem, pelo índice de marcagem e densidade óptica. RESULTADOS: Imunomarcou-se 25 lâminas para caspase-3 e 34 para CD-34. As quantificações da caspase-3 para o índice de marcagem foram acima de 50 em 76% e, para densidade óptica, abaixo de 50 para 96%. Em relação ao CD-34, índice de marcagem foi acima de 50 em 59% e densidade óptica abaixo de 50 em 56%. As correlações entre expressões dos marcadores e a gravidade do tumor, assim como entre os marcadores, não evidenciaram significância estatística. Não se mostrou relação de expressão com o score de Gleason. CONCLUSÃO: A presença caspase-3 e CD-34 foi de 73,5% e 100%, respectivamente; 2. caspase-3 e CD-34 apresentaram alta expressão do índice de marcagem, e baixa para densidade óptica; 3. não houve correlação entre as quantificações com a classificação de Gleason; 4. não houve correlação das expressões dos dois marcadores entre si.

  18. Caspase-1 deficient mice are more susceptible to influenza A virus infection with PA variation.

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    Huang, Chih-Heng; Chen, Chi-Jene; Yen, Chia-Tsui; Yu, Cheng-Ping; Huang, Peng-Nien; Kuo, Rei-Lin; Lin, Sue-Jane; Chang, Cheng-Kai; Shih, Shin-Ru

    2013-12-01

    Reassortment within polymerase genes causes changes in the pathogenicity of influenza A viruses. We previously reported that the 2009 pH1N1 PA enhanced the pathogenicity of seasonal H1N1. We examined the effects of the PA gene from the HPAI H5N1 following its introduction into currently circulating seasonal influenza viruses. To evaluate the role of H5N1 PA in altering the virulence of seasonal influenza viruses, we generated a recombinant seasonal H3N2 (3446) that expressed the H5N1 PA protein (VPA) and evaluated the RNP activity, growth kinetics, and pathogenicity of the reassortant virus in mice. Compared with the wild-type 3446 virus, the substitution of the H5N1 PA gene into the 3446 virus (VPA/3446) resulted in increased RNP activity and an increased replication rate in A549 cells. The recombinant VPA/3446 virus also caused more severe pneumonia in Casp 1(-/-) mice than in IL1β(-/-) and wild-type B6 mice. Although the PA from H5N1 is incidentally compatible with a seasonal H3N2 backbone, the H5N1 PA affected the virulence of seasonal H3N2, particularly in inflammasome-related innate immunity deficient mice. These findings highlight the importance of monitoring PA reassortment in seasonal flu, and confirm the role of the Caspase-1 gene in influenza pathogenesis.

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

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    Bacqueville, Daniel; Mavon, Alain

    2008-01-01

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

  20. Automatic morphological subtyping reveals new roles of caspases in mitochondrial dynamics.

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    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.

  1. Serotonin transporter protects the placental cells against apoptosis in caspase 3-independent pathway.

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    Hadden, Coedy; Fahmi, Tariq; Cooper, Anthonya; Savenka, Alena V; Lupashin, Vladimir V; Roberts, Drucilla J; Maroteaux, Luc; Hauguel-de Mouzon, Sylvie; Kilic, Fusun

    2017-12-01

    Serotonin (5-HT) and its specific transporter, SERT play important roles in pregnancy. Using placentas dissected from 18d gestational SERT-knock out (KO), peripheral 5-HT (TPH1)-KO, and wild-type (WT) mice, we explored the role of 5-HT and SERT in placental functions in detail. An abnormal thick band of fibrosis and necrosis under the giant cell layer in SERT-KO placentas appeared only moderately in TPH1-KO and minimally present in WT placentas. The majority of the changes were located at the junctional zone of the placentas in SERT. The etiology of these findings was tested with TUNEL assays. The placentas from SERT-KO and TPH1-KO showed 49- and 8-fold increase in TUNEL-positive cells without a concurrent change in the DNA repair or cell proliferation compared to WT placentas. While the proliferation rate in the embryos of TPH1-KO mice was 16-fold lower than the rate in gestational age matched embryos of WT or SERT-KO mice. These findings highlight an important role of continuous 5-HT signaling on trophoblast cell viability. SERT may contribute to protecting trophoblast cells against cell death via terminating the 5-HT signaling which changes cell death ratio in trophoblast as well as proliferation rate in embryos. However, the cell death in SERT-KO placentas is in caspase 3-independent pathway. © 2017 Wiley Periodicals, Inc.

  2. Preserved otolith organ function in caspase-3-deficient mice with impaired horizontal semicircular canal function.

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    Armstrong, Patrick A; Wood, Scott J; Shimizu, Naoki; Kuster, Kael; Perachio, Adrian; Makishima, Tomoko

    2015-06-01

    Genetically engineered mice are valuable models for elucidation of auditory and vestibular pathology. Our goal was to establish a comprehensive vestibular function testing system in mice using: (1) horizontal angular vestibulo-ocular reflex (hVOR) to evaluate semicircular canal function and (2) otolith-ocular reflex (OOR) to evaluate otolith organ function and to validate the system by characterizing mice with vestibular dysfunction. We used pseudo off-vertical axis rotation to induce an otolith-only stimulus using a custom-made centrifuge. For the OOR, horizontal slow-phase eye velocity and vertical eye position were evaluated as a function of acceleration. Using this system, we characterized hVOR and OOR in the caspase-3 (Casp3) mutant mice. Casp3 (-/-) mice had severely impaired hVOR gain, while Casp3 (+/-) mice had an intermediate response compared to WT mice. Evaluation of OOR revealed that at low-to-mid frequencies and stimulus intensity, Casp3 mutants and WT mice had similar responses. At higher frequencies and stimulus intensity, the Casp3 mutants displayed mildly reduced otolith organ-related responses. These findings suggest that the Casp3 gene is important for the proper function of the semicircular canals but less important for the otolith organ function.

  3. Preserved otolith organ function in caspase-3 deficient mice with impaired horizontal semicircular canal function

    Science.gov (United States)

    Armstrong, Patrick A; Wood, Scott J; Shimizu, Naoki; Kuster, Kael; Perachio, Adrian; Makishima, Tomoko

    2015-01-01

    Genetically engineered mice are valuable models for elucidation of auditory and vestibular pathology. Our goal was to establish a comprehensive vestibular function testing system in mice using: 1) horizontal angular vestibular-ocular reflex (hVOR) to evaluate semicircular canal function, and 2) otolith-ocular reflex (OOR) to evaluate otolith organ function, and to validate the system by characterizing mice with vestibular dysfunction. We used pseudo-off vertical axis rotation (pOVAR) to induce an otolith-only stimulus using a custom-made centrifuge. For the OOR, horizontal slow phase eye velocity (HEV) and vertical eye position (VEP) was evaluated as a function of acceleration. Using this system, we characterized hVOR and OOR in the caspase-3 (Casp3) mutant mice. Casp3 −/− mice had severely impaired hVOR gain, while Casp3 +/− mice had an intermediate response compared to WT mice. Evaluation of OOR revealed that at low to mid frequencies and stimulus intensity, Casp3 mutants and WT mice had similar responses. At higher frequencies and stimulus intensity, the Casp3 mutants displayed mildly reduced otolith organ related responses. These findings suggest that the Casp3 gene is important for the proper function of the semicircular canals but less important for the otolith organ function. PMID:25827332

  4. In Vitro Antitumor Activity of Aloperine on Human Thyroid Cancer Cells through Caspase-Dependent Apoptosis

    Directory of Open Access Journals (Sweden)

    Ying-Ray Lee

    2018-01-01

    Full Text Available The global incidence of thyroid cancer, one of the most common endocrine malignancies, is especially high among women. Although most patients with thyroid cancers exhibit a good prognosis with standard treatment, there are no effective therapies for patients with anaplastic thyroid cancers or cancers that have reached an advanced or recurrent level. Therefore, it is important to develop highly effective compounds for treating such patients. Aloperine, a natural compound isolated from Sophora alopecuroides, has been reported to possess antioxidant, anti-inflammatory, anti-neuronal injury, anti-renal injury, antitumor, anti-allergic, and antiviral properties. In this study, we show that aloperine can inhibit cell growth in human anaplastic thyroid cancers and multidrug-resistant papillary thyroid cancers. Moreover, it could suppress in vitro tumorigenesis and promote cellular apoptosis. Further analysis demonstrated the involvement of caspase-dependent apoptosis, including intrinsic and/or extrinsic pathways, in aloperine-induced cellular apoptosis. However, cell cycle regulation was not detected with aloperine treatment. This study suggests the potential therapeutic use of aloperine in human anaplastic thyroid cancers and multidrug-resistant papillary thyroid cancers.

  5. Caspase-1 dependent IL-1β secretion is critical for host defense in a mouse model of Chlamydia pneumoniae lung infection.

    Directory of Open Access Journals (Sweden)

    Kenichi Shimada

    Full Text Available Chlamydia pneumoniae (CP is an important human pathogen that causes atypical pneumonia and is associated with various chronic inflammatory disorders. Caspase-1 is a key component of the 'inflammasome', and is required to cleave pro-IL-1β to bioactive IL-1β. Here we demonstrate for the first time a critical requirement for IL-1β in response to CP infection. Caspase-1⁻/⁻ mice exhibit delayed cytokine production, defective clearance of pulmonary bacteria and higher mortality in response to CP infection. Alveolar macrophages harbored increased bacterial numbers due to reduced iNOS levels in Caspase-1⁻/⁻ mice. Pharmacological blockade of the IL-1 receptor in CP infected wild-type mice phenocopies Caspase-1-deficient mice, and administration of recombinant IL-1β rescues CP infected Caspase-1⁻/⁻ mice from mortality, indicating that IL-1β secretion is crucial for host immune defense against CP lung infection. In vitro investigation reveals that CP-induced IL-1β secretion by macrophages requires TLR2/MyD88 and NLRP3/ASC/Caspase-1 signaling. Entry into the cell by CP and new protein synthesis by CP are required for inflammasome activation. Neither ROS nor cathepsin was required for CP infection induced inflammasome activation. Interestingly, Caspase-1 activation during CP infection occurs with mitochondrial dysfunction indicating a possible mechanism involving the mitochondria for CP-induced inflammasome activation.

  6. Efek Perlakuan Logam Berat Kadmium Terhadap Apoptosis Melalui Aktivasi Caspase-3 Bulu Babi Deadema Setosum: Aplikasi Biomonitoring Pencemaran Di Perairan Laut (Effect of Cadmium Heavy Metal Treatment Toward Apoptosis Through Caspase-3 Activation of Sea)

    OpenAIRE

    Rumahlatu, Dominggus; Corebima, Aloysius Duran; Amin, Mohamad; Rohman, Fatchur

    2014-01-01

    Cadmium which accumulated in animals could cause carcinogenic, mutagenic and teratogenic. In this research, non factorial experiments conducted in Complete Random Design (CRD) 6 level with 7 replication for 4 weeks, in order to examine effect of Cd heavy metal treatment toward apoptosis through caspase-3 protein activation in sea urchin Deadema setosum. Research conducted in LIPI Ambon laboratory using 6 aquarium tank size 100 x 60 x 70 cm3. Each tank was filled with 200 litres of sea waters...

  7. EFEK PERLAKUAN LOGAM BERAT KADMIUM TERHADAP APOPTOSIS MELALUI AKTIVASI CASPASE-3 BULU BABI Deadema setosum: APLIKASI BIOMONITORING PENCEMARAN DI PERAIRAN LAUT (Effect of Cadmium Heavy Metal Treatment Toward Apoptosis Through Caspase-3 Activation of Sea)

    OpenAIRE

    Rumahlatu, Dominggus; Corebima, Aloysius Duran; Amin, Mohamad; Rohman, Fatchur

    2014-01-01

    ABSTRAK Kadmium yang terakumulasi pada hewan dapat menyebabkan karsinogenik, mutagenik, dan teratogenik. Pada penelitian ini, dilakukan secara eksperimen nonfaktorial dalam RAL 6 level dengan 7 ulangan selama 4 minggu, untuk mengkaji efek perlakuan logam berat Cd terhadap apoptosis melalui aktivasi protein caspase-3 bulu babi Deadema setosum. Penelitian dilakukan di laboratorium Balai LIPI Ambon dalam 6 bak aquarium berukuran 100 x 60 x 70 cm3.Tiap bak perlakuan diisi 200 L air laut yang ...

  8. 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.

  9. Smac Mimetic Bypasses Apoptosis Resistance in FADD- or Caspase-8-Deficient Cells by Priming for Tumor Necrosis Factor α-Induced Necroptosis12

    Science.gov (United States)

    Laukens, Bram; Jennewein, Claudia; Schenk, Barbara; Vanlangenakker, Nele; Schier, Alexander; Cristofanon, Silvia; Zobel, Kerry; Deshayes, Kurt; Vucic, Domagoj; Jeremias, Irmela; Bertrand, Mathieu JM; Vandenabeele, Peter; Fulda, Simone

    2011-01-01

    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. PMID:22028622

  10. Smac mimetic bypasses apoptosis resistance in FADD- or caspase-8-deficient cells by priming for tumor necrosis factor α-induced necroptosis.

    Science.gov (United States)

    Laukens, Bram; Jennewein, Claudia; Schenk, Barbara; Vanlangenakker, Nele; Schier, Alexander; Cristofanon, Silvia; Zobel, Kerry; Deshayes, Kurt; Vucic, Domagoj; Jeremias, Irmela; Bertrand, Mathieu J M; Vandenabeele, Peter; Fulda, Simone

    2011-10-01

    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.

  11. Caspase-independent cell death mediated by apoptosis-inducing factor (AIF) nuclear translocation is involved in ionizing radiation induced HepG2 cell death

    International Nuclear Information System (INIS)

    Sun, Hengwen; Yang, Shana; Li, Jianhua; Zhang, Yajie; Gao, Dongsheng; Zhao, Shenting

    2016-01-01

    Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. The aim of radiotherapy is to eradicate cancer cells with ionizing radiation. Except for the caspase-dependent mechanism, several lines of evidence demonstrated that caspase-independent mechanism is directly involved in the cell death responding to irradiation. For this reason, defining the contribution of caspase-independent molecular mechanisms represents the main goal in radiotherapy. In this study, we focused on the role of apoptosis-inducing factor (AIF), the caspase-independent molecular, in ionizing radiation induced hepatocellular carcinoma cell line (HepG2) cell death. We found that ionizing radiation has no function on AIF expression in HepG2 cells, but could induce AIF release from the mitochondria and translocate into nuclei. Inhibition of AIF could reduce ionizing radiation induced HepG2 cell death. These studies strongly support a direct relationship between AIF nuclear translocation and radiation induced cell death. What's more, AIF nuclear translocation is caspase-independent manner, but not caspase-dependent manner, in this process. These new findings add a further attractive point of investigation to better define the complex interplay between caspase-independent cell death and radiation therapy. - Highlights: • AIF nuclear translocation is involved in ionizing radiation induced hepatocellular carcinoma cell line HepG2 cell death. • AIF mediated cell death induced by ionizing radiation is caspase-independent. • Caspase-independent pathway is involved in ionzing radiation induced HepG2 cell death.

  12. Caspase-independent cell death mediated by apoptosis-inducing factor (AIF) nuclear translocation is involved in ionizing radiation induced HepG2 cell death

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Hengwen [Department of Radiation, Cancer Center of Guangdong General Hospital (Guangdong Academy of Medical Science), Guangzhou, 510080, Guangdong (China); Yang, Shana; Li, Jianhua [Department of Physiology, Guangzhou Medical University, Guangzhou, 510182, Guangdong (China); Zhang, Yajie [Department of Pathology, Guangzhou Medical University, Guangzhou, 510182, Guangdong (China); Gao, Dongsheng [Department of Oncology, Guangdong Medical College Affiliated Pengpai Memorial Hospital, Hai Feng, 516400, Gungdong (China); Zhao, Shenting, E-mail: zhaoshenting@126.com [Department of Physiology, Guangzhou Medical University, Guangzhou, 510182, Guangdong (China)

    2016-03-25

    Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. The aim of radiotherapy is to eradicate cancer cells with ionizing radiation. Except for the caspase-dependent mechanism, several lines of evidence demonstrated that caspase-independent mechanism is directly involved in the cell death responding to irradiation. For this reason, defining the contribution of caspase-independent molecular mechanisms represents the main goal in radiotherapy. In this study, we focused on the role of apoptosis-inducing factor (AIF), the caspase-independent molecular, in ionizing radiation induced hepatocellular carcinoma cell line (HepG2) cell death. We found that ionizing radiation has no function on AIF expression in HepG2 cells, but could induce AIF release from the mitochondria and translocate into nuclei. Inhibition of AIF could reduce ionizing radiation induced HepG2 cell death. These studies strongly support a direct relationship between AIF nuclear translocation and radiation induced cell death. What's more, AIF nuclear translocation is caspase-independent manner, but not caspase-dependent manner, in this process. These new findings add a further attractive point of investigation to better define the complex interplay between caspase-independent cell death and radiation therapy. - Highlights: • AIF nuclear translocation is involved in ionizing radiation induced hepatocellular carcinoma cell line HepG2 cell death. • AIF mediated cell death induced by ionizing radiation is caspase-independent. • Caspase-independent pathway is involved in ionzing radiation induced HepG2 cell death.

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Mingjia Tan

    2006-12-01

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

  15. Confocal microscopy and image analysis indicates a region-specific relation between active caspases and cytoplasm in ejaculated and epididymal sperm.

    Directory of Open Access Journals (Sweden)

    Susana García Vazquez

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

  16. The caspase 3 sensor Phiphilux G2D2 is activated non-specifically in S1 renal proximal tubules.

    Science.gov (United States)

    Hato, Takashi; Sandoval, Ruben; Dagher, Pierre C

    2015-01-01

    Tubular cell apoptosis is a major phenotype of cell death in various forms of acute kidney injury. Quantifying apoptosis in fixed tissues is problematic because apoptosis evolves over time and dead cells are rapidly cleared by the phagocytic system. Phiphilux is a fluorescent probe that is activated specifically by caspase 3 and does not inhibit the subsequent activity of this effector caspase. It has been used successfully to quantify apoptosis in cell culture. Here we examined the feasibility of using Phiphilux to measure renal tubular apoptosis progression over time in live animals using intravital 2-photon microscopy. Our results show that Phiphilux can detect apoptosis in S2 tubules but is activated non-specifically in S1 tubules.

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

    Science.gov (United States)

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

    2009-11-30

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

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

    Science.gov (United States)

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

    2017-01-28

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

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

    DEFF Research Database (Denmark)

    Larsen, Anna K; Hall, Arnaldur; Lundsgart, Henrik

    2013-01-01

    and design of safe and efficient nucleic acid delivery systems. Numerous methods are presently available to detect and delineate cytotoxicity and cell death-mediated signals in cell cultures. Activation of caspases is part of the classical apoptosis program and increased caspase activity is therefore a well......Cationic polyplexes and lipoplexes are widely used as artificial systems for nucleic acid delivery into the cells, but they can also induce cell death. Mechanistic understanding of cell toxicity and biological side effects of these cationic entities is essential for optimization strategies......-established hallmark of programmed cell death. Additional methods to monitor cell death-related signals must, however, also be carried out to fully define the type of cell toxicity in play. These may include methods that detect plasma membrane damage, loss of mitochondrial membrane potential, phosphatidylserine...

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

    Science.gov (United States)

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

    2006-09-01

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

  1. The non-receptor tyrosine kinase Tec controls assembly and activity of the noncanonical caspase-8 inflammasome.

    Science.gov (United States)

    Zwolanek, Florian; Riedelberger, Michael; Stolz, Valentina; Jenull, Sabrina; Istel, Fabian; Köprülü, Afitap Derya; Ellmeier, Wilfried; Kuchler, Karl

    2014-12-01

    Tec family kinases are intracellular non-receptor tyrosine kinases implicated in numerous functions, including T cell and B cell regulation. However, a role in microbial pathogenesis has not been described. Here, we identified Tec kinase as a novel key mediator of the inflammatory immune response in macrophages invaded by the human fungal pathogen C. albicans. Tec is required for both activation and assembly of the noncanonical caspase-8, but not of the caspase-1 inflammasome, during infections with fungal but not bacterial pathogens, triggering the antifungal response through IL-1β. Furthermore, we identify dectin-1 as the pathogen recognition receptor being required for Syk-dependent Tec activation. Hence, Tec is a novel innate-specific inflammatory kinase, whose genetic ablation or inhibition by small molecule drugs strongly protects mice from fungal sepsis. These data demonstrate a therapeutic potential for Tec kinase inhibition to combat invasive microbial infections by attenuating the host inflammatory response.

  2. MicroRNA-7 Enhances Subventricular Zone Neurogenesis by Inhibiting NLRP3/Caspase-1 Axis in Adult Neural Stem Cells.

    Science.gov (United States)

    Fan, Zheng; Lu, Ming; Qiao, Chen; Zhou, Yan; Ding, Jian-Hua; Hu, Gang

    2016-12-01

    α-Synuclein (α-syn) has been recognized to induce neuroinflammation and to disturb nerve repair process in Parkinson's disease. However, the potential mechanisms underlying α-syn-induced impairment of adult neurogenesis remain unclear. In the present study, A53T mutant α--synuclein transgenic (A53T tg/tg ) mice, caspase-1 knockout mice, and A53T tg/tg ;caspase-1 -/- double transgenic mice were used to prepare adult neural stem cells (ANSCs) and to investigate inflammasome-related mechanism for α-syn-impaired neurogenesis in mouse subventricular zone (SVZ). We showed that α-syn inhibited neurogenesis in the SVZ of A53T tg/tg mice and impaired proliferation and differentiation in ANSCs cultured in vitro, accompanied by reduced microRNA-7 (miR-7) expression levels. We further found that ANSC expressed NLRP3-containing inflammasome and α-syn activated both TLR4/NF-κB and NLRP3/caspase-1 signals in ANSCs. Either Nlrp3 knockdown or Caspase-1 knockout could attenuate the inhibition of proliferation in ANSCs induced by α-syn. Furthermore, we demonstrated that miR-7 post-transcriptionally controlled Nlrp3 expression besides targeting α-syn. Most notably, stereotactic injection of miR-7 mimics into lateral ventricles significantly inhibited NLRP3 inflammasome activation and improved adult neurogenesis in mouse SVZ. Our study provides a direct link between NLRP3 inflammasome activation and α-syn-impaired neurogenesis in the pathogenesis of α-synucleinopathies.

  3. 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

  4. 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

  5. Modulation of Caspase Activity Regulates Skeletal Muscle Regeneration and Function in Response to Vasopressin and Tumor Necrosis Factor

    Science.gov (United States)

    Moresi, Viviana; Garcia-Alvarez, Gisela; Pristerà, Alessandro; Rizzuto, Emanuele; Albertini, Maria C.; Rocchi, Marco; Marazzi, Giovanna; Sassoon, David; Adamo, Sergio; Coletti, Dario

    2009-01-01

    Muscle homeostasis involves de novo myogenesis, as observed in conditions of acute or chronic muscle damage. Tumor Necrosis Factor (TNF) triggers skeletal muscle wasting in several pathological conditions and inhibits muscle regeneration. We show that intramuscular treatment with the myogenic factor Arg8-vasopressin (AVP) enhanced skeletal muscle regeneration and rescued the inhibitory effects of TNF on muscle regeneration. The functional analysis of regenerating muscle performance following TNF or AVP treatments revealed that these factors exerted opposite effects on muscle function. Principal component analysis showed that TNF and AVP mainly affect muscle tetanic force and fatigue. Importantly, AVP counteracted the effects of TNF on muscle function when delivered in combination with the latter. Muscle regeneration is, at least in part, regulated by caspase activation, and AVP abrogated TNF-dependent caspase activation. The contrasting effects of AVP and TNF in vivo are recapitulated in myogenic cell cultures, which express both PW1, a caspase activator, and Hsp70, a caspase inhibitor. We identified PW1 as a potential Hsp70 partner by screening for proteins interacting with PW1. Hsp70 and PW1 co-immunoprecipitated and co-localized in muscle cells. In vivo Hsp70 protein level was upregulated by AVP, and Hsp70 overexpression counteracted the TNF block of muscle regeneration. Our results show that AVP counteracts the effects of TNF through cross-talk at the Hsp70 level. Therefore, muscle regeneration, both in the absence and in the presence of cytokines may be enhanced by increasing Hsp70 expression. PMID:19440308

  6. Modulation of caspase activity regulates skeletal muscle regeneration and function in response to vasopressin and tumor necrosis factor.

    Directory of Open Access Journals (Sweden)

    Viviana Moresi

    Full Text Available Muscle homeostasis involves de novo myogenesis, as observed in conditions of acute or chronic muscle damage. Tumor Necrosis Factor (TNF triggers skeletal muscle wasting in several pathological conditions and inhibits muscle regeneration. We show that intramuscular treatment with the myogenic factor Arg(8-vasopressin (AVP enhanced skeletal muscle regeneration and rescued the inhibitory effects of TNF on muscle regeneration. The functional analysis of regenerating muscle performance following TNF or AVP treatments revealed that these factors exerted opposite effects on muscle function. Principal component analysis showed that TNF and AVP mainly affect muscle tetanic force and fatigue. Importantly, AVP counteracted the effects of TNF on muscle function when delivered in combination with the latter. Muscle regeneration is, at least in part, regulated by caspase activation, and AVP abrogated TNF-dependent caspase activation. The contrasting effects of AVP and TNF in vivo are recapitulated in myogenic cell cultures, which express both PW1, a caspase activator, and Hsp70, a caspase inhibitor. We identified PW1 as a potential Hsp70 partner by screening for proteins interacting with PW1. Hsp70 and PW1 co-immunoprecipitated and co-localized in muscle cells. In vivo Hsp70 protein level was upregulated by AVP, and Hsp70 overexpression counteracted the TNF block of muscle regeneration. Our results show that AVP counteracts the effects of TNF through cross-talk at the Hsp70 level. Therefore, muscle regeneration, both in the absence and in the presence of cytokines may be enhanced by increasing Hsp70 expression.

  7. 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

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

    Czech Academy of Sciences Publication Activity Database

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

    2017-01-01

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

  9. Caspase-2 and oxidative stress underlie the immunogenic potential of high hydrostatic pressure-induced cancer cell death

    Czech Academy of Sciences Publication Activity Database

    Moserová, I.; Truxová, I.; Garg, A.D.; Tomala, Jakub; Agostinis, P.; Cartron, P.F.; Vošahlíková, Š.; Kovář, Marek; Spíšek, R.; Fučíková, J.

    2017-01-01

    Roč. 6, č. 1 (2017), s. 1-35, č. článku e1258505. ISSN 2162-4011 R&D Projects: GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : Caspases * ecto-CALR * ER stress Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology

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

    Science.gov (United States)

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

    2008-11-01

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

  11. Scutellaria barbate extract induces apoptosis of hepatoma H22 cells via the mitochondrial pathway involving caspase-3.

    Science.gov (United States)

    Dai, Zhi-Jun; Wang, Xi-Jing; Li, Zong-Fang; Ji, Zong-Zheng; Ren, Hong-Tao; Tang, Wei; Liu, Xiao-Xu; Kang, Hua-Feng; Guan, Hai-Tao; Song, Ling-Qin

    2008-12-28

    To study the growth inhibitory and apoptotic effects of Scutellaria barbata D.Don (S. barbata) and to determine the underlying mechanism of its antitumor activity in mouse liver cancer cell line H22. Proliferation of H22 cells was examined by MTT assay. Cellular morphology of PC-2 cells was observed under fluorescence microscope and transmission electron microscope (EM). Mitochondrial transmembrane potential was determined under laser scanning confocal microscope (LSCM) with rhodamine 123 staining. Flow cytometry was performed to analyze the cell cycle of H22 cells with propidium iodide staining. Protein level of cytochrome C and caspase-3 was measured by semi-quantitive RT-PCR and Western blot analysis. Activity of caspase-3 enzyme was measured by spectrofluorometry. MTT assay showed that extracts from S. barbata (ESB) could inhibit the proliferation of H22 cells in a time-dependent manner. Among the various phases of cell cycle, the percentage of cells in S phase was significantly decreased, while the percentage of cells in G(1) phase was increased. Flow cytometry assay also showed that ESB had a positive effect on apoptosis. Typical apoptotic morphologies such as condensation and fragmentation of nuclei and blebbing membrane of apoptotic cells could be observed under transmission electron microscope and fluorescence microscope. To further investige the molecular mechanism behind ESB-induced apoptosis, ESB-treated cells rapidly lost their mitochondrial transmembrane potential, released mitochondrial cytochrome C into cytosol, and induced caspase-3 activity in a dose-dependent manner. ESB can effectively inhibit the proliferation and induce apoptosis of H22 cells involving loss of mitochondrial transmembrane potential, release of cytochrome C, and activation of caspase-3.

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Shivakumar Subbanna

    2018-02-01

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

  14. 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

  15. Antioxidants Impair Anti-Tumoral Effects of Vorinostat, but Not Anti-Neoplastic Effects of Vorinostat and Caspase-8 Downregulation

    OpenAIRE

    Bergadà, Laura; Yeramian, Andree; Sorolla, Annabel; Matias-Guiu, Xavier; Dolcet, Xavier

    2014-01-01

    We have recently demonstrated that histone deacetylase inhibitor, Vorinostat, applied as a single therapy or in combination with caspase-8 downregulation exhibits high anti-tumoral activity on endometrial carcinoma cell lines. In the present study, we have assessed the signalling processes underlying anti-tumoral effects of Vorinostat. Increasing evidence suggests that reactive oxygen species are responsible for histone deacetylase inhibitor-induced cell killing. We have found that Vorinostat...

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

    Czech Academy of Sciences Publication Activity Database

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

    2017-01-01

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

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

    Science.gov (United States)

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

    2014-07-01

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

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  19. Antioxidants Impair Anti-Tumoral Effects of Vorinostat, but Not Anti-Neoplastic Effects of Vorinostat and Caspase-8 Downregulation

    Science.gov (United States)

    Bergadà, Laura; Yeramian, Andree; Sorolla, Annabel

    2014-01-01

    We have recently demonstrated that histone deacetylase inhibitor, Vorinostat, applied as a single therapy or in combination with caspase-8 downregulation exhibits high anti-tumoral activity on endometrial carcinoma cell lines. In the present study, we have assessed the signalling processes underlying anti-tumoral effects of Vorinostat. Increasing evidence suggests that reactive oxygen species are responsible for histone deacetylase inhibitor-induced cell killing. We have found that Vorinostat induces formation of reactive oxygen species and DNA damage. To investigate the role of oxidative stress as anti-neoplastic mechanism, we have evaluated the effects of different antioxidants (Bha, Nac and Tiron) on endometrial carcinoma cell line Ishikawa treated with Vorinostat. We show that Bha, Nac and Tiron markedly inhibited the cytotoxic effects of Vorinostat, increasing cell viability in vitro. We found that all three antioxidants did not inhibited accumulation of acetyl Histone H4, so that antioxidants did not inhibit Vorinostat activity. Finally, we have evaluated the effects of antioxidants on anti-tumoral activity of Vorinostat as monotherapy or in combination with caspase-8 downregulation in vivo. Interestingly, antioxidants blocked the reduction of tumour growth caused by Vorinostat, but they were unable to inhibit anti-tumoral activity of Vorinostat plus caspase-8 inhibition. PMID:24651472

  20. Phosphorylation of Smac by Akt promotes the caspase-3 activation during etoposide-induced apoptosis in HeLa cells.

    Science.gov (United States)

    Jeong, Chul-Ho; Chun, Kyung-Soo; Kundu, Juthika; Park, Byoungduck

    2015-02-01

    The Akt, family of serine/threonine protein kinases functions as key regulators of multiple aspects of cell behavior, such as survival, proliferation, migration, and carcinogenesis. Notably, Akt exerts its anti-apoptotic effects through the phosphorylation of numerous substrates related with cell cycle, genome stability, and cancer development. In this report, nevertheless, we focused our view on the novel role of Akt which involves in a pro-apoptotic action by phosphorylating second mitochondria derived activator of caspases (Smac) protein during etoposide-induced apoptotic processes. Our data reveals that Akt could bind to and phosphorylate Smac at serine residue 67, which enhances the ability of Smac to interact with the cytosolic X-chromosome linked IAP (XIAP) protein. The cellular interaction of wild-type Smac with XIAP was enhanced with similar activation kinetics of Akt activity, while this interaction was markedly attenuated in cells expressing the phosphorylation-defective mutant S67A-Smac during etoposide-induced apoptosis. Moreover, we provide the evidence indicating that the phosphorylation of Smac at ser-67 markedly upregulates the caspase-3 activity by promoting the interaction of Smac with XIAP. Taken together, we propose that the phosphorylation of Smac by Akt might be a novel mechanism that involves in amplification of caspase cascade pathway during etoposide-induced apoptosis in HeLa cells. © 2013 Wiley Periodicals, Inc.

  1. Dryocrassin ABBA Induces Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells Through a Caspase-Dependent Mitochondrial Pathway.

    Science.gov (United States)

    Jin, Zhe; Wang, Wen-Fei; Huang, Jian-Ping; Wang, He-Meng; Ju, Han-Xun; Chang, Ying

    2016-01-01

    Biological and pharmacological activities of dryocrassin ABBA, a phloroglucinol derivative extracted from Dryopteris crassirhizoma, have attracted attention. In this study, the apoptotic effect of dryocrassin ABBA on human hepatocellular carcinoma HepG2 cells was investigated. We tested the effects of dryocrassin ABBA on HepG2 in vitro by MTT, flow cytometry, real-time PCR, and Western blotting. KM male mice were used to detect the effect of dryocrassin ABBA on H22 cells in vivo. Dryocrassin ABBA inhibited the growth of HepG2 cells in a concentration-dependent manner. After treatment with 25, 50, and 75 μg/mL dryocrassin ABBA, the cell viability was 68%, 60% and 49%, respectively. Dryocrassin ABBA was able to induce apoptosis, measured by propidium iodide (PI)/annexin V-FITC double staining. The results of real-time PCR and Western ting showed that dryocrassin ABBA up-regulated p53 and Bax expression and inhibited Bcl-2 expression which led to an activation of caspase-3 and caspase-7 in the cytosol, and then induction of cell apoptosis. In vivo experiments also showed that dryocrassin ABBA treatment significantly suppressed tumor growth, without major side effects. Overall, these findings provide evidence that dryocrassin ABBA may induce apoptosis in human hepatocellular carcinoma cells through a caspase-mediated mitochondrial pathway.

  2. 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.

  3. Knockdown of LRP/LR induces apoptosis in pancreatic cancer and neuroblastoma cells through activation of caspases.

    Science.gov (United States)

    Chetty, Carryn J; Ferreira, Eloise; Jovanovic, Katarina; Weiss, Stefan F T

    2017-11-15

    The 37kDa/67kDa laminin receptor (LRP/LR) serves various physiological and pathological roles such as enhancing tumour-related processes including metastasis, angiogenesis, cellular viability and telomerase activation in cancerous cell lines. The present study investigates the effect of siRNA mediated downregulation of LRP/LR on pancreatic cancer (AsPC-1) and neuroblastoma (IMR-32) cells. MTT and BrdU assays revealed that siRNA mediated downregulation of LRP resulted in a significant reduction in cell viability and cell proliferation. In addition, knock-down of LRP resulted in phosphatidylserine externalization, diminished nuclear integrity and significantly enhanced caspase-3 activity, which is indicative of apoptosis. LRP downregulation resulted in a significant increase in caspase-8 activity in IMR-32 cells and enhanced caspase-8 and 9 activity in AsPC-1 cells. These data recommend siRNA mediated knock-down of LRP as a potential therapeutic avenue for the treatment of pancreatic cancer and neuroblastoma. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Purification and characterization of a cytochrome c with novel caspase-3 activation activity from the pathogenic fungus Rhizopus arrhizus.

    Science.gov (United States)

    Saxena, Manoj; Sharma, Rohit Kumar; Ramirez-Paz, Josell; Tinoco, Arthur D; Griebenow, Kai

    2015-09-03

    Members of Rhizopus species are the most common cause of mucormycosis, a rare but often fatal fungal infection. Host induced pathogen apoptosis and pathogen induced host cell apoptosis are often involved in fungal infections. In many organisms, the release of mitochondrial cytochrome c can trigger apoptosis by activating caspase proteases, but the role of fungal cytochrome c in apoptosis remains unknown. DNA sequence encoding Rhizopus arrhizus cytochrome c was cloned and expressed in E. coli. Both native and recombinant cytochrome c were purified using ion exchange followed by gel filtration chromatography. The identities of purified proteins were confirmed by MALDI-MS and UV-Visible spectroscopy. For the first time, we demonstrated that Rhizopus arrhizus cytochrome c could activate human capspase-3 in HeLa cell extracts. We also found that Rhizopus arrhizus cytochrome c has redox potential, peroxidase activity, and spectral properties similar to human and horse cytochrome c proteins. Rhizopus arrhizus cytochrome c can activate human caspase-3 in HeLa cell extracts and it possesses similar physical and spectral properties as human and horse cytochrome c. This protein was found to have a previously unknown potential to activate human caspase-3, an important step in the apoptosis cascade.

  5. Cannabidiol-induced apoptosis in primary lymphocytes is associated with oxidative stress-dependent activation of caspase-8

    International Nuclear Information System (INIS)

    Wu, H.-Y.; Chu, R.-M.; Wang, C.-C.; Lee, C.-Y.; Lin, S.-H.; Jan, T.-R.

    2008-01-01

    We recently reported that cannabidiol (CBD) exhibited a generalized suppressive effect on T-cell functional activities in splenocytes directly exposed to CBD in vitro or isolated from CBD-administered mice. To investigate the potential mechanisms of CBD effects on T cells, we characterized the pro-apoptotic effect of CBD on primary lymphocytes. The apoptosis of splenocytes was markedly enhanced following CBD exposure in a time- and concentration-dependent manner, as evidenced by nuclear hypodiploidity and DNA strand breaks. Exposure of splenocytes to CBD elicited an early production of reactive oxygen species (ROS) with the peak response at 1 h post CBD treatment. In parallel with the ROS production, a gradual diminishment in the cellular glutathione (GSH) content was detected in CBD-treated splenocytes. Both CBD-mediated ROS production and GSH diminishment were remarkably attenuated by the presence of N-acetyl-L-cysteine (NAC), a thiol antioxidant. In addition, CBD treatment significantly stimulated the activation of caspase-8, which was abrogated in the presence of NAC or GSH. Pretreatment of splenocytes with a cell-permeable inhibitor for caspase-8 significantly attenuated, in a concentration-dependent manner, CBD-mediated apoptosis, but not ROS production. Collectively, the present study demonstrated that the apoptotic effect of CBD in primary lymphocytes is closely associated with oxidative stress-dependent activation of caspase-8

  6. 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.

  7. 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.

  8. Antioxidants impair anti-tumoral effects of Vorinostat, but not anti-neoplastic effects of Vorinostat and caspase-8 downregulation.

    Science.gov (United States)

    Bergadà, Laura; Yeramian, Andree; Sorolla, Annabel; Matias-Guiu, Xavier; Dolcet, Xavier

    2014-01-01

    We have recently demonstrated that histone deacetylase inhibitor, Vorinostat, applied as a single therapy or in combination with caspase-8 downregulation exhibits high anti-tumoral activity on endometrial carcinoma cell lines. In the present study, we have assessed the signalling processes underlying anti-tumoral effects of Vorinostat. Increasing evidence suggests that reactive oxygen species are responsible for histone deacetylase inhibitor-induced cell killing. We have found that Vorinostat induces formation of reactive oxygen species and DNA damage. To investigate the role of oxidative stress as anti-neoplastic mechanism, we have evaluated the effects of different antioxidants (Bha, Nac and Tiron) on endometrial carcinoma cell line Ishikawa treated with Vorinostat. We show that Bha, Nac and Tiron markedly inhibited the cytotoxic effects of Vorinostat, increasing cell viability in vitro. We found that all three antioxidants did not inhibited accumulation of acetyl Histone H4, so that antioxidants did not inhibit Vorinostat activity. Finally, we have evaluated the effects of antioxidants on anti-tumoral activity of Vorinostat as monotherapy or in combination with caspase-8 downregulation in vivo. Interestingly, antioxidants blocked the reduction of tumour growth caused by Vorinostat, but they were unable to inhibit anti-tumoral activity of Vorinostat plus caspase-8 inhibition.

  9. 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. Copyright © 2012 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    Caro-Astorga, Joaquín; Fajardo, Ignacio; Ruiz-Pérez, María Victoria; Sánchez-Jiménez, Francisca; Urdiales, José Luis

    2014-09-05

    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. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Development of an inducible caspase-9 safety switch for pluripotent stem cell–based therapies

    Directory of Open Access Journals (Sweden)

    Chuanfeng Wu

    2014-01-01

    Full Text Available Induced pluripotent stem cell (iPSC therapies offer a promising path for patient-specific regenerative medicine. However, tumor formation from residual undifferentiated iPSC or transformation of iPSC or their derivatives is a risk. Inclusion of a suicide gene is one approach to risk mitigation. We introduced a dimerizable-“inducible caspase-9” (iCasp9 suicide gene into mouse iPSC (miPSC and rhesus iPSC (RhiPSC via a lentivirus, driving expression from either a cytomegalovirus (CMV, elongation factor-1 α (EF1α or pluripotency-specific EOS-C(3+ promoter. Exposure of the iPSC to the synthetic chemical dimerizer, AP1903, in vitro induced effective apoptosis in EF1α-iCasp9-expressing (EF1α-iPSC, with less effective killing of EOS-C(3+-iPSC and CMV-iPSC, proportional to transgene expression in these cells. AP1903 treatment of EF1α-iCasp9 miPSC in vitro delayed or prevented teratomas. AP1903 administration following subcutaneous or intravenous delivery of EF1α-iPSC resulted in delayed teratoma progression but did not ablate tumors. EF1α-iCasp9 expression was downregulated during in vitro and in vivo differentiation due to DNA methylation at CpG islands within the promoter, and methylation, and thus decreased expression, could be reversed by 5-azacytidine treatment. The level and stability of suicide gene expression will be important for the development of suicide gene strategies in iPSC regenerative medicine.

  12. Lactobacillus johnsonii N6.2 diminishes caspase-1 maturation in the gastrointestinal system of diabetes prone rats.

    Science.gov (United States)

    Teixeira, L D; Kling, D N; Lorca, G L; Gonzalez, C F

    2018-04-10

    The cells of the gastrointestinal (GI) epithelium are the first to contact the microbiota and food components. As a direct consequence of this, these cells are the first line of defence and key players in priming the immune response. One of the first responses against GI insults is the formation of the inflammasome, a multiprotein complex assembled in response to environmental threats. The formation of the inflammasome regulates caspase-1 by cleaving it into its active form. Once activated, caspase-1 can cleave interleukin-1β (IL-1β), which promotes adaptive and humoral immunity. Some strains, like Lactobacillus johnsonii N6.2, are able to modulate the biosynthesis of important host metabolites mediating inflammation. Of these metabolites are the pro-inflammatory kynurenines. L. johnsonii N6.2 is able to downregulate kynurenines biosynthesis via a redox active mechanism negatively affecting indoleamine 2,3-dioxygenase activity. In this study, we evaluated the effects of L. johnsonii N6.2 combined with the natural antioxidant and anti-inflammatory molecule rosmarinic acid (RA). Inflammasome assembly and the kynurenine pathway were evaluated in GI samples of BioBreeding diabetes-prone (BB-DP) rats. In this work, BB-DP rats were fed daily with RA, L. johnsonii N6.2; or both combined. The transcriptional rate and proteins levels of inflammasome and kynurenine pathway components in ileum tissue were evaluated. Elevated levels of pro-caspase-1 were observed in rats fed with L. johnsonii, while RA had no effect on pro-caspase-1 expression. Western blot assays demonstrated that L. johnsonii fed rats showed lower levels of mature caspase-1, when compared to the other treatments. Furthermore, IL-1β maturation followed a similar pattern across the treatments. Differences were also observed between treatments in expression levels of key enzymes in the kynurenine pathway. These findings support the role of L. johnsonii in modulating the assembly of the inflammasome as well

  13. Cordycepin enhances cisplatin apoptotic effect through caspase/MAPK pathways in human head and neck tumor cells

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    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

  14. Decreased expression of caspase3 in penis and prostate tissues of rat after the treatment with buceng (Pimpinella alpina Molk & Euricoma longifolia Jack

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    Taufiqurrachman Taufiqurrachman

    2013-02-01

    Full Text Available Background: Buceng {combination of pasak bumi (Eurycoma longifolia Jack and purwoceng (Pimpinella alpine Molk} has been proven to increase testosterone (Te level and decrease apoptosis. Unfortunately, there is no evidence whether these effects are mediated by the declining of caspase3. Objective of this study was to evaluate whether buceng could decrease the expression of caspase3 of penis and prostate cells in Sprague Dawley male rats.Methods: Twenty four Sprague Dawley male rats weighing 300 g (90 days old were randomly assigned into 4 groups of 6 male rats. Group A, rats were castrated and received buceng 50 mg. Group B, rats were not castrated, sacrifices as positive control. Group C, rats were castrated and given 2 mL aquadest as negative control. Group D, rats were castrated and got of 6.75 mg mesterolone, dissolved in 2 mL water. MANOVA statistical analysis was adopted to examine the difference expression of caspase3 in all groups. The comparison of caspase3 expression between two groups exhibiting difference values were evaluated by Post Hoc test.Results: MANOVA revealed statistically significant differences in the expression of caspase3 of penis and prostate tissues among the four groups. Post Hoct test also indicated that expression of caspase3 in group A (buceng (33.56; 35.83 was significantly lower compared to group C (negative control (54.33; 60.07 and group D (mesterolone (51.91;56.21, p = 0.000, and higher compared than group B or normal rats (29.40; 27.72, but statistically not significant (p = 0.826.Conclusion: The treatment of 50 mg buceng/day for 30 consecutive days could decrease caspase3 expression in penis and prostate cells. (Med J Indones. 2013;22:2-8Keywords: Apoptosis, buceng (Pimpinella alpine Molk – Eurycoma longifolia Jack, caspase

  15. Intervention with a caspase-1 inhibitor reduces obesity-associated hyperinsulinemia, non-alcoholic steatohepatitis and hepatic fibrosis in LDLR-/-.Leiden mice.

    Science.gov (United States)

    Morrison, M C; Mulder, P; Salic, K; Verheij, J; Liang, W; van Duyvenvoorde, W; Menke, A; Kooistra, T; Kleemann, R; Wielinga, P Y

    2016-09-01

    Non-alcoholic steatohepatitis (NASH) is a serious liver condition, closely associated with obesity and insulin resistance. Recent studies have suggested an important role for inflammasome/caspase-1 in the development of NASH, but the potential therapeutic value of caspase-1 inhibition remains unclear. Therefore, we aimed to investigate the effects of caspase-1 inhibition in the ongoing disease process, to mimic the clinical setting. To investigate effects of caspase-1 inhibition under therapeutic conditions, male LDLR-/-.Leiden mice were fed a high-fat diet (HFD) for 9 weeks to induce a pre-diabetic state before start of treatment. Mice were then continued on HFD for another 12 weeks, without (HFD) or with (HFD-YVAD) treatment with the caspase-1 inhibitor Ac-YVAD-cmk (40 mg kg(-1) per day). Nine weeks of HFD feeding resulted in an obese phenotype, with obesity-associated hypertriglyceridemia, hypercholesterolemia, hyperglycemia and hyperinsulinemia. Treatment with Ac-YVAD-cmk did not affect further body weight gain or dyslipidemia, but did attenuate further progression of insulin resistance. Histopathological analysis of livers clearly demonstrated prevention of NASH development in HFD-YVAD mice: livers were less steatotic and neutrophil infiltration was strongly reduced. In addition, caspase-1 inhibition had a profound effect on hepatic fibrosis, as assessed by histological quantification of collagen staining and gene expression analysis of fibrosis-associated genes Col1a1, Acta2 and Tnfa. Intervention with a caspase-1 inhibitor attenuated the development of NASH, liver fibrosis and insulin resistance. Our data support the importance of inflammasome/caspase-1 in the development of NASH and demonstrate that therapeutic intervention in the already ongoing disease process is feasible.

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

    Science.gov (United States)

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

    2017-01-01

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

  17. Bacterial GroEL-like heat shock protein 60 protects epithelial cells from stress-induced death through activation of ERK and inhibition of caspase 3.

    Science.gov (United States)

    Zhang, Liangxuan; Pelech, Steven; Uitto, Veli-Jukka

    2004-01-01

    Bacterial heat shock proteins (hsps) can have various effects on human cells. We investigated whether bacterial hsp60s can protect epithelial cells from cell death by affecting the mitogen-activated protein kinase (MAPK) signal pathways. Cell protection was studied by adding bacterial hsp60s to skin keratinocyte cultures (HaCaT cell line) before UV radiation. The results show that hsp60 significantly protected against UV radiation-induced cell death. Effects of UV radiation and exogenous hsp60 on phosphorylation of MAPKs and on activation of caspase 3 were examined by Western blot analysis. UV radiation strongly induced phosphorylation of p38 MAPK and formation of active caspase 3. A p38 inhibitor, SB 203580, totally blocked UV radiation-mediated activation of caspase 3. Preincubation with hsp60 strongly induced phosphorylation of ERK1/2 and inhibited UV radiation-mediated activation of caspase 3. PD 98059, a specific inhibitor of the ERK1/2 pathway, blocked this inhibitory effect of exogenous hsp60. Studies on the association between activity of MAPKs or caspase 3 and cell death showed that the ERK1/2 pathway inhibitor reversed protective effect of hsp60 while specific inhibition of p38 and caspase 3 reduced cell death. These results indicate that in HaCaT cells UV radiation mediates cell death through activation of p38 followed by caspase 3 activation. Exogenous hsp60 partially protects against UV radiation-mediated epithelial cell death through activation of ERK1/2, which inhibits caspase 3 activation.

  18. 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

  19. Predictive value of serum gelsolin in hepatitis B virus (HBV)-related ...

    African Journals Online (AJOL)

    Gelsolin, an actin-binding protein, which serves as a substrate of caspase in tissue injury has been proposed as a prognostic marker in acute liver injury, but its relationship with human hepatitis B virus (HBV)-related hepatitis at various stages is still unclear. This study was conducted in order to investigate the predictive ...

  20. 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

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    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.

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

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    Yang Liu

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

  2. 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.

  3. Artemisinin induces A549 cell apoptosis dominantly via a reactive oxygen species-mediated amplification activation loop among caspase-9, -8 and -3.

    Science.gov (United States)

    Gao, Weijie; Xiao, Fenglian; Wang, Xiaoping; Chen, Tongsheng

    2013-10-01

    This report is designed to explore the roles of caspase-8, -9 and -3 in artemisinin (ARTE)-induced apoptosis in non-small cell lung cancer cells (A549 cells). ARTE induced reactive oxygen species (ROS)-mediated apoptosis in dose- and time-dependent fashion. Although ARTE treatment did not induce Bid cleavage and significant loss of mitochondrial membrane potential, it induced release of Smac and AIF but not cytochrome c from mitochondria, and silencing of Bak but not Bax significantly prevented ARTE-induced cytotoxicity. Moreover, ARTE treatment induced ROS-dependent activation of caspase-9, -8 and -3. Of the utmost importance, silencing or inhibiting any one of caspase-8, -9 and -3 almost completely prevented ARTE-induced activation of all the three caspases and remarkably abrogated the cytotoxicity of ARTE, suggesting that ARTE triggered an amplification activation loop among caspase-9, -8 and -3. Collectively, our data demonstrate that ARTE induces a ROS-mediated amplification activation loop among caspase-9, -8 and -3 to dominantly mediate the apoptosis of A549 cells.

  4. Apoptosis-Inducing Factor Participation in Bovine Macrophage Mycobacterium bovis-Induced Caspase-Independent Cell Death▿

    Science.gov (United States)

    Vega-Manriquez, X.; López-Vidal, Y.; Moran, J.; Adams, L. G.; Gutiérrez-Pabello, J. A.

    2007-01-01

    Mycobacterium tuberculosis complex species survive and replicate in phagosomes of the host cell. Cell death (CD) has been highlighted as one of the probable outcomes in this host-pathogen interaction. Previously, our group demonstrated macrophage apoptosis as a consequence of Mycobacterium bovis infection. In this study, we aimed to identify the contribution of apoptotic effector elements in M. bovis-induced CD. Bovine macrophages were either infected with M. bovis (multiplicity of infection, 10:1) or treated with an M. bovis cell extract (CFE). Structural changes compatible with CD were evaluated. Chromatin condensation was increased three times by the CFE. On the other hand, a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay demonstrated that levels of DNA fragmentation induced by M. bovis and CFE were 53.7% ± 24% and 38.9% ± 14%, respectively, whereas control cells had a basal proportion of 8.9% ± 4.1%. Rates of DNA fragmentation were unaffected by the presence of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (z-VAD). Cells treated with 100 μg of CFE for 12 h had a fivefold decrease in the level of mitochondrial outer membrane permeabilization compared to that of untreated cells. Neither M. bovis infection nor CFE treatment induced activation of caspase 3, 8, or 9. Translocation of apoptosis-inducing factor (AIF) to the nucleus was identified in 32% ± 3.5% and 26.3% ± 4.9% of M. bovis-infected and CFE-treated cells, respectively. Incubation of macrophages with z-VAD prior to infection did not alter the percentage of cells showing AIF translocation. Our data suggest that M. bovis-induced CD in bovine macrophages is caspase independent with AIF participation. PMID:17158896

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

    International Nuclear Information System (INIS)

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

    2015-01-01

    Angelica sinensis polysaccharide (ASP) is purified from the fresh roots of Angelica sinensis (AS). This traditional Chinese medicine has been used for thousands of years for treating gynecological diseases and used in functional foods for the prevention and treatment of various diseases, such as inflammation and cancer. The antitumor activity of ASP is related to its biological activities, because it suppresses a variety of pro-proliferative or anti-apoptotic factors that are dramatically expressed in cancer cells of given types. In this study, we show that angelica sinensis polysaccharide induced apoptosis in breast cancer cells of T47D over-expressing the Cyclic AMP response element binding protein (CREB), inducing apoptosis-related signaling pathway activity. The result also found that ASP caused cell death was linked to caspase activity, accompanied by the loss of mitochondrial membrane potential, cytochrome c release, and Bax translocation from the cytosol to the mitochondria. We found that ASP significantly affected the poly-ADP-ribose polymerase (PARP), Bcl-2 Associated X Protein (Bax), Bcl-2, Bcl-xL and apoptotic protease activating facter-1 (Apaf1) protein expression in a dose- and time-dependent manner. DAPI staining and Flow cytometry were used to analyze apoptosis. The nude mice xenograft model was used to evaluate the antitumor effect of ASP in vivo. ASP has profound antitumor effect on T47D cells, probably by inducing apoptosis through CREB signaling pathway. Thus, these results suggest that ASP would be a promising therapeutic agent for breast cancer. - Highlights: • CREB and Caspase-3 signaling pathways are involved in the ASP induced breast cancer cells apoptosis. • ROCK1/Mlc signaling pathway plays a critical role in this ASP-mediated apoptosis. • Angelica sinensis polysaccharide (ASP) affected the PARP, Bax, Bcl-2, Bcl-xL and Apaf1 protein expression. • The activation of CREB and ROCK1 promotes caspase-3 activation and apoptosis induced

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-11-20

    Angelica sinensis polysaccharide (ASP) is purified from the fresh roots of Angelica sinensis (AS). This traditional Chinese medicine has been used for thousands of years for treating gynecological diseases and used in functional foods for the prevention and treatment of various diseases, such as inflammation and cancer. The antitumor activity of ASP is related to its biological activities, because it suppresses a variety of pro-proliferative or anti-apoptotic factors that are dramatically expressed in cancer cells of given types. In this study, we show that angelica sinensis polysaccharide induced apoptosis in breast cancer cells of T47D over-expressing the Cyclic AMP response element binding protein (CREB), inducing apoptosis-related signaling pathway activity. The result also found that ASP caused cell death was linked to caspase activity, accompanied by the loss of mitochondrial membrane potential, cytochrome c release, and Bax translocation from the cytosol to the mitochondria. We found that ASP significantly affected the poly-ADP-ribose polymerase (PARP), Bcl-2 Associated X Protein (Bax), Bcl-2, Bcl-xL and apoptotic protease activating facter-1 (Apaf1) protein expression in a dose- and time-dependent manner. DAPI staining and Flow cytometry were used to analyze apoptosis. The nude mice xenograft model was used to evaluate the antitumor effect of ASP in vivo. ASP has profound antitumor effect on T47D cells, probably by inducing apoptosis through CREB signaling pathway. Thus, these results suggest that ASP would be a promising therapeutic agent for breast cancer. - Highlights: • CREB and Caspase-3 signaling pathways are involved in the ASP induced breast cancer cells apoptosis. • ROCK1/Mlc signaling pathway plays a critical role in this ASP-mediated apoptosis. • Angelica sinensis polysaccharide (ASP) affected the PARP, Bax, Bcl-2, Bcl-xL and Apaf1 protein expression. • The activation of CREB and ROCK1 promotes caspase-3 activation and apoptosis induced

  7. Ouabain Induces Apoptotic Cell Death Through Caspase- and Mitochondria-dependent Pathways in Human Osteosarcoma U-2 OS Cells.

    Science.gov (United States)

    Chou, Wen-Hsiang; Liu, Ko-Lin; Shih, Yung-Luen; Chuang, Ying-Ying; Chou, Jason; Lu, Hsu-Feng; Jair, Herng-Woei; Lee, Ming-Zhe; Au, Man-Kuan; Chung, Jing-Gung

    2018-01-01

    Ouabain, a plant-derived product/substance with Na + /K + -ATPase inhibiting properties, has been shown to exert anti-cancer activity on human cancer cells. This is the first study to investigate the effect of ouabain on apoptotic cell death of human osteosarcoma-derived U-2 OS cells. Flow cytometry was used to examine cell viability, cell cycle, and reactive oxygen species (ROS), Ca 2+ , mitochondrial membrane potential (MMP) and caspase activity. Morphological changes were examined by contrast-phase microscopy, while apoptosis-associated protein levels were analyzed by western blot. Ouabain, at concentrations of 5-60 μM, significantly decreased the total viable cells and induced cell morphological changes in a time-dependent manner. It also time-dependently decreased G 0 /G 1 phase and increased S and G 2 /M phase in U-2 OS cells. The production of ROS and the levels of MMPs (ΔΨ m ) were inhibited, while Ca 2+ production in U-2 OS cells was increased. Regarding cell apoptosis, flow cytometry assay revealed increased caspase-3, -8, and -9 activities in U-2 OS cells. Moreover, western blot results showed that ouabain increased the expression of pro-apoptotic protein Bax and decreased the expression of anti-apoptotic protein Bcl-2 in U-2 OS cells. Furthermore, results also showed that ouabain increased cytochrome c release, apoptosis-inducing factor (AIF) and endonuclease (Endo) G that is associated with apoptosis through caspase-dependent and -independent pathway in U-2 OS cells. Our findings provide important insight into the cytotoxic effects of ouabain on U-2 OS cells, in vitro, which are mediated at least partly via cell apoptosis induction. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  8. Human Innate Immunity to Toxoplasma gondii Is Mediated by Host Caspase-1 and ASC and Parasite GRA15

    Science.gov (United States)

    Gov, Lanny; Karimzadeh, Alborz; Ueno, Norikiyo; Lodoen, Melissa B.

    2013-01-01

    ABSTRACT   Interleukin-1β (IL-1β) functions as a key regulator of inflammation and innate immunity. The protozoan parasite Toxoplasma gondii actively infects human blood monocytes and induces the production of IL-1β; however, the host and parasite factors that mediate IL-1β production during T. gondii infection are poorly understood. We report that T. gondii induces IL-1β transcript, processing/cleavage, and release from infected primary human monocytes and THP-1 cells. Treating monocytes with the caspase-1 inhibitor Ac-YVAD-CMK reduced IL-1β release, suggesting a role for the inflammasome in T. gondii-induced IL-1β production. This was confirmed by performing short hairpin RNA (shRNA) knockdown of caspase-1 and of the inflammasome adaptor protein ASC. IL-1β induction required active parasite invasion of monocytes, since heat-killed or mycalolide B-treated parasites did not induce IL-1β. Among the type I, II, and III strains of T. gondii, the type II strain induced substantially more IL-1β mRNA and protein release than did the type I and III strains. Since IL-1β transcript is known to be induced downstream of NF-κB signaling, we investigated a role for the GRA15 protein, which induces sustained NF-κB signaling in a parasite strain-specific manner. By infecting human monocytes with a GRA15-knockout type II strain and a type I strain stably expressing type II GRA15, we determined that GRA15 is responsible for IL-1β induction during T. gondii infection of human monocytes. This research defines a pathway driving human innate immunity by describing a role for the classical inflammasome components caspase-1 and ASC and the parasite GRA15 protein in T. gondii-induced IL-1β production. PMID:23839215

  9. 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

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

  11. Estudo da expressão citofotométrica do marcador tumoral Caspase-3 no adenocarcinoma de cólon Citophotometric expression study of tumoral marker Caspase-3 on colon adenocarcinoma

    Directory of Open Access Journals (Sweden)

    João Batista Monteiro Tajra

    2007-12-01

    Full Text Available RACIONAL: O adenocarcinoma de cólon é a segunda causa mais comum de morte por câncer em homens e mulheres, sendo responsável por mais de cinco milhões de mortes por ano. No momento do diagnóstico apenas 70% dos tumores são ressecáveis, 75% são curáveis e 25% poderão ter recorrência da doença. A apoptose é uma das responsáveis pelo equilíbrio homeostático entre as células. Durante o desenvolvimento do processo de degeneração maligna celular o desequilíbrio na apoptose é considerado um dos principais marcos neoplásicos. A caspase-3 é uma das mais importantes moléculas na apoptose, sendo sua efetora principal. Sua expressão e prognóstico têm sido relatados em vários estudos e revisões com seu papel valorizado desde o surgimento do pólipo até a sua transformação maligna, com a taxa de apoptose diminuindo progressivamente. OBJETIVOS: Avaliar a expressão citofotométrica computadorizada do marcador Caspase-3 no adenocarcinoma de cólon; avaliá-lo nas fases evolutivas na classificação modificada de Dukes e comparar sua expressão nos tumores do lado direito e esquerdo do cólon. MÉTODOS: Utilizaram-se 19 casos de câncer recuperados de blocos de parafina confirmados por hematoxilina-eosina e submetidos à técnica imunoistoquímica da estreptavidina-biotina com anticorpo policlonal anti-caspase-3. Após este processo as lâminas marcadas foram submetidas à leitura pelo sistema SAMBA com o software IMUNNO 4.00. Foram analisados três índices: marcagem (Label index, heterogeneidade e densidade óptica. Utilizaram-se a marcagem individual, avaliação da expressão do marcador e grupos definidos de tumores com classificação Dukes e pelo lado do tumor. RESULTADOS: A média do índice de marcagem da caspase-3 foi de 85,24 e da densidade óptica de 39,55. Na classificação Dukes de 12 tipos B tiveram índice de marcagem de 86,20 e a densidade óptica de 37,72 e para os 7 tipos C a área de marcagem foi de 85,66 e a

  12. Caspase-2-dependent dendritic cell death, maturation, and priming of T cells in response to Brucella abortus infection.

    Science.gov (United States)

    Li, Xinna; He, Yongqun

    2012-01-01

    Smooth virulent Brucella abortus strain 2308 (S2308) causes zoonotic brucellosis in cattle and humans. Rough B. abortus strain RB51, derived from S2308, is a live attenuated cattle vaccine strain licensed in the USA and many other countries. Our previous report indicated that RB51, but not S2308, induces a caspase-2-dependent apoptotic and necrotic macrophage cell death. Dendritic cells (DCs) are professional antigen presenting cells critical for bridging innate and adaptive immune responses. In contrast to Brucella-infected macrophages, here we report that S2308 induced higher levels of apoptotic and necrotic cell death in wild type bone marrow-derived DCs (WT BMDCs) than RB51. The RB51 and S2308-induced BMDC cell death was regulated by caspase-2, indicated by the minimal cell death in RB51 and S2308-infected BMDCs isolated from caspase-2 knockout mice (Casp2KO BMDCs). More S2308 bacteria were taken up by Casp2KO BMDCs than wild type BMDCs. Higher levels of S2308 and RB51 cells were found in infected Casp2KO BMDCs compared to infected WT BMDCs at different time points. RB51-infected wild type BMDCs were mature and activated as shown by significantly up-regulated expression of CD40, CD80, CD86, MHC-I, and MHC-II. RB51 induced the production of cytokines TNF-α, IL-6, IFN-γ and IL12/IL23p40 in infected BMDCs. RB51-infected WT BMDCs also stimulated the proliferation of CD4(+) and CD8(+) T cells compared to uninfected WT BMDCs. However, the maturation, activation, and cytokine secretion are significantly impaired in Casp2KO BMDCs infected with RB51 or Salmonella (control). S2308-infected WT and Casp2KO BMDCs were not activated and could not induce cytokine production. These results demonstrated that virulent smooth strain S2308 induced more apoptotic and necrotic dendritic cell death than live attenuated rough vaccine strain RB51; however, RB51, but not its parent strain S2308, induced caspase-2-mediated DC maturation, cytokine production, antigen presentation, and T

  13. The Effects of Heavy Metal Cadmium Against Apoptosis Through Activation of Caspase-3 Protein on Sea Urchins Diadema Setosum

    OpenAIRE

    Rumahlatu, Dominggus

    2013-01-01

    Efek Logam Berat Kadmium Terhadap Apoptosis Melalui Aktivasi Protein Caspase-3 pada Bulu Babi Diadema setosum Abstract: This study is the Cd metal effect on apoptosis in cells D. setosum urchins. The study iss conducted in the laboratory of LIPI Ambon (Indonesia).  TheDoses of the treatment is dissolved 0.0, 1.0, 3.0, 6.0, 9.0, and 12.0 mg / L Cd.  Each treatment was repeated 7 times. The age of animal is about 8 months weighs 90 grams with a circumference of 15 cm body. The Experimental...

  14. 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 - others:GA ČR(CZ) GAP502/12/1285 Program:GA 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

  15. Caspase-2-dependent dendritic cell death, maturation, and priming of T cells in response to Brucella abortus infection.

    Directory of Open Access Journals (Sweden)

    Xinna Li

    Full Text Available Smooth virulent Brucella abortus strain 2308 (S2308 causes zoonotic brucellosis in cattle and humans. Rough B. abortus strain RB51, derived from S2308, is a live attenuated cattle vaccine strain licensed in the USA and many other countries. Our previous report indicated that RB51, but not S2308, induces a caspase-2-dependent apoptotic and necrotic macrophage cell death. Dendritic cells (DCs are professional antigen presenting cells critical for bridging innate and adaptive immune responses. In contrast to Brucella-infected macrophages, here we report that S2308 induced higher levels of apoptotic and necrotic cell death in wild type bone marrow-derived DCs (WT BMDCs than RB51. The RB51 and S2308-induced BMDC cell death was regulated by caspase-2, indicated by the minimal cell death in RB51 and S2308-infected BMDCs isolated from caspase-2 knockout mice (Casp2KO BMDCs. More S2308 bacteria were taken up by Casp2KO BMDCs than wild type BMDCs. Higher levels of S2308 and RB51 cells were found in infected Casp2KO BMDCs compared to infected WT BMDCs at different time points. RB51-infected wild type BMDCs were mature and activated as shown by significantly up-regulated expression of CD40, CD80, CD86, MHC-I, and MHC-II. RB51 induced the production of cytokines TNF-α, IL-6, IFN-γ and IL12/IL23p40 in infected BMDCs. RB51-infected WT BMDCs also stimulated the proliferation of CD4(+ and CD8(+ T cells compared to uninfected WT BMDCs. However, the maturation, activation, and cytokine secretion are significantly impaired in Casp2KO BMDCs infected with RB51 or Salmonella (control. S2308-infected WT and Casp2KO BMDCs were not activated and could not induce cytokine production. These results demonstrated that virulent smooth strain S2308 induced more apoptotic and necrotic dendritic cell death than live attenuated rough vaccine strain RB51; however, RB51, but not its parent strain S2308, induced caspase-2-mediated DC maturation, cytokine production, antigen

  16. The Value of Caspase-3 after the Application of Annona muricata Leaf Extract in COLO-205 Colorectal Cancer Cell Line

    OpenAIRE

    Abdullah, Murdani; Syam, Ari Fahrial; Meilany, Sofy; Laksono, Bayu; Prabu, Oryza Gryagus; Bekti, Heri Setiyo; Indrawati, Lili; Makmun, Dadang

    2017-01-01

    Annona muricata, commonly known as soursop, contains annonacin, acetogenin, and polyphenol which are known to have chemopreventive effects on cancer. In this study, we tend to evaluate the apoptosis-inducing effect of soursop (Annona muricata) leaf extract on the colorectal cancer cell line COLO-205 through the activities of caspase-3 which is a marker of cell apoptosis. Cell cultures were incubated with soursop leaf with a concentration of 10 μg/ml and then compared with those of the incubat...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-01-01

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

  18. IMMUNEPOTENT CRP induces cell cycle arrest and caspase-independent regulated cell death in HeLa cells through reactive oxygen species production.

    Science.gov (United States)

    Martínez-Torres, Ana Carolina; Reyes-Ruiz, Alejandra; Benítez-Londoño, Milena; Franco-Molina, Moises Armides; Rodríguez-Padilla, Cristina

    2018-01-03

    Regulated cell death (RCD) is a mechanism by which the cell activates its own machinery to self-destruct. RCD is important for the maintenance of tissue homeostasis and its deregulation is involved in diseases such as cervical cancer. IMMUNEPOTENT CRP (I-CRP) is a dialyzable bovine leukocyte extract that contains transfer factors and acts as an immunomodulator, and can be cytotoxic to cancer cell lines and reduce tumor burden in vivo. Although I-CRP has shown to improve or modulate immune response in inflammation, infectious diseases and cancer, its widespread use has been limited by the absence of conclusive data on the molecular mechanism of its action. In this study we analyzed the mechanism by which I-CRP induces cytotoxicity in HeLa cells. We assessed cell viability, cell death, cell cycle, nuclear morphology and DNA integrity, caspase dependence and activity, mitochondrial membrane potential, and reactive oxygen species production. I-CRP diminishes cell viability in HeLa cells through a RCD pathway and induces cell cycle arrest in the G2/M phase. We show that the I-CRP induces caspase activation but cell death induction is independent of caspases, as observed by the use of a pan-caspase inhibitor, which blocked caspase activity but not cell death. Moreover, we show that I-CRP induces DNA alterations, loss of mitochondrial membrane potential, and production of reactive-oxygen species. Finally, pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger, prevented both ROS generation and cell death induced by I-CRP. Our data indicate that I-CRP treatment induced cell cycle arrest in G2/M phase, mitochondrial damage, and ROS-mediated caspase-independent cell death in HeLa cells. This work opens the way to the elucidation of a more detailed cell death pathway that could potentially work in conjunction with caspase-dependent cell death induced by classical chemotherapies.

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

    Directory of Open Access Journals (Sweden)

    Zhang YueMei

    2005-02-01

    Full Text Available Abstract Background Apoptosis plays a key role in cell death observed in neurodegenerative diseases marked by a progressive loss of neurons as seen in Alzheimer's disease. Although the exact cause of apoptosis is not known, a number of factors such as free radicals, insufficient levels of nerve growth factors and excessive levels of glutamate have been implicated. We and others, have previously reported that in a stable HT22 neuronal cell line, glutamate induces apoptosis as indicated by DNA fragmentation and up- and down-regulation of Bax (pro-apoptotic, and Bcl-2 (anti-apoptotic genes respectively. Furthermore, these changes were reversed/inhibited by estrogens. Several lines of evidence also indicate that a family of cysteine proteases (caspases appear to play a critical role in neuronal apoptosis. The purpose of the present study is to determine in primary cultures of cortical cells, if glutamate-induced neuronal apoptosis and its inhibition by estrogens involve changes in caspase-3 protease and whether this process is mediated by Fas receptor and/or mitochondrial signal transduction pathways involving release of cytochrome c. Results In primary cultures of rat cortical cells, glutamate induced apoptosis that was associated with enhanced DNA fragmentation, morphological changes, and up-regulation of pro-caspase-3. Exposure of cortical cells to glutamate resulted in a time-dependent cell death and an increase in caspase-3 protein levels. Although the increase in caspase-3 levels was evident after 3 h, cell death was only significantly increased after 6 h. Treatment of cells for 6 h with 1 to 20 mM glutamate resulted in a 35 to 45% cell death that was associated with a 45 to 65% increase in the expression of caspase-3 protein. Pretreatment with caspase-3-protease inhibitor z-DEVD or pan-caspase inhibitor z-VAD significantly decreased glutamate-induced cell death of cortical cells. Exposure of cells to glutamate for 6 h in the presence or

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

    Science.gov (United States)

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

    2017-07-01

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

  1. Caspase-4 may play a role in loss of proximal tubules and renal injury in nephropathic cystinosis.

    Science.gov (United States)

    Sansanwal, Poonam; Kambham, Neeraja; Sarwal, Minnie M

    2010-01-01

    Nephropathic cystinosis is characterized clinically by generalized proximal renal tubular dysfunction, renal Fanconi Syndrome and progressive renal failure. Glomerular-proximal tubule disconnection has been noted in renal biopsies from patients with nephropathic cystinosis. In vitro studies performed in cystinotic fibroblasts and renal proximal tubular cells support a role for apoptosis of the glomerulotubular junction, and we have further extended these studies to human native cystinotic kidney specimens. We performed semi-quantitative analysis of tubular density in kidney biopsies from patients with nephropathic cystinosis and demonstrated a significant reduction (p=0.0003) in the number of proximal tubules in the kidney tissue of patients with cystinosis compared to normal kidneys and kidneys with other causes of renal injury; this reduction appears to be associated with the over-expression of caspase-4. This study provides the first quantitative evidence of a loss of proximal tubules in nephropathic cystinosis and suggests a possible role of caspase-4 in the apoptotic loss of proximal tubular cells. Further work is needed to elucidate if this injury mechanism may be causative for the progression of renal functional decline in nephropathic cystinosis.

  2. Second mitochondria-derived activator of caspase (SMAC) mimetic potentiates tumor susceptibility toward natural killer cell-mediated killing.

    Science.gov (United States)

    Brinkmann, Kerstin; Hombach, Andreas; Seeger, Jens Michael; Wagner-Stippich, Diana; Klubertz, Daniela; Krönke, Martin; Abken, Hinrich; Kashkar, Hamid

    2014-03-01

    Resistance to apoptosis is a hallmark of cancer, and represents an important mechanism of how tumor cells resist immune cell destruction. Mitochondria are the central regulators of the apoptotic machinery by releasing pro-apoptotic factors including cytochrome c and second mitochondria-derived activator of caspase (SMAC) upon mitochondrial outer membrane permeabilization (MOMP). Small molecules activating MOMP such as BH3 mimetics or antagonizers of the inhibitor of apoptosis proteins (IAPs) such as SMAC mimetics have recently engendered new optimism for a more individualized and effective cancer therapy. Here we show that a SMAC mimetic potentiates cancer cell killing by natural killer (NK) cells through reactivation of tumor cell apoptosis. Specifically, the SMAC mimetic enhances the susceptibility of tumor cells toward NK cell-mediated effector mechanisms involving death receptors and cytolytic granules containing perforin and granzymes by relieving caspase activity. Our data highlight for the first time the specific use of SMAC mimetics for boosting immune cell-mediated immunotherapy, representing a novel and promising approach in the treatment of cancer.

  3. Antiplatelet Aggregation Activity of Walnut Hull Extract via Suppression of Reactive Oxygen Species Generation and Caspase Activation.

    Science.gov (United States)

    Meshkini, Azadeh; Tahmasbi, Masoumeh

    2017-06-01

    Walnut hull (wal hull) is an agricultural by-product that is widely used in traditional medicine for alleviating pain and treating skin diseases, however, recently it has gained much attention in modern pharmacology due to its antioxidant properties. The current study was aimed to determine the total phenolic, flavonoid, and tannin content of Persian wal hull extract and evaluate its biological effects on platelet function. Experimental data showed that acetone extract of wal hulls has a high content of polyphenolic compounds and antioxidant properties. The analytical study of crude extract by gas chromatography-mass spectrometry demonstrated different types of high- and low-molecular-weight compounds that are basically and biologically important. Moreover, an in vitro study revealed that wal hull extract at a concentration of 50 μg/mL inhibited thrombin-induced platelet aggregation and protein secretion by 50%, without any cytotoxic effects on platelets. The examined extract suppressed reactive oxygen species generation and also caspase activation in thrombin-stimulated platelets. Identically, N-acetylcysteine inhibited the increase of reactive oxygen species level induced by thrombin in platelets, and supported a link between cellular redox status and caspase activation in activated platelets. Presumably, the antiplatelet activity of wal hull extract is related to its polyphenolic compounds and their antioxidant properties. Therefore, wal hulls can be considered as a candidate for thrombotic disorders. Copyright © 2017. Published by Elsevier B.V.

  4. Melatonin affects membrane integrity, intracellular reactive oxygen species, caspase3 activity and AKT phosphorylation in frozen thawed human sperm.

    Science.gov (United States)

    Najafi, Atefeh; Adutwum, Emmanuel; Yari, Abazar; Salehi, Ensieh; Mikaeili, Saideh; Dashtestani, Fariba; Abolhassani, Farid; Rashki, Leila; Shiasi, Setareh; Asadi, Ebrahim

    2018-04-01

    Cryopreservation is known to induce oxidative stress in spermatozoa. Although melatonin has powerful antioxidant properties, little is known about its effects on human sperm quality during cryopreservation. The present study was undertaken to investigate the effects of melatonin treatment on human sperm parameters essential for fertilization. We first evaluated the effects of various concentrations of melatonin (0-15 mM) on human sperm parameters such as motility, viability and levels of intracellular reactive oxygen species during cryopreservation in order to identify an optimal dose with the greatest effects for further studies. Liquefied semen samples were then divided into three aliquots: cryopreserved without melatonin (control), cryopreserved with 3 mM melatonin and fresh groups. After being thawed, samples were evaluated for motility, viability, membrane integrity, intracellular reactive oxygen species levels, caspase-3 activity and AKT phosphorylation. Treatment of