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Sample records for caspase-8s increased sensitivity

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

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    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.

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

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    D. M. Heijink

    2007-01-01

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

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

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

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

    2004-01-01

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

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

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

    2017-10-15

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

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

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

    2009-08-17

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

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

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    Flavell Richard A

    2003-02-01

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

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

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    Ainhoa Mielgo

    2009-11-01

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

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Angela M Keuling

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

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

    Science.gov (United States)

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

    2016-07-01

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

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

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    Sebastian Horn

    2017-04-01

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

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

    NARCIS (Netherlands)

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

    2013-01-01

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

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

    NARCIS (Netherlands)

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

    2011-01-01

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

  16. 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

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    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.

  17. 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.

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

    International Nuclear Information System (INIS)

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

    2014-01-01

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

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

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

    2003-11-14

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

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

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    Vagner, Tatyana; Mouravlev, Alexandre; Young, Deborah

    2015-01-01

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

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

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    Bram Laukens

    2011-10-01

    Full Text Available Searching for new strategies to bypass apoptosis resistance, we investigated the potential of the Smac mimetic BV6 in Jurkat leukemia cells deficient in key molecules of the death receptor pathway. Here, we demonstrate for the first time that Smac mimetic primes apoptosis-resistant, FADD- or caspase-8-deficient leukemia cells for TNFα-induced necroptosis in a synergistic manner. In contrast to TNFα, Smac mimetic significantly enhances CD95-induced apoptosis in wild-type but not in FADD-deficient cells. Interestingly, Smac mimetic- and TNFα-mediated cell death occurs without characteristic features of apoptosis (i.e., caspase activation, DNA fragmentation in FADD-deficient cells. By comparison, Smac mimetic and TNFα trigger activation of caspase-8, -9, and -3 and DNA fragmentation in wild-type cells. Consistently, the caspase inhibitor zVAD.fmk fails to block Smac mimetic- and TNFα-triggered cell death in FADD- or caspase-8-deficient cells, while it confers protection in wild-type cells. By comparison, necrostatin-1, an RIP1 kinase inhibitor, abolishes Smac mimetic- and TNFα-induced cell death in FADD- or caspase-8-deficient. Thus, Smac mimetic enhances TNFα-induced cell death in leukemia cells via two distinct pathways in a context-dependent manner: it primes apoptosis-resistant cells lacking FADD or caspase-8 to TNFα-induced, RIP1-dependent and caspase-independent necroptosis, whereas it sensitizes apoptosis-proficient cells to TNFα-mediated, caspase-dependent apoptosis. These findings have important implications for the therapeutic exploitation of necroptosis as an alternative cell death program to overcome apoptosis resistance.

  2. Histone deacetylase inhibitors strongly sensitise neuroblastoma cells to TRAIL-induced apoptosis by a caspases-dependent increase of the pro- to anti-apoptotic proteins ratio

    International Nuclear Information System (INIS)

    Mühlethaler-Mottet, Annick; Flahaut, Marjorie; Bourloud, Katia Balmas; Auderset, Katya; Meier, Roland; Joseph, Jean-Marc; Gross, Nicole

    2006-01-01

    Neuroblastoma (NB) is the second most common solid childhood tumour, an aggressive disease for which new therapeutic strategies are strongly needed. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in most tumour cells, but not in normal tissues and therefore represents a valuable candidate in apoptosis-inducing therapies. Caspase-8 is silenced in a subset of highly malignant NB cells, which results in full TRAIL resistance. In addition, despite constitutive caspase-8 expression, or its possible restoration by different strategies, NB cells remain weakly sensitive to TRAIL indicating a need to develop strategies to sensitise NB cells to TRAIL. Histone deacetylase inhibitors (HDACIs) are a new class of anti-cancer agent inducing apoptosis or cell cycle arrest in tumour cells with very low toxicity toward normal cells. Although HDACIs were recently shown to increase death induced by TRAIL in weakly TRAIL-sensitive tumour cells, the precise involved sensitisation mechanisms have not been fully identified. NB cell lines were treated with various doses of HDACIs and TRAIL, then cytotoxicity was analysed by MTS/PMS proliferation assays, apoptosis was measured by the Propidium staining method, caspases activity by colorimetric protease assays, and (in)activation of apoptotic proteins by immunoblotting. Sub-toxic doses of HDACIs strongly sensitised caspase-8 positive NB cell lines to TRAIL induced apoptosis in a caspases dependent manner. Combined treatments increased the activation of caspases and Bid, and the inactivation of the anti-apoptotic proteins XIAP, Bcl-x, RIP, and survivin, thereby increasing the pro- to anti-apoptotic protein ratio. It also enhanced the activation of the mitochondrial pathway. Interestingly, the kinetics of caspases activation and inactivation of anti-apoptotic proteins is accelerated by combined treatment with TRAIL and HDACIs compared to TRAIL alone. In contrast, cell surface expression of TRAIL

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

    Directory of Open Access Journals (Sweden)

    Katsuya Kominami

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

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

    Science.gov (United States)

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

    2015-03-01

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

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

    Science.gov (United States)

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

    2016-09-01

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

  6. 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.

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

    Science.gov (United States)

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

    2015-03-12

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

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

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    Xiao-Xi Guo

    2012-11-01

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

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

    Science.gov (United States)

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

    2013-08-01

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

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

    Science.gov (United States)

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

    2016-11-01

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

  11. Caspase-8 acts as a key upstream executor of mitochondria during justicidin A-induced apoptosis in human hepatoma cells.

    Science.gov (United States)

    Su, Chun-Li; Huang, Lynn L H; Huang, Li-Min; Lee, Jenq-Chang; Lin, Chun-Nan; Won, Shen-Jeu

    2006-05-29

    Justicia procumbens is a traditional Taiwanese herbal remedy used to treat fever, pain, and cancer. Justicidin A, isolated from Justicia procumbens, has been reported to suppress in vitro growth of several tumor cell lines as well as hepatoma cells. In this study, justicidin A activated caspase-8 to increase tBid, disrupted mitochondrial membrane potential (Delta psi(m)), and caused the release of cytochrome c and Smac/DIABLO in Hep 3B and Hep G2 cells. Justicidin A also reduced Bcl-x(L) and increased Bax and Bak in mitochondria. Caspase-8 inhibitor (Z-IETD) attenuated the justicidin A-induced disruption of Delta psi(m). Growth of Hep 3B implanted in NOD-SCID mice was suppressed significantly by oral justicidin A (20 mg/kg/day). These results indicate that justicidin A-induced apoptosis in these cells proceeds via caspase-8 and is followed by mitochondrial disruption.

  12. 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.

  13. [Effect of Recombinant Adenovirus AdE-SH2-Caspase 8 on the Apoptosis of Imatinib-resistant K562/G01 Cell Line].

    Science.gov (United States)

    Wang, Lin; Fei, Chang; Huang, Zheng-Lan; Li, Hui; Liu, Zhang-Lin; Feng, Wen-Li

    2015-08-01

    To investigate the effect of SH2-Caspase 8 fusion protein expressed by recombinant adenovirus AdE-SH2-Caspase8-HA-GFP (SC) on the apoptosis of K562/G01 cell line, which is a BCR/ABL positive chronic myeloid leukemia cell line and resistant to imatinib. The K562/G01 cell line was infected with AdE-SH2-Caspase 8-HA-GFP adenovirus (SC), then the cells were divided into 3 groups: AdE-SH2m-Caspase 8-HA-GFP (SmC) group, AdE-GFP (CMV) group and PBS group as control. The infection efficiency was observed under fluorescent microscopy and by flow cytometry. The expression of fusion protein SH2-Caspase 8-HA was measured by Western blot. The morphology of the cells detected by Wright's staining. The apoptosis of the cells were detected by flow cytometry and DNA ladder. The expression of Caspase 3 and PARP were detected by Western blot. The infection efficiency of SC on K562/G01 cells was high which was confirmed by fluorescent microscopy and FCM. SH2-Caspase 8-HA fusion protein were expressed correctly in K562/G01 cells. After treatment with SC the apoptosis of K562/G01 cells could be observed by microscopy. The result of FCM showed that early apoptosis of K562/G01 cells increased significantly as compared with control groups (P SH2-Caspase 8 fusion protein can induces the apoptosis of K562/G01 cells.

  14. Suppressed translation as a mechanism of initiation of CASP8 (caspase 8)-dependent apoptosis in autophagy-deficient NSCLC cells under nutrient limitation.

    Science.gov (United States)

    Allavena, Giulia; Cuomo, Francesca; Baumgartner, Georg; Bele, Tadeja; Sellgren, Alexander Yarar; Oo, Kyaw Soe; Johnson, Kaylee; Gogvadze, Vladimir; Zhivotovsky, Boris; Kaminskyy, Vitaliy O

    2018-01-01

    Macroautophagy/autophagy inhibition under stress conditions is often associated with increased cell death. We found that under nutrient limitation, activation of CASP8/caspase-8 was significantly increased in autophagy-deficient lung cancer cells, which precedes mitochondria outer membrane permeabilization (MOMP), CYCS/cytochrome c release, and activation of CASP9/caspase-9, indicating that under such conditions the activation of CASP8 is a primary event in the initiation of apoptosis as well as essential to reduce clonogenic survival of autophagy-deficient cells. Starvation leads to suppression of CFLAR proteosynthesis and accumulation of CASP8 in SQSTM1 puncta. Overexpression of CFLARs reduces CASP8 activation and apoptosis during starvation, while its silencing promotes efficient activation of CASP8 and apoptosis in autophagy-deficient U1810 lung cancer cells even under nutrient-rich conditions. Similar to starvation, inhibition of protein translation leads to efficient activation of CASP8 and cell death in autophagy-deficient lung cancer cells. Thus, here for the first time we report that suppressed translation leads to activation of CASP8-dependent apoptosis in autophagy-deficient NSCLC cells under conditions of nutrient limitation. Our data suggest that targeting translational machinery can be beneficial for elimination of autophagy-deficient cells via the CASP8-dependent apoptotic pathway.

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

    Science.gov (United States)

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

    2018-05-16

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

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

    Directory of Open Access Journals (Sweden)

    Carole Minker

    2015-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-10-15

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

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

  19. 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...

  20. Apoptose e expressão de Bcl-2 e das caspases 3 e 8 em placenta bovina, em diferentes estádios de gestação Apoptosis and expression of Bcl-2 and caspases 3 and 8 in placenta of cows at different pregnancy stages

    Directory of Open Access Journals (Sweden)

    K.K.O.L. Meça

    2010-04-01

    Full Text Available Apoptose e seus mecanismos reguladores são eventos fisiológicos cruciais para a manutenção da homeostase placentária, e o desequilíbrio desses processos pode comprometer a função placentária e, consequentemente, o sucesso da gravidez. Neste estudo, investigou-se a apoptose utilizando-se histomorfometria em lâminas coradas em HE e submetidas à reação de TUNEL. Além disso, avaliou-se a expressão de Bcl-2 e das caspases 8 e 3, pela reação de polimerase em cadeia em tempo real, em placentas saudáveis em diferentes estádios de gestação. Amostras de placentônios de vacas com quatro, seis e nove meses de gestação foram colhidas e processadas. O índice apoptótico aumentou progressivamente com o avanço da gestação. Tanto o Bcl-2 quanto as caspases 3 e 8 foram expressas nos três períodos estudados, sendo a expressão de Bcl-2 menor que a de caspase 8, que é menor que a de caspase 3. Estes resultados indicam que essas moléculas estão envolvidas na via apoptótica ativada na maturação placentária, exibindo um padrão de expressão ao longo da gestação e contribuindo para o equilíbrio fisiológico da celularidade e renovação celular na placenta bovina.Apoptosis and its regulating mechanisms are crucial physiological events for the maintenance of the placental homostasis; and disequilibrium of these processes may compromise placental function and the success of the pregnancy. In this study, apoptosis was investigated by histomorphometry using slides stained with HE and TUNEL reaction. Besides that, Bcl-2 and caspases 8 and 3 expression were evaluated by real time polymerase chain reaction in healthy placentas under different gestacional ages. Samples of placentones of cows at 4th, 6th, and 9th months of gestation were harvested and processed. The apoptotic index gradually increased with the advance of the gestation. Bcl-2 and caspases 3 and 8 were expressed in all the studied periods, being the expression of Bcl-2

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

    Science.gov (United States)

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

    2018-01-01

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

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

  3. Antioxidants impair anti-tumoral effects of Vorinostat, but not anti-neoplastic effects of Vorinostat and caspase-8 downregulation.

    Science.gov (United States)

    Bergadà, Laura; Yeramian, Andree; Sorolla, Annabel; Matias-Guiu, Xavier; Dolcet, Xavier

    2014-01-01

    We have recently demonstrated that histone deacetylase inhibitor, Vorinostat, applied as a single therapy or in combination with caspase-8 downregulation exhibits high anti-tumoral activity on endometrial carcinoma cell lines. In the present study, we have assessed the signalling processes underlying anti-tumoral effects of Vorinostat. Increasing evidence suggests that reactive oxygen species are responsible for histone deacetylase inhibitor-induced cell killing. We have found that Vorinostat induces formation of reactive oxygen species and DNA damage. To investigate the role of oxidative stress as anti-neoplastic mechanism, we have evaluated the effects of different antioxidants (Bha, Nac and Tiron) on endometrial carcinoma cell line Ishikawa treated with Vorinostat. We show that Bha, Nac and Tiron markedly inhibited the cytotoxic effects of Vorinostat, increasing cell viability in vitro. We found that all three antioxidants did not inhibited accumulation of acetyl Histone H4, so that antioxidants did not inhibit Vorinostat activity. Finally, we have evaluated the effects of antioxidants on anti-tumoral activity of Vorinostat as monotherapy or in combination with caspase-8 downregulation in vivo. Interestingly, antioxidants blocked the reduction of tumour growth caused by Vorinostat, but they were unable to inhibit anti-tumoral activity of Vorinostat plus caspase-8 inhibition.

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

    Science.gov (United States)

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

    2016-05-01

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

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

    International Nuclear Information System (INIS)

    Oh, Seon-Hee; Lee, Byung-Hoon

    2004-01-01

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

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

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

    International Nuclear Information System (INIS)

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

    2009-01-01

    Compound K [20-O-β-(D-glucopyranosyl)-20(S)-protopanaxadiol], a metabolite of the protopanaxadiol-type saponins of Panax ginseng C.A. Meyer, has been reported to possess anti-tumor properties to inhibit angiogenesis and to induce tumor apoptosis. In the present study, we investigated the effect of Compound K on apoptosis and explored the underlying mechanisms involved in HL-60 human leukemia cells. We examined the effect of Compound K on the viabilities of various cancer cell lines using MTT assays. DAPI assay, Annexin V and PI double staining, Western blot assay and immunoprecipitation were used to determine the effect of Compound K on the induction of apoptosis. Compound K was found to inhibit the viability of HL-60 cells in a dose- and time-dependent manner with an IC 50 of 14 μM. Moreover, this cell death had typical features of apoptosis, that is, DNA fragmentation, DNA ladder formation, and the externalization of Annexin V targeted phosphatidylserine residues in HL-60 cells. In addition, compound-K induced a series of intracellular events associated with both the mitochondrial- and death receptor-dependent apoptotic pathways, namely, (1) the activation of caspases-3, -8, and -9; (2) the loss of mitochondrial membrane potential; (3) the release of cytochrome c and Smac/DIABLO to the cytosol; (4) the translocation of Bid and Bax to mitochondria; and (5) the downregulations of Bcl-2 and Bcl-xL. Furthermore, a caspase-8 inhibitor completely abolished caspase-3 activation, Bid cleavage, and subsequent DNA fragmentation by Compound K. Interestingly, the activation of caspase-3 and -8 and DNA fragmentation were significantly prevented in the presence of cycloheximide, suggesting that Compound K-induced apoptosis is dependent on de novo protein synthesis. The results indicate that caspase-8 plays a key role in Compound K-stimulated apoptosis via the activation of caspase-3 directly or indirectly through Bid cleavage, cytochrome c release, and caspase-9 activation

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

    Directory of Open Access Journals (Sweden)

    Choi Jung-Hye

    2009-12-01

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

  9. 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.

  10. G2019S LRRK2 mutant fibroblasts from Parkinson’s disease patients show increased sensitivity to neurotoxin 1-methyl-4-phenylpyridinium dependent of autophagy

    International Nuclear Information System (INIS)

    Yakhine-Diop, Sokhna M.S.; Bravo-San Pedro, José M.; Gómez-Sánchez, Rubén; Pizarro-Estrella, Elisa; Rodríguez-Arribas, Mario; Climent, Vicente; Aiastui, Ana; López de Munain, Adolfo

    2014-01-01

    Parkinson’s disease (PD) is a neurodegenerative disorder of unknown etiology. It is considered as a multifactorial disease dependent on environmental and genetic factors. Deregulation in cell degradation has been related with a significant increase in cell damage, becoming a target for studies on the PD etiology. In the present study, we have characterized the parkinsonian toxin 1-methyl-4-phenylpyridinium ion (MPP + )-induced damage in fibroblasts from Parkinson’s patients with the mutation G2019S in leucine-rich repeat kinase 2 protein (LRRK2) and control individuals without this mutation. The results reveal that MPP + induces mTOR-dependent autophagy in fibroblasts. Moreover, the effects of caspase-dependent cell death to MPP + were higher in cells with the G2019S LRRK2 mutation, which showed basal levels of autophagy due to the G2019S LRRK2 mutation (mTOR-independent). The inhibition of autophagy by 3-methyladenine (3-MA) treatment reduces these sensitivity differences between both cell types, however, the inhibition of autophagosome–lysosome fusion by bafilomycin A1 (Baf A1) increases these differences. This data confirm the importance of the combination of genetic and environmental factors in the PD etiology. Thereby, the sensitivity to the same damage may be different in function of a genetic predisposition, reason why individuals with certain mutations can develop some early-onset diseases, such as individuals with G2019S LRRK2 mutation and PD

  11. Inclusion Complex of Zerumbone with Hydroxypropyl-β-Cyclodextrin Induces Apoptosis in Liver Hepatocellular HepG2 Cells via Caspase 8/BID Cleavage Switch and Modulating Bcl2/Bax Ratio

    Directory of Open Access Journals (Sweden)

    Nabilah Muhammad Nadzri

    2013-01-01

    Full Text Available Zerumbone (ZER isolated from Zingiber zerumbet was previously encapsulated with hydroxypropyl-β-cyclodextrin (HPβCD to enhance ZER’s solubility in water, thus making it highly tolerable in the human body. The anticancer effects of this new ZER-HPβCD inclusion complex via apoptosis cell death were assessed in this study for the first time in liver hepatocellular cells, HepG2. Apoptosis was ascertained by morphological study, nuclear stain, and sub-G1 cell population accumulation with G2/M arrest. Further investigations showed the release of cytochrome c and loss of mitochondrial membrane potential, proving mitochondrial dysfunction upon the ZER-HPβCD treatment as well as modulating proapoptotic and anti-apototic Bcl-2 family members. A significant increase in caspase 3/7, caspase 9, and caspase 8 was detected with the depletion of BID cleaved by caspase 8. Collectively, these results prove that a highly soluble inclusion complex of ZER-HPβCD could be a promising anticancer agent for the treatment of hepatocellular carcinoma in humans.

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

    NARCIS (Netherlands)

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

    1998-01-01

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

  13. Expression levels of cleaved caspase-3 and caspase-3 in tumorigenesis and prognosis of oral tongue squamous cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Pei-Feng Liu

    Full Text Available Apoptosis plays a dual role in cancer development and malignancy. The role of apoptosis-related caspases in cancer remains controversial, particularly in oral tongue squamous cell carcinoma (OTSCC. In this study, we examined the protein levels of cleaved caspase-3, caspase-3, caspase-8, and caspase-9 on tissue microarrays consisting of samples from 246 OTSCC patients by immunohistochemistry. Wilcoxon signed-rank test indicated that the protein levels of cleaved caspase-3, caspase-3, caspase-8, and caspase-9 in tumor tissues were significantly higher compared to those in adjacent normal tissues (all p<0.001. The expression level of caspase-8 in tumors was elevated in patients with lymph node invasion. Moreover, positive expression of cleaved caspase-3 was associated with shorter disease-free survival (DFS in OTSCC patients with moderate differentiation and lymph node invasion. Combination of either positive cleaved caspase-3 or higher caspase-3 expression or both was associated with poor DFS. Interestingly, stratification analysis showed that co-expression levels of positive cleaved caspase-3 or/and higher caspase-3 were associated with better disease-specific survival in patients with advanced stages of the disease, such as large tumor size and lymph node invasion, whereas it was associated with poor DFS in OTSCC patients with moderate cell differentiation and small tumor size. Taken together, cleaved caspase-3 and caspase-3/8/9 could be biomarkers for tumorigenesis in OTSCC patients. The co-expression level of cleaved caspase-3 and caspase-3 might be a prognostic biomarker for OTSCC patients, particular in those patients with certain tumor stages and cell differentiation status.

  14. Y-27632 Increases Sensitivity of PANC-1 Cells to Epigallocatechin Gallate (EGCG) in Regulating Cell Proliferation and Migration

    Science.gov (United States)

    Liu, Xing; Bi, Yongyi

    2016-01-01

    Background The study aimed to investigate the inhibitory effect of (1R,4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl) cyclohexanecarboxamide (Y-27632) and (−)-epigallocatechin-3-gallate (EGCG) on the proliferation and migration of PANC-1 cells. EGCG, found in green tea, has been previously shown to be one of the most abundant and powerful catechins in cancer prevention and treatment. Y-27632, a selective inhibitor of rho-associated protein kinase 1, is widely used in treating cardiovascular disease, inflammation, and cancer. Material/Methods PANC-1 cells, maintained in Dulbecco’s Modified Eagle’s Medium, were treated with dimethyl sulfoxide (control) as well as different concentrations (20, 40, 60, and 80 μg/mL) of EGCG for 48 h. In addition, PANC-1 cells were treated separately with 60 μg/mL EGCG, 20 μM Y-27632, and EGCG combined with Y-27632 (60 μg/mL EGCG + 20 μM Y-27632) for 48 h. The effect of EGCG and Y-27632 on the proliferation and migration of PANC-1 cells was evaluated using Cell Counting Kit-8 and transwell migration assays. The expression of peroxisome proliferator–activated receptor alpha (PPARα) and Caspase-3 mRNA was determined by Quantitative real-time polymerase chain reaction (RT-qPCR). Results EGCG (20–80 μg/mL) inhibited cell viability in a dose-dependent manner. Y-27632 enhanced the sensitivity of PANC-1 cells to EGCG (by increasing the expression of PPARα and Caspase-3 mRNA) and suppressed cell proliferation. PANC-1 cell migration was inhibited by treatment with a combination of EGCG and Y-27632. Conclusions Y-27632 increases the sensitivity of PANC-1 cells to EGCG in regulating cell proliferation and migration, which is likely to be related to the expression of PPARα mRNA and Caspase-3 mRNA. PMID:27694793

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

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

    OpenAIRE

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

    2013-01-01

    Hyperlipidemia may lead to endothelial injury, due to its effects on homocysteine and vascular endothelial growth factor in the serum, and the mRNA expression levels of peroxisome proliferator-activated receptor-? (PPAR?), and caspase-3 and -8 in the vascular wall. In order to prevent and mitigate the high-fat state that results from endothelial injury, this study examined the effect of folic acid (FA) and vitamin B12 (VB12) on the expression of PPAR? and caspase-3 and -8 mRNA in the abdomina...

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

    Science.gov (United States)

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

    2018-02-23

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

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

    Directory of Open Access Journals (Sweden)

    Mohamed Hassan

    2009-01-01

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

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

    Czech Academy of Sciences Publication Activity Database

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

    2013-01-01

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

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

    Czech Academy of Sciences Publication Activity Database

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

    2013-01-01

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

  1. Sustained high serum caspase-3 concentrations and mortality in septic patients.

    Science.gov (United States)

    Lorente, L; Martín, M M; Pérez-Cejas, A; González-Rivero, A F; López, R O; Ferreres, J; Solé-Violán, J; Labarta, L; Díaz, C; Palmero, S; Jiménez, A

    2018-02-01

    Caspase-3 is the main executor of the apoptotic process. Higher serum caspase-3 concentrations in non-survivor compared to survivor septic patients have been found. The objectives of this work (with the increase of sample size to 308 patients, and the determination of serum caspase-3 concentrations also on days 4 and 8 of diagnosis of severe sepsis) were to know whether an association between serum caspase-3 concentrationss during the first week, degree of apoptosis, sepsis severity, and sepsis mortality exists. We collected serum samples of 308 patients with severe sepsis from eight intensive care units on days 1, 4 and 8 to measure concentrations of caspase-3 and caspase-cleaved cytokeratin (CCCK)-18 (to assess degree of apoptosis). End point was 30-day mortality. We found higher serum concentrations of caspase-3 and CCCK-18 in non-survivors compared to survivors on days 1 (p < 0.001), 4 (p < 0.001), and 8 (p < 0.001). We found an association between serum caspase-3 concentrations on days 1, 4 and 8 of severe sepsis diagnosis and serum CCCK-18 concentrations (p < 0.001), SOFA (p < 0.001), serum acid lactic concentrations (p < 0.001), and 30-day sepsis mortality (p < 0.001). The new findings of this work were that an association between serum caspase-3 concentrations during the first week, apoptosis degree, sepsis severity, and sepsis mortality exists.

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

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

    2010-01-26

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

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

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

    2009-01-01

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

  4. Antioxidants Impair Anti-Tumoral Effects of Vorinostat, but Not Anti-Neoplastic Effects of Vorinostat and Caspase-8 Downregulation

    OpenAIRE

    Bergadà, Laura; Yeramian, Andree; Sorolla, Annabel; Matias-Guiu, Xavier; Dolcet, Xavier

    2014-01-01

    We have recently demonstrated that histone deacetylase inhibitor, Vorinostat, applied as a single therapy or in combination with caspase-8 downregulation exhibits high anti-tumoral activity on endometrial carcinoma cell lines. In the present study, we have assessed the signalling processes underlying anti-tumoral effects of Vorinostat. Increasing evidence suggests that reactive oxygen species are responsible for histone deacetylase inhibitor-induced cell killing. We have found that Vorinostat...

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

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    Ju-Ha Kim

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

  6. Sox11 Reduces Caspase-6 Cleavage and Activity.

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    Elaine Waldron-Roby

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

  7. 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.

  8. The emphysematous lung is abnormally sensitive to TRAIL-mediated apoptosis

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    Milot Julie

    2011-08-01

    Full Text Available Abstract Background Alveolar apoptosis is increased in the emphysematous lung. However, mechanisms involved are not fully understood. Recently, we demonstrated that levels of TRAIL receptor 1 and 2, levels of p53, and Bax/Bcl-xL ratio were elevated in the lung of subjects with emphysema, despite smoking cessation. Thus, we postulate that due to chronic pulmonary oxidative stress, the emphysematous lung would be abnormally sensitive to TRAIL-mediated apoptosis. Methodology A549 cells were exposed to rTRAIL, cigarette smoke extract, and/or H2O2 prior to caspase-3 activity measurement and annexin V staining assessment. In addition, freshly resected lung samples were obtained from non-emphysematous and emphysematous subjects and exposed ex vivo to rTRAIL for up to 18 hours. Lung samples were harvested and levels of active caspase-3 and caspase-8 were measured from tissue lysates. Results Both cigarette smoke extract and H2O2 were able to sensitize A549 cells to TRAIL-mediated apoptosis. Moreover, following exposure to rTRAIL, caspase-3 and -8 were activated in lung explants from emphysematous subjects while being decreased in lung explants from non-emphysematous subjects. Significance of the study Alveolar sensitivity to TRAIL-mediated apoptosis is strongly increased in the emphysematous lung due to the presence of oxidative stress. This might be a new mechanism leading to increased alveolar apoptosis and persistent alveolar destruction following smoking cessation.

  9. Y-27632 Increases Sensitivity of PANC-1 Cells to EGCG in Regulating Cell Proliferation and Migration.

    Science.gov (United States)

    Liu, Xing; Bi, Yongyi

    2016-10-03

    BACKGROUND The study aimed to investigate the inhibitory effect of (1R,4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl) cyclohexanecarboxamide (Y-27632) and (-)-epigallocatechin-3-gallate (EGCG) on the proliferation and migration of PANC-1 cells. EGCG, found in green tea, has been previously shown to be one of the most abundant and powerful catechins in cancer prevention and treatment. Y-27632, a selective inhibitor of rho-associated protein kinase 1, is widely used in treating cardiovascular disease, inflammation, and cancer. MATERIAL AND METHODS PANC-1 cells, maintained in Dulbecco's Modified Eagle's Medium, were treated with dimethyl sulfoxide (control) as well as different concentrations (20, 40, 60, and 80 μg/mL) of EGCG for 48 h. In addition, PANC-1 cells were treated separately with 60 μg/mL EGCG, 20 μM Y-27632, and EGCG combined with Y-27632 (60 μg/mL EGCG + 20 μM Y-27632) for 48 h. The effect of EGCG and Y-27632 on the proliferation and migration of PANC-1 cells was evaluated using Cell Counting Kit-8 and transwell migration assays. The expression of peroxisome proliferator-activated receptor alpha (PPARα) and Caspase-3 mRNA was determined by Quantitative real-time polymerase chain reaction (RT-qPCR). RESULTS EGCG (20-80 μg/mL) inhibited cell viability in a dose-dependent manner. Y-27632 enhanced the sensitivity of PANC-1 cells to EGCG (by increasing the expression of PPARa and Caspase-3 mRNA) and suppressed cell proliferation. PANC-1 cell migration was inhibited by treatment with a combination of EGCG and Y-27632. CONCLUSIONS Y-27632 increases the sensitivity of PANC-1 cells to EGCG in regulating cell proliferation and migration, which is likely to be related to the expression of PPARa mRNA and Caspase-3 mRNA.

  10. S-glutathionylation of troponin I (fast) increases contractile apparatus Ca2+ sensitivity in fast-twitch muscle fibres of rats and humans.

    Science.gov (United States)

    Mollica, J P; Dutka, T L; Merry, T L; Lamboley, C R; McConell, G K; McKenna, M J; Murphy, R M; Lamb, G D

    2012-03-15

    Oxidation can decrease or increase the Ca2+ sensitivity of the contractile apparatus in rodent fast-twitch (type II) skeletal muscle fibres, but the reactions and molecular targets involved are unknown. This study examined whether increased Ca2+ sensitivity is due to S-glutathionylation of particular cysteine residues. Skinned muscle fibres were directly activated in heavily buffered Ca2+ solutions to assess contractile apparatus Ca2+ sensitivity. Rat type II fibres were subjected to S-glutathionylation by successive treatments with 2,2′-dithiodipyridine (DTDP) and glutathione (GSH), and displayed a maximal increase in pCa50 (−log10 [Ca2+] at half-maximal force) of ∼0.24 pCa units, with little or no effect on maximum force or Hill coefficient. Partial similar effect was produced by exposure to oxidized gluthathione (GSSG, 10 mM) for 10 min at pH 7.1, and near-maximal effect by GSSG treatment at pH 8.5. None of these treatments significantly altered Ca2+ sensitivity in rat type I fibres. Western blotting showed that both the DTDP–GSH and GSSG–pH 8.5 treatments caused marked S-glutathionylation of the fast troponin I isoform (TnI(f)) present in type II fibres, but not of troponin C (TnC) or myosin light chain 2. Both the increased Ca2+ sensitivity and glutathionylation of TnI(f) were blocked by N-ethylmaleimide (NEM). S-nitrosoglutathione (GSNO) also increased Ca2+ sensitivity, but only in conditions where it caused S-glutathionylation of TnI(f). In human type II fibres from vastus lateralis muscle, DTDP–GSH treatment also caused similar increased Ca2+ sensitivity and S-glutathionylation of TnI(f). When the slow isoform of TnI in type I fibres of rat was partially substituted (∼30%) with TnI(f), DTDP–GSH treatment caused a significant increase in Ca2+ sensitivity (∼0.08 pCa units). TnIf in type II fibres from toad and chicken muscle lack Cys133 present in mammalian TnIf, and such fibres showed no change in Ca2+ sensitivity with DTDP–GSH nor any S

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

    Science.gov (United States)

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

    2017-12-01

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

  12. Higher insulin sensitivity in EDL muscle of rats fed a low-protein, high-carbohydrate diet inhibits the caspase-3 and ubiquitin-proteasome proteolytic systems but does not increase protein synthesis.

    Science.gov (United States)

    Dos Santos, Maísa Pavani; Batistela, Emanuele; Pereira, Mayara Peron; Paula-Gomes, Silvia; Zanon, Neusa Maria; Kettelhut, Isis do Carmo; Karatzaferi, Christina; Andrade, Claudia Marlise Balbinotti; de França, Suélem Aparecida; Baviera, Amanda Martins; Kawashita, Nair Honda

    2016-08-01

    Compared with the extensor digitorum longus (EDL) muscle of control rats (C), the EDL muscle of rats fed a low-protein, high-carbohydrate diet (LPHC) showed a 36% reduction in mass. Muscle mass is determined by the balance between protein synthesis and proteolysis; thus, the aim of this work was to evaluate the components involved in these processes. Compared with the muscle from C rats, the EDL muscle from LPHC diet-fed rats showed a reduction (34%) in the in vitro basal protein synthesis and a 22% reduction in the in vitro basal proteolysis suggesting that the reduction in the mass can be associated with a change in the rate of the two processes. Soon after euthanasia, in the EDL muscles of the rats fed the LPHC diet for 15days, the activity of caspase-3 and that of components of the ubiquitin-proteasome system (atrogin-1 content and chymotrypsin-like activity) were decreased. The phosphorylation of p70(S6K) and 4E-BP1, proteins involved in protein synthesis, was also decreased. We observed an increase in the insulin-stimulated protein content of p-Akt. Thus, the higher insulin sensitivity in the EDL muscle of LPHC rats seemed to contribute to the lower proteolysis in LPHC rats. However, even with the higher insulin sensitivity, the reduction in p-E4-BP1 and p70(S6K) indicates a reduction in protein synthesis, showing that factors other than insulin can have a greater effect on the control of protein synthesis. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2008-12-01

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

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

    International Nuclear Information System (INIS)

    Kobayashi, Daisuke; Tokino, Takashi; Watanabe, Naoki

    2001-01-01

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

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

    Science.gov (United States)

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

    2012-04-01

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

  16. 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.

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

    Science.gov (United States)

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

    2004-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Jun-Ho Lee

    2015-01-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

  20. Eating high fat chow increases the sensitivity of rats to 8-OH-DPAT-induced lower lip retraction.

    Science.gov (United States)

    Li, Jun-Xu; Ju, Shutian; Baladi, Michelle G; Koek, Wouter; France, Charles P

    2011-12-01

    Eating high fat food can alter sensitivity to drugs acting on dopamine systems; this study examined whether eating high fat food alters sensitivity to a drug acting on serotonin (5-HT) systems. Sensitivity to (+)-8-hydroxy-2-(dipropylamino) tetralin hydrobromide (8-OH-DPAT; 5-HT1A receptor agonist)-induced lower lip retraction was examined in separate groups (n=8-9) of rats with free access to standard (5.7% fat) or high fat (34.3% fat) chow; sensitivity to quinpirole (dopamine D3/D2 receptor agonist)-induced yawning was also examined. Rats eating high fat chow gained more body weight than rats eating standard chow and, after 6 weeks of eating high fat chow, they were more sensitive to 8-OH-DPAT (0.01-0.1 mg/kg)-induced lower lip retraction and quinpirole (0.0032-0.32 mg/kg)-induced yawning. These changes were not reversed when rats that previously ate high fat chow were switched to eating standard chow and sensitivity to 8-OH-DPAT and quinpirole increased when rats that previously ate standard chow ate high fat chow. These data extend previous results showing changes in sensitivity to drugs acting on dopamine systems in animals eating high fat chow to a drug acting at 5-HT1A receptors and they provide support for the notion that eating certain foods impacts sensitivity to drugs acting on monoamine systems.

  1. 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.

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

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    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.

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

    Science.gov (United States)

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

    2016-01-01

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

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

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    Ki Sung Kang

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

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

    International Nuclear Information System (INIS)

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

    2005-01-01

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

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

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    Fengmei Song

    2018-04-01

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

  7. Ecstasy-Induced Caspase Expression Alters Following Ginger Treatment

    Directory of Open Access Journals (Sweden)

    Sara Soleimani Asl

    2013-11-01

    Full Text Available Introduction: Exposure to 3-4, methylenedioxymethamphetamine (MDMA leads to cell death. Herein, we studied the protective effects of ginger on MDMA- induced apoptosis. Methods: 15 Sprague dawley male rats were administrated with 0, 10 mg/kg MDMA, or MDMA along with 100mg/kg ginger, IP for 7 days. Brains were removed to study the caspase 3, 8, and 9 expressions in the hippocampus by RT-PCR. Data was analyzed by SPSS 16 software using the one-way ANOVA test. Results: MDMA treatment resulted in a significant increase in caspase 3, 8, and 9 as compared to the sham group (p<0.001. Ginger administration however, appeared to significantly decrease the same (p<0.001. Discussion: Our findings suggest that ginger consumption may lead to the improvement of MDMA-induced neurotoxicity.

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  9. Autoregulatory Feedback Mechanism of P38MAPK/Caspase-8 in Photodynamic Therapy-Hydrophilic/Lipophilic Tetra-α-(4-carboxyphenoxy Phthalocyanine Zinc-Induced Apoptosis of Human Hepatocellular Carcinoma Bel-7402 Cells

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    Yu Wang

    2014-01-01

    Full Text Available Photodynamic therapy (PDT is a novel and promising antitumor treatment. Our previous study showed that hydrophilic/lipophilic tetra-α-(4-carboxyphenoxy phthalocyanine zinc- (TαPcZn- mediated PDT (TαPcZn-PDT inhibits the proliferation of human hepatocellular carcinoma Bel-7402 cells by triggering apoptosis and arresting cell cycle. However, mechanisms of TαPcZn-PDT-induced apoptosis of Bel-7402 cells have not been fully clarified. In the present study, therefore, effect of TαPcZn-PDT on apoptosis, P38MAPK, p-P38MAPK, Caspase-8, Caspase-3, Bcl-2, Bid, Cytochrome c, and mitochondria membrane potential in Bel-7402 cells without or with P38MAPK inhibitor SB203580 or Caspase-8 inhibitor Ac-IEFD-CHO was investigated by haematoxylin and eosin (HE staining assay, flow cytometry analysis of annexin V-FITC/propidium iodide (PI double staining cells and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1, and immunoblot assay. We found that TαPcZn-PDT resulted in apoptosis induction, activation of P38MAPK, Caspase-8, Caspase-3, and Bid, downregulation of Bcl-2, release of Cytochrome c from mitochondria, and disruption of mitochondrial membrane potential in TαPcZn-PDT-treated Bel-7402 cells. In contrast, SB203580 or Ac-IEFD-CHO attenuated induction of apoptosis, activation of P38MAPK, Caspase-8, Caspase-3, and Bid, downregulation of Bcl-2, release of Cytochrome c from mitochondria, and disruption of mitochondrial membrane potential in TαPcZn-PDT-treated Bel-7402 cells. Taken together, we conclude that Caspase-3, Bcl-2, Bid, and mitochondria are involved in autoregulatory feedback of P38MAPK/Caspase-8 during TαPcZn-PDT-induced apoptosis of Bel-7402 cells.

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

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    Salmi Jussi

    2010-06-01

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

  11. Pharmacological caspase inhibitors: Research towards therapeutic perspectives

    Czech Academy of Sciences Publication Activity Database

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2010-07-01

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

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

    Science.gov (United States)

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

    2014-10-01

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

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

    Science.gov (United States)

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

    2004-06-01

    Caspases are key molecules in the control of apoptosis, but relatively little is known about their contribution to eosinophil apoptosis. We examined caspase-3, -8, and -9 activities in receptor ligation-dependent apoptosis induction in the differentiated human eosinophilic cell line EoL-1. Differentiated EoL-1 exhibited bi-lobed nuclei, eosinophil-associated membrane receptors, and basic granule proteins. Annexin-V fluorescein isothiocyanate binding to EoL-1 revealed significant (PEoL-1 but had no effect on constitutive (baseline) apoptosis at 16 and 20 h. Caspase activity was analyzed using the novel CaspaTag trade mark technique and flow cytometry. EoL-1 treated with pan-CD45, CD45RA, CD45RB, and CD95 mAb exhibited caspase-3 and -9 activation at 12 h post-treatment, which increased at 16 and 20 h. Activated caspase-8 was detected 12 and 16 h after ligation with CD45, CD45RA, CD45RB, and CD95 mAb followed by a trend toward basal levels at 20 h. CD69 ligation resulted in caspase-3 activation, a modest but significant activation of caspase-8, and a loss in mitochondrial transmembrane potential but had no significant effect on activation of caspase-9. Thus, the intrinsic and extrinsic caspase pathways are involved in controlling receptor ligation-mediated apoptosis induction in human eosinophils, findings that may aid the development of a more targeted, anti-inflammatory therapy for asthma.

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

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    Yuxia Zhang

    2018-04-01

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

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

    Science.gov (United States)

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

    2017-11-15

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

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

    Science.gov (United States)

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

    2017-06-01

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

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

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

    International Nuclear Information System (INIS)

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

    2007-01-01

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

  1. Sensitivity to Fas-mediated apoptosis in high-risk HPV-positive human cervical cancer cells : Relationship with Fas, caspase-8, and Bid

    NARCIS (Netherlands)

    Hougardy, BMT; van der Zee, AGJ; van den Heuvel, FAJ; Timmer, T; de Vries, EGE; de Jong, S

    Objective. Binding of Fas ligand or agonistic anti-Fas antibody to the death receptor Fas can activate a caspase-cascade resulting in apoptosis. In the present study, the functionality of the Fas pathway was studied in human cervical cancer cells with different HPV and p53 status. Methods. HeLa

  2. Caspase 8 and maspin are downregulated in breast cancer cells due to CpG site promoter methylation

    International Nuclear Information System (INIS)

    Wu, Yanyuan; Alvarez, Monica; Slamon, Dennis J; Koeffler, Phillip; Vadgama, Jaydutt V

    2010-01-01

    Epigenetic changes associated with promoter DNA methylation results in silencing of several tumor suppressor genes that lead to increased risk for tumor formation and for progression of the cancer. Methylation specific PCR (MSP) and bisulfite sequencing were used for determination of proapoptotic gene Caspase 8 (CASP8) and the tumor suppressor gene maspin promoter methylation in four breast cancer and two non-tumorigenic breast cell lines. Involvement of histone H3 methylation in those cell lines were examined by CHIP assay. The CpG sites in the promoter region of CASP8 and maspin were methylated in all four breast cancer cell lines but not in two non-tumorigenic breast cell lines. Demethylation agent 5-aza-2'-deoxycytidine (5-aza-dc) selectively inhibits DNA methyltransferases, DNMT3a and DNMT3b, and restored CASP8 and maspin gene expression in breast cancer cells. 5-aza-dc also reduced histone H3k9me2 occupancy on CASP8 promoter in SKBR3cells, but not in MCF-7 cells. Combination of histone deacetylase inhibitor Trichostatin A (TSA) and 5-aza-dc significant decrease in nuclear expression of Di-methyl histone H3-Lys27 and slight increase in acetyl histone H3-Lys9 in MCF-7 cells. CASP8 mRNA and protein level in MCF-7 cells were increased by the 5-aza-dc in combination with TSA. Data from our study also demonstrated that treatment with 5-FU caused a significant increase in unmethylated CASP8 and in CASP8 mRNA in all 3 cancer lines. CASP8 and maspin expression were reduced in breast cancer cells due to promoter methylation. Selective application of demethylating agents could offer novel therapeutic opportunities in breast cancer

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

    Science.gov (United States)

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

    2008-11-01

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

  4. Expression of Fas, FasL, caspase-8 and other factors of the extrinsic apoptotic pathway during the onset of interdigital tissue elimination.

    Science.gov (United States)

    Svandova, E Budisova; Vesela, B; Lesot, H; Poliard, A; Matalova, E

    2017-04-01

    Elimination of the interdigital web is considered to be the classical model for assessing apoptosis. So far, most of the molecules described in the process have been connected to the intrinsic (mitochondrial) pathway. The extrinsic (receptor mediated) apoptotic pathway has been rather neglected, although it is important in development, immunomodulation and cancer therapy. This work aimed to investigate factors of the extrinsic apoptotic machinery during interdigital regression with a focus on three crucial initiators: Fas, Fas ligand and caspase-8. Immunofluorescent analysis of mouse forelimb histological sections revealed abundant expression of these molecules prior to digit separation. Subsequent PCR Array analyses indicated the expression of several markers engaged in the extrinsic pathway. Between embryonic days 11 and 13, statistically significant increases in the expression of Fas and caspase-8 were observed, along with other molecules involved in the extrinsic apoptotic pathway such as Dapk1, Traf3, Tnsf12, Tnfrsf1A and Ripk1. These results demonstrate for the first time the presence of extrinsic apoptotic components in mouse limb development and indicate novel candidates in the molecular network accompanying the regression of interdigital tissue during digitalisation.

  5. The Enigmatic Roles of Caspases in Tumor Development

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-11-24

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

  6. The Enigmatic Roles of Caspases in Tumor Development

    International Nuclear Information System (INIS)

    Jäger, Richard; Zwacka, Ralf M.

    2010-01-01

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

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

    Science.gov (United States)

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

    2017-12-26

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

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

    NARCIS (Netherlands)

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

    2009-01-01

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

  9. LyGDI expression in HeLa cells increased its sensitivity to radiation-induced apoptosis

    International Nuclear Information System (INIS)

    Zhou Xinwen; Xu Yaxiang

    2006-01-01

    Objective: In order to confirm whether LyGDI has apoptotic signal transduction function and can increase the apoptotic rate of radiation-induced cell death, the lyGDI and mutant D19lyGDI gene, which constructed with the pCDNA3. 1 His A, were transfected into no-endogenous lyGDI HeLa cells. Methods Transient expressions of lyGDI and D19lyGDI in HeLa cells were analyzed by Western blot using anti-mono antibody of LyGDI and Xpress tag. Cell apoptosis was assayed with Annexin V-FITC apoptosis kit. To select stable clone, the transferred HeLa cells had been maintained in G418 medium for 3 weeks, then a cell line, which stably expressed LyGDI and mutant D19lyGDI, was selected. The selected cell line was irradiated with 12 Gy 60 Co y-rays. Caspase-3 activity of the cells was determined by Western blot and cell viability by clone-forming assay after 48 hours post-irradiation culture. Results: Western blot and Annexin V-FITC apoptotic analysis revealed that lyGDI and D19lyGDI transient expressions in HeLa cells induced apoptosis; Caspase-3 activity measurement and clone-forming assay showed that lyGDI increased sensitivity to radiation-induced cell apoptosis. Conclusions: lyGDI performs function in apoptosis signal transduction, its expression in HeLa cells can increase the sensitivity to radiation-induced cell apoptosis. (authors)

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-02-10

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

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

    Science.gov (United States)

    Andersen, Joshua L; Johnson, Carrie E; Freel, Christopher D; Parrish, Amanda B; Day, Jennifer L; Buchakjian, Marisa R; Nutt, Leta K; Thompson, J Will; Moseley, M Arthur; Kornbluth, Sally

    2009-01-01

    The apoptotic initiator caspase-2 has been implicated in oocyte death, in DNA damage- and heat shock-induced death, and in mitotic catastrophe. We show here that the mitosis-promoting kinase, cdk1–cyclin B1, suppresses apoptosis upstream of mitochondrial cytochrome c release by phosphorylating caspase-2 within an evolutionarily conserved sequence at Ser 340. Phosphorylation of this residue, situated in the caspase-2 interdomain, prevents caspase-2 activation. S340 was susceptible to phosphatase 1 dephosphorylation, and an interaction between phosphatase 1 and caspase-2 detected during interphase was lost in mitosis. Expression of S340A non-phosphorylatable caspase-2 abrogated mitotic suppression of caspase-2 and apoptosis in various settings, including oocytes induced to undergo cdk1-dependent maturation. Moreover, U2OS cells treated with nocodazole were found to undergo mitotic catastrophe more readily when endogenous caspase-2 was replaced with the S340A mutant to lift mitotic inhibition. These data demonstrate that for apoptotic stimuli transduced by caspase-2, cell death is prevented during mitosis through the inhibitory phosphorylation of caspase-2 and suggest that under conditions of mitotic arrest, cdk1–cyclin B1 activity must be overcome for apoptosis to occur. PMID:19730412

  12. Pharmacological caspase inhibitors: Research towards therapeutic perspectives

    Czech Academy of Sciences Publication Activity Database

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2013-02-01

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

  14. BmDredd is an initiator caspase and participates in Emodin-induced apoptosis in the silkworm, Bombyx mori.

    Science.gov (United States)

    Wang, La; Song, Juan; Bao, Xi-Yan; Chen, Peng; Yi, Hua-Shan; Pan, Min-Hui; Lu, Cheng

    2016-10-15

    The identification and analysis of the caspases is essential to research into apoptosis in lepidoptera insects. The domesticated silkworm, Bombyx mori, is the model system for lepidopterans. In this study, we cloned and characterized a B. mori Dredd gene, BmDredd, the proposed insect homologue of human caspase-8, which encoded a polypeptide of 543 amino acids. BmDredd possesses a long N-terminal prodomain, a p20 domain, and a p10 domain. When transiently expressed in Escherichia coli cells, BmDredd underwent spontaneous cleavage and exhibited high proteolytic activity for caspase-8 substrate but relatively low for caspase-3 or -9 substrate. In addition, BmDredd induced apoptosis when transiently expressed in BmN-SWU1 cells, an ovarian cell line of B. mori. Moreover, after the treatment of Emodin, a novel apoptosis inducer, endogenous BmDredd expression level, the caspase-8 activity and the apoptotic rate increased notably in BmN-SWU1 cells. When BmDredd was subjected to interference in BmN-SWU1 cells and Emodin treatment, BmDredd expression levels decreased and the apoptotic rate also decreased significantly. These results suggest BmDredd is the homologue of human caspase-8 and plays a role in Emodin-induced apoptosis in BmN-SWU1 cells of B. mori. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Host cell killing by the West Nile Virus NS2B-NS3 proteolytic complex: NS3 alone is sufficient to recruit caspase-8-based apoptotic pathway

    International Nuclear Information System (INIS)

    Ramanathan, Mathura P.; Chambers, Jerome A.; Pankhong, Panyupa; Chattergoon, Michael; Attatippaholkun, Watcharee; Dang, Kesen; Shah, Neelima; Weiner, David B.

    2006-01-01

    The West Nile Virus (WNV) non-structural proteins 2B and 3 (NS2B-NS3) constitute the proteolytic complex that mediates the cleavage and processing of the viral polyprotein. NS3 recruits NS2B and NS5 proteins to direct protease and replication activities. In an effort to investigate the biology of the viral protease, we cloned cDNA encoding the NS2B-NS3 proteolytic complex from brain tissue of a WNV-infected dead crow, collected from the Lower Merion area (Merion strain). Expression of the NS2B-NS3 gene cassette induced apoptosis within 48 h of transfection. Electron microscopic analysis of NS2B-NS3-transfected cells revealed ultra-structural changes that are typical of apoptotic cells including membrane blebbing, nuclear disintegration and cytoplasmic vacuolations. The role of NS3 or NS2B in contributing to host cell apoptosis was examined. NS3 alone triggers the apoptotic pathways involving caspases-8 and -3. Experimental results from the use of caspase-specific inhibitors and caspase-8 siRNA demonstrated that the activation of caspase-8 was essential to initiate apoptotic signaling in NS3-expressing cells. Downstream of caspase-3 activation, we observed nuclear membrane ruptures and cleavage of the DNA-repair enzyme, PARP in NS3-expressing cells. Nuclear herniations due to NS3 expression were absent in the cells treated with a caspase-3 inhibitor. Expression of protease and helicase domains themselves was sufficient to trigger apoptosis generating insight into the apoptotic pathways triggered by NS3 from WNV

  16. α-Hispanolol sensitizes hepatocellular carcinoma cells to TRAIL-induced apoptosis via death receptor up-regulation

    Energy Technology Data Exchange (ETDEWEB)

    Mota, Alba, E-mail: amota@iib.uam.es [Unidad de Terapias Farmacológicas, Área de Genética Humana, Instituto de Investigación de Enfermedades Raras (IIER), Instituto de Salud Carlos III, Madrid (Spain); Jiménez-Garcia, Lidia, E-mail: ljimenez@isciii.es [Unidad de Terapias Farmacológicas, Área de Genética Humana, Instituto de Investigación de Enfermedades Raras (IIER), Instituto de Salud Carlos III, Madrid (Spain); Herránz, Sandra, E-mail: sherranz@isciii.es [Unidad de Terapias Farmacológicas, Área de Genética Humana, Instituto de Investigación de Enfermedades Raras (IIER), Instituto de Salud Carlos III, Madrid (Spain); Heras, Beatriz de las, E-mail: lasheras@ucm.es [Departamento de Farmacología, Facultad de Farmacia, Universidad Complutense de Madrid (UCM), Madrid (Spain); Hortelano, Sonsoles, E-mail: shortelano@isciii.es [Unidad de Terapias Farmacológicas, Área de Genética Humana, Instituto de Investigación de Enfermedades Raras (IIER), Instituto de Salud Carlos III, Madrid (Spain)

    2015-08-01

    Hispanolone derivatives have been previously described as anti-inflammatory and antitumoral agents. However, their effects on overcoming Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance remain to be elucidated. In this study, we analyzed the cytotoxic effects of the synthetic hispanolone derivative α-hispanolol (α-H) in several tumor cell lines, and we evaluated the induction of apoptosis, as well as the TRAIL-sensitizing potential of α-H in the hepatocellular carcinoma cell line HepG2. Our data show that α-H decreased cell viability in a dose-dependent manner in HeLa, MDA-MB231, U87 and HepG2 cell lines, with a more prominent effect in HepG2 cells. Interestingly, α-H had no effect on non-tumoral cells. α-H induced activation of caspase-8 and caspase-9 and also increased levels of the proapoptotic protein Bax, decreasing antiapoptotic proteins (Bcl-2, X-IAP and IAP-1) in HepG2 cells. Specific inhibition of caspase-8 abrogated the cascade of caspase activation, suggesting that the extrinsic pathway has a critical role in the apoptotic events induced by α-H. Furthermore, combined treatment of α-H with TRAIL enhanced apoptosis in HepG2 cells, activating caspase-8 and caspase-9. This correlated with up-regulation of both the TRAIL death receptor DR4 and DR5. DR4 or DR5 neutralizing antibodies abolished the effect of α-H on TRAIL-induced apoptosis, suggesting that sensitization was mediated through the death receptor pathway. Our results demonstrate that α-H induced apoptosis in the human hepatocellular carcinoma cell line HepG2 through activation of caspases and induction of the death receptor pathway. In addition, we describe a novel function of α-H as a sensitizer on TRAIL-induced apoptotic cell death in HepG2 cells. - Highlights: • α-Hispanolol induced apoptosis in the human hepatocellular carcinoma cell line HepG2. • α-Hispanolol induced activation of caspases and the death receptor pathway. • α-Hispanolol enhanced

  17. α-Hispanolol sensitizes hepatocellular carcinoma cells to TRAIL-induced apoptosis via death receptor up-regulation

    International Nuclear Information System (INIS)

    Mota, Alba; Jiménez-Garcia, Lidia; Herránz, Sandra; Heras, Beatriz de las; Hortelano, Sonsoles

    2015-01-01

    Hispanolone derivatives have been previously described as anti-inflammatory and antitumoral agents. However, their effects on overcoming Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance remain to be elucidated. In this study, we analyzed the cytotoxic effects of the synthetic hispanolone derivative α-hispanolol (α-H) in several tumor cell lines, and we evaluated the induction of apoptosis, as well as the TRAIL-sensitizing potential of α-H in the hepatocellular carcinoma cell line HepG2. Our data show that α-H decreased cell viability in a dose-dependent manner in HeLa, MDA-MB231, U87 and HepG2 cell lines, with a more prominent effect in HepG2 cells. Interestingly, α-H had no effect on non-tumoral cells. α-H induced activation of caspase-8 and caspase-9 and also increased levels of the proapoptotic protein Bax, decreasing antiapoptotic proteins (Bcl-2, X-IAP and IAP-1) in HepG2 cells. Specific inhibition of caspase-8 abrogated the cascade of caspase activation, suggesting that the extrinsic pathway has a critical role in the apoptotic events induced by α-H. Furthermore, combined treatment of α-H with TRAIL enhanced apoptosis in HepG2 cells, activating caspase-8 and caspase-9. This correlated with up-regulation of both the TRAIL death receptor DR4 and DR5. DR4 or DR5 neutralizing antibodies abolished the effect of α-H on TRAIL-induced apoptosis, suggesting that sensitization was mediated through the death receptor pathway. Our results demonstrate that α-H induced apoptosis in the human hepatocellular carcinoma cell line HepG2 through activation of caspases and induction of the death receptor pathway. In addition, we describe a novel function of α-H as a sensitizer on TRAIL-induced apoptotic cell death in HepG2 cells. - Highlights: • α-Hispanolol induced apoptosis in the human hepatocellular carcinoma cell line HepG2. • α-Hispanolol induced activation of caspases and the death receptor pathway. • α-Hispanolol enhanced

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

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

    2015-10-01

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

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

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    Kelly A Shipkowski

    Full Text Available Multi-walled carbon nanotubes (MWCNTs represent a human health risk as mice exposed by inhalation display pulmonary fibrosis. Production of IL-1β via inflammasome activation is a mechanism of MWCNT-induced acute inflammation and has been implicated in chronic fibrogenesis. Mice sensitized to allergens have elevated T-helper 2 (Th2 cytokines, IL-4 and IL-13, and are susceptible to MWCNT-induced airway fibrosis. We postulated that Th2 cytokines would modulate MWCNT-induced inflammasome activation and IL-1β release in vitro and in vivo during allergic inflammation.THP-1 macrophages were primed with LPS, exposed to MWCNTs and/or IL-4 or IL-13 for 24 hours, and analyzed for indicators of inflammasome activation. C57BL6 mice were sensitized to house dust mite (HDM allergen and MWCNTs were delivered to the lungs by oropharyngeal aspiration. Mice were euthanized 1 or 21 days post-MWCNT exposure and evaluated for lung inflammasome components and allergic inflammatory responses.Priming of THP-1 macrophages with LPS increased pro-IL-1β and subsequent exposure to MWCNTs induced IL-1β secretion. IL-4 or IL-13 decreased MWCNT-induced IL-1β secretion by THP-1 cells and reduced pro-caspase-1 but not pro-IL-1β. Treatment of THP-1 cells with STAT6 inhibitors, either Leflunomide or JAK I inhibitor, blocked suppression of caspase activity by IL-4 and IL-13. In vivo, MWCNTs alone caused neutrophilic infiltration into the lungs of mice 1 day post-exposure and increased IL-1β in bronchoalveolar lavage fluid (BALF and pro-caspase-1 immuno-staining in macrophages and airway epithelium. HDM sensitization alone caused eosinophilic inflammation with increased IL-13. MWCNT exposure after HDM sensitization increased total cell numbers in BALF, but decreased numbers of neutrophils and IL-1β in BALF as well as reduced pro-caspase-1 in lung tissue. Despite reduced IL-1β mice exposed to MWCNTs after HDM developed more severe airway fibrosis by 21 days and had increased

  20. Function of caspase-14 in trophoblast differentiation

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    Charles Adrian K

    2009-09-01

    Full Text Available Abstract Background Within the human placenta, the cytotrophoblast consists of a proliferative pool of progenitor cells which differentiate to replenish the overlying continuous, multi-nucleated syncytiotrophoblast, which forms the barrier between the maternal and fetal tissues. Disruption to trophoblast differentiation and function may result in impaired fetal development and preeclampsia. Caspase-14 expression is limited to barrier forming tissues. It promotes keratinocyte differentiation by cleaving profilaggrin to stabilise keratin intermediate filaments, and indirectly providing hydration and UV protection. However its role in the trophoblast remains unexplored. Methods Using RNA Interference the reaction of control and differentiating trophoblastic BeWo cells to suppressed caspase-14 was examined for genes pertaining to hormonal, cell cycle and cytoskeletal pathways. Results Transcription of hCG, KLF4 and cytokeratin-18 were increased following caspase-14 suppression suggesting a role for caspase-14 in inhibiting their pathways. Furthermore, hCG, KLF4 and cytokeratin-18 protein levels were disrupted. Conclusion Since expression of these molecules is normally increased with trophoblast differentiation, our results imply that caspase-14 inhibits trophoblast differentiation. This is the first functional study of this unusual member of the caspase family in the trophoblast, where it has a different function than in the epidermis. This knowledge of the molecular underpinnings of trophoblast differentiation may instruct future therapies of trophoblast disease.

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

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    Jerry W. Marlin

    2010-04-01

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

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

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    Mingjia Tan

    2006-12-01

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

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

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

    2009-09-01

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

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

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    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

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

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    CAIPING ZHUANG

    2013-07-01

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

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

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

    2010-06-01

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

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

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

    2017-11-01

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

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

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    Min Zhang

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

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

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

    2016-11-15

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

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

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

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    Li Chen

    2014-04-01

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

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

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

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

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

    2014-02-01

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

  15. mTOR inhibition sensitizes ONC201-induced anti-colorectal cancer cell activity.

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    Jin, Zhe-Zhu; Wang, Wei; Fang, Di-Long; Jin, Yong-Jun

    2016-09-30

    We here tested the anti-colorectal cancer (CRC) activity by a first-in-class small molecule TRAIL inducer ONC201. The potential effect of mTOR on ONC201's actions was also examined. ONC201 induced moderate cytotoxicity against CRC cell lines (HT-29, HCT-116 and DLD-1) and primary human CRC cells. Significantly, AZD-8055, a mTOR kinase inhibitor, sensitized ONC201-induced cytotoxicity in CRC cells. Meanwhile, ONC201-induced TRAIL/death receptor-5 (DR-5) expression, caspase-8 activation and CRC cell apoptosis were also potentiated with AZD-8055 co-treatment. Reversely, TRAIL sequestering antibody RIK-2 or the caspase-8 specific inhibitor z-IETD-fmk attenuated AZD-8055 plus ONC201-induced CRC cell death. Further, mTOR kinase-dead mutation (Asp-2338-Ala) or shRNA knockdown significantly sensitized ONC201's activity in CRC cells, leading to profound cell death and apoptosis. On the other hand, expression of a constitutively-active S6K1 (T389E) attenuated ONC201-induced CRC cell apoptosis. For the mechanism study, we showed that ONC201 blocked Akt, but only slightly inhibited mTOR in CRC cells. Co-treatment with AZD-8055 also concurrently blocked mTOR activation. These results suggest that mTOR could be a primary resistance factor of ONC201 in CRC cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Improving Blast Resistance of a Thermo-Sensitive Genic Male Sterile Rice Line GD-8S by Molecular Marker-Assisted Selection

    Directory of Open Access Journals (Sweden)

    Wu-ge LIU

    2008-09-01

    Full Text Available The broad-spectrum blast resistance gene Pi-1, from donor line BL122, was introduced into a thermo-sensitive genic male sterile rice line GD-8S, which possessed good grain quality but high susceptibility to rice blast, by using backcross breeding and molecular marker-assisted selection. Five elite improved male sterile lines, RGD8S-1, RGD8S-2, RGD8S-3, RGD8S-4 and RGD8S-5, were selected based on the results of molecular marker analysis, spikelet sterility, recovery rate of genetic background and agronomic traits. Thirty-three representative blast isolates collected from Guangdong Province, China were used to inoculate the improved lines and the original line GD-8S artificially. The resistance frequencies of the improved lines ranged from 76.47% to 100%, much higher than that of the original line GD-8S (9.09%. On the agronomic characters, there were no significant differences between the improved lines and GD-8S except for flag leaf length and panicle number per plant. The improved lines could be used for breeding hybrid rice with high blast resistance.

  17. 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.

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

    Science.gov (United States)

    Liu, Lei; Zhang, Yingjie; Wang, Xianwang

    2009-02-01

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

  19. Caspases and osteogenic markers-in vitro screening of inhibition impact

    Czech Academy of Sciences Publication Activity Database

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

    2016-01-01

    Roč. 52, č. 2 (2016), s. 144-148 ISSN 1071-2690 R&D Projects: GA ČR(CZ) GB14-37368G; GA ČR(CZ) GA14-28254S Institutional support: RVO:67985904 ; RVO:68081715 Keywords : osteogenesis * chondrogenesis * caspases * caspase-3 * gene expression Subject RIV: EA - Cell Biology; CB - Analytical Chemistry, Separation (UIACH-O) Impact factor: 0.897, year: 2016

  20. The PARP inhibitor PJ-34 sensitizes cells to UVA-induced phototoxicity by a PARP independent mechanism

    International Nuclear Information System (INIS)

    Lakatos, Petra; Hegedűs, Csaba; Salazar Ayestarán, Nerea; Juarranz, Ángeles; Kövér, Katalin E.; Szabó, Éva; Virág, László

    2016-01-01

    Highlights: • PARP-1 is not a key regulator of photochemotherapy. • The PARP inhibitor PJ-34 sensitizes cells to UVA-induced phototoxicity by a PARP independent mechanism. • Photosensitization by PJ-34 is associated with increased ROS production and DNA damage. • Cells sensitized by PJ-34 undergo caspase-mediated apoptosis. - Abstract: A combination of a photosensitizer with light of matching wavelength is a common treatment modality in various diseases including psoriasis, atopic dermatitis and tumors. DNA damage and production of reactive oxygen intermediates may impact pathological cellular functions and viability. Here we set out to investigate the role of the nuclear DNA nick sensor enzyme poly(ADP-ribose) polymerase 1 in photochemical treatment (PCT)-induced tumor cell killing. We found that silencing PARP-1 or inhibition of its enzymatic activity with Veliparib had no significant effect on the viability of A431 cells exposed to 8-methoxypsoralen (8-MOP) and UVA (2.5 J/cm"2) indicating that PARP-1 is not likely to be a key player in either cell survival or cell death of PCT-exposed cells. Interestingly, however, another commonly used PARP inhibitor PJ-34 proved to be a photosensitizer with potency equal to 8-MOP. Irradiation of PJ-34 with UVA caused changes both in the UV absorption and in the 1H NMR spectra of the compound with the latter suggesting UVA-induced formation of tautomeric forms of the compound. Characterization of the photosensitizing effect revealed that PJ–34 + UVA triggers overproduction of reactive oxygen species, induces DNA damage, activation of caspase 3 and caspase 8 and internucleosomal DNA fragmentation. Cell death in this model could not be prevented by antioxidants (ascorbic acid, trolox, glutathione, gallotannin or cell permeable superoxide dismutase or catalase) but could be suppressed by inhibitors of caspase-3 and −8. In conclusion, PJ-34 is a photosensitizer and PJ–34 + UVA causes DNA damage and caspase

  1. The PARP inhibitor PJ-34 sensitizes cells to UVA-induced phototoxicity by a PARP independent mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Lakatos, Petra; Hegedűs, Csaba [Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, Debrecen (Hungary); Salazar Ayestarán, Nerea; Juarranz, Ángeles [Department of Biology, Faculty of Sciences, Universidad Autónoma of Madrid, 28049-Madrid (Spain); Kövér, Katalin E. [Department of Inorganic and Analytical Chemistry, Faculty of Sciences, University of Debrecen, Debrecen (Hungary); Szabó, Éva [Department of Dermatology, Faculty of Medicine, University of Debrecen, Debrecen (Hungary); Virág, László, E-mail: lvirag@med.unideb.hu [Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, Debrecen (Hungary); MTA-DE Cell Biology and Signaling Research Group, Debrecen (Hungary)

    2016-08-15

    Highlights: • PARP-1 is not a key regulator of photochemotherapy. • The PARP inhibitor PJ-34 sensitizes cells to UVA-induced phototoxicity by a PARP independent mechanism. • Photosensitization by PJ-34 is associated with increased ROS production and DNA damage. • Cells sensitized by PJ-34 undergo caspase-mediated apoptosis. - Abstract: A combination of a photosensitizer with light of matching wavelength is a common treatment modality in various diseases including psoriasis, atopic dermatitis and tumors. DNA damage and production of reactive oxygen intermediates may impact pathological cellular functions and viability. Here we set out to investigate the role of the nuclear DNA nick sensor enzyme poly(ADP-ribose) polymerase 1 in photochemical treatment (PCT)-induced tumor cell killing. We found that silencing PARP-1 or inhibition of its enzymatic activity with Veliparib had no significant effect on the viability of A431 cells exposed to 8-methoxypsoralen (8-MOP) and UVA (2.5 J/cm{sup 2}) indicating that PARP-1 is not likely to be a key player in either cell survival or cell death of PCT-exposed cells. Interestingly, however, another commonly used PARP inhibitor PJ-34 proved to be a photosensitizer with potency equal to 8-MOP. Irradiation of PJ-34 with UVA caused changes both in the UV absorption and in the 1H NMR spectra of the compound with the latter suggesting UVA-induced formation of tautomeric forms of the compound. Characterization of the photosensitizing effect revealed that PJ–34 + UVA triggers overproduction of reactive oxygen species, induces DNA damage, activation of caspase 3 and caspase 8 and internucleosomal DNA fragmentation. Cell death in this model could not be prevented by antioxidants (ascorbic acid, trolox, glutathione, gallotannin or cell permeable superoxide dismutase or catalase) but could be suppressed by inhibitors of caspase-3 and −8. In conclusion, PJ-34 is a photosensitizer and PJ–34 + UVA causes DNA damage and caspase

  2. Two different avian cold-sensitive sensory neurons: Transient receptor potential melastatin 8 (TRPM8)-dependent and -independent activation mechanisms.

    Science.gov (United States)

    Yamamoto, A; Takahashi, K; Saito, S; Tominaga, M; Ohta, T

    2016-12-01

    Sensing the ambient temperature is an important function for survival in animals. Some TRP channels play important roles as detectors of temperature and irritating chemicals. There are functional differences of TRP channels among species. TRPM8 in mammals is activated by cooling compounds and cold temperature, but less information is available on the functional role of TRPM8 in avian species. Here we investigated the pharmacological properties and thermal sensitivities of chicken TRPM8 (cTRPM8) and cold-sensitive mechanisms in avian sensory neurons. In heterologously expressed cTRPM8, menthol and its derivative, WS-12 elicited [Ca 2+ ] i increases, but icilin did not. In chicken sensory neurons, icilin increased [Ca 2+ ] i, in a TRPA1-dependent manner. Icilin selectively stimulated heterologously expressed chicken TRPA1 (cTRPA1). Similar to mammalian orthologue, cTRPM8 was activated by cold. Both heterologous and endogenous expressed cTRPM8 were sensitive to mammalian TRPM8 antagonists. There are two types of cold-sensitive cells regarding menthol sensitivity in chicken sensory neurons. The temperature threshold of menthol-insensitive neurons was significantly lower than that of menthol-sensitive ones. The population of menthol-insensitive neurons was large in chicken but almost little in mammals. The cold-induced [Ca 2+ ] i increases were not abolished by the external Ca 2+ removal or by blockades of PLC-IP 3 pathways and ryanodine channels. The cold stimulation failed to evoke [Ca 2+ ] i increases after intracellular Ca 2+ store-depletion. These results indicate that cTRPM8 acts as a cold-sensor similar to mammals. It is noteworthy that TRPM8-independent cold-sensitive neurons are abundant in chicken sensory neurons. Our results suggest that most of the cold-induced [Ca 2+ ] i increases are mediated via Ca 2+ release from intracellular stores and that these mechanisms may be specific to avian species. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2013-05-01

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

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

    Science.gov (United States)

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

    2017-09-26

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

  5. 3-Monochloro-1,2-propanediol (3-MCPD) induces apoptosis via mitochondrial oxidative phosphorylation system impairment and the caspase cascade pathway.

    Science.gov (United States)

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

    2016-11-30

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

  6. The inhibition of human T cell proliferation by the caspase inhibitor z-VAD-FMK is mediated through oxidative stress

    Energy Technology Data Exchange (ETDEWEB)

    Rajah, T.; Chow, S.C., E-mail: chow.sek.chuen@monash.edu

    2014-07-15

    The caspase inhibitor benzyloxycarbony (Cbz)-L-Val-Ala-Asp (OMe)-fluoromethylketone (z-VAD-FMK) has recently been shown to inhibit T cell proliferation without blocking caspase-8 and caspase-3 activation in primary T cells. We showed in this study that z-VAD-FMK treatment leads to a decrease in intracellular glutathione (GSH) with a concomitant increase in reactive oxygen species (ROS) levels in activated T cells. The inhibition of anti-CD3-mediated T cell proliferation induced by z-VAD-FMK was abolished by the presence of low molecular weight thiols such as GSH, N-acetylcysteine (NAC) and L-cysteine, whereas D-cysteine which cannot be metabolised to GSH has no effect. These results suggest that the depletion of intracellular GSH is the underlying cause of z-VAD-FMK-mediated inhibition of T cell activation and proliferation. The presence of exogenous GSH also attenuated the inhibition of anti-CD3-induced CD25 and CD69 expression mediated by z-VAD-FMK. However, none of the low molecular weight thiols were able to restore the caspase-inhibitory properties of z-VAD-FMK in activated T cells where caspase-8 and caspase-3 remain activated and processed into their respective subunits in the presence of the caspase inhibitor. This suggests that the inhibition of T cell proliferation can be uncoupled from the caspase-inhibitory properties of z-VAD-FMK. Taken together, the immunosuppressive effects in primary T cells mediated by z-VAD-FMK are due to oxidative stress via the depletion of GSH.

  7. The inhibition of human T cell proliferation by the caspase inhibitor z-VAD-FMK is mediated through oxidative stress

    International Nuclear Information System (INIS)

    Rajah, T.; Chow, S.C.

    2014-01-01

    The caspase inhibitor benzyloxycarbony (Cbz)-L-Val-Ala-Asp (OMe)-fluoromethylketone (z-VAD-FMK) has recently been shown to inhibit T cell proliferation without blocking caspase-8 and caspase-3 activation in primary T cells. We showed in this study that z-VAD-FMK treatment leads to a decrease in intracellular glutathione (GSH) with a concomitant increase in reactive oxygen species (ROS) levels in activated T cells. The inhibition of anti-CD3-mediated T cell proliferation induced by z-VAD-FMK was abolished by the presence of low molecular weight thiols such as GSH, N-acetylcysteine (NAC) and L-cysteine, whereas D-cysteine which cannot be metabolised to GSH has no effect. These results suggest that the depletion of intracellular GSH is the underlying cause of z-VAD-FMK-mediated inhibition of T cell activation and proliferation. The presence of exogenous GSH also attenuated the inhibition of anti-CD3-induced CD25 and CD69 expression mediated by z-VAD-FMK. However, none of the low molecular weight thiols were able to restore the caspase-inhibitory properties of z-VAD-FMK in activated T cells where caspase-8 and caspase-3 remain activated and processed into their respective subunits in the presence of the caspase inhibitor. This suggests that the inhibition of T cell proliferation can be uncoupled from the caspase-inhibitory properties of z-VAD-FMK. Taken together, the immunosuppressive effects in primary T cells mediated by z-VAD-FMK are due to oxidative stress via the depletion of GSH

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

    Directory of Open Access Journals (Sweden)

    Harald F. Krug

    2012-01-01

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

  9. FADD cleavage by NK cell granzyme M enhances its self-association to facilitate procaspase-8 recruitment for auto-processing leading to caspase cascade.

    Science.gov (United States)

    Wang, S; Xia, P; Shi, L; Fan, Z

    2012-04-01

    Granzyme M (GzmM), an orphan Gzm, is constitutively and abundantly expressed in innate effector natural killer cells. We previously demonstrated that GzmM induces caspase (casp)-dependent apoptosis and cytochrome c release from mitochondria. We also resolved the crystal structure for GzmM and generated its specific inhibitor. However, how GzmM causes casp activation has not been defined. Here we found that casp-8 is an initiator caspase in GzmM-induced casp cascade, which causes other casp activation and Bid cleavage. GzmM does not directly cleave procaspase-3 and Bid, whose processing is casp dependent. Casp-8 knockdown or deficient cells attenuate or abolish GzmM-induced proteolysis of procaspase-3 and Bid. Extrinsic death receptor pathway adaptor Fas-associated protein with death domain (FADD) contributes to GzmM-induced casp-8 activation. GzmM specifically cleaves FADD after Met 196 to generate truncated FADD (tFADD) that enhances its self-association for oligomerization. The oligomerized tFADD facilitates procaspase-8 recruitment to promote its auto-processing leading to casp activation cascade. FADD-deficient cells abrogate GzmM-induced activation of casp-8 and apoptosis as well as significantly inhibit lymphokine-activated killer cell-mediated cytotoxicity. FADD processing by GzmM can potentiate killing efficacy against tumor cells and intracellular pathogens.

  10. Gamma irradiation increase the sensitivity of Salmonella to antibiotics

    International Nuclear Information System (INIS)

    Ben Miloud, Najla; Barkallah, Insaf

    2008-01-01

    In order to study the effect of ionizing radiation on the resistance of Salmonella to antibiotics, four strains of Salmonella were isolated from foods, The different strains used in the present study are (S. Hadar isolate 287, S. Hadar isolate 63, S. Cerro isolate 291, S. Zanzibar isolate 1103), antibiogram analyses were made to test the in vitro-sensitivity of irradiated Salmonella isolates to different antibiotics.The analyse of Control and exposed antibiograms showed that gamma radiation have increased the sensitivity of Salmonella isolates to Cefalotin, Chloramphenicol, Nalidixic acid, Spiramycin and Gentamycin excepted S. Hadar isolate 287 that was resistant to Cefalotin and became sensitive after irradiation. Statistical analyses showed that the effect of different irradiation dose treatment on the antibiotic sensitivity is increasingly significant. The irradiation didn't induce modifications of the sensitivity to other antibiotics,probably because of their nature, of their penetration mode inside the cell or their action way

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

    Science.gov (United States)

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

    1999-09-01

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

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

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

    Science.gov (United States)

    Eeva, J; Nuutinen, U; Ropponen, A; Mättö, M; Eray, M; Pellinen, R; Wahlfors, J; Pelkonen, J

    2009-12-01

    During the germinal centre reaction (GC), B cells with non-functional or self-reactive antigen receptors are negatively selected by apoptosis to generate B cell repertoire with appropriate antigen specificities. We studied the molecular mechanism of Fas/CD95- and B cell receptor (BCR)-induced apoptosis to shed light on the signalling events involved in the negative selection of GC B cells. As an experimental model, we used human follicular lymphoma (FL) cell line HF1A3, which originates from a GC B cell, and transfected HF1A3 cell lines overexpressing Bcl-x(L), c-FLIP(long) or dominant negative (DN) caspase-9. Fas-induced apoptosis was dependent on the caspase-8 activation, since the overexpression of c-FLIP(long), a natural inhibitor of caspase-8 activation, blocked apoptosis induced by Fas. In contrast, caspase-9 activation was not involved in Fas-induced apoptosis. BCR-induced apoptosis showed the typical characteristics of mitochondria-dependent (intrinsic) apoptosis. Firstly, the activation of caspase-9 was involved in BCR-induced DNA fragmentation, while caspase-8 showed only marginal role. Secondly, overexpression of Bcl-x(L) could block all apoptotic changes induced by BCR. As a novel finding, we demonstrate that caspase-9 can enhance the cytochrome-c release and collapse of mitochondrial membrane potential (DeltaPsi(m)) during BCR-induced apoptosis. The requirement of different signalling pathways in apoptosis induced by BCR and Fas may be relevant, since Fas- and BCR-induced apoptosis can thus be regulated independently, and targeted to different subsets of GC B cells.

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

    Science.gov (United States)

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

    2013-01-01

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

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

    International Nuclear Information System (INIS)

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

    2001-01-01

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

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

    International Nuclear Information System (INIS)

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

    2006-01-01

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

  17. Increased Sensitivity to Angiotensin II is Present Postpartum in Women with History of Hypertensive Pregnancy

    Science.gov (United States)

    Saxena, Aditi R.; Karumanchi, S. Ananth; Brown, Nancy J.; Royle, Caroline M.; McElrath, Thomas F.; Seely, Ellen W.

    2010-01-01

    Pregnancies complicated by new onset hypertension are associated with increased sensitivity to angiotensin II, but it is unclear if this sensitivity persists postpartum. We studied pressor response to infused angiotensin II in 25 normotensive postpartum women in both high and low sodium balance. Ten women had history of hypertensive pregnancy (five with preeclampsia; five with transient hypertension of pregnancy) and 15 women had history of uncomplicated, normotensive pregnancy. Systolic and diastolic blood pressures, aldosterone and soluble fms-like tyrosine kinase 1 (sFlt-1) levels were measured before and after angiotensin II infusion in both dietary phases. In high sodium balance, women with history of hypertensive pregnancy were normotensive but had significantly higher systolic and diastolic blood pressures than controls (115 vs. 104 mmHg and 73 vs. 65 mmHg, respectively, ppregnancy had pressor response to salt loading, demonstrated by increase in systolic blood pressure on high salt diet. They also had greater systolic pressor response (10 vs. 2 mmHg, p=0.03), greater increase in aldosterone (56.8 vs. 30.8 ng/dL, p=0.03) and increase in sFlt-1 levels (11.0 vs. -18.9 pg/mL, p=0.02) after infusion of angiotensin II in low sodium balance, compared with controls. Thus, women with history of hypertensive pregnancy demonstrated salt sensitivity of blood pressure and had increased pressor, adrenal and sFlt-1 responses to infused angiotensin II in low sodium balance. Increased sensitivity to angiotensin II observed during pregnancy in women with hypertensive pregnancy is present postpartum; this feature may contribute to future cardiovascular risk in these women. PMID:20308605

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

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

    Science.gov (United States)

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

    2018-05-23

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

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

    African Journals Online (AJOL)

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

  1. Human eosinophils are direct targets to nanoparticles: Zinc oxide nanoparticles (ZnO) delay apoptosis and increase the production of the pro-inflammatory cytokines IL-1β and IL-8.

    Science.gov (United States)

    Silva, Luis Rafael; Girard, Denis

    2016-09-30

    Zinc oxide NPs (ZnO) have been recently proposed as novel candidates for the treatment of allergic inflammatory diseases. Paradoxically, recent data suggested that ZnO could cause eosinophilic airway inflammation in rodents. Despite the above observations, there are currently no studies reporting direct interaction between a given NP and human eosinophils themselves. In this study, freshly isolated human eosinophils were incubated with ZnO and several cellular functions were studied. We found that ZnO delay human eosinophil apoptosis, partially by inhibiting caspases and by preventing caspase-4 and Bcl-xL degradation. ZnO do not induce production of reactive oxygen species but increase de novo protein synthesis. In addition, ZnO were found to increase the production of the proinflammatory IL-1β and IL-8 cytokines. Using a pharmacological approach, we demonstrated that inhibition of caspase-1 reversed the ability of ZnO to induce IL-1β and IL-8 production, whereas inhibition of caspase-4 only reversed that of IL-8. Our results indicate the necessity of conducting studies to determine the potential of using NP as nanotherapies, particularly in diseases in which eosinophils may be involved. We conclude that, indeed, human eosinophils represent potential new direct targets to NPs, ZnO in the present case. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  2. 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.

  3. Anti-apoptotic effect of caspase inhibitors on H₂O₂-treated HeLa cells through early suppression of its oxidative stress.

    Science.gov (United States)

    Park, Woo Hyun

    2014-05-01

    Oxidative stress-induced cytotoxicity in cervical cancer cells may be of toxicological interest. In the present study, the effects of exogenous H2O2 on cell growth and death in HeLa cervical cancer cells were investigated, and the anti-apoptotic effects of various caspase (pan-caspase, caspase-3, -8 or -9) inhibitors on H2O2-treated HeLa cells were also evaluated with regard to reactive oxygen species (ROS) and glutathione (GSH) levels. Based on MTT assays, H2O2 inhibited the growth of HeLa cells with an IC50 value of ~75 µM at 24 h. H2O2 increased the number of dead cells and Annexin V-FITC-positive cells in the HeLa cells, which was accompanied by the activation of caspase-3 and the loss of mitochondrial membrane potential (MMP; ΔΨm). However, relatively higher doses of H2O2 induced necrosis in HeLa cells. Caspase inhibitors significantly prevented H2O2-induced HeLa cell death. H2O2 increased ROS including O2•- at 24 h and increased the activity of catalase in HeLa cells. H2O2 also increased the ROS level at 1 h, and several caspase inhibitors attenuated the increased level at 1 h but not at 6, 12 and 24 h. H2O2 decreased the GSH level in HeLa cells at 1 h, and several caspase inhibitors attenuated the decreased level of GSH at this time. H2O2 induced GSH depletion at 24 h. In conclusion, H2O2 inhibited the growth of HeLa cells via apoptosis and/or necrosis, which was accompanied by intracellular increases in ROS levels and GSH depletion. Caspase inhibitors are suggested to suppress H2O2-induced oxidative stress to rescue HeLa cells at the early time point of 1 h.

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

    Science.gov (United States)

    Moorwood, Catherine; Barton, Elisabeth R.

    2014-01-01

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

  5. The interaction between the light source dose and caspase-dependent and -independent apoptosis in human SK-MEL-3 skin cancer cells following photodynamic therapy with zinc phthalocyanine: A comparative study.

    Science.gov (United States)

    Doustvandi, Mohammad Amin; Mohammadnejad, Fateme; Mansoori, Behzad; Mohammadi, Ali; Navaeipour, Farzaneh; Baradaran, Behzad; Tajalli, Habib

    2017-11-01

    The aim of this study is to determine the behavior of relative expression of Bcl-2, caspase-8, caspase-9, and caspase-3 genes of/in SK-MEL-3 cancer cells and explore molecular mechanisms responsible for the apoptosis response during an in vitro photodynamic therapy (PDT) with Zinc Phthalocyanine (ZnPc) using different doses of the light source. In this study, firstly the cytotoxic effects of ZnPc-PDT on SK-MEL-3 cells were evaluated. By irradiating the laser, ZnPc induced a significant amount of apoptosis on SK-MEL-3 cells in three IC 50 s including 0.064±0.01, 0.043±0.01, and 0.036±0.01μg/mL at the doses of 8, 16, and 24J/cm 2 , respectively. Moreover, flow cytometry and QRT-PCR experiments were done. The high percentage of apoptotic cells was seen in the early apoptosis stage. The expression of Bcl-2 and caspase-8 genes at all doses of laser experienced an obvious reduction in comparison to the control group. On the other hand, although the expression of caspase-9 and caspase-3 genes remains almost constant at 8J/cm 2 , but they faced an increment at 16 and 24J/cm 2 doses. These data reveal caspase-dependent apoptosis in high and caspase-independent apoptosis in low doses of laser. Based on the results of present work, it can be suggested that the dose of the light source is a key factor in induction of caspase-dependent and caspase-independent apoptosis pathways following PDT. Copyright © 2017. Published by Elsevier B.V.

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

    Science.gov (United States)

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

    2017-03-01

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

  7. 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.

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

    Science.gov (United States)

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

    2006-09-01

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

  9. Fascaplysin sensitizes cells to TRAIL-induced apoptosis through upregulating DR5 expression

    Science.gov (United States)

    Wang, Feng; Chen, Haimin; Yan, Xiaojun; Zheng, Yanling

    2013-05-01

    This study investigated the molecular mechanism of anti-tumor effect of fascaplysin, a nitrogenous red pigment firstly isolated from a marine sponge. Microarray analysis show that the TNF and TNF receptor superfamily in human umbilical vein endothelial cells (HUVEC) and human hepatocarcinoma cells (BEL-7402) were significantly regulated by fascaplysin. Western Blot results reveal that fascaplysin increased the expression of cleaved caspase-9, active caspase-3, and decreased the level of procaspase-8 and Bid. Flow cytometry and cytotoxicity tests indicate that fascaplysin sensitized cells to tumor necrosis-related apoptosisinducing ligand-(TRAIL) induced apoptosis, which was markedly blocked by TRAIL R2/Fc chimera, a dominant negative form of TRAIL receptor DR5. Therefore, our results demonstrate that fascaplysin promotes apoptosis through the activation of TRAIL signaling pathway by upregulating DR5 expression.

  10. Citric acid induces cell-cycle arrest and apoptosis of human immortalized keratinocyte cell line (HaCaT) via caspase- and mitochondrial-dependent signaling pathways.

    Science.gov (United States)

    Ying, Tsung-Ho; Chen, Chia-Wei; Hsiao, Yu-Ping; Hung, Sung-Jen; Chung, Jing-Gung; Yang, Jen-Hung

    2013-10-01

    Citric acid is an alpha-hydroxyacid (AHA) widely used in cosmetic dermatology and skincare products. However, there is concern regarding its safety for the skin. In this study, we investigated the cytotoxic effects of citric acid on the human keratinocyte cell line HaCaT. HaCaT cells were treated with citric acid at 2.5-12.5 mM for different time periods. Cell-cycle arrest and apoptosis were investigated by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, flow cytometry, western blot and confocal microscopy. Citric acid not only inhibited proliferation of HaCaT cells in a dose-dependent manner, but also induced apoptosis and cell cycle-arrest at the G2/M phase (before 24 h) and S phase (after 24 h). Citric acid increased the level of Bcl-2-associated X protein (BAX) and reduced the levels of B-cell lymphoma-2 (BCL-2), B-cell lymphoma-extra large (BCL-XL) and activated caspase-9 and caspase-3, which subsequently induced apoptosis via caspase-dependent and caspase-independent pathways. Citric acid also activated death receptors and increased the levels of caspase-8, activated BH3 interacting-domain death agonist (BID) protein, Apoptosis-inducing factor (AIF), and Endonuclease G (EndoG). Therefore, citric acid induces apoptosis through the mitochondrial pathway in the human keratinocyte cell line HaCaT. The study results suggest that citric acid is cytotoxic to HaCaT cells via induction of apoptosis and cell-cycle arrest in vitro.

  11. Chronic Obstructive Pulmonary Disease-Derived Circulating Cells Release IL-18 and IL-33 under Ultrafine Particulate Matter Exposure in a Caspase-1/8-Independent Manner

    Directory of Open Access Journals (Sweden)

    Gianluigi De Falco

    2017-10-01

    Full Text Available Chronic obstructive pulmonary disease (COPD is considered the fourth-leading causes of death worldwide; COPD is caused by inhalation of noxious indoor and outdoor particles, especially cigarette smoke that represents the first risk factor for this respiratory disorder. To mimic the effects of particulate matter on COPD, we isolated peripheral blood mononuclear cells (PBMCs and treated them with combustion-generated ultrafine particles (UFPs obtained from two different fuel mixtures, namely, pure ethylene and a mixture of ethylene and dimethylfuran (the latter mimicking the combustion of biofuels. UFPs were separated in two fractions: (1 sub-10 nm particles, named nano organic carbon (NOC particles and (2 primarily soot particles of 20–40 nm and their agglomerates (200 nm. We found that both NOC and soot UFPs induced the release of IL-18 and IL-33 from unstable/exacerbated COPD-derived PBMCs. This effect was associated with higher levels of mitochondrial dysfunction and derived reactive oxygen species, which were higher in PBMCs from unstable COPD patients after combustion-generated UFP exposure. Moreover, lower mRNA expression of the repairing enzyme OGG1 was associated with the higher levels of 8-OH-dG compared with non-smoker and smokers. It was interesting that IL-18 and IL-33 release from PBMCs of unstable COPD patients was not NOD-like receptor 3/caspase-1 or caspase-8-dependent, but rather correlated to caspase-4 release. This effect was not evident in stable COPD-derived PBMCs. Our data suggest that combustion-generated UFPs induce the release of caspase-4-dependent inflammasome from PBMCs of COPD patients compared with healthy subjects, shedding new light into the biology of this key complex in COPD.

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

    Science.gov (United States)

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

    2013-09-15

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

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

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

    Science.gov (United States)

    Park, Woo Hyun

    2018-04-01

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

  15. 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.

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2017-08-01

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

  18. Increased spontaneous mitotic segregation in MMS-sensitive mutants of Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Prakash, S.; Prakash, L.

    1977-01-01

    Methyl methanesulfonate (MMS)-sensitive mutants of Saccharomyces cerevisiae belonging to four different complementation groups, when homozygous, increase the rate of spontaneous mitotic segregation to canavanine resistance from heterozygous sensitive (can/sup r//+) diploids by 13- to 170-fold. The mms8-1 mutant is MMS and x-ray sensitive and increases the rate of spontaneous mitotic segregation 170-fold. The mms9-1 and mms13-1 mutants are sensitive to x rays and uv, respectively, in addition to MMS, and increase the rate of spontaneous mitotic segregation by 13-fold and 85-fold, respectively. The mutant mms21-1 is sensitive to MMS, x rays and uv and increases the rate of spontaneous mitotic segregation 23-fold

  19. Short-chain C6 ceramide sensitizes AT406-induced anti-pancreatic cancer cell activity

    International Nuclear Information System (INIS)

    Zhao, Xiaoguang; Sun, Baoyou; Zhang, Jingjing; Zhang, Ruishen; Zhang, Qing

    2016-01-01

    Our previous study has shown that AT406, a first-in-class small molecular antagonist of IAPs (inhibitor of apoptosis proteins), inhibits pancreatic cancer cell proliferation in vitro and in vivo. The aim of this research is to increase AT406's sensitivity by adding short-chain C6 ceramide. We show that co-treatment of C6 ceramide dramatically potentiated AT406-induced caspase/apoptosis activation and cytotoxicity in established (Panc-1 and Mia-PaCa-2 lines) and primary human pancreatic cancer cells. Reversely, caspase inhibitors largely attenuated C6 ceramide plus AT406-induced above cancer cell death. Molecularly, C6 ceramide downregulated Bcl-2 to increase AT406's sensitivity in pancreatic cancer cells. Intriguingly, C6 ceramide-mediated AT406 sensitization was nullified with Bcl-2 shRNA knockdown or pretreatment of the Bcl-2 inhibitor ABT-737. In vivo, liposomal C6 ceramide plus AT406 co-administration dramatically inhibited Panc-1 xenograft tumor growth in severe combined immunodeficient (SCID) mice. The combined anti-tumor activity was significantly more potent than either single treatment. Expressions of IAPs (cIAP1/XIAP) and Bcl-2 were downregulated in Panc-1 xenografts with the co-administration. Together, we demonstrate that C6 ceramide sensitizes AT406-mediated anti-pancreatic cancer cell activity possibly via downregulating Bcl-2. - Highlights: • C6 ceramide dramatically potentiates AT406-induced pancreatic cancer cell death. • C6 ceramide facilitates AT406-induced pancreatic cancer cell apoptosis. • C6 ceramide downregulates Bcl-2 to increase AT406's sensitivity in pancreatic cancer cells. • Liposomal C6 ceramide enhances AT406-induced anti-pancreatic cancer activity in vivo.

  20. An in vitro test to screen skin sensitizers using a stable THP-1-derived IL-8 reporter cell line, THP-G8.

    Science.gov (United States)

    Takahashi, Toshiya; Kimura, Yutaka; Saito, Rumiko; Nakajima, Yoshihiro; Ohmiya, Yoshihiro; Yamasaki, Kenshi; Aiba, Setsuya

    2011-12-01

    Several studies have suggested that interleukin (IL)-8 can serve as a biomarker for discrimination of skin sensitizers from nonsensitizers. We established a stable THP-1-derived IL-8 reporter cell line, THP-G8, which harbors SLO and SLR luciferase genes under the control of IL-8 and glyceraldehyde 3-phosphate dehydrogenase promoters, respectively. After 6 h treatment with chemicals, normalized SLO luciferase activity (nSLO-LA) was calculated by dividing SLO-LA by SLR-LA, and the fold induction of nSLO-LA (FInSLO-LA) was calculated by dividing nSLO-LA of chemically treated cells by that of nontreated cells. The nSLO-LA of THP-G8 cells increased in response to lipopolysaccharide (LPS) and several sensitizers. The FInSLO-LA in THP-G8 cells induced by LPS or sensitizers positively correlated with their induction of IL-8 messenger RNA in THP-1 cells. The nSLO-LA value of THP-G8 cells was significantly increased (FInSLO-LA ≥ 1.4) by 13 of the 15 sensitizers as well as by 5 of the 7 nonsensitizers. Interestingly, pretreatment with N-acetylcysteine suppressed the increase in FInSLO-LA induced by all sensitizers (inhibition index (II) ≤ 0.8) but did not suppress that induced by most of the nonsensitizers. We then evaluated the performance of this assay using values of FInSLO-LA ≥ 1.4 and II ≤ 0.8 in at least two of three independent experiments as the criteria of a sensitizer, which resulted in test accuracies of 82% for the 22 chemicals used and of 88% for the chemicals proposed by European Center for the Validation of Alternative Methods. This newly developed assay is a candidate replacement for animal tests of skin sensitization because of its accuracy, convenience, and high throughput performance.

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

    Science.gov (United States)

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

    2016-04-01

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

  2. Interferon-gamma sensitizes colonic epithelial cell lines to physiological and therapeutic inducers of colonocyte apoptosis.

    LENUS (Irish Health Repository)

    O'Connell, J

    2012-02-03

    Homeostasis in the colonic epithelium is achieved by a continuous cycle of proliferation and apoptosis, in which imbalances are associated with disease. Inflammatory bowel disease (IBD) and colon cancer are associated with either excessive or insufficient apoptosis of colonic epithelial cells, respectively. By using two colonic epithelial cell lines, HT29 and SW620, we investigated how the epithelial cell\\'s sensitivity to apoptosis was regulated by the proinflammatory cytokine interferon-gamma (IFN-gamma). We found that IFN-gamma sensitized HT29 cells, and to a lesser extent SW620, to diverse inducers of apoptosis of physiologic or therapeutic relevance to the colon. These apoptosis inducers included Fas (CD95\\/APO-1) ligand (FasL), short-chain fatty acids, and chemotherapeutic drugs. The extent of IFN-gamma-mediated apoptosis sensitization in these two cell lines correlated well with the degree of IFN-gamma-mediated upregulation of the proapoptotic protease caspase-1. Although IFN-gamma alone effectively sensitized HT29 cells to apoptosis, inclusion of the protein synthesis inhibitor cyclohexamide (CHX) during apoptotic challenge was necessary for maximal sensitization of SW620. The requirement of CHX to sensitize SW620 cells to apoptosis implies a need to inhibit translation of antiapoptotic proteins absent from HT29. In particular, the antiapoptotic protein Bcl-2 was strongly expressed in SW620 cells but absent from HT29. Our results indicate that IFN-gamma increases the sensitivity of colonic epithelial cells to diverse apoptotic stimuli in concert, via upregulation of caspase-1. Our findings implicate caspase-1 and Bcl-2 as important central points of control determining the general sensitivity of colonic epithelial cells to apoptosis.

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

    Directory of Open Access Journals (Sweden)

    Sebastian Heikaus

    2010-01-01

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

  4. Expression of Caspase-3, P53 in EL-4 cells induced by ionizing radiation and its biological implications

    International Nuclear Information System (INIS)

    Ju Guizhi; Shen Bo; Sun Shilong; Yan Fengqin; Fu Shibo; Li Pengwu

    2006-01-01

    Objective: To investigate the effect of ionizing radiation on the expressions of Caspase-3 and P53 proteins in EL-4 cells and its implications in the induction of apoptosis and polyploid cells. Methods: EL- 4 cells were irradiated with 4.0 Gy X-rays (180 kV, 15 mA, 0.287 Gy/min). Fluorescent staining and flow cytometry analysis were used to measure protein expression, apoptosis and polyploid cells. Results: It was found that the expression of Caspase-3 protein was increased significantly at 8 h and 12 h after the irradiation compared with sham-irradiated control (P<0.05), and the expression of P53 protein was also increased significantly at 2,4,8,12 and 24 h after the irradiation compared with sham-irradiated control (P<0.05 or P<0.01). The results showed that apoptosis of EL-4 cells was increased significantly at 2,4,8,12,24,48, and 72 h after 4.0 Gy irradiation compared with sham-irradiated control (P<0.05 or P<0.01 or P<0.001). However, no significant change in the number of polyploidy cells was found during the period from 2 to 48 h after the irradiation with 4.0 Gy X-rays. Conclusions: It is indicated that the expressions of Caspase-3 and P53 protein in EL-4 cells can be induced by ionizing radiation, and play an important role in the induction of apoptosis; the molecular pathway for polyploid formation might be P53-independent. (authors)

  5. 20(s)-Protopanaxadiol (PPD) increases the radiotherapy sensitivity of laryngeal carcinoma.

    Science.gov (United States)

    Teng, Bo; Zhao, Lijing; Gao, Jing; He, Peng; Li, Hejie; Chen, Junyu; Feng, Qingjie; Yi, Chunhui

    2017-12-13

    Laryngeal carcinoma (LC) is one of the most prevalent malignant tumors in the head and neck area. Due to its high morbidity and mortality, LC poses a serious threat to human life and health. Even with surgical removal, some patients were not sensitive to radiotherapy or experienced transfer or recurrence. 20(s)-Protopanaxadiol (PPD), a natural product from Panax ginseng, has been reported to have cytotoxic effects against several cancer cell lines. However, whether it can improve the radiation sensitivity and the underlying mechanism of PPD's sensitization effect is still unknown. Herein, from in vitro and in vivo experiments, we found that the combination of PPD and radiation not only significantly inhibited proliferation and induced apoptosis, but also suppressed the tumor growth in mouse models. These findings confirmed the role of PPD in enhancing the sensitivity of radiotherapy. Moreover, our work showed that the expression levels of mTOR and its downstream effectors decreased remarkably after PPD addition when compared to radiation only. This result suggested that PPD's excellent synergistic effects with radiation might be associated with the down-regulation of the mTOR signaling pathway in Hep-2 cells.

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

    International Nuclear Information System (INIS)

    Li Xiaoming; Song Tianbao

    1999-01-01

    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

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

    Science.gov (United States)

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

    2012-10-10

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

  8. The in vitro immunogenic potential of caspase-3 proficient breast cancer cells with basal low immunogenicity is increased by hypofractionated irradiation.

    Science.gov (United States)

    Kötter, Bernhard; Frey, Benjamin; Winderl, Markus; Rubner, Yvonne; Scheithauer, Heike; Sieber, Renate; Fietkau, Rainer; Gaipl, Udo S

    2015-09-17

    Radiotherapy is an integral part of breast cancer treatment. Immune activating properties of especially hypofractionated irradiation are in the spotlight of clinicians, besides the well-known effects of radiotherapy on cell cycle and the reduction of the clonogenic potential of tumor cells. Especially combination of radiotherapy with further immune stimulation induces immune-mediated anti-tumor responses. We therefore examined whether hypofractionated irradiation alone or in combination with hyperthermia as immune stimulants is capable of inducing breast cancer cells with immunogenic potential. Clonogenic assay, AnnexinA5-FITC/Propidium iodide assay and ELISA analyses of heat shock protein 70 and high mobility group box 1 protein were applied to characterize colony forming capability, cell death induction, cell death forms and release of danger signals by breast cancer cells in response to hypofractionated radiation (4x4Gy, 6x3Gy) alone and in combination with hyperthermia (41.5 °C for 1 h). Caspase-3 deficient, hormone receptor positive, p53 wild type MCF-7 and caspase-3 intact, hormone receptor negative, p53 mutated MDA-MB231 breast cancer cells, the latter in absence or presence of the pan-caspase inhibitor zVAD-fmk, were used. Supernatants of the treated tumor cells were analyzed for their potential to alter the surface expression of activation markers on human-monocyte-derived dendritic cells. Irradiation reduced the clonogenicity of caspase deficient MCF-7 cells more than of MDA-B231 cells. In contrast, higher amounts of apoptotic and necrotic cells were induced in MDA-B231 cells after single irradiation with 4Gy, 10Gy, or 20Gy or after hypofractionated irradiation with 4x4Gy or 6x3Gy. MDA-B231 cells consecutively released higher amounts of Hsp70 and HMGB1 after hypofractionated irradiation. However, only the release of Hsp70 was further increased by hyperthermia. Both, apoptosis induction and release of the danger signals, was dependent on caspase-3. Only

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

    Science.gov (United States)

    Bidle, Kay D; Kwityn, Clifford J

    2012-10-01

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

  10. Alterations in blood pressure, antioxidant status and caspase 8 expression in cobalt chloride-induced cardio-renal dysfunction are reversed by Ocimum gratissimum and gallic acid in Wistar rats.

    Science.gov (United States)

    Akinrinde, A S; Oyagbemi, A A; Omobowale, T O; Asenuga, E R; Ajibade, T O

    2016-07-01

    The protective abilities of the chloroform extract of Ocimum gratissimum (COG) and gallic acid against cobalt chloride (CoCl2) - induced cardiac and renal toxicity were evaluated. Rats were exposed to CoCl2 (350ppm) for 7 days, either alone, or in combination with COG (100 and 200mg/kg) or gallic acid (120mg/kg). CoCl2 given alone, caused significant increases (pgallic acid treatment significantly reduced (pgallic acid by modulation of CoCl2-induced alterations in blood pressure, antioxidant status and pro-apoptotic caspase 8 in Wistar rats. Copyright © 2016 Elsevier GmbH. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Zhang YueMei

    2005-02-01

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

  12. Parkinson’s Disease: Low-Dose Haloperidol Increases Dopamine Receptor Sensitivity and Clinical Response

    Directory of Open Access Journals (Sweden)

    Craig J. Hudson

    2014-01-01

    Full Text Available Background. It is known that ultra-low doses of haloperidol can cause dopamine supersensitivity of dopamine D2 receptors and related behaviour in animals. Objective. The objective was to determine whether a daily ultra-low dose of 40 micrograms of haloperidol could enhance the clinical action of levodopa in Parkinson’s disease patients. Method. While continuing their daily treatment with levodopa, 16 patients with Parkinson’s disease were followed weekly for six weeks. They received an add-on daily dose of 40 micrograms of haloperidol for the first two weeks only. The SPES/SCOPA scale (short scale for assessment of motor impairments and disabilities in Parkinson’s disease was administered before treatment and weekly throughout the trial. Results. The results showed a mean decrease in SPES/SCOPA scores after one week of the add-on treatment. Conclusion. SCOPA scores decreased after the addition of low-dose haloperidol to the standard daily levodopa dose. This finding is consistent with an increase in sensitivity of dopamine D2 receptors induced by haloperidol. Such treatment for Parkinson’s disease may possibly permit the levodopa dose to be reduced and, thus, delay the onset of levodopa side effects.

  13. Increased Levels of S100A8/A9 in Patients with Peritonsillar Abscess: A New Promising Diagnostic Marker to Differentiate between Peritonsillar Abscess and Peritonsillitis.

    Science.gov (United States)

    Spiekermann, Christoph; Russo, Antonella; Stenner, Markus; Rudack, Claudia; Roth, Johannes; Vogl, Thomas

    2017-01-01

    Peritonsillar abscess (PTA) is a very frequent reason for urgent outpatient consultation and otolaryngological hospital admission. Early, correct diagnosis and therapy of peritonsillar abscess are important to prevent possible life-threatening complications. Based on physical examinations, a reliable differentiation between peritonsillar cellulitis and peritonsillar abscess is restricted. A heterodimeric complex called calprotectin consists of the S100 proteins A8 and A9 (S100A8/A9) and is predominantly expressed not only in monocytes and neutrophils but also in epithelial cells. Due to its release by activated phagocytes at local sites of inflammation, we assumed S100A8/A9 to be a potential biomarker for peritonsillar abscess. We examined serum and saliva of patients with peritonsillitis, acute tonsillitis, peritonsillar abscess, and healthy controls and found significantly increased levels of S100A8/A9 in patients with PTA. Furthermore, we could identify halitosis, trismus, uvula edema, and unilateral swelling of the arched palate to be characteristic symptoms for PTA. Using a combination of these characteristic symptoms and S100A8/A9 levels, we developed a PTA score as an objective and appropriate tool to differentiate between peritonsillitis and peritonsillar abscess with a sensitivity of 92% and specificity of 93%.

  14. Increased Levels of S100A8/A9 in Patients with Peritonsillar Abscess: A New Promising Diagnostic Marker to Differentiate between Peritonsillar Abscess and Peritonsillitis

    Directory of Open Access Journals (Sweden)

    Christoph Spiekermann

    2017-01-01

    Full Text Available Peritonsillar abscess (PTA is a very frequent reason for urgent outpatient consultation and otolaryngological hospital admission. Early, correct diagnosis and therapy of peritonsillar abscess are important to prevent possible life-threatening complications. Based on physical examinations, a reliable differentiation between peritonsillar cellulitis and peritonsillar abscess is restricted. A heterodimeric complex called calprotectin consists of the S100 proteins A8 and A9 (S100A8/A9 and is predominantly expressed not only in monocytes and neutrophils but also in epithelial cells. Due to its release by activated phagocytes at local sites of inflammation, we assumed S100A8/A9 to be a potential biomarker for peritonsillar abscess. We examined serum and saliva of patients with peritonsillitis, acute tonsillitis, peritonsillar abscess, and healthy controls and found significantly increased levels of S100A8/A9 in patients with PTA. Furthermore, we could identify halitosis, trismus, uvula edema, and unilateral swelling of the arched palate to be characteristic symptoms for PTA. Using a combination of these characteristic symptoms and S100A8/A9 levels, we developed a PTA score as an objective and appropriate tool to differentiate between peritonsillitis and peritonsillar abscess with a sensitivity of 92% and specificity of 93%.

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

    Science.gov (United States)

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

    2011-06-01

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

  16. Sensitization of interferon-γ induced apoptosis in human osteosarcoma cells by extracellular S100A4

    International Nuclear Information System (INIS)

    Pedersen, Kjetil Boye; Andersen, Kristin; Fodstad, Øystein; Mælandsmo, Gunhild Mari

    2004-01-01

    S100A4 is a small Ca 2+ -binding protein of the S100 family with metastasis-promoting properties. Recently, secreted S100A4 protein has been shown to possess a number of functions, including induction of angiogenesis, stimulation of cell motility and neurite extension. Cell cultures from two human osteosarcoma cell lines, OHS and its anti-S100A4 ribozyme transfected counterpart II-11b, was treated with IFN-γ and recombinant S100A4 in order to study the sensitizing effects of extracellular S100A4 on IFN-γ mediated apoptosis. Induction of apoptosis was demonstrated by DNA fragmentation, cleavage of poly (ADP-ribose) polymerase and Lamin B. In the present work, we found that the S100A4-expressing human osteosarcoma cell line OHS was more sensitive to IFN-γ-mediated apoptosis than the II-11b cells. S100A4 protein was detected in conditioned medium from OHS cells, but not from II-11b cells, and addition of recombinant S100A4 to the cell medium sensitized II-11b cells to apoptosis induced by IFN-γ. The S100A4/IFN-γ-mediated induction of apoptosis was shown to be independent of caspase activation, but dependent on the formation of reactive oxygen species. Furthermore, addition of extracellular S100A4 was demonstrated to activate nuclear factor-κB (NF-κB). In conclusion, we have shown that S100A4 sensitizes osteosarcoma cells to IFN-γ-mediated induction of apoptosis. Additionally, extracellular S100A4 activates NF-κB, but whether these events are causally related remains unknown

  17. Collateral sensitivity to cisplatin in KB-8-5-11 drug-resistant cancer cells.

    Science.gov (United States)

    Doherty, Ben; Lawlor, Denise; Gillet, Jean-Pierre; Gottesman, Michael; O'Leary, John J; Stordal, Britta

    2014-01-01

    KB-8-5-11 cells are a drug-resistant cervical cell model that overexpresses ABCB1 (P-glycoprotein). KB-8-5-11 has become sensitive to non-ABCB1 substrate cisplatin. Understanding the mechanism of collateral sensitivity to cisplatin may lead to biomarker discovery for platinum sensitivity in patients with cancer. A Taqman low-density array was used to characterize the expression of 380 genes previously associated with chemoresistance. Identified pathways were further analyzed using cytotoxicity assays, metabolomics and western blots. KB-8-5-11 cells were sensitive to CuSO4 and the glutathione inhibitor buthionine sulphoximine. Expression of ATPase, Cu(2+) transporting alpha (ATP7A) and ATP7B were decreased at the protein and gene levels respectively in KB-8-5-11. KB-8-5-11 had decreased gene expression of glutathione S-transferase pi 1 (GSTP1), GSTA4 and GSTK1. Cisplatin treatment significantly lowered total cellular glutathione in parental KB-3-1 cells. Glutathione also tended to be lower in KB-8-5-11 cells compared to KB-3-1 cells. KB-8-5-11 cells have alterations in their copper transporters and glutathione metabolism, contributing to their cisplatin-sensitive phenotype.

  18. Osteogenic Potential of Caspases Related to Endochondral Ossification

    Czech Academy of Sciences Publication Activity Database

    Janečková, E.; Bíliková, P.; Matalová, Eva

    2018-01-01

    Roč. 66, č. 1 (2018), s. 47-58 ISSN 0022-1554 Institutional support: RVO:67985904 Keywords : caspases * endochronal ossification * growth plate Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Developmental biology Impact factor: 2.511, year: 2016

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

    Science.gov (United States)

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

    2002-01-01

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

  20. Increased insulin sensitivity in intrauterine growth retarded newborns--do thyroid hormones play a role?

    Science.gov (United States)

    Setia, Sajita; Sridhar, M G; Koner, B C; Bobby, Zachariah; Bhat, Vishnu; Chaturvedula, Lata

    2007-02-01

    Thyroid hormones are necessary for normal brain development. We studied thyroid hormone profile and insulin sensitivity in intrauterine growth retarded (IUGR) newborns to find correlation between insulin sensitivity and thyroid status in IUGR newborns. Fifty IUGR and fifty healthy control infants were studied at birth. Cord blood was collected for determination of T(3), T(4), TSH, glucose and insulin levels. IUGR newborns had significantly lower insulin, mean+/-S.D., 5.25+/-2.81 vs. 11.02+/-1.85microU/ml, but significantly higher insulin sensitivity measured as glucose to insulin ratio (G/I), 9.80+/-2.91 vs. 6.93+/-1.08 compared to healthy newborns. TSH was also significantly higher 6.0+/-2.70 vs. 2.99+/-1.05microU/ml with significantly lower T(4), 8.65+/-1.95 vs. 9.77+/-2.18microg/dl, but similar T(3) levels, 100.8+/-24.36 vs. 101.45+/-23.45ng/dl. On stepwise linear regression analysis in IUGR infants, insulin sensitivity was found to have a significant negative association with T(4) and significant positive association with TSH. Thyroid hormones may play a role in increased insulin sensitivity at birth in IUGR.

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

    Science.gov (United States)

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

    2018-06-01

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

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  3. Caspase-8 Deficiency Presenting as Late-Onset Multi-Organ Lymphocytic Infiltration with Granulomas in two Adult Siblings.

    Science.gov (United States)

    Niemela, Julie; Kuehn, Hye Sun; Kelly, Corin; Zhang, Mingchang; Davies, Joie; Melendez, Jose; Dreiling, Jennifer; Kleiner, David; Calvo, Katherine; Oliveira, João B; Rosenzweig, Sergio D

    2015-05-01

    Caspase-8 deficiency (CED) was originally described in 2002 in two pediatric patients presenting with clinical manifestations resembling autoimmune lymphoproliferative syndrome (ALPS) accompanied by infections, and T, B and NK cell defects. Since then, no new CED patients were published. Here we report two adult siblings (Pt1 and Pt2) presenting in their late thirties with pulmonary hypertension leading to lung transplant (Pt1), and a complex neurological disease leading to multiple cranial nerves palsies (Pt2) as their main manifestations. A thorough clinical and immunological evaluation was performed at the Primary Immunodeficiency Clinic at NIH, followed by whole exome sequencing. The patients had multiorgan lymphocytic infiltration and granulomas, as well as clinical signs of immune deficiency/ immune dysregulation. Both siblings carried homozygous mutations in CASP8, c.1096C > T, p.248R > W. This was the same mutation described on the previously published CED patients, to whom these new patients were likely distantly related. We report two new CED patients presenting during adulthood with life-threatening end-organ lymphocyte infiltrates affecting the lungs, liver, spleen, bone marrow and central nervous system. This phenotype broadens the clinical spectrum of manifestations associated with this disease and warrants the search of CASP8 mutations in other cohorts of patients.

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

    Science.gov (United States)

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

    2012-01-01

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

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

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

    International Nuclear Information System (INIS)

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

    2010-01-01

    Previous work by our group demonstrated that homomeric α7 nicotinic acetylcholine receptors (nAChR) play a role in the neurotoxicity induced by 3,4-methylenedioxymethamphetamine (MDMA), as well as the binding affinity of this drug to these receptors. Here we studied the effect of MDMA on the activation of nAChR subtypes, the consequent calcium mobilization, and calpain/caspase 3 activation because prolonged Ca 2+ increase could contribute to cytotoxicity. As techniques, we used fluorimetry in Fluo-4-loaded PC12 cells and electrophysiology in Xenopus oocytes. MDMA produced a rapid and sustained increase in calcium without reaching the maximum effect induced by ACh. It also concentration-dependently inhibited the response induced by ACh, nicotine, and the specific α7 agonist PNU 282987 with IC 50 values in the low micromolar range. Similarly, MDMA induced inward currents in Xenopus oocytes transfected with human α7 but not with α4β2 nAChR and inhibited ACh-induced currents in both receptors in a concentration-dependent manner. The calcium response was inhibited by methyllycaconitine (MLA) and α-bungarotoxin but not by dihydro-β-erythroidine. These results therefore indicate that MDMA acts as a partial agonist on α7 nAChRs and as an antagonist on the heteromeric subtypes. Subsequently, calcium-induced Ca 2+ release from the endoplasmic reticulum and entry through voltage-operated calcium channels are also implicated as proved using specific antagonists. In addition, treatment with MDMA for 24 h significantly increased basal Ca 2+ levels and induced an increase in α-spectrin breakdown products, which indicates that calpain and caspase 3 were activated. These effects were inhibited by pretreatment with MLA. Moreover, pretreatment with MDMA induced functional upregulation of calcium responses to specific agonists of both heteromeric and α7 nAChR. Sustained calcium entry and calpain activation could favor the activation of Ca 2+ -dependent enzymes such as

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Liang Pang

    2015-05-01

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

  9. Cadmium-sensitive electrode based on tetracetone derivatives of p-tert-butylcalix[8]arene

    Energy Technology Data Exchange (ETDEWEB)

    Dernane, C. [Université de Jijel, Laboratoire de Matériaux: Elaborations-Propriétés-Applications, BP 98, Ouled Aissa, 18000 Jijel (Algeria); Zazoua, A., E-mail: azazoua@yahoo.fr [Université de Jijel, Laboratoire de Matériaux: Elaborations-Propriétés-Applications, BP 98, Ouled Aissa, 18000 Jijel (Algeria); Kazane, I. [Université de Jijel, Laboratoire de Matériaux: Elaborations-Propriétés-Applications, BP 98, Ouled Aissa, 18000 Jijel (Algeria); Jaffrezic-Renault, N. [Université de Lyon, LSA-UMR 5180 CNRS, Université Claude Bernard Lyon 1, 69622 Villeurbanne cedex (France)

    2013-10-15

    The performance of a cadmium-sensitive electrode based on the tetracetone derivatives of p-tert butylcalix[8]arene was investigated. The ion-sensitivity of the calix[8]arene was examined via cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), X-ray photoelectron spectrometry, UV/Vis spectroscopy and FT-IR spectroscopy. The sensitive membrane containing the active ionophore was cast onto the surface of a gold electrode. The electrode exhibited a linear relationship between the charge transfer resistance (R{sub ct}) and the logarithm of the detected ion concentration. The cathodic peak at a potential of 0.56 V increased linearly as the Cd{sup 2+} ion concentration increased. The detection limit of the device reached 10{sup −7} M with high sensitivity toward cadmium. - Highlights: • The performances of cadmium-sensitive electrode were investigated. • The sensitive membrane was casted on the surface of a gold electrode. • The electrode showed a linear relationship between the R{sub ct} and the ion concentration. • The detection limit of the device was 10{sup −7} M with high sensitivity toward cadmium.

  10. Increased soluble serum markers caspase-cleaved cytokeratin-18, histones, and ST2 indicate apoptotic turnover and chronic immune response in COPD.

    Science.gov (United States)

    Hacker, Stefan; Lambers, Christopher; Pollreisz, Andreas; Hoetzenecker, Konrad; Lichtenauer, Michael; Mangold, Andreas; Niederpold, Tina; Hacker, Andreas; Lang, György; Dworschak, Martin; Vukovich, Thomas; Gerner, Christopher; Klepetko, Walter; Ankersmit, Hendrik Jan

    2009-01-01

    Chronic obstructive pulmonary disease (COPD) is a worldwide burden and a major cause of death. The disease is accompanied by chronic inflammation and increased cellular turnover that is partly due to an overwhelming induction of apoptosis. In this study, we hypothesized that systemic markers of apoptosis are altered in patients with mild-to-severe COPD. A total number of 64 patients and controls were enrolled in the study. Lung function parameters of all groups (nonsmoker, healthy smoker, COPD GOLD I&II, COPD GOLD III&IV) were evaluated at the time of inclusion. Enzyme-linked immunosorbent assays were used to quantify protein levels in serum samples. Serum contents of apoptotic end-products caspase-cleaved cytokeratin-18 and histone-associated-DNA-fragments were increased in patients with COPD, whereas anti-inflammatory soluble ST2 showed a peak in patients with COPD I&II (P=0.031) compared to healthy smokers. Levels of pro-inflammatory caspase-1/ ICE correlated significantly with the number of pack years (R=0.337; P=0.007). Our results indicate a systemic release of apoptosis-specific proteins as markers for increased cellular turnover accompanied by progression of COPD. Furthermore, soluble ST2 seems to have a critical role in the anti-inflammatory regulatory mechanism at early stages of the disease.

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

    Science.gov (United States)

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

    2017-10-01

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

  12. Caspases : more than just killers?

    OpenAIRE

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

    2001-01-01

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

  13. Caspases: more than just killers?

    Science.gov (United States)

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

    2001-01-01

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

  14. TRAIL-induced cleavage and inactivation of SPAK sensitizes cells to apoptosis

    International Nuclear Information System (INIS)

    Polek, Tara C.; Talpaz, Moshe; Spivak-Kroizman, Taly R.

    2006-01-01

    Ste20-related proline-alanine-rich kinase (SPAK) has been linked to various cellular processes, including proliferation, differentiation, and ion transport regulation. Recently, we showed that SPAK mediates signaling by the TNF receptor, RELT. The presence of a caspase cleavage site in SPAK prompted us to study its involvement in apoptotic signaling induced by another TNF member, TRAIL. We show that TRAIL stimulated caspase 3-like proteases that cleaved SPAK at two distinct sites. Cleavage had little effect on the activity of SPAK but removed its substrate-binding domain. In addition, TRAIL reduced the activity of SPAK in HeLa cells in a caspase-independent manner. Thus, TRAIL inhibited SPAK by two mechanisms: activation of caspases, which removed its substrate-binding domain, and caspase-independent down-regulation of SPAK activity. Furthermore, reducing the amount of SPAK by siRNA increased the sensitivity of HeLa cells to TRAIL-induced apoptosis. Thus, TRAIL down-regulation of SPAK is an important event that enhances its apoptotic effects

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

    Directory of Open Access Journals (Sweden)

    Banun Kusumawardani

    2016-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Fauzi Yusuf

    2017-02-01

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

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

    Science.gov (United States)

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

    2017-05-01

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

  19. Rocaglamide overcomes tumor necrosis factor-related apoptosis-inducing ligand resistance in hepatocellular carcinoma cells by attenuating the inhibition of caspase-8 through cellular FLICE-like-inhibitory protein downregulation.

    Science.gov (United States)

    Luan, Zhou; He, Ying; He, Fan; Chen, Zhishui

    2015-01-01

    The enhancement of apoptosis is a therapeutic strategy used in the treatment of cancer. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising antitumor agent. However, hepatocellular carcinoma (HCC) cells exhibit marked resistance to the induction of cell death by TRAIL. The present study investigated whether rocaglamide, a naturally occurring product isolated from the genus Aglaia, is able to sensitize resistant HCC cells to TRAIL-mediated apoptosis. Two HCC cell lines, HepG2 and Huh-7, were treated with rocaglamide and/or TRAIL and the induction of apoptosis and effects on the TRAIL signaling pathway were investigated. The in vivo efficacy of rocaglamide was determined in TRAIL-resistant Huh-7-derived tumor xenografts. Rocaglamide significantly sensitized the TRAIL-resistant HCC cells to apoptosis by TRAIL, which resulted from the rocaglamide-mediated downregulation of cellular FLICE-like inhibitory protein and subsequent caspase-8 activation. Furthermore, rocaglamide markedly inhibited tumor growth from Huh-7 cells propagated in severe combined immunodeficient mice, suggesting that chemosentization also occurred in vivo. These data suggest that rocaglamide acted synergistically with TRAIL against the TRAIL-resistant HCC cells. Thus, it is concluded that rocaglamide as an adjuvant to TRAIL-based therapy may present a promising therapeutic approach for the treatment of HCC.

  20. Caspase-2-dependent dendritic cell death, maturation, and priming of T cells in response to Brucella abortus infection.

    Directory of Open Access Journals (Sweden)

    Xinna Li

    Full Text Available Smooth virulent Brucella abortus strain 2308 (S2308 causes zoonotic brucellosis in cattle and humans. Rough B. abortus strain RB51, derived from S2308, is a live attenuated cattle vaccine strain licensed in the USA and many other countries. Our previous report indicated that RB51, but not S2308, induces a caspase-2-dependent apoptotic and necrotic macrophage cell death. Dendritic cells (DCs are professional antigen presenting cells critical for bridging innate and adaptive immune responses. In contrast to Brucella-infected macrophages, here we report that S2308 induced higher levels of apoptotic and necrotic cell death in wild type bone marrow-derived DCs (WT BMDCs than RB51. The RB51 and S2308-induced BMDC cell death was regulated by caspase-2, indicated by the minimal cell death in RB51 and S2308-infected BMDCs isolated from caspase-2 knockout mice (Casp2KO BMDCs. More S2308 bacteria were taken up by Casp2KO BMDCs than wild type BMDCs. Higher levels of S2308 and RB51 cells were found in infected Casp2KO BMDCs compared to infected WT BMDCs at different time points. RB51-infected wild type BMDCs were mature and activated as shown by significantly up-regulated expression of CD40, CD80, CD86, MHC-I, and MHC-II. RB51 induced the production of cytokines TNF-α, IL-6, IFN-γ and IL12/IL23p40 in infected BMDCs. RB51-infected WT BMDCs also stimulated the proliferation of CD4(+ and CD8(+ T cells compared to uninfected WT BMDCs. However, the maturation, activation, and cytokine secretion are significantly impaired in Casp2KO BMDCs infected with RB51 or Salmonella (control. S2308-infected WT and Casp2KO BMDCs were not activated and could not induce cytokine production. These results demonstrated that virulent smooth strain S2308 induced more apoptotic and necrotic dendritic cell death than live attenuated rough vaccine strain RB51; however, RB51, but not its parent strain S2308, induced caspase-2-mediated DC maturation, cytokine production, antigen

  1. Cordycepin enhances cisplatin apoptotic effect through caspase/MAPK pathways in human head and neck tumor cells

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    Chen YH

    2013-07-01

    Full Text Available Ying-Hui Chen,1,2,* Jo-Yu Wang,3,* Bo-Syong Pan,3,4 Yi-Fen Mu,3 Meng-Shao Lai,3,4 Edmund Cheung So,5 Thian-Sze Wong,6 Bu-Miin Huang3,4 1Department of Anesthesia, Chi-Mei Medical Center, Liouying, 2Department of Nursing, Min-Hwei College of Health Care Management, 3Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, 4The Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, 5Department of Anesthesia, An Nan Hospital, China Medical University, Tainan, Taiwan; 6Department of Surgery, University of Hong Kong Medical Center, Faculty of Medicine, The University of Hong Kong, Hong Kong *Authors contributed equally to this work Purpose: The present study aims to investigate whether the combination treatment of cordycepin (an extracted pure compound from Cordyceps sinensis and cisplatin (a platinum-based chemotherapy drug has better apoptotic effect in head and neck squamous cell carcinoma (HNSCC. Methods: The apoptotic influences of cordycepin and/or cisplatin treatments to human OC3, OEC-M1, and FaDu HNSCC cells were investigated by morphological observations, viability assay, flow cytometry assay, and Western blotting methods. Results: Data showed that the cell death phenomenon increased as the dosage of cordycepin or cisplatin increased, and it appeared more in cordycepin plus cisplatin cotreatment among three cell lines. Cell survival rates significantly decreased as the dosage of cordycepin or cisplatin increased, and the better apoptotic effects were observed in cotreatment. Cell cycle analysis further demonstrated that percentages of subG1 cells in cordycepin or cisplatin treatments significantly increased, suggesting that cells underwent apoptosis, and cordycepin plus cisplatin induced many more subG1 cells. Furthermore, cordycepin or cisplatin induced caspase-8, caspase-9, caspase-3, and poly adenosine diphosphate-ribose polymerase protein cleavages, and stimulated c

  2. Inhibition of Histone Deacetylases Permits Lipopolysaccharide-Mediated Secretion of Bioactive IL-1β via a Caspase-1-Independent Mechanism.

    Science.gov (United States)

    Stammler, Dominik; Eigenbrod, Tatjana; Menz, Sarah; Frick, Julia S; Sweet, Matthew J; Shakespear, Melanie R; Jantsch, Jonathan; Siegert, Isabel; Wölfle, Sabine; Langer, Julian D; Oehme, Ina; Schaefer, Liliana; Fischer, Andre; Knievel, Judith; Heeg, Klaus; Dalpke, Alexander H; Bode, Konrad A

    2015-12-01

    Histone deacetylase (HDAC) inhibitors (HDACi) are clinically approved anticancer drugs that have important immune-modulatory properties. We report the surprising finding that HDACi promote LPS-induced IL-1β processing and secretion in human and murine dendritic cells and murine macrophages. HDACi/LPS-induced IL-1β maturation and secretion kinetics differed completely from those observed upon inflammasome activation. Moreover, this pathway of IL-1β secretion was dependent on caspase-8 but was independent of the inflammasome components NACHT, LRR, and PYD domains-containing protein 3, apoptosis-associated speck-like protein containing a carboxyl-terminal caspase-recruitment domain, and caspase-1. Genetic studies excluded HDAC6 and HDAC10 as relevant HDAC targets in this pathway, whereas pharmacological inhibitor studies implicated the involvement of HDAC11. Treatment of mice with HDACi in a dextran sodium sulfate-induced colitis model resulted in a strong increase in intestinal IL-1β, confirming that this pathway is also operative in vivo. Thus, in addition to the conventional inflammasome-dependent IL-1β cleavage pathway, dendritic cells and macrophages are capable of generating, secreting, and processing bioactive IL-1β by a novel, caspase-8-dependent mechanism. Given the widespread interest in the therapeutic targeting of IL-1β, as well as the use of HDACi for anti-inflammatory applications, these findings have substantial clinical implications. Copyright © 2015 by The American Association of Immunologists, Inc.

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

    Science.gov (United States)

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

    2017-05-18

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

  4. Natural indoles, indole-3-carbinol (I3C and 3,3'-diindolylmethane (DIM, attenuate staphylococcal enterotoxin B-mediated liver injury by downregulating miR-31 expression and promoting caspase-2-mediated apoptosis.

    Directory of Open Access Journals (Sweden)

    Philip B Busbee

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

  5. 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.

  6. High Ca2+ Influx During Traumatic Brain Injury Leads to Caspase-1-Dependent Neuroinflammation and Cell Death.

    Science.gov (United States)

    Abdul-Muneer, P M; Long, Mathew; Conte, Adriano Andrea; Santhakumar, Vijayalakshmi; Pfister, Bryan J

    2017-08-01

    We investigated the hypothesis that high Ca 2+ influx during traumatic brain injury induces the activation of the caspase-1 enzyme, which triggers neuroinflammation and cell apoptosis in a cell culture model of neuronal stretch injury and an in vivo model of fluid percussion injury (FPI). We first established that stretch injury causes a rapid increase in the intracellular Ca 2+ level, which activates interleukin-converting enzyme caspase-1. The increase in the intracellular Ca 2+ level and subsequent caspase-1 activation culminates into neuroinflammation via the maturation of IL-1β. Further, we analyzed caspase-1-mediated apoptosis by TUNEL staining and PARP western blotting. The voltage-gated sodium channel blocker, tetrodotoxin, mitigated the stretch injury-induced neuroinflammation and subsequent apoptosis by blocking Ca 2+ influx during the injury. The effect of tetrodotoxin was similar to the caspase-1 inhibitor, zYVAD-fmk, in neuronal culture. To validate the in vitro results, we demonstrated an increase in caspase-1 activity, neuroinflammation and neurodegeneration in fluid percussion-injured animals. Our data suggest that neuronal injury/traumatic brain injury (TBI) can induce a high influx of Ca 2+ to the cells that cause neuroinflammation and cell death by activating caspase-1, IL-1β, and intrinsic apoptotic pathways. We conclude that excess IL-1β production and cell death may contribute to neuronal dysfunction and cognitive impairment associated with TBI.

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-12-15

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

  9. Androgens increase lws opsin expression and red sensitivity in male three-spined sticklebacks.

    Directory of Open Access Journals (Sweden)

    Yi Ta Shao

    Full Text Available Optomotor studies have shown that three-spined sticklebacks (Gasterosteus aculeatus are more sensitive to red during summer than winter, which may be related to the need to detect the red breeding colour of males. This study aimed to determine whether this change of red light sensitivity is specifically related to reproductive physiology. The mRNA levels of opsin genes were examined in the retinae of sexually mature and immature fish, as well as in sham-operated males, castrated control males, or castrated males implanted with androgen 11-ketoandrostenedione (11 KA, maintained under stimulatory (L16:D8 or inhibitory (L8:D16 photoperiods. In both sexes, red-sensitive opsin gene (lws mRNA levels were higher in sexually mature than in immature fish. Under L16:D8, lws mRNA levels were higher in intact than in castrated males, and were up-regulated by 11 KA treatment in castrated males. Moreover, electroretinogram data confirmed that sexual maturation resulted in higher relative red spectral sensitivity. Mature males under L16:D8 were more sensitive to red light than males under L8:D16. Red light sensitivity under L16:D8 was diminished by castration, but increased by 11 KA treatment. Thus, in sexually mature male sticklebacks, androgen is a key factor in enhancing sensitivity to red light via regulation of opsin gene expression. This is the first study to demonstrate that sex hormones can regulate spectral vision sensitivity.

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

    Directory of Open Access Journals (Sweden)

    Bhavnani Bhagu R

    2006-06-01

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

  11. A caspase-2-RFXANK interaction and its implication for MHC class II expression.

    Science.gov (United States)

    Forsberg, Jeremy; Li, Xinge; Akpinar, Birce; Salvatori, Roger; Ott, Martin; Zhivotovsky, Boris; Olsson, Magnus

    2018-01-23

    Despite recent achievements implicating caspase-2 in tumor suppression, the enzyme stands out from the apoptotic caspase family as a factor whose function requires further clarification. To specify enzyme characteristics through the definition of interacting proteins in apoptotic or non-apoptotic settings, a yeast 2-hybrid (Y2H) screen was performed using the full-length protein as bait. The current report describes the analysis of a captured prey and putative novel caspase-2 interacting factor, the regulatory factor X-associated ankyrin-containing protein (RFXANK), previously associated with CIITA, the transactivator regulating cell-type specificity and inducibility of MHC class II gene expression. The interaction between caspase-2 and RFXANK was verified by co-immunoprecipitations using both exogenous and endogenous proteins, where the latter approach suggested that binding of the components occurs in the cytoplasm. Cellular co-localization was confirmed by transfection of fluorescently conjugated proteins. Enhanced caspase-2 processing in RFXANK-overexpressing HEK293T cells treated with chemotherapeutic agents further supported Y2H data. Yet, no distinct differences with respect to MHC class II expression were observed in plasma membranes of antigen-presenting cells derived from wild type and caspase-2 -/- mice. In contrast, increased levels of the total MHC class II protein was evident in protein lysates from caspase-2 RNAi-silenced leukemia cell lines and B-cells isolated from gene-targeted mice. Together, these data identify a novel caspase-2-interacting factor, RFXANK, and indicate a potential non-apoptotic role for the enzyme in the control of MHC class II gene regulation.

  12. Acrylamide induces immunotoxicity through reactive oxygen species production and caspase-dependent apoptosis in mice splenocytes via the mitochondria-dependent signaling pathways.

    Science.gov (United States)

    Zamani, Ehsan; Shaki, Fatemeh; AbedianKenari, Saeid; Shokrzadeh, Mohammad

    2017-10-01

    Acrylamide (AA), a well-known food neo-contamination, can be produced during food preparing at high temperature. The immunotoxicity of AA have been revealed in the experimental animals. In this study, we explored the molecular mechanism responsible for the immunotoxicity of AA. The mice splenocytes exposed to AA concentrations (0,5,10 and 25 mM) and apoptosis cell death was measured through Annexin V/Propidium Iodide staining by flow cytometry method. The role of extrinsic and intrinsic pathways were evaluated respectively by activity of caspase-8 and-9. Furthermore, the spleen mitochondria were obtained using differential centrifugation from mice and mitochondrial toxicity endpoints were determined after AA exposure. Exposure of splenocytes to AA increased the splenocytes' apoptotic cell death. Also, increased activation of both caspase-8 and-9 were observed in mice splenocytes after AA exposure. Treatment of isolated mitochondria with AA lead to disturbance in activity of complex I and III of mitochondrial electron transfer chain that result in increased reactive oxygen species (ROS) production, lipid peroxidation and glutathione oxidation. These events were accompanied by mitochondrial membrane swelling, collapse of mitochondrial membrane potential and significant falling of mitochondrial activity. AA-mediated mitochondrial dysfunction along with mitochondrial oxidative damage seems to be critical events leading to activation of caspase cascade and apoptotic cell death in spleen that finally can attenuate immune system's function. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Tamayev Robert

    2012-12-01

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

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

    Science.gov (United States)

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

    2012-11-01

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

  15. Deep time evidence for climate sensitivity increase with warming

    DEFF Research Database (Denmark)

    Shaffer, Gary; Huber, Matthew; Rondanelli, Roberto

    2016-01-01

    warming analogue. We obtain constrained estimates of CO2 and climate sensitivity before and during the PETM and of the PETM carbon input amount and nature. Sensitivity increased from 3.3-5.6 to 3.7-6.5K (Kelvin) into the PETM. When taken together with Last Glacial Maximum and modern estimates, this result...... world, but past warming events may provide insight. Here we employ paleoreconstructions and new climate-carbon model simulations in a novel framework to explore a wide scenario range for the Paleocene-Eocene Thermal Maximum (PETM) carbon release and global warming event 55.8Ma ago, a possible future...

  16. Cell Morphological Change and Caspase-3 Protein Expression on Epithelial Cells under Stimulation of Oral Bacterium Streptococcus sanguinis

    Directory of Open Access Journals (Sweden)

    Suryani Hutomo

    2015-07-01

    Full Text Available Oral commensal bacterium Streptococcus sanguinis may find in periodontal lesions, deep seated infection, and infective endocarditis that are usually dominated by anaerobes. This bacterium caused cell death on some cells but host responses to this species remained unclear. Objective: This study was aimed to detect cell morphologica change and role of caspase-3 in cell death mechanism induced by S. sanguinis. Methods: HeLa cells as representative model for oral epithelial cells were exposed to 107 cells/ml bacteria for 48 h. Morphological change was observed microscopically after hematoxyline-eosin staining. Expression of active caspase-3 was examined by immunocytochemical analysis after cell stimulation for 36 and 48 h with wild type supragingival S. sanguinis. Doxorubicin (0.5625 μg/ml was used as positive control for caspase-3 activation. Results: The results showed cell shrinkage of bacterial-treated cells; and active caspase-3 molecules were detected after 36 and 48 hours cell stimulation. Conclusion: This study would suggest cell shrinkage and caspase-3-dependent apoptotic cell death induced by S. sanguinis.DOI: 10.14693/jdi.v22i1.375

  17. 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.

  18. Caspase 8/10 are not mediating apoptosis in neuroblastoma cells treated with CDK inhibitory drugs

    OpenAIRE

    Ribas i Fortuny, Judit; Gómez Arbonés, Javier; Boix Torras, Jacint

    2005-01-01

    Olomoucine and Roscovitine are pharmacological inhibitors of cyclin-dependent kinases (CDK) displaying a promising profile as anticancer agents. Both compounds are effective inductors of apoptosis in a human neuroblastoma cell line, SH-SY5Y. The characterization of this process had suggested the involvement of an extrinsic pathway [Ribas, J., Boix, J., 2004. Cell differentiation, Caspase inhibition, and macromolecular synthesis blockage, but not Bcl-2 or Bcl-XL proteins, protect SH-SY5Y cells...

  19. Increased Angiotensin II Sensitivity Contributes to Microvascular Dysfunction in Women Who Have Had Preeclampsia.

    Science.gov (United States)

    Stanhewicz, Anna E; Jandu, Sandeep; Santhanam, Lakshmi; Alexander, Lacy M

    2017-08-01

    Women who have had preeclampsia have increased cardiovascular disease risk; however, the mechanism(s) responsible for this association remain unclear. Microvascular damage sustained during a preeclamptic pregnancy may persist postpartum. The putative mechanisms mediating this dysfunction include a reduction in NO-dependent dilation and an increased sensitivity to angiotensin II. In this study, we evaluated endothelium-dependent dilation, angiotensin II sensitivity, and the therapeutic effect of angiotensin II receptor blockade (losartan) on endothelium-dependent dilation in vivo in the microvasculature of women with a history of preeclampsia (n=12) and control women who had a healthy pregnancy (n=12). We hypothesized that preeclampsia would have (1) reduced endothelium-dependent dilation, (2) reduced NO-mediated dilation, and (3) increased sensitivity to angiotensin II. We further hypothesized that localized losartan would increase endothelium-dependent vasodilation in preeclampsia. We assessed microvascular endothelium-dependent vasodilator function by measurement of cutaneous vascular conductance responses to graded infusion of acetylcholine (acetylcholine; 10 -7 -102 mmol/L) and a standardized local heating protocol in control sites and sites treated with 15 mmol/L L-NAME ( N G -nitro-l-arginine methyl ester; NO-synthase inhibitor) or 43 µmol/L losartan. Further, we assessed microvascular vasoconstrictor sensitivity to angiotensin II (10 -20 -10 -4 mol/L). Preeclampsia had significantly reduced endothelium-dependent dilation (-0.3±0.5 versus -1.0±0.4 log EC50 ; P Preeclampsia also had augmented vasoconstrictor sensitivity to angiotensin II (-10.2±1.3 versus -8.3±0.5; P =0.006). Angiotensin II type I receptor inhibition augmented endothelium-dependent vasodilation and NO-dependent dilation in preeclampsia but had no effect in healthy pregnancy. These data suggest that women who have had preeclampsia have persistent microvascular dysfunction postpartum

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

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    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.

  1. Responses of heat shock protein 70 and caspase-3/7 to dietary selenomethionine in juvenile white sturgeon

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    Weifang Wang

    2016-03-01

    Full Text Available An 8-week feeding trial was conducted to investigate the responses of juvenile white sturgeon (Acipenser transmontanus to elevated dietary selenium (Se based on the determination of the RNA/DNA ratio in muscle, heat shock protein 70 (Hsp70, and caspase-3/7 in muscle and/or liver tissues. Four semi-purified test diets were prepared by adding different levels of L-selenomethionine (0, 50, 100, and 200 mg/kg diet. The analytical determinations of total Se were 2.2, 19.7, 40.1, and 77.7 mg/kg diet. The sturgeon (initial body weight: 30 ± 2 g; mean ± SEM were raised in indoor tanks provided with flow through freshwater (18–19 °C. There were three replicates for each dietary treatment with 25 fish per replicate. The liver and muscle tissues were collected at 4 and 8 weeks after feeding the test diets. A significant interaction between duration and levels of dietary Se exposures on RNA/DNA ratio in the muscle tissue was detected (P < 0.05. Although there was no significant main effect due to the duration of dietary Se exposures (i.e., 4 weeks versus 8 weeks on muscle RNA/DNA ratio (P ≥ 0.05, the ratio was significantly decreased with increasing dietary Se levels. Significant main effects were caused by the duration and levels of dietary Se exposures on Hsp70 in both the muscle and liver tissues, with significant increases in Hsp70 due to a longer exposure (8 weeks and higher levels (40.1 and 77.7 mg Se/kg diet of dietary Se. The caspase-3/7 activity in the liver were significantly higher in fish fed the diets containing 40.1 and 77.7 mg Se/kg diet than those fed the other diets. The toxic thresholds of Se in the muscle were estimated to be 32.2 and 26.6 mg Se/kg for the depressed specific growth rate and the induced Hsp70 response in muscle, respectively. This result indicated that the Hsp70 response in muscle is a more sensitive biomarker than the SGR of sturgeon for evaluating Se toxicity in white sturgeon. Results of the

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

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

    2013-01-01

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

  3. Induction of Fas mediated caspase-8 independent apoptosis in immune cells by Armigeres subalbatus saliva.

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    Shanshan Liu

    Full Text Available BACKGROUND: It is widely recognized that the introduction of saliva of bloodsucking arthropods at the site of pathogen transmission might play a central role in vector-borne infections. However, how the interaction between salivary components and the host immune system takes place and which physiological processes this leads to has yet to be investigated. Armigeres subalbatus is one of the prominent types of mosquitoes involved in the transmission of parasitic and viral diseases in humans and animals. METHODOLOGY/PRINCIPAL FINDINGS: Using murine peritoneal macrophages and lymphocytes, and human peripheral mononuclear cells (PBMCs, this study shows that saliva of the female Ar. subalbatus induces apoptosis via interaction with the Fas receptor within a few hours but without activating caspase-8. The process further activates downstream p38 MAPK signaling, a cascade that leads to the induction of apoptosis in capase-3 dependent manner. We further illustrate that Ar. subalbatus saliva suppresses proinflammatory cytokines without changing IL-10 levels, which might happen as a result of apoptosis. CONCLUSIONS: Our study shows for the first time that saliva-induced apoptosis is the leading phenomenon exerted by Ar.subalbatus that impede immune cells leading to the suppression of their effecter mechanism.

  4. Caspase-1 from Human Myeloid-Derived Suppressor Cells Can Promote T Cell-Independent Tumor Proliferation.

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    Zeng, Qi; Fu, Juan; Korrer, Michael; Gorbounov, Mikhail; Murray, Peter J; Pardoll, Drew; Masica, David L; Kim, Young J

    2018-05-01

    Immunosuppressive myeloid-derived suppressive cells (MDSCs) are characterized by their phenotypic and functional heterogeneity. To better define their T cell-independent functions within the tumor, sorted monocytic CD14 + CD11b + HLA-DR low/- MDSCs (mMDSC) from squamous cell carcinoma patients showed upregulated caspase-1 activity, which was associated with increased IL1β and IL18 expression. In vitro studies demonstrated that mMDSCs promoted caspase-1-dependent proliferation of multiple squamous carcinoma cell lines in both human and murine systems. In vivo , growth rates of B16, MOC1, and Panc02 were significantly blunted in chimeric mice adoptively transferred with caspase-1 null bone marrow cells under T cell-depleted conditions. Adoptive transfer of wild-type Gr-1 + CD11b + MDSCs from tumor-bearing mice reversed this antitumor response, whereas caspase-1 inhibiting thalidomide-treated MDSCs phenocopied the antitumor response found in caspase-1 null mice. We further hypothesized that MDSC caspase-1 activity could promote tumor-intrinsic MyD88-dependent carcinogenesis. In mice with wild-type caspase-1, MyD88-silenced tumors displayed reduced growth rate, but in chimeric mice with caspase-1 null bone marrow cells, MyD88-silenced tumors did not display differential tumor growth rate. When we queried the TCGA database, we found that caspase-1 expression is correlated with overall survival in squamous cell carcinoma patients. Taken together, our findings demonstrated that caspase-1 in MDSCs is a direct T cell-independent mediator of tumor proliferation. Cancer Immunol Res; 6(5); 566-77. ©2018 AACR . ©2018 American Association for Cancer Research.

  5. Measuring T cell-mediated cytotoxicity using fluorogenic caspase substrates.

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    Chahroudi, A; Silvestri, G; Feinberg, M B

    2003-10-01

    Cytotoxic T lymphocytes (CTLs) play a major role in the immune response against viruses and other intracellular pathogens. In addition, CTLs are implicated in the control of tumor cells in certain settings. Accurate measures of CTL function are of critical importance to study the pathogenesis of infectious diseases and to evaluate the efficacy of new vaccines and immunotherapies. To this end, we have recently developed a flow cytometry-based CTL (FCC) assay that measures the CTL-induced caspase activation within target cells using cell permeable fluorogenic caspase substrates. This novel assay reliably detects, by flow cytometry or fluorescence/confocal microscopy, antigen-specific CTLs in a wide variety of human and murine systems, and is safer and more informative than the standard 51Cr-release assay. In addition, the flow cytometric CTL (FCC) assay provides an alternative method that is often more sensitive and physiologically informative when compared to previously described FCC assays, as it measures a biological indicator of apoptosis within the target cell. The FCC assay may thus represent a useful tool to further understand the molecular and cellular mechanisms that underlie CTL-mediated killing during tumorigenesis or following infection with viruses or other intracellular pathogens.

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

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

    2017-07-15

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

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

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

    2017-06-05

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

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

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    Indranila KS

    2016-04-01

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

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

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

    2008-03-01

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

  10. Heterogeneity reduces sensitivity of cell death for TNF-Stimuli

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    Schliemann Monica

    2011-12-01

    Full Text Available Abstract Background Apoptosis is a form of programmed cell death essential for the maintenance of homeostasis and the removal of potentially damaged cells in multicellular organisms. By binding its cognate membrane receptor, TNF receptor type 1 (TNF-R1, the proinflammatory cytokine Tumor Necrosis Factor (TNF activates pro-apoptotic signaling via caspase activation, but at the same time also stimulates nuclear factor κB (NF-κB-mediated survival pathways. Differential dose-response relationships of these two major TNF signaling pathways have been described experimentally and using mathematical modeling. However, the quantitative analysis of the complex interplay between pro- and anti-apoptotic signaling pathways is an open question as it is challenging for several reasons: the overall signaling network is complex, various time scales are present, and cells respond quantitatively and qualitatively in a heterogeneous manner. Results This study analyzes the complex interplay of the crosstalk of TNF-R1 induced pro- and anti-apoptotic signaling pathways based on an experimentally validated mathematical model. The mathematical model describes the temporal responses on both the single cell level as well as the level of a heterogeneous cell population, as observed in the respective quantitative experiments using TNF-R1 stimuli of different strengths and durations. Global sensitivity of the heterogeneous population was quantified by measuring the average gradient of time of death versus each population parameter. This global sensitivity analysis uncovers the concentrations of Caspase-8 and Caspase-3, and their respective inhibitors BAR and XIAP, as key elements for deciding the cell's fate. A simulated knockout of the NF-κB-mediated anti-apoptotic signaling reveals the importance of this pathway for delaying the time of death, reducing the death rate in the case of pulse stimulation and significantly increasing cell-to-cell variability. Conclusions Cell

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

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    Darin Bloemberg

    2016-06-01

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

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

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    Elkington Paul T

    2011-05-01

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

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

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    Pearce, John A.

    2013-02-01

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

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

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    Daniel P Denning

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

  15. Gram-scale synthesis of catalytic Co9S8 nanocrystal ink as a cathode material for spray-deposited, large-area dye-sensitized solar cells.

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    Chang, Shu-Hao; Lu, Ming-De; Tung, Yung-Liang; Tuan, Hsing-Yu

    2013-10-22

    We report the development of Co9S8 nanocrystals as a cost-effective cathode material that can be readily combined with spraying techniques to fabricate large-area dye-sensitized solar cell (DSSC) devices and can be further connected with series or parallel cell architectures to obtain a relatively high output voltage or current. A gram-scale synthesis of Co9S8 nanocrystal is carried out via a noninjection reaction by mixing anhydrous CoCl2 with trioctylphosphine (TOP), dodecanethiol and oleylamine (OLA) at 250 °C. The Co9S8 nanocrystals possess excellent catalytic ability with respect to I(-)/I3(-) redox reactions. The Co9S8 nanocrystals are prepared as nanoinks to fabricate uniform, crack-free Co9S8 thin films on different substrates by using a spray deposition technique. These Co9S8 films are used as counter electrodes assembled with dye-adsorbed TiO2 photoanodes to fabricate DSSC devices having a working area of 2 cm(2) and an average power conversion efficiency (PCE) of 7.02 ± 0.18% under AM 1.5 solar illumination, which is comparable with the PCE of 7.2 ± 0.12% obtained using a Pt cathode. Furthermore, six 2 cm(2)-sized DSSC devices connected in series output an open-circuit voltage of 4.2 V that can power a wide range of electronic devices such as LED arrays and can charge commercial lithium ion batteries.

  16. Decreased expression of caspase3 in penis and prostate tissues of rat after the treatment with buceng (Pimpinella alpina Molk & Euricoma longifolia Jack

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    Taufiqurrachman Taufiqurrachman

    2013-02-01

    Full Text Available Background: Buceng {combination of pasak bumi (Eurycoma longifolia Jack and purwoceng (Pimpinella alpine Molk} has been proven to increase testosterone (Te level and decrease apoptosis. Unfortunately, there is no evidence whether these effects are mediated by the declining of caspase3. Objective of this study was to evaluate whether buceng could decrease the expression of caspase3 of penis and prostate cells in Sprague Dawley male rats.Methods: Twenty four Sprague Dawley male rats weighing 300 g (90 days old were randomly assigned into 4 groups of 6 male rats. Group A, rats were castrated and received buceng 50 mg. Group B, rats were not castrated, sacrifices as positive control. Group C, rats were castrated and given 2 mL aquadest as negative control. Group D, rats were castrated and got of 6.75 mg mesterolone, dissolved in 2 mL water. MANOVA statistical analysis was adopted to examine the difference expression of caspase3 in all groups. The comparison of caspase3 expression between two groups exhibiting difference values were evaluated by Post Hoc test.Results: MANOVA revealed statistically significant differences in the expression of caspase3 of penis and prostate tissues among the four groups. Post Hoct test also indicated that expression of caspase3 in group A (buceng (33.56; 35.83 was significantly lower compared to group C (negative control (54.33; 60.07 and group D (mesterolone (51.91;56.21, p = 0.000, and higher compared than group B or normal rats (29.40; 27.72, but statistically not significant (p = 0.826.Conclusion: The treatment of 50 mg buceng/day for 30 consecutive days could decrease caspase3 expression in penis and prostate cells. (Med J Indones. 2013;22:2-8Keywords: Apoptosis, buceng (Pimpinella alpine Molk – Eurycoma longifolia Jack, caspase

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

    Science.gov (United States)

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

    2013-01-01

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

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

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

    2015-09-01

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

  19. Involvement of proinflammatory S100A9/A8 in the atherocalcinosis of aortic valves

    Directory of Open Access Journals (Sweden)

    R. А. Moskalenko

    2017-04-01

    Full Text Available According to the results of the Euro-Heart Survey on Vascular Heart Disease the most common pathology is nonrheumatic aortic stenosis, it is also called as calcific aortic valve stenosis (CAVS, as in its pathogenesis the process of biomineralization of valve cusps and ring plays the main role. The aim of the work is the immunohistochemical study of mineralized tissue of aortic heart valves, which are affected by atherocalcinosis. Materials and methods. 30 samples of mineralized aortic valves (I group and 10 samples of aortic valve without evidence of biomineralization (II group -– control were studied. Immunohistochemical study of expression of collagen (Collagen I, CD68, myeloperoxidase (MPO, calgranulin A (S100A8, calgranulin B (S100A9, caspase 3 (Casp 3 and osteopontin (OPN was conducted in AV tissue of both groups. Results. In CAV tissues the fibrillar component (collagen I growths was found, but the quantitative and qualitative compositions of CD68+ circulating inflammatory cells are not significantly different from the control group. CAVs contain much more MPO+ -cells (p <0.001 in comparison to the group of AVs without biomineralization. Our data show a significant increase of the S100A9 and OPN expression in the mineralized tissue of AVs (p < 0.01. Also, a higher expression level of Casp3 and MPO was found in CAVs (p < 0.05. Comparing the first and the second groups of AVs connection between the expression of S100A8 was not determined. Conclusion. High Casp 3 expression confirms the increased level of cell elimination in the CAVs tissue, which is obviously connected with the impact of high local concentrations of S100A9. These facts can contribute to the development of pathological biomineralization of AV. Since osteopontin inhibits the hydroxyapatite formation by binding to the surface of the crystals, its hyperproduction is a counteracting factor against biomineralization in AV tissue.

  20. Role of mitochondrial ATP-sensitive potassium channel-mediated PKC-ε in delayed protection against myocardial ischemia/reperfusion injury in isolated hearts of sevoflurane-preconditioned rats

    Energy Technology Data Exchange (ETDEWEB)

    Wang, C. [Department of Anesthesiology and Critical Care, The Second Affiliate Hospital, Soochow University, Suzhou (China); Institute of Neuroscience, Soochow University, Suzhou (China); Hu, S.M. [Institute of Neuroscience, Soochow University, Suzhou (China); Xie, H.; Qiao, S.G. [Department of Anesthesiology and Critical Care, The Second Affiliate Hospital, Soochow University, Suzhou (China); Liu, H. [Department of Anesthesiology and Pain Medicine, University of California Davis Health System, Davis, CA (United States); Liu, C.F. [Institute of Neuroscience, Soochow University, Suzhou (China)

    2015-03-27

    This study aimed to determine the role of mitochondrial adenosine triphosphate-sensitive potassium (mitoK{sub ATP}) channels and protein kinase C (PKC)-ε in the delayed protective effects of sevoflurane preconditioning using Langendorff isolated heart perfusion models. Fifty-four isolated perfused rat hearts were randomly divided into 6 groups (n=9). The rats were exposed for 60 min to 2.5% sevoflurane (the second window of protection group, SWOP group) or 33% oxygen inhalation (I/R group) 24 h before coronary occlusion. The control group (CON) and the sevoflurane group (SEVO) group were exposed to 33% oxygen and 2.5% sevoflurane for 60 min, respectively, without coronary occlusion. The mitoK{sub ATP} channel inhibitor 5-hydroxydecanoate (5-HD) was given 30 min before sevoflurane preconditioning (5-HD+SWOP group). Cardiac function indices, infarct sizes, serum cardiac troponin I (cTnI) concentrations, and the expression levels of phosphorylated PKC-ε (p-PKC-ε) and caspase-8 were measured. Cardiac function was unchanged, p-PKC-ε expression was upregulated, caspase-8 expression was downregulated, cTnI concentrations were decreased, and the infarcts were significantly smaller (P<0.05) in the SWOP group compared with the I/R group. Cardiac function was worse, p-PKC-ε expression was downregulated, caspase-8 expression was upregulated, cTnI concentration was increased and infarcts were larger in the 5-HD+SWOP group (P<0.05) compared with the SWOP group. The results suggest that mitoK{sub ATP} channels are involved in the myocardial protective effects of sevoflurane in preconditioning against I/R injury, by regulating PKC-ε phosphorylation before ischemia, and by downregulating caspase-8 during reperfusion.

  1. Increased skeletal muscle capillarization enhances insulin sensitivity

    DEFF Research Database (Denmark)

    Åkerström, Thorbjörn; Laub, Lasse; Vedel, Kenneth

    2014-01-01

    Increased skeletal muscle capillarization is associated with improved glucose tolerance and insulin sensitivity. However, a possible causal relationship has not previously been identified. We therefore investigated whether increased skeletal muscle capillarization increases insulin sensitivity....... Skeletal muscle specific angiogenesis was induced by adding the α1-adrenergic receptor antagonist Prazosin to the drinking water of Sprague Dawley rats (n=33) while 34 rats served as controls. Insulin sensitivity was measured ≥40 h after termination of the 3-week Prazosin treatment, which ensured...... that Prazosin was cleared from the blood stream. Whole-body insulin sensitivity was measured in conscious, unrestrained rats by hyperinsulinemic euglycemic clamp. Tissue specific insulin sensitivity was assessed by administration of 2-deoxy-[(3)H]-Glucose during the plateau phase of the clamp. Whole...

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

    Directory of Open Access Journals (Sweden)

    Victoria M Virador

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

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

    International Nuclear Information System (INIS)

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

    2014-01-01

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

  4. 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.

  5. 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.

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

    Directory of Open Access Journals (Sweden)

    Saif Khan

    2015-01-01

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

  7. Resveratrol sensitizes melanomas to TRAIL through modulation of antiapoptotic gene expression

    International Nuclear Information System (INIS)

    Ivanov, Vladimir N.; Partridge, Michael A.; Johnson, Geoffrey E.; Huang, Sarah X.L.; Zhou, Hongning; Hei, Tom K.

    2008-01-01

    Although many human melanomas express the death receptors TRAIL-R2/DR5 or TRAIL-R1/DR4 on cell surface, they often exhibit resistance to exogenous TRAIL. One of the main contributors to TRAIL-resistance of melanoma cells is upregulation of transcription factors STAT3 and NF-κB that control the expression of antiapoptotic genes, including cFLIP and Bcl-xL. On the other hand, the JNK-cJun pathway is involved in the negative regulation of cFLIP (a caspase-8 inhibitor) expression. Our observations indicated that resveratrol, a polyphenolic phytoalexin, decreased STAT3 and NF-κB activation, while activating JNK-cJun that finally suppressed expression of cFLIP and Bcl-xL proteins and increased sensitivity to exogenous TRAIL in DR5-positive melanomas. Interestingly, resveratrol did not increase surface expression of DR5 in human melanomas, while γ-irradiation or sodium arsenite treatment substantially upregulated DR5 expression. Hence, an initial increase in DR5 surface expression (either by γ-irradiation or arsenite), and subsequent downregulation of antiapoptotic cFLIP and Bcl-xL (by resveratrol), appear to constitute an efficient approach to reactivate apoptotic death pathways in TRAIL-resistant human melanomas. In spite of partial suppression of mitochondrial function and the mitochondrial death pathway, melanoma cells still retain the potential to undergo the DR5-mediated, caspase-8-dependent death pathway that could be accelerated by either an increase in DR5 surface expression or suppression of cFLIP. Taken together, these results suggest that resveratrol, in combination with TRAIL, may have a significant efficacy in the treatment of human melanomas

  8. 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.

  9. Calcium signals and caspase-12 participated in paraoxon-induced apoptosis in EL4 cells.

    Science.gov (United States)

    Li, Lan; Cao, Zhiheng; Jia, Pengfei; Wang, Ziren

    2010-04-01

    In order to investigate whether calcium signals participate in paraoxon (POX)-induced apoptosis in EL4 cells, real-time laser scanning confocal microscopy (LSCM) was used to detect Ca(2+) changes during the POX application. Apoptotic rates of EL4 cells and caspase-12 expression were also evaluated. POX (1-10nM) increased intracellular calcium concentration ([Ca(2+)]i) in EL4 cells in a dose-dependent manner at early stage (0-2h) of POX application, and apoptotic rates of EL4 cells after treatment with POX for 16h were also increased in a dose-dependent manner. Pre-treatment with EGTA, heparin or procaine attenuated POX-induced [Ca(2+)]i elevation and apoptosis. Additionally, POX up-regulated caspase-12 expression in a dose-dependent manner, and pre-treatment with EGTA, heparin or procaine significantly inhibited POX-induced increase of caspase-12 expression. Our results suggested that POX induced [Ca(2+)]i elevation in EL4 cells at the early stage of POX-induced apoptosis, which might involve Ca(2+) efflux from the endoplasmic reticulum (ER) and Ca(2+) influx from extracellular medium. Calcium signals and caspase-12 were important upstream messengers in POX-induced apoptosis in EL4 cells. The ER-associated pathway possibly operated in this apoptosis. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    2003-01-01

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

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

    Science.gov (United States)

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

    2017-12-01

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

  12. Effect of simvastatin in the autonomic system is dependent on the increased gain/sensitivity of the baroreceptors

    Science.gov (United States)

    Moreira, Edson D; Mostarda, Cristiano T; Moraes-Silva, Ivana C; Ferreira, Janaina B; dos Santos, Fernando; Lacchini, Silvia; De Angelis, Kátia; Rodrigues, Bruno; Irigoyen, Maria Cláudia

    2013-01-01

    A number of mechanisms have been proposed to explain the pleiotropic effect of statin therapy to reduce sympathetic outflow in cardiovascular disease. We tested the hypothesis that statin treatment could improve baroreflex gain-sensitivity triggered by morphological adaptations in the mechanoreceptor site, thus reducing sympathetic activity, regardless of arterial pressure (AP) level reduction. Male spontaneously hypertensive rats (SHR) were divided into control (SHR, n = 8) and SHR-simvastatin (5 mg/kg/day, for 7 days) (SHR-S, n = 8). After treatment, AP, baroreflex sensitivity (BRS) in response to AP-induced changes, aortic depressor nerve activity, and spectral analyses of pulse interval (PI) and AP variabilities were performed. Internal and external carotids were prepared for morphoquantitative evaluation. Although AP was similar between groups, sympathetic modulation, represented by the low frequency band of PI (SHR: 6.84 ± 3.19 vs. SHR-S: 2.41 ± 0.96 msec2) and from systolic AP variability (SHR: 3.95 ± 0.36 vs. SHR-S: 2.86 ± 0.18 mmHg2), were reduced in treated animals. In parallel, simvastatin induced an increase of 26% and 21% in the number of elastic lamellae as well as a decrease of 9% and 25% in the carotid thickness in both, external and internal carotid, respectively. Moreover, improved baroreceptor function (SHR: 0.78 ± 0.03 vs. SHR-S: 1.06 ± 0.04% mv/mmHg) was observed in addition to a 115% increase in aortic depressor nerve activity in SHR-S rats. Therefore, our data suggest that the reduction of sympathetic outflow in hypertension by simvastatin treatment may be triggered by structural changes in the carotid arteries and increased BRS in response to an improvement of the baroreceptors discharge and consequently of the afferent pathway of the baroreflex arch. PMID:24303130

  13. Rats exposed to 2.45GHz of non-ionizing radiation exhibit behavioral changes with increased brain expression of apoptotic caspase 3.

    Science.gov (United States)

    Varghese, Rini; Majumdar, Anuradha; Kumar, Girish; Shukla, Amit

    2018-03-01

    In recent years there has been a tremendous increase in use of Wi-Fi devices along with mobile phones, globally. Wi-Fi devices make use of 2.4GHz frequency. The present study evaluated the impact of 2.45GHz radiation exposure for 4h/day for 45days on behavioral and oxidative stress parameters in female Sprague Dawley rats. Behavioral tests of anxiety, learning and memory were started from day 38. Oxidative stress parameters were estimated in brain homogenates after sacrificing the rats on day 45. In morris water maze, elevated plus maze and light dark box test, the 2.45GHz radiation exposed rats elicited memory decline and anxiety behavior. Exposure decreased activities of super oxide dismutase, catalase and reduced glutathione levels whereas increased levels of brain lipid peroxidation was encountered in the radiation exposed rats, showing compromised anti-oxidant defense. Expression of caspase 3 gene in brain samples were quantified which unraveled notable increase in the apoptotic marker caspase 3 in 2.45GHz radiation exposed group as compared to sham exposed group. No significant changes were observed in histopathological examinations and brain levels of TNF-α. Analysis of dendritic arborization of neurons showcased reduction in number of dendritic branching and intersections which corresponds to alteration in dendritic structure of neurons, affecting neuronal signaling. The study clearly indicates that exposure of rats to microwave radiation of 2.45GHz leads to detrimental changes in brain leading to lowering of learning and memory and expression of anxiety behavior in rats along with fall in brain antioxidant enzyme systems. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Caspases in retinal ganglion cell death and axon regeneration

    Science.gov (United States)

    Thomas, Chloe N; Berry, Martin; Logan, Ann; Blanch, Richard J; Ahmed, Zubair

    2017-01-01

    Retinal ganglion cells (RGC) are terminally differentiated CNS neurons that possess limited endogenous regenerative capacity after injury and thus RGC death causes permanent visual loss. RGC die by caspase-dependent mechanisms, including apoptosis, during development, after ocular injury and in progressive degenerative diseases of the eye and optic nerve, such as glaucoma, anterior ischemic optic neuropathy, diabetic retinopathy and multiple sclerosis. Inhibition of caspases through genetic or pharmacological approaches can arrest the apoptotic cascade and protect a proportion of RGC. Novel findings have also highlighted a pyroptotic role of inflammatory caspases in RGC death. In this review, we discuss the molecular signalling mechanisms of apoptotic and inflammatory caspase responses in RGC specifically, their involvement in RGC degeneration and explore their potential as therapeutic targets. PMID:29675270

  15. 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.

  16. Increased sensitivity to ET-1 in rat cerebral arteries following organ culture

    DEFF Research Database (Denmark)

    Hansen-Schwartz, J; Edvinsson, L

    2000-01-01

    Endothelin-1 (ET-1) is recognized as being involved in the pathophysiology of cerebrovascular diseases. Using organ culture as a model for possible pathological changes we studied changes in ET(A) and ETB receptor function using a sensitive in vitro method. We observed an up-regulation of the ET......(B) receptor and an amazingly increased sensitivity to ET-1 by 3 log units in pEC50; pEC50(fresh) was 8.7 +/- 0.1, and pEC50(cultured) was 11.7 +/- 0.3. pA2 for FR139317 in the fresh vessel was 7.0 +/- 0.2 whereas it could not be obtained for the cultured vessel, indicating a possible cross-talk between the ET......(A) and ET(B) receptors. The increased sensitivity to ET-1 could also take place during cerebrovascular disease such as stroke or haemorrhage rendering the vessels considerably more sensitive to ET-1....

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2013-05-10

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

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

    Science.gov (United States)

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

    2013-01-01

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

  20. N-acetylphytosphingosine enhances the radiosensitivity of tumor cells by increasing apoptosis

    International Nuclear Information System (INIS)

    Han, Y.; Kim, Y.; Yun, Y.; Jeon, S.; Kim, K.; Song, J.; Hong, S.H.; Park, C.

    2005-01-01

    Ceramides are well-known second messengers which mediate apoptosis, proliferation, differentiation in mammalian cells, but the physiological roles of phytosphingosines are poorly understood. We hypothesized that one of the phytosphingosine derivatives, N-acetylphytosphingosine (NAPS) can induce apoptosis in human leukemia Jurkat cell line and increase apoptosis in irradiated MDA-MB-231 cells. We first examined the effect of NAPS on apoptosis of Jurkat cells. NAPS had a more rapid and stronger apoptotic effect than C 2 -ceramide in Jurkat cells and significant increase of apoptosis was observed at 3 h after treatment. In contrast, the apoptosis induced by C2-ceramide was observed only after 16 h of treatment. NAPS induced apoptosis was mediated by caspase 3 and 8 activation and inhibited by z-VAD-fmk. Ceramide plays a pivotal role in radiation induced apoptosis. We postulated that exogenous treatment of NAPS sensitizes tumor cells to ionizing radiation, since NAPS might be used as a more effective alternative to C2-ceramide. As expected, NAPS decreased clonogenic survival of irradiated MDA-MB-231 cells dose dependently, and apoptosis of irradiated cells in the presence of NAPS was increased through the caspase activation. Taken together, NAPS is an effective apoptosis-inducing agent, which can be readily synthesized from yeast sources, and is a potent alternative to ceramide for the further study of ceramide associated signaling and the development of radiosensitizing agent. (orig.)

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

    Science.gov (United States)

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

    2017-10-01

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

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

    Science.gov (United States)

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

    2012-01-01

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

  3. Increase in pollen sensitization in Swedish adults and protective effect of keeping animals in childhood.

    Science.gov (United States)

    Bjerg, A; Ekerljung, L; Eriksson, J; Näslund, J; Sjölander, S; Rönmark, E; Dahl, Å; Holmberg, K; Wennergren, G; Torén, K; Borres, M P; Lötvall, J; Lundbäck, B

    2016-10-01

    To date, most studies of the 'allergy epidemic' have been based on self-reported data. There is still limited knowledge on time trends in allergic sensitization, especially among adults. To study allergic sensitization, its risk factors and time trends in prevalence. Within West Sweden Asthma Study (WSAS), a population-based sample of 788 adults (17-60 years) underwent skin prick tests (SPTs) for 11 aeroallergens 2009-2012. Specific IgE was analysed in 750 of the participants. Those aged 20-46 years (n = 379) were compared with the European Community Respiratory Health Survey sample aged 20-46 year from the same area (n = 591) in 1991-1992. Among those aged 20-46 years, the prevalence of positive SPT to pollen increased, timothy from 17.1% to 29.0% (P < 0.001) and birch from 15.6% to 23.7% (P = 0.002) between 1991-1992 and 2009-2012. Measurements of specific IgE confirmed these increases. Prevalence of sensitization to all other tested allergens was unchanged. In the full WSAS sample aged 17-60 years, any positive SPT was seen in 41.9%, and the dominating sensitizers were pollen (34.3%), animals (22.8%) and mites (12.6%). Pollen sensitization was strongly associated with rhinitis, whereas indoor allergens were more associated with asthma. Growing up with livestock or furred pets decreased the risk of sensitization, adjusted odds ratio 0.53 (0.28-0.995) and 0.68 (0.47-0.98), respectively. Pollen sensitization has increased in Swedish adults since the early 1990s, while the prevalence of sensitization to other allergens has remained unchanged. This is one plausible explanation for the increase in rhinitis 1990-2008 in Swedish adults, during which time the prevalence of asthma, which is more associated with perennial allergens, was stable. Contact with animals in childhood seems to reduce the risk of sensitization well into adulthood. One major factor contributing to the rise in pollen allergy is a significant increase in levels of birch and grass pollen over the past

  4. Expression of caspase-3 predicts prognosis in advanced noncardia gastric cancer.

    Science.gov (United States)

    Amptoulach, Sousana; Lazaris, Andreas C; Giannopoulou, Ioanna; Kavantzas, Nikolaos; Patsouris, Efstratios; Tsavaris, Nikolaos

    2015-01-01

    There is strong evidence that tumor growth is not only a result of uncontrolled cell proliferation but also of decreased apoptosis. Caspase-3 is a member of interleukin-1 beta-converting enzyme which is involved in the induction of apoptosis. Data on the expression of caspase-3 in patients with gastric cancer and its association with patient outcome are somewhat contradictory. We aimed to investigate the potential relation of the expression of caspase-3 protein with response to therapy and overall survival in patients with advanced noncardia gastric cancer. Tumor tissue samples collected from 359 consecutive patients with gastric cancer stage IV were retrospectively analyzed for the expression of caspase-3 in the primary tumor. The DNA apoptotic index assessed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling method. All patients were followed up until death. Caspase-3 was expressed in 43.5 % of tumors. Caspase-3 expression compared to no expression was related with a higher DNA apoptotic index (p gastric cancer is a predictor of poor response to treatment and survival. Further studies are needed to fully elucidate the prognostic value of caspase-3 expression in these patients.

  5. [3-bromopyruvate enhances cisplatin sensitivity of hepatocellular carcinoma cells in vitro].

    Science.gov (United States)

    Zhao, Surong; Zhang, Yuanyuan; Wu, Chengzhu; Li, Hongmei; Jiang, Chenchen; Jiang, Zhiwen; Liu, Hao

    2014-01-01

    To investigate the effect of 3-bromopyruvate (3-BP) in sensitizing hepatocellular carcinoma cells to cisplatin-induced apoptosis and its possible mechanism. The growth inhibition of HepG2 and SMMC7721 cells following exposures to different concentrations of 3-BP and cisplatin was measured by MTT assay. The apoptosis of cells treated with 100 µmol/L 3-BP with or without 8 µmol/L cisplatin was assessed using flow cytometry with PI staining, and the activity of caspase-3 and intracellular ATP level were detected using commercial detection kits; the expression of XIAP and PARP was analyzed using Western blotting. 3-BP produced obvious inhibitory effects on HepG2 and SMMC7721 cells at the concentrations of 50-400 µmol/L with IC50 values of 238.9∓13.9 µmol/L and 278.7∓11.7 µmol/L for a 48-h treatment, respectively. Cisplatin also inhibited the growth of HepG2 and SMMC7721 cells at the concentrations of 2-32 µmol/L, with IC50 values of 16.4∓0.9 µmol/L and 20.9∓1.8 µmol/L after a 48-h treatment, respectively. Treatment with 100 µmol/L 3-BP combined with 8 µmol/L cisplatin for 48 h resulted in a growth inhibition rate of (60.6∓2.2)% in HepG2 cells and (56.8∓2.3)% in SMMC7721 cells, which were significantly higher than those in cells treated with 3-BP or cisplatin alone. The combined treatment for 48 h induced an apoptotic rate of (51.1∓4.3)% in HepG2 cells and (46.5∓3.9)% in SMMC7721 cells, which were also markedly higher than those in cells with 3-BP or cisplatin treatment alone. 3-BP can sensitize HepG2 and SMMC7721 cells to cisplatin-induced apoptosis possibly by causing intracellular ATP deficiency, down-regulating XIAP, and increasing caspase-3 activity.

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

    Science.gov (United States)

    Pirger, Zsolt; Rácz, Boglárka; Kiss, Tibor

    2009-02-01

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

  7. Roles of p53 and caspases in induction of apoptosis in MCF- 7 breast cancer cells treated with a methanolic extract of Nigella sativa seeds.

    Science.gov (United States)

    Alhazmi, Mohammed I; Hasan, Tarique N; Shafi, Gowhar; Al-Assaf, Abdullah H; Alfawaz, Mohammed A; Alshatwi, Ali A

    2014-01-01

    Nigella Sativa (NS) is an herb from the Ranunculaceae family that exhibits numerous medicinal properties and has been used as important constituent of many complementary and alternative medicines (CAMs). The ability of NS to kill cancer cells such as PC3, HeLa and hepatoma cells is well established. However, our understanding of the mode of death caused by NS remains nebulous. The objective of this study was to gain further insight into the mode and mechanism of death caused by NS in breast cancer MCF-7 cells. Human breast cancer cells (MCF-7) were treated with a methanolic extract of NS, and a dose- and time-dependent study was performed. The IC50 was calculated using a Cell Titer Blue® viability assay assay, and evidence for DNA fragmentation was obtained by fluorescence microscopy TUNEL assay. Gene expression was also profiled for a number of apoptosis-related genes (Caspase-3, -8, -9 and p53 genes) through qPCR. The IC50 of MCF-7 cells was 62.8 μL/mL. When MCF-7 cells were exposed to 50 μL/mL and 100 μL/mL NS for 24 h, 48 h and 72 h, microscopic examination (TUNEL assay) revealed a dose- and time-dependent increase in apoptosis. Similarly, the expression of the Caspase-3, -8, -9 and p53 genes increased significantly according to the dose and time. NS induced apoptosis in MCF-7 cells through both the p53 and caspase pathways. NS could potentially represent an alternative source of medicine for breast cancer therapy.

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

    Directory of Open Access Journals (Sweden)

    Yiying Zhang

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

  9. A cell-permeable dominant-negative survivin protein induces apoptosis and sensitizes prostate cancer cells to TNF-α therapy

    Directory of Open Access Journals (Sweden)

    Kanwar Jagat R

    2010-10-01

    Full Text Available Abstract Background Survivin is a member of the inhibitor-of-apoptosis (IAP family which is widely expressed by many different cancers. Overexpression of survivin is associated with drug resistance in cancer cells, and reduced patient survival after chemotherapy and radiotherapy. Agents that antagonize the function of survivin hold promise for treating many forms of cancer. The purpose of this study was to investigate whether a cell-permeable dominant-negative survivin protein would demonstrate bioactivity against prostate and cervical cancer cells grown in three dimensional culture. Results A dominant-negative survivin (C84A protein fused to the cell penetrating peptide poly-arginine (R9 was expressed in E. coli and purified by affinity chromatography. Western blot analysis revealed that dNSurR9-C84A penetrated into 3D-cultured HeLa and DU145 cancer cells, and a cell viability assay revealed it induced cancer cell death. It increased the activities of caspase-9 and caspase-3, and rendered DU145 cells sensitive to TNF-α via by a mechanism involving activation of caspase-8. Conclusions The results demonstrate that antagonism of survivin function triggers the apoptosis of prostate and cervical cancer cells grown in 3D culture. It renders cancer cells sensitive to the proapoptotic affects of TNF-α, suggesting that survivin blocks the extrinsic pathway of apoptosis. Combination of the biologically active dNSurR9-C84A protein or other survivin antagonists with TNF-α therapy warrants consideration as an approach to cancer therapy.

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

    Czech Academy of Sciences Publication Activity Database

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

    2013-01-01

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

  11. Down-regulation of procaspase-8 expression by focal adhesion kinase protects HL-60 cells from TRAIL-induced apoptosis

    International Nuclear Information System (INIS)

    Tamagiku, Yuji; Sonoda, Yoshiko; Kunisawa, Mari; Ichikawa, Daiju; Murakami, Yayoi; Aizu-Yokota, Eriko; Kasahara, Tadashi

    2004-01-01

    We have demonstrated that focal adhesion kinase (FAK)-overexpressed (HL-60/FAK) cells have marked resistance against various apoptotic stimuli such as hydrogen peroxide, etoposide, and ionizing radiation compared with the vector-transfected (HL-60/Vect) cells. HL-60/FAK cells are highly resistant to TRAIL-induced apoptosis, while original HL-60 or HL-60/Vect cells were sensitive. TRAIL at 500 ng/ml induced significant DNA fragmentation, activation of caspase-8 and 3, the processing of a proapoptotic BID, and mitochondrial release of cytochrome c in HL-60/Vect cells, whereas no such events were observed in the HL-60/FAK cells. In particular, the expression of procaspase-8 gene and subsequent cleavage of caspase-8 were markedly reduced in HL-60/FAK cells, while expression of TRAIL-receptor 2 and 3, TRADD, and FADD was equivalent in both types of cells. In HL-60/FAK cells, the phosphoinositide 3 (PI3)-kinase/Akt survival pathway was constitutively activated, accompanied by significant induction of inhibitor-of-apoptosis proteins, XIAP, RIP, and Bcl-XL. The introduction of FAK siRNA in HL-60/FAK cells sensitized them against TRAIL-induced apoptosis, confirming that overexpressed FAK downregulates procaspase-8 expression, which subsequently inhibits downstream apoptosis pathway in the HL-60/FAK cells

  12. Mind Bomb Regulates Cell Death during TNF Signaling by Suppressing RIPK1’s Cytotoxic Potential

    Directory of Open Access Journals (Sweden)

    Rebecca Feltham

    2018-04-01

    Full Text Available Summary: Tumor necrosis factor (TNF is an inflammatory cytokine that can signal cell survival or cell death. The mechanisms that switch between these distinct outcomes remain poorly defined. Here, we show that the E3 ubiquitin ligase Mind Bomb-2 (MIB2 regulates TNF-induced cell death by inactivating RIPK1 via inhibitory ubiquitylation. Although depletion of MIB2 has little effect on NF-κB activation, it sensitizes cells to RIPK1- and caspase-8-dependent cell death. We find that MIB2 represses the cytotoxic potential of RIPK1 by ubiquitylating lysine residues in the C-terminal portion of RIPK1. Our data suggest that ubiquitin conjugation of RIPK1 interferes with RIPK1 oligomerization and RIPK1-FADD association. Disruption of MIB2-mediated ubiquitylation, either by mutation of MIB2’s E3 activity or RIPK1’s ubiquitin-acceptor lysines, sensitizes cells to RIPK1-mediated cell death. Together, our findings demonstrate that Mind Bomb E3 ubiquitin ligases can function as additional checkpoint of cytokine-induced cell death, selectively protecting cells from the cytotoxic effects of TNF. : Feltham et al. show that MIB2 directly ubiquitylates RIPK1 upon TNF stimulation, suppressing the cytotoxic potential of RIPK1 and acting as a checkpoint within the TNF signaling pathway. Keywords: MIB2, RIPK1, TNF, cell death, caspase-8, IAPs, ubiquitin

  13. Depletion of intrinsic expression of Interleukin-8 in prostate cancer cells causes cell cycle arrest, spontaneous apoptosis and increases the efficacy of chemotherapeutic drugs

    Directory of Open Access Journals (Sweden)

    Lokeshwar Bal L

    2009-07-01

    Full Text Available Abstract Background The progression of all cancers is characterized by increased-cell proliferation and decreased-apoptosis. The androgen-independent prostate cancer (AIPC is the terminal stage of the disease. Many chemokines and cytokines are suspects to cause this increased tumor cell survival that ultimately leads to resistance to therapy and demise of the host. The AIPC cells, but not androgen-responsive cells, constitutively express abundant amount of the pro-inflammatory chemokine, Interleukin-8 (IL-8. The mechanism of IL-8 mediated survival and therapeutic resistance in AIPC cells is unclear at present. The purpose of this report is to show the pervasive role of IL-8 in malignant progression of androgen-independent prostate cancer (AIPC and to provide a potential new therapeutic avenue, using RNA interference. Results The functional consequence of IL-8 depletion in AIPC cells was investigated by RNA interference in two IL-8 secreting AIPC cell lines, PC-3 and DU145. The non-IL-8 secreting LNCaP and LAPC-4 cells served as controls. Cells were transfected with RISC-free siRNA (control or validated-pool of IL-8 siRNA. Transfection with 50 nM IL-8 siRNA caused >95% depletion of IL-8 mRNA and >92% decrease in IL-8 protein. This reduction in IL-8 led to cell cycle arrest at G1/S boundary and decreases in cell cycle-regulated proteins: Cyclin D1 and Cyclin B1 (both decreased >50% and inhibition of ERK1/2 activity by >50%. Further, the spontaneous apoptosis was increased by >43% in IL-8 depleted cells, evidenced by increases in caspase-9 activation and cleaved-PARP. IL-8 depletion caused significant decreases in anti-apoptotic proteins, BCL-2, BCL-xL due to decrease in both mRNA and post-translational stability, and increased levels of pro-apoptotic BAX and BAD proteins. More significantly, depletion of intracellular IL-8 increased the cytotoxic activity of multiple chemotherapeutic drugs. Specifically, the cytotoxicity of Docetaxel

  14. Ethanol Extract of Evodia rutaecarpa Attenuates Cell Growth through Caspase-Dependent Apoptosis in Benign Prostatic Hyperplasia-1 Cells

    Directory of Open Access Journals (Sweden)

    Eunsook Park

    2018-04-01

    Full Text Available The dried fruits of Evodia rutaecarpa Bentham have been used widely as a herbal medicine for the treatment of inflammatory disorders and abdominal pain. Benign prostatic hyperplasia (BPH is a nonmalignant disease characterized by overgrowth of prostates. Despite the pharmacological efficacy of the fruits of E. rutaecarpa against various diseases, their effects against BPH have not been reported. Here, we investigated the inhibitory activity of a 70% ethanol extract of E. rutaecarpa (EEER against BPH, and its underlying mechanisms regarding cell growth of BPH using BPH-1 cells. An in vitro 5α-reductase activity assay showed that EEER exhibited inhibitory activity against 5α-reductase. In BPH-1 cells, EEER treatment inhibited cell viability and reduced the expression of the proliferating cell nuclear antigen proliferating cell nuclear antigen (PCNA, cyclin D1, and phosphor-ERK1/2 proteins. Moreover, EEER also induced apoptosis, with chromatin condensation, apoptotic bodies, and internucleosomal DNA fragmentation. Regarding its underlying mechanisms, EEER exacerbated the activation of caspase-8 and caspase-3 in a concentration-dependent manner and eventually caused the cleavage of PARP. Taken together, these data demonstrated that EEER had a potent 5α-reductase inhibitory activity and that EEER treatment in BPH-1 cells inhibited cell viability via caspase-8- and caspase-3-dependent apoptosis. Therefore, EEER may be a potential phytotherapeutic agent for the treatment of BPH.

  15. 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.

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

    Science.gov (United States)

    Grimstad, Øystein; Husebye, Harald; Espevik, Terje

    2013-10-01

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

  17. Expression of Caspase-1 in breast cancer tissues and its effects on cell proliferation, apoptosis and invasion.

    Science.gov (United States)

    Sun, Yanxia; Guo, Yingzhen

    2018-05-01

    The present study aimed to detect the expression of Caspase-1 in the tumor tissues and tumor-adjacent tissues of patients with breast cancer, and to investigate the effects of Caspase-1 on the proliferation, apoptosis and invasion of breast cancer cells. Reverse transcription-quantitative polymerase chain reaction was used to detect Caspase-1 mRNA expression in breast cancer tissues and tumor-adjacent tissues from patients. Additionally, the human breast cancer MDA-MB-231 cell line was treated with the Caspase-1 small molecule inhibitor Ac-YVAD-CMK, following which the changes to Caspase-1 protein expression were detected via western blotting. The MTT method detected the changes to cell proliferation, flow cytometry detected the rate of apoptosis, and a Transwell assay was employed to assess invasion. Caspase-1 mRNA expression was significantly decreased in the breast cancer tissues of patients, compared with in the tumor-adjacent tissues, a difference that was statistically significant (P<0.05). Treatment with the Ac-YVAD-CMK markedly decreased the protein expression of Caspase-1 in MDA-MB-231 cells, and the difference was statistically significant (P<0.05). Following this treatment of Ac-YVAD-CMK cells, the proliferation and invasion abilities markedly increased, while the apoptotic levels significantly decreased (P<0.05). In conclusion, the expression of Caspase-1 is low in breast cancer tissues, which may promote the proliferation and invasion of breast cancer cells and could be closely associated with the occurrence and development of breast cancer.

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

    Directory of Open Access Journals (Sweden)

    Toshihiko Suzuki

    2007-08-01

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

  19. Modulation of TRAIL resistance in colon carcinoma cells: Different contributions of DR4 and DR5

    International Nuclear Information System (INIS)

    Geelen, Caroline MM van; Pennarun, Bodvael; Le, Phuong TK; Vries, Elisabeth GE de; Jong, Steven de

    2011-01-01

    rhTRAIL is a therapeutic agent, derived from the TRAIL cytokine, which induces apoptosis in cancer cells by activating the membrane death receptors 4 and 5 (DR4 and DR5). Here, we investigated each receptor's contribution to rhTRAIL sensitivity and rhTRAIL resistance. We assessed whether agonistic DR4 or DR5 antibodies could be used to circumvent rhTRAIL resistance, alone or in combination with various chemotherapies. Our study was performed in an isogenic model comprised of the SW948 human colon carcinoma cell line and its rhTRAIL resistant sub-line SW948-TR. Effects of rhTRAIL and agonistic DR4/DR5 antibodies on cell viability were measured using MTT assays and identification of morphological changes characteristic of apoptosis, after acridine orange staining. Sensitivity to the different death receptor ligands was stimulated using pretreatment with the cytokine IFN-gamma and the proteasome inhibitor MG-132. To investigate the mechanisms underlying the changes in rhTRAIL sensitivity, alterations in expression levels of targets of interest were measured by Western blot analysis. Co-immunoprecipitation was used to determine the composition of the death-inducing signalling complex at the cell membrane. SW948 cells were sensitive to all three of the DR-targeting agents tested, although the agonistic DR5 antibody induced only weak caspase 8 cleavage and limited apoptosis. Surprisingly, agonistic DR4 and DR5 antibodies induced equivalent DISC formation and caspase 8 cleavage at the level of their individual receptors, suggesting impairment of further caspase 8 processing upon DR5 stimulation. SW948-TR cells were cross-resistant to all DR-targeting agents as a result of decreased caspase 8 expression levels. Caspase 8 protein expression was restored by MG-132 and IFN-gamma pretreatment, which also re-established sensitivity to rhTRAIL and agonistic DR4 antibody in SW948-TR. Surprisingly, MG-132 but not IFN-gamma could also increase DR5-mediated apoptosis in SW948

  20. Omalizumab Increases the Intrinsic Sensitivity of Human Basophils to IgE-Mediated Stimulation

    Science.gov (United States)

    MacGlashan, Donald; Saini, Sarbjit S.

    2013-01-01

    Background Treatment of allergic patients with omalizumab results in a paradoxical increase in their basophil histamine release response, ex vivo, to crosslinking anti-IgE antibody. It is not known whether this change in response is associated with an increase in intrinsic cellular sensitivity, which would be a paradoxical response. Objective To determine if the increase in response to anti-IgE Ab is a reflection of an increased cellular sensitivity, expressed as molecules of antigen-specific IgE per basophil required to produce a 50% of maximal response. Methods Patients were treated with omalizumab or placebo agent for 12 weeks (NCT01003301 at ClinicalTrials.gov) and the metric of basophil sensitivity was assessed at 4 time points, baseline, 6–8 weeks, 12 weeks (after which treatment stopped) and 24 weeks (12 weeks after the end of treatment). Results As observed previously, treatment with omalizumab resulted in a marked increase in the maximal histamine release induced by crosslinking anti-IgE Ab. This change was accompanied by a marked shift in intrinsic basophil sensitivity, ranging from 2.5 to 125 fold, with an average of 6 fold at the midpoint of the treatment to 12 fold after 12 weeks. The magnitude of the increase in cellular sensitivity was inversely related to the starting sensitivity or the starting maximum histamine release. The increased cellular sensitivity also occurred when using LTC4 secretion as a metric of the basophil response. 12 weeks after the end of treatment, cellular sensitivity was found to shift towards the baseline level although the return to baseline was not yet complete at this time point. Conclusions Treatment with omalizumab results in a markedly increased sensitivity of basophils to IgE-mediated stimulation, in terms of the number of IgE molecules required to produce a given response. These results provide a better quantitative sense of the phenotypic change that occurs in basophils during omalizumab treatment which has

  1. Effect of low dose radiation on cytochrome c and caspase-3 protein expressions in spermatogenic cells of mouse testis

    International Nuclear Information System (INIS)

    Wang Zhicheng; Zhao Hongguang; Piao Chunnan; Liu Guangwei; Liu Shuchun; Lv Zhe; Gong Shouliang

    2006-01-01

    Objective: To investigate the effect of low dose radiation on the expressions of cytochrome e (Cyt c) and caspase-3 proteins in spermatogenic cells of mouse testis. Methods: The relationships of dose- and time-effect of Cyt c and caspase-3 protein expressions with different dose of X-rays were observed in the spermatogenic cells of mouse testis with immunohistochemical technique (SABC). Results: After irradiation with 0, 0.025, 0.05, 0.075, 0.1 and 0.2 Gy, Cyt c and caspase-3 proteins expressed differently in all kinds of spermatogenic cells, and principally in spermatogonia and spermatocytes, and less in spermatids and spermatozoa. And the expressions increased with the increasing of irradiation dose. The expressions of both proteins after irradiation with 0.075 Gy increased with the lapse of time and reached to the peak at 12 h, and then decreased. Conclusion: Dose-and time-effect exists on the low-dose irradiation induced expressions of Cyt e and caspase-3 proteins in spermatogenic cells of mouse testis. (authors)

  2. OSU-2S/sorafenib synergistic antitumor combination against hepatocellular carcinoma: The role of PKCδ/p53

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    Hany A Omar

    2016-11-01

    Full Text Available Background: Sorafenib (Nexavar® is an FDA-approved systemic therapy for advanced hepatocellular carcinoma (HCC. However, the low efficacy and adverse effects at high doses limit the clinical application of sorafenib and strongly recommend its combination with other agents aiming at ameliorating its drawbacks. OSU-2S, a PKCδ activator, was selected as a potential candidate anticancer agent to be combined with sorafenib to promote the anti-cancer activity through synergistic interaction. Methods: The antitumor effects of sorafenib, OSU-2S and their combination were assessed by MTT assay, caspase activation, Western blotting, migration/invasion assays in four different HCC cell lines. The synergistic interactions were determined by Calcusyn analysis. PKCδ knockdown was used to elucidate the role of PKCδ activation as a mechanism for the synergy. The knockdown/over-expression of p53 was used to explain the differential sensitivity of HCC cell lines to sorafenib and/or OSU-2S. Results: OSU-2S synergistically enhanced the anti-proliferative effects of sorafenib in the four used HCC cell lines with combination indices < 1. This effect was accompanied by parallel increases in caspase 3/7 activity, PARP cleavage, PKCδ activation and HCC cell migration/invasion. In addition, PKCδ knockdown abolished the synergy between sorafenib and OSU-2S. Furthermore, p53 restoration in Hep3B cells through the over-expression rendered them more sensitive to both agents while p53 knockdown from HepG2 cells increased their resistance to both agents. Conclusions: OSU-2S augments the anti-proliferative effect of sorafenib in HCC cell lines, in part, through the activation of PKCδ. The p53 status in HCC cells predicts their sensitivity towards both sorafenib and OSU-2S. The proposed combination represents a therapeutically relevant approach that can lead to a new HCC therapeutic protocol.

  3. Metallic nickel nano- and fine particles induce JB6 cell apoptosis through a caspase-8/AIF mediated cytochrome c-independent pathway

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    Castranova Vincent

    2009-04-01

    Full Text Available Abstract Background Carcinogenicity of nickel compounds has been well documented. However, the carcinogenic effect of metallic nickel is still unclear. The present study investigates metallic nickel nano- and fine particle-induced apoptosis and the signal pathways involved in this process in JB6 cells. The data obtained from this study will be of benefit for elucidating the pathological and carcinogenic potential of metallic nickel particles. Results Using 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay, we found that metallic nickel nanoparticles exhibited higher cytotoxicity than fine particles. Both metallic nickel nano- and fine particles induced JB6 cell apoptosis. Metallic nickel nanoparticles produced higher apoptotic induction than fine particles. Western-blot analysis showed an activation of proapoptotic factors including Fas (CD95, Fas-associated protein with death domain (FADD, caspase-8, death receptor 3 (DR3 and BID in apoptotic cells induced by metallic nickel particles. Immunoprecipitation (IP western blot analysis demonstrated the formation of the Fas-related death-inducing signaling complex (DISC in the apoptotic process. Furthermore, lamin A and beta-actin were cleaved. Moreover, we found that apoptosis-inducing factor (AIF was up-regulated and released from mitochondria to cytoplasm. Interestingly, although an up-regulation of cytochrome c was detected in the mitochondria of metallic nickel particle-treated cells, no cytochrome c release from mitochondria to cytoplasm was found. In addition, activation of antiapoptotic factors including phospho-Akt (protein kinase B and Bcl-2 was detected. Further studies demonstrated that metallic nickel particles caused no significant changes in the mitochondrial membrane permeability after 24 h treatment. Conclusion In this study, metallic nickel nanoparticles caused higher cytotoxicity and apoptotic induction than fine particles in JB6 cells. Apoptotic cell death

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

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    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.

  5. 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.

  6. Rational approach to identify newer caspase-1 inhibitors using pharmacophore based virtual screening, docking and molecular dynamic simulation studies.

    Science.gov (United States)

    Patel, Shivani; Modi, Palmi; Chhabria, Mahesh

    2018-05-01

    Caspase-1 is a key endoprotease responsible for the post-translational processing of pro-inflammatory cytokines IL-1β, 18 & 33. Excessive secretion of IL-1β leads to numerous inflammatory and autoimmune diseases. Thus caspase-1 inhibition would be considered as an important therapeutic strategy for development of newer anti-inflammatory agents. Here we have employed an integrated virtual screening by combining pharmacophore mapping and docking to identify small molecules as caspase-1 inhibitors. The ligand based 3D pharmacophore model was generated having the essential structural features of (HBA, HY & RA) using a data set of 27 compounds. A validated pharmacophore hypothesis (Hypo 1) was used to screen ZINC and Minimaybridge chemical databases. The retrieved virtual hits were filtered by ADMET properties and molecular docking analysis. Subsequently, the cross-docking study was also carried out using crystal structure of caspase-1, 3, 7 and 8 to identify the key residual interaction for specific caspase-1 inhibition. Finally, the best mapped and top scored (ZINC00885612, ZINC72003647, BTB04175 and BTB04410) molecules were subjected to molecular dynamics simulation for accessing the dynamic structure of protein after ligand binding. This study identifies the most promising hits, which can be leads for the development of novel caspase-1 inhibitors as anti-inflammatory agents. Copyright © 2018 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    Wilkinson, Derek; Ramsdale, Mark

    2011-10-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2007-03-09

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

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

    Czech Academy of Sciences Publication Activity Database

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

    2013-01-01

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

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

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    Noushin Gashmardi

    2017-11-01

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

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

    Science.gov (United States)

    Han, P; Lucero, M T

    2005-01-01

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

  13. Nitrosative stress induces DNA strand breaks but not caspase mediated apoptosis in a lung cancer cell line

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    Bentz Brandon G

    2004-12-01

    Full Text Available Abstract Background Key steps crucial to the process of tumor progression are genomic instability and escape from apoptosis. Nitric oxide and its interrelated reactive intermediates (collectively denoted as NOX have been implicated in DNA damage and mutational events leading to cancer development, while also being implicated in the inhibition of apoptosis through S-nitrosation of key apoptotic enzymes. The purpose of this study was to explore the interrelationship between NOX-mediated DNA strand breaks (DSBs and apoptosis in cultured tumor cell lines. Methods Two well-characterized cell lines were exposed to increasing concentrations of exogenous NOX via donor compounds. Production of NOX was quantified by the Greiss reaction and spectrophotometery, and confirmed by nitrotyrosine immunostaining. DSBs were measured by the alkaline single-cell gel electrophoresis assay (the COMET assay, and correlated with cell viability by the MTT assay. Apoptosis was analyzed both by TUNEL staining and Annexin V/propidium iodine FACS. Finally, caspase enzymatic activity was measured using an in-vitro fluorogenic caspase assay. Results Increases in DNA strand breaks in our tumor cells, but not in control fibroblasts, correlated with the concentration as well as rate of release of exogenously administered NOX. This increase in DSBs did not correlate with an increase in cell death or apoptosis in our tumor cell line. Finally, this lack of apoptosis was found to correlate with inhibition of caspase activity upon exposure to thiol- but not NONOate-based NOX donor compounds. Conclusions Genotoxicity appears to be highly interrelated with both the concentration and kinetic delivery of NOX. Moreover, alterations in cell apoptosis can be seen as a consequence of the explicit mechanisms of NOX delivery. These findings lend credence to the hypothesis that NOX may play an important role in tumor progression, and underscores potential pitfalls which should be considered when

  14. Apoptosis induced by lipid-associated membrane proteins from Mycoplasma hyopneumoniae in a porcine lung epithelial cell line with the involvement of caspase 3 and the MAPK pathway.

    Science.gov (United States)

    Ni, B; Bai, F F; Wei, Y; Liu, M J; Feng, Z X; Xiong, Q Y; Hua, L Z; Shao, G Q

    2015-09-25

    Lipid-associated membrane proteins (LAMPs) are important in the pathogenicity of the Mycoplasma genus of bacteria. We investigated whether Mycoplasma hyopneumoniae LAMPs have pathogenic potential by inducing apoptosis in a St. Jude porcine lung epithelial cell line (SJPL). LAMPs from a pathogenic strain of M. hyopneumoniae (strain 232) were used in the research. Our investigation made use of diamidino-phenylindole (DAPI) and acridine orange/ethidium bromide (AO/EB) staining, terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) analysis, and Annexin-V-propidium iodide staining. After LAMP treatment for 24 h, typical changes were induced, chromosomes were concentrated, apoptotic bodies were observed, the 3'-OH groups of cleaved genomes were exposed, and the percentage of apoptotic cells reached 36.5 ± 11.66%. Caspase 3 and caspase 8 were activated and cytochrome c (cyt c) was released from the mitochondria into the cytoplasm; poly ADP ribose polymerase (PARP) was digested into two fragments; p38 mitogen-activated protein kinase (MAPK) was phosphorylated; and the expression of pro-apoptosis protein Bax increased while the anti-apoptosis protein Bcl-2 decreased. LAMPs also stimulated SJPL cells to produce nitric oxide (NO) and superoxide. This study demonstrated that LAMPs from M. hyopneumoniae can induce apoptosis in SJPL cells through the activation of caspase 3, caspase 8, cyt c, Bax, and p38 MAPK, thereby contributing to our understanding of the pathogenesis of M. hyopneumoniae, which should improve the treatment of M. hyopneumoniae infections.

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

    DEFF Research Database (Denmark)

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

    2007-01-01

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

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

    Science.gov (United States)

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

    2017-08-01

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

  17. Sensitivity of Mesoporous CoSb2O6 Nanoparticles to Gaseous CO and C3H8 at Low Temperatures

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    Héctor Guillén-Bonilla

    2015-01-01

    Full Text Available Mesoporous CoSb2O6 nanoparticles, synthesized through a nonaqueous method (using cobalt nitrate, antimony trichloride, ethylenediamine, and ethanol as a solvent, were tested to establish their sensitivity to CO and C3H8 atmospheres at relatively low temperatures. The precursor material was dried at 200°C and calcined at 600°C. X-ray diffraction and scanning electron microscopy were employed to verify the existence of crystal phases (P42/mnm and the morphology of this trirutile-type CoSb2O6 oxide. Pyramidal and cubic shaped crystals (average size: 41.1 nm, embedded in the material’s surface, were identified. Mesopores (average size: 6.5 nm on the nanoparticles’ surface were observed by means of transmission electron microscopy. The best sensitivity of the CoSb2O6 in a CO atmosphere was at the relatively low temperatures of 250 and 350°C, whereas, in a C3H8 atmosphere, the sensitivity increased uniformly with temperature. These results encourage using the CoSb2O6 nanoparticles as gas sensors.

  18. Increasing Early Childhood Preservice Teachers' Intercultural Sensitivity through the ABCs

    Science.gov (United States)

    Monroe, Lisa; Ruan, Jiening

    2018-01-01

    While the early childhood student population has become increasingly diverse in the U.S., its teaching force remains primarily European American. The diverse student population demands that early childhood educators possess intercultural sensitivity in order to teach their culturally diverse learners effectively. This study examined the…

  19. Inflammation and pancreatic cancer: molecular and functional interactions between S100A8, S100A9, NT-S100A8 and TGFβ1.

    Science.gov (United States)

    Basso, Daniela; Bozzato, Dania; Padoan, Andrea; Moz, Stefania; Zambon, Carlo-Federico; Fogar, Paola; Greco, Eliana; Scorzeto, Michele; Simonato, Francesca; Navaglia, Filippo; Fassan, Matteo; Pelloso, Michela; Dupont, Sirio; Pedrazzoli, Sergio; Fassina, Ambrogio; Plebani, Mario

    2014-03-26

    In order to gain further insight on the crosstalk between pancreatic cancer (PDAC) and stromal cells, we investigated interactions occurring between TGFβ1 and the inflammatory proteins S100A8, S100A9 and NT-S100A8, a PDAC-associated S100A8 derived peptide, in cell signaling, intracellular calcium (Cai2+) and epithelial to mesenchymal transition (EMT). NF-κB, Akt and mTOR pathways, Cai2+ and EMT were studied in well (Capan1 and BxPC3) and poorly differentiated (Panc1 and MiaPaCa2) cell lines. NT-S100A8, one of the low molecular weight N-terminal peptides from S100A8 to be released by PDAC-derived proteases, shared many effects on NF-κB, Akt and mTOR signaling with S100A8, but mainly with TGFβ1. The chief effects of S100A8, S100A9 and NT-S100A8 were to inhibit NF-κB and stimulate mTOR; the molecules inhibited Akt in Smad4-expressing, while stimulated Akt in Smad4 negative cells. By restoring Smad4 expression in BxPC3 and silencing it in MiaPaCa2, S100A8 and NT-S100A8 were shown to inhibit NF-κB and Akt in the presence of an intact TGFβ1 canonical signaling pathway. TGFβ1 counteracted S100A8, S100A9 and NT-S100A8 effects in Smad4 expressing, not in Smad4 negative cells, while it synergized with NT-S100A8 in altering Cai2+ and stimulating PDAC cell growth. The effects of TGFβ1 on both EMT (increased Twist and decreased N-Cadherin expression) and Cai2+ were antagonized by S100A9, which formed heterodimers with TGFβ1 (MALDI-TOF/MS and co-immuno-precipitation). The effects of S100A8 and S100A9 on PDAC cell signaling appear to be cell-type and context dependent. NT-S100A8 mimics the effects of TGFβ1 on cell signaling, and the formation of complexes between TGFβ1 with S100A9 appears to be the molecular mechanism underlying the reciprocal antagonism of these molecules on cell signaling, Cai2+ and EMT.

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

    Czech Academy of Sciences Publication Activity Database

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

    2015-01-01

    Roč. 46, 4-5 (2015), s. 443-455 ISSN 1567-2379 R&D Projects: GA ČR GB14-37368G Institutional support: RVO:67985904 Keywords : caspase-7 * development * hair follicles Subject RIV: EA - Cell Biology Impact factor: 2.221, year: 2015

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

    Science.gov (United States)

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

    2018-01-01

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

  2. Increase of carbon cycle feedback with climate sensitivity: results from a coupled climate and carbon cycle model

    International Nuclear Information System (INIS)

    Govindasamy, B.; Thompson, S.; Mirin, A.; Wickett, M.; Caldeira, K.; Delire, C.

    2005-01-01

    Coupled climate and carbon cycle modelling studies have shown that the feedback between global warming and the carbon cycle, in particular the terrestrial carbon cycle, could accelerate climate change and result in greater warming. In this paper we investigate the sensitivity of this feedback for year 2100 global warming in the range of 0 to 8 K. Differing climate sensitivities to increased CO 2 content are imposed on the carbon cycle models for the same emissions. Emissions from the SRES A2 scenario are used. We use a fully coupled climate and carbon cycle model, the INtegrated Climate and CArbon model (INCCA), the NCAR/DOE Parallel Climate Model coupled to the IBIS terrestrial biosphere model and a modified OCMIP ocean biogeochemistry model. In our integrated model, for scenarios with year 2100 global warming increasing from 0 to 8 K, land uptake decreases from 47% to 29% of total CO 2 emissions. Due to competing effects, ocean uptake (16%) shows almost no change at all. Atmospheric CO 2 concentration increases are 48% higher in the run with 8 K global climate warming than in the case with no warming. Our results indicate that carbon cycle amplification of climate warming will be greater if there is higher climate sensitivity to increased atmospheric CO 2 content; the carbon cycle feedback factor increases from 1.13 to 1.48 when global warming increases from 3.2 to 8 K

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

    Science.gov (United States)

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

    2017-05-01

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

  4. Rapid generation of mitochondrial superoxide induces mitochondrion-dependent but caspase-independent cell death in hippocampal neuronal cells that morphologically resembles necroptosis

    International Nuclear Information System (INIS)

    Fukui, Masayuki; Choi, Hye Joung; Zhu, Bao Ting

    2012-01-01

    Studies in recent years have revealed that excess mitochondrial superoxide production is an important etiological factor in neurodegenerative diseases, resulting from oxidative modifications of cellular lipids, proteins, and nucleic acids. Hence, it is important to understand the mechanism by which mitochondrial oxidative stress causes neuronal death. In this study, the immortalized mouse hippocampal neuronal cells (HT22) in culture were used as a model and they were exposed to menadione (also known as vitamin K 3 ) to increase intracellular superoxide production. We found that menadione causes preferential accumulation of superoxide in the mitochondria of these cells, along with the rapid development of mitochondrial dysfunction and cellular ATP depletion. Neuronal death induced by menadione is independent of the activation of the MAPK signaling pathways and caspases. The lack of caspase activation is due to the rapid depletion of cellular ATP. It was observed that two ATP-independent mitochondrial nucleases, namely, AIF and Endo G, are released following menadione exposure. Silencing of their expression using specific siRNAs results in transient suppression (for ∼ 12 h) of mitochondrial superoxide-induced neuronal death. While suppression of the mitochondrial superoxide dismutase expression markedly sensitizes neuronal cells to mitochondrial superoxide-induced cytotoxicity, its over-expression confers strong protection. Collectively, these findings showed that many of the observed features associated with mitochondrial superoxide-induced cell death, including caspase independency, rapid depletion of ATP level, mitochondrial release of AIF and Endo G, and mitochondrial swelling, are distinctly different from those of apoptosis; instead they resemble some of the known features of necroptosis. -- Highlights: ► Menadione causes mitochondrial superoxide accumulation and injury. ► Menadione-induced cell death is caspase-independent, due to rapid depletion of ATP

  5. Rapid generation of mitochondrial superoxide induces mitochondrion-dependent but caspase-independent cell death in hippocampal neuronal cells that morphologically resembles necroptosis

    Energy Technology Data Exchange (ETDEWEB)

    Fukui, Masayuki; Choi, Hye Joung; Zhu, Bao Ting, E-mail: BTZhu@kumc.edu

    2012-07-15

    Studies in recent years have revealed that excess mitochondrial superoxide production is an important etiological factor in neurodegenerative diseases, resulting from oxidative modifications of cellular lipids, proteins, and nucleic acids. Hence, it is important to understand the mechanism by which mitochondrial oxidative stress causes neuronal death. In this study, the immortalized mouse hippocampal neuronal cells (HT22) in culture were used as a model and they were exposed to menadione (also known as vitamin K{sub 3}) to increase intracellular superoxide production. We found that menadione causes preferential accumulation of superoxide in the mitochondria of these cells, along with the rapid development of mitochondrial dysfunction and cellular ATP depletion. Neuronal death induced by menadione is independent of the activation of the MAPK signaling pathways and caspases. The lack of caspase activation is due to the rapid depletion of cellular ATP. It was observed that two ATP-independent mitochondrial nucleases, namely, AIF and Endo G, are released following menadione exposure. Silencing of their expression using specific siRNAs results in transient suppression (for ∼ 12 h) of mitochondrial superoxide-induced neuronal death. While suppression of the mitochondrial superoxide dismutase expression markedly sensitizes neuronal cells to mitochondrial superoxide-induced cytotoxicity, its over-expression confers strong protection. Collectively, these findings showed that many of the observed features associated with mitochondrial superoxide-induced cell death, including caspase independency, rapid depletion of ATP level, mitochondrial release of AIF and Endo G, and mitochondrial swelling, are distinctly different from those of apoptosis; instead they resemble some of the known features of necroptosis. -- Highlights: ► Menadione causes mitochondrial superoxide accumulation and injury. ► Menadione-induced cell death is caspase-independent, due to rapid depletion of

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

    Science.gov (United States)

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

    2016-05-13

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

  7. Photoelectrolchemical performance of PbS/CdS quantum dots co-sensitized TiO2 nanosheets array film photoelectrodes

    International Nuclear Information System (INIS)

    Yao, Huizhen; Li, Xue; Liu, Li; Niu, Jiasheng; Ding, Dong; Mu, Yannan; Su, Pengyu; Wang, Guangxia; Fu, Wuyou; Yang, Haibin

    2015-01-01

    Herein, PbS/CdS quantum dots (QDs) co-sensitized titanium dioxide nanosheets array (TiO 2 NSs) films were reported for the first time. The TiO 2 NSs films exposed {001} facets were vertically grown on transparent conductive fluorine-doped tin oxide (FTO) glass substrates by a facile hydrothermal method. The PbS/CdS QDs were assembled on TiO 2 NSs photoelectrode by successive ionic layer adsorption and reaction (SILAR). The X-ray diffraction pattern (XRD) and transmission electron microscopy (TEM) verified that QDs with a diameter less than 20 nm were uniformly anchored on the surface of the TiO 2 NSs films. The QDs co-sensitization can significantly extend the absorption range and increase the absorption property of the photoelectrode by UV–vis absorption spectra. The optimal photoelectrolchemical (PEC) performance of PbS/CdS QDs co-sensitization TiO 2 NSs was with photocurrent density of 6.12 mA cm −2 under an illumination of AM 1.5 G, indicating the TiO 2 NSs films co-sensitized by PbS/CdS QDs have potential applications in solar cells. - Highlights: • TiO 2 nanosheets films were fabricated by a simple hydrothermal. • TiO 2 nanosheets film exposed high energy facets was with gaps. • PbS/CdS co-sensitized TiO 2 nanosheets film was obtained for the first time. • Photocurrent intensity of the novel photoelectrode increased to 6.12 mA cm −2

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-08-15

    Benzene is a well-known environmental pollutant that can induce hematotoxicity, aplastic anemia, acute myelogenous leukemia, and lymphoma. However, although benzene metabolites are known to induce oxidative stress and disrupt the cell cycle, the mechanism underlying lympho/leukemogenicity is not fully understood. Caspase-4 (alias caspase-11) and -12 are inflammatory caspases implicated in inflammation and endoplasmic reticulum stress-induced apoptosis. The objectives of this study were to investigate the altered expression of caspase-4 and -12 in mouse bone marrow after benzene exposure and to determine whether their alterations are associated with benzene-induced bone marrow toxicity, especially cellular apoptosis. In addition, we evaluated whether the p53 gene is involved in regulating the mechanism, using both wild-type (WT) mice and mice lacking the p53 gene. For this study, 8-week-old C57BL/6 mice [WT and p53 knockout (KO)] were administered a benzene solution (150 mg/kg diluted in corn oil) via oral gavage once daily, 5 days/week, for 1 or 2 weeks. Blood and bone marrow cells were collected and cell counts were measured using a Coulter counter. Total mRNA and protein extracts were prepared from the harvested bone marrow cells. Then qRT-PCR and Western blotting were performed to detect changes in the caspases at the mRNA and protein level, respectively. A DNA fragmentation assay and Annexin-V staining were carried out on the bone marrow cells to detect apoptosis. Results indicated that when compared to the control, leukocyte number and bone marrow cellularity decreased significantly in WT mice. The expression of caspase-4 and -12 mRNA increased significantly after 12 days of benzene treatment in the bone marrow cells of benzene-exposed p53KO mice. However, apoptosis detection assays indicated no evidence of apoptosis in p53KO or WT mice. In addition, no changes of other apoptosis-related caspases, such as caspase-3 and -9, were found in WT or p53KO mice at the

  9. A Real-Time PCR Assay Based on 5.8S rRNA Gene (5.8S rDNA) for Rapid Detection of Candida from Whole Blood Samples.

    Science.gov (United States)

    Guo, Yi; Yang, Jing-Xian; Liang, Guo-Wei

    2016-06-01

    The prevalence of Candida in bloodstream infections (BSIs) has increased. To date, the identification of Candida in BSIs still mainly relies on blood culture and serological tests, but they have various limitations. Therefore, a real-time PCR assay for the detection of Candida from whole blood is presented. The unique primers/probe system was designed on 5.8S rRNA gene (5.8S rDNA) of Candida genus. The analytical sensitivity was determined by numbers of positive PCRs in 12 repetitions. At the concentration of 10(1) CFU/ml blood, positive PCR rates of 100 % were obtained for C. albicans, C. parapsilosis, C. tropicalis, and C. krusei. The detection rate for C. glabrata was 75 % at 10(1) CFU/ml blood. The reaction specificity was 100 % when evaluating the assay using DNA samples from clinical isolates and human blood. The maximum CVs of intra-assay and inter-assay for the detection limit were 1.22 and 2.22 %, respectively. To assess the clinical applicability, 328 blood samples from 82 patients were prospectively tested and real-time PCR results were compared with results from blood culture. Diagnostic sensitivity of the PCR was 100 % using as gold standard blood culture, and specificity was 98.4 %. Our data suggest that the developed assay can be used in clinical laboratories as an accurate and rapid screening test for the Candida from whole blood. Although further evaluation is warranted, our assay holds promise for earlier diagnosis of candidemia.

  10. The neuroprotective effect of nicotine in Parkinson’s disease models is associated with inhibiting PARP-1 and caspase-3 cleavage

    Directory of Open Access Journals (Sweden)

    Justin Y.D. Lu

    2017-10-01

    Full Text Available Clinical evidence points to neuroprotective effects of smoking in Parkinson’s disease (PD, but the molecular mechanisms remain unclear. We investigated the pharmacological pathways involved in these neuroprotective effects, which could provide novel ideas for developing targeted neuroprotective treatments for PD. We used the ETC complex I inhibitor methylpyridinium ion (MPP+ to induce cell death in SH-SY5Y cells as a cellular model for PD and found that nicotine inhibits cell death. Using choline as a nicotinic acetylcholine receptor (nAChR agonist, we found that nAChR stimulation was sufficient to protect SH-SY5Y cells against cell death from MPP+. Blocking α7 nAChR with methyllycaconitine (MLA prevented the protective effects of nicotine, demonstrating that these receptors are necessary for the neuroprotective effects of nicotine. The neuroprotective effect of nicotine involves other pathways relevant to PD. Cleaved Poly (ADP-ribose polymerase-1 (PARP-1 and cleaved caspase-3 were decreased by nicotine in 6-hydroxydopamine (6-OHDA lesioned mice and in MPP+-treated SH-SY5Y cells. In conclusion, our data indicate that nicotine likely exerts neuroprotective effects in PD through the α7 nAChR and downstream pathways including PARP-1 and caspase-3. This knowledge could be pursued in future research to develop neuroprotective treatments for PD.

  11. Identification of RIP1 as a critical mediator of Smac mimetic-mediated sensitization of glioblastoma cells for Drozitumab-induced apoptosis.

    Science.gov (United States)

    Cristofanon, S; Abhari, B A; Krueger, M; Tchoghandjian, A; Momma, S; Calaminus, C; Vucic, D; Pichler, B J; Fulda, S

    2015-04-16

    This study aims at evaluating the combination of the tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL)-receptor 2 (TRAIL-R2)-specific antibody Drozitumab and the Smac mimetic BV6 in preclinical glioblastoma models. To this end, the effect of BV6 and/or Drozitumab on apoptosis induction and signaling pathways was analyzed in glioblastoma cell lines, primary glioblastoma cultures and glioblastoma stem-like cells. Here, we report that BV6 and Drozitumab synergistically induce apoptosis and reduce colony formation in several glioblastoma cell lines (combination indextrigger the formation of a cytosolic receptor-interacting protein (RIP) 1/Fas-associated via death domain (FADD)/caspase-8-containing complex and subsequent activation of caspase-8 and -3. BV6- and Drozitumab-induced apoptosis is blocked by the caspase inhibitor zVAD.fmk, pointing to caspase-dependent apoptosis. RNA interference-mediated silencing of RIP1 almost completely abolishes the BV6-conferred sensitization to Drozitumab-induced apoptosis, indicating that the synergism critically depends on RIP1 expression. In contrast, both necrostatin-1, a RIP1 kinase inhibitor, and Enbrel, a TNFα-blocking antibody, do not interfere with BV6/Drozitumab-induced apoptosis, demonstrating that apoptosis occurs independently of RIP1 kinase activity or an autocrine TNFα loop. In conclusion, the rational combination of BV6 and Drozitumab presents a promising approach to trigger apoptosis in glioblastoma, which warrants further investigation.

  12. Fisetin-induced apoptosis of human oral cancer SCC-4 cells through reactive oxygen species production, endoplasmic reticulum stress, caspase-, and mitochondria-dependent signaling pathways.

    Science.gov (United States)

    Su, Chen-Hsuan; Kuo, Chao-Lin; Lu, Kung-Wen; Yu, Fu-Shun; Ma, Yi-Shih; Yang, Jiun-Long; Chu, Yung-Lin; Chueh, Fu-Shin; Liu, Kuo-Ching; Chung, Jing-Gung

    2017-06-01

    Oral cancer is one of the cancer-related diseases in human populations and its incidence rates are rising worldwide. Fisetin, a flavonoid from natural products, has been shown to exhibit anticancer activities in many human cancer cell lines but the molecular mechanism of fisetin-induced apoptosis in human oral cancer cells is still unclear; thus, in this study, we investigated fisetin-induced cell death and associated signal pathways on human oral cancer SCC-4 cells in vitro. We examined cell morphological changes, total viable cells, and cell cycle distribution by phase contrast microscopy and flow cytometry assays. Reactive oxygen species (ROS), Ca 2+ , mitochondria membrane potential (ΔΨ m ), and caspase-8, -9, and -3 activities were also measured by flow cytometer. Results indicate that fisetin induced cell death through the cell morphological changes, caused G2/M phase arrest, induction of apoptosis, promoted ROS and Ca 2+ production, and decreased the level of ΔΨ m and increased caspase-3, -8, and -9 activities in SCC-4 cells. DAPI staining and DNA gel electrophoresis were also used to confirm fisetin-induced cell apoptosis in SCC-4 cells. Western blotting also found out that Fisetin increased the proapoptotic proteins such as Bax and Bid and decreased the antiapoptotic proteins such as Bcl-2. Furthermore, results also showed that Fisetin increased the cytochrome c, AIF, and Endo G release from mitochondria in SCC-4 cells. We also used ATF-6α, ATF-6β, GADD153, and GRP78 which indicated that fisetin induced cell death through ER stress. Based on those observations, we suggest that fisetin induced cell apoptosis through ER stress, mitochondria-, and caspase-dependent pathways. © 2017 Wiley Periodicals, Inc.

  13. Cerebrosides from Sea Cucumber Protect Against Oxidative Stress in SAMP8 Mice and PC12 Cells.

    Science.gov (United States)

    Che, Hongxia; Du, Lei; Cong, Peixu; Tao, Suyuan; Ding, Ning; Wu, Fengjuan; Xue, Changhu; Xu, Jie; Wang, Yuming

    2017-04-01

    Alzheimer's disease (AD) is a neurodegenerative disorder. Emerging evidence implicates β-amyloid (Aβ) plays a critical role in the progression of AD. In this study, we investigated the protective effect of cerebrosides obtained from sea cucumber against senescence-accelerated mouse prone 8 (SAMP8) mice in vivo. We also studied the effect of cerebrosides on Aβ-induced cytotoxicity on the rat pheochromocytoma cell (PC12) and the underlying molecular mechanisms. Cerebrosides ameliorated learning and memory deficits and the Aβ accumulation in demented mice, decreased the content of malondialdehyde (MDA), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OHdG), 8-hydroxy-2'-deoxyguanosine (8-oxo-G), and nitric oxide (NO), and enhanced the superoxide dismutase (SOD) activity significantly. The neuroprotective effect of sea cucumber cerebrosides (SCC) was also verified in vitro: the cerebrosides increased the survival rate of PC12 cells, recovered the cellular morphology, downregulated the protein levels of Caspase-9, cleaved Caspase-3, total Caspase-3, and Bax, and upregulated the protein level of Bcl-2, revealing that cerebrosides could inhibit Aβ-induced cell apoptosis. The results showed the protective effect of SCC was regulated by the mitochondria-dependent apoptotic pathway. Our results provide a new approach to developing the marine organisms as functional foods for neuroprotection.

  14. Effect of 5-bromouracil and 5-bromo-2-deoxyuridine in combination with 8-azaadenine on UV sensitivity of bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Jacob, H.E. (Akademie der Wissenschaften der DDR, Jena. Zentralinstitut fuer Mikrobiologie und Experimentelle Therapie); Golovinsky, E. (Bylgarska Akademiya na Naukite, Sofia)

    1983-01-01

    The presence of 5-bromouracil (BU) as well as 5-bromo-2-deoxyuridine (BUdR) in the cultivation media of bacteria results in the distinct increase of UV sensitivity. With the nucleic acid base analogue 8-azaadenine (8-AA) a similar effect was confirmed, however, not so pronounced. The combined action of BU or BUdR and 8-AA on Escherichia coli, Proteus mirabilis, Bacillus subtilis and Bacillus cereus was investigated. The sensitization effect of BUdR does not increase if 8-AA is present additionally during cultivation. On the contrary, a decrease of sensibilization occurs. This may be caused by the protective effect of the adenine derivative against UV irradiation, if it is present in the cell, but not incorporated into the DNA.

  15. Effect of 5-bromouracil and 5-bromo-2-deoxyuridine in combination with 8-azaadenine on UV sensitivity of bacteria

    International Nuclear Information System (INIS)

    Jacob, H.E.; Golovinsky, E.

    1983-01-01

    The presence of 5-bromouracil (BU) as well as 5-bromo-2-deoxyuridine (BUdR) in the cultivation media of bacteria results in the distinct increase of UV sensitivity. With the nucleic acid base analogue 8-azaadenine (8-AA) a similar effect was confirmed, however, not so pronounced. The combined action of BU or BUdR and 8-AA on Escherichia coli, Proteus mirabilis, Bacillus subtilis and Bacillus cereus was investigated. The sensitization effect of BUdR does not increase if 8-AA is present additionally during cultivation. On the contrary, a decrease of sensibilization occurs. This may be caused by the protective effect of the adenine derivative against UV irradiation, if it is present in the cell, but not incorporated into the DNA. (author)

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

    Science.gov (United States)

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

    2014-08-28

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

  17. Inhibition of Notch1 increases paclitaxel sensitivity to human breast cancer

    Institute of Scientific and Technical Information of China (English)

    Zhao Li; Ma Yongjie; Gu Feng; Fu Li

    2014-01-01

    Background Paclitaxel (PAC) is the first-line chemotherapy drug for most breast cancer patients,but clinical studies showed that some breast cancer patients were insensitive to PAC,which led to chemotherapy failure.It was reported that Notch1 signaling participated in drug resistance of breast cancer.Here,we show whether Notch1 expression is related to PAC sensitivity of breast cancer.Methods We employed Notch1 siRNA and Notch1 inhibitor,N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butylester (DAPT),to down regulate Notch1 expression in human breast cancer cells MDA-MB-231,and detected the inhibition effect by Western blotting and reverse trans cription-polymerase chain reaction,respectively.After 24 hours exposure to different concentration of PAC (0,1,5,10,15,20,and 25 μg/ml),the viability of the control group and experimental group cells was tested by MTT.We also examined the expression of Notch1 in PAC sensitive and nonsensitive breast cancer patients,respectively by immunohistochemistry (IHC).The PAC sensitivity of breast cancer patients were identified by collagen gel droplet embedded culture-drug sensitivity test (CD-DST).Results Down regulation of Notch1 expression by Notch1siRNA interference or Notch1 inhibitor increased the PAC sensitivity in MDA-MB-231 cells (P <0.05).Also,the expression of Notch1 in PAC sensitive patients was much lower than that of PAC non-sensitive patients (P <0.01).Conclusion Notch1 expression has an effect on PAC sensitivity in breast cancer patients,and the inhibition of Notch1 increases paclitaxel sensitivity to human breast cancer.

  18. 1800MHz Microwave Induces p53 and p53-Mediated Caspase-3 Activation Leading to Cell Apoptosis In Vitro.

    Directory of Open Access Journals (Sweden)

    Fuqiang Xing

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

  19. Collateral sensitivity to cisplatin in KB-8-5-11 drug-resistant cancer cells.

    LENUS (Irish Health Repository)

    Doherty, Ben

    2014-01-01

    KB-8-5-11 cells are a drug-resistant cervical cell model that overexpresses ABCB1 (P-glycoprotein). KB-8-5-11 has become sensitive to non-ABCB1 substrate cisplatin. Understanding the mechanism of collateral sensitivity to cisplatin may lead to biomarker discovery for platinum sensitivity in patients with cancer.

  20. Licochalcone A induces apoptosis in KB human oral cancer cells via a caspase-dependent FasL signaling pathway.

    Science.gov (United States)

    Kim, Jae-Sung; Park, Mi-Ra; Lee, Sook-Young; Kim, Do Kyoung; Moon, Sung-Min; Kim, Chun Sung; Cho, Seung Sik; Yoon, Goo; Im, Hee-Jeong; You, Jae-Seek; Oh, Ji-Su; Kim, Su-Gwan

    2014-02-01

    Licochalcone A (Lico-A) is a natural phenol licorice compound with multiple bioactivities, including anti-inflammatory, anti-microbial, anti-fungal and osteogenesis-inducing properties. In the present study, we investigated the Lico-A-induced apoptotic effects and examined the associated apoptosis pathway in KB human oral cancer cells. Lico-A decreased the number of viable KB oral cancer cells. However, Lico-A did not have an effect on primary normal human oral keratinocytes. In addition, the IC50 value of Lico-A was determined to be ~50 µM following dose-dependent stimulation. KB oral cancer cells stimulated with Lico-A for 24 h showed chromatin condensation by DAPI staining, genomic DNA fragmentation by agarose gel electrophoresis and a gradually increased apoptotic cell population by FACS analysis. These data suggest that Lico-A induces apoptosis in KB oral cancer cells. Additionally, Lico‑A‑induced apoptosis in KB oral cancer cells was mediated by the expression of factor associated suicide ligand (FasL) and activated caspase-8 and -3 and poly(ADP-ribose) polymerase (PARP). Furthermore, in the KB oral cancer cells co-stimulation with a caspase inhibitor (Z-VAD-fmk) and Lico-A significantly abolished the apoptotic phenomena. Our findings demonstrated that Lico‑A-induced apoptosis in KB oral cancer cells involves the extrinsic apoptotic signaling pathway, which involves a caspase-dependent FasL-mediated death receptor pathway. Our data suggest that Lico-A be developed as a chemotherapeutic agent for the management of oral cancer.

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

    International Nuclear Information System (INIS)

    Jin, Tengchuan; Huang, Mo; Smith, Patrick; Jiang, Jiansheng; Xiao, T. Sam

    2013-01-01

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

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

    Science.gov (United States)

    Yamaguchi, Yoshifumi; Miura, Masayuki

    2015-01-01

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

  3. Increased Susceptibility to Apoptosis and Growth Arrest of Human Breast Cancer Cells Treated by a Snake Venom-Loaded Silica Nanoparticles

    Directory of Open Access Journals (Sweden)

    Gamal Badr

    2014-11-01

    Full Text Available Background: The development of effective treatments against metastatic cancers, including breast cancer, is among the most important challenges in current experimental and clinical cancer research. We recently demonstrated that Walterinnesia aegyptia venom (WEV, either alone or in combination with silica nanoparticles (WEV+NP, resulted in the growth arrest and apoptosis of different cancer cell lines. Aims: In the present study, we evaluated the impact of WEV alone and WEV+NP on human breast cancer cells isolated from cancer biopsies. Methods: The potential effects of WEV alone and WEV+NP on the proliferation, induction of apoptosis and generation of free radicals in breast cancer cells isolated from 80 patients clinically diagnosed with breast cancer were evaluated by flow cytometry and ELISA. Results: WEV alone and WEV+NP inhibited the proliferation, altered the cell cycle and enhanced the induction of apoptosis of the breast cancer cells by increasing the activities of caspase-3, caspase-8 and caspase-9. In addition, the combination of WEV and NP robustly sensitized the breast cancer cells to growth arrest and apoptosis by increasing the generation of free radicals, including reactive oxygen species (ROS, hydroperoxide and nitric oxide. The combination of WEV with NP significantly enhanced the anti-tumor effect of WEV in breast cancer cells. Conclusion: Our data indicate the therapeutic potential of the nanoparticle-sustained delivery of snake venom for the treatment of breast cancer.

  4. Osabc1k8, an abc1-like kinase gene, mediates abscisic acid sensitivity and dehydration tolerance response in rice seedlings

    International Nuclear Information System (INIS)

    Liu, Y.; Li, T.; Yang, C.

    2015-01-01

    The activity of bc1 complex kinase (ABC1K) protein family, which widely exists in prokaryotes and eukaryotes, consists of 15 members in rice, and the role of this family in plants has not yet been studied in details. In this study, a novel function of OsABC1K8 (LOC-Os06g48770), a member of rice ABC1K family, was characterized. The transcript level of OsABC1K8 changes in response to salt, dehydration, cold, PEG, oxidative (H/sub 2/O/sub 2/) stresses, or abscisic acid (ABA) treatment. Overexpression of OsABC1K8 significantly increased sensitivity to dehydration and reduced sensitivity to ABA. In the contrast, RNAi transgenic lines displayed significantly reduced sensitivity to dehydration stress and increased sensitivity to ABA. Furthermore, the transcriptional levels of several ABA/stress-regulated responsive genes were suppressed in OsABC1K8 over-expressing plants under dehydration stress. In conclusion, our results suggested that OsABC1K8 is a negative regulator in response to dehydration stress through an ABA-dependent pathway. (author)

  5. Chikungunya virus–induced autophagy delays caspase-dependent cell death

    Science.gov (United States)

    Joubert, Pierre-Emmanuel; Werneke, Scott W.; de la Calle, Claire; Guivel-Benhassine, Florence; Giodini, Alessandra; Peduto, Lucie; Levine, Beth; Schwartz, Olivier; Lenschow, Deborah J.

    2012-01-01

    Autophagy is an important survival pathway and can participate in the host response to infection. Studying Chikungunya virus (CHIKV), the causative agent of a major epidemic in India, Southeast Asia, and southern Europe, we reveal a novel mechanism by which autophagy limits cell death and mortality after infection. We use biochemical studies and single cell multispectral assays to demonstrate that direct infection triggers both apoptosis and autophagy. CHIKV-induced autophagy is mediated by the independent induction of endoplasmic reticulum and oxidative stress pathways. These cellular responses delay apoptotic cell death by inducing the IRE1α–XBP-1 pathway in conjunction with ROS-mediated mTOR inhibition. Silencing of autophagy genes resulted in enhanced intrinsic and extrinsic apoptosis, favoring viral propagation in cultured cells. Providing in vivo evidence for the relevance of our findings, Atg16LHM mice, which display reduced levels of autophagy, exhibited increased lethality and showed a higher sensitivity to CHIKV-induced apoptosis. Based on kinetic studies and the observation that features of apoptosis and autophagy were mutually exclusive, we conclude that autophagy inhibits caspase-dependent cell death but is ultimately overwhelmed by viral replication. Our study suggests that inducers of autophagy may limit the pathogenesis of acute Chikungunya disease. PMID:22508836

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

    Science.gov (United States)

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

    2017-01-05

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

  7. TUCAN/CARDINAL/CARD8 and apoptosis resistance in non-small cell lung cancer cells

    International Nuclear Information System (INIS)

    Checinska, Agnieszka; Giaccone, Giuseppe; Hoogeland, Bas SJ; Ferreira, Carlos G; Rodriguez, Jose A; Kruyt, Frank AE

    2006-01-01

    Activation of caspase-9 in response to treatment with cytotoxic drugs is inhibited in NSCLC cells, which may contribute to the clinical resistance to chemotherapy shown in this type of tumor. The aim of the present study was to investigate the mechanism of caspase-9 inhibition, with a focus on a possible role of TUCAN as caspase-9 inhibitor and a determinant of chemosensitivity in NSCLC cells. Caspase-9 processing and activation were investigated by Western blot and by measuring the cleavage of the fluorogenic substrate LEHD-AFC. Proteins interaction assays, and RNA interference in combination with cell viability and apoptosis assays were used to investigate the involvement of TUCAN in inhibition of caspase-9 and chemosensitivity NSCLC. Analysis of the components of the caspase-9 activation pathway in a panel of NSCLC and SCLC cells revealed no intrinsic defects. In fact, exogenously added cytochrome c and dATP triggered procaspase-9 cleavage and activation in lung cancer cell lysates, suggesting the presence of an inhibitor. The reported inhibitor of caspase-9, TUCAN, was exclusively expressed in NSCLC cells. However, interactions between TUCAN and procaspase-9 could not be demonstrated by any of the assays used. Furthermore, RNA interference-mediated down-regulation of TUCAN did not restore cisplatin-induced caspase-9 activation or affect cisplatin sensitivity in NSCLC cells. These results indicate that procaspase-9 is functional and can undergo activation and full processing in lung cancer cell extracts in the presence of additional cytochrome c/dATP. However, the inhibitory protein TUCAN does not play a role in inhibition of procaspase-9 and in determining the sensitivity to cisplatin in NSCLC

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-06-01

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

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

    Science.gov (United States)

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

    2004-08-01

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

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

    Directory of Open Access Journals (Sweden)

    Paul B. Tchounwou

    2006-03-01

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

  11. Blood pressure is reduced and insulin sensitivity increased in glucose-intolerant, hypertensive subjects after 15 days of consuming high-polyphenol dark chocolate.

    Science.gov (United States)

    Grassi, Davide; Desideri, Giovambattista; Necozione, Stefano; Lippi, Cristina; Casale, Raffaele; Properzi, Giuliana; Blumberg, Jeffrey B; Ferri, Claudio

    2008-09-01

    Flavanols from chocolate appear to increase nitric oxide bioavailability, protect vascular endothelium, and decrease cardiovascular disease (CVD) risk factors. We sought to test the effect of flavanol-rich dark chocolate (FRDC) on endothelial function, insulin sensitivity, beta-cell function, and blood pressure (BP) in hypertensive patients with impaired glucose tolerance (IGT). After a run-in phase, 19 hypertensives with IGT (11 males, 8 females; 44.8 +/- 8.0 y) were randomized to receive isocalorically either FRDC or flavanol-free white chocolate (FFWC) at 100 g/d for 15 d. After a wash-out period, patients were switched to the other treatment. Clinical and 24-h ambulatory BP was determined by sphygmometry and oscillometry, respectively, flow-mediated dilation (FMD), oral glucose tolerance test, serum cholesterol and C-reactive protein, and plasma homocysteine were evaluated after each treatment phase. FRDC but not FFWC ingestion decreased insulin resistance (homeostasis model assessment of insulin resistance; P < 0.0001) and increased insulin sensitivity (quantitative insulin sensitivity check index, insulin sensitivity index (ISI), ISI(0); P < 0.05) and beta-cell function (corrected insulin response CIR(120); P = 0.035). Systolic (S) and diastolic (D) BP decreased (P < 0.0001) after FRDC (SBP, -3.82 +/- 2.40 mm Hg; DBP, -3.92 +/- 1.98 mm Hg; 24-h SBP, -4.52 +/- 3.94 mm Hg; 24-h DBP, -4.17 +/- 3.29 mm Hg) but not after FFWC. Further, FRDC increased FMD (P < 0.0001) and decreased total cholesterol (-6.5%; P < 0.0001), and LDL cholesterol (-7.5%; P < 0.0001). Changes in insulin sensitivity (Delta ISI - Delta FMD: r = 0.510, P = 0.001; Delta QUICKI - Delta FMD: r = 0.502, P = 0.001) and beta-cell function (Delta CIR(120) - Delta FMD: r = 0.400, P = 0.012) were directly correlated with increases in FMD and inversely correlated with decreases in BP (Delta ISI - Delta 24-h SBP: r = -0.368, P = 0.022; Delta ISI - Delta 24-h DBP r = -0.384, P = 0.017). Thus, FRDC

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

    OpenAIRE

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2017-08-24

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

  14. Photoelectrolchemical performance of PbS/CdS quantum dots co-sensitized TiO{sub 2} nanosheets array film photoelectrodes

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Huizhen; Li, Xue; Liu, Li; Niu, Jiasheng; Ding, Dong [National Key Lab of Superhard Materials, Jilin University, Changchun 130012 (China); Mu, Yannan [National Key Lab of Superhard Materials, Jilin University, Changchun 130012 (China); Department of Physics and Chemistry, Heihe University, Heihe 164300 (China); Su, Pengyu; Wang, Guangxia; Fu, Wuyou [National Key Lab of Superhard Materials, Jilin University, Changchun 130012 (China); Yang, Haibin, E-mail: yanghb@jlu.edu.cn [National Key Lab of Superhard Materials, Jilin University, Changchun 130012 (China)

    2015-10-25

    Herein, PbS/CdS quantum dots (QDs) co-sensitized titanium dioxide nanosheets array (TiO{sub 2}NSs) films were reported for the first time. The TiO{sub 2}NSs films exposed {001} facets were vertically grown on transparent conductive fluorine-doped tin oxide (FTO) glass substrates by a facile hydrothermal method. The PbS/CdS QDs were assembled on TiO{sub 2}NSs photoelectrode by successive ionic layer adsorption and reaction (SILAR). The X-ray diffraction pattern (XRD) and transmission electron microscopy (TEM) verified that QDs with a diameter less than 20 nm were uniformly anchored on the surface of the TiO{sub 2}NSs films. The QDs co-sensitization can significantly extend the absorption range and increase the absorption property of the photoelectrode by UV–vis absorption spectra. The optimal photoelectrolchemical (PEC) performance of PbS/CdS QDs co-sensitization TiO{sub 2}NSs was with photocurrent density of 6.12 mA cm{sup −2} under an illumination of AM 1.5 G, indicating the TiO{sub 2}NSs films co-sensitized by PbS/CdS QDs have potential applications in solar cells. - Highlights: • TiO{sub 2} nanosheets films were fabricated by a simple hydrothermal. • TiO{sub 2} nanosheets film exposed high energy facets was with gaps. • PbS/CdS co-sensitized TiO{sub 2} nanosheets film was obtained for the first time. • Photocurrent intensity of the novel photoelectrode increased to 6.12 mA cm{sup −2}.

  15. Annonaceous acetogenin mimic AA005 induces cancer cell death via apoptosis inducing factor through a caspase-3-independent mechanism.

    Science.gov (United States)

    Han, Bing; Wang, Tong-Dan; Shen, Shao-Ming; Yu, Yun; Mao, Chan; Yao, Zhu-Jun; Wang, Li-Shun

    2015-03-18

    Annonaceous acetogenins are a family of natural products with antitumor activities. Annonaceous acetogenin mimic AA005 reportedly inhibits mammalian mitochondrial NADH-ubiquinone reductase (Complex I) and induces gastric cancer cell death. However, the mechanisms underlying its cell-death-inducing activity are unclear. We used SW620 colorectal adenocarcinoma cells to study AA005 cytotoxic activity. Cell deaths were determined by Trypan blue assay and flow cytometry, and related proteins were characterized by western blot. Immunofluorescence and subcellular fractionation were used to evaluate AIF nuclear translocation. Reactive oxygen species were assessed by using redox-sensitive dye DCFDA. AA005 induces a unique type of cell death in colorectal adenocarcinoma cells, characterized by lack of caspase-3 activation or apoptotic body formation, sensitivity to poly (ADP-ribose) polymerase inhibitor Olaparib (AZD2281) but not pan-caspase inhibitor Z-VAD.fmk, and dependence on apoptosis-inducing factor (AIF). AA005 treatment also reduced expression of mitochondrial Complex I components, and leads to accumulation of intracellular reactive oxygen species (ROS) at the early stage. Blocking ROS formation significantly suppresses AA005-induced cell death in SW620 cells. Moreover, blocking activation of RIP-1 by necroptosis inhibitor necrotatin-1 inhibits AIF translocation and partially suppresses AA005-induced cell death in SW620 cells demonstrating that RIP-1 protein may be essential for cell death. AA005 may trigger the cell death via mediated by AIF through caspase-3 independent pathway. Our work provided new mechanisms for AA005-induced cancer cell death and novel clues for cancer treatment via AIF dependent cell death.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-03-25

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

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

    Science.gov (United States)

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

    2013-05-31

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

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

    Science.gov (United States)

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

    2013-01-01

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

  19. Sensitization to Gliadin Induces Moderate Enteropathy and Insulitis in Nonobese Diabetic-DQ8 Mice

    Science.gov (United States)

    Galipeau, Heather J.; Rulli, Nestor E.; Jury, Jennifer; Huang, Xianxi; Araya, Romina; Murray, Joseph A.; David, Chella S.; Chirdo, Fernando G.; McCoy, Kathy D.; Verdu, Elena F.

    2012-01-01

    Celiac disease (CD) is frequently diagnosed in patients with type 1 diabetes (T1D), and T1D patients can exhibit Abs against tissue transglutaminase, the auto-antigen in CD. Thus, gliadin, the trigger in CD, has been suggested to have a role in T1D pathogenesis. The objective of this study was to investigate whether gliadin contributes to enteropathy and insulitis in NOD-DQ8 mice, an animal model that does not spontaneously develop T1D. Gliadin-sensitized NOD-DQ8 mice developed moderate enteropathy, intraepithelial lymphocytosis, and barrier dysfunction, but not insulitis. Administration of anti-CD25 mAbs before gliadin-sensitization induced partial depletion of CD25+Foxp3+ T cells and led to severe insulitis, but did not exacerbate mucosal dysfunction. CD4+ T cells isolated from pancreatic lymph nodes of mice that developed insulitis showed increased proliferation and proinflammatory cytokines after incubation with gliadin but not with BSA. CD4+ T cells isolated from nonsensitized controls did not response to gliadin or BSA. In conclusion, gliadin sensitization induced moderate enteropathy in NOD-DQ8 mice. However, insulitis development required gliadin-sensitization and partial systemic depletion of CD25+Foxp3+ T cells. This humanized murine model provides a mechanistic link to explain how the mucosal intolerance to a dietary protein can lead to insulitis in the presence of partial regulatory T cell deficiency. PMID:21911598

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

    Science.gov (United States)

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

    2016-07-01

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

  1. Sensitivity analysis on uncertainty variables affecting the NPP's LUEC with probabilistic approach

    International Nuclear Information System (INIS)

    Nuryanti; Akhmad Hidayatno; Erlinda Muslim

    2013-01-01

    One thing that is quite crucial to be reviewed prior to any investment decision on the nuclear power plant (NPP) project is the calculation of project economic, including calculation of Levelized Unit Electricity Cost (LUEC). Infrastructure projects such as NPP’s project are vulnerable to a number of uncertainty variables. Information on the uncertainty variables which makes LUEC’s value quite sensitive due to the changes of them is necessary in order the cost overrun can be avoided. Therefore this study aimed to do the sensitivity analysis on variables that affect LUEC with probabilistic approaches. This analysis was done by using Monte Carlo technique that simulate the relationship between the uncertainty variables and visible impact on LUEC. The sensitivity analysis result shows the significant changes on LUEC value of AP1000 and OPR due to the sensitivity of investment cost and capacity factors. While LUEC changes due to sensitivity of U 3 O 8s price looks not quite significant. (author)

  2. Caspase-2 cleavage of tau reversibly impairs memory.

    Science.gov (United States)

    Zhao, Xiaohui; Kotilinek, Linda A; Smith, Benjamin; Hlynialuk, Chris; Zahs, Kathleen; Ramsden, Martin; Cleary, James; Ashe, Karen H

    2016-11-01

    In Alzheimer's disease (AD) and other tauopathies, the tau protein forms fibrils, which are believed to be neurotoxic. However, fibrillar tau has been dissociated from neuron death and network dysfunction, suggesting the involvement of nonfibrillar species. Here we describe a novel pathological process in which caspase-2 cleavage of tau at Asp314 impairs cognitive and synaptic function in animal and cellular models of tauopathies by promoting the missorting of tau to dendritic spines. The truncation product, Δtau314, resists fibrillation and is present at higher levels in brains from cognitively impaired mice and humans with AD. The expression of tau mutants that resisted caspase-2 cleavage prevented tau from infiltrating spines, dislocating glutamate receptors and impairing synaptic function in cultured neurons, and it prevented memory deficits and neurodegeneration in mice. Decreasing the levels of caspase-2 restored long-term memory in mice that had existing deficits. Our results suggest an overall treatment strategy for re-establishing synaptic function and restoring memory in patients with AD by preventing tau from accumulating in dendritic spines.

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

    NARCIS (Netherlands)

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

    2013-01-01

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

  4. Study of apoptosis and Caspase-3, Fas expression in rat glioma after treatment with gamma knife

    International Nuclear Information System (INIS)

    Zhao Qingqiu; Zhao Wenqing; Yue Xiangyong; Du Yali; Dong Liying; Zhou Lixia

    2003-01-01

    Objective: To investigate the apoptosis and Caspase-3, Fas expression in rat glioma after treatment with gamma knife. Methods: Setting up C6 glioma model with 60 rats, which were divided into a treatment group ( n= 30) and a control group (n=30). On the 14 th day after planting glioma cells, rats of the treatment group were subjected to gamma knife irradiation. At the 12 th hr, 24 th hr, 48 th hr, 7 th day, 14 th day, 21 st day, flow cytometry was performed to estimate the glioma cells' apoptosis and the expression of Caspase-3 and Fas. The relation between apoptosis and the two kinds of proteins was analysed. Results: Compared with the control group, the apoptosis rate of the glioma cells in the treatment group increased obviously (P th hr reached its peak, then decreased gradually. The expression of Caspase-3 and Fas was positively correlated with apoptosis (r 1 =0.928, r 2 =0.916). Conclusion: The apoptosis of the tumor cells is a kind of effect of gamma knife treatment. Caspase-3 and Fas gene may take part in the regulation of apoptosis

  5. Rapid generation of mitochondrial superoxide induces mitochondrion-dependent but caspase-independent cell death in hippocampal neuronal cells that morphologically resembles necroptosis☆

    Science.gov (United States)

    Fukui, Masayuki; Choi, Hye Joung; Zhu, Bao Ting

    2013-01-01

    Studies in recent years have revealed that excess mitochondrial superoxide production is an important etiological factor in neurodegenerative diseases, resulting from oxidative modifications of cellular lipids, proteins, and nucleic acids. Hence, it is important to understand the mechanism by which mitochondrial oxidative stress causes neuronal death. In this study, the immortalized mouse hippocampal neuronal cells (HT22) in culture were used as a model and they were exposed to menadione (also known as vitamin K3) to increase intracellular superoxide production. We found that menadione causes preferential accumulation of superoxide in the mitochondria of these cells, along with the rapid development of mitochondrial dysfunction and cellular ATP depletion. Neuronal death induced by menadione is independent of the activation of the MAPK signaling pathways and caspases. The lack of caspase activation is due to the rapid depletion of cellular ATP. It was observed that two ATP-independent mitochondrial nucleases, namely, AIF and Endo G, are released following menadione exposure. Silencing of their expression using specific siRNAs results in transient suppression (for ~12 h) of mitochondrial superoxide-induced neuronal death. While suppression of the mitochondrial superoxide dismutase expression markedly sensitizes neuronal cells to mitochondrial superoxide-induced cytotoxicity, its over-expression confers strong protection. Collectively, these findings showed that many of the observed features associated with mitochondrial superoxide-induced cell death, including caspase independency, rapid depletion of ATP level, mitochondrial release of AIF and Endo G, and mitochondrial swelling, are distinctly different from those of apoptosis; instead they resemble some of the known features of necroptosis. PMID:22575170

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

    Czech Academy of Sciences Publication Activity Database

    Matalová, Eva; Kovářů, František; Míšek, Ivan

    2006-01-01

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

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

    Science.gov (United States)

    Ieraci, Alessandro; Herrera, Daniel G

    2018-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Marco A Ataide

    2014-01-01

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

  9. Inner ear dysfunction in caspase-3 deficient mice

    Directory of Open Access Journals (Sweden)

    Woo Minna

    2011-10-01

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

  10. Study on radiation protective effect of resveratrol and its molecular mechanism

    International Nuclear Information System (INIS)

    Lv Qiujun; Wen Liqing; Zhang Min; Guo Shaoming; Chen Yuanyuan; Wu Zuze

    2004-01-01

    Objective: To investigate radiation-protective effect of resveratrol and its molecular mechanism. Methods: Kunming mice were administered with resveratrol before 60 Co γ-irradiation. Thirty-day survival rate and the average life span of dead mice post-irradiation were observed. The apoptosis of spleen from irradiated mice was detected by FACS and in situ terminal labeling method. The effect of resveratrol on the activities of Caspase-3 and Caspase-8, and the expression levels of Bcl-2 and Fas were examined. Results: Administration with resveratrol resulted in increases of 30-day survival rate and prolongation of average life span of the dead mice. Apoptotic rate of spleen cells decreased, expression level of bcl-2 increased, the expression of Fas did not change, and the activities of Caspase-3 and Caspase-8 increased in spleen cells of irradiation groups. Conclusion: The results indicate that resveratrol has radiation-protective effect and its mechanism might be related with its suppression of apoptosis of radiation-sensitive cells

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

    Directory of Open Access Journals (Sweden)

    Takahiro Chihara

    2014-06-01

    Full Text Available Sensory and cognitive performance decline with age. Neural dysfunction caused by nerve death in senile dementia and neurodegenerative disease has been intensively studied; however, functional changes in neural circuits during the normal aging process are not well understood. Caspases are key regulators of cell death, a hallmark of age-related neurodegeneration. Using a genetic probe for caspase-3-like activity (DEVDase activity, we have mapped age-dependent neuronal changes in the adult brain throughout the lifespan of Drosophila. Spatio-temporally restricted caspase activation was observed in the antennal lobe and ellipsoid body, brain structures required for olfaction and visual place memory, respectively. We also found that caspase was activated in an age-dependent manner in specific subsets of Drosophila olfactory receptor neurons (ORNs, Or42b and Or92a neurons. These neurons are essential for mediating innate attraction to food-related odors. Furthermore, age-induced impairments of neural transmission and attraction behavior could be reversed by specific inhibition of caspase in these ORNs, indicating that caspase activation in Or42b and Or92a neurons is responsible for altering animal behavior during normal aging.

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

    Science.gov (United States)

    Chihara, Takahiro; Kitabayashi, Aki; Morimoto, Michie; Takeuchi, Ken-ichi; Masuyama, Kaoru; Tonoki, Ayako; Davis, Ronald L; Wang, Jing W; Miura, Masayuki

    2014-06-01

    Sensory and cognitive performance decline with age. Neural dysfunction caused by nerve death in senile dementia and neurodegenerative disease has been intensively studied; however, functional changes in neural circuits during the normal aging process are not well understood. Caspases are key regulators of cell death, a hallmark of age-related neurodegeneration. Using a genetic probe for caspase-3-like activity (DEVDase activity), we have mapped age-dependent neuronal changes in the adult brain throughout the lifespan of Drosophila. Spatio-temporally restricted caspase activation was observed in the antennal lobe and ellipsoid body, brain structures required for olfaction and visual place memory, respectively. We also found that caspase was activated in an age-dependent manner in specific subsets of Drosophila olfactory receptor neurons (ORNs), Or42b and Or92a neurons. These neurons are essential for mediating innate attraction to food-related odors. Furthermore, age-induced impairments of neural transmission and attraction behavior could be reversed by specific inhibition of caspase in these ORNs, indicating that caspase activation in Or42b and Or92a neurons is responsible for altering animal behavior during normal aging.

  13. The role of interleukin-8 (IL-8) and IL-8 receptors in platinum response in high grade serous ovarian carcinoma.

    Science.gov (United States)

    Stronach, Euan A; Cunnea, Paula; Turner, Christina; Guney, Tankut; Aiyappa, Radhika; Jeyapalan, Senthuran; de Sousa, Camila H; Browne, Alacoque; Magdy, Nesreen; Studd, James B; Sriraksa, Ruethairat; Gabra, Hani; El-Bahrawy, Mona

    2015-10-13

    Platinum based drugs are the cornerstone of chemotherapy for ovarian cancer, however the development of chemoresistance hinders its success. IL-8 is involved in regulating several pro-survival pathways in cancer. We studied the expression of IL-8 and IL-8 receptors in platinum sensitive and resistant cell lines. Using qRT-PCR and immunohistochemistry, both platinum sensitive (PEA1, PEO14) and resistant (PEA2, PEO23) show increased expression of IL-8 and IL-8 receptors. IL-8RA shows nuclear and cytoplasmic expression, whilst IL-8RB is present solely in the cytoplasm. Knockdown of IL-8 increased sensitivity to cisplatin in platinum sensitive and reversed platinum resistance in resistant cell lines, decreased the expression of anti-apoptotic Bcl-2 and decreased inhibitory phosphorylation of pro-apoptotic Bad. IL-8 receptor antagonist treatment also enhanced platinum sensitivity. Nuclear localisation of IL-8RA was only detected in platinum resistant tumours. Inhibition of IL-8 signalling can enhance response in platinum sensitive and resistant disease. Nuclear IL-8RA may have potential as a biomarker of resistant disease.

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

    Science.gov (United States)

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

    2012-04-01

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

  15. Licochalcone A induces apoptosis in KB human oral cancer cells via a caspase-dependent FasL signaling pathway

    Science.gov (United States)

    KIM, JAE-SUNG; PARK, MI-RA; LEE, SOOK-YOUNG; KIM, DO KYOUNG; MOON, SUNG-MIN; KIM, CHUN SUNG; CHO, SEUNG SIK; YOON, GOO; IM, HEE-JEONG; YOU, JAE-SEEK; OH, JI-SU; KIM, SU-GWAN

    2014-01-01

    Licochalcone A (Lico-A) is a natural phenol licorice compound with multiple bioactivities, including anti-inflammatory, anti-microbial, anti-fungal and osteogenesis-inducing properties. In the present study, we investigated the Lico-A-induced apoptotic effects and examined the associated apoptosis pathway in KB human oral cancer cells. Lico-A decreased the number of viable KB oral cancer cells. However, Lico-A did not have an effect on primary normal human oral keratinocytes. In addition, the IC50 value of Lico-A was determined to be ~50 μM following dose-dependent stimulation. KB oral cancer cells stimulated with Lico-A for 24 h showed chromatin condensation by DAPI staining, genomic DNA fragmentation by agarose gel electrophoresis and a gradually increased apoptotic cell population by FACS analysis. These data suggest that Lico-A induces apoptosis in KB oral cancer cells. Additionally, Lico-A-induced apoptosis in KB oral cancer cells was mediated by the expression of factor associated suicide ligand (FasL) and activated caspase-8 and −3 and poly(ADP-ribose) polymerase (PARP). Furthermore, in the KB oral cancer cells co-stimulation with a caspase inhibitor (Z-VAD-fmk) and Lico-A significantly abolished the apoptotic phenomena. Our findings demonstrated that Lico-A-induced apoptosis in KB oral cancer cells involves the extrinsic apoptotic signaling pathway, which involves a caspase-dependent FasL-mediated death receptor pathway. Our data suggest that Lico-A be developed as a chemotherapeutic agent for the management of oral cancer. PMID:24337492

  16. Ultraviolet-Ray-Induced Sea Cucumber (Stichopus japonicus) Melting Is Mediated by the Caspase-Dependent Mitochondrial Apoptotic Pathway.

    Science.gov (United States)

    Su, Li; Yang, Jing-Feng; Fu, Xi; Dong, Liang; Zhou, Da-Yong; Sun, Li-Ming; Gong, Zhenwei

    2018-01-10

    Sea cucumber body-wall melting occurs under certain circumstances. We have shown that apoptosis but not autolysis plays a critical role in the initial stage. However, it is still unclear how apoptosis is triggered in this process. In this study, we examined the levels of reactive oxygen species (ROS), the levels of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax) proteins, the depolarization of mitochondrial transmembrane potentials, and cytochrome c (Cyt c) release during sea cucumber melting induced by ultraviolet (UV) exposure. We also investigated the contribution of caspase in this process by injecting a pan-caspase inhibitor. Our data showed that UV exposure stimulates ROS production, dysfunction of mitochondria, and the release of Cyt c in sea cucumber coelomic fluid cells and body walls. We found a decrease of Bcl-2 and increase of Bax in the mitochondria after UV exposure. We also demonstrated that these changes are associated with elevated caspase-9 and -3 activity. Finally, our data showed that the inhibition of caspases-9 and -3 using an inhibitor suppresses UV-induced sea cucumber melting. These results suggest that apoptosis during sea cucumber melting is mediated by mitochondrial dysfunction and follows the activation of the caspase-signaling pathway. This study presents a novel insight into the mechanism of sea cucumber melting.

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

    Science.gov (United States)

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

    2013-12-01

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

  18. A strategy of combining SILAR with solvothermal process for In2S3 sensitized quantum dot-sensitized solar cells

    International Nuclear Information System (INIS)

    Yang, Peizhi; Tang, Qunwei; Ji, Chenming; Wang, Haobo

    2015-01-01

    Graphical abstract: - Highlights: • In 2 S 3 sensitized TiO 2 anode is prepared by combining SILAR with solvothermal process. • The deposition cycle in SILAR process has an impact on cell performances. • A promising conversion efficiency of 1.39% is obtained for the optimal device. • The cell performances are markedly increased compared with SILAR technique. - Abstract: Pursuit of an efficient strategy for quantum dot-sensitized photoanode has been a persistent objective for enhancing photovoltaic performances of quantum dot-sensitized solar cell (QDSC). We present here the fabrication of the indium sulfide (In 2 S 3 ) quantum dot-sensitized titanium dioxide (TiO 2 ) photoanode by combining successive ionic layer adsorption and reaction (SILAR) with solvothermal processes. The resultant QDSC consists of an In 2 S 3 sensitized TiO 2 photoanode, a liquid polysulfide electrolyte, and a Co 0.85 Se counter electrode. The optimized QDSC with photoanode prepared with the help of a SILAR method at 20 deposition cycles and solvothermal method yields a maximum power conversion efficiency of 1.39%.

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

    Directory of Open Access Journals (Sweden)

    Antje Bast

    2014-03-01

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

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

  1. A highly sensitive fluorescence quenching method for perphenazine detection based on its catalysis of K{sub 2}S{sub 2}O{sub 8} oxidizing rhodamine 6G

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Lihong; Huang, Qitong; Lin, Changqing [Department of Food and Biological Engineering, Zhangzhou Institute of Technology, Zhangzhou, 363000 (China); Lin, Xiaofeng [College of Chemistry and Environment, Minnan Normal University, Zhangzhou, 363000 (China); Huang, Yiqun [Zhangzhou Affiliated Hospital of Fujian Medical University, Zhangzhou 363000 (China); Liu, Jiaming, E-mail: mnsdljm@163.com [College of Chemistry and Environment, Minnan Normal University, Zhangzhou, 363000 (China); Ma, Xudong, E-mail: maxudong005@hotmail.com [Zhangzhou Affiliated Hospital of Fujian Medical University, Zhangzhou 363000 (China)

    2014-12-15

    In this paper, the fluorescence spectra of Rhod 6G (rhodamine 6G)–K{sub 2}S{sub 2}O{sub 8}–PPH (perphenazine) were studied. We found that Rhod 6G existed in the form of Rhod 6G{sup +} under the conditions of 60 °C, 10 min and pH 5.42, and Rhod 6G{sup +} can emit strong and stable fluorescence. Further study showed that when PPH and Rhod 6G{sup +} coexisted, the ester exchange reaction carried out between -OH of PPH and -COOC{sub 2}H{sub 5} of Rhod 6G{sup +} to produced Rhod 6G{sup +}–PPH compound. More interestingly, K{sub 2}S{sub 2}O{sub 8} could oxidize Rhod 6G{sup +} and quench its RTP signal, while PPH was oxidized to red compound PPH′ by K{sub 2}S{sub 2}O{sub 8}, and Rhod 6G{sup +}–PPH′ and PPH were produced in the ester exchange reaction between the -OH of PPH′ and the -COOC{sub 2}H{sub 5} of Rhod 6G{sup +}–PPH. In the above process, PPH catalyzed K{sub 2}S{sub 2}O{sub 8} oxidizing Rhod 6G, which caused the fluorescence signal of the system to quench sharply. Hence, a catalytic fluorescence quenching method for the determination of residual PPH has been developed based on the its catalyzing K{sub 2}S{sub 2}O{sub 8} oxidize rhodamine 6G. This sensitive, accurate, simple and selective fluorescence quenching method was used to determine residual PPH in biological samples with the results consisting with those obtained by high performance liquid chromatography (HPLC), showing good accuracy. The structures of Rhod 6G{sup +}, PPH and Rhod 6G{sup +}–PPH were characterized by infrared spectra. The reaction mechanism of the determination of PPH was also discussed. - Highlights: • Fluorescence for the determination of perphenazine (PPH) had been established. • This method had high sensitivity (limit of detection was 3.3×10{sup −14} g mL{sup −1}). • This method had been applied to determination of PPH in biological samples. • Structures of Rhod 6G{sup +}, PPH and Rhod 6G{sup +}–PPH were characterized by infrared spectra. • Mechanism

  2. Increased sensitivity to angiotensin II is present postpartum in women with a history of hypertensive pregnancy.

    Science.gov (United States)

    Saxena, Aditi R; Karumanchi, S Ananth; Brown, Nancy J; Royle, Caroline M; McElrath, Thomas F; Seely, Ellen W

    2010-05-01

    Pregnancies complicated by new-onset hypertension are associated with increased sensitivity to angiotensin II, but it is unclear whether this sensitivity persists postpartum. We studied pressor response to infused angiotensin II in 25 normotensive postpartum women in both high- and low-sodium balance. Ten women had a history of hypertensive pregnancy (5 with preeclampsia; 5 with transient hypertension of pregnancy), and 15 women had a history of uncomplicated, normotensive pregnancy. Systolic and diastolic blood pressures, aldosterone, and soluble fms-like tyrosine kinase 1 levels were measured before and after angiotensin II infusion in both dietary phases. In high sodium balance, women with a history of hypertensive pregnancy were normotensive but had significantly higher systolic and diastolic blood pressures than controls (115 versus 104 mm Hg and 73 versus 65 mm Hg, respectively; Ppregnancy had a pressor response to salt loading, demonstrated by an increase in systolic blood pressure on a high-salt diet. They also had greater systolic pressor response (10 versus 2 mm Hg; P=0.03), greater increase in aldosterone (56.8 versus 30.8 ng/dL; P=0.03), and increase in soluble fms-like tyrosine kinase 1 levels (11.0 versus -18.9 pg/mL; P=0.02) after infusion of angiotensin II in low-sodium balance compared with controls. Thus, women with a history of hypertensive pregnancy demonstrated salt sensitivity of blood pressure and had increased pressor, adrenal, and soluble fms-like tyrosine kinase 1 responses to infused angiotensin II in low-sodium balance. Increased sensitivity to angiotensin II observed during pregnancy in women with hypertensive pregnancy is present postpartum; this feature may contribute to future cardiovascular risk in these women.

  3. Skin sensitizer identification by IL-8 secretion and CD86 expression on THP-1 cells.

    Science.gov (United States)

    Parise, Carolina Bellini; Sá-Rocha, Vanessa Moura; Moraes, Jane Zveiter

    2015-12-25

    Substantial progress has been made in the development of alternative methods for skin sensitization in the last decade in several countries around the world. Brazil is experiencing an increasing concern about using animals for product development, since the publication of the Law 9605/1998, which prohibits the use of animals when an alternative method is available. In this way, an in vitro test to evaluate allergenic potential is a pressing need.This preliminary study started setting the use of myelomonocytic THP-1 cell line, according to the human cell line activation test (h-CLAT), already under validation process. We found that 48-h chemical exposure was necessary to identify 22 out of 23 sensitizers by the analyses of CD86 expression. In addition, the CD54 expression analyses presented a poor efficiency to discriminate sensitizers from non-sensitizers in our conditions. In view of these results, we looked for changes of pro-inflammatory interleukin profile. The IL-8 secretion analyses after 24-h chemical incubation seemed to be an alternative for CD54 expression assessing.Altogether, our findings showed that the combination of the analyses of CD86 expression and IL-8 secretion allowed predicting allergenicity.

  4. Increasing quinolone resistance in Salmonella enterica serotype enteritidis

    DEFF Research Database (Denmark)

    Mølbak, K.; Gerner-Smidt, P.; Wegener, Henrik Caspar

    2002-01-01

    Until recently, Salmonella enterica serotype Enteritidis has remained sensitive to most antibiotics. However, national surveillance data from Denmark show that quinolone resistance in S. Enteritidis has increased from 0.8% in 1995 to 8.5% in 2000. These data support concerns that the current use...... of quinolone in food animals leads to increasing resistance in S. Enteritidis and that action should be taken to limit such use....

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

    Directory of Open Access Journals (Sweden)

    Suzana Makpol

    2012-01-01

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

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

    Science.gov (United States)

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

    2013-05-28

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

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

    Czech Academy of Sciences Publication Activity Database

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Zamboni Dario S

    2010-10-01

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

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

    Science.gov (United States)

    Qi, Jian Hua; Anand-Apte, Bela

    2015-04-01

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

  10. Secretion of S100A8, S100A9, and S100A12 by Neutrophils Involves Reactive Oxygen Species and Potassium Efflux

    Directory of Open Access Journals (Sweden)

    Mélanie R. Tardif

    2015-01-01

    Full Text Available S100A8/A9 (calprotectin and S100A12 proinflammatory mediators are found at inflammatory sites and in the serum of patients with inflammatory or autoimmune diseases. These cytoplasmic proteins are secreted by neutrophils at sites of inflammation via alternative secretion pathways of which little is known. This study examined the nature of the stimuli leading to S100A8/A9 and S100A12 secretion as well as the mechanism involved in this alternative secretion pathway. Chemotactic agents, cytokines, and particulate molecules were used to stimulate human neutrophils. MSU crystals, PMA, and H2O2 induced the release of S100A8, S100A9, and S100A12 homodimers, as well as S100A8/A9 heterodimer. High concentrations of S100A8/A9 and S100A12 were secreted in response to nanoparticles like MSU, silica, TiO2, fullerene, and single-wall carbon nanotubes as well as in response to microbe-derived molecules, such as zymosan or HKCA. However, neutrophils exposed to the chemotactic factors fMLP failed to secrete S100A8/A9 or S100A12. Secretion of S100A8/A9 was dependent on the production of reactive oxygen species and required K+ exchanges through the ATP-sensitive K+ channel. Altogether, these findings suggest that S100A12 and S100A8/A9 are secreted independently either via distinct mechanisms of secretion or following the activation of different signal transduction pathways.

  11. Enhanced proapoptotic gene expression of XAF1, CASP8 and TNFSF10 in the bovine endometrium during early pregnancy is not correlated with augmented apoptosis.

    Science.gov (United States)

    Groebner, A E; Schulke, K; Unterseer, S; Reichenbach, H D; Reichenbach, M; Büttner, M; Wolf, E; Meyer, H H D; Ulbrich, S E

    2010-03-01

    Bovine trophoblast cells release interferon-tau (IFNT), a type I IFN, as the pregnancy recognition signal. Since type I IFNs exert growth inhibitory and proapoptotic actions, the effect of the conceptus on components of the apoptosis pathways was determined in the bovine endometrium during the periimplantation period. Uteri of Simmental heifers were flushed post mortem at days 12, 15, and 18 of cycle or pregnancy for the recovery of conceptuses and the sampling of ipsilateral endometrial tissue at slaughter for quantitative RT-PCR, immunohistochemistry, caspase activity and TUNEL assays. Endometrium samples of pregnant animals revealed increased transcript levels for the proapoptotic genes XAF1 (day 15: 2.9-fold; day 18: 15.1-fold; p=0.005) and CASP8 (day 18: 2.4-fold; p=0.007). The mRNA expression increased significantly with the day of the cycle for the proapoptotic genes FASLG, TNFSF10, TNF and TNFSF1A (p=0.004, p=0.006, p=0.001 and p=0.007) and the antiapoptotic gene BIRC4 (p=0.03). We detected high amounts of FASLG transcripts in day 18 conceptuses (16-fold higher than day 18 endometria). This finding was validated at the protein level by immunohistochemistry. To further analyse the endometrial activation of the caspase cascade, the activities of initiator caspase 8 and effector caspases 3/7 were determined luminometrically. No difference between pregnant and cyclic animals was found for either caspase activity. Additionally, a TUNEL assay showed no increase of apoptotic cells in the pregnant endometrium. In conclusion, although the bovine conceptus induces the expression of proapoptotic genes, neither an activation of a caspase cascade nor an increase of apoptotic cells was noticed. These results suggest inhibitory mechanisms preventing endometrial cells from programmed cell death. Copyright 2009 Elsevier Ltd. All rights reserved.

  12. Flavopiridol induces apoptosis in glioma cell lines independent of retinoblastoma and p53 tumor suppressor pathway alterations by a caspase-independent pathway.

    Science.gov (United States)

    Alonso, Michelle; Tamasdan, Cristina; Miller, Douglas C; Newcomb, Elizabeth W

    2003-02-01

    Flavopiridol is a synthetic flavone, which inhibits growth in vitro and in vivo of several solid malignancies such as renal, prostate, and colon cancers. It is a potent cyclin-dependent kinase inhibitor presently in clinical trials. In this study, we examined the effect of flavopiridol on a panel of glioma cell lines having different genetic profiles: five of six have codeletion of p16(INK4a) and p14(ARF); three of six have p53 mutations; and one of six shows overexpression of mouse double minute-2 (MDM2) protein. Independent of retinoblastoma and p53 tumor suppressor pathway alterations, flavopiridol induced apoptosis in all cell lines but through a caspase-independent mechanism. No cleavage products for caspase 3 or its substrate poly(ADP-ribose) polymerase or caspase 8 were detected. The pan-caspase inhibitor Z-VAD-fmk did not inhibit flavopiridol-induced apoptosis. Mitochondrial damage measured by cytochrome c release and transmission electron microscopy was not observed in drug-treated glioma cells. In contrast, flavopiridol treatment induced translocation of apoptosis-inducing factor from the mitochondria to the nucleus. The proteins cyclin D(1) and MDM2 involved in the regulation of retinoblastoma and p53 activity, respectively, were down-regulated early after flavopiridol treatment. Given that MDM2 protein can confer oncogenic properties under certain circumstances, loss of MDM2 expression in tumor cells could promote increased chemosensitivity. After drug treatment, a low Bcl-2/Bax ratio was observed, a condition that may favor apoptosis. Taken together, the data indicate that flavopiridol has activity against glioma cell lines in vitro and should be considered for clinical development in the treatment of glioblastoma multiforme.

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

    Science.gov (United States)

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

    2014-12-05

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

  14. Increasing sensitivity of MOS dosemeters in cascade connection

    International Nuclear Information System (INIS)

    Vychytil, F.; Cechak, T.; Gerndt, J.; Petr, I.

    1978-01-01

    The possibilities of increasing the sensitivity of MOS transistors in their cascade connection were studied theoretically and experimentally. The measurements confirmed the presumption that the instability of cascade-connected MOS transistors increased with the square of the number of transistors in the system. This allows systems to be formed with different sensitivity to ionizing radiation by encasing 10 to 10 4 transistors connected in cascade, which is technologically feasible. The procedure is also acceptable from the point of view of cost. (Z.M.)

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

    Directory of Open Access Journals (Sweden)

    Alexandre Tourigny

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

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

    Science.gov (United States)

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

    2015-02-01

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

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

    Czech Academy of Sciences Publication Activity Database

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

    2007-01-01

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

  18. Biological Characteristics of Caspase-14 and Its Expression in Neoplastic Diseases in the View of Translational Medicine

    Directory of Open Access Journals (Sweden)

    Kang-sheng LIU

    2016-06-01

    Full Text Available Caspase-14, a member of caspase family, only exists in mammals. As the most divergent member in the family of mammalian caspases, caspase-14 displays a variety of unique characteristics. It is expressed in a limited number of tissues and has the shortest amino acid sequence within the caspase protein family. At present, it has been found that caspase-14 is functionally different from the inflammatory reaction group of typical caspase family members. It exerts a certain effect in the promotion of final differentiation of epidermal cells and hydration of stratum corneum so as to maintain the steady state of skin barrier. In recent years, caspase-14 expression has been discovered in neoplastic diseases. Translational medicine integrates experimental research results and clinical guidance into the optimal implementation criteria for promoting the prediction, prevention and treatment of diseases. Via human genomics and molecular biology, translational medicine offers a possibility of screening molecular markers so that it can be used to diagnose the neoplastic diseases. In this article, the biological characteristics and substrates of caspase-14 as well as its expression in embryonic period and neoplastic diseases were reviewed.

  19. Reported symptoms to peanut between 4 and 8 years among children sensitized to peanut and birch pollen - results from the BAMSE birth cohort.

    Science.gov (United States)

    Asarnoj, A; Ostblom, E; Ahlstedt, S; Hedlin, G; Lilja, G; van Hage, M; Wickman, M

    2010-02-01

    Specific IgE tests are sometimes difficult to interpret due to structural similarities between certain food and pollen allergens. This may be the reason why concomitant sensitization to peanut and birch pollen is frequently seen. The aim of this study was to investigate reported symptoms to peanut- and birch pollen in relation to sensitization. The data originate from 1928 children in the BAMSE birth cohort. Background factors and clinical parameters were obtained and the levels of IgE antibodies to peanut and birch pollen measured at 4 and 8 years. IgE antibodies to peanut were found in 5.5% and 7.4% of the children at 4 and 8 years, respectively. The IgE antibody levels to peanut were higher in children sensitized to peanut but not birch than in children sensitized to peanut and birch among both 4- and 8-year-olds (P = 0.093 and P = 0.003, respectively). Eight-year-olds sensitized to peanut but not birch, more often reported symptoms to peanut than children sensitized to both peanut and birch pollen (76%vs 46%, P = 0.002). The probability of reported symptoms to peanut increased significantly with increasing IgE levels to peanut, especially in 8-year-olds not sensitized to birch. Children sensitized to both peanut and birch pollen are less likely to report symptoms to peanut than children sensitized to peanut but not to birch pollen at 8 years. This is likely due to cross reactions between birch pollen and peanut and can explain the high sensitization rate to peanut in areas where birch trees are common.

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

    Czech Academy of Sciences Publication Activity Database

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

    2017-01-01

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

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

    Czech Academy of Sciences Publication Activity Database

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

    2017-01-01

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

  2. Two Chitotriose-Specific Lectins Show Anti-Angiogenesis, Induces Caspase-9-Mediated Apoptosis and Early Arrest of Pancreatic Tumor Cell Cycle.

    Directory of Open Access Journals (Sweden)

    Ruby Singh

    Full Text Available The antiproliferative activity of two chito-specific agglutinins purified from Benincasa hispida (BhL and Datura innoxia (DiL9 of different plant family origin was investigated on various cancer cell lines. Both lectins showed chitotriose specificity, by inhibiting lectin hemagglutinating activity. On further studies, it was revealed that these agglutinins caused remarkable concentration-dependent antiproliferative effect on human pancreatic cancerous cells but not on the normal human umbilical vein endothelial cells even at higher doses determined using MTT assay. The GI50 values were approximately 8.4 μg ml(-1 (0.247 μM and 142 μg ml(-1 (14.8 μM for BhL and DiL9, respectively, against PANC-1 cells. The growth inhibitory effect of these lectins on pancreatic cancer cells were shown to be a consequence of lectin cell surface binding and triggering G0/G1 arrest, mitochondrial membrane depolarization, sustained increase of the intracellular calcium release and the apoptotic signal is amplified by activation of caspases executing cell death. Interestingly, these lectins also showed anti-angiogenic activity by disrupting the endothelial tubulogenesis. Therefore, we report for the first time two chito-specific lectins specifically binding to tumor glycans; they can be considered to be a class of molecules with antitumor activity against pancreatic cancer cells mediated through caspase dependent mitochondrial apoptotic pathway.

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

    Science.gov (United States)

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

    2014-06-01

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

  4. Role of ZnO photoanode nanostructures and sensitizer deposition approaches on the photovoltaic properties of CdS/CdSe and CdS1-xSex quantum dot-sensitized solar cells

    Science.gov (United States)

    Şişman, İlkay; Tekir, Oktay; Karaca, Hüseyin

    2017-02-01

    Hierarchical bundle-like ZnO nanorod arrays (BNRs) were synthesized by a one-pot hydrothermal method based on two consecutive temperature steps for cascade CdS/CdSe and ternary CdS1-xSex alloy quantum dot-sensitized solar cells (QDSSCs) as photoanode. The CdS/CdSe and CdS1-xSex QDs were deposited on the surface of the ZnO BNRs by conventional and modified successive ionic-layer adsorption and reaction (SILAR) methods, respectively. Using the ZnO BNRs/CdS/CdSe photoanode, the power conversion efficiency reaches 2.08%, which is 1.8 times higher than that of pristine ZnO nanorods/CdS/CdSe photoanode, while by applying ZnO BNRs/CdS1-xSex, the power conversion efficiency improves 2.52%. The remarkably improved photovoltaic performance is mainly derived from the bundle-like nanorod arrays structure, which increases the QDs loading amount and the scattering effect for light absorption, and the appropriate conduction band energy, sufficient Se amount and well coverage of the ternary CdS1-xSex QDs result in enhanced photogenerated electron injection, high light absorption and reduced recombination, respectively. As a result, ZnO BNRs/CdS1-xSex combination can significantly improve performance of QDSSCs.

  5. A hybrid of coumarin and phenylsulfonylfuroxan induces caspase-dependent apoptosis and cytoprotective autophagy in lung adenocarcinoma cells.

    Science.gov (United States)

    Wang, Qian; Guo, Yalan; Jiang, Shanshan; Dong, Mengxue; Kuerban, Kudelaidi; Li, Jiyang; Feng, Meiqing; Chen, Ying; Ye, Li

    2018-01-15

    Lung adenocarcinoma is the most primary histologic subtype of non-small cell lung cancer (NSCLC). Compound 8b, a novel coumarin derivative with phenylsulfonylfuroxan group, shows significant antiproliferation activity against lung adenocarcinoma cell with low toxicity. This study aims to uncover the potential of compound 8b in relation to apoptosis as well as autophagy induction in lung adenocarcinoma cells. The cytotoxicity and apoptosis of A549 and H1299 cells induced by compound 8b were detected by MTT, microscope and western blot analysis. Autophagy was determined by TEM, confocal microscopy and western blot analysis. Akt/mTOR and Erk signaling pathway were also examined by western blot analysis. First, significant growth inhibition and caspase-dependent apoptosis were observed in compound 8b-treated A549 and H1299 cells. Then, we confirmed compound 8b-induced autophagy by autophagosomes formation, upregulated expression of autophagy-related protein LC3-II and autophagic flux. Importantly, abolishing autophagy using inhibitors and ATG5 siRNA enhanced the cytotoxicity of compound 8b, indicating the cytoprotective role of autophagy in lung adenocarcinoma. Further mechanistic investigations suggested that Akt/mTOR and Erk signaling pathways contributed to autophagy induction by compound 8b. This results demonstrate that compound 8b induces caspase-dependent apoptosis as well as cytoprotective autophagy in lung adenocarcinoma cells, which may provide scientific evidence for developing this furoxan-based NO-releasing coumarin derivative as a potential anti-lung adenocarcinoma therapeutic agents. Copyright © 2017 Elsevier GmbH. All rights reserved.

  6. Caspase-Mediated Anti-Apoptotic Effect of Ginsenoside Rg5, a Main Rare Ginsenoside, on Acetaminophen-Induced Hepatotoxicity in Mice.

    Science.gov (United States)

    Wang, Zi; Hu, Jun-Nan; Yan, Meng-Han; Xing, Jing-Jing; Liu, Wen-Cong; Li, Wei

    2017-10-25

    Frequent overdose of acetaminophen (APAP) is one of the most common and important incentives of acute hepatotoxicity. Prior to this work, our research group confirmed that black ginseng (Panax ginseng, BG) showed powerful protective effects on APAP-induced ALI. However, it is not clear which kind of individual ginsenoside from BG plays such a liver protection effect. The objective of the current investigation was to evaluate whether ginsenoside Rg5 (G-Rg5) protected against APAP-induced hepatotoxicity and the involved action mechanisms. Mice were administrated with G-Rg5 at two dosages of 10 or 20 mg/kg for 7 consecutive days. After the last treatment, all of the animals that received a single intraperitoneal injection of APAP (250 mg/kg) showed severe liver toxicity after 24 h, and the liver protection effects of G-Rg5 were examined. The results clearly indicated that pretreatment with G-Rg5 remarkably inhibited the production of serum tumor necrosis factor (TNF-α) and interleukin-1β (IL-1β) compared with the APAP group. Meanwhile, G-Rg5 decreased the hepatic malondialdehyde (MDA) content, the protein expression levels of 4-hydroxynonenal (4-HNE) and cytochrome P450 2E1 (CYP2E1) in the liver tissues. G-Rg5 decreased APAP caused the hepatic overexpression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Furthermore, analysis of immunohistochemistry and Western blotting also indicated that G-Rg5 pretreatment inhibited activation of apoptotic pathways mainly via increasing the expression of Bcl-2 protein, decreasing the expression of Bax protein, proliferating cell nuclear antigen (PCNA), cytochrome c, caspase-3, caspase-8, and caspase-9. Liver histopathological observation provided further evidence that pretreatment with G-Rg5 could significantly inhibit hepatocyte necrosis, inflammatory cell infiltration, and apoptosis caused by APAP. In conclusion, the present study clearly demonstrates that G-Rg5 exerts a liver protection effect against

  7. Breath condensate levels of 8-isoprostane and leukotriene B4 after ozone inhalation are greater in sensitive versus nonsensitive subjects.

    Science.gov (United States)

    Alfaro, Mario F; Walby, William F; Adams, William C; Schelegle, Edward S

    2007-01-01

    Ozone (O3) inhalation induces pulmonary function decrements and inflammation. The present study was designed to determine if a relationship exists between O3 induced pulmonary function changes and the presence of inflammatory markers as measured in exhaled breath condensates (EBCs) obtained from O3-sensitive and nonsensitive human subjects. Eight healthy adult volunteers (4 males/4 females, age 18 to 30 years) were studied, characterized as to their ozone sensitivity and placed into 2 groups (sensitive and nonsensitive) with each group having 2 males and 2 females. Subjects completed a 20-minute EBC collection and pulmonary function test (PFT) prior to a single 60-minute bout of cycle ergometer exercise (V(E) = 50-55 L/min) while breathing filtered air (FA) or 0.35 ppm O3. Subjective symptom scores (SSSs) were collected at 6, 20, 40, and 60 minutes during exposure. An immediate postexposure PFT was performed followed by an EBC collection. Subjective symptom scores, EBCs, and PFTs were collected at 1, 4 and 8 hours post exposure. EBCs were analyzed for prostaglandin E2 (PGE2), leukotriene B4 (LTB4), 8-isoprostane, and total nitric oxide (NO) metabolites (nitrate + nitrite content). Sensitive subjects, breathing O3, had significantly greater functional decrements in PFTs, increased SSSs, and increased rapid shallow breathing as well as elevated levels of 8-isoprostane and LTB4 in EBCs compared to those breathing FA. In addition, there were significant increases in nitrate + nitrite content in both sensitive and nonsensitive subjects breathing O3 compared to FA. These results indicate that sensitive subjects have elevated arachidonic acid metabolites in EBCs compared to nonsensitive subjects after O3 inhalation.

  8. Paris Saponin I Sensitizes Gastric Cancer Cell Lines to Cisplatin via Cell Cycle Arrest and Apoptosis.

    Science.gov (United States)

    Song, Shuichuan; Du, Leiwen; Jiang, Hao; Zhu, Xinhai; Li, Jinhui; Xu, Ji

    2016-10-18

    BACKGROUND Dose-related toxicity is the major restriction of cisplatin and cisplatin-combination chemotherapy, and is a challenge for advanced gastric cancer treatment. We explored the possibility of using Paris saponin I as an agent to sensitize gastric cancer cells to cisplatin, and examined the underlying mechanism. MATERIAL AND METHODS Growth inhibition was detected by MTT assay. The cell cycle and apoptosis were detected using flow cytometry and Annexin V/PI staining. The P21waf1/cip1, Bcl-2, Bax, and caspase-3 protein expression were detected using Western blot analysis. RESULTS The results revealed that PSI sensitized gastric cancer cells to cisplatin, with low toxicity. The IC50 value of cisplatin in SGC-7901 cell lines was decreased when combined with PSI. PSI promoted cisplatin-induced G2/M phase arrest and apoptosis in a cisplatin concentration-dependent manner. Bcl-2 protein expression decreased, but Bax, caspase-3, and P21waf1/cip1 protein expression increased with PSI treatment. CONCLUSIONS The underlying mechanism of Paris saponin I may be related to targeting the apoptosis pathway and cell cycle blocking, which suggests that PSI is a potential therapeutic sensitizer for cisplatin in treating gastric cancer.

  9. FigS8.txt

    Data.gov (United States)

    U.S. Environmental Protection Agency — This is an ASCII file with georeferencing information containing data plotted in Figure S8 of the supplemental information section of the manuscript. This dataset is...

  10. Study of the back recombination processes of PbS quantum dots sensitized solar cells

    Science.gov (United States)

    Badawi, Ali; Al-Hosiny, N.; Merazga, Amar; Albaradi, Ateyyah M.; Abdallah, S.; Talaat, H.

    2016-12-01

    In this study, the back recombination processes of PbS quantum dots sensitized solar cells (QDSSCs) has been investigated. PbS QDs were adsorbed onto titania electrodes to act the role of sensitizers using successive ionic layer adsorption and reaction (SILAR) technique. The energy band gaps of the synthesized PbS QDs/titania are ranged from 1.64 eV (corresponding to 756 nm) to 3.12 eV (397 nm) matching the whole visible solar spectrum. The hyperbolic band model (HBM) was used to calculate PbS QDs size and it ranges from 1.76 to 3.44 nm. The photovoltaic parameters (open circuit voltage Voc, short circuit current density Jsc, fill factor FF and efficiency η) of the assembled PbS QDs sensitized solar cells (QDSSCs) were determined under a solar illumination of 100 mW/cm2 (AM 1.5 conditions). The open circuit voltage-decay (OCVD) rates of the assembled PbS QDSSCs were measured. The time constant (τ) for PbS QDSSCs (4 SILAR cycles) shows one order of magnitude larger than that of PbS QDSSCs (8 SILAR cycles) as a result of a decreased electron-hole back recombination.

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

    Czech Academy of Sciences Publication Activity Database

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

    2014-01-01

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

  12. Proton pump inhibitors induce apoptosis of human B-cell tumors through a caspase-independent mechanism involving reactive oxygen species.

    Science.gov (United States)

    De Milito, Angelo; Iessi, Elisabetta; Logozzi, Mariantonia; Lozupone, Francesco; Spada, Massimo; Marino, Maria Lucia; Federici, Cristina; Perdicchio, Maurizio; Matarrese, Paola; Lugini, Luana; Nilsson, Anna; Fais, Stefano

    2007-06-01

    Proton pumps like the vacuolar-type H+ ATPase (V-ATPase) are involved in the control of cellular pH in normal and tumor cells. Treatment with proton pump inhibitors (PPI) induces sensitization of cancer cells to chemotherapeutics via modifications of cellular pH gradients. It is also known that low pH is the most suitable condition for a full PPI activation. Here, we tested whether PPI treatment in unbuffered culture conditions could affect survival and proliferation of human B-cell tumors. First, we showed that PPI treatment increased the sensitivity to vinblastine of a pre-B acute lymphoblastic leukemia (ALL) cell line. PPI, per se, induced a dose-dependent inhibition of proliferation of tumor B cells, which was associated with a dose- and time-dependent apoptotic-like cytotoxicity in B-cell lines and leukemic cells from patients with pre-B ALL. The effect of PPI was mediated by a very early production of reactive oxygen species (ROS), that preceded alkalinization of lysosomal pH, lysosomal membrane permeabilization, and cytosol acidification, suggesting an early destabilization of the acidic vesicular compartment. Lysosomal alterations were followed by mitochondrial membrane depolarization, release of cytochrome c, chromatin condensation, and caspase activation. However, inhibition of caspase activity did not affect PPI-induced cell death, whereas specific inhibition of ROS by an antioxidant (N-acetylcysteine) significantly delayed cell death and protected both lysosomal and mitochondrial membranes. The proapoptotic activity of PPI was consistent with a clear inhibition of tumor growth following PPI treatment of B-cell lymphoma in severe combined immunodeficient mice. This study further supports the importance of acidity and pH gradients in tumor cell homeostasis and suggests new therapeutic approaches for human B-cell tumors based on PPI.

  13. Co-sensitization of ZnO by CdS quantum dots in natural dye-sensitized solar cells with polymeric electrolytes to improve the cell stability

    Energy Technology Data Exchange (ETDEWEB)

    Junhom, W.; Magaraphan, R. [Polymer Processing and Polymer Nanomaterials Research Unit, Petroleum and Petrochemical College, Chulalongkorn University, Bangkok (Thailand)

    2015-05-22

    The CdS quantum dots (QDs) were deposited on ZnO layer by chemical bath deposition method to absorb light in the shorter wavelength region and used as photoanode in the dye sensitized solar cell (DSSCs) with natural dye extracted from Noni leaves. Microstructures of CdS-ZnO from various dipping time were characterized by XRD, FE-SEM and EDX. The results showed that the CdS is hexagonal structure and the amount of CdS increases when the dipping time increases. The maximal conversion efficiency of 0.292% was achieved by the DSSCs based on Cd