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Sample records for caspase dcp-1 reveals

  1. 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

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

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2014-05-26

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

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

    Science.gov (United States)

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

    2014-05-26

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

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

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

    2006-10-01

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

  6. Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis

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    Hou, Ying-Chen Claire; Chittaranjan, Suganthi; Barbosa, Sharon González; McCall, Kimberly; Gorski, Sharon M.

    2008-01-01

    A complex relationship exists between autophagy and apoptosis, but the regulatory mechanisms underlying their interactions are largely unknown. We conducted a systematic study of Drosophila melanogaster cell death–related genes to determine their requirement in the regulation of starvation-induced autophagy. We discovered that six cell death genes—death caspase-1 (Dcp-1), hid, Bruce, Buffy, debcl, and p53—as well as Ras–Raf–mitogen activated protein kinase signaling pathway components had a r...

  7. Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis.

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    Hou, Ying-Chen Claire; Chittaranjan, Suganthi; Barbosa, Sharon González; McCall, Kimberly; Gorski, Sharon M

    2008-09-22

    A complex relationship exists between autophagy and apoptosis, but the regulatory mechanisms underlying their interactions are largely unknown. We conducted a systematic study of Drosophila melanogaster cell death-related genes to determine their requirement in the regulation of starvation-induced autophagy. We discovered that six cell death genes--death caspase-1 (Dcp-1), hid, Bruce, Buffy, debcl, and p53-as well as Ras-Raf-mitogen activated protein kinase signaling pathway components had a role in autophagy regulation in D. melanogaster cultured cells. During D. melanogaster oogenesis, we found that autophagy is induced at two nutrient status checkpoints: germarium and mid-oogenesis. At these two stages, the effector caspase Dcp-1 and the inhibitor of apoptosis protein Bruce function to regulate both autophagy and starvation-induced cell death. Mutations in Atg1 and Atg7 resulted in reduced DNA fragmentation in degenerating midstage egg chambers but did not appear to affect nuclear condensation, which indicates that autophagy contributes in part to cell death in the ovary. Our study provides new insights into the molecular mechanisms that coordinately regulate autophagic and apoptotic events in vivo. PMID:18794330

  8. Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis.

    Science.gov (United States)

    Hou, Ying-Chen Claire; Chittaranjan, Suganthi; Barbosa, Sharon González; McCall, Kimberly; Gorski, Sharon M

    2008-09-22

    A complex relationship exists between autophagy and apoptosis, but the regulatory mechanisms underlying their interactions are largely unknown. We conducted a systematic study of Drosophila melanogaster cell death-related genes to determine their requirement in the regulation of starvation-induced autophagy. We discovered that six cell death genes--death caspase-1 (Dcp-1), hid, Bruce, Buffy, debcl, and p53-as well as Ras-Raf-mitogen activated protein kinase signaling pathway components had a role in autophagy regulation in D. melanogaster cultured cells. During D. melanogaster oogenesis, we found that autophagy is induced at two nutrient status checkpoints: germarium and mid-oogenesis. At these two stages, the effector caspase Dcp-1 and the inhibitor of apoptosis protein Bruce function to regulate both autophagy and starvation-induced cell death. Mutations in Atg1 and Atg7 resulted in reduced DNA fragmentation in degenerating midstage egg chambers but did not appear to affect nuclear condensation, which indicates that autophagy contributes in part to cell death in the ovary. Our study provides new insights into the molecular mechanisms that coordinately regulate autophagic and apoptotic events in vivo.

  9. The role of the effector caspases drICE and dcp-1 for cell death and corpse clearance in the developing optic lobe in Drosophila.

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    Akagawa, Hiromi; Hara, Yusuke; Togane, Yu; Iwabuchi, Kikuo; Hiraoka, Tsuyoshi; Tsujimura, Hidenobu

    2015-08-15

    In the developing Drosophila optic lobe, cell death occurs via apoptosis and in a distinctive spatio-temporal pattern of dying cell clusters. We analyzed the role of effector caspases drICE and dcp-1 in optic lobe cell death and subsequent corpse clearance using mutants. Neurons in many clusters required either drICE or dcp-1 and each one is sufficient. This suggests that drICE and dcp-1 function in cell death redundantly. However, dying neurons in a few clusters strictly required drICE but not dcp-1, but required drICE and dcp-1 when drICE activity was reduced via hypomorphic mutation. In addition, analysis of the mutants suggests an important role of effecter caspases in corpse clearance. In both null and hypomorphic drICE mutants, greater number of TUNEL-positive cells were observed than in wild type, and many TUNEL-positive cells remained until later stages. Lysotracker staining showed that there was a defect in corpse clearance in these mutants. All the results suggested that drICE plays an important role in activating corpse clearance in dying cells, and that an additional function of effector caspases is required for the activation of corpse clearance as well as that for carrying out cell death. PMID:26022392

  10. The role of the effector caspases drICE and dcp-1 for cell death and corpse clearance in the developing optic lobe in Drosophila.

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    Akagawa, Hiromi; Hara, Yusuke; Togane, Yu; Iwabuchi, Kikuo; Hiraoka, Tsuyoshi; Tsujimura, Hidenobu

    2015-08-15

    In the developing Drosophila optic lobe, cell death occurs via apoptosis and in a distinctive spatio-temporal pattern of dying cell clusters. We analyzed the role of effector caspases drICE and dcp-1 in optic lobe cell death and subsequent corpse clearance using mutants. Neurons in many clusters required either drICE or dcp-1 and each one is sufficient. This suggests that drICE and dcp-1 function in cell death redundantly. However, dying neurons in a few clusters strictly required drICE but not dcp-1, but required drICE and dcp-1 when drICE activity was reduced via hypomorphic mutation. In addition, analysis of the mutants suggests an important role of effecter caspases in corpse clearance. In both null and hypomorphic drICE mutants, greater number of TUNEL-positive cells were observed than in wild type, and many TUNEL-positive cells remained until later stages. Lysotracker staining showed that there was a defect in corpse clearance in these mutants. All the results suggested that drICE plays an important role in activating corpse clearance in dying cells, and that an additional function of effector caspases is required for the activation of corpse clearance as well as that for carrying out cell death.

  11. Morphological irregularities and features of resistance to apoptosis in the dcp-1/pita double mutated egg chambers during Drosophila oogenesis.

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    Nezis, Ioannis P; Stravopodis, Dimitrios J; Papassideri, Issidora S; Stergiopoulos, Costas; Margaritis, Lukas H

    2005-01-01

    In the present study, we demonstrate the most novel characteristic morphological features of Drosophila egg chambers lacking both dcp-1 and pita functions in the germline cells. Dcp-1 is an effector caspase and it has been previously shown to play an important role during Drosophila oogenesis [McCall and Steller, 1998 : Science 279 : 230-234; Laundrie et al., 2003 : Genetics 165 : 1881-1888; Peterson et al., 2003 : Dev Biol 260 : 113-123]. The completion of sequencing and annotation of the Drosophila genome has revealed that the dcp-1 gene is nested within an intron of another distinct gene, called pita, a member of the C2H2 zinc finger protein family that regulates transcriptional initiation. The dcp-1(-/-)/pita(-/-) nurse cells exhibit euchromatic nuclei (delay of apoptosis) during the late stages of oogenesis, as revealed by conventional light and electron microscopy. The phalloidin-FITC staining discloses significant defects in actin cytoskeleton arrangement. The actin bundles fail to organize properly and the distribution of actin filaments in the ring canals is changed compared to the wild type. The oocyte and the chorion structures have been also modified. The oocyte nucleus is out of position and the chorion appears to contain irregular foldings, while the respiratory filaments obtain an altered morphology. The dcp-1(-/-)/pita(-/-) egg chambers do not exhibit the rare events of spontaneously induced apoptosis, observed for the wild type flies, during mid-oogenesis. Interestingly, the mutated egg chambers are protected by staurosporine-induced apoptosis in a percentage of 40%, strongly suggesting the essential role of dcp-1 and/or pita during mid-oogenesis.

  12. Germline cell death is inhibited by P-element insertions disrupting the dcp-1/pita nested gene pair in Drosophila.

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    Laundrie, Bonni; Peterson, Jeanne S; Baum, Jason S; Chang, Jeffrey C; Fileppo, Dana; Thompson, Sharona R; McCall, Kimberly

    2003-12-01

    Germline cell death in Drosophila oogenesis is controlled by distinct signals. The death of nurse cells in late oogenesis is developmentally regulated, whereas the death of egg chambers during mid-oogenesis is induced by environmental stress or developmental abnormalities. P-element insertions in the caspase gene dcp-1 disrupt both dcp-1 and the outlying gene, pita, leading to lethality and defective nurse cell death in late oogenesis. By isolating single mutations in the two genes, we have found that the loss of both genes contributes to this ovary phenotype. Mutants of pita, which encodes a C2H2 zinc-finger protein, are homozygous lethal and show dumpless egg chambers and premature nurse cell death in germline clones. Early nurse cell death is not observed in the dcp-1/pita double mutants, suggesting that dcp-1+ activity is required for the mid-oogenesis cell death seen in pita mutants. dcp-1 mutants are viable and nurse cell death in late oogenesis occurs normally. However, starvation-induced germline cell death during mid-oogenesis is blocked, leading to a reduction and inappropriate nuclear localization of the active caspase Drice. These findings suggest that the combinatorial loss of pita and dcp-1 leads to the increased survival of abnormal egg chambers in mutants bearing the P-element alleles and that dcp-1 is essential for cell death during mid-oogenesis.

  13. Analysis list: DCP1A [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DCP1A Uterus + hg19 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/DCP1A.1....tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/DCP1A.5.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/DCP...1A.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/DCP1A.Uterus.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/Uterus.gml ...

  14. Structural basis of dcp2 recognition and activation by dcp1

    OpenAIRE

    She, M.; Decker, C J; Svergun, D.I.; Round, A; N. Chen; Muhlrad, D; Parker, R.; Song, H

    2008-01-01

    A critical step in mRNA degradation is the removal of the 5' cap structure, which is catalyzed by the Dcp1-Dcp2 complex. The crystal structure of an S. pombe Dcp1p-Dcp2n complex combined with small-angle X-ray scattering analysis (SAXS) reveals that Dcp2p exists in open and closed conformations, with the closed complex being, or closely resembling, the catalytically more active form. This suggests that a conformational change between these open and closed complexes might control decapping. A ...

  15. The activation of the decapping enzyme DCP2 by DCP1 occurs on the EDC4 scaffold and involves a conserved loop in DCP1

    OpenAIRE

    Chang, Chung-Te; Bercovich, Natalia; Loh, Belinda; Jonas, Stefanie; Izaurralde, Elisa

    2014-01-01

    The removal of the 5′-cap structure by the decapping enzyme DCP2 and its coactivator DCP1 shuts down translation and exposes the mRNA to 5′-to-3′ exonucleolytic degradation by XRN1. Although yeast DCP1 and DCP2 directly interact, an additional factor, EDC4, promotes DCP1–DCP2 association in metazoan. Here, we elucidate how the human proteins interact to assemble an active decapping complex and how decapped mRNAs are handed over to XRN1. We show that EDC4 serves as a scaffold for complex assem...

  16. DCP1 forms asymmetric trimers to assemble into active mRNA decapping complexes in metazoa

    OpenAIRE

    Tritschler, Felix; Braun, Joerg E.; Motz, Carina; Igreja, Catia; Haas, Gabrielle; Truffault, Vincent; Izaurralde, Elisa; Weichenrieder, Oliver

    2009-01-01

    DCP1 stimulates the decapping enzyme DCP2, which removes the mRNA 5′ cap structure committing mRNAs to degradation. In multicellular eukaryotes, DCP1-DCP2 interaction is stabilized by additional proteins, including EDC4. However, most information on DCP2 activation stems from studies in S. cerevisiae, which lacks EDC4. Furthermore, DCP1 orthologs from multicellular eukaryotes have a C-terminal extension, absent in fungi. Here, we show that in metazoa, a conserved DCP1 C-terminal domain drives...

  17. Isolation and characterization of Dcp1p, the yeast mRNA decapping enzyme.

    OpenAIRE

    LaGrandeur, T E; Parker, R.

    1998-01-01

    A major mechanism of mRNA decay occurs by the process of deadenylation, decapping and 5' --> 3' exonucleolytic degradation. Recently, the product of the DCP1 gene has been shown to be required for decapping mRNAs in vivo and co-purifies with decapping activity in vitro. We have purified Dcp1p to homogeneity and shown that it is sufficient for decapping, thereby indicating that Dcp1p is the decapping enzyme. Characterization of Dcp1p activity in vitro indicated that the 7-methyl group of the c...

  18. Crystal structure of Dcp1p and its functional implications in mRNA decapping

    OpenAIRE

    She, Meipei; Decker, Carolyn J.; Sundramurthy, Kumar; Liu, Yuying; Chen, Nan; Parker, Roy; Song, Haiwei

    2004-01-01

    A major pathway of eukaryotic mRNA turnover begins with deadenylation, followed by decapping and 5′→3′ exonucleolytic degradation. A critical step in this pathway is decapping, which is carried out by an enzyme composed of Dcp1p and Dcp2p. The crystal structure of Dcp1p shows that it markedly resembles the EVH1 family of protein domains. Comparison of the proline-rich sequence (PRS)-binding sites in this family of proteins with Dcp1p indicates that it belongs to a novel class of EVH1 domains....

  19. Dcp1 links coactivators of mRNA decapping to Dcp2 by proline recognition

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    Borja, Mark S.; Piotukh, Kirill; Freund, Christian; Gross, John D.

    2011-01-01

    Cap hydrolysis is a critical step in several eukaryotic mRNA decay pathways and is carried out by the evolutionarily conserved decapping complex containing Dcp2 at the catalytic core. In yeast, Dcp1 is an essential activator of decapping and coactivators such as Edc1 and Edc2 are thought to enhance activity, though their mechanism remains elusive. Using kinetic analysis we show that a crucial function of Dcp1 is to couple the binding of coactivators of decapping to activation of Dcp2. Edc1 and Edc2 bind Dcp1 via its EVH1 proline recognition site and stimulate decapping by 1000-fold, affecting both the KM for mRNA and rate of the catalytic step. The C-terminus of Edc1 is necessary and sufficient to enhance the catalytic step, while the remainder of the protein likely increases mRNA binding to the decapping complex. Lesions in the Dcp1 EVH1 domain or the Edc1 proline-rich sequence are sufficient to block stimulation. These results identify a new role of Dcp1, which is to link the binding of coactivators to substrate recognition and activation of Dcp2. PMID:21148770

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

    Directory of Open Access Journals (Sweden)

    Jyh-Ying Peng

    2011-10-01

    Full Text Available Morphological dynamics of mitochondria is associated with key cellular processes related to aging and neuronal degenerative diseases, but the lack of standard quantification of mitochondrial morphology impedes systematic investigation. This paper presents an automated system for the quantification and classification of mitochondrial morphology. We discovered six morphological subtypes of mitochondria for objective quantification of mitochondrial morphology. These six subtypes are small globules, swollen globules, straight tubules, twisted tubules, branched tubules and loops. The subtyping was derived by applying consensus clustering to a huge collection of more than 200 thousand mitochondrial images extracted from 1422 micrographs of Chinese hamster ovary (CHO cells treated with different drugs, and was validated by evidence of functional similarity reported in the literature. Quantitative statistics of subtype compositions in cells is useful for correlating drug response and mitochondrial dynamics. Combining the quantitative results with our biochemical studies about the effects of squamocin on CHO cells reveals new roles of Caspases in the regulatory mechanisms of mitochondrial dynamics. This system is not only of value to the mitochondrial field, but also applicable to the investigation of other subcellular organelle morphology.

  1. c-Jun N-terminal kinase phosphorylates DCP1a to control formation of P bodies

    OpenAIRE

    Rzeczkowski, Katharina; Beuerlein, Knut; Müller, Helmut; Dittrich-Breiholz, Oliver; Schneider, Heike; Kettner-Buhrow, Daniela; Holtmann, Helmut; Kracht, Michael

    2011-01-01

    Cytokines and stress-inducing stimuli signal through c-Jun N-terminal kinase (JNK) using a diverse and only partially defined set of downstream effectors. In this paper, the decapping complex subunit DCP1a was identified as a novel JNK target. JNK phosphorylated DCP1a at residue S315 in vivo and in vitro and coimmunoprecipitated and colocalized with DCP1a in processing bodies (P bodies). Sustained JNK activation by several different inducers led to DCP1a dispersion from P bodies, whereas IL-1...

  2. Dcp1 links coactivators of mRNA decapping to Dcp2 by proline recognition

    OpenAIRE

    Borja, Mark S.; Piotukh, Kirill; Freund, Christian; Gross, John D.

    2011-01-01

    Cap hydrolysis is a critical step in several eukaryotic mRNA decay pathways and is carried out by the evolutionarily conserved decapping complex containing Dcp2 at the catalytic core. In yeast, Dcp1 is an essential activator of decapping and coactivators such as Edc1 and Edc2 are thought to enhance activity, though their mechanism remains elusive. Using kinetic analysis we show that a crucial function of Dcp1 is to couple the binding of coactivators of decapping to activation of Dcp2. Edc1 an...

  3. Dehydration stress activates Arabidopsis MPK6 to signal DCP1 phosphorylation

    OpenAIRE

    Xu, Jun; Chua, Nam-Hai

    2012-01-01

    The pathways regulating mRNA stability in response to environment stress in plants are not well understood. The stress responsive MAPK, MPK6, phosphorylates the mRNA decapping enzyme DCP1 to enhance its activity and regulate mRNA stability.

  4. 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.

  5. The P Body Protein Dcp1a Is Hyper-phosphorylated during Mitosis

    OpenAIRE

    Adva Aizer; Pinhas Kafri; Alon Kalo; Yaron Shav-Tal

    2013-01-01

    Processing bodies (PBs) are non-membranous cytoplasmic structures found in all eukaryotes. Many of their components such as the Dcp1 and Dcp2 proteins are highly conserved. Using live-cell imaging we found that PB structures disassembled as cells prepared for cell division, and then began to reassemble during the late stages of cytokinesis. During the cell cycle and as cells passed through S phase, PB numbers increased. However, there was no memory of PB numbers between mother and daughter ce...

  6. Interaction study of a novel Macrobrachium rosenbergii effector caspase with B2 and capsid proteins of M. rosenbergii nodavirus reveals their roles in apoptosis.

    Science.gov (United States)

    Youngcharoen, Supak; Senapin, Saengchan; Lertwimol, Tareerat; Longyant, Siwaporn; Sithigorngul, Paisarn; Flegel, Timothy W; Chaivisuthangkura, Parin

    2015-08-01

    Apoptosis is an essential immune response to protect invertebrates from virus infected cells. In shrimp, virus infection has been reported to induce apoptosis. Macrobrachium rosenbergii (Mr) was considered to be a disease-resistant host when compared to penaeid shrimps. Caspase-3 was classified as an executioner caspase which played a key role in virus-induced apoptosis. In this study, an effector caspase gene of M. rosenbergii (Mrcasp) was cloned and characterized. The open reading frame (ORF) of Mrcasp was 957 nucleotide encoding 318 amino acid with a deduced molecular mass of 35.87 kDa. RT-PCR analysis showed the presence of Mrcasp in all examined tissues. The phylogenetic tree indicated that Mrcasp was closely related with caspase 3 of shrimp. The functions of the Mrcasp, B2 and capsid proteins of M. rosenbergii nodavirus (MrNV) were assayed in Sf-9 cells. The results showed that Mrcasp induce apoptotic morphology cells; however, capsid protein of MrNV could inhibit apoptotic cells whereas B2 could neither induce nor inhibit apoptotic cells by DAPI staining. The protein interaction between Mrcasp and viral MrNV structure revealed that Mrcasp did not bind to B2 or capsid protein whereas B2 and capsid proteins could bind directly to each other. This study reported a novel sequence of a full-length Mrcasp and its functional studies indicated that Mrcasp could induce apoptotic cells. Our data is the first report demonstrating the direct protein-protein interaction between capsid protein and B2 protein of MrNV.

  7. The structural basis of Edc3- and Scd6-mediated activation of the Dcp1:Dcp2 mRNA decapping complex

    OpenAIRE

    Fromm, Simon A; Truffault, Vincent; Kamenz, Julia; Braun, Joerg E.; Hoffmann, Niklas A.; Izaurralde, Elisa; Sprangers, Remco

    2011-01-01

    5′ mRNA caps are removed by the Dcp1:Dcp2 complex. This study provides the structural basis of the competitive interaction of two activators, Edc3 and Scd6, with Dcp2. The activators are required for full decapping activity of the complex and its localization to P-bodies.

  8. Cell survival and proliferation in Drosophila S2 cells following apoptotic stress in the absence of the APAF-1 homolog, ARK, or downstream caspases.

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    Kiessling, S; Green, D R

    2006-04-01

    In Drosophila, the APAF-1 homolog ARK is required for the activation of the initiator caspase DRONC, which in turn cleaves the effector caspases DRICE and DCP-1. While the function of ARK is important in stress-induced apoptosis in Drosophila S2 cells, as its removal completely suppresses cell death, the decision to undergo apoptosis appears to be regulated at the level of caspase activation, which is controlled by the IAP proteins, particularly DIAP1. Here, we further dissect the apoptotic pathways induced in Drosophila S2 cells in response to stressors and in response to knock-down of DIAP1. We found that the induction of apoptosis was dependent in each case on expression of ARK and DRONC and surviving cells continued to proliferate. We noted a difference in the effects of silencing the executioner caspases DCP-1 and DRICE; knock-down of either or both of these had dramatic effects to sustain cell survival following depletion of DIAP1, but had only minor effects following cellular stress. Our results suggest that the executioner caspases are essential for death following DIAP1 knock-down, indicating that the initiator caspase DRONC may lack executioner functions. The apparent absence of mitochondrial outer membrane permeabilization (MOMP) in Drosophila apoptosis may permit the cell to thrive when caspase activation is disrupted.

  9. A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing

    OpenAIRE

    Rehwinkel, Jan; BEHM-ANSMANT, ISABELLE; Gatfield, David; Izaurralde, Elisa

    2005-01-01

    In eukaryotic cells degradation of bulk mRNA in the 5′ to 3′ direction requires the consecutive action of the decapping complex (consisting of DCP1 and DCP2) and the 5′ to 3′ exonuclease XRN1. These enzymes are found in discrete cytoplasmic foci known as P-bodies or GW-bodies (because of the accumulation of the GW182 antigen). Proteins acting in other post-transcriptional processes have also been localized to P-bodies. These include SMG5, SMG7, and UPF1, which function in nonsense-mediated mR...

  10. Caspases: An apoptosis mediator

    Directory of Open Access Journals (Sweden)

    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.

  11. Mechanistic and structural understanding of uncompetitive inhibitors of caspase-6.

    Directory of Open Access Journals (Sweden)

    Christopher E Heise

    Full Text Available Inhibition of caspase-6 is a potential therapeutic strategy for some neurodegenerative diseases, but it has been difficult to develop selective inhibitors against caspases. We report the discovery and characterization of a potent inhibitor of caspase-6 that acts by an uncompetitive binding mode that is an unprecedented mechanism of inhibition against this target class. Biochemical assays demonstrate that, while exquisitely selective for caspase-6 over caspase-3 and -7, the compound's inhibitory activity is also dependent on the amino acid sequence and P1' character of the peptide substrate. The crystal structure of the ternary complex of caspase-6, substrate-mimetic and an 11 nM inhibitor reveals the molecular basis of inhibition. The general strategy to develop uncompetitive inhibitors together with the unique mechanism described herein provides a rationale for engineering caspase selectivity.

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2011-01-01

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

  14. Allosteric modulation of caspases.

    Science.gov (United States)

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

    2011-11-01

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

  15. Caspase Family Proteases and Apoptosis

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  16. Dual Role of Caspase-11 in Mediating Activation of Caspase-1 and Caspase-3 under Pathological Conditions

    OpenAIRE

    Kang, Shin-Jung; Wang, Suyue; Hara, Hideaki; Peterson, Erin P.; Namura, Shobu; Amin-Hanjani, Sepideh; Huang, Zhihong; Srinivasan, Anu; Tomaselli, Kevin J.; Thornberry, Nancy A.; Moskowitz, Michael A; Yuan, Junying

    2000-01-01

    Caspase-11, a member of the murine caspase family, has been shown to be an upstream activator of caspase-1 in regulating cytokine maturation. We demonstrate here that in addition to its defect in cytokine maturation, caspase-11–deficient mice have a reduced number of apoptotic cells and a defect in caspase-3 activation after middle cerebral artery occlusion (MCAO), a mouse model of stroke. Recombinant procaspase-11 can autoprocess itself in vitro. Purified active recombinant caspase-11 cleave...

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

  18. Caspase exploitation by Legionella pneumophila

    OpenAIRE

    Kathrin eKrause; Amer, Amal O.

    2016-01-01

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

  19. Caspase Exploitation by Legionella pneumophila

    OpenAIRE

    Krause, Kathrin; Amer, Amal O.

    2016-01-01

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

  20. Structural Features of Caspase-Activating Complexes

    Directory of Open Access Journals (Sweden)

    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.

  1. Molecular cloning, immunohistochemical localization, characterization and expression analysis of caspase-8 from the blunt snout bream (Megalobrama amblycephala) exposed to ammonia.

    Science.gov (United States)

    Sun, Shengming; Ge, Xianping; Zhu, Jian; Zhang, Wuxiao; Zhang, Qiong

    2015-12-01

    Caspase-8 is an initiator caspase that plays a crucial role in some cases of apoptosis by extrinsic and intrinsic pathways. Caspase-8 structure and function have been extensively studied in mammals, but in fish the characterization of that initiator caspase is still scarce. In this study, we isolated the caspase-8 gene from Megalobrama amblycephala, one of the most important industrial aquatic animals in China using rapid amplification of cDNA ends (RACE). The 2034 bp full-length M. amblycephala caspase-8 cDNA sequence contained an ORF of 1467 bp encoding a polypeptide of 489 amino acid residues, a 5'-UTR of 102 bp and a 3'-UTR of 462 bp. The caspase-8 amino acid sequences contained two highly conservative death effector domains (DEDs) at N-terminal, the caspase family domains P20 and P10, caspase-8 active-site pentapeptide and potential aspartic acid cleavage sites. Phylogenetic analysis revealed that M. amblycephala caspase-8 were clustered with the caspase-8 from other vertebrate. Real-time quantitative PCR analysis revealed that caspase-8 transcripts were detected in liver after exposure to ammonia. Meanwhile using Western blot analysis, caspase-8 cleaved fragment was detected and significant alteration of procaspase-8 level was found with the same ammonia treatment condition. Furthermore, the result of immunohistochemical detection showed that remarkable changes of immunopositive staining were observed after ammonia treatment. Accordingly, the results signify that caspase-8 of fish may play an essential role in ammonia induced apoptosis.

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

    Science.gov (United States)

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

    2016-01-01

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

  3. Design and Synthesis of Caspase Inhibitors

    Institute of Scientific and Technical Information of China (English)

    BAI; Xu

    2001-01-01

    Apoptosis (programmed cell death) is an evolutionarily conserved process of cell suicide. It requires specialized machinery which involving a family of proteases named caspases. Manipulation of apoptosis through inhibiting or activating caspases has been of great therapeutic interests in the pharmaceutical industry.  Using substrate based approach, a systematic investigation of conformationally constrained peptidomimetic inhibitors has led to the discovery of highly selective ones against selected members of the caspase family. It also resulted novel dipeptide inhibitors as useful tools and possible therapeutic agents against diseases caused by excessive apoptotic cell death. This presentation will focus on the design, synthesis and application of novel caspase inhibitors.  ……

  4. Design and Synthesis of Caspase Inhibitors

    Institute of Scientific and Technical Information of China (English)

    BAI Xu

    2001-01-01

    @@ Apoptosis (programmed cell death) is an evolutionarily conserved process of cell suicide. It requires specialized machinery which involving a family of proteases named caspases. Manipulation of apoptosis through inhibiting or activating caspases has been of great therapeutic interests in the pharmaceutical industry. Using substrate based approach, a systematic investigation of conformationally constrained peptidomimetic inhibitors has led to the discovery of highly selective ones against selected members of the caspase family. It also resulted novel dipeptide inhibitors as useful tools and possible therapeutic agents against diseases caused by excessive apoptotic cell death. This presentation will focus on the design, synthesis and application of novel caspase inhibitors.

  5. Caspase Exploitation by Legionella pneumophila

    Science.gov (United States)

    Krause, Kathrin; Amer, Amal O.

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Nukman Moeloek

    2008-09-01

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

  7. Dcp1-Bodies in Mouse Oocytes

    OpenAIRE

    Swetloff, Adam; Conne, Beatrice; Huarte, Joachim; Pitetti, Jean-Luc; Nef, Serge; Vassalli, Jean-Dominique

    2009-01-01

    Processing bodies (P-bodies) are cytoplasmic granules involved in the storage and degradation of mRNAs. In somatic cells, their formation involves miRNA-mediated mRNA silencing. Many P-body protein components are also found in germ cell granules, such as in mammalian spermatocytes. In fully grown mammalian oocytes, where changes in gene expression depend entirely on translational control, RNA granules have not as yet been characterized. Here we show the presence of P-body-like foci in mouse o...

  8. Inactivation of Effector Caspases through Nondegradative Polyubiquitylation

    DEFF Research Database (Denmark)

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

    2008-01-01

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

  9. KIPase activity is a novel caspase-like activity associated with cell proliferation.

    Science.gov (United States)

    Medina-Palazon, Cahora; Bernard, Emmanuelle; Frost, Victoria; Morley, Simon; Sinclair, Alison J

    2004-07-01

    A novel caspase-like activity, which is directly regulated with cell proliferation is a candidate to regulate the abundance of the cyclin-dependent kinase inhibitor, p27(KIP1), in human lymphoid cells. This activity, which we term KIPase activity, can also cleave a subset of caspase substrates. Here we demonstrate that KIPase is a novel enzyme distinct from any of the previously characterized human caspases. We show that KIPase is active in a variety of cell lineages, its activity is associated with the proliferation of the human T-cell line, Jurkat, and is not inhibited by the broad spectrum caspase inhibitor z-VAD-fmk. Gel filtration analysis revealed that KIPase has a native molecular mass of approximately 100-200 kDa. Furthermore, the activity of KIPase does not change during apoptosis induced by either ligation of FAS or exposure of cells to etoposide. The uniqueness of KIPase is demonstrated by the fact that none of the human caspases tested (1-10) are able to cleave a specific KIPase substrate (Ac-DPSD-AMC) and that an aldehyde modified derivative of the DPSD tetra peptide is unable to inhibit caspases, but is a good inhibitor of KIPase activity. This supports a hypothesis whereby KIPase is a currently unidentified caspase-like enzyme which regulates the abundance of p27(KIP1) in a proliferation-dependent manner.

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

    Directory of Open Access Journals (Sweden)

    Eli Arama

    2007-10-01

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

  11. 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.

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

    Directory of Open Access Journals (Sweden)

    Daniel P Denning

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

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

    Science.gov (United States)

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

    2016-08-12

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-10-15

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

  15. SRSF1 (SRp30a) regulates the alternative splicing of caspase 9 via a novel intronic splicing enhancer affecting the chemotherapeutic sensitivity of non-small cell lung cancer cells

    OpenAIRE

    Shultz, Jacqueline C.; Rachel W Goehe; Murudkar, Charuta S.; Wijesinghe, Dayanjan S.; Mayton, Eric K.; Massiello, Autumn; Hawkins, Amy J.; Mukerjee, Prabhat; Pinkerman, Ryan L.; Park, Margaret A; Chalfant, Charles E.

    2011-01-01

    Increasing evidence points to the functional importance of alternative splice variations in cancer pathophysiology with the alternative pre-mRNA processing of caspase 9 as one example. In this study, we delve into the underlying molecular mechanisms that regulate the alternative splicing of caspase 9. Specifically, the pre-mRNA sequence of caspase 9 was analyzed for RNA cis-elements known to interact with SRSF1, a required enhancer for caspase 9 RNA splicing. This analysis revealed thirteen p...

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

    Directory of Open Access Journals (Sweden)

    I. I. Tokin

    2015-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Alejandro Romero

    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.

  19. 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.

  20. Krebs Cycle Moonlights in Caspase Regulation.

    Science.gov (United States)

    Minis, Adi; Steller, Hermann

    2016-04-01

    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.

  1. Coxsackievirus B3-induced apoptosis and Caspase-3

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

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

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

    Science.gov (United States)

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

    1999-11-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Arvind Suresh

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

  5. The unconventional myosin CRINKLED and its mammalian orthologue MYO7A regulate caspases in their signalling roles.

    Science.gov (United States)

    Orme, Mariam H; Liccardi, Gianmaria; Moderau, Nina; Feltham, Rebecca; Wicky-John, Sidonie; Tenev, Tencho; Aram, Lior; Wilson, Rebecca; Bianchi, Katiuscia; Morris, Otto; Monteiro Domingues, Celia; Robertson, David; Tare, Meghana; Wepf, Alexander; Williams, David; Bergmann, Andreas; Gstaiger, Matthias; Arama, Eli; Ribeiro, Paulo S; Meier, Pascal

    2016-03-10

    Caspases provide vital links in non-apoptotic regulatory networks controlling inflammation, compensatory proliferation, morphology and cell migration. How caspases are activated under non-apoptotic conditions and process a selective set of substrates without killing the cell remain enigmatic. Here we find that the Drosophila unconventional myosin CRINKLED (CK) selectively interacts with the initiator caspase DRONC and regulates some of its non-apoptotic functions. Loss of CK in the arista, border cells or proneural clusters of the wing imaginal discs affects DRONC-dependent patterning. Our data indicate that CK acts as substrate adaptor, recruiting SHAGGY46/GSK3-β to DRONC, thereby facilitating caspase-mediated cleavage and localized modulation of kinase activity. Similarly, the mammalian CK counterpart, MYO7A, binds to and impinges on CASPASE-8, revealing a new regulatory axis affecting receptor interacting protein kinase-1 (RIPK1)>CASPASE-8 signalling. Together, our results expose a conserved role for unconventional myosins in transducing caspase-dependent regulation of kinases, allowing them to take part in specific signalling events.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2016-09-01

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

  8. Caspases regulate VAMP-8 expression and phagocytosis in dendritic cells.

    Science.gov (United States)

    Ho, Yong Hou Sunny; Cai, Deyu Tarika; Huang, Dachuan; Wang, Cheng Chun; Wong, Siew Heng

    2009-09-18

    During an inflammation and upon encountering pathogens, immature dendritic cells (DC) undergo a maturation process to become highly efficient in presenting antigens. This transition from immature to mature state is accompanied by various physiological, functional and morphological changes including reduction of caspase activity and inhibition of phagocytosis in the mature DC. Caspases are cysteine proteases which play essential roles in apoptosis, necrosis and inflammation. Here, we demonstrate that VAMP-8, (a SNARE protein of the early/late endosomes) which has been shown previously to inhibit phagocytosis in DC, is a substrate of caspases. Furthermore, we identified two putative conserved caspase recognition/cleavage sites on the VAMP-8 protein. Consistent with the up-regulation of VAMP-8 expression upon treatment with caspase inhibitor (CI), immature DC treated with CI exhibits lower phagocytosis activity. Thus, our results highlight the role of caspases in regulating VAMP-8 expression and subsequently phagocytosis during maturation of DC.

  9. 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.

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

    Science.gov (United States)

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

    2016-07-01

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

  11. Caspase-3 and its inhibitor Ac-DEVD-CHO in rat lens epithelial cell apoptosis induced by hydrogen in vitro

    Institute of Scientific and Technical Information of China (English)

    姚克; 王凯军; 徐雯; 孙朝晖; 申屠形超; 邱培瑾

    2003-01-01

    Objective To investigate the role of caspase-3 and its inhibitor Ac-DEVD-CHO in rat lens epithelial cell apoptosis induced by hydrogen peroxide (H2O2) in vitro.Methods Rat lenses were incubated in modified Eagle' s medium containing 2 mmol/L H2O2 to induce apoptosis in vitro. Apoptosis in lens epithelial cells was assessed by transmission electron microscopy and annexin V-propidium iodide (PI) double staining flow cytometry after 12, 24 and 48 h of incubation. The activity of caspase-3 was analyzed by western blotting.Results Observations under transmission electron microscopy revealed that 2 mmol/L H2O2 could effectively induce lens epithelial cell apoptosis in vitro. Caspase-3 activity increased during cell apoptosis and the peak measurement occurred at 24 h after treatment with H2O2. Cell apoptosis was blocked by caspase-3 inhibitor Ac-DEVD-CHO.Conclusions The activation of caspase-3 plays an important role in executing apoptosis in H2O2-treated lens epithelial cells and in the formation of cataract. The caspase-3 inhibitor Ac-DEVD-CHO may effectively prevent lens epithelial cell apoptosis caused by oxidative injury.

  12. Molecular and acute temperature stress response characterizations of caspase-8 gene in two mussels, Mytilus coruscus and Mytilus galloprovincialis.

    Science.gov (United States)

    Zhang, Duo; Wang, Hong-Wei; Yao, Cui-Luan

    2014-01-01

    The caspase family represents aspartate-specific cysteine proteases that play key roles in initiation of apoptosis in various cells response to environmental stress. In this study, two caspase-8 cDNA sequences were cloned from two Mytilus mussels, Mytilus coruscus (Mccaspase-8) and Mytilus galloprovincialis (Mgcaspase-8), respectively. The full-length cDNA of Mccaspase-8 was 1884bp, including a 5'-terminal untranslated region (UTR) of 140bp, a 3'-terminal UTR of 238bp and an open reading frame (ORF) of 1506bp encoding a polypeptide of 501 amino acids. The 1775bp full-length Mg caspase-8 cDNA sequence contained an ORF of 1488bp encoding a polypeptide of 495 amino acid residues, a 5'-UTR of 51bp and a 3'-UTR of 236bp. Both the Mccaspase-8 and Mgcaspase-8 amino acid sequences contained two highly conservative death effector domains (DEDs) at N-terminal, the caspase family domains P20 and P10 and the caspase family cysteine active site 'KPKLFFIQACQG'. Phylogenetic analysis revealed that Mccaspase-8 and Mgcaspase-8 were clustered with the caspase-8 from other organisms, with the close relationship with caspase-8 from mollusk. Quantitative real-time reverse transcription PCR (qRT-PCR) analysis indicated that the predominant transcripts of Mccaspase-8 were in mantle and gonad tissue of M. coruscus and the high expression levels of Mgcaspase-8 were in digestive gland and gill tissue of M. galloprovincialis, respectively. The impacts of temperature stress on Mccaspase-8 and Mgcaspase-8 expressions were tested in gill tissue and hemocytes of both species. Our results showed that both Mccaspase-8 and Mgcaspase-8 transcripts and caspase-8 activity in gill tissue and hemocytes could be induced significantly after cold and heat stress (pgalloprovincialis.

  13. 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.

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

    International Nuclear Information System (INIS)

    Caspases are central players in proteolytic pathways that regulate cellular processes such as apoptosis and differentiation. To accelerate the discovery of novel caspase substrates we developed a method combining in silico screening and in vitro validation. With this approach, we identified TAF15 as a novel caspase substrate in a trial study. We find that TAF15 was specifically cleaved by caspases-3 and -7. Site-directed mutagenesis revealed the consensus sequence 106DQPD/Y110 as the only site recognized by these caspases. Surprisingly, TAF15 was cleaved at more than one site in staurosporine-treated Jurkat cells. In addition, we generated two oncogenic TAF15-CIZ/NMP4-fused proteins which have been found in acute myeloid leukemia and demonstrate that caspases-3 and -7 cleave the fusion proteins at one single site. Broad application of this combination approach should expedite identification of novel caspase-interacting proteins and provide new insights into the regulation of caspase pathways leading to cell death in normal and cancer cells.

  15. Skin presenting a higher level of caspase-14 is better protected from UVB irradiation according to in vitro and in vivo studies.

    Science.gov (United States)

    Bergeron, Laurine; Gondran, Catherine; Oberto, Gilles; Garcia, Noelle; Botto, Jean Marie; Cucumel, Karine; Dal Farra, Claude; Domloge, Nouha

    2012-06-01

    Caspase-14, a cysteine endoproteinase belonging to the conserved family of aspartate-specific proteinases, was shown to play an important role in the terminal differentiation of keratinocytes and barrier function of the skin. In the present study, we developed a biofunctional compound that we described as a modulator of caspase-14 expression. Using normal human keratinocytes (NHK) in culture and human skin biopsies, this compound was shown to increase caspase-14 expression and partially reverse the effect of caspase-14-specific siRNA on NHK. Moreover, the increase in filaggrin expression visualized on skin biopsies and the recovery of the barrier structure after tape-stripping indicated that this compound could exhibit a beneficial effect on the skin barrier function. Considering the possible link between caspase-14 and the barrier function, a UVB irradiation on NHK and skin biopsies previously treated with the caspase-14 inducer, was performed. Results indicated that pretreated skin biopsies exhibited less signs of UV damage such as active caspase-3 and cyclobutane pyrimidine dimers (CPDs). Likewise, pretreated NHK were protected from UV-induced genomic DNA damage, as revealed by the Comet Assay. Finally, a clinical test showed a reduction of transepidermal water loss (TEWL) on the treated skin compared with placebo, under UV stress condition, confirming a protecting effect. Taken together, these results strongly suggest that, by increasing caspase-14 expression, the biofunctional compound could exhibit a protective effect on the skin barrier function, especially in case of barrier damage and UV irradiation. PMID:22672275

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    梁赤周; 马志章

    2001-01-01

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

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

    OpenAIRE

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

    2014-01-01

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

  19. 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.

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

    Science.gov (United States)

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

    2013-01-01

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

  1. High throughput ratio imaging to profile caspase activity: potential application in multiparameter high content apoptosis analysis and drug screening.

    Directory of Open Access Journals (Sweden)

    Jeena Joseph

    Full Text Available Recent advancement in the area of green fluorescent protein techniques coupled with microscopic imaging has significantly contributed in defining and dissecting subcellular changes of apoptosis with high spatio-temporal resolution. Although single cell based studies using EGFP and associated techniques have provided valuable information of initiation and hierarchical changes of apoptosis, they are yet to be exploited for multiparameter cell based real time analysis for possible drug screening or pathway defining in a high throughput manner. Here we have developed multiple cancer cell lines expressing FRET sensors for active caspases and adapted them for high throughput live cell ratio imaging, enabling high content image based multiparameter analysis. Sensitivity of the system to detect live cell caspase activation was substantiated by confocal acceptor bleaching as well as wide field FRET imaging. Multiple caspase-specific activities of DEVDase, IETDase and LEHDase were analysed simultaneously with other decisive events of cell death. Through simultaneous analysis of caspase activation by FRET ratio change coupled with detection of mitochondrial membrane potential loss or superoxide generation, we identified several antitumor agents that induced caspase activation with or without membrane potential loss or superoxide generation. Also, cells that escaped the initial drug-induced caspase activation could be easily followed up for defining long term fate. Employing such a revisit imaging strategy of the same area, we have tracked the caspase surviving fractions with multiple drugs and its subsequent response to retreatment, revealing drug-dependent diverging fate of surviving cells. This thereby indicates towards a complex control of drug induced tumor resistance. The technique described here has wider application in both screening of compound libraries as well as in defining apoptotic pathways by linking multiple signaling to identify non

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

    Science.gov (United States)

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

    2016-07-01

    Diatoms are an important class of unicellular algae that produce bioactive secondary metabolites with cytotoxic activity collectively termed oxylipins, including polyunsaturated aldehydes (PUAs), hydroxyacids (HEPEs), oxo-acids and epoxyalcohols. Previous results showed that at higher concentrations, the PUA decadienal induced apoptosis on copepods and sea urchin embryos via caspase-3 activation; at lower concentrations decadienal affected the expression levels of the caspase-8 gene in embryos of the sea urchin Paracentrotus lividus. In the present work, we studied the effects of other common oxylipins produced by diatoms: two PUAs (heptadienal and octadienal) and four hydroxyacids (5-, 9- 11- and 15-HEPE) on P. lividus cell death and caspase activities. Our results showed that (i) at higher concentrations PUAs and HEPEs induced apoptosis in sea urchin embryos, detected by microscopic observation and through the activation of caspase-3/7 and caspase-8 measured by luminescent assays; (ii) at low concentrations, PUAs and HEPEs affected the expression levels of caspase-8 and caspase-3/7 (isolated for the first time here in P. lividus) genes, detected by Real Time qPCR. These findings have interesting implications from the ecological point of view, given the importance of diatom blooms in nutrient-rich aquatic environments. PMID:27130972

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

    Directory of Open Access Journals (Sweden)

    Li Chen

    2014-04-01

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

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

    Science.gov (United States)

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

    2003-07-10

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

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

    OpenAIRE

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

    2008-01-01

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

  6. SVM-based prediction of caspase substrate cleavage sites

    OpenAIRE

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

    2006-01-01

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

  7. 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.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Mathieu Kerbiriou

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

  10. Analysis of death receptor 5 and caspase-8 expression in primary and metastatic head and neck squamous cell carcinoma and their prognostic impact.

    Directory of Open Access Journals (Sweden)

    Heath A Elrod

    Full Text Available Death receptor 5 (DR5 and caspase-8 are major components in the extrinsic apoptotic pathway. The alterations of the expression of these proteins during the metastasis of head and neck squamous cell carcinoma (HNSCC and their prognostic impact have not been reported. The present study analyzes the expression of DR5 and caspase-8 by immunohistochemistry (IHC in primary and metastatic HNSCCs and their impact on patient survival. Tumor samples in this study included 100 primary HNSCC with no evidence of metastasis, 100 primary HNSCC with lymph node metastasis (LNM and 100 matching LNM. IHC analysis revealed a significant loss or downregulation of DR5 expression in primary tumors with metastasis and their matching LNM compared to primary tumors with no evidence of metastasis. A similar trend was observed in caspase-8 expression although it was not statistically significant. Downregulation of caspase-8 and DR5 expression was significantly correlated with poorly differentiated tumors compared to moderately and well differentiated tumors. Univariate analysis indicates that, in HNSCC with no metastasis, higher expression of caspase-8 significantly correlated with better disease-free survival and overall survival. However, in HNSCC with LNM, higher caspase-8 expression significantly correlated with poorer disease-free survival and overall survival. Similar results were also generated when we combined both DR5 and caspase-8. Taken together, we suggest that both DR5 and caspase-8 are involved in regulation of HNSCC metastasis. Our findings warrant further investigation on the dual role of caspase-8 in cancer development.

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

    Science.gov (United States)

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

    2016-04-01

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

  12. Caspase-14 expression impairs retinal pigment epithelium barrier function: potential role in diabetic macular edema.

    Science.gov (United States)

    Beasley, Selina; El-Sherbiny, Mohamed; Megyerdi, Sylvia; El-Shafey, Sally; Choksi, Karishma; Kaddour-Djebbar, Ismail; Sheibani, Nader; Hsu, Stephen; Al-Shabrawey, Mohamed

    2014-01-01

    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. PMID:25121097

  13. 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.

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

    Science.gov (United States)

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

    2015-09-17

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

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

    Science.gov (United States)

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

    2015-09-17

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

  16. Caspase-2 deficiency accelerates chemically induced liver cancer in mice.

    Science.gov (United States)

    Shalini, S; Nikolic, A; Wilson, C H; Puccini, J; Sladojevic, N; Finnie, J; Dorstyn, L; Kumar, S

    2016-10-01

    Aberrant cell death/survival has a critical role in the development of hepatocellular carcinoma (HCC). Caspase-2, a cell death protease, limits oxidative stress and chromosomal instability. To study its role in reactive oxygen species (ROS) and DNA damage-induced liver cancer, we assessed diethylnitrosamine (DEN)-mediated tumour development in caspase-2-deficient (Casp2(-/-)) mice. Following DEN injection in young animals, tumour development was monitored for 10 months. We found that DEN-treated Casp2(-/-) mice have dramatically elevated tumour burden and accelerated tumour progression with increased incidence of HCC, accompanied by higher oxidative damage and inflammation. Furthermore, following acute DEN injection, liver injury, DNA damage, inflammatory cytokine release and hepatocyte proliferation were enhanced in mice lacking caspase-2. Our study demonstrates for the first time that caspase-2 limits the progression of tumourigenesis induced by an ROS producing and DNA damaging reagent. Our findings suggest that after initial DEN-induced DNA damage, caspase-2 may remove aberrant cells to limit liver damage and disease progression. We propose that Casp2(-/-) mice, which are more susceptible to genomic instability, are limited in their ability to respond to DNA damage and thus carry more damaged cells resulting in accelerated tumourigenesis.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2009-01-01

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

  19. Nitric oxide induces caspase activity in boar spermatozoa.

    Science.gov (United States)

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

    2008-07-01

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

  20. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-12-31

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

  2. MAPKs and Mst1/Caspase-3 pathways contribute to H2B phosphorylation during UVB-induced apoptosis

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Apoptosis is a highly coordinated or programmed cell suicide mechanism in eukaryotes.Histone modification is associated with nuclear events in apoptotic cells.Specifically H2B phosphorylation at serine 14 (Ser14) catalyzed by Mst1 kinase has been linked to chromatin condensation during apoptosis.We report that activation of MAPKs (ERK1/2,JNK1/2 and p38) together with Mst1 and caspase-3 is required for phosphorylation of H2B (Ser14) during ultraviolet B light (UVB)-induced apoptosis.UVB can trigger activation of MAPKs and induce H2B phosphorylation at Ser14 but not acetylation in a time-dependent manner.Inhibition of ERK1/2,JNK1/2 or p38 activity blocked H2B phosphorylation (Ser14).Furthermore,caspase-3 was activated by UVB to regulate Mst1 activity,which phosphorylates H2B at Ser14,leading to chromatin condensation.Full inhibition of caspase-3 activity reduced Mst1 activation and partially inhibited H2B phosphorylation (Ser14),but ERK1/2,JNK1/2 and p38 activities were not affected.Taken together,these data revealed that H2B phosphorylation is regulated by both MAPKs and caspase-3/Mst1 pathways during UVB-induced apoptosis.

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

    Science.gov (United States)

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

    2016-08-01

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

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

    NARCIS (Netherlands)

    Gaussand, Gwénael Martial Daniel Jean-Marie

    2007-01-01

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

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

    Science.gov (United States)

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

    2002-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Tao Qu

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

  7. Early activation of caspases during T lymphocyte stimulation results in selective substrate cleavage in nonapoptotic cells.

    Science.gov (United States)

    Alam, A; Cohen, L Y; Aouad, S; Sékaly, R P

    1999-12-20

    Apoptosis induced by T cell receptor (TCR) triggering in T lymphocytes involves activation of cysteine proteases of the caspase family through their proteolytic processing. Caspase-3 cleavage was also reported during T cell stimulation in the absence of apoptosis, although the physiological relevance of this response remains unclear. We show here that the caspase inhibitor benzyloxycarbonyl (Cbz)-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD) blocks proliferation, major histocompatibility complex class II expression, and blastic transformation during stimulation of peripheral blood lymphocytes. Moreover, T cell activation triggers the selective processing and activation of downstream caspases (caspase-3, -6, and -7), but not caspase-1, -2, or -4, as demonstrated even in intact cells using a cell-permeable fluorescent substrate. Caspase-3 processing occurs in different T cell subsets (CD4(+), CD8(+), CD45RA(+), and CD45RO(+)), and in activated B lymphocytes. The pathway leading to caspase activation involves death receptors and caspase-8, which is also processed after TCR triggering, but not caspase-9, which remains as a proenzyme. Most importantly, caspase activity results in a selective substrate specificity, since poly(ADP-ribose) polymerase (PARP), lamin B, and Wee1 kinase, but not DNA fragmentation factor (DFF45) or replication factor C (RFC140), are processed. Caspase and substrate processing occur in nonapoptotic lymphocytes. Thus, caspase activation is an early and physiological response in viable, stimulated lymphocytes, and appears to be involved in early steps of lymphocyte activation. PMID:10601362

  8. Pseudolaric Acid B Induces Caspase-Dependent and Caspase-Independent Apoptosis in U87 Glioblastoma Cells

    Directory of Open Access Journals (Sweden)

    Muhammad Khan

    2012-01-01

    Full Text Available Pseudolaric acid B (PLAB is one of the major bioactive components of Pseudolarix kaempferi. It has been reported to exhibit inhibitory effect on cell proliferation in several types of cancer cells. However, there is no report elucidating its effect on glioma cells and organ toxicity in vivo. In the present study, we found that PLAB inhibited growth of U87 glioblastoma cells in a dose-dependent manner with IC50~10 μM. Flow cytometry analysis showed that apoptotic cell death mediated by PLAB was accompanied with cell cycle arrest at G2/M phase. Using Western blot, we found that PLAB induced G2/M phase arrest by inhibiting tubulin polymerization in U87 cells. Apoptotic cell death was only partially inhibited by pancaspase inhibitor, z-VAD-fmk, which suggested that PLAB-induced apoptosis in U87 cells is partially caspase-independent. Further mechanistic study demonstrated that PLAB induced caspase-dependent apoptosis via upregulation of p53, increased level of proapoptotic protein Bax, decreased level of antiapoptotic protein Bcl-2, release of cytochrome c from mitochondria, activation of caspase-3 and proteolytic cleavage of poly (ADP-ribose polymerase (PARP and caspase-independent apoptosis through apoptosis inducing factor (AIF. Furthermore, in vivo toxicity study demonstrated that PLAB did not induce significant structural and biochemical changes in mouse liver and kidneys at a dose of 25 mg/kg. Therefore, PLAB may become a potential lead compound for future development of antiglioma therapy.

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

    International Nuclear Information System (INIS)

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-02-15

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

  11. 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

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

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

  14. Tumor promotion by caspase-resistant retinoblastoma protein

    Science.gov (United States)

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

    2005-01-01

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

  15. 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.

  16. Alternative germ cell death pathway in Drosophila involves HtrA2/Omi, lysosomes, and a caspase-9 counterpart.

    Science.gov (United States)

    Yacobi-Sharon, Keren; Namdar, Yuval; Arama, Eli

    2013-04-15

    In both flies and mammals, almost one-third of the newly emerging male germ cells are spontaneously eliminated before entering meiosis. Here, we show that in Drosophila, germ cell death (GCD) involves the initiator caspase Dronc independently of the apoptosome and the main executioner caspases. Electron microscopy of dying germ cells revealed mixed morphologies of apoptosis and necrosis. We further show that the lysosomes and their catabolic enzymes, but not macroautophagy, are involved in the execution of GCD. We then identified, in a screen, the Parkinson's disease-associated mitochondrial protease, HtrA2/Omi, as an important mediator of GCD, acting mainly through its catalytic activity rather than by antagonizing inhibitor of apoptosis proteins. Concomitantly, other mitochondrial-associated factors were also implicated in GCD, including Pink1 (but not Parkin), the Bcl-2-related proteins, and endonuclease G, which establish the mitochondria as central mediators of GCD. These findings uncover an alternative developmental cell death pathway in metazoans.

  17. Apoptosis-associated speck-like protein containing CARD forms specks but does not activate caspase-1 in the absence of NLRP3 during macrophage swelling

    Science.gov (United States)

    Compan, Vincent; Martín-Sánchez, Fátima; Baroja-Mazo, Alberto; López-Castejón, Gloria; Gomez, Ana I.; Verkhratsky, Alexei; Brough, David; Pelegrín, Pablo

    2016-01-01

    Apoptosis-associated speck-like protein containing a CARD (ASC) is a key adaptor molecule required for inflammatory processes. ASC acts by bridging NLRP proteins, such as NLRP3, with pro-caspase-1 within the inflammasome complex that subsequently results in the activation of caspase-1 and the secretion of interleukin (IL)-1β and IL-18. In response to bacterial infection, ASC also forms specks by self-oligomerization to activate caspase-1 and induce pyroptosis. Hitherto the role of these specks in NLRP3 inflammasome activation in response to danger signals is largely unexplored. Here we report that under hypotonic conditions, ASC formed specks independently of NLRP3 that did not activate caspase-1. These specks were not associated with pyroptosis and were controlled by Transient Receptor Potential Vanilloid 2 channel mediated signaling. However, interaction with NLRP3 enhanced ASC speck formation leading to fully functional inflammasomes and caspase-1 activation. This study reveals that the ASC speck could present different oligomerization assemblies and represents an essential step in the activation of functional NLRP3 inflammasomes. PMID:25552542

  18. Role of Caspase and MMPs in Amniochorionic during PROM

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

  1. Effect of Exogenous Erythropoietin on Caspase-9 and Caspase-3 Expression in Newborn Rats after Hypoxia-ischemia Brain Damage%EPO对新生大鼠缺氧缺血性脑损伤时脑组织Caspase-9和Caspase-3的影响

    Institute of Scientific and Technical Information of China (English)

    白丹; 阴怀清; 阴崇娟; 武师润

    2011-01-01

    目的 观察新生大鼠缺氧缺血性脑损伤(HIBD)时脑组织Caspase-9和Caspase-3的表达变化及促红细胞细胞生成素(EPO)对其表达的影响,从而探讨EPO发挥脑保护作用的可能机制.方法 将新生7 d SD大鼠120只随机分成3组,假手术组、HIBD组、rhEPO治疗组,每组根据不同时间点又分为5个亚组:6 h组、12 h组、24 h组、48 h组、72 h组,每组8只,用免疫组化的方法观察各组脑组织Caspase-9和Caspase-3的表达.结果 Caspase-9的表达在缺氧缺血6 h即增强,12 h时逐渐升高,24 h~72 h维持在高峰水平(P<0.01);Caspase-3的表达也在缺氧缺血6 h即增强,12 h时逐渐升高,24 h~48 h达高峰,72 h稍有降低(P<0.01);rhEPO治疗组各时间点Caspase-9和Caspase-3的表达水平较HIBD组均明显降低(P<0.01).结论 Caspase-9和Caspase-3参与了新生大鼠脑组织HIBD的发生发展过程,EPO可能通过降低新生大鼠缺氧缺血性脑损伤时脑组织Caspase-9和Caspase-3的表达发挥其脑保护作用.%Objective To observe the expression of caspase - 9 and caspase - 3 in brain tissue of neonatal rats after hypoxia - ischemia brain damage (HIBD) and the effects of exogenous erythropoietin (EPO) on the expression of caspase- 9 and caspase- 3. Methods One hundred and twenty seven - day - old neonatal Sprague - Dawley rats were randomly divided into three groups: Sham - operated group, HIBD group and rhEPO treatment group. Each group was further divided into five sub-groups(n= 8) based on different time points after H IBD(6 h, 12 h, 24 h, 48 h, 72 h), respectively. Immunohistochemical technique was used to determine the expression of caspase - 9 and caspase - 3 in brain tissues. Results The expression of caspase - 9 in HIBD group was increased at 6 h after HIBD,increased gradually at 12 h,and maintained the peak level 24 h to 72 h (P<0.01). The expression of caspase-3 in HIBD group was increased at 6 h after HIBD,increased gradually at 12 h,reached the peak at 24 h

  2. Matrine inhibited the growth of rat osteosarcoma UMR-108 cells by inducing apoptosis in a mitochondrial-caspase-dependent pathway.

    Science.gov (United States)

    Yan, Feng; Liu, Yang; Wang, Wenbo

    2013-08-01

    Matrine, one of the main active components of the extracts from the dry roots of Sophora flavescens, has a potent antitumor activity in vitro and in vivo. However, the molecular mechanism of cell apoptosis induced by matrine remains elusive. Here, we investigated the apoptosis in matrine-treated rat osteosarcoma UMR-108 cells. The results showed that matrine could inhibit cell proliferation and induce apoptosis in a dose- and time-dependent manner. Further investigation revealed a disruption of mitochondrial transmembrane potential and an upregulation of reactive oxygen species in matrine-treated cells. By western blot analysis, we found the upregulation of cleaved poly(ADP-ribose) polymerase, cleaved caspase-3, and cleaved caspase-9 and the downregulation of Bax/Bcl-2 with different concentrations of matrine. These protein interactions may play a pivotal role in the regulation of apoptosis. Taken together, these results overall indicate that matrine could be used as an effective antitumor agent in therapy of osteosarcoma targets the caspase-dependent signaling pathway.

  3. Dasatinib promotes paclitaxel-induced necroptosis in lung adenocarcinoma with phosphorylated caspase-8 by c-Src.

    Science.gov (United States)

    Diao, Yan; Ma, Xiaobin; Min, WeiLi; Lin, Shuai; Kang, HuaFeng; Dai, ZhiJun; Wang, Xijing; Zhao, Yang

    2016-08-28

    Cisplatin and paclitaxel are considered to be the backbone of chemotherapy in lung adenocarcinoma. These agents show pleiotropic effects on cell death. However, the precise mechanisms remain unclear. The present study reported that phosphorylated caspase-8 at tyrosine 380 (p-Casp8) was characterized as a biomarker of chemoresistance to TP regimen (cisplatin and paclitaxel) in patients with resectable lung adenocarcinoma with significantly poorer 5-year disease-free survival (DFS) and overall survival (OS). Cisplatin killed lung adenocarcinoma cells regardless of c-Src-induced caspase-8 phosphorylation at tyrosine 380. Subsequently, we identified a novel mechanism by which paclitaxel induced necroptosis in lung adenocarcinoma cells that was dependent upon p-Casp8, receptor-interacting protein kinase 1 (RIPK1), and RIPK3. Moreover, dasatinib, a c-Src inhibitor, dephosphorylated caspase-8 to facilitate necroptosis, rather than apoptosis, in paclitaxel-treated p-Casp8-expressing lung adenocarcinoma cells. The data from our study revealed previously unrecognized roles of p-Casp8 as a positive effector in the initiation of necroptosis and as a negative effector in the repression of the interaction between RIPK1 and RIPK3. Moreover, these outcomes supported the need for further clinical studies with the goal of evaluating the efficacy of dasatinib plus paclitaxel in the treatment of lung adenocarcinoma. PMID:27195913

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

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

  6. Ecstasy-Induced Caspase Expression Alters Following Ginger Treatment

    OpenAIRE

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

    2013-01-01

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

  7. 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

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

    Institute of Scientific and Technical Information of China (English)

    林若庭; 蔡若蔚; 张鹏飞; 林元相

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Achmad Hidayat

    2011-12-01

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

  10. ER stress does not cause upregulation and activation of caspase-2 to initiate apoptosis.

    Science.gov (United States)

    Sandow, J J; Dorstyn, L; O'Reilly, L A; Tailler, M; Kumar, S; Strasser, A; Ekert, P G

    2014-03-01

    A recent report claimed that endoplasmic reticulum (ER) stress activates the ER trans-membrane receptor IRE1α, leading to increased caspase-2 levels via degradation of microRNAs, and consequently induction of apoptosis. This observation casts caspase-2 into a central role in the apoptosis triggered by ER stress. We have used multiple cell types from caspase-2-deficient mice to test this hypothesis but failed to find significant impact of loss of caspase-2 on ER-stress-induced apoptosis. Moreover, we did not observe increased expression of caspase-2 protein in response to ER stress. Our data strongly argue against a critical role for caspase-2 in ER-stress-induced apoptosis.

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

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

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

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

    Science.gov (United States)

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

    2010-09-15

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

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

    OpenAIRE

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

    2011-01-01

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

  14. Early Activation of Caspases during T Lymphocyte Stimulation Results in Selective Substrate Cleavage in Nonapoptotic Cells

    OpenAIRE

    Alam, Antoine; Cohen, Luchino Y.; Aouad, Salah; Sékaly, Rafick-Pierre

    1999-01-01

    Apoptosis induced by T cell receptor (TCR) triggering in T lymphocytes involves activation of cysteine proteases of the caspase family through their proteolytic processing. Caspase-3 cleavage was also reported during T cell stimulation in the absence of apoptosis, although the physiological relevance of this response remains unclear. We show here that the caspase inhibitor benzyloxycarbonyl (Cbz)-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD) blocks proliferation, major histocompatibility complex...

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

    OpenAIRE

    Odonkor, Charles Amoatey; Achilefu, Samuel

    2008-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    赵树鹏; 靳彩玲; 赵新利; 周文科

    2012-01-01

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

  18. EXPRESSIONS OF survivin, bax AND caspase-3 IN MISSED ABORTION PATIENTS%稽留流产病人survivin、bax及caspase-3的表达

    Institute of Scientific and Technical Information of China (English)

    张娟; 王蓁

    2011-01-01

    目的 探讨survivin、bax及caspase-3的表达与稽留流产的关系.方法 以35例稽留流产病人为病例组,30例正常妊娠者为对照组.采用免疫组织化学sP法检测绒毛细胞滋养细胞和合体滋养细胞及蜕膜组织蜕膜细胞中survivin、bax和caspase-3的表达.结果 病例组妊娠组织中survivin的表达明显低于对照组,bax及caspase-3的表达明显高于对照组(u=2.87~5.29,P<0.01).病例组survivin与bax及caspase-3的表达均呈负相关(r=-0.59、-0.71,P<0.05、0.01),bax与caspase-3的表达呈正相关(r=0.79,P<0.01).结论 survivin的低表达、bax及caspase-3的高表达在稽留流产的发生中起重要作用.%Objective To study the relationship of expressions of survivin, bax and caspase-3 with missed abortion.Methods Thirty-five missed abortion patients were assigned as study group, and 30 with normal pregnancy as controls. Immunohistochemistry technique was employed to detect the expressions of survivin, bax and caspase-3 in cytotrophoblast and syncytiotrophoblast of villi and decidual cells of decidua. Results Compared with that of the controls, the expression of survivin in the study group was significantly lower, while the expressions of bax and caspase-3 were higher (uc = 2. 87-5.29, P<0.01 ). In the study group, the survivin expression was negatively correlated wit h t he expressions of bax and caspase-3 (r = - 0. 59,0. 71; P< 0. 05,0.01), respectively, and the expression of bax was found to be positively correlated with caspase-3 (r=0. 79,P<0.01). Conclusion Low expression of survivin and high expressions of bax and caspase-3 play an important role in the occurrence of missed abortion.

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

    Institute of Scientific and Technical Information of China (English)

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

    2012-01-01

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

  20. Activated caspase-9 immunoreactivity in glial and neuronal cytoplasmic inclusions in multiple system atrophy.

    Science.gov (United States)

    Kawamoto, Yasuhiro; Ayaki, Takashi; Urushitani, Makoto; Ito, Hidefumi; Takahashi, Ryosuke

    2016-08-15

    The mitochondria play an important role in apoptotic cell death, and the released cytochrome c from the mitochondria promotes the formation of the apoptosome, which contains cytochrome c, Apaf-1 and caspase-9, resulting in the activation of caspase-9 and the promotion of the apoptotic cascade. To investigate the role of mitochondria-dependent apoptotic cell death in patients with multiple system atrophy (MSA), we performed immunohistochemical studies on apoptosome-related proteins in formalin-fixed, paraffin-embedded sections from 8 normal subjects and 10 patients with MSA. We then performed double-labeling immunohistochemistry for activated caspase-9 and α-synuclein in some sections from 10 patients with MSA. In the brains with MSA, glial cytoplasmic inclusions (GCIs) and neuronal cytoplasmic inclusions (NCIs) were intensely immunoreactive for cytochrome c, Apaf-1 and caspase-9. Activated caspase-9 immunoreactivities were also confirmed to be densely localized to both GCIs and NCIs using two types of anti-cleaved caspase-9 antibodies. The semiquantitative analyses using the upper pontine sections double-immunostained with cleaved caspase-9 and α-synuclein demonstrated that approximately 80% of GCIs and NCIs were immunopositive for cleaved caspase-9. Our results suggest that the formation of the apoptosome accompanied by the activation of caspase-9 may occur in brains affected by MSA, and that a mitochondria-dependent apoptotic pathway may be partially associated with the pathogenesis of MSA. PMID:27345387

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

    Institute of Scientific and Technical Information of China (English)

    Jens Staal; Tine Bekaert; Rudi Beyaert

    2011-01-01

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

  2. 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.

  3. pRb/E2F-1-mediated caspase-dependent induction of Noxa amplifies the apoptotic effects of the Bcl-2/Bcl-xL inhibitor ABT-737.

    Science.gov (United States)

    Bertin-Ciftci, J; Barré, B; Le Pen, J; Maillet, L; Couriaud, C; Juin, P; Braun, F

    2013-05-01

    Although Bcl-2 family members control caspase activity by regulating mitochondrial permeability, caspases can, in turn, amplify the apoptotic process upstream of mitochondria by ill-characterized mechanisms. We herein show that treatment with a potent inhibitor of Bcl-2 and Bcl-xL, ABT-737, triggers caspase-dependent induction of the BH3-only protein, Mcl-1 inhibitor, Noxa. RNA interference experiments reveal that induction of Noxa, and subsequent cell death, rely not only on the transcription factor E2F-1 but also on its regulator pRb. In response to ABT-737, pRb is cleaved by caspases into a p68Rb form that still interacts with E2F-1. Moreover, pRb occupies the noxa promoter together with E2F-1, in a caspase-dependent manner upon ABT-737 treatment. Thus, caspases contribute to trigger the mitochondrial apoptotic pathway by coupling Bcl-2/Bcl-xL inhibition to that of Mcl-1, via the pRb/E2F-1-dependent induction of Noxa.

  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. Phylogenomics of caspase-activated DNA fragmentation factor

    International Nuclear Information System (INIS)

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

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

    Science.gov (United States)

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

    2006-11-01

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

  7. Caspase Induction and BCL2 Inhibition in Human Adipose Tissue

    Science.gov (United States)

    Tinahones, Francisco José; Coín Aragüez, Leticia; Murri, Mora; Oliva Olivera, Wilfredo; Mayas Torres, María Dolores; Barbarroja, Nuria; Gomez Huelgas, Ricardo; Malagón, Maria M.; El Bekay, Rajaa

    2013-01-01

    OBJECTIVE Cell death determines the onset of obesity and associated insulin resistance. Here, we analyze the relationship among obesity, adipose tissue apoptosis, and insulin signaling. RESEARCH DESIGN AND METHODS The expression levels of initiator (CASP8/9) and effector (CASP3/7) caspases as well as antiapoptotic B-cell lymphoma (BCL)2 and inflammatory markers were assessed in visceral (VAT) and subcutaneous (SAT) adipose tissue from patients with different degrees of obesity and without insulin resistance or diabetes. Adipose tissue explants from lean subjects were cultured with TNF-α or IL-6, and the expression of apoptotic and insulin signaling components was analyzed and compared with basal expression levels in morbidly obese subjects. RESULTS SAT and VAT exhibited increased CASP3/7 and CASP8/9 expression levels and decreased BCL2 expression with BMI increase. These changes were accompanied by increased inflammatory cytokine mRNA levels and macrophage infiltration markers. In obese subjects, CASP3/7 activation and BCL2 downregulation correlated with the IRS-1/2–expression levels. Expression levels of caspases, BCL2, p21, p53, IRS-1/2, GLUT4, protein tyrosine phosphatase 1B, and leukocyte antigen-related phosphatase in TNF-α– or IL-6–treated explants from lean subjects were comparable with those found in adipose tissue samples from morbidly obese subjects. These insulin component expression levels were reverted with CASP3/7 inhibition in these TNF-α– or IL-6–treated explants. CONCLUSIONS Body fat mass increase is associated with CASP3/7 and BCL2 expression in adipose tissue. Moreover, this proapoptotic state correlated with insulin signaling, suggesting its potential contribution to the development of insulin resistance. PMID:23193206

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

    Science.gov (United States)

    Levayer, Romain; Dupont, Carole; Moreno, Eduardo

    2016-03-01

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

  9. Effect of NF-κB, survivin, Bcl-2 and Caspase3 on apoptosis of gastric cancer cells induced by tumor necrosis factor related apoptosis inducing ligand

    Institute of Scientific and Technical Information of China (English)

    Liu-Qin Yang; Dian-Chun Fang; Rong-Quan Wang; Shi-Ming Yang

    2004-01-01

    AIM: To study the effect of NF-κB, survivin, Bcl-2 and Caspase3 on tumor necrosis factors related apoptosis inducing ligand (TRAIL) induced apoptosis of gastric cancer cells.METHODS: Gastric cancer cells of SGC-7901, MKN28,MKN45 and AGS lines were cultured in PRMI-1640 medium and the apoptosis rates of the cells of 4 lines were observed after treatment of tumor necrosis factors related apoptosis inducing ligand (TRAIL) with a flow cytometer. The expression of NF-κB, survivin, Bcl-2 and Caspase3 in gastric cancer cells of 4 lines was analyzed with Western blot.RESULTS: After the gastric cancer cells were exposed to TRAIL 300 ng/ml for 24 hours, the apoptosis rate was 36.05%, 20.27%, 16.50% and 11.80% in MKN28, MKN45,AGS and SGC-7901cells respectively. Western blot revealed that the expressions of NF-κB and survivin were lower in MKN28 cells than in MKN45, AGS and SGC-7901 cells. In contrast, the expression of Caspase3 was higher in MKN28 cells than in MKN45, AGS and SGC-7901 cells.CONCLUSION: There is a selectivity of TRAIL potency to induce apoptosis in gastric cancer cells of different cell lines.The anticancer potency of TRAIL is associated with the decreased expression of NF-κB and survivin and increased expression of Caspase3 of gastric cancer cells.

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    孙红; 惠延年; 王立勤; 马吉献

    2003-01-01

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

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

    NARCIS (Netherlands)

    Hervella, M.; Plantinga, T.S.; Alonso, S.; Ferwerda, B.; Izagirre, N.; Fontecha, L.; Fregel, R.; Meer, J.W.M. van der; de-la-Rúa, C.; Netea, M.G.

    2012-01-01

    BACKGROUND: 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 inacti

  13. Expression and effect of Caspase-3 in neurons after tractive spinal cord injury in rats

    Institute of Scientific and Technical Information of China (English)

    LIU Lei; PEI Fu-xing; TANG Kang-lai; XU Jian-zhong; LI Qi-hong

    2005-01-01

    Objective: To investigate Caspase-3 expression and its role in neuronal apoptosis.Methods: The T13-L2 spinal cord of rats was injured by traction after the amplitude of P1-N1 wave, monitored by a cortical somatosensory evoked potential (CSEP) monitor, decreased to seventy percent of that before operation. Then rats were killed in 6 h, 1 d, 4 d, 7 d, 14 d and 21 d respectively after operation. Flow cytometer terminal deoxynucleotldyl transferease-mediated biotinylated deoxynuridine triphosphate nick end labeling (TUNEL), Caspase-3 activity assay and immunohistochemical method were applied to investigate Caspase-3 expression in the spinal cord tissue and to study neuronal apoptosis in rats. Results: After spinal cord injury, apoptotic cells detected by flow cytometry and TUNEL-positive cells were significantly more, and positive immunohistochemical staining of Caspase-3 and Caspase-3 activity were significantly higher in Group injury than in Groups control and laminectomy, respectively (P>0.05, P>0.01). Similar trend of changes was noticed in apoptotic cells, TUNEL-positive cells and positive immunohistochemical staining of Caspase-3, all of which reached their respective peak 7 days after operation. Caspase-3 activity reached its peak, however, 4 days postoperatively. Conclusions: Increased expression and activity of Caspase-3 protein in neurons after tractive spinal cord injury is the biochemical signal of early spinal cell apoptosis. It is of great significance for understanding the mechanism of spinal cord injury.

  14. 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 activa...

  15. The effect of K(+) on caspase activity of corneal epithelial cells exposed to UVB.

    Science.gov (United States)

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

    2016-10-01

    Exposure of human corneal limbal epithelial (HCLE) cells to UVB triggers rapid loss of K(+) and apoptosis via activation of caspases -9, -8 and -3. It has been shown that preventing loss of intracellular K(+) can inhibit apoptosis. The goal of this study was to investigate the effect of K(+) on the UVB-induced caspase activity. HCLE cells were exposed to 150 mJ/cm(2) UVB, followed by measurement of caspase activity in cell lysates. Caspase activity was measured in the presence and absence of 100 mM K(+) in the reaction buffer. UVB-induced activity of caspases -9, -8 and -3 all decreased in the presence of 100 mM K(+). These results suggest that a role of high [K(+)] in the cell is to inhibit caspase activity. Therefore, when cells lose K(+) in response to UVB, caspases are activated and cells go into apoptosis. This supports our hypothesis that K(+) inhibits caspase activity.

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

    Directory of Open Access Journals (Sweden)

    Marta I R Reis

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

  17. Octreotide induces caspase activation and apoptosis inhuman hepatoma HepG2 cells

    Institute of Scientific and Technical Information of China (English)

    Nikos J Tsagarakis; Ioannis Drygiannakis; Antonis G Batistakis; George Kolios; Elias A Kouroumalis

    2011-01-01

    AIM: To investigate the role of octreotide on cellular proliferation and apoptosis of human hepatoma (HepG2) cells.METHODS: We studied cellular proliferation, apoptosis and the possible internal caspase-mediated apoptosis pathway involved, after treatment of HepG2 carcinomacells with octreotide in comparison with the apoptosis caused by tumor necrosis factor-α (TNF-α). Activities of caspase-3, caspase-9, caspase-8 and caspase-2 were studied, while apoptosis was investigated through detection of DNA fragmentation and through identification of apoptotic cells with the annexin-V/propidium iodide flow cytometric method.RESULTS: After an initial increase in HepG2 cellular proliferation, a significant inhibition was observed with 10-8 mol/L octreotide, while TNF-α dose-dependentlydecreased proliferation. Early and late apoptosis was significantly increased with both substances. Octreotide significantly increased caspase-3, caspase-8 andcaspase-2 activity. TNF-α significantly increased only caspase-2. Cellular proliferation was decreased after treatment with octreotide or TNF-α alone but, in contrast to TNF-α, octreotide decreased proliferation onlyat concentrations of 10-8 mol/L, while lower concentrations increased proliferation.CONCLUSION: Our findings are suggestive of caspasemediated signaling pathways of octreotide antitumor activity in HepG2 cells, and indicate that measurementsof serum octreotide levels may be important, at least in clinical trials, to verify optimal therapeutic drug concentrations.

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

    OpenAIRE

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

    2012-01-01

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

  19. 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.

  20. CLINICOPATHOLOGICAL SIGNIFICANCE OF PTEN AND CASPASE-3 EXPRESSIONS IN BREAST CANCER

    Institute of Scientific and Technical Information of China (English)

    Xue-fei Yang; Yan Xin; Li-li Mao

    2008-01-01

    Objective To investigate the expressions of PTEN and Caspase-3 proteins in human breast carcinoma, and to evaluate their clinicopathological implications during the tumorigenesis and progression of breast cancer.Methods The expressions of PTEN and Caspase-3 proteins in 95 cases of breast cancer and 15 cases of benignbreast diseases were investigated immunohistochemically. Correlations between the expression of PTEN protein,Caspase-3 protein, and clinicopathological features of breast cancers were analyzed.Results The loss expression rate of PTEN protein in tumor tissues was significantly higher than that in benignbreast diseases (33.7% vs. 0, P 0. 05). In addition,the expression of PTEN protein had significantly positive correlation with the expression of Caspase-3 protein in breast cancer (P <0.01 ).Conclusion The combination detection of PTEN and Caspase-3 may serve as an important index to estimate the pathobiological behavior and pognosis of breast cancer.

  1. 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

    a role in the peripheral phenotypes, such as muscle wasting observed in HD. We assessed skeletal muscle tissue from HD patients and well-characterized mouse models of HD. Cleavage of the caspase-6 specific substrate lamin A is significantly increased in skeletal muscle obtained from HD patients as well...... 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......-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...

  2. Blockage of caspase-1 activation ameliorates bone marrow inflammation in mice after hematopoietic stem cell transplantation.

    Science.gov (United States)

    Qiao, Jianlin; Wu, Jinyan; Li, Yuanyuan; Xia, Yuan; Chu, Peipei; Qi, Kunming; Yan, Zhiling; Yao, Haina; Liu, Yun; Xu, Kailin; Zeng, Lingyu

    2016-01-01

    Conditioning regimens before hematopoietic stem cell transplantation (HSCT), cause damage to bone marrow and inflammation. Whether inflammasomes are involved in bone marrow inflammation remains unclear. The study aims to evaluate the role of inflammasomes in bone marrow inflammation after HSCT. On days 7, 14, 21 and 28 after HSCT, mice were sacrificed for analysis of bone marrow inflammation, pro-inflammatory cytokines secretion, inflammasomes expression and caspase-1 activation. Bone marrow inflammation with neutrophils and macrophages infiltration was observed after HSCT. Secretion of IL-1β, IL-18, TNF-α and IL-6 were elevated, with increased caspase-1 activation and inflammasomes expression. Caspase-1 inhibitor administration after HSCT significantly reduced infiltration of neutrophils and macrophages into bone marrow and increased the numbers of megakaryocytes and platelets. In conclusion, inflammasomes activation is involved in bone marrow inflammation after HSCT and caspase-1 inhibition attenuates bone marrow inflammation and promoted hematopoietic reconstitution, suggesting targeting caspase-1 might be beneficial for improving HSCT outcomes.

  3. Castration-induced expression of caspase-1 in epithelia of accessory sex organs in male rats

    Institute of Scientific and Technical Information of China (English)

    Masao Izawa; Mitunori Kimura; Tomiko Yamada; Makoto Saji

    2001-01-01

    Aim: As an attempt to clarify the molecular basis of castration-induced apoptosis, this study was undertaken to demonstrate the expression of caspase-1 in male accessory sex organs of rats. Methods and results: cDNA of rat caspase-1 was cloned by reverse transcription-polymerase chain reaction from the ventral prostates. The open reading frame predicts 402 amino acids, which shows more than 91% and 63 % identity to those of mouse and human, respec tively. Northern analyses demonstrated the presence of castration-induced up-regulation of the 1.6 kb transcript in the ventral prostate and the seminal vesicles. Finally, the authors demonstrated the caspase-1 transcripts in the epithelia of these tissues by in situ hybridization analyses. Conclusion: Castration induces the expression of caspase-1 tran scripts in the epithelia of ventral prostate and seminal vesicle. These observations suggest a possible role of caspase-1 in apoptosis in male accessory sex organs.

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

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

    Science.gov (United States)

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

    2015-06-30

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

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

    Directory of Open Access Journals (Sweden)

    Darren Finlay

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

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

    Institute of Scientific and Technical Information of China (English)

    LU Shi; ZHANG Baohua; WANG Zehua

    2005-01-01

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

  8. 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.

  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. Induction of Apoptosis by Green Synthesized Gold Nanoparticles Through Activation of Caspase-3 and 9 in Human Cervical Cancer Cells

    Science.gov (United States)

    Baharara, Javad; Ramezani, Tayebe; Divsalar, Adeleh; Mousavi, Marzieh; Seyedarabi, Arefeh

    2016-01-01

    Background: Gold Nanoparticles (GNPs) are used in imaging and molecular diagnostic applications. As the development of a novel approach in the green synthesis of metal nanoparticles is of great importance and a necessity, a simple and safe method for the synthesis of GNPs using plant extracts of Zataria multiflora leaves was applied in this study and the results on GNPs’ anticancer activity against HeLa cells were reported. Methods: The GNPs were characterized by UV-visible spectroscopy, FTIR, TEM, DLS and Zeta-potential measurements. In addition, the cellular up-take of nanoparticles was investigated using Dark Field Microscopy (DFM). Induction of apoptosis by high dose of GNPs in HeLa cells was assessed by MTT assay, Acridin orange, DAPI staining, Annexin V/PI double-labeling flow cytometry and caspase activity assay. Results: UV-visible spectroscopy results showed a surface plasmon resonance band for GNPs at 530 nm. FTIR results demonstrated an interaction between plant extract and nanoparticles. TEM images revealed different shapes for GNPs and DLS results indicated that the GNPs range in size from 10 to 42 nm. The Zeta potential values of the synthesized GNPs were between 30 to 50 Mev, indicating the formation of stable particles. As evidenced by MTT assay, GNPs inhibit proliferation of HeLa cells in dose-dependent GNPs and cytotoxicity of GNPs in Bone Marrow Mesenchymal Stem Cell (BMSCs) was lower than cancerous cells. At nontoxic concentrations, the cellular up-take of the nanoparticles took place. Acridin orange and DAPI staining showed morphological changes in the cell’s nucleus due to apoptosis. Finally, caspase activity assay demonstrated HeLa cell’s apoptosis through caspase activation. Conclusion: The results showed that GNPs have the ability to induce apoptosis in HeLa cells. PMID:27141266

  11. 活化 Caspase 9在牛磺酸保护神经细胞中的保护作用%Taurine protects neuronal cells by suppressing Caspase 9 activation

    Institute of Scientific and Technical Information of China (English)

    刘阳; 王李瑶; 张庆华; 夏鹤春; 孙涛

    2014-01-01

    detected by immunofluorescence and Western blot. Results The injury and apoptosis group showed a poor proliferation of the hippocampal neuronal cells and decreased cell viability (A=0.102 ±0.025), significantly lower than the control group (relative A=0.643 ± 0.013), the low-dose taurine group (relative A=0.504 ±0.072), and the high-dose taurine group (relative A=0.452 ±0.029) ( all P<0 .05 ) .Immunofluorescence assay revealed significantly increased Caspase 9 activation in the injury and apoptosis group (A=61386.8 ±10083.6) compared with the control (A=4502.2 ±2518.1), the low-dose taurine (A=20077.4 ±4187.5), and the high-dose taurine group (A=13976.2 ±7044.1) (all P<0.05).Western blot showed a remarkably higher expression of Caspase 9 in in the injury and apoptosis group (A=1.23) than in the control (relative A=0.17), the low-dose taurine (A=0.21), and the high-dose taurine group (A=0.19) (all P<0.05). Conclusion Taurine can protect neuronal cells by inhibi-ting Caspase 9 activation.

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

    Science.gov (United States)

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

    2016-01-15

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

  13. Acteoside Binds to Caspase-3 and Exerts Neuroprotection in the Rotenone Rat Model of Parkinson's Disease

    Science.gov (United States)

    Wang, Ying; He, Xiao; Zhao, Yuwu

    2016-01-01

    Parkinson’s disease (PD) is characterized by the progressive degeneration of the dopaminergic neurons in the substantia nigra (SN) region. Acteoside has displayed multiple biological functions. Its potential role against PD and the underlying signaling mechanisms are largely unknown. Here, we showed that oral administration of acteoside significantly attenuated parkinsonism symptoms in rotenone-induced PD rats. Further, acteoside inhibited rotenone-induced α-synuclein, caspase-3 upregulation and microtubule-associated protein 2 (MAP2) downregulation in PD rats. The molecular docking and molecular dynamics (MD) simulation results indicated that acteoside may directly bind to and inhibit caspase-3. Acteoside formed hydrogen bonds with at least six residues of caspase-3: ThrA177, SerA178, GlyA238, SerB339, ArgB341 and TrpB348. In addition, a pi-pi interaction was formed between acteoside and caspase-3’s HisA237, which might further stabilize the complex. MD simulation results demonstrated that the binding affinity of the caspase-3-acteoside complex was higher than that of caspase-3 and its native ligand inhibitor. Together, we show that acteoside binds to caspase-3 and exerts neuroprotection in the rotenone rat model of PD. PMID:27632381

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

    Directory of Open Access Journals (Sweden)

    Sonu Kumar

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

  15. Caspase-3 and survivin expression in pediatric neuroblastoma and their roles in apoptosis

    Institute of Scientific and Technical Information of China (English)

    王家祥; 郑树

    2004-01-01

    Background Neuroblastoma, one of the common tumors in children, possesses the feature of natural regression that might be related to apoptosis caspase-3 and survivin are believed to respectively induce and inhibit apoptosis. We investigated the expression of caspase-3 and survivin in pediatric neuroblastoma and the role that these genes played in apoptosis.Methods The expression of caspase-3 and survivin in pediatric neuroblastoma tissue samples was detected using in situ hybridization, ter mintuesal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), and immunohistochemical staining. The role that these genes played in apoptosis was then evaluated.Results A converse correlation was observed between the expression of survivin and caspase-3. When survivin was expressed at high levels in neuroblastoma samples, caspase-3 expression was downregulated, and the apoptotic index decreased simultaneously.Conclusion There is a converse correlation between the expression of caspase-3 and the expression of survivin in neuroblastoma cells, indicating that caspase-3 might induce apoptosis, and survivin may inhibit this process.

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

    Science.gov (United States)

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

    2016-01-15

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

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

    Lifescience Database Archive (English)

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

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

    International Nuclear Information System (INIS)

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

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

    Directory of Open Access Journals (Sweden)

    Mei‑Hsuan Chen

    2013-11-01

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

  20. Genetic deletion of caspase-2 accelerates MMTV/c-neu-driven mammary carcinogenesis in mice.

    Science.gov (United States)

    Parsons, M J; McCormick, L; Janke, L; Howard, A; Bouchier-Hayes, L; Green, D R

    2013-09-01

    Despite being the most evolutionarily conserved of the mammalian caspases, little is understood about the cellular function of caspase-2 in normal tissues or what role caspase-2 may have in the progression of human disease. It has been reported that deletion of the caspase-2 gene (Casp2), accelerates Eμ-myc lymphomagenesis in mice, and thus caspase-2 may act as a tumor suppressor in hematological malignancies. Here, we sought to extend these findings to epithelial cancers by examining the potential role of caspase-2 as a tumor suppressor in the mouse mammary carcinogenesis model; MMTV/c-neu. The rate of tumor acquisition was significantly higher in multiparous Casp2(-/-)/MMTV mice compared with Casp2(+/+)/MMTV and Casp2(+/-)/MMTV mice. Cells from Casp2(-/-)/MMTV tumors were often multinucleated and displayed bizarre mitoses and karyomegaly, while cells from Casp2(+/+)/MMTV and Casp2(+/-)/MMTV tumors never displayed this phenotype. Tumors from Casp2(-/-)/MMTV animals had a significantly higher mitotic index than tumors from Casp2(+/+)/MMTV and Casp2(+/-)/MMTV animals. Cell cycle analysis of Casp2(-/-) E1A/Ras-transformed mouse embryonic fibroblasts (MEF) also indicated a higher proliferative rate in the absence of caspase-2. In vitro assays further illustrated that MEF had increased genomic instability in the absence of caspase-2. This appears to be due to disruption of the p53 pathway because we observed a concomitant decrease in the induction of the p53 target genes, Pidd, p21 and Mdm2. Thus caspase-2 may function as a tumor suppressor, in part, through regulation of cell division and genomic stability.

  1. Norcantharidin induces apoptosis in HeLa cells through caspase, MAPK, and mitochondrial pathways

    Institute of Scientific and Technical Information of China (English)

    Wei-weiAN; Xian-fengGONG; Min-weiWANG; Shin-ichiTASHIRO; SatoshiONODERA; TakashiIKEJIMA

    2004-01-01

    AIM: To investigate the mechanism of norcantharidin (NCTD)-induced HeLa cell apoptosis. METHODS: HeLa cell growth inhibition was measured by MTT method. Apoptosis was detected by Hoechst 33258 staining and agarose gel electrophoresis. Caspase activities were assayed using caspase apoptosis detection kit. Western blot analysis was used to evaluate the level of ICAD, ERK/p-ERK, JNK/p-JNK, and Bcl-X.L/Bax expression. RESULTS: Norcantharidin inhibited HeLa cell growth in a time- and dose-dependent manner. HeLa cells treated with norcantharidin showed typical characteristics of apoptosis including the morphological changes and DNA fragmentation. Caspase family inhibitor (z-VAD-fmk), caspase-8, -9 inhibitor (z-IETD-fmk, Ac-LEHD-CHO, respectively) and caspase-3 inhibitor (z-DEVD-fmk) partially prevent norcantharidin-induced apoptosis, but initiator caspase-1 inhibitor (Ac-YVAD-fmk) did not. The activities of caspase-3, -8, and -9 were up-regulated after norcantharidin treatment. Furthermore, NCTD-induced activation of caspase-3 resulted in the degradation of the inhibitor of caspase-activated DNase (ICAD). Up-regulation of mitochondrial Bax expression and down-regulation of Bcl-xLexpression also participated in the apoptosis induced by NCTD. Although p38 MAPK inhibitor (SB203580) failed to block cell death, ERK MAPK inhibitor (PD98059) and JNK MAPK inhibitor (SP600125) had marked inhibitory effects on norcantharidin-induced apoptosis. Moreover, the phosphorylation of JNK were up-regulated followed by delayed ERK phosphorylation after treatment with NCTD, suggesting that ERK and JNK were both responsible for NCTD-induced apoptosis in HeLa cells and worked at different stages. CONCLUSION: The cytotoxic effect of NCTD on HeLa cells was mainly due to apoptosis. The anti-tumor mechanism of NCTD might involve caspses, mitochondrial, and MAPKs pathways.

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

    OpenAIRE

    Kranz, Dominique; Boutros, Michael

    2014-01-01

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

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

    DEFF Research Database (Denmark)

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

    2007-01-01

    In this study we investigated chronological onset and involvement of active caspase-3, apoptotic nuclear morphology, and TUNEL-labeling, as well as ultrastructural evidence of apoptosis, in both spontaneous and induced cell death during pre-implantation development of bovine in vitro produced...... staining for detection of apoptotic nuclear morphology, and subjected to fluorescence microscopy. Additionally, treated and untreated blastocysts were fixed and processed for ultrastructural identification of apoptosis. Untreated embryos revealed no apoptotic features at 2- and 4-cell stages. However......, active caspase-3 and apoptotic nuclear morphology were observed in an untreated 8-cell stage, and TUNEL-labeling was observed from the 16-cell stage. Blastomeres concurrently displaying all apoptotic features were present in a few embryos at 16-cell and morula stages and in all blastocysts. All three...

  4. Bcl-2、Caspase-3、Survivin与银屑病的研究进展%Research Progress of Bcl-2,Caspase-3,Survivin and Psoriasis

    Institute of Scientific and Technical Information of China (English)

    秦兰英; 邢卫斌; 叶文静

    2013-01-01

    Bcl-2, caspase-3, survivin genes are important genes in the process of apoptosis, playing important roles in psoriasis keratinocyte apoptosis. Bcl-2 is a kind of apoptosis suppressor gene, which can prolong life period of cells. Caspase-3 can promote cell apoptosis. Survivin is one of the strongest anti-apopto-sis factor discovered so far,which can inhibit cell apoptosis and promote cell proliferation. Psoriasis lesions contain less Bcl-2, more caspase-3 and survivin. Interaction between them may result in the shortened life period and fastened apoptosis in psoriasis keratinocy,and cells proliferation is obvious,which maintains the benign proliferative state of psoriasis epidermis.%Bcl-2、caspase-3、survivin是细胞凋亡过程中重要的调控基因,在银屑病角质形成细胞凋亡中,三种蛋白起着非常重要的作用.Bcl-2是一种凋亡抑制基因,可延长细胞的生存期,caspase-3可促进细胞凋亡,survivin是迄今发现最强的凋亡抑制因子,具有抑制细胞凋亡、促进细胞增殖的作用,在银屑病皮损中Bcl-2呈低表达,caspase-3、survivin呈高表达,三种蛋白的相互作用,可能导致银屑病角质形成细胞的生存期缩短、凋亡速度加快,同时细胞增殖明显,从而维持银屑病表皮的良性增生状态.

  5. Colonization of epidermal tissue by Staphylococcus aureus produces localized hypoxia and stimulates secretion of antioxidant and caspase-14 proteins.

    Science.gov (United States)

    Lone, Abdul G; Atci, Erhan; Renslow, Ryan; Beyenal, Haluk; Noh, Susan; Fransson, Boel; Abu-Lail, Nehal; Park, Jeong-Jin; Gang, David R; Call, Douglas R

    2015-08-01

    A partial-thickness epidermal explant model was colonized with green fluorescent protein (GFP)-expressing Staphylococcus aureus, and the pattern of S. aureus biofilm growth was characterized using electron and confocal laser scanning microscopy. The oxygen concentration in explants was quantified using microelectrodes. The relative effective diffusivity and porosity of the epidermis were determined using magnetic resonance imaging, while hydrogen peroxide (H2O2) concentration in explant media was measured by using microelectrodes. Secreted proteins were identified and quantified using elevated-energy mass spectrometry (MS(E)). S. aureus biofilm grows predominantly in lipid-rich areas around hair follicles and associated skin folds. Dissolved oxygen was selectively depleted (2- to 3-fold) in these locations, but the relative effective diffusivity and porosity did not change between colonized and control epidermis. Histological analysis revealed keratinocyte damage across all the layers of colonized epidermis after 4 days of culture. The colonized explants released significantly (P < 0.01) more antioxidant proteins of both epidermal and S. aureus origin, consistent with elevated H2O2 concentrations found in the media from the colonized explants (P< 0.001). Caspase-14 was also elevated significantly in the media from the colonized explants. While H2O2 induces primary keratinocyte differentiation, caspase-14 is required for terminal keratinocyte differentiation and desquamation. These results are consistent with a localized biological impact from S. aureus in response to colonization of the skin surface.

  6. Colonization of Epidermal Tissue by Staphylococcus aureus Produces Localized Hypoxia and Stimulates Secretion of Antioxidant and Caspase-14 Proteins

    Science.gov (United States)

    Lone, Abdul G.; Atci, Erhan; Renslow, Ryan; Beyenal, Haluk; Noh, Susan; Fransson, Boel; Abu-Lail, Nehal; Park, Jeong-Jin; Gang, David R.

    2015-01-01

    A partial-thickness epidermal explant model was colonized with green fluorescent protein (GFP)-expressing Staphylococcus aureus, and the pattern of S. aureus biofilm growth was characterized using electron and confocal laser scanning microscopy. The oxygen concentration in explants was quantified using microelectrodes. The relative effective diffusivity and porosity of the epidermis were determined using magnetic resonance imaging, while hydrogen peroxide (H2O2) concentration in explant media was measured by using microelectrodes. Secreted proteins were identified and quantified using elevated-energy mass spectrometry (MSE). S. aureus biofilm grows predominantly in lipid-rich areas around hair follicles and associated skin folds. Dissolved oxygen was selectively depleted (2- to 3-fold) in these locations, but the relative effective diffusivity and porosity did not change between colonized and control epidermis. Histological analysis revealed keratinocyte damage across all the layers of colonized epidermis after 4 days of culture. The colonized explants released significantly (P < 0.01) more antioxidant proteins of both epidermal and S. aureus origin, consistent with elevated H2O2 concentrations found in the media from the colonized explants (P< 0.001). Caspase-14 was also elevated significantly in the media from the colonized explants. While H2O2 induces primary keratinocyte differentiation, caspase-14 is required for terminal keratinocyte differentiation and desquamation. These results are consistent with a localized biological impact from S. aureus in response to colonization of the skin surface. PMID:25987705

  7. Colonization of epidermal tissue by Staphylococcus aureus produces localized hypoxia and stimulates secretion of antioxidant and caspase-14 proteins

    Energy Technology Data Exchange (ETDEWEB)

    Lone , Abdul G.; Atci, Erhan; Renslow, Ryan S.; Beyenal, Haluk; Noh, S.; Fransson, B.; Abu-Lail, Nehal; Park, Jeong-Jin; Gang, David R.; Call, Douglas R.

    2015-08-31

    A partial-thickness epidermal explant model was colonized with GFP-expressing S. aureus and the pattern of S. aureus biofilm growth was characterized using electron and confocal laser scanning microscopy. Oxygen concentration in explants was quantified using microelectrodes. The relative effective diffusivity and porosity of the epidermis were determined using magnetic resonance imaging, while hydrogen peroxide (H2O2) concentration in explant media was measured by using microelectrodes. Secreted proteins were identified and quantified using MSE mass spectrometry. We found that S. aureus biofilm grows predominantly in sebum-rich areas around hair follicles and associated skin folds. Dissolved oxygen was selectively depleted (2-3 fold) in these locations, but the relative effective diffusivity and porosity did not change between colonized and control epidermis. Histological analysis revealed keratinocyte damage across all the layers of colonized epidermis after four days of culture. The colonized explants released significantly (P< 0.01) more anti-oxidant proteins of both epidermal and S. aureus origin, consistent with elevated H2O2 concentration found in the media from the colonized explants (P< 0.001). Caspase-14 was also elevated significantly in media from infected explants. While H2O2 induces primary keratinocyte differentiation, caspase-14 is required for terminal keratinocyte differentiation and desquamation. These results are consistent with a localized biological impact from S. aureus in response to colonization of the skin surface.

  8. Evidence of caspase-mediated apoptosis induced by l-amino acid oxidase isolated from Bothrops atrox snake venom.

    Science.gov (United States)

    Alves, Raquel Melo; Antonucci, Gilmara Ausech; Paiva, Helder Henrique; Cintra, Adélia Cristina Oliveira; Franco, João José; Mendonça-Franqueiro, Elaine Paula; Dorta, Daniel Junqueira; Giglio, José Roberto; Rosa, José César; Fuly, André Lopes; Dias-Baruffi, Marcelo; Soares, Andreimar Martins; Sampaio, Suely Vilela

    2008-12-01

    The aim of this work was to investigate the involvement of caspases in apoptosis induced by l-amino acid oxidase isolated from Bothrops atrox snake venom. The isolation of LAAO involved three chromatographic steps: molecular exclusion on a G-75 column; ion exchange column by HPLC and affinity chromatography on a Lentil Lectin column. SDS-PAGE was used to confirm the expected high purity level of BatroxLAAO. It is a glycoprotein with 12% sugar and an acidic character, as confirmed by its amino acid composition, rich in "Asp and Glu" residues. It displays high specificity toward hydrophobic l-amino acids. The N-terminal amino acid sequence and internal peptide sequences showed close structural homology to other snake venom LAAOs. This enzyme induces in vitro platelet aggregation, which may be due to H2O2 production by LAAOs, since the addition of catalase completely inhibited the aggregation effect. It also showed cytotoxicity towards several cancer cell lines: HL60, Jurkat, B16F10 and PC12. The cytotoxicity activity was abolished by catalase. A fluorescence microscopy evaluation revealed a significant increase in the apoptotic index of these cells after BatroxLAAO treatment. This observation was confirmed by phosphatidyl serine exposure and activation of caspases. BatroxLAAO is a protein with various biological functions that can be involved in envenomation. Further investigations of its function will contribute to toxicology advances. PMID:18804547

  9. Equine torovirus (BEV induces caspase-mediated apoptosis in infected cells.

    Directory of Open Access Journals (Sweden)

    Ana M Maestre

    Full Text Available Toroviruses are gastroenteritis causing agents that infect different animal species and humans. To date, very little is known about how toroviruses cause disease. Here, we describe for the first time that the prototype member of this genus, the equine torovirus Berne virus (BEV, induces apoptosis in infected cells at late times postinfection. Observation of BEV infected cells by electron microscopy revealed that by 24 hours postinfection some cells exhibited morphological characteristics of apoptotic cells. Based on this finding, we analyzed several apoptotic markers, and observed protein synthesis inhibition, rRNA and DNA degradation, nuclear fragmentation, caspase-mediated cleavage of PARP and eIF4GI, and PKR and eIF2α phosphorylation, all these processes taking place after peak virus production. We also determined that both cell death receptor and mitochondrial pathways are involved in the apoptosis process induced by BEV. BEV-induced apoptosis at late times postinfection, once viral progeny are produced, could facilitate viral dissemination in vivo and contribute to viral pathogenesis.

  10. Caspase 9 promoter polymorphisms confer increased susceptibility to breast cancer.

    Science.gov (United States)

    Theodoropoulos, George E; Michalopoulos, Nikolaos V; Pantou, Malena P; Kontogianni, Panagiota; Gazouli, Maria; Karantanos, Theodoros; Lymperi, Maria; Zografos, George C

    2012-10-01

    Caspases (CASPs), play a crucial role in the development and progression of cancer. We evaluated the association between two polymorphisms (rs4645978 and rs4645981) of the CASP9 gene and the risk of breast cancer (BC). Genotypes and allelic frequencies for the two polymorphisms were determined in 261 patients with breast cancer and 480 healthy controls. Polymerase chain reaction-restriction fragment length polymorphisms were used, and statistical significance was determined by the χ(2) test. Carriers of the rs4645978G allele (AG and GG genotypes) were at higher risk for BC than individuals with other genotypes (odds ratio (OR) 1.59, 95% confidence interval (CI) 1.07-2.37, P = 0.022). The rs4645978GG genotype, in particular, was associated with the highest risk for BC development (OR 2.25, 95% CI 1.45-3.49, P = 0.0003). Similarly, individuals with at least one rs4645981T allele were at a significantly increased risk of developing BC compared with those harboring the CC genotype (OR 2.75, 95% CI 1.99-3.78, P < 0.0001), and the risk of BC increased with increasing numbers of rs4645981T alleles (OR 2.66, 95% CI 1.91-3.69, P < 0.0001 for the CT genotype; OR 3.95, 95% CI 1.58-9.88, P = 0.004 for the TT genotype). The CASP9 promoter polymorphisms rs4645978 and rs4645981 are associated with BC susceptibility and suggest that CASP9 transcriptional regulation is an important factor during BC development.

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

    Science.gov (United States)

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

    2015-11-01

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

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

    DEFF Research Database (Denmark)

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

    2001-01-01

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

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

    Directory of Open Access Journals (Sweden)

    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

  14. Caspase-3 activation and increased procollagen type I in irradiated hearts

    Directory of Open Access Journals (Sweden)

    Samara C. Ferreira-Machado

    2013-03-01

    Full Text Available The caspase-3-cleaved presence was evaluated in this study in the heart of irradiated rats, during the decline of ventricular function. Female Wistar rats were irradiated with a single dose of radiation (15 Gy delivered directly to the heart and the molecular, histological and physiological evaluations were performed at thirteen months post-irradiation. The expressions of procollagen type I, TGF-ß1 and caspase-3-cleaved were analyzed using Western blotting. Cardiac structural and functional alterations were investigated by echocardiography and electron microscopy. In the irradiated group, the levels of procollagen type I, TGF-ß1 and caspase-3-cleaved are increased. Significant histological changes (degeneration of heart tissue and collagen deposition and functional (reduced ejection fraction were observed. Data suggest that the cardiac function decline after exposure to ionizing radiation is related, in part, to increased collagen and increased caspase-3-cleaved.

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

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

    International Nuclear Information System (INIS)

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

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

    Directory of Open Access Journals (Sweden)

    VelvetLee Finckbone

    2009-05-01

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

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

    Directory of Open Access Journals (Sweden)

    Shikha Snigdha

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

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

    OpenAIRE

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

    2008-01-01

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

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

    OpenAIRE

    Bin Zhi; Lei Wang; Guangyi Wang; Xiaobo Zhang

    2011-01-01

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

  1. 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

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

    Science.gov (United States)

    Liu, Xue-lan; Shan, Wen-jie; Jia, Li-juan; Yang, Xu; Zhang, Jin-jing; Wu, Ya-rong; Xu, Fa-zhi; Li, Jin-nian

    2016-04-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

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

  4. Inhibition of caspase-9 aggravates acute liver injury through suppression of cytoprotective autophagy

    Science.gov (United States)

    Guo, Rui; Lin, Bin; Pan, Jing Fei; Liong, Emily C.; Xu, Ai Min; Youdim, Moussa; Fung, Man Lung; So, Kwok Fai; Tipoe, George L.

    2016-01-01

    Acute liver disease is characterized by inflammation, oxidative stress and necrosis, which can greatly influence the long term clinical outcome and lead to liver failure or cancer. Here, we initially demonstrated the beneficial role of caspase-9-dependent autophagy in acute liver injury. Treatment with caspase-9 inhibitor z-LEHD-FMK in HepG2 cells, AML12 cells and C57BL/b6N mice exacerbated CCl4-induced acute hepatocellular damage, and also down-regulated autophagy markers expression levels, indicating that caspase-9 inhibition may aggravate acute liver damage by suppressing cytoprotective autophagy. CCl4 was used as an acute liver injury inducer which caused oxidative stress and apoptosis through up-regulation of HIF-1α, as well as triggered hepatic inflammation and necroptosis via TLR4/NF-κB pathway. Caspase-9 Thr125 site was firstly phosphorylated by ERK1/2 which subsequently activated the cytoprotective autophagy process to attenuate acute CCl4 injury. Caspase-9 inhibition further aggravated hepatic necroptosis through NF-κB expression, leading to increased pro-inflammatory mediators levels, suggesting a protective role of caspase-9-dependent autophagy in the inflammatory process as well as its possibility being a new therapeutic target for the treatment of acute liver injury. PMID:27580936

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

    International Nuclear Information System (INIS)

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

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

    International Nuclear Information System (INIS)

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

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

    Science.gov (United States)

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

    2016-09-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

  9. Crystal structure of MC159 reveals molecular mechanism of DISC assembly and FLIP inhibition.

    Science.gov (United States)

    Yang, Jin Kuk; Wang, Liwei; Zheng, Lixin; Wan, Fengyi; Ahmed, Misonara; Lenardo, Michael J; Wu, Hao

    2005-12-22

    The death-inducing signaling complex (DISC) comprising Fas, Fas-associated death domain (FADD), and caspase-8/10 is assembled via homotypic associations between death domains (DDs) of Fas and FADD and between death effector domains (DEDs) of FADD and caspase-8/10. Caspase-8/10 and FLICE/caspase-8 inhibitory proteins (FLIPs) that inhibit caspase activation at the DISC level contain tandem DEDs. Here, we report the crystal structure of a viral FLIP, MC159, at 1.2 Angstroms resolution. It reveals a noncanonical fold of DED1, a dumbbell-shaped structure with rigidly associated DEDs and a different mode of interaction in the DD superfamily. Whereas the conserved hydrophobic patch of DED1 interacts with DED2, the corresponding region of DED2 mediates caspase-8 recruitment and contributes to DISC assembly. In contrast, MC159 cooperatively assembles with Fas and FADD via an extensive surface that encompasses the conserved charge triad. This interaction apparently competes with FADD self-association and disrupts higher-order oligomerization required for caspase activation in the DISC. PMID:16364918

  10. Effects of Modified Gandou Decoction on Protein and mRNA Expression of X-linked Inhibitor of Apoptosis, Caspase-9, and Caspase-3 in Brain Tissue of TX Mice%肝豆汤改良方对TX小鼠脑组织XIAP、Caspase-9和Caspase-3蛋白及其mRNA表达的影响

    Institute of Scientific and Technical Information of China (English)

    赵雯; 马艳红; 韩咏竹; 程楠; 饶娆; 王训

    2015-01-01

    目的 探究肝豆汤改良方抑制肝豆状核变性(Wilson's disease,WD)脑神经细胞凋亡的分子机制.方法 选取1月龄TX小鼠120只,随机分为TX模型组(40只)、肝豆汤改良方组(40只)、丁苯酞组(20只),另选DL小鼠40只作为正常组;每组再分成2组,各20只,分别予以生理盐水、肝豆汤改良方、丁苯酞灌胃2个月和4个月.分别采用免疫组织化学法和Western blot法检测小鼠脑组织中X-连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis,XIAP)、胱冬肽酶-9(Caspase-9)及胱冬肽酶-3(Caspase-3)蛋白表达水平,采用RT-PCR技术检测小鼠脑组织中XIAP、Caspase-9及Caspase-3 mRNA表达水平.结果 免疫组织化学检测显示,肝豆汤改良方组XIAP表达水平较同月龄模型组明显增多,Caspase-9和Caspase-3表达水平较同月龄模型组下降,5月龄和3月龄肝豆汤改良方组XIAP和Caspase-3表达水平有所差异.Western blot检测结果显示,月龄因素对脑组织XIAP、Caspase-9、Caspase-3蛋白表达水平的主效应均无统计学意义(P>0.05);月龄因素和分组因素对3种蛋白表达水平的交互作用均无统计学意义(P>0.05);对于同月龄小鼠,模型组脑组织XIAP表达水平显著低于正常组(P<0.05),Caspase-9和Caspase-3蛋白表达水平显著高于正常组(P<0.05);肝豆汤改良方组XIAP表达水平显著高于模型组(P<0.05),Caspase-9和Caspase-3蛋白表达水平显著低于模型组(P<0.05).RT-PCR检测结果显示,月龄因素对小鼠脑组织XIAP mRNA表达水平的主效应无统计学意义(P>0.05),但对Caspase-9、Caspase-3 mRNA表达水平的主效应具有统计学意义(P<0.05);月龄因素和分组因素对XIAP、Caspase-9、Caspase-3 mRNA表达水平的交互作用均无统计学意义(P>0.05).对于相同月龄小鼠,模型组脑组织XIAP mRNA表达水平显著低于正常组(P<0.05),而Caspase-9、Caspase-3 mRNA表达水平显著高于正常组(P<0.05);与模型组比较,丁苯酞

  11. Truncation of Caspase-3 on Phosphorylated tau%Caspase-3对磷酸化tau蛋白截断作用的研究

    Institute of Scientific and Technical Information of China (English)

    段萍; 李夏春; 邓艳秋; 张蕲; 王建枝

    2005-01-01

    磷酸化tau是阿尔茨海默病(Alzheimer's disease,AD)的特征性病理改变--神经原纤维缠结(neurofibrillarytangles,NFTs)的主要组成部分.最近的研究显示:NFT存在Glu391和Asp421位点被截断的tau片段,然而,tau蛋白的磷酸化是否会影响caspase-3的切割作用尚不清楚.首先纯化重组tau蛋白,然后利用蛋白激酶A(PKA)、钙/钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)和乳鼠海马组织抽提液对其磷酸化,并用caspase-3对不同磷酸化的tau蛋白进行切割,比较caspase-3对非磷酸化和不同蛋白激酶磷酸化的tau蛋白的切割特性.结果显示:除切割非磷酸化tau蛋白外,caspase-3在体外可分别切割被PKA、CaMKⅡ和乳鼠海马组织抽提液磷酸化的tau蛋白.这一结果提示:磷酸化修饰的tau蛋白仍然是caspase-3的底物.

  12. Levofloxacin increases the effect of serum deprivation on anoikis of rat nucleus pulposus cells via Bax/Bcl-2/caspase-3 pathway.

    Science.gov (United States)

    Yang, Si-Dong; Bai, Zhi-Long; Zhang, Feng; Ma, Lei; Yang, Da-Long; Ding, Wen-Yuan

    2014-12-01

    Levofloxacin, a fluoroquinolone, is a widely-used and effective antibiotic. However, various adverse side effects are associated with levofloxacin. The purpose of this study was to further explore the effects of levofloxacin on rat nucleus pulposus cells (NPCs). Inverted phase-contrast microscopy, flow cytometry and caspase-3 activity assays were used and revealed that serum deprivation induced apoptosis, which was markedly increased by levofloxacin in a dose-dependent manner. Simultaneously, levofloxacin decreased cell binding to type II collagen (COL2). Thus, levofloxacin-induced apoptosis exhibits characteristics of anoikis, the process by which cell death is triggered by separation from the extracellular matrix, which contains COL2. Furthermore, real-time quantitative RT-PCR was used to further confirm that levofloxacin downregulates COL2 expression in a dose-dependent manner. At last, western blot was used to find that levofloxacin increased the ratio of Bax/Bcl-2 and active caspase-3 in a dose-dependent manner. Levofloxacin therefore increases the effects of serum deprivation on anoikis by downregulating COL2 in rat NPCs in vitro via Bax/Bcl-2/caspase-3 pathway. This research provides a novel insight into the mechanisms of levofloxacin-induced toxicity and may potentially lead to a better understanding of the clinical effects of levofloxacin, especially in terms of intervertebral disc degeneration. PMID:25224805

  13. Dioscin-induced apoptosis of human LNCaP prostate carcinoma cells through activation of caspase-3 and modulation of Bcl-2 protein family.

    Science.gov (United States)

    Chen, Jing; Li, Hui-min; Zhang, Xue-nong; Xiong, Chao-mei; Ruan, Jin-lan

    2014-02-01

    Dioscin is a natural steroid saponin derived from several plants, showing potent anti-cancer effect against a variety of tumor cell lines. In the present study, we investigated the anti-cancer activity of dioscin against human LNCaP cells, and evaluated the possible mechanism involved in its antineoplastic action. It was found that dioscin (1, 2 and 4 μmol/L) could significantly inhibit the viability of LNCaP cells in a time- and concentration-dependent manner. Flow cytometry revealed that the apoptosis rate was increased after treatment of LNCaP cells with dioscin for 24 h, indicating that apoptosis was an important mechanism by which dioscin inhibited cancer. Western blotting was employed to detect the expression of caspase-3, Bcl-2 and Bax in LNCaP cells. The expression of cleaved caspase-3 was significantly increased, and meanwhile procaspase-3 was markedly decreased. The expression of anti-apoptotic protein Bcl-2 was down-regulated, whereas the pro-apoptotic protein Bax was up-regulated. Moreover, the Bcl-2/Bax ratio was drastically decreased. These results suggested that dioscin possessed potential anti-tumor activity in human LNCaP cells through the apoptosis pathway, which might be associated with caspase-3 and Bcl-2 protein family. PMID:24496691

  14. 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.

  15. 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.

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

    International Nuclear Information System (INIS)

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

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

    Directory of Open Access Journals (Sweden)

    Jazir Haneef

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

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

    Science.gov (United States)

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

    2012-01-01

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

  19. ROLE OF CASPASE-3 IN ACUTE LIGHT DAMAGE TO RETINA OF RATS

    Institute of Scientific and Technical Information of China (English)

    Xiao Wang; Shi-xing Hu; Wei Li; Shao-chun Lin

    2007-01-01

    Objective To investigate the role of Caspase-3 in retinal damage caused by light exposure in rats.Methods Light injury to retina was induced by persistent exposure to illumination (intensity; 30000±50 lux) of operating microscope for 30 minutes in the right eyes of Sprague-Dawley rats. The pathological changes of retina were observed under optical and electron microscopies at different time points, which were 6 hours, 1,3,7,and 15 days after the light exposure. Apoptosis of retinal cells was analyzed by flow cytometry. The activity of Caspase-3 was evaluated by using the Caspase-3 assay kit. At the same time, the expression of Caspase-3 protease was determined with Western blot analysis.Results The examination results of optical and transmission electron microscopes showed that edema of inner and outer segments of the retina, espectially the chondriosome inside the inner segment, became obvious 6 hours after the light exposure. The change was deteriorated along with the increasing time. The structures of the discoidal valve dissociated in the outer segment simultaneously. Disorderly arranged nuclei, karyopycnosis, and thinning in the outer nuclear layer were observed. The retinal pogment epithelium almost disappeared during the later stage. The staining results of Annexin-V combined with PI demonstrated that the proportion of apoptotic cells increased with time. The proportion between 7th day (82.7%) and 15th day (80.4%), however, showed no significant difference. Caspase-3 became remarkably active with the lapse of time, which increased from 0.02 at 6th hour to the peak of 9.8 at 7th day before it started to descend. The Western blot detected a expression of the active form of Caspase-3 at 7th day and 15th day.Conclusion Apoptosis of photoreceptor cells is markedly involved in the light damage and Caspase-3 protease may play an important role in the apoptotic process of the retina after light exposure in rats.

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

    International Nuclear Information System (INIS)

    Quinolones (QNs)-induced arthropathy is an important toxic effect in immature animals leading to restriction of their therapeutic use in pediatrics. However, the exact mechanism still remains unclear. Recently, we have demonstrated that ofloxacin, a typical QN, induces apoptosis of alginate microencapsulated juvenile rabbit joint chondrocytes by disturbing the β1 integrin functions and inactivating the ERK/MAPK signaling pathway. In this study, we extend our initial observations to further elucidate the mechanism(s) of ofloxacin-induced apoptosis by utilizing specific caspase inhibitors. Pretreatment with both caspase-9-specific inhibitor zLEHD-fmk and caspase-8 inhibitor zIETD-fmk attenuated ofloxacin-induced apoptosis and activation of caspase-3 of chondrocyte in a concentration-dependent manner, as determined by fluorescent dye staining, enzyme activity assay and immunoblotting. Furthermore, the activation of caspase-9, -8 and -3 stimulated by ofloxacin was significantly inhibited in the presence of zIETD-fmk while pretreatment with zLEHD-fmk only blocked the activation of caspase-9 and -3. Ofloxacin also stimulated a concentration-dependent translocation of cytochrome c from mitochondria into the cytosol and a decrease of mitochondrial transmembrane potential, which was completely inhibited by zIETD-fmk. In addition, ofloxacin was found to increase the level of Bax, tBid, p53 in a concentration- and time-dependent manner. Taken together, The current results indicate that the caspase-8-dependent mitochondrial pathway is primarily involved in the ofloxacin-induced apoptosis of microencapsulated juvenile rabbit joint chondrocytes

  1. 宫颈癌术前介入化疗前后组织中Survivin、Caspase-3和Caspase-7的表达变化及意义%The significance of different expression of survivin,caspase-3 and caspase-7 in cervical carcinoma cells before and after the ameba femoralis interventional chemotherapy

    Institute of Scientific and Technical Information of China (English)

    杨斌; 陈赛英; 史佃云; 张佃乾

    2009-01-01

    目的:研究宫颈癌术前行动脉介入化疗前后组织中细胞凋亡相关蛋白Survivin、caspase-3、Caspase-7的表达变化及其临床意义.方法:对54例宫颈癌患者术前行介入化疗前宫颈活检组织和介入化疗后手术切除的标本,免疫组化检测Survivin、Caspase-3、Caspase-7的表达,同时检测38例正常宫颈组织以及28例宫颈上皮内瘤变组织作为对照.结果:Survivin在介入化疗前宫颈癌组织中呈高表达,介入化疗后Survivin呈现不同程度的下降,而凋亡相关蛋白Caspase-3、Caspase-7的表达则呈不同程度的上升.相关性分析表明,介入化疗后Survivin的表达变化与Caspase-3及Capase-7的表达变化呈负相关,介入前后3种蛋白表达变化越大,临床有效率越高.结论:Survivin高表达及Caspase-3、Caspase-7低表达在官颈癌的发生发展中有一定作用,宫颈癌患者术前动脉介入化疗可以通过促进肿瘤细胞的凋亡来达到抑制肿瘤生长、缩小肿瘤体积进而抑制其远处转移.

  2. 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.

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

    Science.gov (United States)

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

    2016-01-01

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

  4. HAMLET triggers apoptosis but tumor cell death is independent of caspases, Bcl-2 and p53.

    Science.gov (United States)

    Hallgren, O; Gustafsson, L; Irjala, H; Selivanova, G; Orrenius, S; Svanborg, C

    2006-02-01

    HAMLET (Human alpha-lactalbumin Made Lethal to Tumor cells) triggers selective tumor cell death in vitro and limits tumor progression in vivo. Dying cells show features of apoptosis but it is not clear if the apoptotic response explains tumor cell death. This study examined the contribution of apoptosis to cell death in response to HAMLET. Apoptotic changes like caspase activation, phosphatidyl serine externalization, chromatin condensation were detected in HAMLET-treated tumor cells, but caspase inhibition or Bcl-2 over-expression did not prolong cell survival and the caspase response was Bcl-2 independent. HAMLET translocates to the nuclei and binds directly to chromatin, but the death response was unrelated to the p53 status of the tumor cells. p53 deletions or gain of function mutations did not influence the HAMLET sensitivity of tumor cells. Chromatin condensation was partly caspase dependent, but apoptosis-like marginalization of chromatin was also observed. The results show that tumor cell death in response to HAMLET is independent of caspases, p53 and Bcl-2 even though HAMLET activates an apoptotic response. The use of other cell death pathways allows HAMLET to successfully circumvent fundamental anti-apoptotic strategies that are present in many tumor cells.

  5. 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

    2016-04-01

    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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Sun Lei

    2009-02-01

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

  8. Human Primary Keratinocytes as a Tool for the Analysis of Caspase-1-Dependent Unconventional Protein Secretion.

    Science.gov (United States)

    Strittmatter, Gerhard E; Garstkiewicz, Martha; Sand, Jennifer; Grossi, Serena; Beer, Hans-Dietmar

    2016-01-01

    Inflammasomes comprise a group of protein complexes, which activate the protease caspase-1 upon sensing a variety of stress factors. Active caspase-1 in turn cleaves and thereby activates the pro-inflammatory cytokines prointerleukin (IL)-1β and -18, and induces unconventional protein secretion (UPS) of mature IL-1β, IL-18, as well as of many other proteins involved in and required for induction of inflammation. Human primary keratinocytes (HPKs) represent epithelial cells able to activate caspase-1 in an inflammasome-dependent manner upon irradiation with a physiological dose of ultraviolet B (UVB) light. Here, we describe the isolation of keratinocytes from human skin, their cultivation, and induction of caspase-1-dependent UPS upon UVB irradiation as well as its siRNA- and chemical-mediated inhibition. In contrast to inflammasome activation of professional immune cells, UVB-irradiated HPKs represent a robust and physiological cell culture system for the analysis of UPS induced by active caspase-1. PMID:27665556

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

    Indian Academy of Sciences (India)

    Entsar Saheb; Wendy Trzyna; John Bush

    2014-12-01

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

  10. Megakaryocytes regulate expression of Pyk2 isoforms and caspase-mediated cleavage of actin in osteoblasts.

    Science.gov (United States)

    Kacena, Melissa A; Eleniste, Pierre P; Cheng, Ying-Hua; Huang, Su; Shivanna, Mahesh; Meijome, Tomas E; Mayo, Lindsey D; Bruzzaniti, Angela

    2012-05-18

    The proliferation and differentiation of osteoblast (OB) precursors are essential for elaborating the bone-forming activity of mature OBs. However, the mechanisms regulating OB proliferation and function are largely unknown. We reported that OB proliferation is enhanced by megakaryocytes (MKs) via a process that is regulated in part by integrin signaling. The tyrosine kinase Pyk2 has been shown to regulate cell proliferation and survival in a variety of cells. Pyk2 is also activated by integrin signaling and regulates actin remodeling in bone-resorbing osteoclasts. In this study, we examined the role of Pyk2 and actin in the MK-mediated increase in OB proliferation. Calvarial OBs were cultured in the presence of MKs for various times, and Pyk2 signaling cascades in OBs were examined by Western blotting, subcellular fractionation, and microscopy. We found that MKs regulate the temporal expression of Pyk2 and its subcellular localization. We also found that MKs regulate the expression of two alternatively spliced isoforms of Pyk2 in OBs, which may regulate OB differentiation and proliferation. MKs also induced cytoskeletal reorganization in OBs, which was associated with the caspase-mediated cleavage of actin, an increase in focal adhesions, and the formation of apical membrane ruffles. Moreover, BrdU incorporation in MK-stimulated OBs was blocked by the actin-polymerizing agent, jasplakinolide. Collectively, our studies reveal that Pyk2 and actin play an important role in MK-regulated signaling cascades that control OB proliferation and may be important for therapeutic interventions aimed at increasing bone formation in metabolic diseases of the skeleton. PMID:22447931

  11. Megakaryocytes Regulate Expression of Pyk2 Isoforms and Caspase-mediated Cleavage of Actin in Osteoblasts*

    Science.gov (United States)

    Kacena, Melissa A.; Eleniste, Pierre P.; Cheng, Ying-Hua; Huang, Su; Shivanna, Mahesh; Meijome, Tomas E.; Mayo, Lindsey D.; Bruzzaniti, Angela

    2012-01-01

    The proliferation and differentiation of osteoblast (OB) precursors are essential for elaborating the bone-forming activity of mature OBs. However, the mechanisms regulating OB proliferation and function are largely unknown. We reported that OB proliferation is enhanced by megakaryocytes (MKs) via a process that is regulated in part by integrin signaling. The tyrosine kinase Pyk2 has been shown to regulate cell proliferation and survival in a variety of cells. Pyk2 is also activated by integrin signaling and regulates actin remodeling in bone-resorbing osteoclasts. In this study, we examined the role of Pyk2 and actin in the MK-mediated increase in OB proliferation. Calvarial OBs were cultured in the presence of MKs for various times, and Pyk2 signaling cascades in OBs were examined by Western blotting, subcellular fractionation, and microscopy. We found that MKs regulate the temporal expression of Pyk2 and its subcellular localization. We also found that MKs regulate the expression of two alternatively spliced isoforms of Pyk2 in OBs, which may regulate OB differentiation and proliferation. MKs also induced cytoskeletal reorganization in OBs, which was associated with the caspase-mediated cleavage of actin, an increase in focal adhesions, and the formation of apical membrane ruffles. Moreover, BrdU incorporation in MK-stimulated OBs was blocked by the actin-polymerizing agent, jasplakinolide. Collectively, our studies reveal that Pyk2 and actin play an important role in MK-regulated signaling cascades that control OB proliferation and may be important for therapeutic interventions aimed at increasing bone formation in metabolic diseases of the skeleton. PMID:22447931

  12. Astragalus saponins induce apoptosis in human gastric adenocarcinoma cells via a caspase 3-dependent pathway

    Institute of Scientific and Technical Information of China (English)

    JOSHUA K S Ko; Kathy K W Auyeung

    2008-01-01

    Objective Many Asian countriea including China, Japan and Korea have very high incidence of gastric cancer, in which about 42 % cases occur in mainland China. The precise targets and underlying mechanisms are not well understood. Our previous study revealed that Astragalus saponins (AST) showed promising effects on the suppression of the growth of HT-29 human colon cancer cells and tumor xenograft by inhibiting cell proliferation and promoting apoptosis. In the present study, we investigated the anti-carcinogenic effects of AST in AGS human gastric adenocarcinoma cells and attempted to elucidate the underlying mechanisms. Methods Growth inhibition of AGS cells was determined by using the MTT viability test. Involvement of different members of the apoptotic cascade and other growth-related factors was explored by assessment of their protein expression using Western blot analysis. Distribution of cells in different phases of the cell cycle was assessed by flow eytometry. Results Our data indicate that AST induced growth-inhibition and apoptosis in AGS cells by activating caspase 3 with subsequent poly (ADP-ribose) polymerase (PARP) cleavage. Cell cycle arrest at the G2/M phase had been observed in AST-treated AGS cells. The anti-proliferative effect of AST was associated with modulation of eydin B1 and p21. We then demonstrate that AST could downregulate the expression of VEGF, of which interaction with its receptors is important for angiogenesis during tumor formation. Conclusions Our findings suggest that AST is an effective agent in gastric cancer treatment by inducing cell cycle arrest and apoptosis, of which anti-angiogenesis could be an alternative mode of action.

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

    OpenAIRE

    Bloemberg, Darin; Quadrilatero, Joe

    2016-01-01

    Investigating cell death signaling using cell culture is commonly performed to examine the effects of novel pharmaceuticals or to further characterize discrete cellular signaling pathways. Here, we provide data regarding the cell death response to either cisplatin or A23187 in sub-confluent C2C12 cells, by utilizing several concentrations and incubation times for each chemical. These data include an assessment of the activation of the proteolytic enzymes caspase-3, caspase-8, caspase-9, calpa...

  14. Safrole oxide induces apoptosis by activating caspase-3, -8, and -9 in A549 human lung cancer cells.

    Science.gov (United States)

    Du, Aiying; Zhao, Baoxiang; Yin, Deling; Zhang, Shangli; Miao, Junying

    2006-01-01

    Previously we found that 3,4-(methylenedioxy)-1-(2',3'-epoxypropyl)-benzene (safrole oxide) induced a typical apoptosis in A549 human lung cancer cells. In this study, we further investigated which caspases were activated by safrole oxide during the apoptosis. The data showed that the activity of caspase-3, -8, and -9 was significantly enhanced by the compound, which suggested that safrole oxide might be used as a caspase promoter to initiate lung cancer cell apoptosis.

  15. Caspase Inhibitors may Attenuate Opioid-induced Hyperalgesia and Tolerance via Inhibiting Microglial Activation and Neuroinflammation

    Directory of Open Access Journals (Sweden)

    Jiancheng Zhang

    2013-07-01

    Full Text Available Prolonged exposure to an opioid induces hyperalgesia and tolerance, which negatively affect pain management in turn and significantly hamper the application of opioids. A growing body of evidence has demonstrated that glial activation contributes to the development of these two side effects. Recent studies have demonstrated that morphine, binding to an accessory protein of Toll-like receptor 4 (TLR4, activates microglia and produces neuroinflammation in amanner parallel to lipopolysaccharide. Meanwhile, lipopolysaccharide activates microglia through TLR4/caspase signalling. Therefore, we hypothesise that morphine may activate microglia throughTLR4/caspase signalling and that caspase inhibitors may attenuate opioid-induced hyperalgesia and tolerance via inhibiting microglial activation and neuroinflammation

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

    Science.gov (United States)

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

    2014-01-01

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

  17. Caspase-3-mediated degradation of condensin Cap-H regulates mitotic cell death.

    Science.gov (United States)

    Lai, S-K; Wong, C-H; Lee, Y-P; Li, H-Y

    2011-06-01

    Mitotic death is a major form of cell death in cancer cells that have been treated with chemotherapeutic drugs. However, the mechanisms underlying this form of cell death is poorly understood. Here, we report that the loss of chromosome integrity is an important determinant of mitotic death. During prolonged mitotic arrest, caspase-3 is activated and it cleaves Cap-H, a subunit of condensin I. The depletion of Cap-H results in the loss of condensin I complex at the chromosomes, thus affecting the integrity of the chromosomes. Consequently, DNA fragmentation by caspase-activated DNase is facilitated, thus driving the cell towards mitotic death. By expressing a caspase-resistant form of Cap-H, mitotic death is abrogated and the cells are able to reenter interphase after a long mitotic delay. Taken together, we provide new insights into the molecular events that occur during mitotic death.

  18. 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....

  19. Fragment N2, a caspase-3-generated RasGAP fragment, inhibits breast cancer metastatic progression.

    Science.gov (United States)

    Barras, David; Lorusso, Girieca; Lhermitte, Benoît; Viertl, David; Rüegg, Curzio; Widmann, Christian

    2014-07-01

    The p120 RasGAP protein negatively regulates Ras via its GAP domain. RasGAP carries several other domains that modulate several signaling molecules such as Rho. RasGAP is also a caspase-3 substrate. One of the caspase-3-generated RasGAP fragments, corresponding to amino acids 158-455 and called fragment N2, was previously reported to specifically sensitize cancer cells to death induced by various anticancer agents. Here, we show that fragment N2 inhibits migration in vitro and that it impairs metastatic progression of breast cancer to the lung. Hence, stress-activated caspase-3 might contribute to the suppression of metastasis through the generation of fragment N2. These results indicate that the activity borne by fragment N2 has a potential therapeutic relevance to counteract the metastatic process.

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

    Institute of Scientific and Technical Information of China (English)

    戚基萍; 吴爱萍; 王德生; 王立峰; 李淑霞; 徐凤琳

    2004-01-01

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

  1. Two new glycosides isolated from Sapindus mukorossi fruits: effects on cell apoptosis and caspase-3 activation in human lung carcinoma cells.

    Science.gov (United States)

    Zhang, Xuan-Ming; Yang, De-Po; Xie, Zhi-Yong; Li, Qing; Zhu, Long-Ping; Zhao, Zhi-Min

    2016-07-01

    Two new glycosides (1, 2) and two saponins (3, 4) were isolated from the fruits of Sapindus mukorossi Gaertn. The two glycosides were designated as sapindoside G (1) and 4'',4'''''-O-diacetylmukurozioside IIa (2). All four compounds exhibited inhibitory effects against A549 human lung adenocarcinoma cells with inhibition rates up to 69.2-83.3% at a concentration of 100 μg/mL. Flow cytometric analysis revealed that compounds 1-4 could suppress A549 cell growth by promoting cell apoptosis, which was related to the activation of caspase-3. PMID:26158392

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

    OpenAIRE

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

    2010-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Ernesto R Bongarzone

    2011-10-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2009-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Yuji Kajiwara

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2015-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-02-10

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

  8. Nucleoside drugs induce cellular differentiation by caspase-dependent degradation of stem cell factors.

    Directory of Open Access Journals (Sweden)

    Tanja Musch

    Full Text Available BACKGROUND: Stem cell characteristics are an important feature of human cancer cells and play a major role in the therapy resistance of tumours. Strategies to target cancer stem cells are thus of major importance for cancer therapy. Differentiation therapy by nucleoside drugs represents an attractive approach for the elimination of cancer stem cells. However, even if it is generally assumed that the activity of these drugs is mediated by their ability to modulate epigenetic pathways, their precise mode of action remains to be established. We therefore analysed the potential of three nucleoside analogues to induce differentiation of the embryonic cancer stem cell line NTERA 2 D1 and compared their effect to the natural ligand retinoic acid. METHODOLOGY/PRINCIPAL FINDINGS: All nucleoside analogues analyzed, but not retinoic acid, triggered proteolytic degradation of the Polycomb group protein EZH2. Two of them, 3-Deazaneplanocin A (DZNep and 2'-deoxy-5-azacytidine (decitabine, also induced a decrease in global DNA methylation. Nevertheless, only decitabine and 1beta-arabinofuranosylcytosine (cytarabine effectively triggered neuronal differentiation of NT2 cells. We show that drug-induced differentiation, in contrast to retinoic acid induction, is caused by caspase activation, which mediates depletion of the stem cell factors NANOG and OCT4. Consistent with this observation, protein degradation and differentiation could be counteracted by co-treatment with caspase inhibitors or by depletion of CASPASE-3 and CASPASE-7 through dsRNA interference. In agreement with this, OCT4 was found to be a direct in-vitro-target of CASPASE-7. CONCLUSIONS/SIGNIFICANCE: We show that drug-induced differentiation is not a consequence of pharmacologic epigenetic modulation, but is induced by the degradation of stem-cell-specific proteins by caspases. Our results thus uncover a novel pathway that induces differentiation of embryonic cancer stem cells and is triggered by

  9. Effect of tenuigenin on the Caspase-3 and Par-4 expression of neural stem cells induced by beta-amyloid protein%远志总皂苷干预β-淀粉样蛋白致伤神经干细胞Caspase-3及Par-4的表达

    Institute of Scientific and Technical Information of China (English)

    张晓梅; 孙光涛; 黄作义; 吴成吉; 戚询中; 朱晓峰

    2011-01-01

    BACKGROUND: The effect of tenuigenin which has a good nerve protection to neural stem cells has not been reported. OBJECTIVE: To investigate the protective effect and mechanism of tenuigenin for neural stem cells in the hippocampus impaired by β-amyloid protein.METHODS: The third passage neural stem cells generated from the hippocampi of Kunming mice which injured by β-amyloid protein in vitro were incubated with different concentrations of tenuigenin.RESULTS AND CONCLUSION: Immunocytochemical technique was used to detect Caspase3-positive neural stem cells. The study revealed that the expression of Par-4 and caspase-3 positive neural stem cells in the tenuigenin group were significantly lower than that in the control group with statistical significance (P < 0.05). Tenuigenin can reduce the expression of Par-4 and Caspase-3 in neural stem cells impaired by β-amyloid protein.%背景:远志总皂苷具有良好的神经保护作用.目的:分析远志总皂苷对β-淀粉样蛋白致伤海马神经干细胞的保护作用及机制.方法:自昆明小鼠海马分离培养神经干细胞,取第3代神经干细胞,用含不同质量浓度远志总皂苷与β-淀粉样蛋白致伤体外培养的神经干细胞共孵育.结果:应用免疫组织化学法检测Caspase-3阳性神经干细胞,与对照组相比,远志总皂苷组Caspase-3阳性细胞率及Par-4的表达明显降低,差异具有显著性意义(P<0.05).提示远志总皂苷能够降低β-淀粉样蛋白致伤神经干细胞中Caspase-3及Par-4的表达.

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

    Science.gov (United States)

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

    2012-02-01

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

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

    Science.gov (United States)

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

    2016-05-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-05-09

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

  13. Low Dose and Long Term Toxicity of Sodium Arsenite Caused Caspase Dependent Apoptosis Based on Morphology and Biochemical Character

    Directory of Open Access Journals (Sweden)

    Mohammad Hussein Abnosi

    2012-01-01

    Full Text Available Objective: Although arsenite is toxic it is currently recommended for the treatment of malignancies. In this study the effects of sub-micromolar concentrations of sodium arsenite on the viability, morphology and mechanism of cell death of rat bone marrow mesenchymal stem cells (BMCs over 21 days was investigated.Materials and Methods: In this experimental study, BMCs were extracted in Dulbecco’s Modified Eagles Medium (DMEM containing 15% of fetal bovine serum (FBS and expanded till the 3rd passage. The cells were treated with 1, 10, 25, 50, 75 and 100 nM of sodium arsenite for 21 days and the viability of the cells estimated using 3-(4, 5-dimethylthiazol-2-yl-2, 5 diphenyl tetrazolium (MTT and trypan blue staining. Cells were then treated with the selected dose (25 nM of sodium arsenite to determine their colony forming ability (CFA and population doubling number (PDN. Morphology of the cells was studied using florescent dyes, and the integrity of the DNA was investigated using the comet assay and agarose gel electrophoresis. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL and the caspase 3 assay were then applied to understand the mechanism of cell death. Data was analyzed using one way ANOVA, Tukey test.Results: A significant reduction of viability, PDN and CFA was found following treatment of BMCs with 25 nM sodium arsenite (p<0.05. Cytoplasm shrinkage and a significant decrease in the diameter of the nuclei were also seen. Comet assay and agarose gel electrophoresis revealed DNA breakage, while positive TUNEL and activated caspase 3 confirmed the apoptosis.Conclusion: A low concentration of sodium arsenite (25 nM caused reduction of viability due to induction of apoptosis. Therefore, long term exposure to low dose of this chemical may have unwanted effects on BMCs.

  14. Oridonin induces apoptosis in gastric cancer through Apaf-1, cytochrome c and caspase-3 signaling pathway

    Institute of Scientific and Technical Information of China (English)

    Ke-Wang Sun; Ying-Yu Ma; Tian-Pei Guan; Ying-Jie Xia; Chang-Ming Shao; Le-Gao Chen; Ya-Jun Ren

    2012-01-01

    /ethidium bromide staining.After treatment with oridonin,the cells became round,shrank,and developed small buds around the nuclear membrane while forming apoptotic bodies.Lactate dehydrogenase (LDH) release assay showed that after treated with 1.25 μg/mL and 20 μg/mL oridonin for 24 h,LDH release of HGC-27 caused by apoptosis increased from 22.94% ± 3.8% to 52.68% ± 2.4% (P < 0.001).However,the change in the release of LDH caused by necrosis was insignificant,suggesting that the major cause of oridonin-induced HGC-27 cell death was apoptosis.Flow cytometric analysis also revealed that oridonin induced significant apoptosis compared with the controls (P < 0.05).And the apoptosis rates of HGC-27 induced by the four different concentrations of oridonin were 5.3% ± 1.02%,12.8% ± 2.53%,28.5% ± 4.23% and 49.6% ± 3.76%,which were in a dose-dependent manner (P < 0.05).After treatment for 24 h,DNA ladder showed that oridonin induced a significant increase in DNA fragmentation in a dosedependent manner.RT-PCR revealed that mRNA expression levels were up-regulated compared with the controls in caspase-3 (0.917 ± 0.103 vs 0.357 ± 0.019,P < 0.05),cytochrome c (1.429 ± 0.111 vs 1.002 ±0.014,P < 0.05),Apaf-1 (0.688 ± 0.101 vs 0.242 ±0.037,P < 0.05) and Bax (0.856 ± 0.101 vs 0.278 ±0.027,P < 0.05) (P < 0.05),whereas down-regulated in Bd-2 (0.085 ± 0.012 vs 0.175 ± 0.030,P < 0.05).Western blotting analysis also confirmed this result.CONCLUSION:Apoptosis of HGC-27 induced by oridonin may be associated with differential expression of Apaf-1,caspase-3 and cytochrome c,which are highly dependent upon the mitochondrial pathway.

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

    Science.gov (United States)

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

    2016-12-01

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

  16. 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...

  17. TRAF2 Sets a threshold for extrinsic apoptosis by tagging caspase-8 with a ubiquitin shutoff timer.

    Science.gov (United States)

    Gonzalvez, Francois; Lawrence, David; Yang, Becky; Yee, Sharon; Pitti, Robert; Marsters, Scot; Pham, Victoria C; Stephan, Jean-Philippe; Lill, Jennie; Ashkenazi, Avi

    2012-12-28

    Apoptotic caspase activation mechanisms are well defined, yet inactivation modes remain unclear. The death receptors (DRs), DR4, DR5, and Fas, transduce cell-extrinsic apoptotic signals by recruiting caspase-8 into a death-inducing signaling complex (DISC). At the DISC, Cullin3-dependent polyubiquitination on the small catalytic subunit of caspase-8 augments stimulation. Here we report that tumor necrosis factor receptor-associated factor 2 (TRAF2) interacts with caspase-8 at the DISC, downstream of Cullin3. TRAF2 directly mediates RING-dependent, K48-linked polyubiquitination on the large catalytic domain of caspase-8. This modification destines activated caspase-8 molecules to rapid proteasomal degradation upon autoprocessing and cytoplasmic translocation. TRAF2 depletion lowers the signal threshold for DR-mediated apoptosis, altering cell life versus death decisions in vitro and in vivo. Thus, TRAF2 sets a critical barrier for cell-extrinsic apoptosis commitment by tagging activated caspase-8 with a K48-ubiquitin shutoff timer. These results may have important implications for caspase regulation mechanisms.

  18. Staphylococcus aureus alpha-toxin-induced cell death : predominant necrosis despite apoptotic caspase activation

    NARCIS (Netherlands)

    Essmann, F; Bantel, H; Totzke, G; Engels, I H; Sinha, B; Schulze-Osthoff, K; Jänicke, R U

    2003-01-01

    Recent data suggest that alpha-toxin, the major hemolysin of Staphylococcus aureus, induces cell death via the classical apoptotic pathway. Here we demonstrate, however, that although zVAD-fmk or overexpression of Bcl-2 completely abrogated caspase activation and internucleosomal DNA fragmentation,

  19. Gamma secretase activating protein is a substrate for caspase-3: implications for Alzheimer’s disease

    Science.gov (United States)

    Chu, Jin; Li, Jian-Guo; Joshi, Yash B.; Giannopoulos, Phillip F.; Hoffman, Nicholas E.; Madesh, Muniswamy; Praticò, Domenico

    2014-01-01

    SUMMARY Background A major hallmark feature of Alzheimer’s disease (AD) is the accumulation of amyloid β (Aβ), whose formation is regulated by the γ-secretase complex and its activating protein (also known as GSAP). Because GSAP interacts with the γ-secretase without affecting the cleavage of Notch, it is an ideal target for a viable anti-Aβ therapy. However, despite much interest in this protein, the mechanisms involved in its neurobiology are not known. Methods Post-mortem brain tissues from AD patients, transgenic mouse models of AD and neuronal cells were used to investigate the molecular mechanism involved in GSAP formation and subsequent amyloidogenesis. Results We identify a caspase-3 processing domain in the GSAP sequence and provide experimental evidence that this caspase is essential for GSAP activation and biogenesis of Aβ peptides. Furthermore, we demonstrate that caspase-3-dependent GSAP formation occurs in brains of individuals with AD and two different mouse models of AD, and that the process is biologically relevant since its pharmacological blockade reduces Aβ pathology in vivo. Interpretation Our data by identifying caspase-3 as the endogenous modulator of GSAP and Aβ production establish it as a novel, attractive and viable Aβ lowering therapeutic target for AD. PMID:25052851

  20. 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...

  1. 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.

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

    Science.gov (United States)

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

    2012-08-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  4. 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.

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

    Directory of Open Access Journals (Sweden)

    Zhang Yiying

    2012-08-01

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

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

    Science.gov (United States)

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

    2008-02-01

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

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

    Institute of Scientific and Technical Information of China (English)

    宁舒鹏; 杨桂兰; 王佳媚; 白景瑞; 龙朝钦; 罗洋

    2012-01-01

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

  8. Effects of methylation status of caspase-8 promoter on antitumor activity of TRAIL to human gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Ru-gang; FANG Dian-chun; YANG Liu-qin; LUO Yuan-gang

    2004-01-01

    Objective: To study the effects of the methylation status of caspase-8 promoter on the antitumor activity of TRAIL to the human gastric cancer cells. Methods: The methylation of caspase-8 was measured with methylation specific PCR (MSP) and the antitomor capability of TRAIL to human gastric cancer cells was determined with MTT. Results: No methylation of caspase-8 in the human gastric cancer cells was found. The sensitivity of 5 lines of gastric cancer cells to the antitumor activity of TRAIL was different. The administration of the demethylation agent 5-Aza-2'-deoxycytidine ( 5-AzaCdR) increased the sensitivity of gastric cancer cells to TRAIL but did not change the methylation status of caspase-8 promoter in gastric cancer cells. Conclusion: 5-Aza-CdR increases the sensitivity of most of gastric cancer cells to TRAIL but caspase-8 is not involved in the antitumor activity of TRAIL.

  9. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Tim Hendrikx

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

  12. miR-155 targets Caspase-3 mRNA in activated macrophages.

    Science.gov (United States)

    De Santis, Rebecca; Liepelt, Anke; Mossanen, Jana C; Dueck, Anne; Simons, Nadine; Mohs, Antje; Trautwein, Christian; Meister, Gunter; Marx, Gernot; Ostareck-Lederer, Antje; Ostareck, Dirk H

    2016-01-01

    To secure the functionality of activated macrophages in the innate immune response, efficient life span control is required. Recognition of bacterial lipopolysaccharides (LPS) by toll-like receptor 4 (TLR4) induces downstream signaling pathways, which merge to induce the expression of cytokine genes and anti-apoptotic genes. MicroRNAs (miRNAs) have emerged as important inflammatory response modulators, but information about their functional impact on apoptosis is scarce. To identify miRNAs differentially expressed in response to LPS, cDNA libraries from untreated and LPS-activated murine macrophages were analyzed by deep sequencing and regulated miRNA expression was verified by Northern blotting and qPCR. Employing TargetScan(TM) we identified CASPASE-3 (CASP-3) mRNA that encodes a key player in apoptosis as potential target of LPS-induced miR-155. LPS-dependent primary macrophage activation revealed TLR4-mediated enhancement of miR-155 expression and CASP-3 mRNA reduction. Endogenous CASP-3 and cleaved CASP-3 protein declined in LPS-activated macrophages. Accumulation of miR-155 and CASP-3 mRNA in miRNA-induced silencing complexes (miRISC) was demonstrated by ARGONAUTE 2 (AGO2) immunoprecipitation. Importantly, specific antagomir transfection effectively reduced mature miR-155 and resulted in significantly elevated CASP-3 mRNA levels in activated macrophages. In vitro translation assays demonstrated that the target site in the CASP-3 mRNA 3'UTR mediates miR-155-dependent Luciferase reporter mRNA destabilization. Strikingly, Annexin V staining of macrophages transfected with antagomir-155 and stimulated with LPS prior to staurosporine (SSP) treatment implied that LPS-induced miR-155 prevents apoptosis through CASP-3 mRNA down-regulation. In conclusion, we report that miR-155-mediated CASP-3 mRNA destabilization in LPS-activated RAW 264.7 macrophages suppresses apoptosis, as a prerequisite to maintain their crucial function in inflammation. PMID:26574931

  13. Study on the enzymatic activity of Caspase-3 in response to alginic acid decomposing bacteria in Laminaria japonica Aresch.(Phaeophyta)

    Institute of Scientific and Technical Information of China (English)

    Wang Gaoge; Lin Wei; Yan Xiaojun; Duan Delin

    2005-01-01

    Caspase-3 is the major factor in apoptosis triggered by various stimuli, and plays a critical role during the apoptosis process. By using CaspGLOWTM fluorescein active caspase-3 staining method, caspase-3 enzymatic activities were detected in response to alginic acid bacteria in Laminaria japonica sporophytic tissues. Results showed that caspase-3 enzymatic activities were observed at 5 min after the infection. Caspase-3 enzymatic activity increased with the infection time, and had a tendency of moving from the infection site to outside. By applying caspase-specific peptide inhibitor Z-VAD-FMK, caspase-3 activation could be effectively abolished in the infected tissues. Our results indicate that programmed cell death (PCD) may be involved in the infected Laminaria japonica sporophytic tissues, and provide the evidence that defense mechanisms in algae may have similar caspase cascade events in animals.

  14. Deficiency in the mitochondrial apoptotic pathway reveals the toxic potential of autophagy under ER stress conditions.

    Science.gov (United States)

    Deegan, Shane; Saveljeva, Svetlana; Logue, Susan E; Pakos-Zebrucka, Karolina; Gupta, Sanjeev; Vandenabeele, Peter; Bertrand, Mathieu J M; Samali, Afshin

    2014-01-01

    Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of the mitochondrial apoptotic pathway, which is characterized by CYCS (cytochrome c, somatic) release, apoptosome formation, and caspase activation, resulting in cell death. In this study, we demonstrate that under conditions of ER stress cells devoid of CASP9/caspase-9 or BAX and BAK1, and therefore defective in the mitochondrial apoptotic pathway, still undergo a delayed form of cell death associated with the activation of caspases, therefore revealing the existence of an alternative stress-induced caspase activation pathway. We identified CASP8/caspase-8 as the apical protease in this caspase cascade, and found that knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel ER stress-induced death pathway. In line with this, we identified a protein complex composed of ATG5, FADD, and pro-CASP8 whose assembly coincides with caspase activation and cell death induction. Together, our results reveal the toxic potential of autophagy in cells undergoing ER stress that are defective in the mitochondrial apoptotic pathway, and suggest a model in which the autophagosome functions as a platform facilitating pro-CASP8 activation. Chemoresistance, a common problem in the treatment of cancer, is frequently caused by the downregulation of key mitochondrial death effector proteins. Alternate stress-induced apoptotic pathways, such as the one described here, may become of particular relevance for tackling the problem of chemoresistance in cancer cells. PMID:25470234

  15. Punicalagin attenuated cerebral ischemia-reperfusion insult via inhibition of proinflammatory cytokines, up-regulation of Bcl-2, down-regulation of Bax, and caspase-3.

    Science.gov (United States)

    Yaidikar, Lavanya; Thakur, Santhrani

    2015-04-01

    Punicalagin (PG) is a hydrolysable tannin compound found in Punica granatum L. The purpose of the present work is to explore the neuroprotective mechanism of PG against ischemia-reperfusion (I/R) injury in rat model of middle cerebral artery occlusion (MCAO). Rats were randomly divided into sham, MCAO, and PG-treated groups. PG (15 and 30 mg/kg), the vehicle was administered orally for 7 days prior to MCAO. Rats were anesthetised with ketamine (100 mg/kg/im), xylazine (10 mg/kg/im) and subjected to 2 h occlusion and 22 h reperfusion. The effects of PG on behavioral deficit and infarct volume, the levels of glutamate and calcium as well as the levels of inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) were evaluated. Moreover, the expressions of caspase-3, Bcl-2, and Bax were detected by Western blotting. As compared with MCAO group, PG-treated rats showed dose-dependent reduction in infarct volume and substantial improvement in behavioral deficit. The levels of glutamate, calcium, TNF-α, IL-1β, and IL-6 were restored significantly. The Western blotting results revealed that the expression of Bcl-2 was up-regulated and that of caspase-3, Bax were down-regulated when exposed to PG. From our results, it can be concluded that PG showed an ameliorative effect against cerebral I/R injury in rats through its anti-inflammatory, antioxidant actions besides it inhibits excitotoxicity. It also suppresses apoptosis through regulating, Bcl-2, caspase-3, and Bax protein expressions, perhaps another mechanism by which PG employs its neuroprotective action. PMID:25555468

  16. Quercetin induces caspase-dependent extrinsic apoptosis through inhibition of signal transducer and activator of transcription 3 signaling in HER2-overexpressing BT-474 breast cancer cells.

    Science.gov (United States)

    Seo, Hye-Sook; Ku, Jin Mo; Choi, Han-Seok; Choi, Youn Kyung; Woo, Jong-Kyu; Kim, Minsoo; Kim, Ilhwan; Na, Chang Hyeok; Hur, Hansol; Jang, Bo-Hyoung; Shin, Yong Cheol; Ko, Seong-Gyu

    2016-07-01

    Flavonoids are assumed to exert beneficial effects in different types of cancers at high concentrations. Yet, their molecular mechanisms of action remain unknown. The present study aimed to examine the effect of quercetin on proliferation and apoptosis in HER2-expressing breast cancer cells. The anti-proliferative effects of quercetin were examined by proliferation, MTT and clonogenic survival assays. The effect of quercetin on expression of apoptotic molecules was determined by western blotting. Luciferase reporter assay was performed to measure signal transducer and activator of transcription 3 (STAT3) transcriptional activity. ELISA assay was performed to measure intracellular MMP-9 levels. Immunocytochemistry was performed to evaluate the nuclear STAT3 level. The results revealed that quercetin inhibited the proliferation of BT-474 cells in a dose- and time-dependent manner. Quercetin also inhibited clonogenic survival (anchorage-dependent and -independent) of BT-474 cells in a dose-dependent manner. These growth inhibitions were accompanied with an increase in sub-G0/G1 apoptotic populations. Quercetin induced caspase-dependent extrinsic apoptosis upregulating the levels of cleaved caspase-8 and cleaved caspase-3, and inducing the cleavage of poly(ADP‑ribose) polymerase (PARP). In contrast, quercetin did not induce apoptosis via intrinsic mitochondrial apoptosis pathway since this compound did not decrease the mitochondrial membrane potential and did not affect the levels of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (BAX). Quercetin reduced the expression of phospho-JAK1 and phospho-STAT3 and decreased STAT3-dependent luciferase reporter gene activity in the BT-474 cells. Quercetin inhibited MMP-9 secretion and decreased the nuclear translocation of STAT3. Our study indicates that quercetin induces apoptosis at concentrations >20 µM through inhibition of STAT3 signaling and could serve as a useful compound to prevent or treat HER2

  17. Pharmacological inhibition of caspase and calpain proteases: a novel strategy to enhance the homing responses of cord blood HSPCs during expansion.

    Directory of Open Access Journals (Sweden)

    V M Sangeetha

    Full Text Available BACKGROUND: Expansion of hematopoietic stem/progenitor cells (HSPCs is a well-known strategy employed to facilitate the transplantation outcome. We have previously shown that the prevention of apoptosis by the inhibition of cysteine proteases, caspase and calpain played an important role in the expansion and engraftment of cord blood (CB derived HSPCs. We hypothesize that these protease inhibitors might have maneuvered the adhesive and migratory properties of the cells rendering them to be retained in the bone marrow for sustained engraftment. The current study was aimed to investigate the mechanism of the homing responses of CB cells during expansion. METHODOLOGY/PRINCIPAL FINDINGS: CB derived CD34(+ cells were expanded using a combination of growth factors with and without Caspase inhibitor -zVADfmk or Calpain 1 inhibitor- zLLYfmk. The cells were analyzed for the expression of homing-related molecules. In vitro adhesive/migratory interactions and actin polymerization dynamics of HSPCs were assessed. In vivo homing assays were carried out in NOD/SCID mice to corroborate these observations. We observed that the presence of zVADfmk or zLLYfmk (inhibitors caused the functional up regulation of CXCR4, integrins, and adhesion molecules, reflecting in a higher migration and adhesive interactions in vitro. The enhanced actin polymerization and the RhoGTPase protein expression complemented these observations. Furthermore, in vivo experiments showed a significantly enhanced homing to the bone marrow of NOD/SCID mice. CONCLUSION/SIGNIFICANCE: Our present study reveals another novel aspect of the regulation of caspase and calpain proteases in the biology of HSPCs. The priming of the homing responses of the inhibitor-cultured HSPCs compared to the cytokine-graft suggests that the modulation of these proteases may help in overcoming the major homing defects prevalent in the expansion cultures thereby facilitating the manipulation of cells for transplant

  18. Caspase-1 activation and mature interleukin-1β release are uncoupled events in monocytes

    Institute of Scientific and Technical Information of China (English)

    Amy; J; Galliher-Beckley; Li-Qiong; Lan; Shelly; Aono; Lei; Wang; Jishu; Shi

    2013-01-01

    AIM:To investigate whether caspase-1 activation/intracellular processing of pro-interleukin-1β(pro-IL-1β) and extracellular release of mature IL-1β from activated monocytes are separable events.METHODS:All experiments were performed on fresh or overnight cultured human peripheral blood monocytes(PBMCs) that were isolated from healthy donors.PBMCs were activated by lipopolysaccharide(LPS) stimulation before being treated with Adenosine triphosphate(ATP,1 mmol/L),human α-defensin-5(HD-5,50 μg/mL),and/or nigericin(Nig,30 μmol/L).For each experiment,the culture supernatants were collected separately from the cells.Cell lysates and supernatants were both subject to immunoprecipitation with anti-IL1β antibodies followed by western blot analysis with anti-caspase-1 and anti-IL-1β antibodies.RESULTS:We found that pro-IL-1β was processed to mature IL-1β in LPS-activated fresh and overnight cultured human monocytes in response to ATP stimulation.In the presence of HD-5,this release of IL-1β,but not the processing of pro-IL-1β to IL-1β,was completely inhibited.Similarly,in the presence of HD-5,the release of IL-1β,but not the processing of IL-1β,was significantly inhibited from LPS-activated monocytes stimulated with Nig.Finally,we treated LPS-activated monocytes with ATP and Nig and collected the supernatants.We found that both ATP and Nig stimulation could activate and release cleaved caspase-1 from the monocytes.Interestingly,and contrary to IL-1β processing and release,caspase-1 cleavage and release was not blocked by HD-5.All images are representative of three independent experiments.CONCLUSION:These data suggest that caspase-1 activation/processing of pro-IL-1β by caspase-1 and the release of mature IL-1β from human monocytes are distinct and separable events.

  19. Therapeutic effect of Caspase-1 inhibitor on liver injury in experimental severe acute pancreatitis

    Institute of Scientific and Technical Information of China (English)

    张晓华; 李兆申; 屠振兴; 许国铭; 龚燕芳; 满晓华

    2004-01-01

    Objective: To assess the therapeutic effect of Caspase-1 inhibitor on liver injury in experimental severe acute pancreatitis (SAP). Methods: Forty-two SD rats were randomly divided into 3 groups: healthy controls (HC, n =6); SAP-S group (n = 18); SAP-ICE-I group (n = 18). SAP was induced by retrograde infusion of 5% sodium taurocholate into the bili-pancreatic duct in SD rats. HC rats underwent same surgical procedures and duct cannulation without sodium taurocholate. In SAP-S group, rats received the first intraperitoneal injection of isotonic saline 2 h after induction of acute pancreatitis, which was repeated after 12 h. In SAP-ICE-I group, rats were firstly given ICE inhibitor intraperitoneally 2 h after induction of pancreatitis. As in SAP-S group, this was repeated at 12 h. Survied rats were killed at certain time points, and all samples were obtained for subsequent analysis. Results: The serum levels of ALT, AST and IL-1β in SAPS group were (215.50 ±58.52) U/L, (372.17 ± 38.05) U/L, (276.77 ±44.92) pg/ml at 6 h, (396.67± 70.29) U/L,(548.50±75.29)U/L, (308.99± 34.95)pg/ml at 12 h, (425.17±86.33)U/L, (665.83±84.05)U/L, (311.60±46.51 ) pg/ml, respectively, which were increased significantly ( P < 0.01, vs HC). In SAP-ICE-I group, their levels were decreased significantly ( P < 0.01, vs SAP-S). Intrahepatic expressions of Caspase-1, IL-1β and IL-18 mRNA could be observed in HC, which were increased significantly in SAP-S group ( P < 0.01, vs HC). The expressions of IL-1β and IL-18mRNA were decreased significantly in SAP-ICE-I group ( P < 0.01, vs SAP-S), whereas Caspase-1 mRNA expressions had no significant differences ( P > 0.05). Caspase-1 inhibition had no effect on the severity of liver tissue damage. Conclusion: Caspase-1 activate cytokines, IL-1β and IL-18, play a pivotal role in the course of liver injury in SAP. Caspase-1 inhibitor can improve liver functions effectively.

  20. Mathematics revealed

    CERN Document Server

    Berman, Elizabeth

    1979-01-01

    Mathematics Revealed focuses on the principles, processes, operations, and exercises in mathematics.The book first offers information on whole numbers, fractions, and decimals and percents. Discussions focus on measuring length, percent, decimals, numbers as products, addition and subtraction of fractions, mixed numbers and ratios, division of fractions, addition, subtraction, multiplication, and division. The text then examines positive and negative numbers and powers and computation. Topics include division and averages, multiplication, ratios, and measurements, scientific notation and estim

  1. Revealed Attention

    OpenAIRE

    Masatlioglu, Yusufcan; NAKAJIMA, Daisuke; Ozbay, Erkut Y

    2012-01-01

    The standard revealed preference argument relies on an implicit assumption that a decision maker considers all feasible alternatives. The marketing and psychology literatures, however, provide wellestablished evidence that consumers do not consider all brands in a given market before making a purchase (Limited Attention). In this paper, we illustrate how one can deduce both the decision maker's preference and the alternatives to which she pays attention and inattention from the observed behav...

  2. Revealed Attention

    OpenAIRE

    Yusufcan Masatlioglu; Daisuke Nakajima; Ozbay, Erkut Y

    2012-01-01

    The standard revealed preference argument relies on an implicit assumption that a decision maker considers all feasible alternatives. The marketing and psychology literatures, however, provide well-established evidence that consumers do not consider all brands in a given market before making a purchase (Limited Attention). In this paper, we illustrate how one can deduce both the decision maker's preference and the alternatives to which she pays attention and inattention from the observed beha...

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

    Institute of Scientific and Technical Information of China (English)

    潘峥; 陈军; 覃冠德; 覃思繁

    2012-01-01

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

  4. Evaluation of caspase3 and 9 gene polymorphisms in gastric cancer patients in Mazandaran province: a brief report

    Directory of Open Access Journals (Sweden)

    Saeid Abediankenari

    2013-11-01

    Full Text Available Background: Gastric cancer is the most prevalent cancer with poor survival in gastrointestinal tract. Caspase 3 and 9 play an important role in the development and progression of cancer. Polymorphisms in the genes for these enzymes can affect gene activity and thus may influence susceptibility to gastric cancer. In this study, caspase 3 and 9 genes polymorphisms in patients with gastric cancer were examined.Methods: In a case - control study, 100 patients with gastric cancer and 100 healthy individuals were evaluated in the region rs4647601: G> T for caspase-3 and -1263 A> G gene promoter for caspase 9. DNA extraction was performed from whole blood according to manufacture protocol. RFLP-PCR method was carrying out for detection of caspase 3 and 9 genes genotype in two groups.Results: In this study, 143 men and 57 women were evaluated. All of them were selected from the same race and geographical area. The results indicated an increase of the mutant G allele in the control group, which leads to a decreasing in the incidence of gastric cancer (P caspase 9 polymorphism could be a useful marker in personal sensitivity to gastric cancer and help to cancer treatment and prevention process. It is concluded that caspase gene variation may be a diagnostic factor in the gastric cancer.

  5. Selective inhibition of caspases in skeletal muscle reverses the apoptotic synaptic degeneration in slow-channel myasthenic syndrome.

    Science.gov (United States)

    Zhu, Haipeng; Pytel, Peter; Gomez, Christopher M

    2014-01-01

    Slow-channel syndrome (SCS) is a congenital myasthenic disorder caused by point mutations in subunits of skeletal muscle acetylcholine receptor leading to Ca(2+) overload and degeneration of the postsynaptic membrane, nuclei and mitochondria of the neuromuscular junction (NMJ). In both SCS muscle biopsies and transgenic mouse models for SCS (mSCS), the endplate regions are shrunken, and there is evidence of DNA damage in the subsynaptic region. Activated caspase-9, -3 and -7 are intensely co-localized at the NMJ, and the Ca(2+)-activated protease, calpain, and the atypical cyclin-dependent kinase (Cdk5) are overactivated in mSCS. Thus, the true mediator(s) of the disease process is not clear. Here, we demonstrate that selective inhibition of effector caspases, caspase-3 and -7, or initiator caspase, caspase-9, in limb muscle in vivo by localized expression of recombinant inhibitor proteins dramatically decreases subsynaptic DNA damage, increases endplate area and improves ultrastructural abnormalities in SCS transgenic mice. Calpain and Cdk5 are not affected by this treatment. On the other hand, inhibition of Cdk5 by expression of a dominant-negative form of Cdk5 has no effect on the degeneration. Together with previous studies, these results indicate that focal activation of caspase activity at the NMJ is the principal pathological process responsible for the synaptic apoptosis in SCS. Thus, treatments that reduce muscle caspase activity are likely to be of benefit for SCS patients.

  6. Cytoprotection against Hypoxic and/or MPP+ Injury: Effect of δ–Opioid Receptor Activation on Caspase 3

    Directory of Open Access Journals (Sweden)

    Yuan Xu

    2016-08-01

    Full Text Available The pathological changes of Parkinson’s disease (PD are, at least partially, associated with the dysregulation of PTEN-induced putative kinase 1 (PINK1 and caspase 3. Since hypoxic and neurotoxic insults are underlying causes of PD, and since δ-opioid receptor (DOR is neuroprotective against hypoxic/ischemic insults, we sought to determine whether DOR activation could protect the cells from damage induced by hypoxia and/or MPP+ by regulating PINK1 and caspase 3 expressions. We exposed PC12 cells to either severe hypoxia (0.5%–1% O2 for 24–48 h or to MPP+ at different concentrations (0.5, 1, 2 mM and then detected the levels of PINK1 and cleaved caspase 3. Both hypoxia and MPP+ reduced cell viability, progressively suppressed the expression of PINK1 and increased the cleaved caspase 3. DOR activation using UFP-512, effectively protected the cells from hypoxia and/or MPP+ induced injury, reversed the reduction in PINK1 protein and significantly attenuated the increase in the cleaved caspase 3. On the other hand, the application of DOR antagonist, naltrindole, greatly decreased cell viability and increased cleaved caspase 3. These findings suggest that DOR is cytoprotective against both hypoxia and MPP+ through the regulation of PINK1 and caspase 3 pathways.

  7. The DEAD box protein Dhh1 stimulates the decapping enzyme Dcp1

    OpenAIRE

    Fischer, Nicole; Weis, Karsten

    2002-01-01

    An important control step in the regulation of cytoplasmic mRNA turnover is the removal of the m7G cap structure at the 5′ end of the message. Here, we describe the functional characterization of Dhh1, a highly conserved member of the family of DEAD box-containing proteins, as a regulator of mRNA decapping in Saccharomyces cerevisiae. Dhh1 is a cytoplasmic protein and is shown to be in a complex with the mRNA degradation factor Pat1/Mtr1 and with the 5′–3′ exoribonuclease Xrn1. Dhh1 specifica...

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

    Directory of Open Access Journals (Sweden)

    Montserrat Hervella

    Full Text Available BACKGROUND: 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. CONCLUSIONS/SIGNIFICANCE: 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.

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

    OpenAIRE

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

    2008-01-01

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

  10. Evodiamine induces caspase-dependent apoptosis and S phase arrest in human colon lovo cells.

    Science.gov (United States)

    Zhang, Chun; Fan, Xia; Xu, Xiang; Yang, Xue; Wang, Xi; Liang, Hua-Ping

    2010-09-01

    Evodiamine, one of the major bioactive components derived from Wu-Chu-Yu, a long-standing Chinese herb, was reported to possess anticancer activity. In this study, we investigated the in-vitro and in-vivo anticancer effects of evodiamine on human colon lovo cells and their potential mechanisms. The 3-(4, 5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay showed that the in-vitro proliferation of lovo cells was inhibited by evodiamine of various concentrations. Flow cytometry showed a time-dependent increase in the percentage of apoptotic cells and cells arrested in the S phase after treatment with 60 micromol/l evodiamine. Western blot indicated that evodiamine treatment decreased the expression of procaspase-8, procaspase-9, and procaspase-3 in lovo cells, accompanied by the activation of caspase-8, caspase-9, and caspase-3. However, the translocation of apoptosis-inducing factor and endonuclease G was not affected by evodiamine. Moreover, western blot assay also suggested that evodiamine-induced S phase arrest in lovo cells was associated with a marked decrease in the protein expression of cyclinA, cyclinA-dependent kinase 2, and cdc25c. In-vivo antineoplastic characteristics of evodiamine were examined in a human colon carcinoma lovo xenograft model and results showed that evodiamine increased the number of TUNEL-positive cells accompanied by the downregulated expression of procaspase-8, procaspase-9, and procaspase-3. In conclusion, these findings indicated that evodiamine could inhibit the in-vitro and in-vivo proliferation of human colon lovo cells by inducing caspase-dependent apoptosis and S phase arrest.

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

    OpenAIRE

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

    2011-01-01

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

  12. Stachyose-induced apoptosis of Caco-2 cells via the caspase-dependent mitochondrial pathway.

    Science.gov (United States)

    Huang, Guidong; Mao, Jian; Ji, Zhongwei; Ailati, Aisikaer

    2015-03-01

    Some studies have shown that stachyose, as prebiotics, can prevent indirectly colon cancer cell growth by promoting the proliferation of probiotics or producing beneficial materials in the intestine. However, its direct inhibitory effects on cancer cells are still unclear. Thus, this study aims to investigate the direct inhibitory effect of stachyose on human colon cancer cells and determine the molecular mechanism underlying this effect. The MTT assay was used to assess the inhibitory effect of stachyose on Caco-2 cells. Apoptosis and mitochondrial membrane potential (ΔΨm) measurements were analyzed using flow cytometry. The activities and mRNA expressions of caspases 3 and 9 were determined using caspase assay kits and quantitative real-time polymerase chain reaction. The apoptotic protein expressions of Bcl-2, Bax, and cytochrome C (Cyt C) were detected through western blotting. Results showed that stachyose inhibits Caco-2 cell proliferation and induces apoptosis in a dose-dependent manner. After pretreatment with 0.4, 0.8, 1.6 and 3.2 mg mL(-1) stachyose, cell inhibitory rates of 15.31% ± 3.20%, 28.45% ± 2.10%, 40.23% ± 5.70%, and 55.67% ± 4.50% were respectively obtained. Compared with the control, decreases in ΔΨm, increases in caspase 3 and 9 activities and mRNA expressions, down-regulation of Bcl-2 protein expression, up-regulation of the Bax protein and Cyt C release of Caco-2 cells were clearly observed upon exposure to different stachyose concentrations. The inhibitory mechanism of stachyose on Caco-2 cells involves the caspase-dependent mitochondrial apoptosis pathway. PMID:25578308

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

    OpenAIRE

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

    2013-01-01

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

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

    OpenAIRE

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

    2011-01-01

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

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

    OpenAIRE

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

    2012-01-01

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

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

    Science.gov (United States)

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

    2009-01-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    Luis Santamaría

    2011-05-01

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

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

    Science.gov (United States)

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

    2010-02-01

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

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

    Institute of Scientific and Technical Information of China (English)

    张丽萍; 张志军; 程萍; 肖劲松

    2010-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    陈雪梅; 杜显刚; 官鹏; 谭志巍; 王亚琴

    2005-01-01

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

  2. Matrine inhibits diethylnitrosamine-induced HCC proliferation in rats through inducing apoptosis via p53, Bax-dependent caspase-3 activation pathway and down-regulating MLCK overexpression.

    Science.gov (United States)

    Zhang, Xiaolin; Yu, Hao

    2016-01-01

    The proliferation of hepatocellular carcinoma (HCC) cells is one of the leading causes of liver cancer mortality in humans. The inhibiting effects of matrine on HCC cell proliferation have been studied, but the mechanism of that inhibition has not been fully elucidated. Since, apoptosis plays an important role in HCC cell proliferation. We examined the apoptosis-inducing effect of matrine on tumor cells. Western blot analysis of p53, Bax, cleaved caspase-3 and myosin light chain kinase (MLCK) revealed that matrine induced tumor cell apoptosis by controlling anoikis. It activated p53, Bax-dependent caspase-3 and blocked the ECM-integrin mediated cell survival pathway through down-regulating MLCK over-expression in the liver of rats with diethyl nitrosamine (DENA)-induced HCC. Our results suggest that matrine can inhibit the proliferation of HCC cells through inducing tumor cell apoptosis via activation of the p53 pathway and inhibition of MLCK overexpression. Matrine may thus be used as a potentially promising reagent to inhibit HCC cell proliferation and MLCK may be a novel target for the treatment of HCC. PMID:27642320

  3. Impact of Curcuma mangga Val. Rhizome Essential Oil to p53, Bcl-2, H-Ras and Caspase-9 expression of Myeloma Cell Line

    Directory of Open Access Journals (Sweden)

    Endang Astuti

    2015-12-01

    Full Text Available Cancer is a disease, a public health problem, which is found in the world as well as in Indonesia. Ingeneral, some of cancer theraphies are ineffective, characterized by the resistance performance of cancer cell line,the exposed normal cell and by the side effects. Nowadays, studies to fi nd the specifi c and safely anti-cancerdrugs were increased by the time. Several studies revealed that Curcuma mangga Val. Rhizome contains somesecondary metabolites, essential or non-essential oil, which has cytotoxic activities to the cancer cells. Basedon these anti-cancer potentials, this study has several aims to recognize anti-cancer selectivity and molecularmechanism by inducting apoptosis and inhibiting myeloma cell proliferation. To C. mangga Val. essential oil,immunocyto chemical test was performed to determine the expression of p53, caspase-9, Bcl-2, H-Ras proteinwhile TUNEL test was performed to determine the number of apoptosis cells.The results of this study shown that anti-cancer molecular mechanism of C. mangga Val. essential oil tomyeloma cell line was performed by increasing apoptosis; by increasing the expression of pro-apoptosis p53,caspase-9 protein and reducing protein which is increasing proliferation Bcl-2 and H-Ras.

  4. Matrine inhibits diethylnitrosamine-induced HCC proliferation in rats through inducing apoptosis via p53, Bax-dependent caspase-3 activation pathway and down-regulating MLCK overexpression.

    Science.gov (United States)

    Zhang, Xiaolin; Yu, Hao

    2016-01-01

    The proliferation of hepatocellular carcinoma (HCC) cells is one of the leading causes of liver cancer mortality in humans. The inhibiting effects of matrine on HCC cell proliferation have been studied, but the mechanism of that inhibition has not been fully elucidated. Since, apoptosis plays an important role in HCC cell proliferation. We examined the apoptosis-inducing effect of matrine on tumor cells. Western blot analysis of p53, Bax, cleaved caspase-3 and myosin light chain kinase (MLCK) revealed that matrine induced tumor cell apoptosis by controlling anoikis. It activated p53, Bax-dependent caspase-3 and blocked the ECM-integrin mediated cell survival pathway through down-regulating MLCK over-expression in the liver of rats with diethyl nitrosamine (DENA)-induced HCC. Our results suggest that matrine can inhibit the proliferation of HCC cells through inducing tumor cell apoptosis via activation of the p53 pathway and inhibition of MLCK overexpression. Matrine may thus be used as a potentially promising reagent to inhibit HCC cell proliferation and MLCK may be a novel target for the treatment of HCC.

  5. Matrine inhibits diethylnitrosamine-induced HCC proliferation in rats through inducing apoptosis via p53, Bax-dependent caspase-3 activation pathway and down-regulating MLCK overexpression

    Science.gov (United States)

    Zhang, Xiaolin; Yu, Hao

    2016-01-01

    The proliferation of hepatocellular carcinoma (HCC) cells is one of the leading causes of liver cancer mortality in humans. The inhibiting effects of matrine on HCC cell proliferation have been studied, but the mechanism of that inhibition has not been fully elucidated. Since, apoptosis plays an important role in HCC cell proliferation. We examined the apoptosis-inducing effect of matrine on tumor cells. Western blot analysis of p53, Bax, cleaved caspase-3 and myosin light chain kinase (MLCK) revealed that matrine induced tumor cell apoptosis by controlling anoikis. It activated p53, Bax-dependent caspase-3 and blocked the ECM-integrin mediated cell survival pathway through down-regulating MLCK over-expression in the liver of rats with diethyl nitrosamine (DENA)-induced HCC. Our results suggest that matrine can inhibit the proliferation of HCC cells through inducing tumor cell apoptosis via activation of the p53 pathway and inhibition of MLCK overexpression. Matrine may thus be used as a potentially promising reagent to inhibit HCC cell proliferation and MLCK may be a novel target for the treatment of HCC. PMID:27642320

  6. Viola plant cyclotide vigno 5 induces mitochondria-mediated apoptosis via cytochrome C release and caspases activation in cervical cancer cells.

    Science.gov (United States)

    Esmaeili, Mohammad Ali; Abagheri-Mahabadi, Nazanin; Hashempour, Hossein; Farhadpour, Mohsen; Gruber, Christian W; Ghassempour, Alireza

    2016-03-01

    Cyclotides describe a unique cyclic peptide family that displays a broad range of biological activities including uterotonic, anti-bacteria, anti-cancer and anti-HIV. The vigno cyclotides consist of vigno 1-10 were reported recently from Viola ignobilis. In the present study, we examined the effects of vigno 5, a natural cyclopeptide from V. ignobilis, on cervical cancer cells and the underlying mechanisms. We found that vigno 5-treated Hela cells were killed off by apoptosis in a dose-dependent manner within 24h, and were characterized by the appearance of nuclear shrinkage, cleavage of poly (ADP-ribose) polymerase (PARP) and DNA fragmentation. The mitochondrial pathway of apoptosis revealed that cytochrome C is released from mitochondria to cytosol, associated with the activation of caspase-9 and -3, and the cleavage of poly (ADP-ribose) polymerase (PARP). Overall, the results indicate that vigno 5 induces apoptosis in part via the mitochondrial pathway, which is associated with a release of cytochrome C and elevated activity of caspase-9 and -3 in Hela cells. PMID:26751970

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

    International Nuclear Information System (INIS)

    Selective serotonin reuptake inhibitors (SSRIs), a group of antidepressants, are generally used for treatment of various mood and anxiety disorders. There has been much research showing the anti-tumor and cytotoxic activities of some antidepressants; but the detailed mechanisms were unclear. In cultured human osteosarcoma cells (MG63), paroxetine reduced cell viability in a concentration- and time-dependent manner. Paroxetine caused apoptosis as assessed by propidium iodide-stained cells and increased caspase-3 activation. Although immunoblotting data revealed that paroxetine could activate the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), only SB203580 (a p38 MAPK inhibitor) partially prevented cells from apoptosis. Paroxetine also induced [Ca2+]i increases which involved the mobilization of intracellular Ca2+ stored in the endoplasmic reticulum and Ca2+ influx from extracellular medium. However, pretreatment with BAPTA/AM, a Ca2+ chelator, to prevent paroxetine-induced [Ca2+]i increases did not protect cells from death. The results suggest that in MG63 cells, paroxetine caused Ca2+-independent apoptosis via inducing p38 MAPK-associated caspase-3 activation

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

    Science.gov (United States)

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

    2016-06-01

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

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

    Directory of Open Access Journals (Sweden)

    KARIN S. CUNHA

    2013-11-01

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

  10. Rational Design of a GFP-Based Fluorogenic Caspase Reporter for Imaging Apoptosis In Vivo.

    Science.gov (United States)

    To, Tsz-Leung; Schepis, Antonino; Ruiz-González, Rubén; Zhang, Qiang; Yu, Dan; Dong, Zhiqiang; Coughlin, Shaun R; Shu, Xiaokun

    2016-07-21

    Fluorescence resonance energy transfer-based executioner caspase reporters using GFP are important tools for imaging apoptosis. While these reporters are useful for imaging apoptosis in cultured cells, their in vivo application has been handicapped by poor signal to noise. Here, we report the design and characterization of a GFP-based fluorogenic protease reporter, dubbed ZipGFP. ZipGFP-based TEV protease reporter increased fluorescence 10-fold after activation by protease. A ZipGFP-based executioner caspase reporter visualized apoptosis in live zebrafish embryos with spatiotemporal resolution. Thus, the ZipGFP-based caspase reporter may be useful for monitoring apoptosis during animal development and for designing reporters of proteases beyond the executioner caspases.

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

    Science.gov (United States)

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

    2016-10-01

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

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

    OpenAIRE

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2016-05-27

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

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

    OpenAIRE

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

    2013-01-01

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

  15. EFFECTS OF ANTISENSE OLIGODEOXYNUCLEOTIDES ON EXPRESSION OF CASPASE-3 IN Γ-RADIATION INDUCED APOPTOTIC HL-60 CELLS

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Objective To study the inhibitory effects of caspase-3 mRNA antisense oligodeoxynucleotides (ASODN) on expressions of caspase-3 and it's mRNA in γ-radiation induced apoptotic HL-60 cells, and screen the effective ASODN. Methods ASODN-1 and ASODN-2 targeting 5′-noncoding region and initial translation region of caspase-3 mRNA were respectively designed, synthesized and introduced into HL-60 cells by means of liposome-mediated transfection followed by 10Gy γ-radiation exposures. TUNEL assay was conducted to investigate the morphologic change and apoptotic percentage of HL-60 cells 18 h later. Immunocytochemical staining and one step RT-PCR were respectively performed to detect the expressions of caspase-3 and it's mRNA. Mismatched oligodeoxynucleotide (MODN) transfected and un-transfected HL-60 cells were taken as control. Results TUNEL assay found that the apoptotic percentages in ASODN-1 and ASODN-2 groups were significantly reduced compared with the control groups (P<0.01) when the final concentration of both ASODNs was ≥3μmol/L. Immunocytochemistry showed that caspase-3 positive cell percentages were reduced but the average gray values increased significantly compared with the control groups (P<0.01). RT-PCR showed expressions of caspase-3 mRNA was decreased after ASODN transfection. Furthermore, ASODN-1 proved more effective in inhibiting HL-60 cell apoptosis than ASODN-2 (P<0.01). Conclusion Caspase-3 mRNA ASODNs can prevent HL-60 cells from apoptosis induced by γ-radiation and reduce expression of caspase-3 and its mRNA. These effects are dose dependent in a certain range.

  16. Revealing Rembrandt

    Directory of Open Access Journals (Sweden)

    Andrew J Parker

    2014-04-01

    Full Text Available The power and significance of artwork in shaping human cognition is self-evident. The starting point for our empirical investigations is the view that the task of neuroscience is to integrate itself with other forms of knowledge, rather than to seek to supplant them. In our recent work, we examined a particular aspect of the appreciation of artwork using present-day functional magnetic resonance imaging (fMRI. Our results emphasised the continuity between viewing artwork and other human cognitive activities. We also showed that appreciation of a particular aspect of artwork, namely authenticity, depends upon the co-ordinated activity between the brain regions involved in multiple decision making and those responsible for processing visual information. The findings about brain function probably have no specific consequences for understanding how people respond to the art of Rembrandt in comparison with their response to other artworks. However, the use of images of Rembrandt’s portraits, his most intimate and personal works, clearly had a significant impact upon our viewers, even though they have been spatially confined to the interior of an MRI scanner at the time of viewing. Neuroscientific studies of humans viewing artwork have the capacity to reveal the diversity of human cognitive responses that may be induced by external advice or context as people view artwork in a variety of frameworks and settings.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-10-24

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

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

    Directory of Open Access Journals (Sweden)

    Park Sung

    2008-12-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-05-27

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    International Nuclear Information System (INIS)

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

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

    Institute of Scientific and Technical Information of China (English)

    汪武; 周远大; 何海霞

    2009-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    高建新; 周玉琴; 张茹华; 马雪莲; 刘克敬

    2005-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    汪立杰; 叶良平; 沈彤; 朱启星

    2009-01-01

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

  7. 甲状腺癌组织中Caspase-9、Livin表达的临床意义%Clinical significance of expression of Caspase-9,Livin in thyroid cancer tissue

    Institute of Scientific and Technical Information of China (English)

    林建山; 郑剑忠; 黄冬凌; 李蜜; 邹桂华

    2015-01-01

    Objective To investigate the clinical significance of expression of cysteine aspartic acid protease(caspase)-9,Livin in thy-roid cancer tissue. Methods The expression of Caspase-9,Livin of thyroid cancer tissue in 65 patients and normal thyroid tissue in 40 patients were detected. The positive rate of expression of Caspase-9,Livin were compared in two different kind of tissues. The relationship between the expression of Livin,Caspase-9 and clinical pathological factors were analyzed withχ2 test. The relationship of expression of Livin,Caspase-9 in thyroid carcinoma tissue were analyzed with Spearman rank correlation test. Results Livin,Caspase-9 positive staining was mainly located in cytoplasm,and it was showed brown yellow patchy distribution or yellow brown granular. The positive expression rate of Caspase-9 was lower than that of normal thyroid tissue in thyroid cancer tissue(χ2 =38. 149,P =0. 000),the positive expression rate of Livin was higher than that of nor-mal thyroid tissue(χ2 =43. 161,P =0. 000). The expression of Livin,Caspase-9 in thyroid cancer tissue had nothing to do with the age,gen-der,tumor size and histologic types of patients( P ﹥0. 05),but it related to the tumor staging,lymph node metastasis and diolame invasion( P﹤0. 05). The expression of Livin,Caspase-9 in thyroid cancer tissues presented negative correlation( r = -0. 346,P ﹤0. 05). Conclusion Expression of Caspase-9 down-regulated in thyroid cancer tissue,while the expression of Livin increased,the abnormal expression participa-ted in the occurrence and development of thyroid cancer. The combined detection of Livin,Caspase-9 can be used as an important reference in-dex in judging the prognosis in patients with thyroid carcinoma.%目的:探讨甲状腺癌组织中半胱氨酸天冬氨酸蛋白酶( caspase)-9和凋亡抑制蛋白因子( Livin)表达的临床意义。方法检测65例甲状腺癌组织和40例正常甲状腺组织中Caspase-9、Livin表达情况,分别比较Caspase

  8. Phyllanthus amarus inhibits cell growth and induces apoptosis in Dalton's lymphoma ascites cells through activation of caspase-3 and downregulation of Bcl-2.

    Science.gov (United States)

    Harikumar, Kuzhuvelil B; Kuttan, Girija; Kuttan, Ramadasan

    2009-06-01

    The authors found in an earlier study that Phyllanthus amarus extract could significantly inhibit the solid and ascites tumor development in mice induced by Dalton's lymphoma ascites (DLA) cells. In the present study, the apoptotic effects of P. amarus against DLA cells in culture was evaluated. P. amarus produced significant reduction in cell viability as determined by the MTT assay. It also induces the formation of apoptotic bodies with characteristic features like plasma membrane invagination, elongation, fragmentation, and chromatin condensation. P. amarus at concentrations of 100 and 200 microg/mL is shown to induce DNA fragmentation. Gene expression analysis reveals that P. amarus induces the expression of caspase-3 and inhibits the expression of Bcl-2, which is an antiapoptotic protein. So the present study provides some insights into the possible mechanism by which P. amarus brings about apoptosis and growth inhibition in DLA cells. PMID:19223368

  9. Influence of ellagic acid on prostate cancer cell proliferation:A caspase-dependent pathway

    Institute of Scientific and Technical Information of China (English)

    Arshi Malik; Sarah Afaq; Mohammad Shahid; Kafil Akhtar; Abdullah Assiri

    2011-01-01

    Objective:To evaluate the effect of allagic acid treatment on the cell viability of human prostate cancer cells.Methods: Ellagic acid (10-100mol/L) treatment (48 h) of human prostate carcinomaPC3 cells was found to result in a dose-dependent inhibition of cell growth and apoptosis ofPC3 cells as assessed by MTTassay, western blotting, flow cytometry and confocal microscopy.Results: We observed that ellagic acid treatment ofPC3 cells resulted in a dose dependent inhibition of cell growth/cell viability. This ellagic acid caused cell growth inhibition was found to be accompanied by induction of apoptosis, as assessed by the cleavage of poly (ADP-ribose) polymerase(PARP) and morphological changes. Further, induction of apoptosis accompanied a decrease in the levels of antiapoptotic protein Bcl-2 and increase in proapoptotic protein Bax, thus shifting the Bax: Bcl-2 ratio in favor of apoptosis. Ellagic acid treatment of PC3 cells was also found to result in significant activation of caspases, as shown by the dose dependent decrease in the protein expression of procaspase-3, -6, -8 and-9. This ellagic acid-mediated induction of apoptosis was significantly (80%-90%) inhibited by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone(Z-VAD-FMK). Thus these data suggested an essential role of caspases in ellagic acid-mediated apoptosis ofPC3 cells.Conclusions:It is tempting to suggest that consumption of tropical pigmented fruits and vegetables could be an effective strategy to combat prostate cancer.

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

    Science.gov (United States)

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

    2011-09-15

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

  11. 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.

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

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Ira O Racoma

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

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

    International Nuclear Information System (INIS)

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

  16. 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...

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

    OpenAIRE

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

    2011-01-01

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

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

    International Nuclear Information System (INIS)

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

  19. Quercetin ameliorates ischemia/reperfusion-induced cognitive deficits by inhibiting ASK1/JNK3/caspase-3 by enhancing the Akt signaling pathway.

    Science.gov (United States)

    Pei, Bing; Yang, Miaomiao; Qi, Xiaoyan; Shen, Xin; Chen, Xing; Zhang, Fayong

    2016-09-01

    Cerebral ischemia/reperfusion (I/R) is a major cause of severe disability and death all worldwide. However, therapeutic options to minimize the detrimental effects of cerebral I/R injury are limited. Recent research has demonstrated that quercetin mediates neuroprotective effects associated with the activation of the Akt signaling pathway in the cerebral I/R brain. Therefore, the aim of this study was to further investigate the mechanisms of cognitive deficits induced by cerebral I/R injury and the effects of quercetin on these mechanisms. First, we assessed anxiety-like behavioral and cognitive impairment using the open field test and the Morris water maze test, respectively. Next, we examined the severity of apoptosis by staining hippocampal neurons by the Cresyl violet method. Third, we used western blot analysis to investigate the expression of total and phosphorylated Akt, ASK1, JNK3, c-Jun and caspase-3 after I/R injury. Our results revealed that mice subjected to bilateral common carotid occlusion exhibited severe anxiety-like behavior, learning and memory impairment, cell damage and apoptosis. These severe effects were attenuated by administration of quercetin. Further, western blot analysis revealed that quercetin increased p-Akt expression and decreased p-ASK1, p-JNK3 and cleaved caspase-3 expression after cerebral I/R injury and led to inhibition of neuronal apoptosis. Conversely, treatment with LY294002 (a selective inhibitor of Akt1) reversed the effects of quercetin. In conclusion, these findings highlight the important role of quercetin in protecting against cognitive deficits and inhibiting neuronal apoptosis via the Akt signaling pathway. We believe that quercetin might prove to be a useful therapeutic component in treating cerebral I/R diseases in the near future. PMID:27450812

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

    Directory of Open Access Journals (Sweden)

    Kessler Thomas

    2009-02-01

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

  1. 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.

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

    Science.gov (United States)

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

    2013-01-15

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

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

    International Nuclear Information System (INIS)

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

  4. The expression of caspase-10 in differentiated thyroid carcinoma and association with its development and metastasis%caspase-10在甲状腺分化癌中的表达及临床意义

    Institute of Scientific and Technical Information of China (English)

    尹大龙; 杨海彦; 刘连新; 陈曦; 田蓝天; 张树庚; 姜洪池

    2009-01-01

    Objective To investigate the expression of caspase-10 in differentiated thyroid carcinoma and association with its development and metastasis. Methods Thyroid samples from 37 patients in a period from January 2006 to December 2007, with differentiated thyroid carcinoma were retrospectively analyzed for caspase-10 by immunohistocbemistry(streptavidin-perosidase, S-P), compared to control group of 46 cases with nodtdar goiter. The relationship between the expression of caspase-10 and the clinical pathologic characteristics of thyroid carcinoma were also explored simultaneously. Results caspase-10 were observed as brown or yellow particles located in the cytoplasm or cell membrane of nodular goiter but there were no significant evidence for its positive expression in thyroid carcinoma, caspase-10 expression was markedly down-regulated in differentiated thyroid carcinoma(29.73%,11/37) compared with benign nodules(71.74%,33/46, χ2=14.528, P 0.05) between the expression of caspase-10 and the clinical pathologic characteristics including male, age, TNM stage and pathologic type. Conclusion Down-regulation of caspase-10 may play a critical role in carcinogenesis and development of differentiated thyroid carcinoma.%目的 探讨caspase-10表达在甲状腺分化癌(DTC)发生、发展及预后中的作用.方法 37例DTC来自于2006年1月至2007年12月行甲状腺切除术患者.取DTC患者病理标本,链霉菌抗生物素蛋白-过氧化物酶连结法(S-P)染色,光镜下检测DTC患者caspase-10表达,分析caspase-10在DTC中的表达与临床病理特征之间的关系.取同期46例结节性甲状腺肿组织标本作为对照.结果 结节性甲状腺肿细胞质或细胞膜,可见到较多的棕色或黄色caspase-10表达颗粒,但在DTC中棕色或黄色表达颗粒不明显.caspase-10表达阳性率在DTC患者中为29.73%(11/37),在结节性甲状腺肿患者中为71.74%(33/46),二者比较差异有统计学意义(χ2=14528,P0.05).结论 caspase-10表达

  5. 寻常型银屑病皮损处ASC和caspase-1的表达%Expressions of apoptosis-associated speck-like protein and caspase-1 in psoriasis vulgaris

    Institute of Scientific and Technical Information of China (English)

    胡坚; 杨闰平; 李恒进; 赵华

    2013-01-01

    Objective To study the role of apoptosis-associated speck-like protein (ASC) and caspase-1 in pathogenesis of psoriasis vulgaris. Methods Expression and distribution of ASC and caspase-1 in 30 psoriasis vulgaris patients were detected by immunohistochemistry. Results The ASC was intensely stained and expressed in cytoplasm and nuclei of psoriasis vulgaris patients and mainly expressed in middle and lower epidermis of normal persons. The caspase-1 was weakly stained and expressed in cytoplasm and full epidermis of psoriasis vulgaris patients and in basal and lower spinous cells of normal persons(t=49, 55, respectively, P<0.01). Conclusion ASC and caspase-1 play a significant role in the inflammation of psoriasis vulgarpsoriasis.%  目的探讨凋亡相关斑点样蛋白(apoptosis-associated speck-like protein,ASC)和半胱氨酸蛋白酶-1(caspase-1)在银屑病发病中的作用。方法采用免疫组化方法检测30例寻常型银屑病患者皮损处ASC和caspase-1的表达和分布。结果银屑病患者皮损处表皮全层强阳性表达ASC,主要分布在胞浆、胞核;正常人对照ASC主要表达于表皮中、下部,以轻、中度表达为主。银屑病患者皮损处caspase-1亦表达于表皮全层,位于胞浆,染色呈强阳性;正常人对照caspase-1表达于基底细胞和棘细胞层下部,染色强度较弱,两组比较差异均有统计学意义(t值分别为49和55,P均<0.01)。结论 ASC和caspase-1在银屑病的炎症过程中可能发挥重要作用。

  6. 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.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-06-01

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

  9. Magnetic resonance imaging of stem cell apoptosis in arthritic joints with a caspase activatable contrast agent.

    Science.gov (United States)

    Nejadnik, Hossein; Ye, Deju; Lenkov, Olga D; Donig, Jessica S; Martin, John E; Castillo, Rostislav; Derugin, Nikita; Sennino, Barbara; Rao, Jianghong; Daldrup-Link, Heike

    2015-02-24

    About 43 million individuals in the U.S. encounter cartilage injuries due to trauma or osteoarthritis, leading to joint pain and functional disability. Matrix-associated stem cell implants (MASI) represent a promising approach for repair of cartilage defects. However, limited survival of MASI creates a significant bottleneck for successful cartilage regeneration outcomes and functional reconstitution. We report an approach for noninvasive detection of stem cell apoptosis with magnetic resonance imaging (MRI), based on a caspase-3-sensitive nanoaggregation MRI probe (C-SNAM). C-SNAM self-assembles into nanoparticles after hydrolysis by caspase-3, leading to 90% amplification of (1)H MR signal and prolonged in vivo retention. Following intra-articular injection, C-SNAM causes significant MR signal enhancement in apoptotic MASI compared to viable MASI. Our results indicate that C-SNAM functions as an imaging probe for stem cell apoptosis in MASI. This concept could be applied to a broad range of cell transplants and target sites.

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

    Science.gov (United States)

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

    2016-06-01

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

  11. The New World arenavirus Tacaribe virus induces caspase-dependent apoptosis in infected cells.

    Science.gov (United States)

    Wolff, Svenja; Groseth, Allison; Meyer, Bjoern; Jackson, David; Strecker, Thomas; Kaufmann, Andreas; Becker, Stephan

    2016-04-01

    The Arenaviridae is a diverse and growing family of viruses that already includes more than 25 distinct species. While some of these viruses have a significant impact on public health, others appear to be non-pathogenic. At present little is known about the host cell responses to infection with different arenaviruses, particularly those found in the New World; however, apoptosis is known to play an important role in controlling infection of many viruses. Here we show that infection with Tacaribe virus (TCRV), which is widely considered the prototype for non-pathogenic arenaviruses, leads to stronger induction of apoptosis than does infection with its human-pathogenic relative Junín virus. TCRV-induced apoptosis occurred in several cell types during late stages of infection and was shown to be caspase-dependent, involving the activation of caspases 3, 7, 8 and 9. Further, UV-inactivated TCRV did not induce apoptosis, indicating that the activation of this process is dependent on active viral replication/transcription. Interestingly, when apoptosis was inhibited, growth of TCRV was not enhanced, indicating that apoptosis does not have a direct negative effect on TCRV infection in vitro. Taken together, our data identify and characterize an important virus-host cell interaction of the prototypic, non-pathogenic arenavirus TCRV, which provides important insight into the growing field of arenavirus research aimed at better understanding the diversity in responses to different arenavirus infections and their functional consequences.

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

    Directory of Open Access Journals (Sweden)

    Aiqi Zhang

    2013-10-01

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

  13. A nonapoptotic role for CASP2/caspase 2: modulation of autophagy.

    Science.gov (United States)

    Tiwari, Meenakshi; Sharma, Lokendra K; Vanegas, Difernando; Callaway, Danielle A; Bai, Yidong; Lechleiter, James D; Herman, Brian

    2014-06-01

    CASP2/caspase 2 plays a role in aging, neurodegeneration, and cancer. The contributions of CASP2 have been attributed to its regulatory role in apoptotic and nonapoptotic processes including the cell cycle, DNA repair, lipid biosynthesis, and regulation of oxidant levels in the cells. Previously, our lab demonstrated CASP2-mediated modulation of autophagy during oxidative stress. Here we report the novel finding that CASP2 is an endogenous repressor of autophagy. Knockout or knockdown of CASP2 resulted in upregulation of autophagy in a variety of cell types and tissues. Reinsertion of Caspase-2 gene (Casp2) in mouse embryonic fibroblast (MEFs) lacking Casp2 (casp2(-/-)) suppresses autophagy, suggesting its role as a negative regulator of autophagy. Loss of CASP2-mediated autophagy involved AMP-activated protein kinase, mechanistic target of rapamycin, mitogen-activated protein kinase, and autophagy-related proteins, indicating the involvement of the canonical pathway of autophagy. The present study also demonstrates an important role for loss of CASP2-induced enhanced reactive oxygen species production as an upstream event in autophagy induction. Additionally, in response to a variety of stressors that induce CASP2-mediated apoptosis, casp2(-/-) cells demonstrate a further upregulation of autophagy compared with wild-type MEFs, and upregulated autophagy provides a survival advantage. In conclusion, we document a novel role for CASP2 as a negative regulator of autophagy, which may provide important insight into the role of CASP2 in various processes including aging, neurodegeneration, and cancer.

  14. 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.

  15. Histone deacetylase inhibitor trichostatin A induced caspase-independent apoptosis in human gastric cancer cell

    Institute of Scientific and Technical Information of China (English)

    WU Zhi-qun; ZHANG Rui; Connie Chao; ZHANG Ji-feng; ZHANG Yuan-qiang

    2007-01-01

    Background Histone deacetylase inhibitors (HDACIs) have been reported to induce apoptosis in cancer cells.The effects of trichostatin A (TSA) on gastric cancer cells have not been well characterized.This study was aimed to explore the effects and mechanisms of TSA on human gastric cancer SGC-7901 cells.Methods The cells were treated with TSA and analyzed by cell proliferation assay,Western blot,TUNEL assay,flow cytometry by fluorescein isothiocyanate (FITC) conjugated with Annexin V and PI staining,immunofluorescence analysis,analysis of subcellular fractionation,gene chips and real time polymerase chain reaction (PCR).Results TSA could inhibit cell growth and induced apoptosis in gastric cancer SGC-7901 cells through the regulation of apoptosis-related genes,such as Bcl-2,Bax and survivin.Further study indicated that the pan-caspase inhibitor z-VAD-fmk did not inhibit the apoptosis induced by TSA,and we did not observe the cleavage of poly ADP ribose polymerase(PARP)after TSA treatment too.In addition,apoptosis inducing factor (AIF) and EndoG were found to translocate from mitochondria to nucleus in the immunofluorescence assay and the Western analysis of subcellular fractionation confirmed the result of immunofluorescence assay.Conclusions The apoptosis induced by TSA in gastric cancer SGC-7901 cells involves a caspase-independent pathway.

  16. Caspase-cleaved cytokeratin-18 and tumour regression in gastro-oesophageal adenocarcinomas treated with neoadjuvant chemotherapy

    Institute of Scientific and Technical Information of China (English)

    Khaleel R Fareed; Irshad N Soomro; Khalid Hameed; Arvind Arora; Dileep N Lobo; Simon L Parsons; Srinivasan Madhusudan

    2012-01-01

    AIM:To examine cytokeratin-18 (CK-18) and caspasecleaved CK-18 expression in tumouts and correlate with clinicopathological outcomes including tumour regression grade (TRG) response.METHODS:Formalin-fixed human gastro-oesophageal cancers were constructed into tissue microarrays.The first set consisted of 122 gastric/gastro-oesophageal cancer cases not exposed to neoadjuvant chemotherapy and the second set consisted of 97 gastric/gastrooesophageal cancer cases exposed to pre-operative platinum-based chemotherapy.Expression of CK-18 and caspase-cleaved CK-18 was investigated using immunohistochemistry.RESULTS:CK18 was commonly expressed in gastrooesophageal tumours (92.6%).Fifty-six point seven percent of tumours previously exposed to neoadjuvant chemotherapy were positive for caspase-cleaved CK-18 expression compared to only 24.6% of tumours not previously exposed to neoadjuvant chemotherapy (P =0.009).In patients who received neoadjuvant chemotherapy,caspase-cleaved cytokeratin-18 expression correlated with favourable TRG response (TRG 1,2 or 3,P =0.043).CONCLUSION:This is the largest study to date of CK-18 and caspase-cleaved CK-18 expression in gastrooesophageal tumours.We provide the first evidence that caspase-cleaved CK-18 predicts tumour regression with neoadjuvant chemotherapy.

  17. Retinoid-dependent growth inhibition, differentiation and apoptosis in acute promyelocytic leukemia cells. Expression and activation of caspases.

    Science.gov (United States)

    Gianni, M; Ponzanelli, I; Mologni, L; Reichert, U; Rambaldi, A; Terao, M; Garattini, E

    2000-05-01

    In the NB4 model of acute promyelocytic leukemia (APL), ATRA, 9-cis retinoic acid (9-cis RA), the pan-RAR and RARalpha-selective agonists, TTNPB and AM580, induce growth inhibition, granulocytic differentiation and apoptosis. By contrast, two RXR agonists, a RARbeta agonist and an anti-AP1 retinoid have very limited activity, ATRA- and AM580-dependent effects are completely inhibited by RAR antagonistic blockade, while 9-cis RA-induced cell-growth-inhibition and apoptosis are equally inhibited by RAR and RXR antagonists. ATRA, 9-cis RA and AM580 cause upregulation of the mRNAs coding for pro-caspase-1, -7, -8, and -9, which, however, results in increased synthesis of only pro-caspase-1 and -7 proteins. These phenomena are associated with activation of pro-caspase-6, -7 and -8, cytochrome c release from the mitochondria, inversion of Bcl-2/Bax ratio and degradation of PML-RARalpha. Caspase activation is fundamental for retinoid-induced apoptosis, which is suppressed by the caspase-inhibitor z-VAD.

  18. 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.

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

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    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.

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

    Science.gov (United States)

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

    2009-07-01

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

  1. 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.

  2. Caspase dependent programmed cell death in developing embryos: a potential target for therapeutic intervention against pathogenic nematodes.

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    Alok Das Mohapatra

    2011-09-01

    Full Text Available BACKGROUND: Successful embryogenesis is a critical rate limiting step for the survival and transmission of parasitic worms as well as pathology mediated by them. Hence, blockage of this important process through therapeutic induction of apoptosis in their embryonic stages offers promise for developing effective anti-parasitic measures against these extra cellular parasites. However, unlike in the case of protozoan parasites, induction of apoptosis as a therapeutic approach is yet to be explored against metazoan helminth parasites. METHODOLOGY/PRINCIPAL FINDINGS: For the first time, here we developed and evaluated flow cytometry based assays to assess several conserved features of apoptosis in developing embryos of a pathogenic filarial nematode Setaria digitata, in-vitro as well as ex-vivo. We validated programmed cell death in developing embryos by using immuno-fluorescence microscopy and scoring expression profile of nematode specific proteins related to apoptosis [e.g. CED-3, CED-4 and CED-9]. Mechanistically, apoptotic death of embryonic stages was found to be a caspase dependent phenomenon mediated primarily through induction of intracellular ROS. The apoptogenicity of some pharmacological compounds viz. DEC, Chloroquine, Primaquine and Curcumin were also evaluated. Curcumin was found to be the most effective pharmacological agent followed by Primaquine while Chloroquine displayed minimal effect and DEC had no demonstrable effect. Further, demonstration of induction of apoptosis in embryonic stages by lipid peroxidation products [molecules commonly associated with inflammatory responses in filarial disease] and demonstration of in-situ apoptosis of developing embryos in adult parasites in a natural bovine model of filariasis have offered a framework to understand anti-fecundity host immunity operational against parasitic helminths. CONCLUSIONS/SIGNIFICANCE: Our observations have revealed for the first time, that induction of apoptosis in

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

    Institute of Scientific and Technical Information of China (English)

    李小琴; 白彩云

    2016-01-01

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

  4. Mifepristone-inducible caspase-1 expression in mouse embryonic stem cells eliminates tumor formation but spares differentiated cells in vitro and in vivo.

    Science.gov (United States)

    Wang, Yi; Yang, Dehua; Song, Lin; Li, Ting; Yang, Juan; Zhang, Xiaojie; Le, Weidong

    2012-02-01

    Embryonic stem cell (ESC)-based therapy is a promising treatment for neurodegenerative diseases. But there is always a risk of tumor formation that is due to contamination of undifferentiated ESCs. To reduce the risk and improve ESC-based therapy, we have established a novel strategy by which we can selectively eliminate tumor cells derived from undifferentiated ESCs but spare differentiated cells. In this study, we generated a caspase-1-ESC line transfected with a mifepristone-regulated caspase-1 expression system. Mifepristone induced caspase-1 overexpression both in differentiated and undifferentiated caspase-1-ESCs. All the undifferentiated caspase-1-ESCs were induced to death after mifepristone treatment. Tumors derived from undifferentiated caspase-1-ESCs were eliminated following 3 weeks of mifepristone treatment in vivo. However, differentiated caspase-1-ESCs survived well under the condition of mifepristone-induced caspase-1 overexpression. To examine in vivo the impact of mifepristone-induced caspase-1 activation on grafted cells, we transplanted wild-type ESCs or caspase-1-ESCs into nude mice brains. After 8 weeks of mifepristone treatment, we could not detect any tumor cells in the caspase-1-ESC grafts in the brains of mice. However, we found that donor dopamine neurons survived in the recipient brains. These data demonstrate that mifepristone-induced caspase-1 overexpression in ESCs can eliminate the potential tumor formation meanwhile spares the differentiated cells in the host brains. These results suggest that this novel ESC-based therapy can be used in Parkinson's disease and other related disorders without the risk of tumor formation.

  5. 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

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

    Institute of Scientific and Technical Information of China (English)

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

    2010-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    张远贵; 段冬; 李攀登; 陈润芳; 李之华; 高新宇; 吴炜

    2016-01-01

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

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

    International Nuclear Information System (INIS)

    appearance of DNA ladders, caspase 3 protein processing, PARP protein cleavage, and increasing p21 protein. These results revealed in vitro, ex vivo, and in vivo antitumor activities of 2'-OH flavanone via apoptosis induction, and indicates that 2'-OH flavanone is an active compound worthy of development for cancer chemotherapy

  9. Enhanced G2/M Arrest, Caspase Related Apoptosis and Reduced E-Cadherin Dependent Intercellular Adhesion by Trabectedin in Prostate Cancer Stem Cells

    Science.gov (United States)

    Uslu, Ruchan; Kara, Mikail; Soner, Burak Cem; Oktem, Gulperi

    2015-01-01

    Trabectedin (Yondelis, ET-743) is a marine-derived tetrahydroisoquinoline alkaloid. It is originally derived from the Caribbean marine tunicate Ecteinascidia turbinata and currently produced synthetically. Trabectedin is active against a variety of tumor cell lines growing in culture. The present study focused on the effect of trabectedin in cell proliferation, cell cycle progression, apoptosis and spheroid formation in prostate cancer stem cells (CSCs). Cluster of differentiation (CD) 133+high/CD44+high prostate CSCs were isolated from the DU145 and PC-3 human prostate cancer cell line through flow cytometry. We studied the growth-inhibitory effects of trabectedin and its molecular mechanisms on human prostate CSCs and non-CSCs. DU-145 and PC-3 CSCs were treated with 0.1, 1, 10 and 100 nM trabectedin for 24, 48 and 72 h and the growth inhibition rates were examined using the sphere-forming assay. Annexin-V assay and immunofluorescence analyses were performed for the detection of the cell death. Concentration-dependent effects of trabectedin on the cell cycle were also evaluated. The cells were exposed to the different doses of trabectedin for 24, 48 and 72 h to evaluate the effect of trabectedin on the number and diameter of spheroids. According to the results, trabectedin induced cytotoxicity and apoptosis at the IC50 dose, resulting in a significant increase expression of caspase-3, caspase-8, caspase-9, p53 and decrease expression of bcl-2 in dose-dependent manner. Cell cycle analyses revealed that trabectedin induces dose-dependent G2/M-phase cell cycle arrest, particularly at high-dose treatments. Three-dimensional culture studies showed that trabectedin reduced the number and diameter of spheroids of DU145 and PC3 CSCs. Furthermore, we have found that trabectedin disrupted cell-cell interactions via E-cadherin in prostasphere of DU-145 and PC-3 CSCs. Our results showed that trabectedin inhibits cellular proliferation and accelerates apoptotic events in

  10. Enhanced G2/M Arrest, Caspase Related Apoptosis and Reduced E-Cadherin Dependent Intercellular Adhesion by Trabectedin in Prostate Cancer Stem Cells.

    Directory of Open Access Journals (Sweden)

    Eda Acikgoz

    Full Text Available Trabectedin (Yondelis, ET-743 is a marine-derived tetrahydroisoquinoline alkaloid. It is originally derived from the Caribbean marine tunicate Ecteinascidia turbinata and currently produced synthetically. Trabectedin is active against a variety of tumor cell lines growing in culture. The present study focused on the effect of trabectedin in cell proliferation, cell cycle progression, apoptosis and spheroid formation in prostate cancer stem cells (CSCs. Cluster of differentiation (CD 133+high/CD44+high prostate CSCs were isolated from the DU145 and PC-3 human prostate cancer cell line through flow cytometry. We studied the growth-inhibitory effects of trabectedin and its molecular mechanisms on human prostate CSCs and non-CSCs. DU-145 and PC-3 CSCs were treated with 0.1, 1, 10 and 100 nM trabectedin for 24, 48 and 72 h and the growth inhibition rates were examined using the sphere-forming assay. Annexin-V assay and immunofluorescence analyses were performed for the detection of the cell death. Concentration-dependent effects of trabectedin on the cell cycle were also evaluated. The cells were exposed to the different doses of trabectedin for 24, 48 and 72 h to evaluate the effect of trabectedin on the number and diameter of spheroids. According to the results, trabectedin induced cytotoxicity and apoptosis at the IC50 dose, resulting in a significant increase expression of caspase-3, caspase-8, caspase-9, p53 and decrease expression of bcl-2 in dose-dependent manner. Cell cycle analyses revealed that trabectedin induces dose-dependent G2/M-phase cell cycle arrest, particularly at high-dose treatments. Three-dimensional culture studies showed that trabectedin reduced the number and diameter of spheroids of DU145 and PC3 CSCs. Furthermore, we have found that trabectedin disrupted cell-cell interactions via E-cadherin in prostasphere of DU-145 and PC-3 CSCs. Our results showed that trabectedin inhibits cellular proliferation and accelerates

  11. 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

    2012-12-01

    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.

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

    Institute of Scientific and Technical Information of China (English)

    Hong LAN; You-yong LU

    2004-01-01

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

  13. Effects of aerobic exercise combined with resveratrol on cardiomyocyte apoptosis factors TNF-αand Caspase-3 in type II diabetic rats%有氧运动联合白藜芦醇对Ⅱ型糖尿病大鼠心肌细胞凋亡因子TNF-α、Caspase-3的影响

    Institute of Scientific and Technical Information of China (English)

    任建厂; 王红丽; 肖国强

    2014-01-01

    the effects of aerobic exercise combined with resveratrol on cardiomyocyte apoptosis factors TNF-αand Caspase-3 in type 2 diabetic rats, the authors prepared 40 2-month old male SD rats, randomly selected 8 rats to form a normal control group (ZA), fed them with standard feedstuff, intraperitoneally injected a small dose of streptozotocin into the remaining rats on the basis of high-fat and high-carbohydrate diet to establish a type II diabetic rat model, divided type II diabetic model rats randomly into 4 groups, namely, a model calm group (TA), a model exercise group (TY), a model resveratrol gavage calm group (TB), and a model resveratrol gavage exercise group (TYB), let the rats in the exercise groups swim in a load free condition 6 days a week, 7 weeks later, checked the fasting blood sugar level, blood fat index, morphological changes of myocardial tissue cells as well as myocardial TNF-αand Caspase-3 con-tents of the rats in various groups, and revealed the following findings:1) in 30, 60 and 120 minutes after OGTT, the blood sugar concentration of the rats in groups TY, TB and TYB decreased significantly (P<0.01) as compared with the rats in group TA;as compared with the rats in group TA, the serum TC, TG and TDL levels of the rats in groups TY, TB and TYB decreased significantly (P<0.05 or P<0.01);as compared with the rats in group ZA, the cardiac muscle of the rats in group TA showed pathological changes;as compared with the rats in group TA, the myocardial pathological changes of the rats in groups TY, TB and TYB were alleviated significantly;2) as compared with the rats in group ZA, TNF-αin myocar-dial tissue of the rats in group TA increased significantly (P<0.01);as compared with the rats in group TA, TNF-αin myo-cardial tissue of the rats in groups TY, TB and TYB decreased significantly (P<0.05);as compared with the rats in group TYB, the level of expression of TNF-α in myocardial tissue of the rats in groups TY and TB increased significantly (P<0

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

    Science.gov (United States)

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

    2016-09-01

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

  15. Expression and Significance of Bcl-2, Bax, Fas and Caspase-3 in Different Phases of Human Hemangioma

    Institute of Scientific and Technical Information of China (English)

    YANG Hong; DENG Chenguo; SHEN Shengguo; ZHANG Duanlian; YUYing

    2006-01-01

    The relationship between Bcl-2, Bax, Fas, caspase-3 and development of hemangioma and the molecular mechanism was investigated. By using immunohistochemical S-P method, proliferating cell nuclear antigen was detected. According to the classification of Mulliken in combination with PCNA expression, 27 cases were identified as proliferating hemangioma and 22 cases as involutive hemangioma. Five normal skin tissues around the tumor tissue served as controls. By using immunohistochemical technique, the expression of Bcl-2, Bax, Fax and Caspase-3 was detected. The cells expressing Bcl-2, Bax, Fax and cappase-3 were identified as hemangioma endothelia by immunohistochemical staining of Ⅷ factor. The average absorbance (A) and average positive area rate of Bcl-2, Bax, Fas and caspase-3 expression were measured by using HPIAS-2000 imaging analysis system. The results showed that the expression of Bcl-2 in the endothelia of proliferating hemangioma was significantly higher that in involutive degenerative hemangioma endothelia and vascular endothelia of normal skin tissue (P<0.01). The expression of Bax, Fas and Caspase-3 in the endothelia of involutive hemangioma was obviously higher than in the endothelia of proliferating hemangioma and normal skin tissue (P<0.01). The expression of BAx and Fas in endothelia of proliferating hemangioma was higher than in those of normal skin tissue (P<0.05). It was suggested that Bcl-2,Bax, Fas and caspase-3 might be involved in the development and involution of hemangioma. Bcl-2 could promote the growth of hemangioma by inhibiting apoptosis of endothelia. Bax, Fas and caspase-3 promote the switch of hemangioma from proliferation to involution by inducing the apoptosis of hemangioma endothelia.

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Blay Jean-Yves

    2006-03-01

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

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

    International Nuclear Information System (INIS)

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

  19. Ritonavir and disulfiram have potential to inhibit caspase-1 mediated inflammation and reduce neurological sequelae after minor blast exposure.

    Science.gov (United States)

    Foley, Kevin; Kast, Richard E; Altschuler, Eric L

    2009-02-01

    Caspase-1 triggers cytokine release following acceleration-induced concussive head injury. Minor blast injury in which no physical tissue injury occurs, results in the release of cytokines in a similar fashion. Ritonavir, a generically available protease inhibitor with a benign short-term side-effect profile, has been shown to inhibit expression of caspase-1. We review the relevant literature and propose that ritonavir may be of benefit in reducing adverse neuropsychiatric outcomes and hastening recovery following mild blast injury. Further research in animal models of blast injury followed by clinical studies would determine whether this therapy is effective.

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Rojanasakul Yon

    2010-05-01

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

  2. Single-cell resolution imaging of retinal ganglion cell apoptosis in vivo using a cell-penetrating caspase-activatable peptide probe.

    Directory of Open Access Journals (Sweden)

    Xudong Qiu

    Full Text Available Peptide probes for imaging retinal ganglion cell (RGC apoptosis consist of a cell-penetrating peptide targeting moiety and a fluorophore-quencher pair flanking an effector caspase consensus sequence. Using ex vivo fluorescence imaging, we previously validated the capacity of these probes to identify apoptotic RGCs in cell culture and in an in vivo rat model of N-methyl- D-aspartate (NMDA-induced neurotoxicity. Herein, using TcapQ488, a new probe designed and synthesized for compatibility with clinically-relevant imaging instruments, and real time imaging of a live rat RGC degeneration model, we fully characterized time- and dose-dependent probe activation, signal-to-noise ratios, and probe safety profiles in vivo. Adult rats received intravitreal injections of four NMDA concentrations followed by varying TcapQ488 doses. Fluorescence fundus imaging was performed sequentially in vivo using a confocal scanning laser ophthalmoscope and individual RGCs displaying activated probe were counted and analyzed. Rats also underwent electroretinography following intravitreal injection of probe. In vivo fluorescence fundus imaging revealed distinct single-cell probe activation as an indicator of RGC apoptosis induced by intravitreal NMDA injection that corresponded to the identical cells observed in retinal flat mounts of the same eye. Peak activation of probe in vivo was detected 12 hours post probe injection. Detectable fluorescent RGCs increased with increasing NMDA concentration; sensitivity of detection generally increased with increasing TcapQ488 dose until saturating at 0.387 nmol. Electroretinography following intravitreal injections of TcapQ488 showed no significant difference compared with control injections. We optimized the signal-to-noise ratio of a caspase-activatable cell penetrating peptide probe for quantitative non-invasive detection of RGC apoptosis in vivo. Full characterization of probe performance in this setting creates an important in

  3. Lipopolysaccharide-Mediated Induction of Concurrent IL-1β and IL-23 Expression in THP-1 Cells Exhibits Differential Requirements for Caspase-1 and Cathepsin B Activity.

    Science.gov (United States)

    Wynick, Christopher; Petes, Carlene; Tigert, Alexander; Gee, Katrina

    2016-08-01

    The inflammasome is a multimeric protein complex required for interleukin (IL)-1β production. Upon lipopolysaccharide (LPS) triggering of toll-like receptor (TLR)-4 and subsequent ATP signaling, the NOD-like receptor containing-pyrin domain 3 (NLRP3) inflammasome is activated to cleave pro-caspase-1 into caspase-1, allowing the secretion of IL-1β. IL-1β is known to function with IL-23 in the regulation of IL-17-producing CD4(+) T cells, Th17 cells, in adaptive immunity. Recently, studies have shown that IL-1β and IL-23 together activate IL-17-producing innate lymphoid cells, demonstrating that the pair may exhibit additional effects on cell differentiation. Using an in vitro model of bacterial infection, LPS treatment of human monocytic cells, we investigated the molecular mechanisms involved in the co-expression of IL-1β and IL-23. We found that IL-1β is partially required for optimal LPS-induced IL-23 production. We also found that IL-23 production was partially dependent on ATP signaling via the P2X7 receptor, whereas IL-1β production required this signaling. Furthermore, we identified a novel role for cathepsin B activity in IL-23 production. Taken together, this study identifies differential requirements for the co-expression of IL-1β and IL-23. Due to their similar roles in Th17 differentiation, characterization of the regulatory mechanisms for LPS-induced IL-1β and IL-23 may reveal novel information into the pathology of the inflammatory response particularly during bacterial infection. PMID:27096899

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

    Directory of Open Access Journals (Sweden)

    Javad Baharara

    2015-02-01

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

  5. Endocytosis of indium-tin-oxide nanoparticles by macrophages provokes pyroptosis requiring NLRP3-ASC-Caspase1 axis that can be prevented by mesenchymal stem cells.

    Science.gov (United States)

    Naji, Abderrahim; Muzembo, Basilua André; Yagyu, Ken-Ichi; Baba, Nobuyasu; Deschaseaux, Frédéric; Sensebé, Luc; Suganuma, Narufumi

    2016-01-01

    The biological effects of indium-tin-oxide (ITO) are of considerable importance because workers exposed to indium compounds have been diagnosed with interstitial lung disease or pulmonary alveolar proteinosis; however, the pathophysiology of these diseases is undefined. Here, mice intraperitoneally inoculated with ITO-nanoparticles (ITO-NPs) resulted in peritonitis dependent in NLRP3 inflammasome, with neutrophils recruitment and interleukin-1β (IL-1β) production. Withal peritoneal macrophages exposed ex vivo to ITO-NPs caused IL-1β secretion and cytolysis. Further, alveolar macrophages exposed to ITO-NPs in vitro showed ITO-NP endocytosis and production of tumor necrosis factor-α (TNF-α) and IL-1β, ensued cell death by cytolysis. This cell death was RIPK1-independent but caspase1-dependent, and thus identified as pyroptosis. Endocytosis of ITO-NPs by activated THP-1 cells induced pyroptosis with IL-1β/TNF-α production and cytolysis, but not in activated THP-1 cells with knockdown of NLRP3, ASC, or caspase1. However, exposing activated THP-1 cells with NLRP3 or ASC knockdown to ITO-NPs resulted in cell death but without cytolysis, with deficiency in IL-1β/TNF-α, and revealing features of apoptosis. While, mesenchymal stem cells (MSCs) co-cultured with macrophages impaired both inflammation and cell death induced by ITO-NPs. Together, our findings provide crucial insights to the pathophysiology of respiratory diseases caused by ITO particles, and identify MSCs as a potent therapeutic. PMID:27194621

  6. An evidence on G2/M arrest, DNA damage and caspase mediated apoptotic effect of biosynthesized gold nanoparticles on human cervical carcinoma cells (HeLa)

    Energy Technology Data Exchange (ETDEWEB)

    Jeyaraj, M. [Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu (India); Arun, R. [Department of Biomedical Sciences, Bharathidasan University, Tiruchirappalli 620024 (India); Sathishkumar, G. [Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu (India); MubarakAli, D. [Central Inter-Disciplinary Research Facility, Mahatma Gandhi Medical College and Research Institute Campus, Pondicherry 607402 (India); Rajesh, M.; Sivanandhan, G.; Kapildev, G.; Manickavasagam, M. [Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu (India); Thajuddin, N. [Department of Microbiology, Bharathidasan University, Tiruchirappalli 620024 (India); Ganapathi, A., E-mail: aganapathi2007@gmail.com [Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu (India)

    2014-04-01

    Highlights: • Gold nanoparticles (AuNPs) have been synthesized using Podophyllum hexandrum L. • AuNPs induces the oxidative stress to cell death in human cervical carcinoma cells. • It activates the caspase-cascade to cellular death. • It is actively blocks G2/M phase of cell cycle. - Abstract: Current prospect of nanobiotechnology involves in the greener synthesis of nanostructured materials particularly noble metal nanoparticles for various biomedical applications. In this study, biologically (Podophyllum hexandrum L.) synthesized crystalline gold nanoparticles (AuNPs) with the size range between 5 and 35 nm were screened for its anticancereous potential against human cervical carcinoma cells (HeLa). Stoichiometric proportion of the reaction mixture and conditions were optimized to attain stable nanoparticles with narrow size range. Different high throughput techniques like transmission electron microscope (TEM), X-ray diffraction (XRD) and UV–vis spectroscopy were adopted for the physio-chemical characterization of AuNPs. Additionally, Fourier transform infrared spectroscopy (FTIR) study revealed that the water soluble fractions present in the plant extract solely influences the reduction of AuNPs. Sublimely, synthesized AuNPs exhibits an effective in vitro anticancer activity against HeLa cells via induction of cell cycle arrest and DNA damage. Furthermore, it was evidenced that AuNPs treated cells are undergone apoptosis through the activation of caspase cascade which subsequently leads to mitochondrial dysfunction. Thereby, this study proves that biogenic colloidal AuNPs can be developed as a promising drug candidature for human cervical cancer therapy.

  7. Caspase-resistant VirD2 protein provides enhanced gene delivery and expression in plants.

    Science.gov (United States)

    Reavy, Brian; Bagirova, Svetlana; Chichkova, Nina V; Fedoseeva, Svetlana V; Kim, Sang Hyon; Vartapetian, Andrey B; Taliansky, Michael E

    2007-08-01

    Agrobacterium tumefaciens VirD2 protein is one of the key elements of Agrobacterium-mediated plant transformation, a process of transfer of T-DNA sequence from the Agrobacterium tumour inducing plasmid into the nucleus of infected plant cells and its integration into the host genome. The VirD2 protein has been shown to be a substrate for a plant caspase-like protease activity (PCLP) in tobacco. We demonstrate here that mutagenesis of the VirD2 protein to prevent cleavage by PCLP increases the efficiency of reporter gene transfer and expression. These results indicate that PCLP cleavage of the Agrobacterium VirD2 protein acts to limit the effectiveness of T-DNA transfer and is a novel resistance mechanism that plants utilise to combat Agrobacterium infection. PMID:17370074

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

    Directory of Open Access Journals (Sweden)

    V. Ramnath

    2009-01-01

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

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

    Science.gov (United States)

    Roth, Susanne; Ruland, Jürgen

    2013-06-01

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

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

    Science.gov (United States)

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

    2016-07-12

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

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

    Directory of Open Access Journals (Sweden)

    Mohammad Ahmed Khan

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    John C Means

    2015-05-01

    Full Text Available While circadian dysfunction and neurodegeneration are correlated, the mechanism for this is not understood. It is not known if age-dependent circadian dysfunction leads to neurodegeneration or vice-versa, and the proteins that mediate the effect remain unidentified. Here, we show that the knock-down of a regulator (spag of the circadian kinase Dbt in circadian cells lowers Dbt levels abnormally, lengthens circadian rhythms and causes expression of activated initiator caspase (Dronc in the optic lobes during the middle of the day or after light pulses at night. Likewise, reduced Dbt activity lengthens circadian period and causes expression of activated Dronc, and a loss-of-function mutation in Clk also leads to expression of activated Dronc in a light-dependent manner. Genetic epistasis experiments place Dbt downstream of Spag in the pathway, and Spag-dependent reductions of Dbt are shown to require the proteasome. Importantly, activated Dronc expression due to reduced Spag or Dbt activity occurs in cells that do not express the spag RNAi or dominant negative Dbt and requires PDF neuropeptide signaling from the same neurons that support behavioral rhythms. Furthermore, reduction of Dbt or Spag activity leads to Dronc-dependent Drosophila Tau cleavage and enhanced neurodegeneration produced by human Tau in a fly eye model for tauopathy. Aging flies with lowered Dbt or Spag function show markers of cell death as well as behavioral deficits and shortened lifespans, and even old wild type flies exhibit Dbt modification and activated caspase at particular times of day. These results suggest that Dbt suppresses expression of activated Dronc to prevent Tau cleavage, and that the circadian clock defects confer sensitivity to expression of activated Dronc in response to prolonged light. They establish a link between the circadian clock factors, light, cell death pathways and Tau toxicity, potentially via dysregulation of circadian neuronal remodeling in

  13. 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

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

    International Nuclear Information System (INIS)

    Highlights: • Nascent histamine alters cyclin expression pattern. • Nascent histamine increases expression of α-synuclein. • Nascent histamine activates caspase-3. - Abstract: Histamine (Hia) is the most multifunctional biogenic amine. It is synthetized by histidine decarboxylase (HDC) in a reduced set of mammalian cell types. Mast cells and histaminergic neurons store Hia in specialized organelles until the amine is extruded by exocytosis; however, other immune and cancer cells are able to produce but not store Hia. The intracellular effects of Hia are still not well characterized, in spite of its physiopathological relevance. Multiple functional relationships exist among Hia metabolism/signaling elements and those of other biogenic amines, including growth-related polyamines. Previously, we obtained the first insights for an inhibitory effect of newly synthetized Hia on both growth-related polyamine biosynthesis and cell cycle progression of non-fully differentiated mammalian cells. In this work, we describe progress in this line. HEK293 cells were transfected to express active and inactive versions of GFP-human HDC fusion proteins and, after cell sorting by flow cytometry, the relative expression of a large number of proteins associated with cell signaling were measured using an antibody microarray. Experimental results were analyzed in terms of protein–protein and functional interaction networks. Expression of active HDC induced a cell cycle arrest through the alteration of the levels of several proteins such as cyclin D1, cdk6, cdk7 and cyclin A. Regulation of α-synuclein and caspase-3 was also observed. The analyses provide new clues on the molecular mechanisms underlying the regulatory effects of intracellular newly synthetized Hia on cell proliferation/survival, cell trafficking and protein turnover. This information is especially interesting for emergent and orphan immune and neuroinflammatory diseases

  15. 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.

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

    Institute of Scientific and Technical Information of China (English)

    王雪; 李静; 祝三平

    2007-01-01

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

  17. 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.

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

    Institute of Scientific and Technical Information of China (English)

    马英桓; 袁正伟

    2012-01-01

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

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

    Science.gov (United States)

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

    2011-01-01

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

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

    International Nuclear Information System (INIS)

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

  1. Location of caspase 3-like protease in the development of sieve element and tracheary element of stem in Cucurbita moschata.

    Science.gov (United States)

    Hao, Xia; Qian, Jie; Xu, Shan; Song, Xin; Zhu, Jian

    2008-12-01

    The casepase is considered to regulate the process of programmed cell death in the development of organisms. In this study, caspase 3-like protease was detected by immunohistochemistry and immunoelectron microscopy during the development of sieve element and tracheary element of stem in Cucurbita moschata Duch. Antibody with brown color (under light microscopy) and gold particles (under transmission electron microscopy) for detecting caspase 3-like protease was mainly displayed in inner phloem, external phloem and xylem in the region close to procambium. From the results it was considered that caspase 3-like protease did exist in vascular elements and played different roles during the development of sieve and tracheary elements, and different types of programmed cell death might be carried out. The caspase 3-like protease mainly participated in making cytoplasmic streaming cease and in degrading P-protein bodies; however, it rarely participated in the function for signal transferring in the developmental sieve element. However, it might induce calcium accumulation for rupturing the tonoplast in the signal of PCD in the developmental tracheary element.

  2. Location of Caspase 3-like Protease in the Development of Sieve Element and Tracheary Element of Stem in Cucurbita moschata

    Institute of Scientific and Technical Information of China (English)

    Xia Hao; Jie Qian; Shan Xu; Xin Song; Jian Zhu

    2008-01-01

    The casepase is considered to regulate the process of programmed cell death in the development of organisms. In this study, caspase 3-like protease was detected by immunohistochemistry and immunoelectron microscopy during the development of sieve element and tracheary element of stem in Cucurbita moschata Duch. Antibody with brown color (under light microscopy) and gold particles (under transmission electron microscopy) for detecting caspase 3-like protease was mainly displayed in inner phloem, extemal phloem and xylem in the region close to procambium. From the results it was considered that caspase 3-like protease did exist in vascular elements and played different roles during the development of sieve and tracheary elements, and different types of programmed cell death might be carried out. The caspase 3-like protease mainly participated in making cytoplasmic streaming cease and in degrading P-protein bodies; however, it rarely participated in the function for signal transferring in the developmental sieve element. However, it might induce calcium accumulation for rupturing the tonoplast in the signal of PCD in the developmental tracheary element.

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

    Directory of Open Access Journals (Sweden)

    Hongru LI

    2008-10-01

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

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

    Science.gov (United States)

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

    2001-05-11

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

  5. 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.

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

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    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

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

    Science.gov (United States)

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

    2016-10-01

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

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

    Science.gov (United States)

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

    2016-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Gu Bin

    2011-12-01

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

  10. 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.

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

    Science.gov (United States)

    Okamoto, Toshihiro; Kawasaki, Toru; Hino, Okio

    2003-02-15

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

  12. Effect of Caspase Inhibitor Ac-DEVD-CHO on Apoptosis of Vascular Smooth Muscle Cells Induced by Artesunate

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    Jingwen Zhang

    2014-05-01

    Full Text Available Numerous studies have shown that the proliferation and apoptosis of vascular smooth muscle cells play a key role in restenosis. Artesunate is a triterpenoid with a peroxide structure and its antimalarial, antitumor, and antiangiogenetic activities can inhibit the proliferation and apoptosis of multifarious cells. Apoptosis is caused by the activation of a series of intracellular proteolytic enzymes, among which caspase-dependent apoptosis was the earliest to be recognized. The purpose of this article is to study the effects of caspase-3 inhibitor Ac-DEVD-CHO on proliferation and apoptosis of vascular smooth muscle cells induced by Artesunate and to explore the mechanism of Artesunate-induced apoptosis of vascular smooth muscle cells. By using the method based on methyl thiazolyl tetrazolium to observe the effects of Artesunate on the growth and proliferation of vascular smooth muscle cells; observing the change in cell shape before and after Artesunate administration by transmission electron microscopy; detecting the changes in cell cycle and apoptosis rates before and after drug administration by flow cytometry; detecting the activity of caspase-3 in the caspase apoptosis pathway by the Western Blot method, we found that Artesunate inhibits the growth and proliferation of vascular smooth muscle cells in a dose- and time-dependent manner within the concentration range of 7.5–120 μg/mL, and the inhibition rate of Artesunate can be as high as 89.49 % at a concentration of 120 μg/mL after acting for 72 hours; vascular smooth muscle cells show a typical apoptosis peak due to the effects of higher concentration of Artesunate. Compared with the control group, the higher-concentration group shows major variability, Ac-DEVD-CHO, however, can significantly decrease this induction; it has been detected by Western Blot that Artesunate can induce caspase-3 activity dramatically in vascular smooth muscle cells, but this activation may be remarkably

  13. Effects of nerve growth factor on the expression of caspase-12 of nerve cells in cerebral ischemia/reperfusion area

    Institute of Scientific and Technical Information of China (English)

    Jiping Yang; Huaijun Liu; Ying Li; Yan Liu; Haiqing Yang

    2006-01-01

    BACKGROUND: Researches suggest that cascade reaction of cysteine protease mediated by caspase-12 can cause apoptosis after cerebral ischemia/reperfusion injury;however, nerve growth factor (NGF) can reduce apoptosis through inhibiting activation of that reaction.OBJECTTVE: To observe the effect of NGF on the expression of caspase-12 in brain tissue of rabbits with cerebral ischemia/reperfusion injury, and elucidate the protective mechanism of NGF on neural apoptosis induced by cerebral ischemia/reperfusion injury.DESIGN: Randomized controlled animal study.SETTING: Department of Image, Second Hospital, Hebei Medical University.MATERIALS: A total of 26 healthy New Zealand rabbits, of clean grade, aged 4.5-5 months, weighing (2.6±0.2) kg, were selected in this study. Reagents: NGF (Xiamen Beida Zhilu Biotechnology Co., Ltd.);caspase-12 (Santa Cruz Biotechnology Company, USA, clone number: SC-12395); caspase-3 (Santa Cruz Biotechnology Company, USA, clone number: SC-7272); biotin-antibody Ⅱ and ABC compound (Wuhan Boster Company); in situ end-labeling (ISEL, Beijing Zhongshan Company).METHODS: The experiment was carried out in the Laboratories of Nerve Molecule Image Science and Neurology of the Second Hospital of Hebei Medical University from May to August 2005. ① All animals were randomly divided into three groups. Ischemia/reperfusion (I/R) group (n=10): Left middle cerebral artery (MCA) was blocked for 2 hours and then blooded for 2 hours in order to establish focal cerebral ischemia/reperfusion models. Sham operation group (n=6): Cork was inserted with 3 cm in depth, and then pulled to common carotid artery. Other procedures were as the same as those in ischemia/reperfusion group.Treatment group (n=10): After modeling, 400 AU (16 μg/L) NGF was inserted into cerebral infarction focus sham operation group and at 3 days after reperfusion in other two groups. In addition, contents of caspase-12 and caspase-3 were measured with immunohistochemical technique; mean

  14. Induction of Caspase 8 by 5-Azacytidine renders NB cells sensitive to TRAIL%5氮杂胞苷诱导NB细胞Caspase 8表达及对TRAIL敏感性的研究

    Institute of Scientific and Technical Information of China (English)

    佟海侠; 张锦华; 张继红; 陆春伟

    2005-01-01

    目的:探讨5氮杂胞苷(5-Azacytidine)对肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor related apoptosis inducing ligand,TRAIL)诱导神经母细胞瘤(neuroblastoma,NB)细胞株SH-SY5Y凋亡的影响及其发生机制.方法:应用RT-PCR方法检测5氮杂胞苷作用前后SY5Y细胞Caspase 8的表达;应用四甲基偶氮唑蓝(MTT)比色法及流式细胞仪(FCM)检测TRAIL、5氮杂胞苷+TRAIL、5氮杂胞苷+Caspase 8抑制剂+TRAIL对SY5Y细胞生长及凋亡的影响.结果:SY5Y细胞不表达Caspase 8,5氮杂胞苷作用1、3和5d的SY5Y细胞Caspase 8表达逐渐增加;SY5Y细胞对TRAIL不敏感,经5氮杂胞苷诱导表达Caspase 8的 SY5Y 细胞对TRAIL敏感,并呈现一定的时间和剂量依赖性.联合用药组细胞凋亡率分别为(28.6±4.0)%、(38.5±4.2)%和(42.3±6.2)%,与单独用TRAIL组[(9.5±2.0)%、(10.7±1.8)%和(11.2±1.5)%]比较,差异有统计学意义,F值分别为41.131、45.014和49.405,P值均为0.000;Caspase 8抑制剂组细胞凋亡率分别为(11.8±2.0)%、(13.5±2.9)%和(15.1±3.4)%,与相应浓度的5氮杂胞苷+TRAIL组相比,细胞凋亡率明显降低,F值分别为41.131、45.014和49.405,P值均为0.000,说明Caspase 8抑制剂可明显降低TRAIL对表达Caspase 8的SY5Y细胞的杀伤作用.结论:5氮杂胞苷能增强SY5Y细胞对TRAIL的敏感性,其发生机制可能与Caspase 8表达上调有关.

  15. Effects of Treadmill Running on the mRNA Expression of Caspase Dependent and Independent Apoptotic Genes in Mice with Sarcopenia%跑台运动对Sarcopeniadx鼠Caspase依赖与非依赖性细胞凋亡基因mRNA表达的影响

    Institute of Scientific and Technical Information of China (English)

    李海鹏; 杨东升; 王立丰; 关尚一; 丁树哲; 卢健

    2011-01-01

    forced to nm on treadmill with the frequency of 5 times per week for 8 weeks. The animals in each group were executed 24 to 48 hours after the last running, and sarcopenia Index (SI) was calculated by the ratio of gastrocnemius muscle mass (mg) to body weight (g). Real-time PCR was used to detect the changes in apoptotic genes, such as Cyt C, apaf-l, caspase-9, caspase-3, PARP, AIF and Endo G in each group. Results ( 1 ) The SI value of gastrocnemius in group OC was significantly lower than that in group YC, while no significant difference were observed between group OE and group OC.(2) Comparing to group YC, the expression of apaf-1, caspase-3 and PARP mRNA increased significantly, the expression of AIF mRNA decreased significantly, whereas Cyt C, caspase-9 and Endo G mRNA remained unchanged in group OC. (3) As compared to group OC, the expression of Cyt C and AIF mRNA increased significantly, and the expressions of caspase-3 and PARP mRNA decreased significantly with no difference in f apaf- 1, caspase-9 and Endo G mRNA in group OE. Conclusion As compared to caspase independent apoptotic signaling pathway, caspase dependent apoptotic signaling pathway plays more important role in the development of sarcopenia. Treadmill running exercise can attenuate the inclination of the apoptosis to some extent mediated by caspase dependent pathway in the senile mice, while the AIF induced caspase independent mRNA expression reveals up regulation.

  16. Caspase-1-like regulation of the proPO-system and role of ppA and caspase-1-like cleaved peptides from proPO in innate immunity.

    Science.gov (United States)

    Jearaphunt, Miti; Noonin, Chadanat; Jiravanichpaisal, Pikul; Nakamura, Seiko; Tassanakajon, Anchalee; Söderhäll, Irene; Söderhäll, Kenneth

    2014-04-01

    Invertebrates rely on innate immunity to respond to the entry of foreign microorganisms. One of the important innate immune responses in arthropods is the activation of prophenoloxidase (proPO) by a proteolytic cascade finalized by the proPO-activating enzyme (ppA), which leads to melanization and the elimination of pathogens. Proteolytic cascades play a crucial role in innate immune reactions because they can be triggered more quickly than immune responses that require altered gene expression. Caspases are intracellular proteases involved in tightly regulated limited proteolysis of downstream processes and are also involved in inflammatory responses to infections for example by activation of interleukin 1ß. Here we show for the first time a link between caspase cleavage of proPO and release of this protein and the biological function of these fragments in response to bacterial infection in crayfish. Different fragments from the cleavage of proPO were studied to determine their roles in bacterial clearance and antimicrobial activity. These fragments include proPO-ppA, the N-terminal part of proPO cleaved by ppA, and proPO-casp1 and proPO-casp2, the fragments from the N-terminus after cleavage by caspase-1. The recombinant proteins corresponding to all three of these peptide fragments exhibited bacterial clearance activity in vivo, and proPO-ppA had antimicrobial activity, as evidenced by a drastic decrease in the number of Escherichia coli in vitro. The bacteria incubated with the proPO-ppA fragment were agglutinated and their cell morphology was altered. Our findings show an evolutionary conserved role for caspase cleavage in inflammation, and for the first time show a link between caspase induced inflammation and melanization. Further we give a more detailed understanding of how the proPO system is regulated in time and place and a role for the peptide generated by activation of proPO as well as for the peptides resulting from Caspase 1 proteolysis.

  17. Caspase-1-like regulation of the proPO-system and role of ppA and caspase-1-like cleaved peptides from proPO in innate immunity.

    Directory of Open Access Journals (Sweden)

    Miti Jearaphunt

    2014-04-01

    Full Text Available Invertebrates rely on innate immunity to respond to the entry of foreign microorganisms. One of the important innate immune responses in arthropods is the activation of prophenoloxidase (proPO by a proteolytic cascade finalized by the proPO-activating enzyme (ppA, which leads to melanization and the elimination of pathogens. Proteolytic cascades play a crucial role in innate immune reactions because they can be triggered more quickly than immune responses that require altered gene expression. Caspases are intracellular proteases involved in tightly regulated limited proteolysis of downstream processes and are also involved in inflammatory responses to infections for example by activation of interleukin 1ß. Here we show for the first time a link between caspase cleavage of proPO and release of this protein and the biological function of these fragments in response to bacterial infection in crayfish. Different fragments from the cleavage of proPO were studied to determine their roles in bacterial clearance and antimicrobial activity. These fragments include proPO-ppA, the N-terminal part of proPO cleaved by ppA, and proPO-casp1 and proPO-casp2, the fragments from the N-terminus after cleavage by caspase-1. The recombinant proteins corresponding to all three of these peptide fragments exhibited bacterial clearance activity in vivo, and proPO-ppA had antimicrobial activity, as evidenced by a drastic decrease in the number of Escherichia coli in vitro. The bacteria incubated with the proPO-ppA fragment were agglutinated and their cell morphology was altered. Our findings show an evolutionary conserved role for caspase cleavage in inflammation, and for the first time show a link between caspase induced inflammation and melanization. Further we give a more detailed understanding of how the proPO system is regulated in time and place and a role for the peptide generated by activation of proPO as well as for the peptides resulting from Caspase 1

  18. 5氮杂胞苷联合干扰素-γ上调神经母细胞瘤细胞Caspase 8表达的研究%5-Azacytidine and IFN-gamma cooperate to upregulate Caspase-8 expression in neuroblastoma cells

    Institute of Scientific and Technical Information of China (English)

    佟海侠; 陆春伟; 王秋实; 王立维

    2011-01-01

    Objective : To investigate if induction of Caspase 8 by demethylation agent 5 - Azacytidine( 5AZA ) and/or γ - interferon ( IFNγ ) renders neuroblastoma ( NB ) cells sensitive to tumor necrosis factor related apoptosis inducing ligand( TRAIL ). Methods: Caspase 8 expression was monitored by Western blot analysis. The effects of 5AZA,IFNγ, TRAIL, 5AZA and/or IFNγ +TRAIL, 5AZA and/or IFNγ + Caspase 8 inhibitor + TRAIL on the growth and apoptosis of NB cells were detected with flow cytometry. The relative Caspase 8 activity was measured with colorimetric assay. Results : Caspase 8 was undetectable in SH - SY5Y( SY5Y ) cells which were resistant to TRAIL , but an increased expression of Caspase 8 was found after treatment with 5AZA and/or IFNγ. The level of Caspase 8 protein in IFNγ +5AZA group was higher than that in single treatment group. Moreover, TRAIL combined with 5AZA and/or IFNγ induced apoptosis in SY5Y cells. The relative Caspase 8 activity of SY5Y cells in SAZA and/or IFNγ + TRAIL group was significantly higher than those of control group, 5AZA group, IFNγ group, TRAIL group and Caspase 8 inhibitor group. Conclusion :5 - AZA and IFNγ could cooperate to upregulate Caspase - 8 expression in NB cells. TRAIL induced apoptosis in NB cells expressing Caspase 8 with an increase of relative Caspase 8 activity.%目的:应用去甲基药5氮杂胞苷(5AZA)和/或干扰素(IFNγ)诱导神经母细胞瘤(neuroblastoma,NB)细胞Caspase 8的表达,并观察是否可以恢复NB细胞对肿瘤坏死因子相关凋亡诱导配体(TRAIL)的敏感性.方法:应用Western blot方法检测5AZA和/或IFNγ作用前后SY5Y细胞Caspase 8蛋白的表达;应用流式细胞术检测5AZA 、IFNγ、TRAIL、5AZA 和/或IFNγ+TRAIL、5AZA和/或IFNγ+Caspase 8抑制剂zIETD-FMK+TRAIL对NB细胞生长及凋亡的影响;应用比色法测定Caspase 8相对活性.结果:对TRAIL耐药的SY5Y细胞不表达Caspase 8,经5AZA 和/或IFNγ处理后Caspase 8 蛋白

  19. Ceramide induces release of mitochondrial proapoptotic proteins in caspase-dependent and -independent manner in HT-29 cells

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    In this study, the release of mitochondrial proapoptotic intermembrane space proteins induced by exogenous C2-ceramide in human colon carcinoma (HT-29) cell line was investigated. HT-29 cells were treated with 12.5, 25 and 50 μmol/L C2-ceramide in vitro. Flow cytometer was used to detect the mitochondrial membrane potential (△Ψm). Subcellular fractions were extracted by Mitochondrial/Cytosol Fractionation Kit after C2-ceramide treatment for 24 h. SDS-PAGE was used to determine the level of cytochrome c (Cyt c), high temperature requirement A2 (HtrA2) and second mitochondrial-derived activator of caspases (Smac) released from mitochondria, the expression of X-linked inhibitor of apoptosis protein (XIAP) and caspase-3 for 24 h. The results showed that △Ψm began to decrease from 6 h after 25 and 50 μmol/L C2-ceramide treatment (P<0.05) and cyclosporin A (CsA) could inhibit the collapse of △Ψm through regulating mitochondrial membrane permeability transition pore. There was no effect of C2-ceramide on the expression of Cyt c, HtrA2 and Smac in the total levels. 12.5, 25 and 50 μmol/L C2-ceramide could induce Cyt c, HtrA2 and Smac to release from mitochondria to cytosol and down-regulate the expression of XIAP (P<0.05). Also there was expression of cleaved caspase-3 with C2-ceramide treatment. After the treatment with caspase inhibitor, C2-ceramide still induced the release of Cyt c and HtrA2, but Smac did not. Therefore, C2-ceramide could induce apoptosis of HT-29 cells through the mitochondria pathway. The release of Cyt c, HtrA2 and Smac from mitochondria did not occur via the same mechanism, the release of Cyt c and HtrA2 was caspase-independent and the release of Smac was caspase-dependent.

  20. Ceramide induces release of mitochondrial proapop-totic proteins in caspase-dependent and -independent manner in HT-29 cells

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    In this study, the release of mitochondrial proapoptotic intermembrane space proteins induced by ex-ogenous C2-ceramide in human colon carcinoma (HT-29) cell line was investigated. HT-29 cells were treated with 12.5, 25 and 50 μmol/L C2-ceramide in vitro. Flow cytometer was used to detect the mito-chondrial membrane potential (ΔΨm). Subcellular fractions were extracted by Mitochondrial/Cytosol Fractionation Kit after C2-ceramide treatment for 24 h. SDS-PAGE was used to determine the level of cytochrome c (Cyt c), high temperature requirement A2 (HtrA2) and second mitochondrial-derived ac-tivator of caspases (Smac) released from mitochondria, the expression of X-linked inhibitor of apop-tosis protein (XIAP) and caspase-3 for 24 h. The results showed that ΔΨm began to decrease from 6 h after 25 and 50 μmol/L C2-ceramide treatment (P<0.05) and cyclosporin A (CsA) could inhibit the col-lapse of ΔΨm through regulating mitochondrial membrane permeability transition pore. There was no effect of C2-ceramide on the expression of Cyt c, HtrA2 and Smac in the total levels. 12.5, 25 and 50 μmol/L C2-ceramide could induce Cyt c, HtrA2 and Smac to release from mitochondria to cytosol and down-regulate the expression of XIAP (P<0.05). Also there was expression of cleaved caspase-3 with C2-ceramide treatment. After the treatment with caspase inhibitor, C2-ceramide still induced the release of Cyt c and HtrA2, but Smac did not. Therefore, C2-ceramide could induce apoptosis of HT-29 cells through the mitochondria pathway. The release of Cyt c, HtrA2 and Smac from mitochondria did not occur via the same mechanism, the release of Cyt c and HtrA2 was caspase-independent and the re-lease of Smac was caspase-dependent.

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

    International Nuclear Information System (INIS)

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

  2. Proton pump inhibitors induce a caspase-independent antitumor effect against human multiple myeloma.

    Science.gov (United States)

    Canitano, Andrea; Iessi, Elisabetta; Spugnini, Enrico Pierluigi; Federici, Cristina; Fais, Stefano

    2016-07-01

    Multiple Myeloma (MM) is the second most common hematological malignancy and is responsive to a limited number of drugs. Unfortunately, to date, despite the introduction of novel drugs, no relevant increase in survival rates has been obtained. Proton pump inhibitors (PPIs) have been shown to have significant antitumor action as single agents as well as in combination with chemotherapy. This study investigates the potential anti-tumor effectiveness of two PPIs, Lansoprazole and Omeprazole, against human MM cells. We found that Lansoprazole exerts straightforward efficacy against myeloma cells, even at suboptimal concentrations (50 µM), while Omeprazole has limited cytotoxic action. The Lansoprazole anti-MM effect was mostly mediated by a caspase-independent apoptotic-like cytotoxicity, with only a secondary anti-proliferative action. This study provides clear evidence supporting the use of Lansoprazole in the strive against MM with an efficacy proven much higher than current therapeutical approaches and without reported side effects. It is however conceivable that, consistent with the results obtained in other human tumors, Lansoprazole may well be combined with existing anti-myeloma therapies with the aim to improve the low level of efficacy of the current strategies. PMID:27084522

  3. Reduced expression of PNUTS leads to activation of Rb-phosphatase and caspase-mediated apoptosis.

    Science.gov (United States)

    De Leon, Gabriel; Sherry, Tara C; Krucher, Nancy A

    2008-06-01

    There is abundant evidence that Retinoblastoma (Rb) activity is important in the control of cell proliferation and apoptosis. Reversible phosphorylation of the Rb protein that is carried out by cyclin dependent kinases and Protein phosphatase 1 (PP1) regulates its functions. A PP1 interacting protein, PNUTS (Phosphatase Nuclear Targeting Subunit) is proposed to be a regulator of Rb phosphorylation. In this study, PNUTS knockdown in MCF7, SKA and HCT116 cancer cells causes a reduction in viability due to increased apoptosis. However, normal cells (MCF10A breast and CCD-18Co colon) do not exhibit reduced viability when PNUTS expression is diminished. PNUTS knockdown has no effect in Rb-null Saos-2 cells. However, when Rb is stably expressed in Saos-2 cells, PNUTS knockdown reduces cell number. Knockdown of PNUTS in p53-/- HCT116 cells indicates that p53 is dispensable for the induction of apoptosis. Loss of PNUTS expression results in increased Rb-phosphatase activity and Rb dephosphorylation. E2F1 dissociates from Rb in cells depleted of PNUTS and the resulting apoptosis is dependent on caspase-8. These results indicate that Rb phosphorylation state can be manipulated by targeting Rb phosphatase activity and suggest that PNUTS may be a potential target for therapeutic pro-apoptotic strategies. PMID:18360108

  4. PLGA-carbon nanotube conjugates for intercellular delivery of caspase-3 into osteosarcoma cells.

    Directory of Open Access Journals (Sweden)

    Qingsu Cheng

    Full Text Available Cancer has arisen to be of the most prominent health care issues across the world in recent years. Doctors have used physiological intervention as well as chemical and radioactive therapeutics to treat cancer thus far. As an alternative to current methods, gene delivery systems with high efficiency, specificity, and safety that can reduce side effects such as necrosis of tissue are under development. Although viral vectors are highly efficient, concerns have arisen from the fact that viral vectors are sourced from lethal diseases. With this in mind, rod shaped nano-materials such as carbon nanotubes (CNTs have become an attractive option for drug delivery due to the enhanced permeability and retention effect in tumors as well as the ability to penetrate the cell membrane. Here, we successfully engineered poly (lactic-co-glycolic (PLGA functionalized CNTs to reduce toxicity concerns, provide attachment sites for pro-apoptotic protein caspase-3 (CP3, and tune the temporal release profile of CP3 within bone cancer cells. Our results showed that CP3 was able to attach to functionalized CNTs, forming CNT-PLGA-CP3 conjugates. We show this conjugate can efficiently transduce cells at dosages as low as 0.05 μg/ml and suppress cell proliferation up to a week with no further treatments. These results are essential to showing the capabilities of PLGA functionalized CNTs as a non-viral vector gene delivery technique to tune cell fate.

  5. Dual role of the caspase enzymes in satellite cells from aged and young subjects.

    Science.gov (United States)

    Fulle, S; Sancilio, S; Mancinelli, R; Gatta, V; Di Pietro, R

    2013-01-01

    Satellite cell (SC) proliferation and differentiation have critical roles in skeletal muscle recovery after injury and adaptation in response to hypertrophic stimuli. Normal ageing hinders SC proliferation and differentiation, and is associated with increased expression of a number of pro-apoptotic factors in skeletal muscle. In light of previous studies that have demonstrated age-related altered expression of genes involved in SC antioxidant and repair activity, this investigation was aimed at evaluating the incidence of apoptotic features in human SCs. Primary cells were obtained from vastus lateralis of nine young (27.3±2.0 years old) and nine old (71.1±1.8 years old) subjects, and cultured in complete medium for analyses at 4, 24, 48, and 72 h. Apoptosis was assessed using AnnexinV/propidium iodide staining, the terminal deoxynucleotidyl transferase dUTP nick-end labelling technique, RT-PCR, DNA microarrays, flow cytometry, and immunofluorescence analysis. There was an increased rate of apoptotic cells in aged subjects at all of the experimental time points, with no direct correlation between AnnexinV-positive cells and caspase-8 activity. On the other hand, CASP2, CASP6, CASP7, and CASP9 and a number of cell death genes were upregulated in the aged SCs. Altogether, our data show age-related enhanced susceptibility of human SCs to apoptosis, which might be responsible for their reduced response to muscle damage. PMID:24336075

  6. Expression of Caspase-3 in Dentate Gyrus of Adult Mice with Chronic Arsenic Poisoning%慢性砷中毒对小鼠齿状回caspase-3表达的影响

    Institute of Scientific and Technical Information of China (English)

    孙宝飞; 康朝胜; 余资江; 李玉飞

    2011-01-01

    目的 观察慢性砷中毒对成年小鼠齿状回神经元的形态学影响,探讨慢性砷中毒对成年小鼠脑部的神经毒性机制.方法 选取健康成年昆明小鼠80只,雌雄各半,分为对照组、高、中、低剂量砷染毒组,每组20只,高、中、低剂量砷染毒组分别以As03的1/5、1/10、1/40 LD50(9、4.5、1.1 mg/kg)灌胃染毒,对照组以蒸馏水灌胃,连续3个月.利用免疫组织化学和蛋白印迹技术观察小鼠齿状回部位神经元半胱氨酸蛋白水解酶-3(caspase-3)蛋白的表达.结果 免疫组化染色显示,与正常对照组比较,砷染毒组小鼠齿状回caspase-3阳性细胞明显增多(P<0.01),阳性反应产物平均光密度增高(P<0.01),同时蛋白印迹结果显示随砷中毒剂量的增加,小鼠齿状回caspase-3蛋白含量随之增加(P<0.01),各剂量组雌雄间各数据差异无统计学意义(P>0.05).结论 慢性砷中毒导致脑齿状回神经元细胞凋亡可能与齿状回细胞caspase-3增加有关,同时脑细胞caspase-3随砷浓度增加而增高.%Objective To investigate the effects of chronic arsenic exposure at different doses on dentate gyrus neurons in adult mice. Methods Eighty healthy adult Kunming mice, 20-22 g, were randomly divided into four groups: normal control group, low-dose group, moderate dose group and high dose group, 20 in each (10 males and 10 females in each group), each group was fed respectively with distilled water, 1/5 LD50, 1/10 LD50 and 1/40 LD50 As203 for 3 consecutive months, and adjusting the dose according to their weight changes. The content of arsenic in brain was determined. The expression of caspase-3 in dentate gyrus neurons was detected by western blotting and immunohistochemistry and analyzed by morphology methods. Results Compared with normal control group, groups of arsenic poisoning had been the main changes: caspase-3 immunohistochemical staining positive cells increased significantly (P0.05). Conclusion The obvious up

  7. 吗啡成瘾时脑内Fas、Bcl-2和Caspase-3蛋白表达的改变%Changes of Fas, Bcl-2 and Caspase-3 protein in rat brain during morphine addiction

    Institute of Scientific and Technical Information of China (English)

    刘立伟; 王新华; 傅舒昆; 吴青华; 傅强

    2012-01-01

    目的 观察吗啡成瘾时脑细胞中凋亡相关蛋白Fas、Caspase-3和Bcl-2表达的改变.方法 将48只体质量为190~210 g的成年SD大鼠随机分为3组:吗啡依赖组、吗啡戒断组和对照组,每组16只.依据药物递增原则,依赖组和戒断组大鼠腹腔内给予吗啡13d,建立吗啡成瘾模型.戒断组大鼠在成瘾后腹腔内注射纳洛酮5 mg/kg,诱导戒断30 min.对照组大鼠在相同的治疗时间腹腔内注射生理盐水.应用免疫组织化学、蛋白印迹分析方法检测大鼠海马区Fas、Bcl-2和Caspase-3蛋白的表达.结果 与对照组比较,吗啡依赖组和戒断组大鼠海马区Fas和Caspase-3的表达增加(P<0.01),而Bcl-2的表达降低(P<0.01).结论 长期应用吗啡可通过Fas、Caspase-3表达的增加和Bcl-2表达的降低诱发脑细胞异常凋亡,这可能是阿片类药物引起神经损害的机制之一.%Objective To investigate the changes of apoptosis-related proteins Fas,Caspase-3 and Bcl-2 expression in rat brain during morphine addiction. Methods A total of 48 adult male Sprague-Dawley rats, weighing 190-210 g, were randomly divided into 3 groups (n=16): chronic morphine-dependent group, chronic morphine-abstinent group and control group. The rats in dependent group and abstinent group were chronically treated with morphine for 13 days to establish morphine dependent model. In the abstinent group, the withdrawal syndromes were induced with intraperitoneal injection of naloxone 5 mg/kg for 30 min. The control group was injected with normal saline. Immunohistochemistry and Western blotting analysis were used to examine the expression of Fas, Bcl-2 and Caspase-3 proteins. Results Compared with the control group, the other two groups had significantly increased expression of Fas and Caspase-3 (P

  8. Harmol induces apoptosis by caspase-8 activation independently of Fas/Fas ligand interaction in human lung carcinoma H596 cells.

    Science.gov (United States)

    Abe, Akihisa; Yamada, Hiroyuki

    2009-06-01

    The beta-carboline alkaloids are naturally existing plant substances. It is known that these alkaloids have a wide spectrum of neuropharmacological, psychopharmacological, and antitumor effects. Therefore, they have been traditionally used in oriental medicine for the treatment of various diseases including cancers and malaria. In this study, harmol and harmalol, which are beta-carboline alkaloids, were examined for their antitumor effect on human lung carcinoma cell lines, and structure-activity relationship was also investigated. H596, H226, and A549 cells were treated with harmol and harmalol, respectively. Apoptosis was induced by harmol only in H596 cells. In contrast, harmalol had negligible cytotoxicity in three cell lines. Harmol induced caspase-3, caspase-8, and caspase-9 activities and caspase-3 activities accompanied by cleavage of poly-(ADP-ribose)-polymerase. Furthermore, harmol treatment decreased the native Bid protein, and induced the release of cytochrome c from mitochondria to cytosol. The apoptosis induced by harmol was completely inhibited by caspase-8 inhibitor and partially inhibited by caspase-9 inhibitor. The antagonistic antibody ZB4 blocked Fas ligand-induced apoptosis, but had no effect on harmol-induced apoptosis. Harmol had no significant effect on the expression of Fas. In conclusion, our results showed that the harmol could cause apoptosis-inducing effects in human lung H596 cells through caspase-8-dependent pathway but independent of Fas/Fas ligand interaction. PMID:19318910

  9. Oridonin induces apoptosis of HeLa cells via altering expres sion of Bcl-2/Bax and activating caspase-3/ICAD pathway

    Institute of Scientific and Technical Information of China (English)

    Chun-ling ZHANG; Li-jun WU; Shin-ichi TASHIRO; Satoshi ONODERA; Takashi IKEJIMA

    2004-01-01

    AIM: To study the mechanisms by which oridonin inhibited HeLa cell growth in vitro. METHODS: Viability of oridonin-induced HeLa cells was measured by MTT assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis.Caspase activity was assayed using fiuorometric protease assay. ICAD, Bcl-2, and Bax proteins expression were detected by Western blot analysis. RESULTS: Oridonin induced oligonucleosomal fragmentation of DNA and increased caspase-3 activity, on the other hand, reduced the expression of inhibitor of caspase-3-activated DNase (ICAD), a caspase-3 substrate, at 12 h in HeLa cells. Oridonin-induced DNA fragmentation, caspase-3 activation and down-regulation of ICAD expression were effectively inhibited by a caspase-3 inhibitor, z-DEVD-fmk (z-AspGlu-Val-Asp-fmk). However, pretreatment with an inhibitor of poly (ADP-ribose) polymerase (PARP), 3, 4-dihydro5-[4-(1-piperidinyl)butoxy]-1 (2H)-isoquinolinone (DPQ), did not suppress oridonin-induced HeLa cell death. In addition, oridonin-induced apoptosis was associated with an increase in the expression of the apoptosis inducer Bax, and a significant reduction in expression of the apoptosis suppressor Bcl-2 in mitochondria. CONCLUSION:Oridonin induces HeLa cells apoptosis by altering balance of Bcl-2 and Bax protein expression and activation of caspase-3/ICAD pathway.

  10. 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.

  11. Caspase-3、DIAPH-3蛋白在浸润性乳腺癌中的作用研究%Role of caspase-3 and DIAPH3 in invasive breast cancer

    Institute of Scientific and Technical Information of China (English)

    张玲玲; 张亚男; 耿翠芝; 丁妍; 杨会钗; 马力; 刘月平

    2016-01-01

    目的:探讨浸润性乳腺癌组织中Caspase-3、DIAPH-3蛋白的表达与临床病理特征及预后的关系。方法采用免疫组织化学EliVision法检测Caspase-3、DIAPH-3在浸润性乳腺癌(=240)及癌旁组织(=44)中的表达情况,并分析其与临床病理特征及预后的关系。结果 Caspase-3、DIAPH-3在浸润性乳腺癌组织中表达的阳性率(36.7%和40.8%)低于癌旁组织(81.8%和86.7%),<0.05。Caspase-3的阳性表达率随淋巴结转移数的增多、肿瘤长径的增加、临床分期的提高、脉管瘤栓的产生及P53的阳性表达的增强而降低。DIAPH-3的表达随淋巴结转移数的增加、脉管瘤栓的产生及临床分期的提高而降低。Kaplan- Meier单因素生存分析显示Caspase-3和DIAPH-3蛋白阳性组患者的5年生存率(95.5%、95.8%),高于阴性组(85.5%、84.5%)。COX多因素生存分析显示DIAPH-3阴性表达与患者不良预后正相关(O?=1.687,95%CI:1.032,2.758)。结论浸润性乳腺癌组织中Caspase-3、DIAPH-3蛋白表达均低于癌旁正常乳腺组织,两者均与乳腺癌的进展及预后密切相关,但两者之间无关联;DIAPH-3的低表达提示患者预后不良,可能是预测乳腺癌患者预后的独立因素之一。%Objective To study the possible association of the expressions of caspase-3 and diaphanous related formin 3 (DIAPH3) protein with clinicopathological features and prognosis in invasive breast cancer. Methods The expressions of caspase-3 and DIAPH3 protein in invasive breast cancer tissues from 240 cases and para-cancerous tissues from 44 cases were detected by immunohistochemical EliVision method, and then their relationships with the clinicopathological features and prognosis were analyzed. Results The positive-expression rates of caspase-3 and DIAPH3 protein in the invasive breast cancer tissues (36.7% and 40.8%respectively) were significantly lower than those in the para-cancerous tissues

  12. TRAIL, DR5 and caspase 3-dependent apoptosis in vessels of diseased human temporomandibular joint disc. An immunohistochemical study

    Directory of Open Access Journals (Sweden)

    C. Loreto

    2010-09-01

    Full Text Available To evaluate the apoptosis involvement in the angiogenesis as a self-limiting process in patients with temporomandibular joint (TMJ degenerated disc vessels, we assessed, by immunohistochemistry, the detection of TRAIL, its death receptor DR5 and caspase 3. TRAIL, its death receptor DR5 and caspase 3 expression were studied by immunohistochemistry in 15 TMJ discs displaced without reduction and in 4 unaffected discs. These apoptosis molecules were detected in the intima and media layers of newly formed vessels affected discs. In conclusion, vessels apoptosis activation in TMJ disc with ID could be regarded as a self-limiting process that try to leads to vessel regression; in this way an inhibition of angiogenic vessels may prove a key strategy in limiting pathological angiogenesis, by cutting off blood supply to tumors, or by reducing harmful inflammation.

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

    Science.gov (United States)

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

    2016-01-01

    In the present study, the apoptotic effect of pycnogenol and its molecular mechanism in human oral squamous cell carcinoma HSC-3 cells were investigated. Pycnogenol significantly inhibited the viability of HSC-3 cells and suppressed neoplastic cell transformation in HSC-3 cells and TPA-treated JB6 cells. It caused caspase-dependent apoptosis evidenced by the increase in cleaved poly (ADP-ribose) polymerase and caspase 3 in a dose-dependent manner. Pycnogenol increased Bak protein by enhancing its protein stability whereas other Bcl-2 family members were not altered. In addition, the treatment with pycnogenol led to the production of reactive oxygen species and N-acetyl-l-cysteine almost blocked pycnogenol-induced reactive oxygen species generation. Taken together, these findings suggest that pycnogenol may be a potential candidate for the chemoprevention or chemotherapy of human oral cancer. PMID:26798196

  14. 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

    2016-01-01

    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.

  15. QSAR Analysis for Some 1, 2-Benzisothiazol-3-one Derivatives as Caspase-3 Inhibitors by Stepwise MLR Method.

    Science.gov (United States)

    Hajimahdi, Zahra; Safizadeh, Fatemeh; Zarghi, Afshin

    2016-01-01

    Caspase-3 inhibitory activities of some 1, 2-benzisothiazol-3-one derivatives were modeled by quantitative structure-activity relationship (QSAR) using stepwise-multiple linear regression (SW-MLR) method. The built model was robust and predictive with correlation coefficient (R(2)) of 0.91 and 0.59 for training and test groups, respectively. The quality of the model was evaluated by leave-one out (LOO) cross validation (LOO correlation coefficient, Q(2)) of 0.80). The results indicate that the descriptors related to the electronegativity, the atomic masses, the atomic van der Waals volumes and R--CX--R Atom-centered fragments play a more significant role in caspase-3 inhibitory activity. PMID:27642314

  16. Caspase-cleaved cytokeratin 18 fragment (M30) as marker of postoperative residual tumor load in colon cancer patients

    OpenAIRE

    Ausch, C.; Buxhofer-Ausch, V; Olszewski, U.; Hinterberger, W.; Ogris, E.; Schiessel, R.; Hamilton, G.

    2009-01-01

    Abstract Background Soluble cytokeratin 18 (CK18; M65) and a caspase-cleaved fragment of CK18 (M30) have been used as biomarkers, corresponding to tumor cell death and apoptosis respectively. Methods In the present study, M30 was quantified for the first time in serum samples of colon cancer patients pre- and postoperatively as well as during chemotherapy. Minimal residual disease (MRD) was assessed preoperatively by detection of pan-cytokeratin ant...

  17. Analysis of macrophage apoptosis induced by Brucella melitensis and the effects of caspases 3,8 and 9

    Institute of Scientific and Technical Information of China (English)

    任晓莉

    2013-01-01

    Objective To determine the difference of macrophage RAW264.7 apoptosis induced by Brucella melitensis virulent strain 16M and attenuated strain M5-90 and elucidate the regulatory role of caspases 3,8 and 9.Methods The best multiplicity of infection (MOI) was determined through kinetic analysis of Brucella melitensis strain 16M and M5-90 induced mouse macrophages apop-

  18. Cytotoxic L-amino-acid oxidases from Amanita phalloides and Clitocybe geotropa induce caspase-dependent apoptosis.

    Science.gov (United States)

    Pišlar, A; Sabotič, J; Šlenc, J; Brzin, J; Kos, J

    2016-01-01

    L-amino-acid oxidases (LAO) purified from fungi induce cell death in various mammalian cells including human tumor cell lines. The mechanism, however, remains poorly understood. In this study, we aimed to define a precise mechanism of cell death induced in Jurkat and MCF7 cancer cell lines by ApLAO and CgLAO, LAOs isolated from Amanita phalloides and Clitocybe geotropa, respectively. Cell death induced by both LAOs is shown to be concentration- and time-dependent, with higher toxic effects in Jurkat cells. LAO activity is required for the cytotoxicity. Detailed study on Jurkat cells further demonstrated that ApLAO and CgLAO both induce the intrinsic mitochondrial pathway of apoptosis, accompanied by a time-dependent depolarization of the mitochondrial membrane through the generation of reactive oxygen species. Treatment with the LAOs resulted in an increased ratio of the expression of proapoptotic Bax to that of antiapoptotic Bcl-2, subsequently leading to the activation of caspase-9 and -3. However, the pancaspase inhibitor, Z-VAD-FMK, did not completely abolish the cell death induced by either ApLAO or CgLAO, suggesting an alternative pathway for LAO-induced apoptosis. Indeed, caspase-8 activity in ApLAO- and CgLAO-treated cells was increased. Further, Fas/FasL (Fas ligand) antagonist caused a slight reduction in toxin-induced cell death, supporting the involvement of ApLAO and CgLAO in death-receptor-mediated apoptosis. These results thus provide new evidence that ApLAO and CgLAO induce apoptosis in Jurkat cells via both the intrinsic and extrinsic pathways, although the significantly higher increase of caspase-9 over caspase-8 activity suggests that it is the intrinsic pathway that is the predominant mode of ApLAO- and CgLAO-induced apoptosis. PMID:27551514

  19. Cleavage of caspases-1, -3, -6, -8 and -9 substrates by proteases in skeletal muscles from mice undergoing cancer cachexia

    OpenAIRE

    Belizário, J E; Lorite, M. J.; Tisdale, M J

    2001-01-01

    A prominent feature of several type of cancer is cachexia. This syndrome causes a marked loss of lean body mass and muscle wasting, and appears to be mediated by cytokines and tumour products. There are several proteases and proteolytic pathways that could be responsible for the protein breakdown. In the present study, we investigated whether caspases are involved in the proteolytic process of skeletal muscle catabolism observed in a murine model of cancer cachexia (MAC16), in comparison with...

  20. Thimerosal induces DNA breaks, caspase-3 activation, membrane damage, and cell death in cultured human neurons and fibroblasts.

    Science.gov (United States)

    Baskin, David S; Ngo, Hop; Didenko, Vladimir V

    2003-08-01

    Thimerosal is an organic mercurial compound used as a preservative in biomedical preparations. Little is known about the reactions of human neuronal and skin cells to its micro- and nanomolar concentrations, which can occur after using thimerosal-containing products. A useful combination of fluorescent techniques for the assessment of thimerosal toxicity is introduced. Short-term thimerosal toxicity was investigated in cultured human cerebral cortical neurons and in normal human fibroblasts. Cells were incubated with 125-nM to 250-microM concentrations of thimerosal for 45 min to 24 h. A 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) dye exclusion test was used to identify nonviable cells and terminal transferase-based nick-end labeling (TUNEL) to label DNA damage. Detection of active caspase-3 was performed in live cell cultures using a cell-permeable fluorescent caspase inhibitor. The morphology of fluorescently labeled nuclei was analyzed. After 6 h of incubation, the thimerosal toxicity was observed at 2 microM based on the manual detection of the fluorescent attached cells and at a 1-microM level with the more sensitive GENios Plus Multi-Detection Microplate Reader with Enhanced Fluorescence. The lower limit did not change after 24 h of incubation. Cortical neurons demonstrated higher sensitivity to thimerosal compared to fibroblasts. The first sign of toxicity was an increase in membrane permeability to DAPI after 2 h of incubation with 250 microM thimerosal. A 6-h incubation resulted in failure to exclude DAPI, generation of DNA breaks, caspase-3 activation, and development of morphological signs of apoptosis. We demonstrate that thimerosal in micromolar concentrations rapidly induce membrane and DNA damage and initiate caspase-3-dependent apoptosis in human neurons and fibroblasts. We conclude that a proposed combination of fluorescent techniques can be useful in analyzing the toxicity of thimerosal.

  1. Mitochondrial calcium uniporter silencing potentiates caspase-independent cell death in MDA-MB-231 breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Curry, Merril C.; Peters, Amelia A. [School of Pharmacy, The University of Queensland, Brisbane, Queensland 4072 (Australia); Kenny, Paraic A. [Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Roberts-Thomson, Sarah J. [School of Pharmacy, The University of Queensland, Brisbane, Queensland 4072 (Australia); Monteith, Gregory R., E-mail: gregm@uq.edu.au [School of Pharmacy, The University of Queensland, Brisbane, Queensland 4072 (Australia)

    2013-05-10

    Highlights: •Some clinical breast cancers are associated with MCU overexpression. •MCU silencing did not alter cell death initiated with the Bcl-2 inhibitor ABT-263. •MCU silencing potentiated caspase-independent cell death initiated by ionomycin. •MCU silencing promoted ionomycin-mediated cell death without changes in bulk Ca{sup 2+}. -- Abstract: The mitochondrial calcium uniporter (MCU) transports free ionic Ca{sup 2+} into the mitochondrial matrix. We assessed MCU expression in clinical breast cancer samples using microarray analysis and the consequences of MCU silencing in a breast cancer cell line. Our results indicate that estrogen receptor negative and basal-like breast cancers are characterized by elevated levels of MCU. Silencing of MCU expression in the basal-like MDA-MB-231 breast cancer cell line produced no change in proliferation or cell viability. However, distinct consequences of MCU silencing were seen on cell death pathways. Caspase-dependent cell death initiated by the Bcl-2 inhibitor ABT-263 was not altered by MCU silencing; whereas caspase-independent cell death induced by the calcium ionophore ionomycin was potentiated by MCU silencing. Measurement of cytosolic Ca{sup 2+} levels showed that the promotion of ionomycin-induced cell death by MCU silencing occurs independently of changes in bulk cytosolic Ca{sup 2+} levels. This study demonstrates that MCU overexpression is a feature of some breast cancers and that MCU overexpression may offer a survival advantage against some cell death pathways. MCU inhibitors may be a strategy to increase the effectiveness of therapies that act through the induction of caspase-independent cell death pathways in estrogen receptor negative and basal-like breast cancers.

  2. Effect of protein kinase C alpha, caspase-3, and survivin on apoptosis of oral cancer cells induced by staurosporine

    Institute of Scientific and Technical Information of China (English)

    Yu-xia ZHANG; Shi-bin YU; Jing-ping OU-YANG; Dong XIA; Min WANG; Jin-rong LI

    2005-01-01

    Aim: To elucidate inhibition of protein kinase C α (PKC α) activity by staurosporine on apoptosis of oral cancer cell line tongue squamous cell carcinoma (TSCCa)cells and to clarify the role of survivin and caspase-3 in mediating apoptosis.Methods: TSCCa cell viability was measured by MTT assay after 100 nmol/L staurosporine treatment. Apoptotic cells were identified by using phase contrast microscopy, acridine orange/ethidium bromide staining, and flow cytometry. Level of PKC α and its subcellular location were investigated using Western blot analysis.Expression of survivin and caspase-3 were evaluated using immunocytochemistry.Results: Staurosporine significantly inhibited the cell viability of TSCCa cells in a dose- and time-dependent manner. Marked cell accumulation in G2/M phase was observed after 100 nmol/L staurosporine exposure for 6 h and 12 h. In addition,the percentage of apoptosis increased in a time-dependent manner, from 2.9% in control cultures to approximately 27.4% at 100 nmol/L staurosporine treatment for24 h. Staurosporine displayed difference in inhibitory efficacy between cytosolic and membrance-derived PKC α. The content of PKCα in membrane versus cytosol decreased quickly, from 0.45 in ethanol-treated control cultures to 0.18 after staurosporine exposure for 24 h (P<0.01). After treatment withstaurosporine, a time-dependent reduction of survivin and an activation of caspase-3 were observed in TSCCa cells. Conclusion: Staurosporine inhibited cell viability and promoted apoptosis in TSCCa cells. Inhibition of PKCα activity might be a potential mechanism for staurosporine to induce apoptosis in this cell line. The cleavage of survivin and activation of caspase-3 signaling pathway might contribute to PKC α inhibition-induced apoptosis.

  3. BARF1 gene silencing triggers caspase-dependent mitochondrial apoptosis in Epstein-Barr virus-positive malignant cells

    Indian Academy of Sciences (India)

    Taznim Begam Mohd Mohidin; Ching Ching Ng

    2015-03-01

    Epstein-Barr virus (EBV)-encoded BARF1 (BamH1-A Rightward Frame-1) is expressed in EBV-positive malignancies such as nasopharyngeal carcinoma, EBV-associated gastric cancer, B-cell lymphoma and nasal NK/T-cell lymphoma, and has been shown to have an important role in oncogenesis. However, the mechanism by which BARF1 elicits its biological effects is unclear. We investigated the effects of BARF1 silencing on cell proliferation and apoptosis in EBV-positive malignant cells. We observed that BARF1 silencing significantly inhibits cell proliferation and induces apoptosis-mediated cell death by collapsing the mitochondrial membrane potential in AG876 and Hone-Akata cells. BARF1 knockdown up-regulates the expression of pro-apoptotic proteins and down-regulates the expression of anti-apoptotic proteins. In BARF1-down-regulated cells, the Bcl-2/BAX ratio is decreased. The caspase inhibitor z-VAD-fmk was found to rescue siBARF1-induced apoptosis in these cells. Immunoblot analysis showed significant increased levels of cleaved caspase 3 and caspase 9. We observed a significant increase in cytochrome c level as well as the formation of apoptosome complex in BARF1-silenced cells. In conclusion, siRNA-mediated BARF1 down-regulation induces caspase-dependent apoptosis via the mitochondrial pathway through modulation of Bcl-2/BAX ratio in AG876 and Hone-Akata cells. Targeting BARF1 using siRNA has the potential to be developed as a novel therapeutic strategy in the treatment of EBV-associated malignancies.

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

    International Nuclear Information System (INIS)

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

  5. Pathological cyclic strain-induced apoptosis in human periodontal ligament cells through the RhoGDIα/caspase-3/PARP pathway.

    Directory of Open Access Journals (Sweden)

    Li Wang

    Full Text Available AIM: Human periodontal ligament (PDL cells incur changes in morphology and express proteins in response to cyclic strain. However, it is not clear whether cyclic strain, especially excessive cyclic strain, induces PDL cell apoptosis and if so, what mechanism(s are responsible. The aim of the present study was to elucidate the molecular mechanisms by which pathological levels of cyclic strain induce human PDL cell apoptosis. MATERIALS AND METHODS: Human PDL cells were obtained from healthy premolar tissue. After three to five passages in culture, the cells were subjected to 20% cyclic strain at a frequency of 0.1 Hz for 6 or 24 h using an FX-5000T system. Morphological changes of the cells were assessed by inverted phase-contrast microscopy, and apoptosis was detected by fluorescein isothiocyanate (FITC-conjugated annexin V and propidium iodide staining followed by flow cytometry. Protein expression was evaluated by Western blot analysis. RESULTS: The number of apoptotic human PDL cells increased in a time-dependent manner in response to pathological cyclic strain. The stretched cells were oriented parallel to each another with their long axes perpendicular to the strain force vector. Cleaved caspase-3 and poly-ADP-ribose polymerase (PARP protein levels increased in response to pathological cyclic strain over time, while Rho GDP dissociation inhibitor alpha (RhoGDIα decreased. Furthermore, knock-down of RhoGDIα by targeted siRNA transfection increased stretch-induced apoptosis and upregulated cleaved caspase-3 and PARP protein levels. Inhibition of caspase-3 prevented stretch-induced apoptosis, but did not change RhoGDIα protein levels. CONCLUSION: The overall results suggest that pathological-level cyclic strain not only influenced morphology but also induced apoptosis in human PDL cells through the RhoGDIα/caspase-3/PARP pathway. Our findings provide novel insight into the mechanism of apoptosis induced by pathological cyclic strain in

  6. 完全睡眠剥夺对大鼠caspase-3表达的影响%Effect of total sleep deprivation on expression of caspase-3 in cortex and hippocampus

    Institute of Scientific and Technical Information of China (English)

    先雄斌; 刘军祥; 先德海; 郑宇杰

    2008-01-01

    目的:探讨完全睡眠剥夺对大鼠caspase-3的影响及其导致记忆、认知等功能障碍的机制.方法:取Sprague-Dawley大鼠35只,随机分为正常对照组、大平台对照组和完全睡眠剥夺组.其中正常对照组(NC)5只大鼠,正常笼养;大平台对照组(TC)只有72、96及120 h 3个时间点,每个时间点5只大鼠;完全睡眠剥夺组(TSD)也有72、96及120 h 3个时间点,每个时间点5只大鼠.采用小平台水环境法建立完全睡眠剥夺模型,在不同的时点处死大鼠后运用免疫组化SP法检测各组大鼠海马和皮质caspase-3的分布规律和动态变化.结果:caspase-3在各组的海马和皮质均有表达,随着睡眠剥夺时间的延长,caspase-3的表达逐渐增加,与正常对照组、大平台对照组差异有显著性(P<0.01).结论:完全睡眠剥夺能引起大鼠海马和皮质caspase-3的表达增加.并随睡眠剥夺时间的延长而渐趋明显.可能是完全睡眠剥夺引起大鼠神经元调亡,进而引起大鼠记忆、认知等功能障碍的机制之一.

  7. Fatal Hepatitis Mediated by TNFα Requires Caspase-8 and Involves the BH3-only Proteins Bid and Bim

    Science.gov (United States)

    Kaufmann, Thomas; Jost, Philipp J; Pellegrini, Marc; Puthalakath, Hamsa; Gugasyan, Raffi; Gerondakis, Steve; Cretney, Erika; Smyth, Mark J; Silke, John; Hakem, Razq; Bouillet, Philippe; Mak, Tak W; Dixit, Vishva M; Strasser, Andreas

    2009-01-01

    SUMMARY Apoptotic death of hepatocytes, a feature and contributing factor of many chronic and acute liver diseases, can be a consequence of over-activation of the immune system. Injection with lipopolysaccharide (LPS) plus the transcriptional inhibitor D(+)-galactosamine (GalN) or mitogenic T cell activation cause fatal hepatocyte apoptosis in mice. In both settings hepatocyte killing is mediated by TNFα/TNF-R signaling but the effector mechanisms remain unclear. Our analysis of gene-targeted mice showed that caspase-8 is essential for hepatocyte killing in both settings. Loss of Bid, the pro-apoptotic BH3-only protein activated by caspase-8, and essential for Fas ligand-induced hepatocyte killing, resulted only in a minor reduction of liver damage. However, combined loss of Bid and another BH3-only protein, Bim, activated by JNK, protected mice from LPS+GalN-induced hepatitis. These observations identify caspase-8 and the BH3-only proteins Bid and Bim as potential therapeutic targets for treatment of inflammatory liver diseases. PMID:19119023

  8. 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.

  9. FG020326 Sensitized Multidrug Resistant Cancer Cells to Docetaxel-Mediated Apoptosis via Enhancement of Caspases Activation

    Directory of Open Access Journals (Sweden)

    Li-Wu Fu

    2012-05-01

    Full Text Available Apoptotic resistance is the main obstacle for treating cancer patients with chemotherapeutic drugs. Multidrug resistance (MDR is often characterized by the expression of P-glycoprotein (P-gp, a 170-KD ATP-dependent drug efflux protein. Functional P-gp can confer resistance to activate caspase-8 and -3 dependent apoptosis induced by a range of different stimuli, including tumor necrosis and chemotherapeutic drugs such as docetaxel and vincristine. We demonstrated here that comparison of sensitive KB cells, P-gp positive (P-gp+ve KBv200 cells were extremely resistant to apoptosis induced by docetaxel. FG020326, a pharmacological inhibitor of P-gp function, could enhance concentration-dependently the effect of docetaxel on cell apoptosis and sensitize caspase-8, -9 and -3 activation in P-gp overexpressing KBv200 cells, but not in KB cells. Therefore, the enhancement of caspase-8, -9 and -3 activation induced by docetaxel may be one of the key mechanisms of the reversal of P-gp mediated docetaxel resistance by FG020326.

  10. NPM-RAR binding to TRADD selectively inhibits caspase activation, while allowing activation of NFκB and JNK.

    Science.gov (United States)

    Chattopadhyay, Anuja; Abecassis, Irina; Redner, Robert L

    2015-01-01

    The t(5;17) variant of acute promeylocytic leukemia (APL) expresses a fusion of nucleophosmin (NPM) with the retinoic acid receptor alpha (RARA). We have previously shown that NPM-RAR is a binding partner of the tumor necrosis factor (TNF) receptor type-I-associated DEATH domain protein, TRADD. Binding of TNF to its receptor, TNF-R, induces recruitment of TRADD, and subsequent recruitment of a cascade of proteins that ultimate activate caspase 3, nuclear factor κB (NFκB) and c-Jun N-terminal kinase (JNK). We have previously shown that NPM-RAR interaction with TRADD blocks TNF activation of caspase 3, caspase 8, poly(ADP-ribose) polymerase (PARP) cleavage and, ultimately, apoptosis. We now report that NPM-RAR expression is permissive for TNF activation of NFκB and JNK. We propose that inhibition of TNF activation of apoptosis, while preserving TNF activation of NFκB and JNK pathways that stimulate cell growth and survival, represents a novel mechanism through which NPM-RAR contributes to development of the leukemic phenotype.

  11. Quercetin induces apoptosis by activating caspase-3 and regulating Bcl-2 and cyclooxygenase-2 pathways in human HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    Guomin Niu; Songmei Yin; Shuangfeng Xie; Yiqing Li; Danian Nie; Liping Ma; Xiuju Wang; Yudan Wu

    2011-01-01

    Quercetin is one of the naturally occurring dietary flavo-nol compounds. It is present abundantly in plants and has chemopreventive and anticancer effects. To investigate its anticancer mechanism, we examined the activity of quercetin against acute leukemia cell line, HL-60. Our results showed that quercetin inhibited cell proliferation and induced apoptosis in a time- and dose-dependent manner. Furthermore, quercetin down-regulated the expression of anti-apoptosis protein Bcl-2 and up-regulated the expression of pro-apoptosis protein Bax. Caspase-3 was also activated by quercetin, which started a caspase-3-depended mitochodrial pathway to induce apoptosis. It was also found that quercetin inhibited the expression of the cycloocygenase-2 (Cox-2) mRNA and Cox-2 protein. Taken together, these findings suggested that quercetin induces apoptosis in a caspase-3-dependent pathway by inhibiting Cox-2 expression and regulates the expression of downstream apoptotic components, including Bcl-2 and Bax. Quercetin can be a potent and promising medicine which might be safely used in leukemia therapy.

  12. 苯并芘对人角质形成细胞Caspase-14和Filaggrin表达的影响

    Institute of Scientific and Technical Information of China (English)

    张芳芳; 车雅敏; 刘原君; 王晶晶; 刘全忠

    2013-01-01

    目的:研究苯并芘(BaP)对永生化人角质形成细胞(HaCaT)中的Caspase-14和Filaggrin mRNA表达的影响,探讨其与BaP所致的细胞损伤的关系.方法:体外培养HaCaT;噻唑蓝法(MTT)检测BaP对HaCaT增殖活性的影响;反转录-聚合酶链反应(RT-PCR)检测不同浓度的BaP对HaCaT中Caspase-14和Filaggrin mRNA表达的影响.结果:不同浓度的BaP对HaCaT细胞体外增殖均有抑制作用,随其浓度增加对细胞的抑制更为明显,且存在量效关系;BaP对Caspase-14和Filaggrin mRNA表达起到明显的上调作用,成浓度依赖性,差异有统计学意义(P<0.01).结论:BaP可以引起皮肤屏障功能的损伤.

  13. Butylphthalide Suppresses Neuronal Cells Apoptosis and Inhibits JNK-Caspase3 Signaling Pathway After Brain Ischemia /Reperfusion in Rats.

    Science.gov (United States)

    Wen, Xiang-Ru; Tang, Man; Qi, Da-Shi; Huang, Xiao-Jing; Liu, Hong-Zhi; Zhang, Fang; Wu, Jian; Wang, Yi-Wen; Zhang, Xun-Bao; Guo, Ji-Qiang; Wang, Shu-Ling; Liu, Yong; Wang, Yu-Lan; Song, Yuan-Jian

    2016-10-01

    Although Butylphthalide (BP) has protective effects that reduce ischemia-induced brain damage and neuronal cell death, little is known about the precise mechanisms occurring during cerebral ischemia/reperfusion (I/R). Therefore, the aim of this study was to investigate the neuroprotective mechanisms of BP against ischemic brain injury induced by cerebral I/R through inhibition of the c-Jun N-terminal kinase (JNK)-Caspase3 signaling pathway. BP in distilled non-genetically modified Soybean oil was administered intragastrically three times a day at a dosage of 15 mg/(kg day) beginning at 20 min after I/R in Sprague-Dawley rats. Immunohistochemical staining and Western blotting were performed to examine the expression of related proteins, and TUNEL-staining was used to detect the percentage of neuronal apoptosis in the hippocampal CA1 region. The results showed that BP could significantly protect neurons against cerebral I/R-induced damage. Furthermore, the expression of p-JNK, p-Bcl2, p-c-Jun, FasL, and cleaved-caspase3 was also decreased in the rats treated with BP. In summary, our results imply that BP could remarkably improve the survival of CA1 pyramidal neurons in I/R-induced brain injury and inhibit the JNK-Caspase3 signaling pathway. PMID:27015680

  14. Bcl-2/caspase 3 mucosal imbalance favors T cell resistance to apoptosis in dogs with inflammatory bowel disease.

    Science.gov (United States)

    Jergens, A; Young, J; Moore, D; Wang, C; Hostetter, J; Augustine, L; Allenspach, K; Schmitz, S; Mosher, C

    2014-04-15

    Canine idiopathic inflammatory bowel disease (IBD) is believed to result from complex interplay between genetic, microbial, and immunologic factors. Abnormal cell death by apoptosis may result in the persistence of activated intestinal T cells that contribute to mucosal inflammation and clinical severity. To test this hypothesis, we investigated the mucosal expression of pro- and anti-apoptotic proteins in different intestinal compartments and their association with inflammatory indices in dogs with IBD. Apoptosis of lamina propria (LP) T cells in duodenal, ileal, and colonic tissues in control and IBD dogs was analyzed by caspase 3/Bcl-2 immunohistochemistry and TUNEL assays. Densities and distributions of LP caspase 3 and Bcl-2 cells were correlated to histopathologic lesions and the clinical activity index (CIBDAI). Compared to control tissues, IBD dogs had significantly (Pdogs, there were significantly greater numbers of Bcl-2 cells at the apical and basilar villus in the duodenum as compared to the colon and to the apical and basilar villus in the ileum (Pdogs compared with controls (Pdogs and the CIBDAI (Pdogs with IBD. Mucosal imbalance of Bcl-2/caspase 3 expression favors T cell resistance to apoptosis which may contribute to T cell accumulation and chronic intestinal inflammation, similar to human IBD.

  15. Tolerance of Mice to Lipopolysaccharide is Correlated with Inhibition of Caspase-3-mediated Apoptosis in Mouse Liver Cells

    Institute of Scientific and Technical Information of China (English)

    Jie LUAN; Bingrong ZHOU; Hui DING; Zhongtian QI

    2007-01-01

    Bacterial endotoxin lipopolysaccharide (LPS) often results in multiple organ failure. However,pre-exposure of mice to a sublethal dose of LPS renders the animal tolerant to a lethal dose of LPS. This study was designed to determine whether pre-exposure of a small dose of LPS was able to suppress apoptosis in mice when challenged with LPS in combination with D-galactosamine, and to investigate the expression changes of the apoptosis-associated molecules. The results showed that a characteristic apoptotic DNA fragmentation existed in mouse livers of the LPS-naive group, but not in control groups; and the mice of the LPS-naive group were all dead after 2 d. However, in the LPS-tolerance groups, both the lethal rate and apoptotic DNA fragmentation were suppressed after the mice were challenged with LPS/D-galactosamine,and the protection against the lethality and apoptotic reaction could be maintained for up to 7 d. In this period, significantly lower levels of caspase-3 and its mRNA appeared in LPS-tolerant groups compared to those of the LPS-naive group (P<0.05), and the caspase-3 activities gradually recovered as the observation was prolonged. Our findings suggest that LPS tolerance could suppress apoptosis in mouse liver cells, and the expression and activity of caspase-3 could be down-regulated.

  16. 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.

  17. 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.

  18. Induction of caspase 8 and reactive oxygen species by ruthenium-derived anticancer compounds with improved water solubility and cytotoxicity.

    Science.gov (United States)

    Vidimar, Vania; Meng, Xiangjun; Klajner, Marcelina; Licona, Cynthia; Fetzer, Ludivine; Harlepp, Sébastien; Hébraud, Pascal; Sidhoum, Marjorie; Sirlin, Claude; Loeffler, Jean-Philippe; Mellitzer, Georg; Sava, Gianni; Pfeffer, Michel; Gaiddon, Christian

    2012-12-01

    Organometallic compounds which contain metals, such as ruthenium or gold, have been investigated as a replacement for platinum-derived anticancer drugs. They often show good antitumor effects, but the identification of their precise mode of action or their pharmacological optimization is still challenging. We have previously described a class of ruthenium(II) compounds with interesting anticancer properties. In comparison to cisplatin, these molecules have lower side effects, a reduced ability to interact with DNA, and they induce cell death in absence of p53 through CHOP/DDIT3. We have now optimized these molecules by improving their cytotoxicity and their water solubility. In this article, we demonstrate that by changing the ligands around the ruthenium we modify the ability of the compounds to interact with DNA. We show that these optimized molecules reduce tumor growth in different mouse models and retain their ability to induce CHOP/DDIT3. However, they are more potent inducers of cancer cell death and trigger the production of reactive oxygen species and the activation of caspase 8. More importantly, we show that blocking reactive oxygen species production or caspase 8 activity reduces significantly the activity of the compounds. Altogether our data suggest that water-soluble ruthenium(II)-derived compounds represent an interesting class of molecules that, depending on their structures, can target several pro-apoptotic signaling pathways leading to reactive oxygen species production and caspase 8 activation. PMID:22964219

  19. Mitochondrial calcium uniporter silencing potentiates caspase-independent cell death in MDA-MB-231 breast cancer cells.

    Science.gov (United States)

    Curry, Merril C; Peters, Amelia A; Kenny, Paraic A; Roberts-Thomson, Sarah J; Monteith, Gregory R

    2013-05-10

    The mitochondrial calcium uniporter (MCU) transports free ionic Ca(2+) into the mitochondrial matrix. We assessed MCU expression in clinical breast cancer samples using microarray analysis and the consequences of MCU silencing in a breast cancer cell line. Our results indicate that estrogen receptor negative and basal-like breast cancers are characterized by elevated levels of MCU. Silencing of MCU expression in the basal-like MDA-MB-231 breast cancer cell line produced no change in proliferation or cell viability. However, distinct consequences of MCU silencing were seen on cell death pathways. Caspase-dependent cell death initiated by the Bcl-2 inhibitor ABT-263 was not altered by MCU silencing; whereas caspase-independent cell death induced by the calcium ionophore ionomycin was potentiated by MCU silencing. Measurement of cytosolic Ca(2+) levels showed that the promotion of ionomycin-induced cell death by MCU silencing occurs independently of changes in bulk cytosolic Ca(2+) levels. This study demonstrates that MCU overexpression is a feature of some breast cancers and that MCU overexpression may offer a survival advantage against some cell death pathways. MCU inhibitors may be a strategy to increase the effectiveness of therapies that act through the induction of caspase-independent cell death pathways in estrogen receptor negative and basal-like breast cancers.

  20. mTOR inhibition by everolimus in childhood acute lymphoblastic leukemia induces caspase-independent cell death.

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    Rana Baraz

    Full Text Available Increasingly, anti-cancer medications are being reported to induce cell death mechanisms other than apoptosis. Activating alternate death mechanisms introduces the potential to kill cells that have defects in their apoptotic machinery, as is commonly observed in cancer cells, including in hematological malignancies. We, and others, have previously reported that the mTOR inhibitor everolimus has pre-clinical efficacy and induces caspase-independent cell death in acute lymphoblastic leukemia cells. Furthermore, everolimus is currently in clinical trial for acute lymphoblastic leukemia. Here we characterize the death mechanism activated by everolimus in acute lymphoblastic leukemia cells. We find that cell death is caspase-independent and lacks the morphology associated with apoptosis. Although mitochondrial depolarization is an early event, permeabilization of the outer mitochondrial membrane only occurs after cell death has occurred. While morphological and biochemical evidence shows that autophagy is clearly present it is not responsible for the observed cell death. There are a number of features consistent with paraptosis including morphology, caspase-independence, and the requirement for new protein synthesis. However in contrast to some reports of paraptosis, the activation of JNK signaling was not required for everolimus-induced cell death. Overall in acute lymphoblastic leukemia cells everolimus induces a cell death that resembles paraptosis.

  1. Clioquinol inhibits zinc-triggered caspase activation in the hippocampal CA1 region of a global ischemic gerbil model.

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    Tao Wang

    Full Text Available BACKGROUND: Excessive release of chelatable zinc from excitatory synaptic vesicles is involved in the pathogenesis of selective neuronal cell death following transient forebrain ischemia. The present study was designed to examine the neuroprotective effect of a membrane-permeable zinc chelator, clioquinol (CQ, in the CA1 region of the gerbil hippocampus after transient global ischemia. METHODOLOGY/PRINCIPAL FINDINGS: The common carotid arteries were occluded bilaterally, and CQ (10 mg/kg, i.p. was injected into gerbils once a day. The zinc chelating effect of CQ was examined with TSQ fluorescence and autometallography. Neuronal death, the expression levels of caspases and apoptosis inducing factor (AIF were evaluated using TUNEL, in situ hybridization and Western blotting, respectively. We were able to show for the first time that CQ treatment attenuates the ischemia-induced zinc accumulation in the CA1 pyramidal neurons, accompanied by less neuronal loss in the CA1 field of the hippocampus after ischemia. Furthermore, the expression levels of caspase-3, -9, and AIF were significantly decreased in the hippocampus of CQ-treated gerbils. CONCLUSIONS/SIGNIFICANCE: The present study indicates that the neuroprotective effect of CQ is related to downregulation of zinc-triggered caspase activation in the hippocampal CA1 region of gerbils with global ischemia.

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

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    Mihael Vucur

    2013-08-01

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

  3. Inhibition of Corydalis decumbens Alkaloids on Hydrogen Peroxideinduced Apoptosis of PC12 Cells through Down-regulating Caspase-3 Expression

    Institute of Scientific and Technical Information of China (English)

    YAN Ren-jie; YANG Yi-fang; LUO Yong-ming; WU Chun-zhen

    2011-01-01

    Objective To extract alkaloids from Corydalis decumbens (AsCD) by supercritical CO2 fluid extraction (SFE) and to evaluate protective effects of AsCD against hydrogen peroxide (H2O2)-induced apoptosis in rat PC12 cells.Methods AsCD were extracted by SFE and oxidative damage PC12 cells model was induced by H2O2.The survival rate of the cells was determined by MTT assay; Lactate dehydrogenase release was determined by ultraviolet spectrophotometry; Flow cytometry was used to detect apoptosis; Caspase-3 mRNA and protein were determined by real-time PCR and Western blotting assay,respectively.Results AsCD remarkably reduced the cytotoxicity,prevented membrane damage,and inhibited cell apoptosis.AsCD inhibited Caspase-3 mRNA and protein expression induced by H2O2 in PC12 cells.Conclusion AsCD possess protective effects against H2O2-induced apoptosis in PC12 cells,and the mechanism of AsCD responsible to the inhibition of apoptosis is possibly attributed to thedown-regulating Caspase-3 expression.AsCD might be useful in the treatment of oxidative stress-related neurodegenerative diseases.

  4. Cooperative Control of Caspase Recruitment Domain-containing Protein 11 (CARD11) Signaling by an Unusual Array of Redundant Repressive Elements.

    Science.gov (United States)

    Jattani, Rakhi P; Tritapoe, Julia M; Pomerantz, Joel L

    2016-04-15

    Several classes of signaling proteins contain autoinhibitory domains that prevent unwarranted signaling and coordinate the induction of activity in response to external cues. CARD11, a scaffold protein critical for antigen receptor signaling to NF-κB, undergoes autoregulation by a poorly understood inhibitory domain (ID), which keeps CARD11 inactive in the absence of receptor triggering through inhibitory intramolecular interactions. This autoinhibitory strategy makes CARD11 highly susceptible to gain-of-function mutations that are frequently observed in diffuse large B cell lymphoma (DLBCL) and that disrupt ID-mediated autoinhibition, leading to constitutive NF-κB activity, which can promote lymphoma proliferation. Although DLBCL-associated CARD11 mutations in the caspase recruitment domain (CARD), LATCH domain, and coiled coil have been shown to disrupt intramolecular ID binding, surprisingly, no gain-of-function mutations in the ID itself have been reported and validated. In this study, we solve this paradox and report that the CARD11 ID contains an unusual array of four repressive elements that function cooperatively with redundancy to prevent spontaneous NF-κB activation. Our quantitative analysis suggests that potent oncogenic CARD11 mutations must perturb autoinhibition by at least three repressive elements. Our results explain the lack of ID mutations in DLBCL and reveal an unusual autoinhibitory domain structure and strategy for preventing unwarranted scaffold signaling to NF-κB.

  5. Radiation-Inducible Caspase-8 Gene Therapy for Malignant Brain Tumors

    International Nuclear Information System (INIS)

    Purpose: Patients with malignant gliomas have a poor prognosis. To explore a novel and more effective approach for the treatment of patients with malignant gliomas, we designed a strategy that combines caspase-8 (CSP8) gene therapy and radiation treatment (RT). In addition, the specificity of the combined therapy was investigated to decrease the unpleasant effects experienced by the surrounding normal tissue. Methods and Materials: We constructed the plasmid pEGR-green fluorescence protein that included the radiation-inducible early growth response gene-1 (Egr-1) promoter and evaluated its characteristics. The pEGR-CSP8 was constructed and included the Egr-1 promoter and CSP8 complementary DNA. Assays that evaluated the apoptosis inducibility and cytotoxicity caused by CSP8 gene therapy combined with RT were performed using U251 and U87 glioma cells. The pEGR-CSP8 was transfected into the subcutaneous U251 glioma cells of nude mice by means of in vivo electroporation. The in vivo effects of CSP8 gene therapy combined with RT were evaluated. Results: The Egr-1 promoter yielded a better response with fractionated RT than with single-dose RT. In the assay of apoptosis inducibility and cytotoxicity, pEGR-CSP8 showed response for RT. The pEGR-CSP8 combined with RT is capable of inducing cell death effectively. In mice treated with pEGR-CSP8 and RT, apoptotic cells were detected in pathologic sections, and a significant difference was observed in tumor volumes. Conclusions: Our results indicate that radiation-inducible gene therapy may have great potential because this can be spatially or temporally controlled by exogenous RT and is safe and specific

  6. HDAC1 inactivation induces mitotic defect and caspase-independent autophagic cell death in liver cancer.

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    Hong Jian Xie

    Full Text Available Histone deacetylases (HDACs are known to play a central role in the regulation of several cellular properties interlinked with the development and progression of cancer. Recently, HDAC1 has been reported to be overexpressed in hepatocellular carcinoma (HCC, but its biological roles in hepatocarcinogenesis remain to be elucidated. In this study, we demonstrated overexpression of HDAC1 in a subset of human HCCs and liver cancer cell lines. HDAC1 inactivation resulted in regression of tumor cell growth and activation of caspase-independent autophagic cell death, via LC3B-II activation pathway in Hep3B cells. In cell cycle regulation, HDAC1 inactivation selectively induced both p21(WAF1/Cip1 and p27(Kip1 expressions, and simultaneously suppressed the expression of cyclin D1 and CDK2. Consequently, HDAC1 inactivation led to the hypophosphorylation of pRb in G1/S transition, and thereby inactivated E2F/DP1 transcription activity. In addition, we demonstrated that HDAC1 suppresses p2