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Sample records for death receptor-mediated apoptosis

  1. USP8 suppresses death receptor-mediated apoptosis by enhancing FLIPL stability.

    PubMed

    Jeong, M; Lee, E-W; Seong, D; Seo, J; Kim, J-H; Grootjans, S; Kim, S-Y; Vandenabeele, P; Song, J

    2017-01-26

    FLICE-like inhibitory protein (FLIP) is a critical regulator of death receptor-mediated apoptosis. Here, we found ubiquitin-specific peptidase 8 (USP8) to be a novel deubiquitylase of the long isoform of FLIP (FLIPL). USP8 directly deubiquitylates and stabilizes FLIPL, but not the short isoform. USP8 depletion induces FLIPL destabilization, promoting anti-Fas-, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)- and tumor necrosis factor alpha-induced extrinsic apoptosis by facilitating death-inducing signaling complex or TNFR1 complex II formation, which results in the activation of caspase-8 and caspase-3. USP8 mRNA levels are elevated in melanoma and cervical cancers, and the protein levels of USP8 and FLIPL are positively correlated in these cancer cell lines. Xenograft analyses using ME-180 cervical cancer cells showed that USP8 depletion attenuated tumor growth upon TRAIL injection. Taken together, our data indicate that USP8 functions as a novel deubiquitylase of FLIPL and inhibits extrinsic apoptosis by stabilizing FLIPL.

  2. Fluid shear stress sensitizes cancer cells to receptor-mediated apoptosis via trimeric death receptors

    NASA Astrophysics Data System (ADS)

    Mitchell, Michael J.; King, Michael R.

    2013-01-01

    Cancer metastasis, the process of cancer cell migration from a primary to distal location, typically leads to a poor patient prognosis. Hematogenous metastasis is initiated by intravasation of circulating tumor cells (CTCs) into the bloodstream, which are then believed to adhere to the luminal surface of the endothelium and extravasate into distal locations. Apoptotic agents such as tumor necrosis factor apoptosis-inducing ligand (TRAIL), whether in soluble ligand form or expressed on the surface of natural killer cells, have shown promise in treating CTCs to reduce the probability of metastasis. The role of hemodynamic shear forces in altering the cancer cell response to apoptotic agents has not been previously investigated. Here, we report that human colon cancer COLO 205 and prostate cancer PC-3 cells exposed to a uniform fluid shear stress in a cone-and-plate viscometer become sensitized to TRAIL-induced apoptosis. Shear-induced sensitization directly correlates with the application of fluid shear stress, and TRAIL-induced apoptosis increases in a fluid shear stress force- and time-dependent manner. In contrast, TRAIL-induced necrosis is not affected by the application fluid shear stress. Interestingly, fluid shear stress does not sensitize cancer cells to apoptosis when treated with doxorubicin, which also induces apoptosis in cancer cells. Caspase inhibition experiments reveal that shear stress-induced sensitization to TRAIL occurs via caspase-dependent apoptosis. These results suggest that physiological fluid shear forces can modulate receptor-mediated apoptosis of cancer cells in the presence of apoptotic agents.

  3. A whole-genome RNAi screen identifies an 8q22 gene cluster that inhibits death receptor-mediated apoptosis.

    PubMed

    Dompe, Nicholas; Rivers, Celina Sanchez; Li, Li; Cordes, Shaun; Schwickart, Martin; Punnoose, Elizabeth A; Amler, Lukas; Seshagiri, Somasekar; Tang, Jerry; Modrusan, Zora; Davis, David P

    2011-10-25

    Deregulation of apoptosis is a common occurrence in cancer, for which emerging oncology therapeutic agents designed to engage this pathway are undergoing clinical trials. With the aim of uncovering strategies to activate apoptosis in cancer cells, we used a pooled shRNA screen to interrogate death receptor signaling. This screening approach identified 16 genes that modulate the sensitivity to ligand induced apoptosis, with several genes exhibiting frequent overexpression and/or copy number gain in cancer. Interestingly, two of the top hits, EDD1 and GRHL2, are found 50 kb apart on chromosome 8q22, a region that is frequently amplified in many cancers. By using a series of silencing and overexpression studies, we show that EDD1 and GRHL2 suppress death-receptor expression, and that EDD1 expression is elevated in breast, pancreas, and lung cancer cell lines resistant to death receptor-mediated apoptosis. Supporting the relevance of EDD1 and GRHL2 as therapeutic candidates to engage apoptosis in cancer cells, silencing the expression of either gene sensitizes 8q22-amplified breast cancer cell lines to death receptor induced apoptosis. Our findings highlight a mechanism by which cancer cells may evade apoptosis, and therefore provide insight in the search for new targets and functional biomarkers for this pathway.

  4. Ursodeoxycholic Acid Induces Death Receptor-mediated Apoptosis in Prostate Cancer Cells

    PubMed Central

    Lee, Won Sup; Jung, Ji Hyun; Panchanathan, Radha; Yun, Jeong Won; Kim, Dong Hoon; Kim, Hye Jung; Kim, Gon Sup; Ryu, Chung Ho; Shin, Sung Chul; Hong, Soon Chan; Choi, Yung Hyun; Jung, Jin-Myung

    2017-01-01

    Background Bile acids have anti-cancer properties in a certain types of cancers. We determined anticancer activity and its underlying molecular mechanism of ursodeoxycholic acid (UDCA) in human DU145 prostate cancer cells. Methods Cell viability was measured with an MTT assay. UDCA-induced apoptosis was determined with flow cytometric analysis. The expression levels of apoptosis-related signaling proteins were examined with Western blotting. Results UDCA treatment significantly inhibited cell growth of DU145 in a dose-dependent manner. It induced cellular shrinkage and cytoplasmic blebs and accumulated the cells with sub-G1 DNA contents. Moreover, UDCA activated caspase 8, suggesting that UDCA-induced apoptosis is associated with extrinsic pathway. Consistent to this finding, UDCA increased the expressions of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, death receptor 4 (DR4) and death receptor 5 (DR5), and TRAIL augmented the UDCA-induced cell death in DU145 cells. In addition, UDCA also increased the expressions of Bax and cytochrome c and decreased the expression of Bcl-xL in DU145 cells. This finding suggests that UDCA-induced apoptosis may be involved in intrinsic pathway. Conclusions UDCA induces apoptosis via extrinsic pathway as well as intrinsic pathway in DU145 prostate cancer cells. UDCA may be a promising anti-cancer agent against prostate cancer. PMID:28382282

  5. Ouabain-induced perturbations in intracellular ionic homeostasis regulate death receptor-mediated apoptosis.

    PubMed

    Panayiotidis, Mihalis I; Franco, Rodrigo; Bortner, Carl D; Cidlowski, John A

    2010-07-01

    Apoptosis is defined by specific morphological and biochemical characteristics including cell shrinkage (termed apoptotic volume decrease), a process that results from the regulation of ion channels and plasma membrane transporter activity. The Na(+)-K(+)-ATPase is the predominant pump that controls cell volume and plasma membrane potential in cells and alterations in its function have been suggested to be associated with apoptosis. We report here that the Na(+)-K(+)-ATPase inhibitor ouabain, potentiates apoptosis in the human lymphoma Jurkat cells exposed to Fas ligand (FasL) or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not other apoptotic agents such as H(2)O(2), thapsigargin or UV-C implicating a role for the Na(+)-K(+)-ATPase in death receptor-induced apoptosis. Interestingly, ouabain also potentiated perturbations in cell Ca(2+) homeostasis only in conjunction with the apoptotic inducer FasL but not TRAIL. Ouabain did not affect alterations in the intracellular Ca(2+) levels in response to H(2)O(2), thapsigargin or UV-C. FasL-induced alterations in Ca(2+) were not abolished in Ca(2+)-free medium but incubation of cells with BAPTA-AM inhibited both Ca(2+) perturbations and the ouabain-induced potentiation of FasL-induced apoptosis. Our data suggest that the impairment of the Na(+)-K(+)-ATPase activity during apoptosis is linked to perturbations in cell Ca(2+) homeostasis that modulate apoptosis induced by the activation of Fas by FasL.

  6. Doxorubicin induces cardiotoxicity through upregulation of death receptors mediated apoptosis in cardiomyocytes

    PubMed Central

    Zhao, Liqun; Zhang, Baolin

    2017-01-01

    Doxorubicin is a highly effective anticancer agent but causes cardiotoxicity in many patients. The mechanisms of doxorubicin-induced cardiotoxicity remain incompletely understood. Here we investigated doxorubicin-induced cytotoxicity in human induced pluripotent stem cells-derived cardiomyocytes (iPS-CMs). We found that doxorubicin and related anthracycline agents (e.g., daunorubicin, idarubicin, and epirubicin) significantly upregulated the expression of death receptors (DRs) (TNFR1, Fas, DR4 and DR5) in iPS-derived cardiomyocytes at both protein and mRNA levels. The resulting iPS-CMs cells underwent spontaneous apoptosis which was further enhanced by physiologically relevant death ligands including TNF-related apoptosis inducing ligand (TRAIL). Furthermore, TRAIL potentiated doxorubicin-induced decrease in beating rate and amplitude of iPS-derived cardiomyocytes. These data demonstrate that the induction of death receptors in cardiomyocytes is likely a critical mechanism by which doxorubicin causes cardiotoxicity. PMID:28300219

  7. Essential oil of Cephalotaxus griffithii needle inhibits proliferation and migration of human cervical cancer cells: involvement of mitochondria-initiated and death receptor-mediated apoptosis pathways.

    PubMed

    Moirangthem, Dinesh Singh; Laishram, Surbala; Rana, Virendra Singh; Borah, Jagat Chandra; Talukdar, Narayan Chandra

    2015-01-01

    This study was conducted to determine the effect of Cephalotaxus griffithii needle essential oil (CGNO) on proliferation and migration of human cervical cancer (HCC) cells. CGNO treatment decreased the viability of all the tested HCC (HeLa, ME-180 and SiHa) cells. Morphological and DNA fragmentation analysis of CGNO-treated HeLa cells indicated the involvement of apoptosis in inducing HCC cell death. CGNO increased mitochondrial membrane depolarisation and upregulated the expression of caspase-9, caspase-8, caspase-3 and cleaved-PARP. The activity of caspase-8 and caspase-9 was also significantly increased. Wound healing and transwell migration assay demonstrated that CGNO significantly inhibited the migration of HeLa cells to close a scratched wound and also inhibited their migration through filter towards a chemotactic stimulus. Taken together, these results indicated that CGNO inhibited the proliferation and migration of HCC cells. Of note, CGNO induced HeLa cell death through mitochondria-initiated and death receptor-mediated apoptosis pathway.

  8. CoCl2 induces apoptosis through the mitochondria- and death receptor-mediated pathway in the mouse embryonic stem cells.

    PubMed

    Lee, Jin-Ha; Choi, Seong-Ho; Baek, Min-Woo; Kim, Mi-Hwa; Kim, Heong-Jun; Kim, Sun-Hun; Oh, Sang-Jin; Park, Hong-Ju; Kim, Won-Jae; Jung, Ji-Yeon

    2013-07-01

    Embryonic hypoxia/ischemia is a major cause of a poor fetal outcome and future neonatal and adult handicaps. However, biochemical cellular events in mouse embryonic stem (mES) cells during hypoxia remains unclear. This study investigated the underlying mechanism of apoptosis in mES cells under CoCl2-induced hypoxic/ischemic conditions. CoCl2 enhanced the expression of hypoxia-inducible factor-1α (HIF-1α) and the accumulation of reactive oxygen species in mES cells. The CoCl2-treated mES cells showed a decrease in cell viability as well as typical apoptotic changes, cell shrinkage, chromatin condensation, and nuclear fragmentation and an extended G2/M phase of the cell cycle. CoCl2 augmented the release of cytochrome c into the cytosol from the mitochondria with a concomitant loss of the mitochondrial transmembrane potential (ΔΨm) and upregulated the voltage-dependent anion channel. In addition, CoCl2-induced caspase-3, -8, and -9 activation and upregulation of p53 level, whereas downregulated Bcl-2 and Bcl-xL, a member of the anti-apoptotic Bcl-2 family in mES cells. Furthermore, CoCl2 led to the upregulation of Fas and Fas-ligand, which are the death receptor assemblies, as well as the cleavage of Bid in mES cells. These results suggest that CoCl2 induces apoptosis through both mitochondria- and death receptor-mediated pathways that are regulated by the Bcl-2 family in mES cells.

  9. Combination Treatment with Sublethal Ionizing Radiation and the Proteasome Inhibitor, Bortezomib, Enhances Death-Receptor Mediated Apoptosis and Anti-Tumor Immune Attack

    PubMed Central

    Cacan, Ercan; Spring, Alexander M.; Kumari, Anita; Greer, Susanna F.; Garnett-Benson, Charlie

    2015-01-01

    Sub-lethal doses of radiation can modulate gene expression, making tumor cells more susceptible to T-cell-mediated immune attack. Proteasome inhibitors demonstrate broad anti-tumor activity in clinical and pre-clinical cancer models. Here, we use a combination treatment of proteasome inhibition and irradiation to further induce immunomodulation of tumor cells that could enhance tumor-specific immune responses. We investigate the effects of the 26S proteasome inhibitor, bortezomib, alone or in combination with radiotherapy, on the expression of immunogenic genes in normal colon and colorectal cancer cell lines. We examined cells for changes in the expression of several death receptors (DR4, DR5 and Fas) commonly used by T cells for killing of target cells. Our results indicate that the combination treatment resulted in increased cell surface expression of death receptors by increasing their transcript levels. The combination treatment further increases the sensitivity of carcinoma cells to apoptosis through FAS and TRAIL receptors but does not change the sensitivity of normal non-malignant epithelial cells. Furthermore, the combination treatment significantly enhances tumor cell killing by tumor specific CD8+ T cells. This study suggests that combining radiotherapy and proteasome inhibition may simultaneously enhance tumor immunogenicity and the induction of antitumor immunity by enhancing tumor-specific T-cell activity. PMID:26703577

  10. Receptor-mediated choreography of life and death.

    PubMed

    Bhardwaj, Anjana; Aggarwal, Bharat B

    2003-09-01

    The cytokine tumor necrosis factor was originally identified as a protein that kills tumor cells. So far, 18 distinct members of this family have been identified. All of them regulate cell survival, proliferation, differentiation, and cell death, also called apoptosis. The apoptosis induced by TNF, and other members of the family, for example, FasL, VEGI, and TRAIL is mediated through death receptors. The apoptotic signals by these cytokines are transduced by eight different death domain- (DD) containing receptors (TNFR1, also called DR1; Fas, also called DR2; DR3, DR4, DR5, DR6, NGFR, and EDAR). The intracellular portion of all these receptors contains a region approximately 80 amino acids long referred to as the "death domain." Upon activation by its ligand, the DD recruits various proteins that mediate both death and proliferation of the cells. These proteins in turn recruit other proteins via their DDs or death effector domains. The actual destruction of the cell, however, is accomplished by serial activation of a family of proteases referred to as caspases. Cell death is negatively regulated by a family of proteins that includes decoy receptors, silencer of DD, sentrin, cellular FLICE inhibitory protein, cellular inhibitors of apoptosis, and survivin. This review is an attempt to describe how these negative and positive players of cell death perform a harmonious dance with each other.

  11. Resveratrol attenuates acute kidney injury by inhibiting death receptor-mediated apoptotic pathways in a cisplatin-induced rat model

    PubMed Central

    Hao, Qiufa; Xiao, Xiaoyan; Zhen, Junhui; Feng, Jinbo; Song, Chun; Jiang, Bei; Hu, Zhao

    2016-01-01

    Acute kidney injury is a clinical syndrome characterized by a loss of renal function and acute tubular necrosis. Resveratrol exerts a wide range of pharmacological effects based on its anti-inflammatory, antioxidant and cytoprotective properties. The present study aimed to evaluate whether resveratrol attenuates acute kidney injury in a cisplatin-induced rat model and to investigate the potential mechanisms involved. Rats were randomly divided into four treatment groups: Control, cisplatin, resveratrol, and cisplatin plus resveratrol. Rats exposed to cisplatin displayed acute kidney injury, identified by analysis of renal function and histopathological observation. Resveratrol significantly ameliorated the increased serum creatinine, blood urea nitrogen, renal index and histopathological damage induced by cisplatin. Furthermore, compared with untreated control animals, cisplatin lead to significantly increased expression of Fas ligand, tumor necrosis factor-α (TNF-α), caspase-8 and Bcl-2 associated protein X apoptosis regulator (Bax), and decreased expression of anti-apoptosis regulators, BH3 interacting domain death agonist (BID) and B cell lymphoma 2 apoptosis regulator (Bcl-2). Administration of resveratrol significantly reversed the cisplatin-induced alteration in these apoptosis-associated proteins. In conclusion, these findings suggest that resveratrol attenuates cisplatin-induced acute kidney injury through inactivation of the death receptor-mediated apoptotic pathway, and may provide a new therapeutic strategy to ameliorate the process of acute kidney injury. PMID:27600998

  12. Genetic disruption of oncogenic Kras sensitizes lung cancer cells to Fas receptor-mediated apoptosis.

    PubMed

    Mou, Haiwei; Moore, Jill; Malonia, Sunil K; Li, Yingxiang; Ozata, Deniz M; Hough, Soren; Song, Chun-Qing; Smith, Jordan L; Fischer, Andrew; Weng, Zhiping; Green, Michael R; Xue, Wen

    2017-04-04

    Genetic lesions that activate KRAS account for ∼30% of the 1.6 million annual cases of lung cancer. Despite clinical need, KRAS is still undruggable using traditional small-molecule drugs/inhibitors. When oncogenic Kras is suppressed by RNA interference, tumors initially regress but eventually recur and proliferate despite suppression of Kras Here, we show that tumor cells can survive knockout of oncogenic Kras, indicating the existence of Kras-independent survival pathways. Thus, even if clinical KRAS inhibitors were available, resistance would remain an obstacle to treatment. Kras-independent cancer cells exhibit decreased colony formation in vitro but retain the ability to form tumors in mice. Comparing the transcriptomes of oncogenic Kras cells and Kras knockout cells, we identified 603 genes that were specifically up-regulated in Kras knockout cells, including the Fas gene, which encodes a cell surface death receptor involved in physiological regulation of apoptosis. Antibodies recognizing Fas receptor efficiently induced apoptosis of Kras knockout cells but not oncogenic Kras-expressing cells. Increased Fas expression in Kras knockout cells was attributed to decreased association of repressive epigenetic marks at the Fas promoter. Concordant with this observation, treating oncogenic Kras cells with histone deacetylase inhibitor and Fas-activating antibody efficiently induced apoptosis, thus bypassing the need to inhibit Kras. Our results suggest that activation of Fas could be exploited as an Achilles' heel in tumors initiated by oncogenic Kras.

  13. Homocysteine-NMDA receptor mediated activation of extracellular-signal regulated kinase leads to neuronal cell death

    PubMed Central

    Poddar, Ranjana; Paul, Surojit

    2009-01-01

    Hyper-homocysteinemia is an independent risk factor for stroke and neurological abnormalities. However the underlying cellular mechanisms by which elevated homocysteine can promote neuronal death is not clear. In the present study we have examined the role of NMDA receptor mediated activation of the extracellular-signal regulated mitogen activated protein (ERK MAP) kinase pathway in homocysteine-dependent neurotoxicity. The study demonstrates that in neurons L-homocysteine-induced cell death is mediated through activation of NMDA receptors. The study also shows that homocysteine-dependent NMDA receptor stimulation and resultant Ca2+ influx leads to rapid and sustained phosphorylation of ERK MAP kinase. Inhibition of ERK phosphorylation attenuates homocysteine mediated neuronal cell death thereby demonstrating that activation of ERK MAP kinase signaling pathway is an intermediate step that couples homocysteine mediated NMDA receptor stimulation to neuronal death. The findings also show that cAMP response-element binding protein (CREB), a pro-survival transcription factor and a downstream target of ERK, is only transiently activated following homocysteine exposure. The sustained activation of ERK but a transient activation of CREB together suggest that exposure to homocysteine initiates a feedback loop that shuts off CREB signaling without affecting ERK phosphorylation and thereby facilitates homocysteine mediated neurotoxicity. PMID:19508427

  14. Role of heme oxygenase 1 in TNF/TNF receptor-mediated apoptosis after hepatic ischemia/reperfusion in rats.

    PubMed

    Kim, Seok-Joo; Eum, Hyun-Ae; Billiar, Timothy R; Lee, Sun-Mee

    2013-04-01

    Hepatocellular apoptosis commonly occurs in ischemia/reperfusion (I/R) injury. The binding of tumor necrosis factor (TNF) to TNF receptor 1 (TNFR1) leads to the formation of a death-inducing signaling complex (DISC), which subsequently initiates a caspase cascade resulting in apoptosis. Heme oxygenase 1 (HO-1) confers cytoprotection against cell death in I/R injury and inhibits stress-induced apoptotic pathways in vitro. This study investigated the role of HO-1 in modulating TNF/TNFR1-mediated cell death pathways in hepatic I/R injury. Rats were pretreated with hemin, an HO-1 inducer, and zinc protoporphyrin (ZnPP), an HO-1 inhibitor, before undergoing hepatic I/R. Heme oxygenase 1 activity increased after reperfusion. Ischemia/reperfusion-induced hepatocellular apoptosis was attenuated by hemin, as determined by the caspase-3 and -8 activity assays and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling). Zinc protoporphyrin eliminated the cytoprotective effect of hemin. Hepatic TNFR1 protein expression was unchanged among the experimental groups, whereas mitochondrial TNFR1 protein increased after I/R. Ischemia/reperfusion increased the quantity of DISC components, including TRADD (TNFR1-associated death domain), FADD (Fas-associated death domain), and caspase-8, as well as the assembly of DISCs within the liver. In the mitochondrial fraction, TNFR1-associated caspase-8 was increased after I/R. These increases were attenuated by hemin; zinc protoporphyrin eliminated this effect. Our findings suggest that the cytoprotective effects of HO-1 are mediated by suppression of TNF/TNFR1-mediated apoptotic signaling, specifically by modulating apoptotic DISC formation and mitochondrial TNFR1 translocation during hepatic I/R.

  15. P2X7 receptor-mediated PARP1 activity regulates astroglial death in the rat hippocampus following status epilepticus

    PubMed Central

    Kim, Ji Yang; Ko, Ah-Reum; Kim, Ji-Eun

    2015-01-01

    Poly(ADP-ribose) polymerase-1 (PARP1) plays a regulatory role in apoptosis, necrosis, and other cellular processes after injury. Recently, we revealed that PARP1 regulates the differential neuronal/astroglial responses to pilocarpine-induced status epilepticus (SE) in the distinct brain regions. In addition, P2X7 receptor (P2X7R), an ATP-gated ion channel, activation accelerates astroglial apoptosis, while it attenuates clasmatodendrosis (lysosome-derived autophagic astroglial death). Therefore, we investigated whether P2X7R regulates regional specific astroglial PARP1 expression/activation in response to SE. In the present study, P2X7R activation exacerbates SE-induced astroglial apoptosis, while P2X7R inhibition attenuates it accompanied by increasing PARP1 activity in the molecular layer of the dentate gyrus following SE. In the CA1 region, however, P2X7R inhibition deteriorates SE-induced clasmatodendrosis via PARP1 activation following SE. Taken together, our findings suggest that P2X7R function may affect SE-induced astroglial death by regulating PARP1 activation/expression in regional-specific manner. Therefore, the selective modulation of P2X7R-mediated PARP1 functions may be a considerable strategy for controls in various types of cell deaths. PMID:26388738

  16. Methamphetamine-induced neuronal apoptosis involves the activation of multiple death pathways. Review.

    PubMed

    Cadet, Jean Lud; Jayanthi, Subramaniam; Deng, Xiaolin

    2005-11-01

    The abuse of the illicit drug methamphetamine (METH) is a major concern because it can cause terminal degeneration and neuronal cell death in the brain. METH-induced cell death occurs via processes that resemble apoptosis. In the present review, we discuss the role of various apoptotic events in the causation of METH-induced neuronal apoptosis in vitro and in vivo. Studies using comprehensive approaches to gene expression profiling have allowed for the identification of several genes that are up-regulated or down-regulated after an apoptosis-inducing dose of the drug. Further experiments have also documented the fact that the drug can cause demise of striatal enkephalinergic neurons by cross-talks between mitochondria-, endoplasmic reticulum- and receptor-mediated apoptotic events. These neuropathological observations have also been reported in models of drug-induced neuroplastic alterations used to mimic drug addiction (Nestler, 2001).

  17. Signaling pathways that regulate life and cell death: evolution of apoptosis in the context of self-defense.

    PubMed

    Muñoz-Pinedo, Cristina

    2012-01-01

    Programmed Cell Death is essential for the life cycle of many organisms. Cell death in multicellular organisms can occur as a consequence of massive damage (necrosis) or in a controlled form, through engagement of diverse biochemical programs. The best well known form of programmed cell death is apoptosis. Apoptosis occurs in animals as a consequence of a variety of stimuli including stress and social signals and it plays essential roles in morphogenesis and immune defense. The machinery of apoptosis is well conserved among animals and it is composed of caspases (the proteases which execute cell death), adapter proteins (caspase activators), Bcl-2 family proteins and Inhibitor of Apoptosis Proteins (IAPs). We will describe in this chapter the main apoptotic pathways in animals: the extrinsic (death receptor-mediated), the intrinsic/mitochondrial and the Granzyme B pathway. Other forms of non-apoptotic Programmed Cell Death which occur in animals will also be discussed. We will summarize the current knowledge about apoptotic-like and other forms of cell death in other organisms such as plants and protists.Additionally, we will discuss the hypothesis that apoptosis originated as part of a host defense mechanism. We will explore the similarities between the protein complexes which mediate apoptosis (apoptosomes) and complexes involved in immunity: inflammasomes. Additional functions of apoptotic proteins related to immune function will be summarized, in an effort to explore the evolutionary origins of cell death.

  18. Lymphotropic Virions Affect Chemokine Receptor-Mediated Neural Signaling and Apoptosis: Implications for Human Immunodeficiency Virus Type 1-Associated Dementia

    PubMed Central

    Zheng, Jialin; Ghorpade, Anuja; Niemann, Douglas; Cotter, Robin L.; Thylin, Michael R.; Epstein, Leon; Swartz, Jennifer M.; Shepard, Robin B.; Liu, Xiaojuan; Nukuna, Adeline; Gendelman, Howard E.

    1999-01-01

    Chemokine receptors pivotal for human immunodeficiency virus type 1 (HIV-1) infection in lymphocytes and macrophages (CCR3, CCR5, and CXCR4) are expressed on neural cells (microglia, astrocytes, and/or neurons). It is these cells which are damaged during progressive HIV-1 infection of the central nervous system. We theorize that viral coreceptors could effect neural cell damage during HIV-1-associated dementia (HAD) without simultaneously affecting viral replication. To these ends, we studied the ability of diverse viral strains to affect intracellular signaling and apoptosis of neurons, astrocytes, and monocyte-derived macrophages. Inhibition of cyclic AMP, activation of inositol 1,4,5-trisphosphate, and apoptosis were induced by diverse HIV-1 strains, principally in neurons. Virions from T-cell-tropic (T-tropic) strains (MN, IIIB, and Lai) produced the most significant alterations in signaling of neurons and astrocytes. The HIV-1 envelope glycoprotein, gp120, induced markedly less neural damage than purified virions. Macrophage-tropic (M-tropic) strains (ADA, JR-FL, Bal, MS-CSF, and DJV) produced the least neural damage, while 89.6, a dual-tropic HIV-1 strain, elicited intermediate neural cell damage. All T-tropic strain-mediated neuronal impairments were blocked by the CXCR4 antibody, 12G5. In contrast, the M-tropic strains were only partially blocked by 12G5. CXCR4-mediated neuronal apoptosis was confirmed in pure populations of rat cerebellar granule neurons and was blocked by HA1004, an inhibitor of calcium/calmodulin-dependent protein kinase II, protein kinase A, and protein kinase C. Taken together, these results suggest that progeny HIV-1 virions can influence neuronal signal transduction and apoptosis. This process occurs, in part, through CXCR4 and is independent of CD4 binding. T-tropic viruses that traffic in and out of the brain during progressive HIV-1 disease may play an important role in HAD neuropathogenesis. PMID:10482576

  19. The aryl hydrocarbon receptor mediates raloxifene-induced apoptosis in estrogen receptor-negative hepatoma and breast cancer cells

    PubMed Central

    O'Donnell, E F; Koch, D C; Bisson, W H; Jang, H S; Kolluri, S K

    2014-01-01

    Identification of new molecular targets for the treatment of breast cancer is an important clinical goal, especially for triple-negative breast cancer, which is refractory to existing targeted treatments. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known primarily as the mediator of dioxin toxicity. However, the AhR can also inhibit cellular proliferation in a ligand-dependent manner and act as a tumor suppressor in mice, and thus may be a potential anticancer target. To investigate the AhR as an anticancer target, we conducted a small molecule screen to discover novel AhR ligands with anticancer properties. We identified raloxifene, a selective estrogen receptor (ER) modulator currently used in the clinic for prevention of ER-positive breast cancer and osteoporosis in post-menopausal women, as an AhR activator. Raloxifene directly bound the AhR and induced apoptosis in ER-negative mouse and human hepatoma cells in an AhR-dependent manner, indicating that the AhR is a molecular target of raloxifene and mediates raloxifene-induced apoptosis in the absence of ER. Raloxifene selectively induced apoptosis of triple-negative MDA-MB-231 breast cancer cells compared with non-transformed mammary epithelial cells via the AhR. Combined with recent data showing that raloxifene inhibits triple-negative breast cancer xenografts in vivo (Int J Oncol. 43(3):785-92, 2013), our results support the possibility of repurposing of raloxifene as an AhR-targeted therapeutic for triple-negative breast cancer patients. To this end, we also evaluated the role of AhR expression on survival of patients diagnosed with breast cancer. We found that higher expression of the AhR is significantly associated with increased overall survival and distant metastasis-free survival in both hormone-dependent (ER-positive) and hormone-independent (ER and progesterone receptor (PR)-negative) breast cancers. Together, our data strongly support the possibility of using the Ah

  20. The aryl hydrocarbon receptor mediates raloxifene-induced apoptosis in estrogen receptor-negative hepatoma and breast cancer cells.

    PubMed

    O'Donnell, E F; Koch, D C; Bisson, W H; Jang, H S; Kolluri, S K

    2014-01-30

    Identification of new molecular targets for the treatment of breast cancer is an important clinical goal, especially for triple-negative breast cancer, which is refractory to existing targeted treatments. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known primarily as the mediator of dioxin toxicity. However, the AhR can also inhibit cellular proliferation in a ligand-dependent manner and act as a tumor suppressor in mice, and thus may be a potential anticancer target. To investigate the AhR as an anticancer target, we conducted a small molecule screen to discover novel AhR ligands with anticancer properties. We identified raloxifene, a selective estrogen receptor (ER) modulator currently used in the clinic for prevention of ER-positive breast cancer and osteoporosis in post-menopausal women, as an AhR activator. Raloxifene directly bound the AhR and induced apoptosis in ER-negative mouse and human hepatoma cells in an AhR-dependent manner, indicating that the AhR is a molecular target of raloxifene and mediates raloxifene-induced apoptosis in the absence of ER. Raloxifene selectively induced apoptosis of triple-negative MDA-MB-231 breast cancer cells compared with non-transformed mammary epithelial cells via the AhR. Combined with recent data showing that raloxifene inhibits triple-negative breast cancer xenografts in vivo (Int J Oncol. 43(3):785-92, 2013), our results support the possibility of repurposing of raloxifene as an AhR-targeted therapeutic for triple-negative breast cancer patients. To this end, we also evaluated the role of AhR expression on survival of patients diagnosed with breast cancer. We found that higher expression of the AhR is significantly associated with increased overall survival and distant metastasis-free survival in both hormone-dependent (ER-positive) and hormone-independent (ER and progesterone receptor (PR)-negative) breast cancers. Together, our data strongly support the possibility of using the Ah

  1. NR2B-NMDA receptor mediated modulation of the tyrosine phosphatase STEP regulates glutamate induced neuronal cell death

    PubMed Central

    Poddar, Ranjana; Deb, Ishani; Mukherjee, Saibal; Paul, Surojit

    2011-01-01

    The present study examines the role of a neuron-specific tyrosine phosphatase (STEP) in excitotoxic cell death. Our findings demonstrate that p38 MAPK, a stress-activated kinase that is known to play a role in the etiology of excitotoxic cell death is a substrate of STEP. Glutamate-mediated NMDA receptor stimulation leads to rapid but transient activation of p38 MAPK, which is primarily dependent on NR2A-NMDA receptor activation. Conversely, activation of NR2B-NMDA receptors leads to dephosphorylation and subsequent activation of STEP, which in turn leads to inactivation of p38 MAPK. Thus during transient NMDA receptor stimulation, increases in STEP activity appears to limit the duration of activation of p38 MAPK and improves neuronal survival. However, if NR2B-NMDA receptor stimulation is sustained, protective effects of STEP activation are lost, as these stimuli cause significant degradation of active STEP, leading to secondary activation of p38 MAP kinase. Consistent with this observation, a cell transducible TAT-STEP peptide that constitutively binds to p38 MAPK attenuated neuronal cell death caused by sustained NMDA receptor stimulation. The findings imply that the activation and levels of STEP are dependent on the duration and magnitude of NR2B-NMDA receptor stimulation and STEP serves as a modulator of NMDA receptor dependent neuronal injury, through its regulation of p38 MAPK. PMID:21029094

  2. Apoptosis, oncosis, and necrosis. An overview of cell death.

    PubMed Central

    Majno, G.; Joris, I.

    1995-01-01

    The historical development of the cell death concept is reviewed, with special attention to the origin of the terms necrosis, coagulation necrosis, autolysis, physiological cell death, programmed cell death, chromatolysis (the first name of apoptosis in 1914), karyorhexis, karyolysis, and cell suicide, of which there are three forms: by lysosomes, by free radicals, and by a genetic mechanism (apoptosis). Some of the typical features of apoptosis are discussed, such as budding (as opposed to blebbing and zeiosis) and the inflammatory response. For cell death not by apoptosis the most satisfactory term is accidental cell death. Necrosis is commonly used but it is not appropriate, because it does not indicate a form of cell death but refers to changes secondary to cell death by any mechanism, including apoptosis. Abundant data are available on one form of accidental cell death, namely ischemic cell death, which can be considered an entity of its own, caused by failure of the ionic pumps of the plasma membrane. Because ischemic cell death (in known models) is accompanied by swelling, the name oncosis is proposed for this condition. The term oncosis (derived from ónkos, meaning swelling) was proposed in 1910 by von Reckling-hausen precisely to mean cell death with swelling. Oncosis leads to necrosis with karyolysis and stands in contrast to apoptosis, which leads to necrosis with karyorhexis and cell shrinkage. Images Figure 1 Figure 2 Figure 3 Figure 5 Figure 6 Figure 7 Figure 8 PMID:7856735

  3. Apoptosis through Death Receptors in Temporal Lobe Epilepsy-Associated Hippocampal Sclerosis

    PubMed Central

    Teocchi, Marcelo Ananias; D'Souza-Li, Lília

    2016-01-01

    Seizure models have demonstrated that neuroinflammation and neurodegeneration are preponderant characteristics of epilepsy. Considering the lack of clinical studies, our aim is to investigate the extrinsic pathway of apoptosis in pharmacoresistant temporal lobe epilepsy (TLE) associated with hippocampal sclerosis (HS) patients, TLE(HS). By a specific death receptor-mediated apoptosis array plate, 31 upregulated targets were revealed in the sclerotic hippocampus from TLE(HS) patients. Amongst them are the encoding genes for ligands (FASLG, TNF, and TNFSF10) and death receptors (FAS, TNFRSF1A, TNFRSF10A, and TNFRSF10B). In addition, we evaluated the hippocampal relative mRNA expression of the two TNF receptors, TNFRSF1A and TNFRSF1B, in patients, being both upregulated (n = 14; P < 0.01 and P < 0.04, resp.) when compared to the post mortem control group (n = 4). Our results have clearly suggested that three different death receptor apoptotic systems may be associated with the maintenance and progression of TLE-associated HS: (1) TNF-TNFRSF1A, (2) FASLG-FAS, and (3) TNFSF10-TNFRSF10A/B. Their effects on epilepsy are still scarcely comprehended. Our study points out to TNF and TNF receptor superfamily pathways as important targets for pharmacological studies regarding the benefits of an anti-inflammatory therapy in these patients. PMID:27006531

  4. Comparison of Types of Cell Death: Apoptosis and Necrosis.

    ERIC Educational Resources Information Center

    Manning, Francis; Zuzel, Katherine

    2003-01-01

    Cell death is an essential factor in many biological processes including development. Discusses two types of cell death: (1) necrosis (induced by sodium azide); and (2) apoptosis (induced by sodium chromate). Illustrates key features that differ between these two types of cells death including loss of membrane integrity and internucleosomal DNA…

  5. Maslinic Acid, a Natural Triterpene, Induces a Death Receptor-Mediated Apoptotic Mechanism in Caco-2 p53-Deficient Colon Adenocarcinoma Cells

    PubMed Central

    Reyes-Zurita, Fernando J.; Rufino-Palomares, Eva E.; García-Salguero, Leticia; Peragón, Juan; Medina, Pedro P.; Parra, Andrés; Cascante, Marta; Lupiáñez, José A.

    2016-01-01

    Maslinic acid (MA) is a natural triterpene present in high concentrations in the waxy skin of olives. We have previously reported that MA induces apoptotic cell death via the mitochondrial apoptotic pathway in HT29 colon cancer cells. Here, we show that MA induces apoptosis in Caco-2 colon cancer cells via the extrinsic apoptotic pathway in a dose-dependent manner. MA triggered a series of effects associated with apoptosis, including the cleavage of caspases -8 and -3, and increased the levels of t-Bid within a few hours of its addition to the culture medium. MA had no effect on the expression of the Bax protein, release of cytochrome-c or on the mitochondrial membrane potential. This suggests that MA triggered the extrinsic apoptotic pathway in this cell type, as opposed to the intrinsic pathway found in the HT29 colon-cancer cell line. Our results suggest that the apoptotic mechanism induced in Caco-2 may be different from that found in HT29 colon-cancer cells, and that in Caco-2 cells MA seems to work independently of p53. Natural antitumoral agents capable of activating both the extrinsic and intrinsic apoptotic pathways could be of great use in treating colon-cancer of whatever origin. PMID:26751572

  6. Signaling pathway for apoptosis: a racetrack for life or death.

    PubMed

    Wang, E; Marcotte, R; Petroulakis, E

    1999-01-01

    Apoptosis, or programmed cell death, is a gene-directed mechanism activated as a suicidal event to get rid of excess, damaged, or infected cells. The recent astounding pace of research in this area has expanded our horizon of understanding that this mechanism is regulated largely by pro- and anti-apoptosis factors acting for or against the final death event. The driving force behind these factors, either pro-apoptosis or pro-survival, is largely determined by signal transduction pathways, starting with the initiation of a death signal at the plasma membrane, and following through a complex cytoplasmic network before reaching the end point of cell demise. Enmeshed in this intricate cytoplasmic network are many checkpoints, where complexes of pro- and anti-apoptosis factors function to facilitate or deter the death signals. The culmination of the balancing act between these two camps of factors at these signal transduction checkpoints may then result in the final decision to die or to live. Thus, the eventual death of a cell may require successful passage through all the checkpoints, a mechanism Nature has provided as a safeguard to prevent erroneous triggering of death. With the advent of a new biotechnology revolution at the dawn of the new millenium, we look forward to an exciting era when we can gain fuller understanding of the operation of all these checkpoints. Ultimately, this gain will pave the way to control the apoptosis event at the checkpoints, and to support the organism's functionality as long as possible. J. Cell. Biochem. Suppls. 32/33:95-102, 1999.

  7. X-ray-induced cell death: Apoptosis and necrosis

    SciTech Connect

    Nakano, Hisako; Shinohara, Kunio

    1994-10-01

    X-ray-induced cell death in MOLT-4N1, a subclone of MOLT-4 cells, and M10 cells was studied with respect to their modes of cell death, apoptosis and necrosis. MOLT-4N1 cells showed radiosensitivity similar to that of M10 cells, a radiosensitive mutant of L5178Y, as determined by the colony formation assay. Analysis of cell size demonstrated that MOLT-4N1 cells increased in size at an early stage after irradiation and then decreased to a size smaller than that of control cells, whereas the size of irradiated M10 cells increased continuously. Apoptosis detected by morphological changes and DNA ladder formation (the cleavage of DNA into oligonucleosomal fragments) occurred in X-irradiated MOLT-4N1 cells but not in M10 cells. Pulsed-field gel electrophoresis showed that the ladder formation involved an intermediate-sized DNA (about 20 kbp). Most of the DNA was detected at the origin in both methods of electrophoresis in the case of M10 cells, though a trace amount of ladder formation was observed. Heat treatment of M10 cells induced apoptosis within 30 min after treatment, in contrast to MOLT-4N1 cells. The results suggest that apoptosis and necrosis are induced by X rays in a manner which is dependent on the cell line irrespective of the capability of the cells to develop apoptosis. DNA fragmentation was the earliest change observed in the development of apoptosis. 27 refs., 8 figs., 1 tab.

  8. Apoptosis: understanding programmed cell death for the CRNA.

    PubMed

    Bennetts, Paul S; Pierce, Janet D

    2010-06-01

    Apoptosis, or programmed cell death, is a physiologic mechanism employed by most multicellular organisms to maintain homeostasis of body tissues. In balance with the production of new cells by mitosis, apoptosis provides for the orderly destruction and removal of cells that are no longer needed by the organism. Apoptosis occurs by complex pathways involving multiple biochemical signals and processes. Dysfunctional apoptotic mechanisms are the pathologic basis for many human diseases, including common disorders of the heart, lungs, brain, and endocrine systems. Researchers have demonstrated in animal models that neurodegenerative changes after the administration of anesthetic drugs are related to apoptosis. Anesthesia drugs have been found to induce apoptosis, perhaps through the production of reactive oxygen species. Propofol is a drug used in anesthesia that has unique antioxidant qualities that may be beneficial. The purpose of this article is to review, for nurse anesthesia providers, current information about the process of apoptosis, the role of apoptosis in comorbid diseases, and the implications of the effects of anesthesia drugs on normal apoptotic mechanisms that need to be evaluated as potential sources of risk or benefit to surgical patients.

  9. Osteoblast cell death on methacrylate polymers involves apoptosis.

    PubMed

    Gough, J E; Downes, S

    2001-12-15

    The success of an implant depends on the implant-tissue interface. There are many causes of implant failure, one of which is tissue necrosis. The aim of this in vitro study was to determine whether cell death of primary human osteoblasts (implant site specific cells) occurred by apoptosis (a form of programmed cell death) on two methacrylate polymers. Cells were cultured on poly(ethyl methacrylate)/tetrahydrofurfuryl methacrylate and poly(methyl methacrylate in the form of 13-mm discs, in conditioned medium containing leachable monomer and in the presence of various concentrations of monomer itself in the culture medium. It was found that monomer and leached monomer caused apoptosis of human osteoblast cells in this system. Tetrahydrofurfuryl methacrylate monomer was found to be more toxic than currently used monomer methylmethacrylate. Preincubation of polymers in serum containing medium was found to increase the biocompatibility of the polymers. High levels of apoptosis occurred on polymer used directly after polymerization. Apoptosis levels were decreased after polymer was incubated at 60 degrees C overnight or for 3 days. Apoptosis therefore may occur in cells at the implant site in vivo.

  10. Carboxylation of multiwalled carbon nanotube attenuated the cytotoxicity by limiting the oxidative stress initiated cell membrane integrity damage, cell cycle arrestment, and death receptor mediated apoptotic pathway.

    PubMed

    Liu, Zhenbao; Liu, Yanfei; Peng, Dongming

    2015-08-01

    In this study, the effects of carboxylated multiwalled carbon nanotubes (MWCNTs-COOH) on human normal liver cell line L02 was compared with that of pristine multiwalled carbon nanotubes (p-MWCNTs). It was shown that compared with MWCNTs-COOH, p-MWCNTs induced apoptosis, reduced the level of intracellular antioxidant glutathione more significantly, and caused severer cell membrane damage as demonstrated by lactate dehydrogenase leakage. Cell cycles were arrested by both MWCNTs, while p-MWCNTs induced higher ratio of G0/G1 phase arrestment as compared with MWCNTs-COOH. Caspase-8 was also activated after both MWCNTs exposure, indicating extrinsic apoptotic pathway was involved in the apoptosis induced by MWCNTs exposure, more importantly, MWCNTs-COOH significantly reduced the activation of caspase-8 as compared with p-MWCNTs. All these results suggested that MWCNTs-COOH might be safer for in vivo application as compared with p-MWCNTs.

  11. Wnt3a mitigates acute lung injury by reducing P2X7 receptor-mediated alveolar epithelial type I cell death

    PubMed Central

    Guo, Y; Mishra, A; Weng, T; Chintagari, N R; Wang, Y; Zhao, C; Huang, C; Liu, L

    2014-01-01

    Acute lung injury (ALI) is characterized by pulmonary endothelial and epithelial cell damage, and loss of the alveolar–capillary barrier. We have previously shown that P2X7 receptor (P2X7R), a cell death receptor, is specifically expressed in alveolar epithelial type I cells (AEC I). In this study, we hypothesized that P2X7R-mediated purinergic signaling and its interaction with Wnt/β-catenin signaling contributes to AEC I death. We examined the effect of P2X7R agonist 2′-3′-O-(4-benzoylbenzoyl)-ATP (BzATP) and Wnt agonist Wnt3a on AEC I death in vitro and in vivo. We also assessed the therapeutic potential of Wnt3a in a clinically relevant ALI model of intratracheal lipopolysaccharide (LPS) exposure in ventilated mice. We found that the activation of P2X7R by BzATP caused the death of AEC I by suppressing Wnt/β-catenin signaling through stimulating glycogen synthase kinase-3β (GSK-3β) and proteasome. On the other hand, the activation of Wnt/β-catenin signaling by Wnt3a, GSK-3β inhibitor, or proteasome inhibitor blocked the P2X7R-mediated cell death. More importantly, Wnt3a attenuated the AEC I damage caused by intratracheal instillation of BzATP in rats or LPS in ventilated mice. Our results suggest that Wnt3a overrides the effect of P2X7R on the Wnt/β-catenin signaling to prevent the AEC I death and restrict the severity of ALI. PMID:24922070

  12. Role of transglutaminase 2 in PAC1 receptor mediated protection against hypoxia-induced cell death and neurite outgrowth in differentiating N2a neuroblastoma cells.

    PubMed

    Algarni, Alanood S; Hargreaves, Alan J; Dickenson, John M

    2017-03-15

    The PAC1 receptor and tissue transglutaminase (TG2) play important roles in neurite outgrowth and modulation of neuronal cell survival. In this study, we investigated the regulation of TG2 activity by the PAC1 receptor in retinoic acid-induced differentiating N2a neuroblastoma cells. TG2 transamidase activity was determined using an amine incorporation and a peptide cross linking assay. In situ TG2 activity was assessed by visualising the incorporation of biotin-X-cadaverine using confocal microscopy. TG2 phosphorylation was monitored via immunoprecipitation and Western blotting. The role of TG2 in PAC1 receptor-induced cytoprotection and neurite outgrowth was investigated by monitoring hypoxia-induced cell death and appearance of axonal-like processes, respectively. The amine incorporation and protein crosslinking activity of TG2 increased in a time and concentration-dependent manner following stimulation with pituitary adenylate cyclase-activating polypeptide-27 (PACAP-27). PACAP-27 mediated increases in TG2 activity were abolished by the TG2 inhibitors Z-DON and R283 and by pharmacological inhibition of protein kinase A (KT 5720 and Rp-cAMPs), protein kinase C (Ro 31-8220), MEK1/2 (PD 98059), and removal of extracellular Ca(2+). Fluorescence microscopy demonstrated PACAP-27 induced in situ TG2 activity. TG2 inhibition blocked PACAP-27 induced attenuation of hypoxia-induced cell death and outgrowth of axon-like processes. TG2 activation and cytoprotection were also observed in human SH-SY5Y cells. Together, these results demonstrate that TG2 activity was stimulated downstream of the PAC1 receptor via a multi protein kinase dependent pathway. Furthermore, PAC1 receptor-induced cytoprotection and neurite outgrowth are dependent upon TG2. These results highlight the importance of TG2 in the cellular functions of the PAC1 receptor.

  13. Receptor-Mediated Tobacco Toxicity

    PubMed Central

    Arredondo, Juan; Chernyavsky, Alexander I.; Marubio, Lisa M.; Beaudet, Arthur L.; Jolkovsky, David L.; Pinkerton, Kent E.; Grando, Sergei A.

    2005-01-01

    Tobacco is a known cause of oral disease but the mechanism remains elusive. Nicotine (Nic) is a likely culprit of pathobiological effects because it displaces the local cytotransmitter acetylcholine from the nicotinic receptors (nAChRs) expressed by oral keratinocytes (KCs). To gain a mechanistic insight into tobacco-induced morbidity in the oral cavity, we studied effects of exposures to environmental tobacco smoke (ETS) versus equivalent concentration of pure Nic on human and murine KCs. Both ETS and Nic up-regulated expression of cell cycle and apoptosis regulators, differentiation marker filaggrin, and signal transduction factors at both the mRNA and protein levels. These changes could be abolished in cultured human oral KCs transfected with anti-α3 small interfering RNA or treated with the α3β2-preferring antagonist α-conotoxin MII. Functional inactivation of α3-mediated signaling in α3−/− mutant KCs prevented most of the ETS/Nic-dependent changes in gene expression. To determine relevance of the in vitro findings to the in vivo situation, we studied gene expression in oral mucosa of neonatal α3+/+ and α3−/− littermates delivered by heterozygous mice soon after their exposures to ETS or equivalent concentration of pure Nic in drinking water. In addition to reverse transcriptase-polymerase chain reaction and Western blot, the ETS/Nic-dependent alterations in gene expression were also detected by semiquantitative immunofluorescence assay directly in KCs comprising murine oral mucosa. Only wild-type mice consistently developed significant (P < 0.05) changes in the gene expression. These results identified α3β2 nAChR as a major receptor mediating effects of tobacco products on KC gene expression. Real-time polymerase chain reaction demonstrated that in all three model systems the common genes targeted by α3β2-mediated ETS/Nic toxicity were p21, Bcl-2, NF-κB, and STAT-1. The expression of the nAChR subunits α5 and β2 and the muscarinic

  14. Stressed to death: implication of lymphocyte apoptosis for psychoneuroimmunology.

    PubMed

    Shi, Yufang; Devadas, Satish; Greeneltch, Kristy M; Yin, Deling; Allan Mufson, R; Zhou, Jian-nian

    2003-02-01

    Psychological and physical stressors best exemplify the intercommunication of the immune and the nervous systems. It has been shown that stress significantly impacts leukocyte cellularity and immune responses and alters susceptibility to various diseases. While acute stress has been shown to enhance immune responses, chronic stress often leads to immunosuppression. Among many criteria examined upon exposure to chronic stress, the reduction in lymphocyte mitogenic response and lymphocyte cellularity are commonly assessed. We have reported that chronic restraint stress could induce lymphocyte reduction, an effect dependent on endogenous opioids. Interestingly, the effect of endogenous opioids was found to be exerted through increasing the expression of a cell death receptor, Fas, and an increased sensitivity of lymphocytes to apoptosis. Stress-induced lymphocyte reduction was not affected by adrenalectomy. In this review, based on available literature and our recent data, we will discuss the role of the hypothalamic-pituitary-adrenal axis and endogenous opioids and examine the mechanisms by which chronic stress modulates lymphocyte apoptosis.

  15. Statins, Bcl-2, and apoptosis: cell death or cell protection?

    PubMed

    Wood, W Gibson; Igbavboa, Urule; Muller, Walter E; Eckert, Gunter P

    2013-10-01

    Statins have proven their effectiveness in the treatment of cardiovascular disease. This class of drugs has also attracted attention as a potential treatment for dissimilar diseases such as certain types of cancers and neurodegenerative diseases. What appears to be a contradiction is that, in the case of cancer, it has been suggested that statins increase apoptosis and alter levels of Bcl-2 family members (e.g., reduce Bcl-2 and increase Bax), whereas studies mainly using noncancerous cells report opposite effects. This review examined studies reporting on the effects of statins on Bcl-2 family members, apoptosis, cell death, and cell protection. Much, but not all, of the evidence supporting the pro-apoptotic effects of statins is based on data in cancer cell lines and the use of relatively high drug concentrations. Studies indicating an anti-apoptotic effect of statins are fewer in number and generally used much lower drug concentrations and normal cells. Those conclusions are not definitive, and certainly, there is a need for additional research to determine if statin repositioning is justified for noncardiovascular diseases.

  16. Stressed to death: implication of lymphocyte apoptosis for psychoneuroimmunology

    NASA Technical Reports Server (NTRS)

    Shi, Yufang; Devadas, Satish; Greeneltch, Kristy M.; Yin, Deling; Allan Mufson, R.; Zhou, Jian-nian

    2003-01-01

    Psychological and physical stressors best exemplify the intercommunication of the immune and the nervous systems. It has been shown that stress significantly impacts leukocyte cellularity and immune responses and alters susceptibility to various diseases. While acute stress has been shown to enhance immune responses, chronic stress often leads to immunosuppression. Among many criteria examined upon exposure to chronic stress, the reduction in lymphocyte mitogenic response and lymphocyte cellularity are commonly assessed. We have reported that chronic restraint stress could induce lymphocyte reduction, an effect dependent on endogenous opioids. Interestingly, the effect of endogenous opioids was found to be exerted through increasing the expression of a cell death receptor, Fas, and an increased sensitivity of lymphocytes to apoptosis. Stress-induced lymphocyte reduction was not affected by adrenalectomy. In this review, based on available literature and our recent data, we will discuss the role of the hypothalamic-pituitary-adrenal axis and endogenous opioids and examine the mechanisms by which chronic stress modulates lymphocyte apoptosis.

  17. Endogenous interleukin 18 regulates testicular germ cell apoptosis during endotoxemia.

    PubMed

    Inoue, Taketo; Aoyama-Ishikawa, Michiko; Kamoshida, Shingo; Nishino, Satoshi; Sasano, Maki; Oka, Nobuki; Yamashita, Hayato; Kai, Motoki; Nakao, Atsunori; Kotani, Joji; Usami, Makoto

    2015-08-01

    Orchitis (testicular swelling) often occurs during systemic inflammatory conditions, such as sepsis. Interleukin 18 (IL18) is a proinflammatory cytokine and is an apoptotic mediator during endotoxemia, but the role of IL18 in response to inflammation in the testes was unclear. WT and IL18 knockout (KO) mice were injected lipopolysaccharide (LPS) to induce endotoxemia and examined 12 and 48  h after LPS administration to model the acute and recovery phases of endotoxemia. Caspase activation was assessed using immunohistochemistry. Protein and mRNA expression were examined by western blot and quantitative real-time RT-PCR respectively. During the acute phase of endotoxemia, apoptosis (as indicated by caspase-3 cleavage) was increased in WT mice but not in IL18 KO mice. The death receptor-mediated and mitochondrial-mediated apoptotic pathways were both activated in the WT mice but not in the KO mice. During the recovery phase of endotoxemia, apoptosis was observed in the IL18 KO mice but not in the WT mice. Activation of the death-receptor mediated apoptotic pathway could be seen in the IL18 KO mice but not the WT mice. These results suggested that endogenous IL18 induces germ cell apoptosis via death receptor mediated- and mitochondrial-mediated pathways during the acute phase of endotoxemia and suppresses germ cell apoptosis via death-receptor mediated pathways during recovery from endotoxemia. Taken together, IL18 could be a new therapeutic target to prevent orchitis during endotoxemia.

  18. Synaptic NMDA Receptors Mediate Hypoxic Excitotoxic Death

    PubMed Central

    Wroge, Christine M.; Hogins, Joshua; Eisenman, Larry; Mennerick, Steven

    2012-01-01

    Excessive NMDA receptor activation and excitotoxicity underlies pathology in many neuropsychiatric and neurological disorders, including hypoxia/ischemia. Thus, the development of effective therapeutics for these disorders demands a complete understanding of NMDA receptor (NMDAR) activation during excitotoxic insults. The extrasynaptic NMDAR hypothesis posits that synaptic NMDARs are neurotrophic/neuroprotective and extrasynaptic NMDARs are neurotoxic. In part, the extrasynaptic hypothesis is built on observed selectivity for extrasynaptic receptors of a neuroprotective use-dependent NMDAR channel blocker, memantine. In rat hippocampal neurons we found that a neuroprotective concentration of memantine shows little selectivity for extrasynaptic NMDARs when all receptors are tonically activated by exogenous glutamate. This led us to test the extrasynaptic NMDAR hypothesis using metabolic challenge, where the source of excitotoxic glutamate buildup may be largely synaptic. Three independent approaches suggest strongly that synaptic receptors participate prominently in hypoxic excitotoxicity. First, block of glutamate transporters with a non-substrate antagonist exacerbated rather than prevented damage, consistent with a primarily synaptic source of glutamate. Second, selective, preblock of synaptic NMDARs with a slowly reversible, use-dependent antagonist protected nearly fully against prolonged hypoxic insult. Third, glutamate pyruvate transaminase (GPT), which degrades ambient but not synaptic glutamate, did not protect against hypoxia but protected against exogenous glutamate damage. Together, these results suggest that synaptic NMDARs can mediate excitotoxicity, particularly when the glutamate source is synaptic and when synaptic receptor contributions are rigorously defined. Moreover, the results suggest that in some situations therapeutically targeting extrasynaptic receptors may be inappropriate. PMID:22573696

  19. Brain death is associated with endoplasmic reticulum stress and apoptosis in rat liver.

    PubMed

    Cao, S; Wang, T; Yan, B; Lu, Y; Zhao, Y; Zhang, S

    2014-12-01

    Cell death pathways initiated by stress on the endoplasmic reticulum (ER) have been implicated in a variety of common diseases, such as ischemia/reperfusion injury, diabetes, heart disease, and neurodegenerative disorders. However, the contribution of ER stress to apoptosis and liver injury after brain death is not known. In the present study, we found that brain death induces a variety of signature ER stress markers, including ER stress-specific X box-binding protein 1 and up-regulation of glucose-regulated protein 78. Furthermore, brain death causes up-regulation of C/EBP homologous protein and caspase-12. Consistent with this, terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labeling assay and transmission electron microscopy confirmed apoptosis in the liver after brain death. Taken together, the present study provides strong evidence supporting the presence and importance of ER stress and response in mediating brain death-induced apoptosis and liver injury.

  20. Aflatoxin B1 invokes apoptosis via death receptor pathway in hepatocytes.

    PubMed

    Mughal, Muhammad Jameel; Xi, Peng; Yi, Zhou; Jing, Fang

    2017-01-31

    The fungal metabolites produced by Aspergillus flavus and Aspergillus parasiticus cause detrimental health effects on humans and animals. Particularly aflatoxin B1 (AFB1) is the most studied and a well-known global carcinogen, producing hepatotoxic, genotoxic and immunotoxic effects in multiple species. AFB1 is shown to provoke liver dysfunctioning by causing hepatocytes apoptosis and disturbing cellular enzymatic activities. In liver, AFB1 causes apoptosis via extrinsic mechanism because of high expression of death receptor pathway. The detailed mechanism of AFB1 induced hepatocytes apoptosis, via death receptor pathway still remains elusive. So the present study was conducted to explore apoptotic mechanism initiated by death receptors and associated genes in aflatoxin B1 induced liver apoptosis in chickens fed with AFB1 for 3 weeks. Results from the present study displayed histopathological and ultrastructural changes in liver such as hydropic degeneration, fatty vacuolar degeneration and proliferation of bile duct in hepatocytes in AFB1 group, along with imbalance between reactive oxygen species (ROS) and antioxidant defense system upon AFB1 ingestion. Moreover, AFB1 intoxicated chickens showed upregulation of death receptors FAS, TNFR1 and associated genes and downregulation of inhibitory apoptotic proteins XIAP and BCL-2. The results obtained from this novel and comprehensive study including histopathological, ultrastructural, flow cytometrical and death receptor pathway gene expression profiles, will facilitate better understanding of mechanisms and involvement of death receptor pathway in hepatocytes apoptosis induced by AFB1 and ultimately may be helpful in bringing down the toxigenic potential of AFB1.

  1. Death Inducer-Obliterator 1 Triggers Apoptosis after Nuclear Translocation and Caspase Upregulation

    PubMed Central

    García-Domingo, David; Ramírez, Dorian; González de Buitrago, Gonzalo; Martínez-A, Carlos

    2003-01-01

    Death inducer-obliterator 1 (DIO-1) is a gene that is upregulated early in apoptosis. Here we report that in healthy cells, the DIO-1 gene product was located in the cytoplasm, where it formed oligomers. After interleukin-3 starvation or c-Myc-induced apoptosis in serum-free conditions, DIO-1 translocated to the nucleus, where it upregulated caspase levels and activity. A nuclear localization signal deletion mutant (DIO-1ΔNLS) was unable to translocate to the nuclear compartment in the absence of interleukin-3 and failed to upregulate procaspase levels or trigger cell death. In addition, cells stably expressing DIO-1ΔNLS were protected from apoptosis induced by interleukin-3 withdrawal. These results indicate that DIO-1 has a relevant role in regulating the early stages of cell death. PMID:12697821

  2. Apoptosis and accidental cell death in cultured human keratinocytes after thermal injury.

    PubMed

    Matylevitch, N P; Schuschereba, S T; Mata, J R; Gilligan, G R; Lawlor, D F; Goodwin, C W; Bowman, P D

    1998-08-01

    The respective roles of apoptosis and accidental cell death after thermal injury were evaluated in normal human epidermal keratinocytes. By coupling the LIVE/DEAD fluorescence viability assay with the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and ultrastructural morphology, these two processes could be distinguished. Cells were grown on glass coverslips with a microgrid pattern so that the results of several staining procedures performed sequentially could be visualized in the same cells after heating at temperatures of up to 72 degrees C for 1 second. After exposure to temperatures of 58 to 59 degrees C, cells died predominantly by apoptosis; viable cells became TUNEL positive, indicating degradation of DNA. After exposure to temperatures of 60 to 66 degrees C, both TUNEL-positive viable cells and TUNEL-positive nonviable cells were observed, indicating that apoptosis and accidental cell death were occurring simultaneously. Cells died almost immediately after exposure to temperatures above 72 degrees C, presumably from heat fixation. The fluorescent mitochondrial probe MitoTracker Orange indicated that cells undergoing apoptosis became TUNEL positive before loss of mitochondrial function. Nucleosomal fragmentation of DNA analyzed by enzyme-linked immunosorbent assay and gel electrophoresis occurred after exposure to temperatures of 58 to 59 degrees C. The characteristic morphological findings of cells undergoing apoptosis, by transmission electron microscopy, included cellular shrinkage, cytoplasmic budding, and relatively intact mitochondria. Depending on temperature and time of exposure, normal human epidermal keratinocytes may die by apoptosis, accidental cell death, or heat fixation.

  3. The mitochondrial and death receptor pathways involved in the thymocytes apoptosis induced by aflatoxin B1

    PubMed Central

    Chi, Xiaofeng; Li, Xiaochong; Jiang, Min; Fang, Jing; Cui, Hengmin; Lai, Weimin; Zhou, Yi; Zhou, Shan

    2016-01-01

    Aflatoxin B1 (AFB1) is a potent immunosuppressive agent in endotherms, which can be related to the up-regulated apoptosis of immune organs. In this study, we investigated the roles of the mitochondrial, death receptor, and endoplasmic reticulum pathways in Aflatoxin B1 induced thymocytes apoptosis. Chickens were fed an aflatoxin B1 containing diet (0.6 mg/kg AFB1) for 3 weeks. Our results showed that (1) AFB1 diet induced the decrease of T-cell subsets, morphological changes, and excessive apoptosis of thymus. (2) The excessive apoptosis involved the mitochondrial pathway (up-regulation of Bax, Bak, cytC and down-regulation of Bcl-2 and Bcl-xL) and death receptor pathway (up-regulation of FasL, Fas and FADD). (3) Oxidative stress, an apoptosis inducer, was confirmed in the thymus. In conclusion, this is the first study to demonstrate that mitochondrial and death receptor pathways involved in AFB1 induced thymocytes apoptosis in broilers. PMID:26933817

  4. Surviving apoptosis: life-death signaling in single cells

    PubMed Central

    Flusberg, Deborah A.; Sorger, Peter K.

    2015-01-01

    Tissue development and homeostasis are regulated by opposing pro-survival and pro-death signals. An interesting feature of the Tumor Necrosis Factor (TNF) family of ligands is that they simultaneously activate opposing signals within a single cell via the same ligand-receptor complex. The magnitude of pro-death events such as caspase activation and pro-survival events such as NF-κB activation vary not only from one cell type to the next but also among individual cells of the same type due to intrinsic and extrinsic noise. The molecules involved in these pro-survival/pro-death pathways, and the different phenotypes that result from their activities, have been recently reviewed. Here we focus on the impact of cell-to-cell variability in the strength of these opposing signals on shaping cell fate decisions. PMID:25920803

  5. Apoptosis and caspases regulate death and inflammation in sepsis.

    PubMed

    Hotchkiss, Richard S; Nicholson, Donald W

    2006-11-01

    Although the prevailing concept has been that mortality in sepsis results from an unbridled hyper-inflammatory cytokine-mediated response, the failure of more than 30 clinical trials to treat sepsis by controlling this cytokine response requires a 'rethink' of the molecular mechanism underpinning the development of sepsis. As we discuss here, remarkable new studies indicate that most deaths from sepsis are actually the result of a substantially impaired immune response that is due to extensive death of immune effector cells. Rectification of this apoptotic-inflammatory imbalance using modulators of caspases and other components of the cell-death pathway have shown striking efficacy in stringent animal models of sepsis, indicating an entirely novel path forward for the clinical treatment of human sepsis.

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

    PubMed

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

    2006-02-01

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

  7. The meaning of death: evolution and ecology of apoptosis in protozoan parasites.

    PubMed

    Reece, Sarah E; Pollitt, Laura C; Colegrave, Nick; Gardner, Andy

    2011-12-01

    The discovery that an apoptosis-like, programmed cell death (PCD) occurs in a broad range of protozoan parasites offers novel therapeutic tools to treat some of the most serious infectious diseases of humans, companion animals, wildlife, and livestock. Whilst apoptosis is an essential part of normal development, maintenance, and defence in multicellular organisms, its occurrence in unicellular parasites appears counter-intuitive and has proved highly controversial: according to the Darwinian notion of "survival of the fittest", parasites are expected to evolve strategies to maximise their proliferation, not death. The prevailing, and untested, opinion in the literature is that parasites employ apoptosis to "altruistically" self-regulate the intensity of infection in the host/vector. However, evolutionary theory tells us that at most, this can only be part of the explanation, and other non-mutually exclusive hypotheses must also be tested. Here, we explain the evolutionary concepts that can explain apoptosis in unicellular parasites, highlight the key questions, and outline the approaches required to resolve the controversy over whether parasites "commit suicide". We highlight the need for integration of proximate and functional approaches into an evolutionary framework to understand apoptosis in unicellular parasites. Understanding how, when, and why parasites employ apoptosis is central to targeting this process with interventions that are sustainable in the face of parasite evolution.

  8. The mitochondria-regulated death pathway mediates asbestos-induced alveolar epithelial cell apoptosis.

    PubMed

    Panduri, Vijayalakshmi; Weitzman, Sigmund A; Chandel, Navdeep; Kamp, David W

    2003-02-01

    The mechanisms underlying asbestos-induced pulmonary toxicity are not fully understood. Alveolar epithelial cell (AEC) apoptosis by iron-derived reactive oxygen species (ROS) is one important mechanism implicated. The two major pathways regulating apoptosis include (i) the mitochondrial death (intrinsic) pathway caused by DNA damage, and (ii) the plasma-membrane death receptor (extrinsic) pathway. However, it is unknown whether asbestos activates either death pathway in AEC. We determined whether asbestos triggers AEC mitochondrial dysfunction by exposing cells (A549 and rat alveolar type II) to amosite asbestos and assessing mitochondrial membrane potential changes (deltapsi(m)) using a fluorometric technique involving tetremethylrhodamine ethyl ester (TMRE) and mitotracker green. Unlike inert particulates (titanium dioxide and glass beads), amosite asbestos caused dose- and time-dependent reductions in deltapsi(m). Asbestos-induced deltapsi(m) was associated with the release of cytochrome c from the mitochondria to the cytoplasm as well as activation of caspase 9, a mitochondrial-activated caspase. In contrast, a lower level of caspase 8, the death receptor-activated caspase, was detected in asbestos-exposed AEC. An iron chelator (phytic acid or deferoxamine) or a hydroxyl radical scavenger (sodium benzoate) each blocked asbestos-induced reductions in deltapsi(m) and caspase 9 activation, suggesting a role for iron-derived ROS. Finally, Bcl-X(L), a mitochondrial antiapoptotic protein that prevents cell death by preserving the outer mitochondrial membrane integrity, blocked asbestos-induced decreases in A549 cell deltapsi(m) and reduced apoptosis as assessed by DNA fragmentation. We conclude that asbestos-induced AEC apoptosis results from mitochondrial dysfunction, in part due to iron-derived ROS, which is followed by the release of cytochrome c and caspase 9 activation. Our findings suggest an important role for the mitochondria-regulated death pathway in the

  9. Death harmony played by nucleus and mitochondria: nuclear apoptosis during conjugation of tetrahymena.

    PubMed

    Endoh, Hiroshi; Kobayashi, Takashi

    2006-01-01

    Tetrahymena programmed nuclear death or nuclear apoptosis is a unique process during conjugation in which only the parental macronucleus is eliminated from the progeny cytoplasm, and other nuclei such as new micro- and macronuclei are unaffected. The nuclear death process consists of three successive steps: chromatin cleavage into high-molecular mass DNA, oligonucleosomal laddering concomitant with nuclear condensation, and complete degradation of the nuclear DNA. Following the first step of the death process, the parental macronucleus is engulfed by a large autophagosome in which many mitochondria are incorporated. Those sequestered mitochondria simply break down and release endonuclease similar to mammalian endonuclease G that is responsible for the generation of the DNA ladder, leading to the conclusion that mitochondria play a crucial role in the execution of the death program. Thus, the parental macronucleus is subject to final death by autophagy in collaboration with caspase-like enzymes, resulting in the ultimate outcome of nuclear resorption.

  10. Necrosis, and then stress induced necrosis-like cell death, but not apoptosis, should be the preferred cell death mode for chemotherapy: clearance of a few misconceptions

    PubMed Central

    Zhang, Ju; Lou, Xiaomin; Jin, Longyu; Zhou, Rongjia; Liu, Siqi; Xu, Ningzhi; Liao, D. Joshua

    2014-01-01

    Cell death overarches carcinogenesis and is a center of cancer researches, especially therapy studies. There have been many nomenclatures on cell death, but only three cell death modes are genuine, i.e. apoptosis, necrosis and stress-induced cell death (SICD). Like apoptosis, SICD is programmed. Like necrosis, SICD is a pathological event and may trigger regeneration and scar formation. Therefore, SICD has subtypes of stress-induced apoptosis-like cell death (SIaLCD) and stress-induced necrosis-like cell death (SInLCD). Whereas apoptosis removes redundant but healthy cells, SICD removes useful but ill or damaged cells. Many studies on cell death involve cancer tissues that resemble parasites in the host patients, which is a complicated system as it involves immune clearance of the alien cancer cells by the host. Cancer resembles an evolutionarily lower-level organism having a weaker apoptosis potential and poorer DNA repair mechanisms. Hence, targeting apoptosis for cancer therapy, i.e. killing via SIaLCD, will be less efficacious and more toxic. On the other hand, necrosis of cancer cells releases cellular debris and components to stimulate immune function, thus counteracting therapy-caused immune suppression and making necrosis better than SIaLCD for chemo drug development. PMID:25594039

  11. Apoptosis is not the major death mechanism induced by celecoxib on rheumatoid arthritis synovial fibroblasts.

    PubMed

    Audo, Rachel; Deschamps, Véronique; Hahne, Michael; Combe, Bernard; Morel, Jacques

    2007-01-01

    Synovial hyperplasia in rheumatoid arthritis (RA) has been associated with apoptosis deficiency of RA fibroblast-like synoviocytes (FLSs). Celecoxib is a non-steroidal anti-inflammatory drug that has been demonstrated to induce apoptosis in some cellular systems. We have therefore examined the dose- and time-dependent effects of celecoxib on RA FLS viability. Treatment of RA FLSs with celecoxib for 24 hours reduced their viability in a dose-dependent manner. Analysis of celecoxib-treated RA FLSs for their content of apoptotic and necrotic cells by Annexin V staining and TO-PRO-3 uptake displayed only few apoptotic cells. Caspase 3, a key mediator of apoptosis, was not activated in celecoxib-treated RA FLSs, and the presence of specific caspase 3 or pan-caspase inhibitors did not affect celecoxib-induced cell death. Moreover, we could not detect other signs of apoptosis, such as cleavage of poly(ADP-ribose) polymerase, caspase 8 or 9, or DNA fragmentation. We therefore conclude that apoptosis is not the major death pathway in celecoxib-treated RA FLSs.

  12. RIPK1 promotes death receptor-independent caspase-8-mediated apoptosis under unresolved ER stress conditions

    PubMed Central

    Estornes, Y; Aguileta, M A; Dubuisson, C; De Keyser, J; Goossens, V; Kersse, K; Samali, A; Vandenabeele, P; Bertrand, M J M

    2014-01-01

    Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER stress and results in the activation of the unfolded protein response (UPR), which aims at restoring ER homeostasis. However, when the stress is too severe the UPR switches from being a pro-survival response to a pro-death one, and the molecular mechanisms underlying ER stress-mediated death have remained incompletely understood. In this study, we identified receptor interacting protein kinase 1 (RIPK1)—a kinase at the crossroad between life and death downstream of various receptors—as a new regulator of ER stress-induced death. We found that Ripk1-deficient MEFs are protected from apoptosis induced by ER stressors, which is reflected by reduced caspase activation and PARP processing. Interestingly, the pro-apoptotic role of Ripk1 is independent of its kinase activity, is not regulated by its cIAP1/2-mediated ubiquitylation, and does not rely on the direct regulation of JNK or CHOP, two reportedly main players in ER stress-induced death. Instead, we found that ER stress-induced apoptosis in these cells relies on death receptor-independent activation of caspase-8, and identified Ripk1 upstream of caspase-8. However, in contrast to RIPK1-dependent apoptosis downstream of TNFR1, we did not find Ripk1 associated with caspase-8 in a death-inducing complex upon unresolved ER stress. Our data rather suggest that RIPK1 indirectly regulates caspase-8 activation, in part via interaction with the ER stress sensor inositol-requiring protein 1 (IRE1). PMID:25476903

  13. Death Associated Protein Kinase 1 (DAPK1): A Regulator of Apoptosis and Autophagy

    PubMed Central

    Singh, Pratibha; Ravanan, Palaniyandi; Talwar, Priti

    2016-01-01

    Death-Associated Protein Kinase 1 (DAPK1) belongs to a family of five serine/threonine (Ser/Thr) kinases that possess tumor suppressive function and also mediate a wide range of cellular processes, including apoptosis and autophagy. The loss and gain-of–function of DAPK1 is associated with various cancer and neurodegenerative diseases respectively. In recent years, mechanistic studies have broadened our knowledge of the molecular mechanisms involved in DAPK1-mediated autophagy/apoptosis. In the present review, we have discussed the structural information and various cellular functions of DAPK1 in a comprehensive manner. PMID:27445685

  14. Role of apoptosis and necrosis in cell death induced by nanoparticle-mediated photothermal therapy

    NASA Astrophysics Data System (ADS)

    Pattani, Varun P.; Shah, Jay; Atalis, Alexandra; Sharma, Anirudh; Tunnell, James W.

    2015-01-01

    Current cancer therapies can cause significant collateral damage due to a lack of specificity and sensitivity. Therefore, we explored the cell death pathway response to gold nanorod (GNR)-mediated photothermal therapy as a highly specific cancer therapeutic to understand the role of apoptosis and necrosis during intense localized heating. By developing this, we can optimize photothermal therapy to induce a maximum of `clean' cell death pathways, namely apoptosis, thereby reducing external damage. GNRs were targeted to several subcellular localizations within colorectal tumor cells in vitro, and the cell death pathways were quantitatively analyzed after photothermal therapy using flow cytometry. In this study, we found that the cell death response to photothermal therapy was dependent on the GNR localization. Furthermore, we demonstrated that nanorods targeted to the perinuclear region irradiated at 37.5 W/cm2 laser fluence rate led to maximum cell destruction with the `cleaner' method of apoptosis, at similar percentages as other anti-cancer targeted therapies. We believe that this indicates the therapeutic potential for GNR-mediated photothermal therapy to treat cancer effectively without causing damage to surrounding tissue.

  15. Autophagy and apoptosis coordinate physiological cell death in larval salivary glands of Apis mellifera (Hymenoptera: Apidae).

    PubMed

    Zacarin, Elaine C M Silva

    2007-01-01

    Larval salivary glands of bees provide a good model for the study of hormone-induced programmed cell death in Hymenoptera because they have a well-defined secretory cycle with a peak of secretory activity phase, prior to cocoon spinning, and a degenerative phase, after the cocoon spinning. Our findings demonstrate that there is a relationship between apoptosis and autophagy during physiological cell death in these larval salivary glands, that adds evidence to the hypothesis of overlap in the regulation pathways of both types of programmed cell death. Features of autophagy include cytoplasm vacuolation, acid phosphatase activity, presence of autophagic vacuoles and multi-lamellar structures, as well as a delay in the collapse of many nuclei. Features of apoptosis include bleb formation in the cytoplasm and nuclei, with release of parts of the cytoplasm into the lumen, chromatin compaction, and DNA and nucleolar fragmentation. We propose a model for programmed cell death in larval salivary glands of Apis mellifera where autophagy and apoptosis function cooperatively for a more efficient degeneration of the gland secretory cells.

  16. CD95 death receptor and epidermal growth factor receptor (EGFR) in liver cell apoptosis and regeneration.

    PubMed

    Reinehr, Roland; Häussinger, Dieter

    2012-02-01

    Recent evidence suggests that signaling pathways towards cell proliferation and cell death are much more interconnected than previously thought. Whereas not only death receptors such as CD95 (Fas, APO-1) can couple to both, cell death and proliferation, also growth factor receptors such as the epidermal growth factor receptor (EGFR) are involved in these opposing kinds of cell fate. EGFR is briefly discussed as a growth factor receptor involved in liver cell proliferation during liver regeneration. Then the role of EGFR in activating CD95 death receptor in liver parenchymal cells (PC) and hepatic stellate cells (HSC), which represent a liver stem/progenitor cell compartment, is described summarizing different ways of CD95- and EGFR-dependent signaling in the liver. Here, depending on the hepatic cell type (PC vs. HSC) and the respective signaling context (sustained vs. transient JNK activation) CD95-/EGFR-mediated signaling ends up in either liver cell apoptosis or cell proliferation.

  17. Cell Death and Inflammatory Bowel Diseases: Apoptosis, Necrosis, and Autophagy in the Intestinal Epithelium

    PubMed Central

    2014-01-01

    Cell death mechanisms have been associated with the development of inflammatory bowel diseases in humans and mice. Recent studies suggested that a complex crosstalk between autophagy/apoptosis, microbe sensing, and enhanced endoplasmic reticulum stress in the epithelium could play a critical role in these diseases. In addition, necroptosis, a relatively novel programmed necrosis-like pathway associated with TNF receptor activation, seems to be also present in the pathogenesis of Crohn's disease and in specific animal models for intestinal inflammation. This review attempts to cover new data related to cell death mechanisms and inflammatory bowel diseases. PMID:25126549

  18. Mechanisms of apoptosis in Crustacea: what conditions induce versus suppress cell death?

    PubMed Central

    Menze, Michael A.; Fortner, Grady; Nag, Suman; Hand, Steven C.

    2014-01-01

    Arthropoda is the largest of all animal phyla and includes about 90% of extant species. Our knowledge about regulation of apoptosis in this phylum is largely based on findings for the fruit fly Drosophila melanogaster. Recent work with crustaceans shows that apoptotic proteins, and presumably mechanisms of cell death regulation, are more diverse in arthropods than appreciated based solely on the excellent work with fruit flies. Crustacean homologs exist for many major proteins in the apoptotic networks of mammals and D. melanogaster, but integration of these proteins into the physiology and pathophysiology of crustaceans is far from complete. Whether apoptosis in crustaceans is mainly transcriptionally regulated as in D. melanogaster (e.g., RHG ‘killer’ proteins), or rather is controlled by pro- and anti-apoptotic Bcl-2 family proteins as in vertebrates needs to be clarified. Some phenomena like the calcium-induced opening of the mitochondrial permeability transition pore (MPTP) are apparently lacking in crustaceans and may represent a vertebrate invention. We speculate that differences in regulation of the intrinsic pathway of crustacean apoptosis might represent a prerequisite for some species to survive harsh environmental insults. Pro-apoptotic stimuli described for crustaceans include UV radiation, environmental toxins, and a diatom-produced chemical that promotes apoptosis in offspring of a copepod. Mechanisms that serve to depress apoptosis include the inhibition of caspase activity by high potassium in energetically healthy cells, alterations in nucleotide abundance during energy-limited states like diapause and anoxia, resistance to opening of the calcium-induced MPTP, and viral accommodation during persistent viral infection. Characterization of the players, pathways, and their significance in the core machinery of crustacean apoptosis is revealing new insights for the field of cell death. PMID:20043212

  19. Mechanisms of apoptosis in Crustacea: What conditions induce versus suppress cell death?

    PubMed

    Menze, Michael A; Fortner, Grady; Nag, Suman; Hand, Steven C

    2010-03-01

    Arthropoda is the largest of all animal phyla and includes about 90% of extant species. Our knowledge about regulation of apoptosis in this phylum is largely based on findings for the fruit fly Drosophila melanogaster. Recent work with crustaceans shows that apoptotic proteins, and presumably mechanisms of cell death regulation, are more diverse in arthropods than appreciated based solely on the excellent work with fruit flies. Crustacean homologs exist for many major proteins in the apoptotic networks of mammals and D. melanogaster, but integration of these proteins into the physiology and pathophysiology of crustaceans is far from complete. Whether apoptosis in crustaceans is mainly transcriptionally regulated as in D. melanogaster (e.g., RHG 'killer' proteins), or rather is controlled by pro- and anti-apoptotic Bcl-2 family proteins as in vertebrates needs to be clarified. Some phenomena like the calcium-induced opening of the mitochondrial permeability transition pore (MPTP) are apparently lacking in crustaceans and may represent a vertebrate invention. We speculate that differences in regulation of the intrinsic pathway of crustacean apoptosis might represent a prerequisite for some species to survive harsh environmental insults. Pro-apoptotic stimuli described for crustaceans include UV radiation, environmental toxins, and a diatom-produced chemical that promotes apoptosis in offspring of a copepod. Mechanisms that serve to depress apoptosis include the inhibition of caspase activity by high potassium in energetically healthy cells, alterations in nucleotide abundance during energy-limited states like diapause and anoxia, resistance to opening of the calcium-induced MPTP, and viral accommodation during persistent viral infection. Characterization of the players, pathways, and their significance in the core machinery of crustacean apoptosis is revealing new insights for the field of cell death.

  20. Bifunctional apoptosis inhibitor (BAR) protects neurons from diverse cell death pathways.

    PubMed

    Roth, W; Kermer, P; Krajewska, M; Welsh, K; Davis, S; Krajewski, S; Reed, J C

    2003-10-01

    The bifunctional apoptosis regulator (BAR) is a multidomain protein that was originally identified as an inhibitor of Bax-induced apoptosis. Immunoblot analysis of normal human tissues demonstrated high BAR expression in the brain, compared to low or absent expression in other organs. Immunohistochemical staining of human adult tissues revealed that the BAR protein is predominantly expressed by neurons in the central nervous system. Immunofluorescence microscopy indicated that BAR localizes mainly to the endoplasmic reticulum (ER) of cells. Overexpression of BAR in CSM 14.1 neuronal cells resulted in significant protection from a broad range of cell death stimuli, including agents that activate apoptotic pathways involving mitochondria, TNF-family death receptors, and ER stress. Downregulation of BAR by antisense oligonucleotides sensitized neuronal cells to induction of apoptosis. Moreover, the search for novel interaction partners of BAR identified several candidate proteins that might contribute to the regulation of neuronal apoptosis (HIP1, Hippi, and Bap31). Taken together, the expression pattern and functional data suggest that the BAR protein is involved in the regulation of neuronal survival.

  1. Myelin-associated glycoprotein modulates apoptosis of motoneurons during early postnatal development via NgR/p75NTR receptor-mediated activation of RhoA signaling pathways

    PubMed Central

    Palandri, A; Salvador, V R; Wojnacki, J; Vivinetto, A L; Schnaar, R L; Lopez, P H H

    2015-01-01

    Myelin-associated glycoprotein (MAG) is a minor constituent of nervous system myelin, selectively expressed on the periaxonal myelin wrap. By engaging multiple axonal receptors, including Nogo-receptors (NgRs), MAG exerts a nurturing and protective effect the axons it ensheaths. Pharmacological activation of NgRs has a modulatory role on p75NTR-dependent postnatal apoptosis of motoneurons (MNs). However, it is not clear whether this reflects a physiological role of NgRs in MN development. NgRs are part of a multimeric receptor complex, which includes p75NTR, Lingo-1 and gangliosides. Upon ligand binding, this multimeric complex activates RhoA/ROCK signaling in a p75NTR-dependent manner. The aim of this study was to analyze a possible modulatory role of MAG on MN apoptosis during postnatal development. A time course study showed that Mag-null mice suffer a loss of MNs during the first postnatal week. Also, these mice exhibited increased susceptibility in an animal model of p75NTR-dependent MN apoptosis induced by nerve-crush injury, which was prevented by treatment with a soluble form of MAG (MAG-Fc). The protective role of MAG was confirmed in in vitro models of p75NTR-dependent MN apoptosis using the MN1 cell line and primary cultures. Lentiviral expression of shRNA sequences targeting NgRs on these cells abolished protection by MAG-Fc. Analysis of RhoA activity using a FRET-based RhoA biosensor showed that MAG-Fc activates RhoA. Pharmacological inhibition of p75NTR/RhoA/ROCK pathway, or overexpression of a p75NTR mutant unable to activate RhoA, completely blocked MAG-Fc protection against apoptosis. The role of RhoA/ROCK signaling was further confirmed in the nerve-crush model, where pretreatment with ROCK inhibitor Y-27632 blocked the pro-survival effect of MAG-Fc. These findings identify a new protective role of MAG as a modulator of apoptosis of MNs during postnatal development by a mechanism involving the p75NTR/RhoA/ROCK signaling pathway. Also, our results

  2. Myelin-associated glycoprotein modulates apoptosis of motoneurons during early postnatal development via NgR/p75(NTR) receptor-mediated activation of RhoA signaling pathways.

    PubMed

    Palandri, A; Salvador, V R; Wojnacki, J; Vivinetto, A L; Schnaar, R L; Lopez, P H H

    2015-09-03

    Myelin-associated glycoprotein (MAG) is a minor constituent of nervous system myelin, selectively expressed on the periaxonal myelin wrap. By engaging multiple axonal receptors, including Nogo-receptors (NgRs), MAG exerts a nurturing and protective effect the axons it ensheaths. Pharmacological activation of NgRs has a modulatory role on p75(NTR)-dependent postnatal apoptosis of motoneurons (MNs). However, it is not clear whether this reflects a physiological role of NgRs in MN development. NgRs are part of a multimeric receptor complex, which includes p75(NTR), Lingo-1 and gangliosides. Upon ligand binding, this multimeric complex activates RhoA/ROCK signaling in a p75(NTR)-dependent manner. The aim of this study was to analyze a possible modulatory role of MAG on MN apoptosis during postnatal development. A time course study showed that Mag-null mice suffer a loss of MNs during the first postnatal week. Also, these mice exhibited increased susceptibility in an animal model of p75(NTR)-dependent MN apoptosis induced by nerve-crush injury, which was prevented by treatment with a soluble form of MAG (MAG-Fc). The protective role of MAG was confirmed in in vitro models of p75(NTR)-dependent MN apoptosis using the MN1 cell line and primary cultures. Lentiviral expression of shRNA sequences targeting NgRs on these cells abolished protection by MAG-Fc. Analysis of RhoA activity using a FRET-based RhoA biosensor showed that MAG-Fc activates RhoA. Pharmacological inhibition of p75(NTR)/RhoA/ROCK pathway, or overexpression of a p75(NTR) mutant unable to activate RhoA, completely blocked MAG-Fc protection against apoptosis. The role of RhoA/ROCK signaling was further confirmed in the nerve-crush model, where pretreatment with ROCK inhibitor Y-27632 blocked the pro-survival effect of MAG-Fc. These findings identify a new protective role of MAG as a modulator of apoptosis of MNs during postnatal development by a mechanism involving the p75(NTR)/RhoA/ROCK signaling pathway

  3. 188Rhenium-induced cell death and apoptosis in a panel of tumor cell lines

    NASA Astrophysics Data System (ADS)

    Antoccia, Antonio; Banzato, Alessandra; Bello, Michele; Bollini, Dante; De Notaristefani, Francesco; Giron, Cecilia; Mazzi, Ulderico; Alafort, Laura Melendez; Moschini, Giuliano; Nadali, Anna; Navarria, Francesco; Perrotta, Andrea; Rosato, Antonio; Tanzarella, Caterina; Uzunov, Nikolay

    2007-02-01

    Assessment of "in vitro" tumor growth inhibition and radiobiological effects, such as apoptosis, have been evaluated in human neoplastic cells of different histotypes (H460 lung cancer cells, U87 glioblastoma, LnCaP prostate tumor cells) treated using solutions of 188Rhenium-perrhenate. The MTT assay, which measures mitochondrial metabolism in the entire cell culture is a recognized test for cytotoxicity and was used in cells exposed 48-72 h to specific activities ranged from 37 to 148 GBq/l. Whereas H460 and LnCaP were particularly sensitive to treatment, U87 glioblastoma cells behaved as radioresistant ones. However, evaluation of 188Re-induced apoptosis indicated that this kind of cell death contributed only marginally to the reduction in cell viability of H460 and LNCaP lines, suggesting the existence of protective mechanisms against apoptosis. In this respect, the membrane receptor, CD44, whose expression is dysregulated in most malignant cell types has proven to alter the response of cancer cells to apoptotic stimuli, including ionizing radiation. Cell samples decorated with a FITC-labelled CD44 antibody indicated, that in H460 and U87 cells the CD44(+) correlated well with an apoptosis-resistant response. Conversely, LnCap cells proven as CD44(-) did not display however sensitivity to radio-induced apoptosis.

  4. [Apoptosis modulation by human papillomavirus].

    PubMed

    Jave-Suárez, Luis Felipe; Ratkovich-González, Sarah; Olimón-Andalón, Vicente; Aguilar-Lemarroy, Adriana

    2015-01-01

    One of the most important processes to keep the homeostasis in organisms is the apoptosis, also called programmed cell death. This mechanism works through two pathways: The intrinsic or mitochondrial, which responds to DNA damage and extern agents like UV radiation; and the extrinsic or receptor-mediated, which binds to their ligands to initiate the apoptotic trail. The evasion of apoptosis is one of the main causes of cellular transformation to malignity. Many viruses had shown capacity to modify the apoptotic process; among them is the human papillomavirus, which, by means of its oncoproteins, interferes in pathways, reacting with the receptors and molecules and participating in the death mechanism. This creates ideal conditions for cancer development.

  5. Untangling the Roles of Anti-Apoptosis in Regulating Programmed Cell Death using Humanized Yeast Cells.

    PubMed

    Clapp, Caitlin; Portt, Liam; Khoury, Chamel; Sheibani, Sara; Eid, Rawan; Greenwood, Matthew; Vali, Hojatollah; Mandato, Craig A; Greenwood, Michael T

    2012-01-01

    Genetically programmed cell death (PCD) mechanisms, including apoptosis, are important for the survival of metazoans since it allows, among things, the removal of damaged cells that interfere with normal function. Cell death due to PCD is observed in normal processes such as aging and in a number of pathophysiologies including hypoxia (common causes of heart attacks and strokes) and subsequent tissue reperfusion. Conversely, the loss of normal apoptotic responses is associated with the development of tumors. So far, limited success in preventing unwanted PCD has been reported with current therapeutic approaches despite the fact that inhibitors of key apoptotic inducers such as caspases have been developed. Alternative approaches have focused on mimicking anti-apoptotic processes observed in cells displaying increased resistance to apoptotic stimuli. Hormesis and pre-conditioning are commonly observed cellular strategies where sub-lethal levels of pro-apoptotic stimuli lead to increased resistance to higher or lethal levels of stress. Increased expression of anti-apoptotic sequences is a common mechanism mediating these protective effects. The relevance of the latter observation is exemplified by the observation that transgenic mice overexpressing anti-apoptotic genes show significant reductions in tissue damage following ischemia. Thus strategies aimed at increasing the levels of anti-apoptotic proteins, using gene therapy or cell penetrating recombinant proteins are being evaluated as novel therapeutics to decrease cell death following acute periods of cell death inducing stress. In spite of its functional and therapeutic importance, more is known regarding the processes involved in apoptosis than anti-apoptosis. The genetically tractable yeast Saccharomyces cerevisiae has emerged as an exceptional model to study multiple aspects of PCD including the mitochondrial mediated apoptosis observed in metazoans. To increase our knowledge of the process of anti-apoptosis

  6. Untangling the Roles of Anti-Apoptosis in Regulating Programmed Cell Death using Humanized Yeast Cells

    PubMed Central

    Clapp, Caitlin; Portt, Liam; Khoury, Chamel; Sheibani, Sara; Eid, Rawan; Greenwood, Matthew; Vali, Hojatollah; Mandato, Craig A.; Greenwood, Michael T.

    2012-01-01

    Genetically programmed cell death (PCD) mechanisms, including apoptosis, are important for the survival of metazoans since it allows, among things, the removal of damaged cells that interfere with normal function. Cell death due to PCD is observed in normal processes such as aging and in a number of pathophysiologies including hypoxia (common causes of heart attacks and strokes) and subsequent tissue reperfusion. Conversely, the loss of normal apoptotic responses is associated with the development of tumors. So far, limited success in preventing unwanted PCD has been reported with current therapeutic approaches despite the fact that inhibitors of key apoptotic inducers such as caspases have been developed. Alternative approaches have focused on mimicking anti-apoptotic processes observed in cells displaying increased resistance to apoptotic stimuli. Hormesis and pre-conditioning are commonly observed cellular strategies where sub-lethal levels of pro-apoptotic stimuli lead to increased resistance to higher or lethal levels of stress. Increased expression of anti-apoptotic sequences is a common mechanism mediating these protective effects. The relevance of the latter observation is exemplified by the observation that transgenic mice overexpressing anti-apoptotic genes show significant reductions in tissue damage following ischemia. Thus strategies aimed at increasing the levels of anti-apoptotic proteins, using gene therapy or cell penetrating recombinant proteins are being evaluated as novel therapeutics to decrease cell death following acute periods of cell death inducing stress. In spite of its functional and therapeutic importance, more is known regarding the processes involved in apoptosis than anti-apoptosis. The genetically tractable yeast Saccharomyces cerevisiae has emerged as an exceptional model to study multiple aspects of PCD including the mitochondrial mediated apoptosis observed in metazoans. To increase our knowledge of the process of anti-apoptosis

  7. VMP1 related autophagy and apoptosis in colorectal cancer cells: VMP1 regulates cell death

    SciTech Connect

    Qian, Qinyi; Zhou, Hao; Chen, Yan; Shen, Chenglong; He, Songbing; Zhao, Hua; Wang, Liang; Wan, Daiwei; Gu, Wen

    2014-01-17

    Highlights: •This research confirmed VMP1 as a regulator of autophagy in colorectal cancer cell lines. •We proved the pro-survival role of VMP1-mediated autophagy in colorectal cancer cell lines. •We found the interaction between VMP1 and BECLIN1 also existing in colorectal cancer cell lines. -- Abstract: Vacuole membrane protein 1 (VMP1) is an autophagy-related protein and identified as a key regulator of autophagy in recent years. In pancreatic cell lines, VMP1-dependent autophagy has been linked to positive regulation of apoptosis. However, there are no published reports on the role of VMP1 in autophagy and apoptosis in colorectal cancers. Therefore, to address this gap of knowledge, we decided to interrogate regulation of autophagy and apoptosis by VMP1. We have studied the induction of autophagy by starvation and rapamycin treatment in colorectal cell lines using electron microscopy, immunofluorescence, and immunoblotting. We found that starvation-induced autophagy correlated with an increase in VMP1 expression, that VMP1 interacted with BECLIN1, and that siRNA mediated down-regulation of VMP1-reduced autophagy. Next, we examined the relationship between VMP1-dependent autophagy and apoptosis and found that VMP1 down-regulation sensitizes cells to apoptosis and that agents that induce apoptosis down-regulate VMP1. In conclusion, similar to its reported role in other cell types, VMP1 is an important regulator of autophagy in colorectal cell lines. However, in contrast to its role in pancreatic cell lines, in colorectal cancer cells, VMP1-dependent autophagy appears to be pro-survival rather than pro-cell death.

  8. Diazene JK-279 induces apoptosis-like cell death in human cervical carcinoma cells.

    PubMed

    Jakopec, S; Dubravcic, K; Polanc, S; Kosmrlj, J; Osmak, M

    2006-03-01

    Diazene N-phenyl-2-(2-pyridinyl)diazenecarboxamide (JK-279) is a newly synthesized compound, cytotoxic for several tumor cell lines and their drug-resistant sublines. In human cervical carcinoma cells (HeLa), this compound reduced intracellular glutathione content and increased sensitivity to cisplatin. The aim of the present study was to elucidate the molecular mechanisms involved in the cytotoxic effect of diazene JK-279 on HeLa cells. Cytotoxicity was determined by the MTT method. Flow cytometry analysis showed that diazene JK-279 induces G(2)/M phase arrest, mediated by the increase in p21 expression, and accompanied by an alteration in the expression of survivin. The highest concentration of JK-279 altered nuclear morphology in intact cells, showing "apoptosis-like" features. No cleavage of procaspase-3, procaspase-9 and PARP, or altered expression of apoptotic proteins Bcl-2 and Bax were detected. At the same time, PS externalization and internucleosomal DNA cleavage were observed. Partial necrosis was detected as well. Our results demonstrate that cytotoxicity of diazene JK-279 is mostly the consequence of caspase-independent cell death, which is in some aspects "apoptosis-like". Taking into account the multiplicity of mechanisms used by cancer cells to prevent apoptosis, the drugs (like diazene JK-279) that would activate alternative cell death pathways could provide a useful tool for new types of cancer therapy.

  9. Apoptosis as a Mechanism for Keratinocyte Death in Canine Toxic Epidermal Necrolysis.

    PubMed

    Banovic, F; Dunston, S; Linder, K E; Rakich, P; Olivry, T

    2017-03-01

    In humans and dogs, toxic epidermal necrolysis (TEN) is a life-threatening dermatosis characterized by sudden epidermal death resulting in extensive skin detachment. There is little information on the pathogenesis of keratinocyte cell death in canine TEN. We studied the occurrence of apoptosis in skin lesions of dogs with TEN to determine if apoptosis contributes to the pathogenesis of this disease. Immunostaining with antibodies to activated caspase-3 and the terminal deoxynucleotidyl-transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling technique revealed positive apoptotic keratinocytes in basal and suprabasal epidermal compartments in 17 biopsy specimens collected from 3 dogs with TEN and 16 from 3 dogs with erythema multiforme (EM). There was no significant difference in the number of positively stained epidermal cells between TEN and EM. These results suggest that apoptosis of epidermal keratinocytes and lymphocytic satellitosis represent one of the early steps in the pathogenesis of canine TEN, as in the human disease counterpart.

  10. [Cell death in inflammatory heart muscle diseases--apoptosis or necrosis?].

    PubMed

    Pankuweit, S; Jobmann, M; Crombach, M; Portig, I; Alter, P; Kruse, T; Hufnagel, G; Maisch, B

    1999-05-01

    Cell death can be induced by 2 different mechanisms: necrosis and apoptosis. Necrosis, on the one hand, is usually caused by unphysiological stress factors such as hyperthermia or hypoxia, apoptosis, on the other hand, is part of the normal organ development and controls for example immune responses. Morphologically, necrosis is characterized by swelling of cells and their organelles leading to the disruption of the cell membrane, which in turn causes an inflammatory reaction in the surrounding tissue. Morphological and biochemical criteria (Figure 1, Table 1) of apoptosis are the condensation of chromatin leading to the development of apoptotic bodies or membrane-enclosed vesicles containing oligonucleosomal DNA fragments. Important diagnostic tools of cell death (Table 2), such as the TUNEL test (Figure 2) or gel electrophoresis of extracted DNA (Figure 3) are based on the above mentioned biochemical characteristics, but a reliable differentiation of apoptotic versus necrotic processes is not always possible. Experimental studies in animals and studies in various diseases of the cardiovascular system were able to show that apoptosis in myocytes can be induced, an issue that has long been discussed controversially. Ischemia, reperfusion, and myocardial infarction were also shown to lead to apoptosis in cardiomyocytes, whereas cell destruction was caused mainly by necrosis. Several authors (Table 3) demonstrated apoptotic indices in cardiomyocytes of patients with dilatated cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy and patients with acute infarction from 0.25 to 35% by the use of the TUNEL test. Others were able to demonstrate an elevated expression of Fas-receptor in cells of atheroslerotic plaques in patients with atherosclerosis and high indices of apoptotic cardiomyocytes in patients with chronic heart failure. We investigated endomyocardial biopsies of patients with inflammatory cardiomyopathy, DCM without inflammatory reaction but the

  11. Human group IIA secretory phospholipase A2 induces neuronal cell death via apoptosis.

    PubMed

    Yagami, Tatsurou; Ueda, Keiichi; Asakura, Kenji; Hata, Satoshi; Kuroda, Takayuki; Sakaeda, Toshiyuki; Takasu, Nobuo; Tanaka, Kazushige; Gemba, Takefumi; Hori, Yozo

    2002-01-01

    Expression of group IIA secretory phospholipase A2 (sPLA2-IIA) is documented in the cerebral cortex (CTX) after ischemia, suggesting that sPLA2-IIA is associated with neurodegeneration. However, how sPLA2-IIA is involved in the neurodegeneration remains obscure. To clarify the pathologic role of sPLA2-IIA, we examined its neurotoxicity in rats that had the middle cerebral artery occluded and in primary cultures of cortical neurons. After occlusion, sPLA2 activity was increased in the CTX. An sPLA2 inhibitor, indoxam, significantly ameliorated not only the elevated activity of the sPLA2 but also the neurodegeneration in the CTX. The neuroprotective effect of indoxam was observed even when it was administered after occlusion. In primary cultures, sPLA2-IIA caused marked neuronal cell death. Morphologic and ultrastructural characteristics of neuronal cell death by sPLA2-IIA were apoptotic, as evidenced by condensed chromatin and fragmented DNA. Before apoptosis, sPLA2-IIA liberated arachidonic acid (AA) and generated prostaglandin D2 (PGD2), an AA metabolite, from neurons. Indoxam significantly suppressed not only AA release, but also PGD2 generation. Indoxam prevented neurons from sPLA2-IIA-induced neuronal cell death. The neuroprotective effect of indoxam was observed even when it was administered after sPLA2-IIA treatment. Furthermore, a cyclooxygenase-2 inhibitor significantly prevented neurons from sPLA2-IIA-induced PGD2 generation and neuronal cell death. In conclusion, sPLA2-IIA induces neuronal cell death via apoptosis, which might be associated with AA metabolites, especially PGD2. Furthermore, sPLA2 contributes to neurodegeneration in the ischemic brain, highlighting the therapeutic potential of sPLA2-IIA inhibitors for stroke.

  12. Receptor-mediated signaling in Aspergillus fumigatus

    PubMed Central

    Grice, C. M.; Bertuzzi, M.; Bignell, E. M.

    2013-01-01

    Aspergillus fumigatus is the most pathogenic species among the Aspergilli, and the major fungal agent of human pulmonary infection. To prosper in diverse ecological niches, Aspergilli have evolved numerous mechanisms for adaptive gene regulation, some of which are also crucial for mammalian infection. Among the molecules which govern such responses, integral membrane receptors are thought to be the most amenable to therapeutic modulation. This is due to the localization of these molecular sensors at the periphery of the fungal cell, and to the prevalence of small molecules and licensed drugs which target receptor-mediated signaling in higher eukaryotic cells. In this review we highlight the progress made in characterizing receptor-mediated environmental adaptation in A. fumigatus and its relevance for pathogenicity in mammals. By presenting a first genomic survey of integral membrane proteins in this organism, we highlight an abundance of putative seven transmembrane domain (7TMD) receptors, the majority of which remain uncharacterized. Given the dependency of A. fumigatus upon stress adaptation for colonization and infection of mammalian hosts, and the merits of targeting receptor-mediated signaling as an antifungal strategy, a closer scrutiny of sensory perception and signal transduction in this organism is warranted. PMID:23430083

  13. Receptor-mediated DNA-targeted photoimmunotherapy.

    PubMed

    Karagiannis, Tom C; Lobachevsky, Pavel N; Leung, Brenda K Y; White, Jonathan M; Martin, Roger F

    2006-11-01

    We show the efficacy of a therapeutic strategy that combines the potency of a DNA-binding photosensitizer, UV(A)Sens, with the tumor-targeting potential of receptor-mediated endocytosis. The photosensitizer is an iodinated bibenzimidazole, which, when bound in the minor groove of DNA and excited by UV(A) irradiation, induces cytotoxic lesions attributed to a radical species resulting from photodehalogenation. Although reminiscent of photochemotherapy using psoralens and UV(A) irradiation, an established treatment modality in dermatology particularly for the treatment of psoriasis and cutaneous T-cell lymphoma, a critical difference is the extreme photopotency of the iodinated bibenzimidazole, approximately 1,000-fold that of psoralens. This feature prompted consideration of combination with the specificity of receptor-mediated targeting. Using two in vitro model systems, we show the UV(A) cytotoxicity of iodo ligand/protein conjugates, implying binding of the conjugate to cell receptors, internalization, and degradation of the conjugate-receptor complex, with release and translocation of the ligand to nuclear DNA. For ligand-transferrin conjugates, phototoxicity was inhibited by coincubation with excess native transferrin. Receptor-mediated UV(A)-induced cytotoxicity was also shown with the iodo ligand conjugate of an anti-human epidermal growth factor receptor monoclonal antibody, exemplifying the potential application of the strategy to other cancer-specific targets to thus improve the specificity of phototherapy of superficial lesions and for extracorporeal treatments.

  14. Detachment of esophageal carcinoma cells from extracellular matrix causes relocalization of death receptor 5 and apoptosis

    PubMed Central

    Liu, Guang-Chao; Zhang, Jun; Liu, Shi-Gui; Gao, Rong; Long, Zhang-Fu; Tao, Ke; Ma, Yuan-Fang

    2009-01-01

    AIM: To investigate the effect of detachment of esophageal cancer cells from extracellular matrix on the localization of death receptor 5 (DR5) and apoptosis. METHODS: Anchorage-dependent EC9706 cells of esophageal squamous cell carcinoma were pretreated or not treated with brefeldin A. Detached cells were harvested by ethylenediaminetetraacetic acid digestion. Expression and localization of DR5 in these cells were determined by immunocytochemical and immunofluorescence assays, as well as flow cytometry analysis. Apoptosis of EC9706 cells was detected by flow cytometry after stained with fluorescein isothiocyanate-labeled annexin V/propidium iodide. Activation of caspase 8 was detected by Western blot analysis. RESULTS: Immunocytochemical assay indicated that DR5 was predominantly perinuclear in adherent cells but was mainly localized in cell membrane in detached cells. In addition, immunofluorescence assay also confirmed the above-mentioned results, and further demonstrated that DR5 was present in the form of coarse granules in detached cells, but in the form of fine granules in adherent cells. Cytometry analysis revealed higher levels of DR5 expression on the surfaces of brefeldin-A-untreated cells than on the surfaces of brefeldin-A-treated cells, but brefeldin A treatment did not affect the total DR5 expression levels. Moreover, nocodazole did not influence the extracelluar DR5 expression levels in EC9706 cells. Apoptosis assay revealed that detached cells were more sensitive to DR5 antibody-induced apoptosis than adherent cells. Western blotting showed that caspase 8 was activated in temporarily detached cells 4 h earlier than in adherent cells. CONCLUSION: Progress from adhesion to detachment of EC9706 cells causes DR5 relocalization, and promotes cytoplasmic translocation of DR5 to cell surfaces via a Golgi-dependent pathway. Moreover, it might also result in DR5 aggregation to render apoptosis of detached cells. PMID:19230044

  15. Increased lymphocyte death by neglect-apoptosis is associated with lymphopenia and autoantibodies in lupus patients presenting with neuropsychiatric manifestations.

    PubMed

    Silva, Lucienir M; Garcia, Aglair B; Donadi, Eduardo A

    2002-08-01

    To evaluate lymphocyte death by neglect-apoptosis features in systemic lupus erythematosus (SLE) patients presenting with neuropsychiatric (NPSLE) involvement we studied 40 SLE patients with active disease, 20 with and 20 without neuropsychiatric manifestations, and 20 control individuals. Lymphocyte apoptosis was evaluated by means of DNA staining using flow cytometry, immediately after cell isolation and after incubation with culture medium or autologous serum. Compared with controls, NPSLE and non-NPSLE patients exhibited increased rates of neglect-apoptosis immediately after cell isolation. Only NPSLE patients exhibited an increased neglect-apoptosis rate after incubation with culture medium; however, the neglect-apoptosis rate was associated with lymphopenia in both series of patients. After lymphocyte incubation with autologous serum, only NPSLE patients exhibited a significant negative correlation between the neglect-apoptosis rate and the number of peripheral lymphocytes. The incubation of lymphocytes with autologous serum containing antiphospholipid or anti-SSA/Ro antibodies significantly increased the neglect-apoptosis in NPSLE when compared with non-NPSLE patients with a similar autoantibody profile. In conclusion, NPSLE and non-NPSLE patients shared several abnormalities in terms of lymphocyte neglect-apoptosis. Peculiar findings were observed in NPSLE patients particularly after incubation with autologous serum, such as the fact that the increased lymphocyte death by neglect-apoptosis was associated with lymphopenia and with the presence of antiphospholipid and anti-SSA/Ro antibodies.

  16. Partial equilibrium approximations in apoptosis. II. The death-inducing signaling complex subsystem.

    PubMed

    Huang, Ya-Jing; Hong, Liu; Yong, Wen-An

    2015-12-01

    This paper is a continuation of our previous work (Huang and Yong, 2013) for simplifying the Fas signaling-induced apoptotic pathway identified by Hua et al. (2005) for human tumor T cells. The previous paper studied the downstream intracelluar-signaling subsystem, while the present one is concerned with the upstream death-inducing signaling complex (DISC) subsystem. Under the assumption that the bind of Fas-associated death domains and FLICE-inhibitory proteins to the DISC is much faster than that of the initiator procaspases, we greatly simplify the upstream subsystem from 35 reactions with 26 species to 6 reactions with 9 species by adopting the classical and recently justified partial equilibrium approximation method. Numerical simulations show that the simplified model is in an excellent agreement with the original model. Most importantly, the simplified model clearly reveals the key reactants and dominated pathways in the Fas signaling process, and thus provides new insights into the apoptosis.

  17. Activated microglia cause reversible apoptosis of pheochromocytoma cells, inducing their cell death by phagocytosis

    PubMed Central

    Hornik, Tamara C.; Vilalta, Anna; Brown, Guy C.

    2016-01-01

    ABSTRACT Some apoptotic processes, such as phosphatidylserine exposure, are potentially reversible and do not necessarily lead to cell death. However, phosphatidylserine exposure can induce phagocytosis of a cell, resulting in cell death by phagocytosis: phagoptosis. Phagoptosis of neurons by microglia might contribute to neuropathology, whereas phagoptosis of tumour cells by macrophages might limit cancer. Here, we examined the mechanisms by which BV-2 microglia killed co-cultured pheochromocytoma (PC12) cells that were either undifferentiated or differentiated into neuronal cells. We found that microglia activated by lipopolysaccharide rapidly phagocytosed PC12 cells. Activated microglia caused reversible phosphatidylserine exposure on and reversible caspase activation in PC12 cells, and caspase inhibition prevented phosphatidylserine exposur and decreased subsequent phagocytosis. Nitric oxide was necessary and sufficient to induce the reversible phosphatidylserine exposure and phagocytosis. The PC12 cells were not dead at the time they were phagocytised, and inhibition of their phagocytosis left viable cells. Cell loss was inhibited by blocking phagocytosis mediated by phosphatidylserine, MFG-E8, vitronectin receptors or P2Y6 receptors. Thus, activated microglia can induce reversible apoptosis of target cells, which is insufficient to cause apoptotic cell death, but sufficient to induce their phagocytosis and therefore cell death by phagoptosis. PMID:26567213

  18. Activated microglia cause reversible apoptosis of pheochromocytoma cells, inducing their cell death by phagocytosis.

    PubMed

    Hornik, Tamara C; Vilalta, Anna; Brown, Guy C

    2016-01-01

    Some apoptotic processes, such as phosphatidylserine exposure, are potentially reversible and do not necessarily lead to cell death. However, phosphatidylserine exposure can induce phagocytosis of a cell, resulting in cell death by phagocytosis: phagoptosis. Phagoptosis of neurons by microglia might contribute to neuropathology, whereas phagoptosis of tumour cells by macrophages might limit cancer. Here, we examined the mechanisms by which BV-2 microglia killed co-cultured pheochromocytoma (PC12) cells that were either undifferentiated or differentiated into neuronal cells. We found that microglia activated by lipopolysaccharide rapidly phagocytosed PC12 cells. Activated microglia caused reversible phosphatidylserine exposure on and reversible caspase activation in PC12 cells, and caspase inhibition prevented phosphatidylserine exposur and decreased subsequent phagocytosis. Nitric oxide was necessary and sufficient to induce the reversible phosphatidylserine exposure and phagocytosis. The PC12 cells were not dead at the time they were phagocytised, and inhibition of their phagocytosis left viable cells. Cell loss was inhibited by blocking phagocytosis mediated by phosphatidylserine, MFG-E8, vitronectin receptors or P2Y6 receptors. Thus, activated microglia can induce reversible apoptosis of target cells, which is insufficient to cause apoptotic cell death, but sufficient to induce their phagocytosis and therefore cell death by phagoptosis.

  19. Apoptosis Cell Death Effect of Scrophularia Variegata on Breast Cancer Cells via Mitochondrial Intrinsic Pathway

    PubMed Central

    Azadmehr, Abbas; Hajiaghaee, Reza; Baradaran, Behzad; Haghdoost-Yazdi, Hashem

    2015-01-01

    Purpose: Scrophularia variegata M. Beib. (Scrophulariaceae) is an Iranian medicinal plant which is used for various inflammatory disorders in traditional medicine. In this study we evaluated the anti-cancer and cytotoxic effects of the Scrophularia variegata (S. variegata) ethanolic extract on the human breast cancer cell line. Methods: The cytotoxicity effect of the extract on MCF-7 cells was evaluated by MTT assay. In addition, Caspase activity, DNA ladder and Cell death were evaluated by ELISA, gel electrophoresis and Annexin V-FITC/PI staining, respectively. Results: The S. variegata extract showed significant effect cytotoxicity on MCF-7 human breast cancer cell line. Treatment with the extract induced apoptosis on the breast cancer cells by cell cycle arrest in G2/M phase. The results indicated that cytotoxicity activity was associated with an increase of apoptosis as demonstrated by DNA fragmentation as well as an increase of the amount of caspase 3 and caspase 9. In addition, the phytochemical assay showed that the extract had antioxidant capacity and also flavonoids, phenolic compounds and phenyl propanoids were presented in the extract. Conclusion: Our findings indicated that S. variegata extract induced apoptosis via mitochondrial intrinsic pathway on breast cancer by cell cycle arrest in G2/M phase and an increase of caspase 3 and caspase 9. However future studies are needed. PMID:26504768

  20. Programmed cell death-10 enhances proliferation and protects malignant T cells from apoptosis.

    PubMed

    Lauenborg, Britt; Kopp, Katharina; Krejsgaard, Thorbjørn; Eriksen, Karsten W; Geisler, Carsten; Dabelsteen, Sally; Gniadecki, Robert; Zhang, Qian; Wasik, Mariusz A; Woetmann, Anders; Odum, Niels

    2010-10-01

    The programmed cell death-10 (PDCD10; also known as cerebral cavernous malformation-3 or CCM3) gene encodes an evolutionarily conserved protein associated with cell apoptosis. Mutations in PDCD10 result in cerebral cavernous malformations, an important cause of cerebral hemorrhage. PDCD10 is associated with serine/threonine kinases and phosphatases and modulates the extracellular signal-regulated kinase pathway suggesting a role in the regulation of cellular growth. Here we provide evidence of a constitutive expression of PDCD10 in malignant T cells and cell lines from peripheral blood of cutaneous T-cell lymphoma (Sezary syndrome) patients. PDCD10 is associated with protein phosphatase-2A, a regulator of mitogenesis and apoptosis in malignant T cells. Inhibition of oncogenic signal pathways [Jak3, Notch1, and nuclear factor-κB (NF-κB)] partly inhibits the constitutive PDCD10 expression, whereas an activator of Jak3 and NF-κB, interleukin-2 (IL-2), enhances PDCD10 expression. Functional data show that PDCD10 depletion by small interfering RNA induces apoptosis and decreases proliferation of the sensitive cells. To our knowledge, these data provide the first functional link between PDCD10 and cancer.

  1. Role of apoptosis-inducing factor (AIF) in programmed nuclear death during conjugation in Tetrahymena thermophila

    PubMed Central

    2010-01-01

    Background Programmed nuclear death (PND), which is also referred to as nuclear apoptosis, is a remarkable process that occurs in ciliates during sexual reproduction (conjugation). In Tetrahymena thermophila, when the new macronucleus differentiates, the parental macronucleus is selectively eliminated from the cytoplasm of the progeny, concomitant with apoptotic nuclear events. However, the molecular mechanisms underlying these events are not well understood. The parental macronucleus is engulfed by a large autophagosome, which contains numerous mitochondria that have lost their membrane potential. In animals, mitochondrial depolarization precedes apoptotic cell death, which involves DNA fragmentation and subsequent nuclear degradation. Results We focused on the role of mitochondrial apoptosis-inducing factor (AIF) during PND in Tetrahymena. The disruption of AIF delays the normal progression of PND, specifically, nuclear condensation and kilobase-size DNA fragmentation. AIF is localized in Tetrahymena mitochondria and is released into the macronucleus prior to nuclear condensation. In addition, AIF associates and co-operates with the mitochondrial DNase to facilitate the degradation of kilobase-size DNA, which is followed by oligonucleosome-size DNA laddering. Conclusions Our results suggest that Tetrahymena AIF plays an important role in the degradation of DNA at an early stage of PND, which supports the notion that the mitochondrion-initiated apoptotic DNA degradation pathway is widely conserved among eukaryotes. PMID:20146827

  2. Praziquantel induced oxidative stress and apoptosis-like cell death in Raillietina echinobothrida.

    PubMed

    Giri, Bikash Ranjan; Roy, Bishnupada

    2016-07-01

    Praziquantel (PZQ) is an anthelmintic drug used against trematode and cestode parasites of humans and veterinary animals. Since praziquantel was introduced as a broadspectrum anthelmintic, numerous studies described its successful use against helminth parasites, but its exact mechanism of action is feebly understood. Therefore, the present study was carried out to evaluate the possible role of PZQ induced oxidative stress in apoptosis-like cell death in the poultry tapeworm Raillietina echinobothrida. Parasite viability assay revealed a time-dependent reduction in the worm viability compared to the control. Transmission electron microscopy showed typical apoptotic features like condensed nucleus, damaged nuclear envelope and altered mitochondrial membrane in PZQ exposed parasites. Results revealed chromatin condensation and DNA fragmentation in PZQ exposed parasites. There was a notable decline in the level of glutathione and glutathione-s-transferase activity leading to the augmented generation of reactive oxygen species. This led to the alterations in the mitochondrial membrane potential with increased active caspase-3/7, confirms the involvement of mitochondria in the event. The present study suggests that PZQ exerts oxidative stress leading to apoptosis-like events in the parasites resulting their death.

  3. Systems modelling methodology for the analysis of apoptosis signal transduction and cell death decisions.

    PubMed

    Rehm, Markus; Prehn, Jochen H M

    2013-06-01

    Systems biology and systems medicine, i.e. the application of systems biology in a clinical context, is becoming of increasing importance in biology, drug discovery and health care. Systems biology incorporates knowledge and methods that are applied in mathematics, physics and engineering, but may not be part of classical training in biology. We here provide an introduction to basic concepts and methods relevant to the construction and application of systems models for apoptosis research. We present the key methods relevant to the representation of biochemical processes in signal transduction models, with a particular reference to apoptotic processes. We demonstrate how such models enable a quantitative and temporal analysis of changes in molecular entities in response to an apoptosis-inducing stimulus, and provide information on cell survival and cell death decisions. We introduce methods for analyzing the spatial propagation of cell death signals, and discuss the concepts of sensitivity analyses that enable a prediction of network responses to disturbances of single or multiple parameters.

  4. Marijuana smoke and Delta(9)-tetrahydrocannabinol promote necrotic cell death but inhibit Fas-mediated apoptosis.

    PubMed

    Sarafian, T A; Tashkin, D P; Roth, M D

    2001-08-01

    Marijuana smoke shares many components in common with tobacco smoke except for the presence of Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the psychotropic compound found only in Cannibis sativa. Delta(9)-THC has been shown to potentiate smoke-induced oxidative stress and necrotic cell death. In the present study, our objective was to determine the effects of Delta(9)-THC on the balance between Fas-induced apoptosis and necrosis in A549 lung tumor cells. We found that Fas-induced activation of caspase-3 was inhibited by whole smoke from both tobacco and marijuana cigarettes. Gas-phase smoke, which generates high levels of intracellular reactive oxygen species, had no effect on caspase-3 activity. However, particulate-phase smoke (tar) was a potent inhibitor of Fas-induced caspase-3 activity, with marijuana tar being more potent than either tobacco or placebo marijuana tar (lacking Delta(9)-THC). Delta(9)-THC also inhibited Fas-induced caspase-3 activity in A549 cells. In contrast, no inhibition was observed when Delta(9)-THC was incubated with activated caspase-3 enzyme, suggesting that Delta(9)-THC acts on the cell pathway(s) leading to caspase-3 activation and not directly on enzyme function. Flow cytometry was used to measure the percentage of cells undergoing apoptosis (staining for annexin V) versus necrosis (staining for propidium iodide) and confirmed that both marijuana tar extract and synthetic Delta(9)-THC inhibit Fas-induced apoptosis while promoting necrosis. These observations suggest that the Delta(9)-THC contained in marijuana smoke disrupts elements of the apoptotic pathway, thereby shifting the balance between apoptotic and necrotic cell death. This shift may affect both the carcinogenic and immunologic consequences of marijuana smoke exposure.

  5. Saikosaponin-d, a novel SERCA inhibitor, induces autophagic cell death in apoptosis-defective cells.

    PubMed

    Wong, V K W; Li, T; Law, B Y K; Ma, E D L; Yip, N C; Michelangeli, F; Law, C K M; Zhang, M M; Lam, K Y C; Chan, P L; Liu, L

    2013-07-11

    Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase pump, leading to autophagy induction through the activation of the Ca(2+)/calmodulin-dependent kinase kinase-AMP-activated protein kinase-mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.

  6. Apoptosis-like cell death pathways in the unicellular parasite Toxoplasma gondii following treatment with apoptosis inducers and chemotherapeutic agents: a proof-of-concept study.

    PubMed

    Ni Nyoman, Ayu Dewi; Lüder, Carsten G K

    2013-06-01

    Ancient pathways of an apoptosis-like cell death have been identified in unicellular eukaryotes including protozoan parasites. Here, we examined programmed cell death in the apicomplexan Toxoplasma gondii which is a common intracellular pathogen of humans and warm-blooded animals. Treatment of extracellular T. gondii with various pro-apoptotic stimuli significantly induced DNA strand breaks as revealed by TUNEL and flow cytometry. Using staurosporine or miltefosine as pro-apoptotic stimuli, parasites also presented a reduced cell size, i.e. pyknosis and externalized phosphatidylserine while the plasma membrane remained intact. Importantly, staurosporine also induced DNA strand breaks in intracellular T. gondii. Data mining of the Toxoplasma genome resource identified 17 putative cell death-associated genes encoding proteases, a nuclease and several apoptosis regulators. Staurosporine-treated parasites but not controls strongly up-regulated several of these genes in a time-dependent fashion with a putative PDCD2 protein being more than 100-fold up-regulated. However, the mitochondrial membrane potential (ΔΨ(m)) remained intact and caspase-like activity increased only slightly during staurosporine-triggered cell death. As compared to staurosporine, the transcriptional response of parasites to miltefosine was more restricted but PDCD2 was again strongly induced. Furthermore, T. gondii lost their ΔΨ(m) and rapidly presented strong caspase-like activity during miltefosine treatment. Consequently, protease inhibitors abrogated miltefosine-induced but not staurosporine-induced Toxoplasma cell death. Finally, toxoplasmacidal drugs triggered DNA strand breaks in extracellular T. gondii. Interestingly, clindamycin also induced markers of an apoptosis-like cell death in intracellular parasites. Together, the data indicate that T. gondii possesses ancient apoptosis-like cell death machinery which can be triggered by chemotherapeutic agents.

  7. Apoptosis in mammalian oocytes: a review.

    PubMed

    Tiwari, Meenakshi; Prasad, Shilpa; Tripathi, Anima; Pandey, Ashutosh N; Ali, Irfan; Singh, Arvind K; Shrivastav, Tulsidas G; Chaube, Shail K

    2015-08-01

    Apoptosis causes elimination of more than 99% of germ cells from cohort of ovary through follicular atresia. Less than 1% of germ cells, which are culminated in oocytes further undergo apoptosis during last phases of oogenesis and depletes ovarian reserve in most of the mammalian species including human. There are several players that induce apoptosis directly or indirectly in oocytes at various stages of meiotic cell cycle. Premature removal of encircling granulosa cells from immature oocytes, reduced levels of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate, increased levels of calcium (Ca(2+)) and oxidants, sustained reduced level of maturation promoting factor, depletion of survival factors, nutrients and cell cycle proteins, reduced meiotic competency, increased levels of proapoptotic as well as apoptotic factors lead to oocyte apoptosis. The BH3-only proteins also act as key regulators of apoptosis in oocyte within the ovary. Both intrinsic (mitochondria-mediated) as well as extrinsic (cell surface death receptor-mediated) pathways are involved in oocyte apoptosis. BID, a BH3-only protein act as a bridge between both apoptotic pathways and its cleavage activates cell death machinery of both the pathways inside the follicular microenvironment. Oocyte apoptosis leads to the depletion of ovarian reserve that directly affects reproductive outcome of various mammals including human. In this review article, we highlight some of the important players and describe the pathways involved during oocyte apoptosis in mammals.

  8. Identification of a novel cyclin required for the intrinsic apoptosis pathway in lymphoid cells.

    PubMed

    Roig, M B; Roset, R; Ortet, L; Balsiger, N A; Anfosso, A; Cabellos, L; Garrido, M; Alameda, F; Brady, H J M; Gil-Gómez, G

    2009-02-01

    We have identified an early step common to pathways activated by different forms of intrinsic apoptosis stimuli. It requires de novo synthesis of a novel cyclin, cyclin O, that forms active complexes primarily with Cdk2 upon apoptosis induction in lymphoid cells. Cyclin O expression precedes glucocorticoid and gamma-radiation-induced apoptosis in vivo in mouse thymus and spleen, and its overexpression induces caspase-dependent apoptosis in cultured cells. Knocking down the endogenous expression of cyclin O by shRNA leads to the inhibition of glucocorticoid and DNA damage-induced apoptosis due to a failure in the activation of apical caspases while leaving CD95 death receptor-mediated apoptosis intact. Our data demonstrate that apoptosis induction in lymphoid cells is one of the physiological roles of cyclin O and it does not act by perturbing a normal cellular process such as the cell cycle, the DNA damage checkpoints or transcriptional response to glucocorticoids.

  9. Role of apoptosis-related miRNAs in resveratrol-induced breast cancer cell death.

    PubMed

    Venkatadri, R; Muni, T; Iyer, A K V; Yakisich, J S; Azad, N

    2016-02-18

    Breast cancer is the most frequently diagnosed cancer in women, and one of the leading causes of cancer-related deaths worldwide. Recent evidences indicate that dietary agents such as resveratrol may inhibit cancer progression through modulation of microRNAs (miRNAs). We demonstrate that resveratrol regulates apoptotic and cell cycle machinery in breast cancer cells by modulating key tumor-suppressive miRNAs including miR-125b-5p, miR-200c-3p, miR-409-3p, miR-122-5p and miR-542-3p. Resveratrol-mediated miRNA modulation regulates key anti-apoptotic and cell cycle proteins including Bcl-2, X-linked inhibitor of apoptosis protein and CDKs, which are critical for its activity. Modulating miRNAs with mimics or inhibitors further validated a key role for miR-542-3p in MCF-7 and miR-122-5p in MDA-MB-231 breast cancer cell death in response to resveratrol. In conclusion, this study reveals novel miRNAs modulated by resveratrol that have a key role in breast cancer cell death.

  10. Targeting Death Receptor TRAIL-R2 by Chalcones for TRAIL-Induced Apoptosis in Cancer Cells

    PubMed Central

    Szliszka, Ewelina; Jaworska, Dagmara; Kłósek, Małgorzata; Czuba, Zenon P.; Król, Wojciech

    2012-01-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in cancer cells without toxicity to normal cells. TRAIL binds to death receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5) expressed on cancer cell surface and activates apoptotic pathways. Endogenous TRAIL plays an important role in immune surveillance and defense against cancer cells. However, as more tumor cells are reported to be resistant to TRAIL mediated death, it is important to search for and develop new strategies to overcome this resistance. Chalcones can sensitize cancer cells to TRAIL-induced apoptosis. We examined the cytotoxic and apoptotic effects of TRAIL in combination with four chalcones: chalcone, isobavachalcone, licochalcone A and xanthohumol on HeLa cancer cells. The cytotoxicity was measured by MTT and LDH assays. The apoptosis was detected using annexin V-FITC staining by flow cytometry and fluorescence microscopy. Death receptor expression was analyzed using flow cytometry. The decreased expression of death receptors in cancer cells may be the cause of TRAIL-resistance. Chalcones enhance TRAIL-induced apoptosis in HeLa cells through increased expression of TRAIL-R2. Our study has indicated that chalcones augment the antitumor activity of TRAIL and confirm their cancer chemopreventive properties. PMID:23203129

  11. Cell Death Mechanisms in Tumoral and Non-Tumoral Human Cell Lines Triggered by Photodynamic Treatments: Apoptosis, Necrosis and Parthanatos.

    PubMed

    Soriano, J; Mora-Espí, I; Alea-Reyes, M E; Pérez-García, L; Barrios, L; Ibáñez, E; Nogués, C

    2017-01-23

    Cell death triggered by photodynamic therapy can occur through different mechanisms: apoptosis, necrosis or autophagy. However, recent studies have demonstrated the existence of other mechanisms with characteristics of both necrosis and apoptosis. These new cell death pathways, collectively termed regulated necrosis, include a variety of processes triggered by different stimuli. In this study, we evaluated the cell death mechanism induced by photodynamic treatments with two photosensitizers, meso-tetrakis (4-carboxyphenyl) porphyrin sodium salt (Na-H2TCPP) and its zinc derivative Na-ZnTCPP, in two human breast epithelial cell lines, a non-tumoral (MCF-10A) and a tumoral one (SKBR-3). Viability assays showed that photodynamic treatments with both photosensitizers induced a reduction in cell viability in a concentration-dependent manner and no dark toxicity was observed. The cell death mechanisms triggered were evaluated by several assays and cell line-dependent results were found. Most SKBR-3 cells died by either necrosis or apoptosis. By contrast, in MCF-10A cells, necrotic cells and another cell population with characteristics of both necrosis and apoptosis were predominant. In this latter population, cell death was PARP-dependent and translocation of AIF to the nucleus was observed in some cells. These characteristics are related with parthanatos, being the first evidence of this type of regulated necrosis in the field of photodynamic therapy.

  12. Cell Death Mechanisms in Tumoral and Non-Tumoral Human Cell Lines Triggered by Photodynamic Treatments: Apoptosis, Necrosis and Parthanatos

    PubMed Central

    Soriano, J.; Mora-Espí, I.; Alea-Reyes, M. E.; Pérez-García, L.; Barrios, L.; Ibáñez, E.; Nogués, C.

    2017-01-01

    Cell death triggered by photodynamic therapy can occur through different mechanisms: apoptosis, necrosis or autophagy. However, recent studies have demonstrated the existence of other mechanisms with characteristics of both necrosis and apoptosis. These new cell death pathways, collectively termed regulated necrosis, include a variety of processes triggered by different stimuli. In this study, we evaluated the cell death mechanism induced by photodynamic treatments with two photosensitizers, meso-tetrakis (4-carboxyphenyl) porphyrin sodium salt (Na-H2TCPP) and its zinc derivative Na-ZnTCPP, in two human breast epithelial cell lines, a non-tumoral (MCF-10A) and a tumoral one (SKBR-3). Viability assays showed that photodynamic treatments with both photosensitizers induced a reduction in cell viability in a concentration-dependent manner and no dark toxicity was observed. The cell death mechanisms triggered were evaluated by several assays and cell line-dependent results were found. Most SKBR-3 cells died by either necrosis or apoptosis. By contrast, in MCF-10A cells, necrotic cells and another cell population with characteristics of both necrosis and apoptosis were predominant. In this latter population, cell death was PARP-dependent and translocation of AIF to the nucleus was observed in some cells. These characteristics are related with parthanatos, being the first evidence of this type of regulated necrosis in the field of photodynamic therapy. PMID:28112275

  13. Monocytes regulate the mechanism of T-cell death by inducing Fas-mediated apoptosis during bacterial infection.

    PubMed

    Daigneault, Marc; De Silva, Thushan I; Bewley, Martin A; Preston, Julie A; Marriott, Helen M; Mitchell, Andrea M; Mitchell, Timothy J; Read, Robert C; Whyte, Moira K B; Dockrell, David H

    2012-01-01

    Monocytes and T-cells are critical to the host response to acute bacterial infection but monocytes are primarily viewed as amplifying the inflammatory signal. The mechanisms of cell death regulating T-cell numbers at sites of infection are incompletely characterized. T-cell death in cultures of peripheral blood mononuclear cells (PBMC) showed 'classic' features of apoptosis following exposure to pneumococci. Conversely, purified CD3(+) T-cells cultured with pneumococci demonstrated necrosis with membrane permeabilization. The death of purified CD3(+) T-cells was not inhibited by necrostatin, but required the bacterial toxin pneumolysin. Apoptosis of CD3(+) T-cells in PBMC cultures required 'classical' CD14(+) monocytes, which enhanced T-cell activation. CD3(+) T-cell death was enhanced in HIV-seropositive individuals. Monocyte-mediated CD3(+) T-cell apoptotic death was Fas-dependent both in vitro and in vivo. In the early stages of the T-cell dependent host response to pneumococci reduced Fas ligand mediated T-cell apoptosis was associated with decreased bacterial clearance in the lung and increased bacteremia. In summary monocytes converted pathogen-associated necrosis into Fas-dependent apoptosis and regulated levels of activated T-cells at sites of acute bacterial infection. These changes were associated with enhanced bacterial clearance in the lung and reduced levels of invasive pneumococcal disease.

  14. Antibacterial active compounds from Hypericum ascyron L. induce bacterial cell death through apoptosis pathway.

    PubMed

    Li, Xiu-Mei; Luo, Xue-Gang; Si, Chuan-Ling; Wang, Nan; Zhou, Hao; He, Jun-Fang; Zhang, Tong-Cun

    2015-01-01

    Hypericum ascyron L. has been used as a traditional medicine for the treatment of wounds, swelling, headache, nausea and abscesses in China for thousands of years. However, modern pharmacological studies are still necessary to provide a scientific basis to substantiate their traditional use. In this study, the mechanism underlying the antimicrobial effect of the antibacterial activity compounds from H. ascyron L. was investigated. Bioguided fractionation of the extract from H. ascyron L. afforded antibacterial activity fraction 8. The results of cup plate analysis and MTT assay showed that the MIC and MBC of fraction 8 is 5 mg/mL. Furthermore, using Annexin V-FITC/PI, TUNEL labeling and DNA gel electrophoresis, we found that cell death with apoptosis features similar to those in eucaryon could be induced in bacteria strains after exposure to the antibacterial activity compounds from H. ascyron L. at moderate concentration. In addition, we further found fraction 8 could disrupt the cell membrane potential indicate that fraction 8 exerts pro-apoptotic effects through a membrane-mediated apoptosis pathway. Finally, quercetin and kaempferol 3-O-β-(2″-acetyl)-galactopyranoside, were identified from fraction 8 by means of Mass spectrometry and Nuclear magnetic resonance. To our best knowledge, this study is the first to show that Kaempferol 3-O-β-(2″-acetyl)-galactopyranoside coupled with quercetin had significant antibacterial activity via apoptosis pathway, and it is also the first report that Kaempferol 3-O-β-(2″-acetyl)-galactopyranoside was found in clusiacea. Our data might provide a rational base for the use of H. ascyron L. in clinical, and throw light on the development of novel antibacterial drugs.

  15. Oxidative stress and apoptosis in a pig model of brain death (BD) and living donation (LD)

    PubMed Central

    2013-01-01

    Background As organ shortage is increasing, the acceptance of marginal donors increases, which might result in poor organ function and patient survival. Mostly, organ damage is caused during brain death (BD), cold ischemic time (CIT) or after reperfusion due to oxidative stress or the induction of apoptosis. The aim of this study was to study a panel of genes involved in oxidative stress and apoptosis and compare these findings with immunohistochemistry from a BD and living donation (LD) pig model and after cold ischemia time (CIT). Methods BD was induced in pigs; after 12 h organ retrieval was performed; heart, liver and kidney tissue specimens were collected in the BD (n = 6) and in a LD model (n = 6). PCR analysis for NFKB1, GSS, SOD2, PPAR-alpha, OXSR1, BAX, BCL2L1, and HSP 70.2 was performed and immunohistochemistry used to show apoptosis and nitrosative stress induced cell damage. Results In heart tissue of BD BAX, BCL2L1 and HSP 70.2 increased significantly after CIT. Only SOD2 was over-expressed after CIT in BD liver tissue. In kidney tissue, BCL2L1, NFKB, OXSR1, SOD2 and HSP 70.2 expression was significantly elevated in LD. Immunohistochemistry showed a significant increase in activated Caspase 3 and nitrotyrosine positive cells after CIT in BD in liver and in kidney tissue but not in heart tissue. Conclusion The up-regulation of protective and apoptotic genes seems to be divergent in the different organs in the BD and LD setting; however, immunohistochemistry revealed more apoptotic and nitrotyrosine positive cells in the BD setting in liver and kidney tissue whereas in heart tissue both BD and LD showed an increase. PMID:24088575

  16. UV irradiation/cold shock-mediated apoptosis is switched to bubbling cell death at low temperatures

    PubMed Central

    Lin, Hsin-Ping; Huang, Shenq-Shyang; Sheu, Hamm-Ming; Hsu, Li-Jin; Chang, Nan-Shan

    2015-01-01

    When COS7 fibroblasts and other cells were exposed to UVC irradiation and cold shock at 4°C for 5 min, rapid upregulation and nuclear accumulation of NOS2, p53, WWOX, and TRAF2 occurred in 10–30 min. By time-lapse microscopy, an enlarging gas bubble containing nitric oxide (NO) was formed in the nucleus in each cell that finally popped out to cause “bubbling death”. Bubbling occurred effectively at 4 and 22°C, whereas DNA fragmentation was markedly blocked at 4°C. When temperature was increased to 37°C, bubbling was retarded and DNA fragmentation occurred in 1 hr, suggesting that bubbling death is switched to apoptosis with increasing temperatures. Bubbling occurred prior to nuclear uptake of propidium iodide and DAPI stains. Arginine analog Nω-LAME inhibited NO synthase NOS2 and significantly suppressed the bubbling death. Unlike apoptosis, there were no caspase activation and flip-over of membrane phosphatidylserine (PS) during bubbling death. Bubbling death was significantly retarded in Wwox knockout MEF cells, as well as in cells overexpressing TRAF2 and dominant-negative p53. Together, UV/cold shock induces bubbling death at 4°C and the event is switched to apoptosis at 37°C. Presumably, proapoptotic WWOX and p53 block the protective TRAF2 to execute the bubbling death. PMID:25779665

  17. A form of cell death with some features resembling apoptosis in the amitochondrial unicellular organism Trichomonas vaginalis.

    PubMed

    Chose, Olivier; Noël, Christophe; Gerbod, Delphine; Brenner, Catherine; Viscogliosi, Eric; Roseto, Alberto

    2002-05-15

    One of hallmarks of apoptosis is the degradation and concomitant compaction of chromatin. It is assumed that caspases and caspase-independent pathways are rate limiting for the development of nuclear apoptosis. The caspase-independent pathway involves apoptosis-inducing factor (AIF) and leads to DNA fragmentation and peripheral chromatin condensation. Both pathways are the result of activation of death signals that the mitochondrion receives, integrates, and responds to with the release of various molecules (e.g., cytochrome c and AIF). In fact, both pathways have in common the final point of the DNA fragmentation and the mitochondrial origin of molecules that initiate the apoptotic events. Here, we examine the question of whether apoptosis or apoptotic-like processes exist in a unicellular organism that lacks mitochondria. We herein show that a form of cell death with some features resembling apoptosis is indeed present in Trichomonas vaginalis. Characterization of morphological aspects implicated in this event together with the preliminary biochemical data provided may lead to new insight about the evolutionary relationships between the different forms of programmed cell death identified so far.

  18. Modified arabinoxylan rice bran (MGN-3/Biobran) sensitizes human T cell leukemia cells to death receptor (CD95)-induced apoptosis.

    PubMed

    Ghoneum, Mamdooh; Gollapudi, Sastry

    2003-11-10

    MGN-3, an arabinoxylan extracted from rice bran that is treated enzymatically with an extract from Shiitaki mushrooms, is an effective biological response modifier that increases NK cell activity, and potentiates the activity of conventional chemotherapeutic agents. In this study, we investigated the effect of MGN-3 on death receptor-induced apoptosis in the human leukemic HUT 78 cell line. HUT 78 cells were pre-treated with MGN-3, and then were incubated with the agonistic antibody against death receptor (Fas, CD95). Apoptosis was determined by the propidium iodide technique using FACScan. Activation of caspase 3, caspase 8, and caspase 9 was determined by flow cytometry. Mitochondrial membrane potential was measured with DIOC(6) dye using FACScan. Expression of CD95 and Bcl-2 were measured by flow cytometry. In a dose-dependent manner, MGN-3 enhanced anti-CD95 antibody-induced apoptosis. Increased cell death was correlated with increased depolarization of mitochondrial membrane potential and increased activation of caspase 3, caspase 8, and caspase 9. MGN-3 treatment had no effect on the level of expression of CD95, but it caused down regulation of Bcl-2 expression. These results suggest that MGN-3 increases the susceptibility of cancer cells to undergo apoptosis mediated by death ligands, which may be relevant for anti-cancer activities.

  19. Ryanodine receptor-mediated arrhythmias and sudden cardiac death

    PubMed Central

    Blayney, Lynda M.; Lai, F. Anthony

    2009-01-01

    The cardiac ryanodine receptor-Ca2+ release channel (RyR2) is an essential sarcoplasmic reticulum (SR) transmembrane protein that plays a central role in excitation–contraction coupling (ECC) in cardiomyocytes. Aberrant spontaneous, diastolic Ca2+ leak from the SR due to dysfunctional RyR2 contributes to the formation of delayed after-depolarisations, which are thought to underlie the fatal arrhythmia that occurs in both heart failure (HF) and in catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT is an inherited disorder associated with mutations in either the RyR2 or a SR luminal protein, calsequestrin. RyR2 shows normal function at rest in CPVT but the RyR2 dysfunction is unmasked by physical exercise or emotional stress, suggesting abnormal RyR2 activation as an underlying mechanism. Several potential mechanisms have been advanced to explain the dysfunctional RyR2 observed in HF and CPVT, including enhanced RyR2 phosphorylation status, altered RyR2 regulation at luminal/cytoplasmic sites and perturbed RyR2 intra/inter-molecular interactions. This review considers RyR2 dysfunction in the context of the structural and functional modulation of the channel, and potential therapeutic strategies to stabilise RyR2 function in cardiac pathology. PMID:19345240

  20. Aloe-emodin induces apoptosis of human nasopharyngeal carcinoma cells via caspase-8-mediated activation of the mitochondrial death pathway.

    PubMed

    Lin, Meng-Liang; Lu, Yao-Cheng; Chung, Jing-Gung; Li, Yi-Chen; Wang, Shyang-Guang; N G, Sue-Hwee; Wu, Chia-Yin; Su, Hong-Lin; Chen, Shih-Shun

    2010-05-01

    Aloe-emodin (AE), a natural, biologically active compound from the rhizome of Rheum palmatum, has been shown to induce apoptosis in several cancer cell lines in vitro. However, its molecular mechanism of action in the apoptosis induction of human nasopharyngeal carcinoma (NPC) cells has not been explored. This study shows that AE induced G(2)/M phase arrest by increasing levels of cyclin B1 bound to Cdc2, and also caused an increase in apoptosis of NPC cells, which was characterized by morphological changes, nuclear condensation, DNA fragmentation, caspase-3 activation, cleavage of poly (ADP-ribose) polymerase (PARP) and increased sub-G(1) population. Treatment of NPC cells with AE also resulted in a decrease in Bcl-X(L) and an increase in Bax expression. Ectopic expression of Bcl-X(L) but not Bcl-2 or small interfering RNA (siRNA)-mediated attenuation of Bax suppressed AE-induced apoptotic cell death. AE-induced loss of mitochondrial membrane potential (MMP) and increase in cellular Ca(++) content, reactive oxygen species (ROS) and apoptotic cell death were suppressed by the treatment of cyclosporin A (CsA) or caspase-8 inhibitor Z-IETD-FMK. Co-treatment with caspase-9 inhibitor Z-LEHD-FMK could inhibit AE-induced cell death and the activation of caspase-3 and -9. In addition, suppression of caspase-8 with the specific inhibitor Z-IETD-FMK inhibited AE-induced the activation of Bax, the cleavage of Bid, the translocation of tBid to the mitochondria and the release of cytochrome c, apoptosis-inducing factor (AIF) and Endo G from the mitochondria and subsequent apoptosis. Taken together, these results indicate that the caspase-8-mediated activation of the mitochondrial death pathway plays a critical role in AE-induced apoptosis of NPC cells.

  1. Glutamate receptor-mediated toxicity in optic nerve oligodendrocytes

    PubMed Central

    Matute, Carlos; Sánchez-Gómez, M. Victoria; Martínez-Millán, Luis; Miledi, Ricardo

    1997-01-01

    In cultured oligodendrocytes isolated from perinatal rat optic nerves, we have analyzed the expression of ionotropic glutamate receptor subunits as well as the effect of the activation of these receptors on oligodendrocyte viability. Reverse transcription–PCR, in combination with immunocytochemistry, demonstrated that most oligodendrocytes differentiated in vitro express the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluR3 and GluR4 and the kainate receptor subunits GluR6, GluR7, KA1 and KA2. Acute and chronic exposure to kainate caused extensive oligodendrocyte death in culture. This effect was partially prevented by the AMPA receptor antagonist GYKI 52466 and was completely abolished by the non-N-methyl-d-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), suggesting that both AMPA and kainate receptors mediate the observed kainate toxicity. Furthermore, chronic application of kainate to optic nerves in vivo resulted in massive oligodendrocyte death which, as in vitro, could be prevented by coinfusion of the toxin with CNQX. These findings suggest that excessive activation of the ionotropic glutamate receptors expressed by oligodendrocytes may act as a negative regulator of the size of this cell population. PMID:9238063

  2. A systems level strategy for analyzing the cell death network: implication in exploring the apoptosis/autophagy connection.

    PubMed

    Zalckvar, E; Yosef, N; Reef, S; Ber, Y; Rubinstein, A D; Mor, I; Sharan, R; Ruppin, E; Kimchi, A

    2010-08-01

    The mammalian cell death network comprises three distinct functional modules: apoptosis, autophagy and programmed necrosis. Currently, the field lacks systems level approaches to assess the extent to which the intermodular connectivity affects cell death performance. Here, we developed a platform that is based on single and double sets of RNAi-mediated perturbations targeting combinations of apoptotic and autophagic genes. The outcome of perturbations is measured both at the level of the overall cell death responses, using an unbiased quantitative reporter, and by assessing the molecular responses within the different functional modules. Epistatic analyses determine whether seemingly unrelated pairs of proteins are genetically linked. The initial running of this platform in etoposide-treated cells, using a few single and double perturbations, identified several levels of connectivity between apoptosis and autophagy. The knock down of caspase3 turned on a switch toward autophagic cell death, which requires Atg5 or Beclin-1. In addition, a reciprocal connection between these two autophagic genes and apoptosis was identified. By applying computational tools that are based on mining the protein-protein interaction database, a novel biochemical pathway connecting between Atg5 and caspase3 is suggested. Scaling up this platform into hundreds of perturbations potentially has a wide, general scope of applicability, and will provide the basis for future modeling of the cell death network.

  3. Type I interferons induce apoptosis by balancing cFLIP and caspase-8 independent of death ligands.

    PubMed

    Apelbaum, Amir; Yarden, Ganit; Warszawski, Shira; Harari, Daniel; Schreiber, Gideon

    2013-02-01

    Interferons induce a pleiotropy of responses through binding the same cell surface receptor. Here we investigated the molecular mechanism driving interferon-induced apoptosis. Using a nonbiased small interfering RNA (siRNA) screen, we show that silencing genes whose products are directly engaged in the initiation of interferon signaling completely abrogate the interferon antiproliferative response. Apoptosis-related genes such as the caspase-8, cFLIP, and DR5 genes specifically interfere with interferon-induced apoptosis, which we found to be independent of the activity of death ligands. The one gene for which silencing resulted in the strongest proapoptotic effect upon interferon signaling is the cFLIP gene, where silencing shortened the time of initiation of apoptosis from days to hours and increased dramatically the population of apoptotic cells. Thus, cFLIP serves as a regulator for interferon-induced apoptosis. A shift over time in the balance between cFLIP and caspase-8 results in downstream caspase activation and apoptosis. While gamma interferon (IFN-γ) also causes caspase-8 upregulation, we suggest that it follows a different path to apoptosis.

  4. The dorsal motor nucleus of the vagus (DMNV) in sudden infant death syndrome (SIDS): pathways leading to apoptosis.

    PubMed

    Bejjani, Charbel; Machaalani, Rita; Waters, Karen A

    2013-01-15

    Sudden infant death syndrome (SIDS) remains the commonest cause of death in the post-neonatal period in the developed world. A leading hypothesis is that an abnormality in the brainstem of infants who succumb to SIDS, either causes or predisposes to failure to respond appropriately to an exogenous stressor. Neuronal apoptosis can lead to loss of cardiorespiratory reflexes, compromise of the infant's ability to respond to stressors such as hypoxia, and ultimately a sleep-related death. The dorsal motor nucleus of the vagus (DMNV) is a medullary autonomic nucleus where abnormalities have regularly been identified in SIDS research. This review collates neurochemical findings documented over the last 30 years, including data from our laboratory focusing on neuronal apoptosis and the DMNV, and provides potential therapeutic interventions targeting neurotransmitters, growth factors and/or genes.

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

    SciTech Connect

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

    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

  6. Apoptosis and Necrosis in the Liver

    PubMed Central

    Guicciardi, Maria Eugenia; Malhi, Harmeet; Mott, Justin L.; Gores, Gregory J.

    2013-01-01

    Because of its unique function and anatomical location, the liver is exposed to a multitude of toxins and xenobiotics, including medications and alcohol, as well as to infection by hepatotropic viruses, and therefore, is highly susceptible to tissue injury. Cell death in the liver occurs mainly by apoptosis or necrosis, with apoptosis also being the physiologic route to eliminate damaged or infected cells and to maintain tissue homeostasis. Liver cells, especially hepatocytes and cholangiocytes, are particularly susceptible to death receptor-mediated apoptosis, given the ubiquitous expression of the death receptors in the organ. In a quite unique way, death receptor-induced apoptosis in these cells is mediated by both mitochondrial and lysosomal permeabilization. Signaling between the endoplasmic reticulum and the mitochondria promotes hepatocyte apoptosis in response to excessive free fatty acid generation during the metabolic syndrome. These cell death pathways are partially regulated by microRNAs. Necrosis in the liver is generally associated with acute injury (i.e., ischemia/reperfusion injury) and has been long considered an unregulated process. Recently, a new form of “programmed” necrosis (named necroptosis) has been described: the role of necroptosis in the liver has yet to be explored. However, the minimal expression of a key player in this process in the liver suggests this form of cell death may be uncommon in liver diseases. Because apoptosis is a key feature of so many diseases of the liver, therapeutic modulation of liver cell death holds promise. An updated overview of these concepts is given in this article. PMID:23720337

  7. p53-independent death and p53-induced protection against apoptosis in fibroblasts treated with chemotherapeutic drugs.

    PubMed Central

    Malcomson, R. D.; Oren, M.; Wyllie, A. H.; Harrison, D. J.

    1995-01-01

    Many recent studies have implicated p53 in the cellular response to injury and induction of cell death by apoptosis. In a rat embryonal fibroblast cell line transformed with c-Ha-ras and a mutant temperature-sensitive p53 (val135), cells were G1 arrested at the permissive temperature of 32 degrees C when overexpressed p53 was in wild-type conformation. In this state cells were resistant to apoptosis induced by etoposide (at up to 50 microM) or bleomycin (15 microU ml-1). Cells at 37 degrees C with overexpressed p53 in mutant conformation were freed from this growth arrest, continued proliferating and showed dose-dependent increases in apoptosis. This death is independent of wild-type p53 function. Control cells containing a non-temperature-sensitive mutant p53 (phe132) were sensitive to both etoposide and bleomycin after 24 h at 32 degrees C and 37 degrees C, indicating that the results are not simply due to temperature effects on pharmacokinetics or DNA damage. Our data show that induction of a stable p53-mediated growth arrest renders these cells much less likely to undergo apoptosis in response to certain anti-cancer drugs, and we conclude that the regulatory role of p53 in apoptosis is influenced by the particular cellular context in which this gene is expressed. PMID:7547247

  8. X-Linked Inhibitor of Apoptosis Protein – A Critical Death Resistance Regulator and Therapeutic Target for Personalized Cancer Therapy

    PubMed Central

    Obexer, Petra; Ausserlechner, Michael J.

    2014-01-01

    Defects in apoptosis regulation are one main cause of cancer development and may result from overexpression of anti-apoptotic proteins such as inhibitor of apoptosis proteins (IAPs). IAPs are cell death regulators that, among other functions, bind caspases, and interfere with apoptotic signaling via death receptors or intrinsic cell death pathways. All IAPs share one to three common structures, the so called baculovirus-IAP-repeat (BIR)-domains that allow them to bind caspases and other proteins. X-linked inhibitor of apoptosis protein (XIAP) is the most potent and best-defined anti-apoptotic IAP family member that directly neutralizes caspase-9 via its BIR3 domain and the effector caspases-3 and -7 via its BIR2 domain. A natural inhibitor of XIAP is SMAC/Diablo, which is released from mitochondria in apoptotic cells and displaces bound caspases from the BIR2/BIR3 domains of XIAP thereby reactivating cell death execution. The central apoptosis-inhibitory function of XIAP and its overexpression in many different types of advanced cancers have led to significant efforts to identify therapeutics that neutralize its anti-apoptotic effect. Most of these drugs are chemical derivatives of the N-terminal part of SMAC/Diablo. These “SMAC-mimetics” either specifically induce apoptosis in cancer cells or act as drug-sensitizers. Several “SMAC-mimetics” are currently tested by the pharmaceutical industry in Phase I and Phase II trials. In this review, we will discuss recent advances in understanding the function of IAPs in normal and malignant cells and focus on approaches to specifically neutralize XIAP in cancer cells. PMID:25120954

  9. Glutathione administration reduces mitochondrial damage and shifts cell death from necrosis to apoptosis in ageing diabetic mice hearts during exercise

    PubMed Central

    Golbidi, S; Botta, A; Gottfred, S; Nusrat, A; Laher, I; Ghosh, S

    2014-01-01

    Background and Purpose The effect of antioxidants on ageing type 2 diabetic (T2D) hearts during exercise is unclear. We hypothesized that GSH therapy during exercise reduces mitochondrial oxidative stress (mOXS) and cell death in ageing db/db mice hearts. Experimental Approach The effect of GSH on cardiac mOXS and cell death was evaluated both in vivo and in vitro. Key Results During exercise, GSH treatment protected db/db hearts from exaggerated mOXS without reducing total cell death. Despite similar cell death, investigations on apoptosis-specific single-stranded DNA breaks and necrosis-specific damage provided the first in vivo evidence of a shift from necrosis to apoptosis, with reduced fibrosis following GSH administration in exercised db/db hearts. Further support for a GSH-regulated ‘switch’ in death phenotypes came from NIH-3T3 fibroblasts and H9c2 cardiomyocytes treated with H2O2, a reactive oxygen species (ROS). Similar to in vivo findings, augmenting GSH by overexpressing glutamyl cysteine ligase (GCLc) protected fibroblasts and cardiomyocytes from necrosis induced by H2O2, but elevated caspase-3 and apoptosis instead. Similar to in vivo findings, where GSH therapy in normoglycaemic mice suppressed endogenous antioxidants and augmented caspase-3 activity, GCLc overexpression during staurosporine-induced death, which was not characterized by ROS, increased GSH efflux and aggravated death in fibroblasts and cardiomyocytes, confirming that oxidative stress is required for GSH-mediated cytoprotection. Conclusions and Implications While GSH treatment is useful for reducing mOXS and attenuating necrosis and fibrosis in ageing T2D hearts during exercise, such antioxidant treatment could be counterproductive in the healthy heart during exercise. PMID:25039894

  10. Akt-phosphorylated mitogen-activated kinase-activating death domain protein (MADD) inhibits TRAIL-induced apoptosis by blocking Fas-associated death domain (FADD) association with death receptor 4.

    PubMed

    Li, Peifeng; Jayarama, Shankar; Ganesh, Lakshmy; Mordi, David; Carr, Ryan; Kanteti, Prasad; Hay, Nissim; Prabhakar, Bellur S

    2010-07-16

    MADD plays an essential role in cancer cell survival. Abrogation of endogenous MADD expression results in significant spontaneous apoptosis and enhanced susceptibility to tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. However, the regulation of MADD function is largely unknown. Here, we demonstrate that endogenous MADD is phosphorylated at three highly conserved sites by Akt, and only the phosphorylated MADD can directly interact with the TRAIL receptor DR4 thereby preventing Fas-associated death domain recruitment. However, in cells susceptible to TRAIL treatment, TRAIL induces a reduction in MADD phosphorylation levels resulting in MADD dissociation from, and Fas-associated death domain association with DR4, which allows death-inducing signaling complex (DISC) formation leading to apoptosis. Thus, the pro-survival function of MADD is dependent upon its phosphorylation by Akt. Because Akt is active in most cancer cells and phosphorylated MADD confers resistance to TRAIL-induced apoptosis, co-targeting Akt-MADD axis is likely to increase efficacy of TRAIL-based therapies.

  11. Apoptosis: A Functional Paradigm for Programmed Plant Cell Death Induced by a Host-Selective Phytotoxin and Invoked during Development.

    PubMed Central

    Wang, H; Li, J; Bostock, RM; Gilchrist, DG

    1996-01-01

    The host-selective AAL toxins secreted by Alternaria alternata f sp lycopersici are primary chemical determinants in the Alternaria stem canker disease of tomato. The AAL toxins are members of a new class of sphinganine analog mycotoxins that cause cell death in both animals and plants. Here, we report detection of stereotypic hallmarks of apoptosis during cell death induced by these toxins in tomato. DNA ladders were observed during cell death in toxin-treated tomato protoplasts and leaflets. The intensity of the DNA ladders was enhanced by Ca2+ and inhibited by Zn2+. The progressive delineation of fragmented DNA into distinct bodies, coincident with the appearance of DNA ladders, also was observed during death of toxin-treated tomato protoplasts. In situ analysis of cells dying during development in both onion root caps and tomato leaf tracheary elements revealed DNA fragmentation localized to the dying cells as well as the additional formation of apoptotic-like bodies in sloughing root cap cells. We conclude that the fundamental elements of apoptosis, as characterized in animals, are conserved in plants. The apoptotic process may be expressed during some developmental transitions and is the functional process by which symptomatic lesions are formed in the Alternaria stem canker disease of tomato. Sphinganine analog mycotoxins may be used to characterize further signaling pathways leading to apoptosis in plants. PMID:12239387

  12. Pulse mode irradiation at Radachlorin PDT shifted cell death to apoptosis in vitro

    NASA Astrophysics Data System (ADS)

    Klimenko, Vladimir V.; Bogdanov, Alexey A.; Knyazev, Nickolay A.; Dubina, Michael V.

    2015-07-01

    Photodynamic therapy (PDT) is a clinically approved treatment that can exhibit onsite cytotoxic activity toward tumor cells. One of the main factors limiting PDT efficiency is tissue hypoxia derived from photodynamic action. PDT with pulse mode irradiation at the same peak fluence rates as in continuous wave (CW) mode and with appropriate irradiation parameters could be more effective in the potency of 1O2 generation and the cytotoxic effect enhancement by tissue reoxygenation. In this study, we demonstrated theoretically that the main parameter of pulse mode irradiation is the intermittency factor, which makes it possible to maintain the intended 3O2 concentration and to regulate the efficiency of 1O2 generation. We also showed experimentally that photodynamic treatment with pulse mode irradiation has congruent cytotoxicity to CW mode but induces preferable cell apoptosis. We assume that not only is cumulative 1O2 concentration is important in photodynamic cytotoxicity, but so is the temporal distribution of 1O2 generation, which determines the types of cell death.

  13. Hepatocyte Death: A Clear and Present Danger

    PubMed Central

    MALHI, HARMEET; GUICCIARDI, MARIA EUGENIA; GORES, GREGORY J.

    2010-01-01

    The hepatocyte is especially vulnerable to injury due to its central role in xenobiotic metabolism including drugs and alcohol, participation in lipid and fatty acid metabolism, its unique role in the enterohepatic circulation of bile acids, the widespread prevalence of hepatotropic viruses, and its existence within a milieu of innate immune responding cells. Apoptosis and necrosis are the most widely recognized forms of hepatocyte cell death. The hepatocyte displays many unique features regarding cell death by apoptosis. It is quite susceptible to death receptor-mediated injury, and its death receptor signaling pathways involve the mitochondrial pathway for efficient cell killing. Also, death receptors can trigger lysosomal disruption in hepatocytes which further promote cell and tissue injury. Interestingly, hepatocytes are protected from cell death by only two anti-apoptotic proteins, Bcl-xL and Mcl-1, which have nonredundant functions. Endoplasmic reticulum stress or the unfolded protein response contributes to hepatocyte cell death during alterations of lipid and fatty acid metabolism. Finally, the current information implicating RIP kinases in necrosis provides an approach to more fully address this mode of cell death in hepatocyte injury. All of these processes contributing to hepatocyte injury are discussed in the context of potential therapeutic strategies. PMID:20664081

  14. Systems biology of death receptor networks: live and let die

    PubMed Central

    Lavrik, I N

    2014-01-01

    The extrinsic apoptotic pathway is initiated by death receptor activation. Death receptor activation leads to the formation of death receptor signaling platforms, resulting in the demolition of the cell. Despite the fact that death receptor-mediated apoptosis has been studied to a high level of detail, its quantitative regulation until recently has been poorly understood. This situation has dramatically changed in the last years. Creation of mathematical models of death receptor signaling led to an enormous progress in the quantitative understanding of the network regulation and provided fascinating insights into the mechanisms of apoptosis control. In the following sections, the models of the death receptor signaling and their biological implications will be addressed. Central attention will be given to the models of CD95/Fas/APO-1, an exemplified member of the death receptor signaling pathways. The CD95 death-inducing signaling complex (DISC) and regulation of CD95 DISC activity by its key inhibitor c-FLIP, have been vigorously investigated by modeling approaches, and therefore will be the major topic here. Furthermore, the non-linear dynamics of the DISC, positive feedback loops and bistability as well as stoichiometric switches in extrinsic apoptosis will be discussed. Collectively, this review gives a comprehensive view how the mathematical modeling supported by quantitative experimental approaches has provided a new understanding of the death receptor signaling network. PMID:24874731

  15. Cell death (apoptosis) in mouse intestine after continuous irradiation with gamma rays and with beta rays from tritiated water

    SciTech Connect

    Ijiri, K.

    1989-04-01

    Apoptosis is a pattern of cell death involving nuclear pycnosis, cytoplasmic condensation, and karyorrhexis. Apoptosis induced by continuous irradiation with gamma rays (externally given by a 137Cs source) or with beta rays (from tritiated water injected ip) was quantified in the crypts of two portions of mouse bowel, the small intestine and descending colon. The time-course change in the incidence of apoptosis after each type of radiation could be explained on the basis of the innate circadian rhythm of the cells susceptible to apoptotic death and of the excretion of tritiated water (HTO) from the body. For 6-h continuous gamma irradiation at various dose rates (0.6-480 mGy/h) and for 6 h after injection of HTO of various radioactivities (0.15-150 GBq per kg body wt), the relationships between dose and incidence of apoptosis were obtained. Survival curves were then constructed from the curves for dose vs incidence of apoptosis. For the calculation of the absorbed dose from HTO, the water content both of the mouse body and of the cells was assumed to be 70%. One megabecquerel of HTO per mouse (i.e., 40 MBq/kg body wt) gave a dose rate of 0.131 mGy/h. The mean lethal doses (D0) were calculated for gamma rays and HTO, and relative biological effectiveness values of HTO relative to gamma rays were obtained. The D0 values for continuous irradiation with gamma rays were 210 mGy for small intestine and 380 mGy for descending colon, and the respective values for HTO were 130 and 280 mGy, indicating the high radiosensitivity of target cells for apoptotic death. The relative biological effectiveness of HTO relative to 137Cs gamma rays for cell killing in both the small intestine and the descending colon in the mouse was 1.4-2.1.

  16. Autophagy and apoptosis have a role in the survival or death of stallion spermatozoa during conservation in refrigeration.

    PubMed

    Gallardo Bolaños, Juan M; Miró Morán, Álvaro; Balao da Silva, Carolina M; Morillo Rodríguez, Antolín; Plaza Dávila, María; Aparicio, Inés M; Tapia, José A; Ortega Ferrusola, Cristina; Peña, Fernando J

    2012-01-01

    Apoptosis has been recognized as a cause of sperm death during cryopreservation and a cause of infertility in humans, however there is no data on its role in sperm death during conservation in refrigeration; autophagy has not been described to date in mature sperm. We investigated the role of apoptosis and autophagy during cooled storage of stallion spermatozoa. Samples from seven stallions were split; half of the ejaculate was processed by single layer centrifugation, while the other half was extended unprocessed, and stored at 5°C for five days. During the time of storage, sperm motility (CASA, daily) and membrane integrity (flow cytometry, daily) were evaluated. Apoptosis was evaluated on days 1, 3 and 5 (active caspase 3, increase in membrane permeability, phosphatidylserine translocation and mitochondrial membrane potential) using flow cytometry. Furthermore, LC3B processing was investigated by western blotting at the beginning and at the end of the period of storage. The decrease in sperm quality over the period of storage was to a large extent due to apoptosis; single layer centrifugation selected non-apoptotic spermatozoa, but there were no differences in sperm motility between selected and unselected sperm. A high percentage of spermatozoa showed active caspase 3 upon ejaculation, and during the period of storage there was an increase of apoptotic spermatozoa but no changes in the percentage of live sperm, revealed by the SYBR-14/PI assay, were observed. LC3B was differentially processed in sperm after single layer centrifugation compared with native sperm. In processed sperm more LC3B-II was present than in non-processed samples; furthermore, in non-processed sperm there was an increase in LC3B-II after five days of cooled storage. These results indicate that apoptosis plays a major role in the sperm death during storage in refrigeration and that autophagy plays a role in the survival of spermatozoa representing a new pro-survival mechanism in

  17. Heat shock induces apoptosis through reactive oxygen species involving mitochondrial and death receptor pathways in corneal cells.

    PubMed

    Hsu, Ya-Ling; Yu, Hsin-Su; Lin, Hsien-Chung; Wu, Kwou-Yeung; Yang, Rei-Cheng; Kuo, Po-Lin

    2011-10-01

    Although many studies have been performed to elucidate the molecular consequences of ultraviolet irradiation, little is known about the effect of infrared radiation on ocular disease. In addition to photons, heat is generated as a consequence of infrared irradiation, and heat shock is widely considered to be an environmental stressor. Here, we are the first to investigate the biological effect of heat shock on Statens Seruminstitut Rabbit Cornea (SIRC) cells. Our results indicate that heat shock exhibits effective cell proliferation inhibition by inducing apoptosis. Heat shock triggers the mitochondrial apoptotic pathway indicated by a change in Bax/Bcl-2 ratios, resulting in caspase-9 activity. In addition, heat shock triggered the death receptor apoptotic pathway indicated by a change in Fas ligand expression, resulting in caspase-8 activity. Furthermore, we also found that generation of reactive oxygen species (ROS) is a critical mediator in heat shock-induced apoptosis. In addition, the antioxidant vitamin C significantly decreased heat shock-mediated apoptosis. Taken together, these findings suggest a critical role for ROS involving mitochondrial and death receptor pathways in heat shock-mediated apoptosis of cornea cells.

  18. Flavanonol taxifolin attenuates proteasome inhibition-induced apoptosis in differentiated PC12 cells by suppressing cell death process.

    PubMed

    Nam, Yoon Jeong; Lee, Da Hee; Shin, Yong Kyoo; Sohn, Dong Suep; Lee, Chung Soo

    2015-03-01

    The proteasomal dysfunction and mitochondrial impairment has been implicated in neuronal degeneration. Taxifolin has antioxidant and anti-inflammatory effects. However, the effect of taxifolin on the neuronal cell death induced by proteasome inhibition has not been studied. Therefore, in the respect of cell death process, we assessed the effect of taxifolin on the proteasome inhibition-induced apoptosis in neuronal cell injury using differentiated PC12 cells. The proteasome inhibitors MG132 and MG115 induced a decrease in Bid, Bcl-2, and survivin protein levels, an increase in Bax, loss of the mitochondrial transmembrane potential, cytochrome c release, activation of caspases(-8, -9 and -3), an increase in the tumor suppressor p53 levels and cleavage of PARP-1. The addition of taxifolin attenuated the proteasome inhibitor-induced changes in the apoptosis-related protein levels, formation of reactive oxygen species, depletion and oxidation of GSH, formations of malondialdehyde and carbonyls, and cell death. The results show that taxifolin may attenuate the proteasome inhibitor-induced apoptosis in PC12 cells by suppressing the activation of the mitochondrial pathway and the caspase-8- and Bid-dependent pathways. The preventive effect of taxifolin appears to be attributed to its inhibitory effect on the formation of reactive oxygen species, and depletion and oxidation of GSH.

  19. T315 Decreases Acute Myeloid Leukemia Cell Viability through a Combination of Apoptosis Induction and Autophagic Cell Death

    PubMed Central

    Chiu, Chang-Fang; Weng, Jing-Ru; Jadhav, Appaso; Wu, Chia-Yung; Sargeant, Aaron M.; Bai, Li-Yuan

    2016-01-01

    T315, an integrin-linked kinase (ILK) inhibitor, has been shown to suppress the proliferation of breast cancer, stomach cancer and chronic lymphocytic leukemia cells. Here we demonstrate that T315 decreases cell viability of acute myeloid leukemia (AML) cell lines (HL-60 and THP-1) and primary leukemia cells from AML patients in a dose-responsive manner. Normal human bone marrow cells are less sensitive than leukemia cells to T315. T315 down regulates protein kinase B (Akt) and p-Akt and induces caspase activation, poly-ADP-ribose polymerase (PARP) cleavage, apoptosis and autophagy through an ILK-independent manner. Interestingly, pretreatment with autophagy inhibitors rescues cells from apoptosis and concomitant PARP cleavage, which implicates a key role of autophagic cell death in T315-mediated cytotoxicity. T315 also demonstrates efficacy in vivo, suppressing the growth of THP-1 xenograft tumors in athymic nude mice when administered intraperitoneally. This study shows that autophagic cell death and apoptosis cooperatively contribute to the anticancer activity of T315 in AML cells. In conclusion, the complementary roles of apoptotic and autophagic cell death should be considered in the future assessment of the translational value of T315 in AML therapy. PMID:27537872

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

    PubMed

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

    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.

  1. ST6Gal-I Regulates Macrophage Apoptosis via α2-6 Sialylation of the TNFR1 Death Receptor*

    PubMed Central

    Liu, Zhongyu; Swindall, Amanda F.; Kesterson, Robert A.; Schoeb, Trenton R.; Bullard, Daniel C.; Bellis, Susan L.

    2011-01-01

    Macrophages play a central role in innate immunity, however mechanisms regulating macrophage survival are not fully understood. Herein we describe a novel apoptotic pathway involving α2-6 sialylation of the TNFR1 death receptor by the ST6Gal-I sialyltransferase. Variant glycosylation of TNFR1 has not previously been implicated in TNFR1 function, and little is known regarding the TNFR1 glycan composition. To study sialylation in macrophages, we treated U937 monocytic cells with PMA, which stimulates both macrophage differentiation and apoptosis. Interestingly, macrophage differentiation induces ST6Gal-I down-regulation, leading to reduced α2-6 sialylation of selected receptors. To prevent loss of α2-6 sialylation, we forced constitutive expression of ST6Gal-I, and found that this strongly inhibited PMA-induced apoptosis. Given that PMA-mediated apoptosis is thought to result from up-regulation of TNFα, which then activates TNFR1, we next evaluated the α2-6 sialylation of TNFR1. U937 cells with forced ST6Gal-I displayed TNFR1 with elevated α2-6 sialylation, and this was associated with diminished TNFα-stimulated apoptosis. Correspondingly, removal of α2-6 sialylation from TNFR1 through either neuraminidase treatment or expression of ST6Gal-I shRNA markedly enhanced TNFα-mediated apoptosis. To confirm the physiologic importance of TNFR1 sialylation, we generated overexpressing ST6Gal-I transgenic mice. Peritoneal macrophages from transgenic lines displayed TNFR1 with elevated α2-6 sialylation, and these cells were significantly protected against TNFα-stimulated apoptosis. Moreover, greater numbers of thioglycollate-induced peritoneal cells were observed in transgenic mice. These collective results highlight a new mechanism of TNFR1 regulation, and further intimate that loss of α2-6 sialylation during macrophage differentiation may limit macrophage lifespan by sensitizing cells to TNFα-stimulated apoptosis. PMID:21930713

  2. Distinct modes of cell death induced by different reactive oxygen species: amino acyl chloramines mediate hypochlorous acid-induced apoptosis.

    PubMed

    Englert, Robert P; Shacter, Emily

    2002-06-07

    Oxidants derived from inflammatory phagocytes compose a key element of the host immune defense system and can kill mammalian cells by one of several different mechanisms. In this report, we compare mechanisms of cell death induced in human B lymphoma cells by the inflammatory oxidants superoxide, H(2)O(2), and HOCl. The results indicate that the mode of cell death induced depends on the nature of the oxidant involved and the medium in which the cells are treated. When human Burkitt's lymphoma cells are exposed to superoxide anion, generated as a flux from xanthine and xanthine oxidase, the cells die by a non-apoptotic mechanism (pyknosis/necrosis) identical to that seen when cells are treated with a bolus of reagent H(2)O(2). Addition of superoxide dismutase has no effect, whereas catalase is completely protective, indicating that exogenously generated superoxide kills cells entirely through its dismutation into H(2)O(2). In contrast, cells treated in culture media with reagent HOCl die largely by apoptosis. HOCl-induced apoptosis is mediated by aminoacyl chloramines generated in the culture media and can be mimicked by treatment of cells with taurine chloramine or with long lived chloramines generated from modified Lys or Arg. The results suggest that in a physiological milieu in which O(2)(-) and H(2)O(2) are the main oxidants being formed, the principal form of cell death may be necrotic, and under inflammatory conditions in which HOCl is generated, apoptotic cell death may predominate.

  3. Molecular Cell Biology of Apoptosis and Necroptosis in Cancer.

    PubMed

    Dillon, Christopher P; Green, Douglas R

    2016-01-01

    Cell death is a major mechanism to eliminate cells in which DNA is damaged, organelles are stressed, or oncogenes are overexpressed, all events that would otherwise predispose cells to oncogenic transformation. The pathways that initiate and execute cell death are complex, genetically encoded, and subject to significant regulation. Consequently, while these pathways are often mutated in malignancy, there is considerable interest in inducing cell death in tumor cells as therapy. This chapter addresses our current understanding of molecular mechanisms contributing to two cell death pathways, apoptotic cell death and necroptosis, a regulated form of necrotic cell death. Apoptosis can be induced by a wide variety of signals, leading to protease activation that dismantles the cell. We discuss the physiological importance of each apoptosis pathway and summarize their known roles in cancer suppression and the current efforts at targeting each pathway therapeutically. The intricate mechanistic link between death receptor-mediated apoptosis and necroptosis is described, as well as the potential opportunities for utilizing necroptosis in the treatment of malignancy.

  4. Apoptosis (cell death) induced in mouse bowel by 1,2-dimethylhydrazine, methylazoxymethanol acetate, and gamma-rays

    SciTech Connect

    Ijiri, K. )

    1989-11-15

    Apoptosis is a pattern of cell death involving nuclear pyknosis, cytoplasmic condensation, and karyorrhexis. The frequency of apoptosis after treatment with two colon carcinogens and radiation was studied in the crypts of five different portions of mouse bowel. When 1,2-dimethylhydrazine (DMH) was injected s.c., the earliest rise in apoptotic incidence after a high dose (200 mg/kg) was noted at 3 h in small intestine and at 6 h in large bowel. After i.p. administration of methylazoxymethanol (MAM) acetate, apoptotic cells were seen in large bowel after 3 h. When the plateau values attained after high doses of DMH were compared, many apoptotic cells were found in the lower part of the large bowel, whereas few such cells were observed in the small intestine and the upper part of the large bowel. This finding was reversed in the case of radiation-induced apoptosis. In the descending colon, a definite circadian rhythm in the apoptotic incidence was observed 6 h after injection of DMH or MAM acetate. Apoptosis showed a high incidence when these drugs were given between 2400 h and 0900 h, but a low incidence after administration between 1200 h and 2100 h. In the small intestine a rhythm was also noted for MAM acetate, but not significantly for DMH.

  5. Colistin-induced apoptosis in PC12 cells: involvement of the mitochondrial apoptotic and death receptor pathways.

    PubMed

    Jiang, Hong; Li, Jichang; Zhou, Tiezhong; Wang, Chunhua; Zhang, Hua; Wang, Hongjun

    2014-05-01

    Colistin, a cyclic cationic polypeptide antibiotic that is used to treat infections, may cause neurotoxicity. However, whether colistin can induce apoptosis and the precise mechanism of apoptosis involved in PC12 cells remains to be determined. The aim of the present study was to determine reactive oxygen species (ROS) level and DNA damage, as well as apoptotic factors such as p53, cytochrome c, Bax, Bcl-2, Fas, Fas-L and caspase family via western blotting in PC12 cells treated with colistin sulfate. The results showed that colistin sulfate increased ROS levels significantly. An increase of ROS levels induces the release of cytochrome c and DNA damage. DNA damage can activate p53, which leads to the upregulation of Bax and downregulation of Bcl-2. The imbalance of Bax/Bcl-2 promotes additional release of cytochrome c. The release of cytochrome c contributes to the activation of caspase-9 and the subsequent activation of caspase-3. An increase of Fas and Fas-L induced the activation of caspase-8 leading to the activation of caspases-3, the latter induces apoptosis. Therefore, these results demonstrate that the apoptotic pathway of colistin-induced apoptosis in PC12 cells is involved in both the mitochondrial and death receptor pathway.

  6. Expression of apoptosis and proliferating cell nuclear antigen (PCNA) in the cardiac conduction system of crib death (SIDS).

    PubMed

    Matturri, L; Ottaviani, G; Lavezzi, A M; Turconi, P; Cazzullo, A; Rossi, L

    2001-07-01

    Aim of this study is to determine the expression of apoptosis and Proliferating Cell Nuclear Antigen (PCNA) in the cardiac conduction system in crib death and explained death (ED) cases. Postnatal morphogenesis of the conducting tissue is an important part of its normal development. In the atrio-ventricular node (AVN) and His bundle (HB) it consists of degeneration, cell death and replacing in an orderly programmed way. However, its nature and its relation to crib death is not yet fully explained. Apoptosis and PCNA were investigated in 8 heart conduction systems of infants dying of crib death and in 3 conduction systems of infants dying of ED as controls. The cardiac conduction system was removed in two blocks: the first included the sino-atrial node (SAN) and the crista terminalis, the second contained the atrio-ventricular node (AVN), His bundle (HB), bifurcation, and bundle branches. In the conduction systems as well as in the common myocardium the PCNA Labeling Index (PCNA-LI) was found to be negative in all cases. The apoptotic indices (AI) in SIDS and in ED were found to have no statistically significant differences (p>0.05). The SAN, in both groups, showed an AI similar to the one detected in common myocardium. In almost all cases, TUNEL labeling was detected in peripheral region of the AVN, close to the atrial myocardium. The AI was higher in the AVN, HB and the initial tract of bundle branches than in the common myocardium (p<0.05; Student's t test).

  7. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces death receptor 5 networks that are highly organized.

    PubMed

    Valley, Christopher C; Lewis, Andrew K; Mudaliar, Deepti J; Perlmutter, Jason D; Braun, Anthony R; Karim, Christine B; Thomas, David D; Brody, Jonathan R; Sachs, Jonathan N

    2012-06-15

    Recent evidence suggests that TNF-related apoptosis-inducing ligand (TRAIL), a death-inducing cytokine with anti-tumor potential, initiates apoptosis by re-organizing TRAIL receptors into large clusters, although the structure of these clusters and the mechanism by which they assemble are unknown. Here, we demonstrate that TRAIL receptor 2 (DR5) forms receptor dimers in a ligand-dependent manner at endogenous receptor levels, and these receptor dimers exist within high molecular weight networks. Using mutational analysis, FRET, fluorescence microscopy, synthetic biochemistry, and molecular modeling, we find that receptor dimerization relies upon covalent and noncovalent interactions between membrane-proximal residues. Additionally, by using FRET, we show that the oligomeric structure of two functional isoforms of DR5 is indistinguishable. The resulting model of DR5 activation should revise the accepted architecture of the functioning units of DR5 and the structurally homologous TNF receptor superfamily members.

  8. Farnesol induces apoptosis-like cell death in the pathogenic fungus Aspergillus flavus.

    PubMed

    Wang, Xiaoyun; Wang, Youzhi; Zhou, Yuguang; Wei, Xinli

    2014-01-01

    Farnesol (FOH) is known to induce apoptosis in some fungi and mammalian cells. We treated Aspergillus flavus, one of the leading causes of human invasive aspergillosis and a key producer of the most potent naturally occurring hepatocarcinogenic compounds, with FOH to assess its effect on the viability of the fungus. FOH strongly inhibited germination and growth of A. flavus and induced markers for apoptosis including nuclear condensation, phosphatidylserine (PS) externalization, DNA fragmentation and intracellular reactive oxygen species (ROS) generation, metacaspase activation and abnormal cellular ultrastructure. Moreover, FOH-induced apoptosis in A. flavus was inhibited by the broad-spectrum caspase inhibitor Z-VAD-fmk and partially inhibited by the ROS scavenger l-proline, which suggests that FOH induces apoptosis in A. flavus via a mechanism involving metacaspase activation and ROS production.

  9. The plant alkaloid voacamine induces apoptosis-independent autophagic cell death on both sensitive and multidrug resistant human osteosarcoma cells.

    PubMed

    Meschini, Stefania; Condello, Maria; Calcabrini, Annarica; Marra, Manuela; Formisano, Giuseppe; Lista, Pasquale; De Milito, Angelo; Federici, Elena; Arancia, Giuseppe

    2008-11-01

    In our previous studies, the bisindolic alkaloid voacamine (VOA), isolated from the plant Peschiera fuchsiaefolia, proved to exert a chemosensitizing effect on cultured multidrug resistant (MDR) osteosarcoma cells exposed to doxorubicin (DOX). In particular, VOA was capable of inhibiting P-glycoprotein action in a competitive way, thus explaining the enhancement of the cytotoxic effect induced by DOX on MDR cells. Afterwards, preliminary observations suggested that such an enhancement did not involve the apoptotic process but was due instead to the induction of autophagic cell death. The results of the present investigation demonstrate that the plant alkaloid VOA is an autophagy inducer able to exert apoptosis-independent cytotoxic effect on both wild-type and MDR tumor cells. In fact, under treatment condition causing about 50 percent of cell death, no evidence of apoptosis could be revealed by microscopical observations, Annexin V-FITC labeling and analysis of PARP cleavage, whereas the same cells underwent apoptosis when treated with apoptosis inducers, such as doxorubicin and staurosporine. Conversely, VOA-induced autophagy was clearly evidentiated by electron microscopy observations, monodansylcadaverine staining, LC3 expression, and conversion. These results were confirmed by the analysis of the modulating effects of the pretreatment with autophagy inhibitors prior to VOA administration. In addition, transfection of osteosarcoma cells with siRNA against ATG genes reduced VOA cytotoxicity. In conclusion, considering the very debated dual role of autophagy in cancer cells (protective or lethal, pro- or anti- apoptotic) our findings seem to demonstrate, at least in vitro, that a natural product able to induce autophagy can be effective against drug resistant tumors, either used alone or in association with conventional chemotherapeutics.

  10. The role of mitochondria-mediated intrinsic death pathway in gingerdione derivative I6-induced neuronal apoptosis.

    PubMed

    Lin, Chia-Ho; Chen, Po-See; Kuo, Sheng-Chu; Huang, Li-Jiau; Gean, Po-Wu; Chiu, Ted-H

    2012-03-01

    Neuronal death induced by I6 displayed apoptotic characteristics but the precise mechanism has not been fully elucidated. In the present studies, I6 at 24 h after intraperitoneal administration significantly decreased the density of surviving neurons and increased caspase-3 activity in frontal cortex, suggesting that peripherally administered I6 may cross BBB to induce CNS toxicity. In rat embryonic primary cortical cells, I6-induced reduction of mitochondrial viability and neuronal apoptosis was inhibited by vitamin E. In addition, I6-induced reactive oxygen species (ROS) caused the disruption of mitochondria membrane potential (MMP), the release of cytochrome c, the activation of caspase-9 and caspase-3, and cleavage of poly(ADP-ribose) polymerase (PARP), resulting in activation of mitochondrial-mediated intrinsic death pathway. Pre-treatment with antioxidant vitamin E or N-acetylcysteine (NAC) completely abolished the I6-induced generation of ROS, loss of MMP, release of cytochrome c, activation of caspase-9 and caspase-3, and cleavage of PARP. Carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), a mitochondrial uncoupler, significantly reduced I6-induced neuronal death as well as caspase-3 activation and PARP cleavage. These results suggest that I6 induces neuronal death by promoting intracellular ROS production to cause a loss of MMP that result in release of cytochrome c and activation of mitochondria-mediated intrinsic death pathway.

  11. Kaempferol induces ATM/p53-mediated death receptor and mitochondrial apoptosis in human umbilical vein endothelial cells.

    PubMed

    Lee, Chiu-Fang; Yang, Jai-Sing; Tsai, Fuu-Jen; Chiang, Ni-Na; Lu, Chi-Cheng; Huang, Yu-Syuan; Chen, Chun; Chen, Fu-An

    2016-05-01

    Kaempferol is a member of the flavonoid compounds found in vegetables and fruits. It is shown to exhibit biological impact and anticancer activity, but no report exists on the angiogenic effect of kaempferol and induction of cell apoptosis in vitro. In this study, we investigated the role of kaempferol on anti-angiogenic property and the apoptotic mechanism of human umbilical vein endothelial cells (HUVECs). Our results demonstrated that kaempferol decreased HUVEC viability in a time- and concentration-dependent manner. Kaempferol also induced morphological changes and sub-G1 phase cell population (apoptotic cells). Kaempferol triggered apoptosis of HUVECs as detecting by DNA fragmentation, comet assay and immunofluorescent staining for activated caspase-3. The caspase signals, including caspase-8, -9 and -3, were time-dependently activated in HUVECs after kaempferol exposure. Furthermore, pre-treatment with a specific inhibitor of caspase-8 (Z-IETD-FMK) significantly reduced the activity of caspase-8, -9 and -3, indicating that extrinsic pathway is a major signaling pathway in kaempferol-treated HUVECs. Importantly, kaempferol promoted reactive oxygen species (ROS) evaluated using flow cytometric assay in HUVECs. We further investigated the upstream extrinsic pathway and showed that kaempferol stimulated death receptor signals [Fas/CD95, death receptor 4 (DR4) and DR5] through increasing the levels of phosphorylated p53 and phosphorylated ATM pathways in HUVECs, which can be individually confirmed by N-acetylcysteine (NAC), ATM specific inhibitor (caffeine) and p53 siRNA. Based on these results, kaempferol-induced HUVEC apoptosis was involved in an ROS-mediated p53/ATM/death receptor signaling. Kaempferol might possess therapeutic effects on cancer treatment in anti-vascular targeting.

  12. Apoptosis and tumor cell death in response to HAMLET (human alpha-lactalbumin made lethal to tumor cells).

    PubMed

    Hallgren, Oskar; Aits, Sonja; Brest, Patrick; Gustafsson, Lotta; Mossberg, Ann-Kristin; Wullt, Björn; Svanborg, Catharina

    2008-01-01

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a molecular complex derived from human milk that kills tumor cells by a process resembling programmed cell death. The complex consists of partially unfolded alpha-lactalbumin and oleic acid, and both the protein and the fatty acid are required for cell death. HAMLET has broad antitumor activity in vitro, and its therapeutic effect has been confirmed in vivo in a human glioblastoma rat xenograft model, in patients with skin papillomas and in patients with bladder cancer. The mechanisms of tumor cell death remain unclear, however. Immediately after the encounter with tumor cells, HAMLET invades the cells and causes mitochondrial membrane depolarization, cytochrome c release, phosphatidyl serine exposure, and a low caspase response. A fraction of the cells undergoes morphological changes characteristic of apoptosis, but caspase inhibition does not rescue the cells and Bcl-2 overexpression or altered p53 status does not influence the sensitivity of tumor cells to HAMLET. HAMLET also creates a state of unfolded protein overload and activates 20S proteasomes, which contributes to cell death. In parallel, HAMLET translocates to tumor cell nuclei, where high-affinity interactions with histones cause chromatin disruption, loss of transcription, and nuclear condensation. The dying cells also show morphological changes compatible with macroautophagy, and recent studies indicate that macroautophagy is involved in the cell death response to HAMLET. The results suggest that HAMLET, like a hydra with many heads, may interact with several crucial cellular organelles, thereby activating several forms of cell death, in parallel. This complexity might underlie the rapid death response of tumor cells and the broad antitumor activity of HAMLET.

  13. Effect of vitamin E on 24(S)-hydroxycholesterol-induced necroptosis-like cell death and apoptosis.

    PubMed

    Nakazawa, Takaya; Miyanoki, Yuta; Urano, Yasuomi; Uehara, Madoka; Saito, Yoshiro; Noguchi, Noriko

    2016-03-04

    24(S)-Hydroxycholesterol (24S-OHC) has diverse physiological and pathological functions. In particular, cytotoxic effects of 24S-OHC in neuronal cells are important in development of neurodegenerative diseases. 24S-OHC induces necroptosis-like cell death in SH-SY5Y cells expressing little caspase-8. In the present study, 24S-OHC was found to induce apoptosis as determined by caspase-3 activation in all-trans-retinoic acid (atRA)-treated SH-SY5Y cells in which expression of caspase-8 was induced. 24S-OHC-induced cell death was inhibited by α-tocopherol (α-Toc) but not by α-tocotrienol (α-Toc3) in SH-SY5Y cells regardless of whether cells were treated with atRA. In contrast, cumene hydroperoxide (CumOOH)-induced cell death was significantly inhibited by α-Toc and α-Toc3. In atRA-treated SH-SY5Y cells, generation of reactive oxygen species (ROS) was induced by stimulation with CumOOH but was not induced by stimulation with 24S-OHC. These results suggest that inhibition of 24S-OHC-induced cell death by α-Toc cannot be explained by its radical scavenging antioxidant activity. Esterification of 24S-OHC followed by lipid droplet (LD) formation due to acyl-CoA:cholesterol acyltransferase 1 (ACAT1) are key events in 24S-OHC-induced cell death in atRA-treated SH-SY5Y cells as demonstrated by inhibition of cell death by ACAT1 inhibitor. LD number was not changed by treatment with either α-Toc or α-Toc3. The different physical properties of α-Toc and α-Toc3 may account for their different inhibitory effects on 24S-OHC-induced cell death.

  14. Receptor-Mediated Drug Delivery Systems Targeting to Glioma

    PubMed Central

    Wang, Shanshan; Meng, Ying; Li, Chengyi; Qian, Min; Huang, Rongqin

    2015-01-01

    Glioma has been considered to be the most frequent primary tumor within the central nervous system (CNS). The complexity of glioma, especially the existence of the blood-brain barrier (BBB), makes the survival and prognosis of glioma remain poor even after a standard treatment based on surgery, radiotherapy, and chemotherapy. This provides a rationale for the development of some novel therapeutic strategies. Among them, receptor-mediated drug delivery is a specific pattern taking advantage of differential expression of receptors between tumors and normal tissues. The strategy can actively transport drugs, such as small molecular drugs, gene medicines, and therapeutic proteins to glioma while minimizing adverse reactions. This review will summarize recent progress on receptor-mediated drug delivery systems targeting to glioma, and conclude the challenges and prospects of receptor-mediated glioma-targeted therapy for future applications.

  15. Unique spectral signatures of the nucleic acid dye acridine orange can distinguish cell death by apoptosis and necroptosis.

    PubMed

    Plemel, Jason R; Caprariello, Andrew V; Keough, Michael B; Henry, Tyler J; Tsutsui, Shigeki; Chu, Tak H; Schenk, Geert J; Klaver, Roel; Yong, V Wee; Stys, Peter K

    2017-03-06

    Cellular injury and death are ubiquitous features of disease, yet tools to detect them are limited and insensitive to subtle pathological changes. Acridine orange (AO), a nucleic acid dye with unique spectral properties, enables real-time measurement of RNA and DNA as proxies for cell viability during exposure to various noxious stimuli. This tool illuminates spectral signatures unique to various modes of cell death, such as cells undergoing apoptosis versus necrosis/necroptosis. This new approach also shows that cellular RNA decreases during necrotic, necroptotic, and apoptotic cell death caused by demyelinating, ischemic, and traumatic injuries, implying its involvement in a wide spectrum of tissue pathologies. Furthermore, cells with pathologically low levels of cytoplasmic RNA are detected earlier and in higher numbers than with standard markers including TdT-mediated dUTP biotin nick-end labeling and cleaved caspase 3 immunofluorescence. Our technique highlights AO-labeled cytoplasmic RNA as an important early marker of cellular injury and a sensitive indicator of various modes of cell death in a range of experimental models.

  16. Crocetin exploits p53-induced death domain (PIDD) and FAS-associated death domain (FADD) proteins to induce apoptosis in colorectal cancer

    PubMed Central

    Ray, Pallab; Guha, Deblina; Chakraborty, Juni; Banerjee, Shuvomoy; Adhikary, Arghya; Chakraborty, Samik; Das, Tanya; Sa, Gaurisankar

    2016-01-01

    Tumor suppressor p53 preserves the genomic integrity by restricting anomaly at the gene level. The hotspots for mutation in half of all colon cancers reside in p53. Hence, in a p53-mutated cellular milieu targeting cancer cells may be achievable by targeting the paralogue(s) of p53. Here we have shown the effectiveness of crocetin, a dietary component, in inducing apoptosis of colon cancer cells with varying p53 status. In wild-type p53-expressing cancer cells, p53 in one hand transactivates BAX and in parallel up-regulates p53-induced death domain protein (PIDD) that in turn cleaves and activates BID through caspase-2. Both BAX and t-BID converge at mitochondria to alter the transmembrane potential thereby leading to caspase-9 and caspase-3-mediated apoptosis. In contrast, in functional p53-impaired cells, this phytochemical exploits p53-paralogue p73, which up-regulates FAS to cleave BID through FAS-FADD-caspase-8-pathway. These findings not only underline the phenomenon of functional switch-over from p53 to p73 in p53-impaired condition, but also validate p73 as a promising and potential target for cancer therapy in absence of functional p53. PMID:27622714

  17. Blockade of the tumor necrosis factor-related apoptosis inducing ligand death receptor DR5 prevents beta-amyloid neurotoxicity.

    PubMed

    Uberti, Daniela; Ferrari-Toninelli, Giulia; Bonini, Sara Anna; Sarnico, Ilenia; Benarese, Marina; Pizzi, Marina; Benussi, Luisa; Ghidoni, Roberta; Binetti, Giuliano; Spano, PierFranco; Facchetti, Fabio; Memo, Maurizio

    2007-04-01

    We originally suggested that inhibition of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) death pathway could be taken into consideration as a potential therapeutic strategy for Alzheimer's disease (AD). However, because the critical role of TRAIL in immune surveillance, the neutralization of TRAIL protein by an antibody to prevent its binding to death receptors is definitely a risky approach. Here, we demonstrated that the blockade of the TRAIL death receptor DR5 with a specific antibody completely prevented amyloid beta peptide (A beta) neurotoxicity in both neuronal cell line and primary cortical neurons. DR5 was demonstrated to be a key factor in TRAIL death pathway. In fact, whereas TRAIL expression was enhanced dose-dependently by concentrations of beta amyloid ranging from 10 nM to 1 microM, only the highest toxic dose of A beta (25 microM) induced the increased expression of DR5 and neuronal cell death. In addition, the increased expression of DR5 receptor after beta amyloid treatment was sustained by p53 transcriptional activity, as demonstrated by the data showing that the p53 inhibitor Pifithrin alpha prevented both beta amyloid-induced DR5 induction and cell death. These data suggest a sequential activation of p53 and DR5 upon beta amyloid exposure. Further insight into the key role of DR5 in AD was suggested by data showing a significant increase of DR5 receptor in cortical slices of AD brain. Thus, these findings may give intracellular TRAIL pathway a role in AD pathophysiology, making DR5 receptor a possible candidate as a pharmacological target.

  18. Cypermethrin Induces Macrophages Death through Cell Cycle Arrest and Oxidative Stress-Mediated JNK/ERK Signaling Regulated Apoptosis

    PubMed Central

    Huang, Fang; Liu, Qiaoyun; Xie, Shujun; Xu, Jian; Huang, Bo; Wu, Yihua; Xia, Dajing

    2016-01-01

    Cypermethrin is one of the most highly effective synthetic pyrethroid insecticides. The toxicity of cypermethrin to the reproductive and nervous systems has been well studied. However, little is known about the toxic effect of cypermethrin on immune cells such as macrophages. Here, we investigated the cytotoxicity of cypermethrin on macrophages and the underlying molecular mechanisms. We found that cypermethrin reduced cell viability and induced apoptosis in RAW 264.7 cells. Cypermethrin also increased reactive oxygen species (ROS) production and DNA damage in a dose-dependent manner. Moreover, cypermethrin-induced G1 cell cycle arrest was associated with an enhanced expression of p21, wild-type p53, and down-regulation of cyclin D1, cyclin E and CDK4. In addition, cypermethrin treatment activated MAPK signal pathways by inducing c-Jun N-terminal kinase (JNK) and extracellular regulated protein kinases 1/2 ERK1/2 phosphorylation, and increased the cleaved poly ADP-ribose polymerase (PARP). Further, pretreatment with antioxidant N-acetylcysteine (NAC) effectively abrogated cypermethrin-induced cell cytotoxicity, G1 cell cycle arrest, DNA damage, PARP activity, and JNK and ERK1/2 activation. The specific JNK inhibitor (SP600125) and ERK1/2 inhibitor (PD98059) effectively reversed the phosphorylation level of JNK and ERK1/2, and attenuated the apoptosis. Taken together, these data suggested that cypermethrin caused immune cell death via inducing cell cycle arrest and apoptosis regulated by ROS-mediated JNK/ERK pathway. PMID:27322250

  19. Cucurbitacin E as Inducer of Cell Death and Apoptosis in Human Oral Squamous Cell Carcinoma Cell Line SAS

    PubMed Central

    Hung, Chao-Ming; Chang, Chi-Chang; Lin, Chen-Wei; Ko, Shun-Yao; Hsu, Yi-Chiang

    2013-01-01

    Human oral squamous cell carcinoma (OSCC) is a common form of malignant cancer, for which radiotherapy or chemotherapy are the main treatment methods. Cucurbitacin E (CuE) is a natural compound previously shown to be an antifeedant as well as a potent chemopreventive agent against several types of cancer. The present study investigates anti-proliferation (using MTT assay, CuE demonstrated cytotoxic activity against SAS cell with IC50 values at 3.69 μM) and induced apoptosis of human oral squamous cell carcinoma SAS cells after 24 h treatment with CuE. Mitochondrial membrane potential (MMP) and caspase activity were studied and our results indicate that CuE inhibits cell proliferation as well as the activation of apoptois in SAS cells. Both effects increased in proportion to the dosage of CuE and apoptosis was induced via mitochondria- and caspase-dependent pathways. CuE can induce cell death by a mechanism that is not dependent on apoptosis induction, and thus represents a promising anticancer agent for prevention and treatment of OSCC. PMID:23965977

  20. Cucurbitacin E as inducer of cell death and apoptosis in human oral squamous cell carcinoma cell line SAS.

    PubMed

    Hung, Chao-Ming; Chang, Chi-Chang; Lin, Chen-Wei; Ko, Shun-Yao; Hsu, Yi-Chiang

    2013-08-20

    Human oral squamous cell carcinoma (OSCC) is a common form of malignant cancer, for which radiotherapy or chemotherapy are the main treatment methods. Cucurbitacin E (CuE) is a natural compound previously shown to be an antifeedant as well as a potent chemopreventive agent against several types of cancer. The present study investigates anti-proliferation (using MTT assay, CuE demonstrated cytotoxic activity against SAS cell with IC50 values at 3.69 µM) and induced apoptosis of human oral squamous cell carcinoma SAS cells after 24 h treatment with CuE. Mitochondrial membrane potential (MMP) and caspase activity were studied and our results indicate that CuE inhibits cell proliferation as well as the activation of apoptois in SAS cells. Both effects increased in proportion to the dosage of CuE and apoptosis was induced via mitochondria- and caspase-dependent pathways. CuE can induce cell death by a mechanism that is not dependent on apoptosis induction, and thus represents a promising anticancer agent for prevention and treatment of OSCC.

  1. D-cyclins Repress Apoptosis in Hematopoietic Cells by Controlling Death Receptor Fas and its Ligand FasL

    PubMed Central

    Choi, Yoon Jong; Saez, Borja; Anders, Lars; Hydbring, Per; Stefano, Joanna; Bacon, Nickolas A.; Cook, Colleen; Kalaszczynska, Ilona; Signoretti, Sabina; Young, Richard A.; Scadden, David T.; Sicinski, Piotr

    2014-01-01

    SUMMARY D-type cyclins (D1, D2 and D3) are components of the mammalian core cell cycle machinery and function to drive cell proliferation. Here we report that D-cyclins perform a rate-limiting anti-apoptotic function in vivo. We found that acute shutdown of all three D-cyclins in bone marrow of adult mice resulted in massive apoptosis of all hematopoietic cell types. We demonstrate that adult hematopoietic stem cells are particularly dependent on D-cyclins for survival, and they are especially sensitive to cyclin D loss. Surprisingly, we found that the anti-apoptotic function of D-cyclins also operates in quiescent hematopoietic stem and progenitor cells. Our analyses revealed that D-cyclins repress the expression of the death receptor Fas and its ligand, FasL. Acute ablation of D-cyclins upregulated these pro-apoptotic genes, and led to Fas- and caspase 8-dependent apoptosis. These results reveal an unexpected function of cell cycle proteins in controlling apoptosis in normal cell homeostasis. PMID:25087893

  2. Bigelovin triggered apoptosis in colorectal cancer in vitro and in vivo via upregulating death receptor 5 and reactive oxidative species

    PubMed Central

    Li, Mingyue; Song, Li-Hua; Yue, Grace Gar-Lee; Lee, Julia Kin-Ming; Zhao, Li-Mei; Li, Lin; Zhou, Xunian; Tsui, Stephen Kwok-Wing; Ng, Simon Siu-Man; Fung, Kwok-Pui; Tan, Ning-Hua; Lau, Clara Bik-San

    2017-01-01

    Colorectal cancer (CRC) is the third most prevalent cancer and the third highest cancer-related mortality in the United States. Bigelovin, a sesquiterpene lactone isolated from Inula helianthus aquatica, has been proven to induce apoptosis and exhibit anti-inflammatory and anti-angiogenic activities. However, the effects of bigelovin on CRC and underlying mechanisms have not been explored. The present study demonstrated that bigelovin exhibited potent anti-tumor activities against CRC in vitro and in vivo. Bigelovin suppressed cell proliferation and colony formation and induced apoptosis in human colorectal cancer HT-29 and HCT 116 cells in vitro. Results also revealed that bigelovin activated caspases, caused the G2/M cell cycle arrest and induced DNA damage through up-regulation of death receptor (DR) 5 and increase of ROS. In HCT 116 xenograft model, bigelovin treatment resulted in suppression of tumor growth. Bigelovin at 20 mg/kg showed more significant tumor suppression and less side effects than conventional FOLFOX (containing folinic acid, 5-fluorouracil and oxaliplatin) treatment. In addition, in vivo data confirmed that anti-tumor activity of bigelovin in CRC was through induction of apoptosis by up-regulating DR5 and increasing ROS. In conclusion, these results strongly suggested that bigelovin has potential to be developed as therapeutic agent for CRC patients. PMID:28181527

  3. miR-613 suppresses ischemia-reperfusion-induced cardiomyocyte apoptosis by targeting the programmed cell death 10 gene.

    PubMed

    Wu, Zhenhua; Qi, Yujuan; Guo, Zhigang; Li, Peijun; Zhou, Ding

    2016-09-05

    MicroRNAs (miRNAs) are important gene regulators in both biological and pathological processes, including myocardial ischemia/reperfusion (I/R) injury. This study investigated the effect of miR-613 on I/R-induced cardiomyocyte apoptosis and its molecular mechanism of action. Hypoxia/reoxygenation (H/R) significantly increased the release of lactate dehydrogenase (LDH), levels of malondialdehyde (MDA), and cardiomyocyte apoptosis, but these effects were attenuated by an miR-613 mimic. Programmed cell death 10 (PDCD10) was identified as a target gene of miR-613. miR-613 significantly increased the phosphorylation of Akt (p-Akt). An miR-613 mimic lowered the level of expression of pro-apoptotic proteins, C/EBP homologous protein (CHOP), and phosphorylated c-Jun N-terminal kinase (p-JNK), and it up-regulated the expression of the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2). All of these effects were reversed by restoration of PDCD10. Taken together, the current findings indicate that miR-613 inhibits I/R-induced cardiomyocyte apoptosis by targeting PDCD10 by regulating the PI3K/AKT signaling pathway.

  4. Melatonin ameliorates oxidative stress, modulates death receptor pathway proteins, and protects the rat cerebrum against bisphenol-A-induced apoptosis.

    PubMed

    El-Missiry, Mohamed A; Othman, Azza I; Al-Abdan, Monera A; El-Sayed, Aml A

    2014-12-15

    Epidemiological reports have indicated a correlation between the increasing of bisphenol-A (BPA) levels in the environment and the incidence of neurodegenerative diseases. In the present study, the protective effect of melatonin on oxidative stress and the death receptor apoptotic proteins in the cerebrum of the bisphenol-A-treated rats were examined. Adult male rats were orally administered melatonin (10mg/kg bw) concurrently with BPA (50mg/kg bw) 3 days a week for 6 weeks. BPA exposure resulted in significant elevations of oxidative stress, as evidenced by the increased malondialdehyde level and the decreased glutathione level and superoxide dismutase activity in the cerebrum. BPA caused an upregulation of p53 and CD95-Fas and activation of capsases-3 and 8, resulting in cerebral cell apoptosis. Melatonin significantly attenuated the BPA-evoked brain oxidative stress, modulated apoptotic-regulating proteins and protected against apoptosis. These data suggest that melatonin modulated important steps in the death receptor apoptotic pathway which likely related to its redox control properties. Melatonin is a promising pharmacological agent for preventing the potential neurotoxicity of BPA following occupational or environmental exposures.

  5. IGF-I maintains calpastatin expression and attenuates apoptosis in several models of photoreceptor cell death.

    PubMed

    Arroba, Ana I; Wallace, Deborah; Mackey, Ashley; de la Rosa, Enrique J; Cotter, Thomas G

    2009-09-01

    Retinitis pigmentosa is a heterogeneous group of inherited retinal dystrophies in which the loss of photoreceptor cells via apoptosis leads to blindness. In this study we have experimentally mimicked this condition by treating 661W cells and wild-type mouse retinal explants with a Ca(2+) ionophore. Ca(2+) overload induced apoptosis, which was correlated with calpain-2 activation, loss of calpastatin, its endogenous inhibitor, as well as the loss of its transcriptional activator, phospho-cAMP response element binding (CREB). All are similar changes to those observed in the rd1 mouse model of retinitis pigmentosa. Insulin like-growth factor-I (IGF-I) attenuated this Ca(2+)-induced apoptosis, as well as decreased the activation of calpain-2 and maintained calpastatin levels through the activation of the Akt-CREB pathway. Similarly, IGF-I decreased photoreceptor apoptosis in rd1 mouse retinal explants in parallel with reduced activation of calpain-2 and increased levels of calpastatin and activation of phospho-CREB. In conclusion, IGF-I seems to protect neural cells following a physiopathological or an experimental increase in intracellular Ca(2+), an observation that may have therapeutic consequences in neurodegenerative diseases such as retinitis pigmentosa.

  6. Analysis of a mathematical model of apoptosis: individual differences and malfunction in programmed cell death.

    PubMed

    Bagci, Elife Zerrin; Sen, S Murat; Camurdan, Mehmet C

    2013-08-01

    Apoptosis is an important area of research because of its role in keeping a mature multicellular organism's number of cells constant, hence, ensuring that the organism does not have cell accumulation that may transform into cancer with additional hallmarks. Firstly, we have carried out sensitivity analysis on an existing mathematical mitochondria-dependent apoptosis model to find out which parameters have a role in causing monostable cell survival, which may, in turn, lead to malfunction in apoptosis. We have then generated three base parameter sets that represent healthy cells. These parameter sets were built by changing the sensitive parameters while preserving the bistability. For each base set, we varied the proapoptotic and antiapoptotic production rates, to yield new sets for the cells that have malfunctioning apoptosis. In a hypothetical cell model, we simulated caspase-3 activation by numerically integrating the governing ordinary differential equations of a mitochondria-dependent apoptosis model. These simulations were carried out for four potential treatments, namely: (1) proteasome inhibitor treatment, (2) Bcl-2 inhibitor treatment, (3) IAP inhibitor treatment, (4) Bid-like synthetic peptides treatment. The results suggest that the proteasome inhibitor treatment is the most effective treatment, though it may have severe side effects. For this treatment, the amount of proteasome inhibitor needed for caspase-3 activation may be different for hypothetical cells with a different pro- or anti-apoptotic protein defect. It is also found that caspase-3 can be activated by Bcl-2 inhibitor treatment only in those hypothetical malfunctioning cells with Bax deficiency but not in others. These results are in line with the view that molecular heterogeneity in individuals may be an important factor in determining the individuals' positive or negative responses to treatments.

  7. Endothelial apoptosis in pulmonary hypertension is controlled by a microRNA/programmed cell death 4/caspase-3 axis.

    PubMed

    White, Kevin; Dempsie, Yvonne; Caruso, Paola; Wallace, Emma; McDonald, Robert A; Stevens, Hannah; Hatley, Mark E; Van Rooij, Eva; Morrell, Nicholas W; MacLean, Margaret R; Baker, Andrew H

    2014-07-01

    Pulmonary endothelial cell apoptosis is a transient, yet defining pathogenic event integral to the onset of many pulmonary vascular diseases such as pulmonary hypertension (PH). However, there is a paucity of information concerning the molecular pathway(s) that control pulmonary arterial endothelial cell apoptosis. Here, we introduce a molecular axis that when functionally active seems to induce pulmonary arterial endothelial cell apoptosis in vitro and PH in vivo. In response to apoptotic stimuli, human pulmonary arterial endothelial cells exhibited robust induction of a programmed cell death 4 (PDCD4)/caspase-3/apoptotic pathway that was reversible by direct PDCD4 silencing. Indirectly, this pathway was also repressed by delivery of a microRNA-21 mimic. In vivo, genetic deletion of microRNA-21 in mice (miR-21(-/-) mice) resulted in functional activation of the PDCD4/caspase-3 axis in the pulmonary tissues, leading to the onset of progressive PH. Conversely, microRNA-21-overexpressing mice (CAG-microRNA-21 mice) exhibited reduced PDCD4 expression in pulmonary tissues and were partially resistant to PH in response to chronic hypoxia plus SU 5416 injury. Furthermore, direct PDCD4 knockout in mice (PDCD4(-/-) mice) potently blocked pulmonary caspase-3 activation and the development of chronic hypoxia plus SU 5416 PH, confirming its importance in disease onset. Broadly, these findings support the existence of a microRNA-21-responsive PDCD4/caspase-3 pathway in the pulmonary tissues that when active serves to promote endothelial apoptosis in vitro and PH in vivo.

  8. Death associated protein 1 (DAP 1) positively regulates virus replication and apoptosis of hemocytes in shrimp Marsupenaeus japonicus.

    PubMed

    Xia, Wen-Li; Kang, Li-Hua; Liu, Chang-Bin; Kang, Cui-Jie

    2017-04-01

    Death-associated protein 1 (DAP1) is a small proline-rich cytoplasmic protein that functions both in the apoptosis and autophage process of mammalian and in the clinical cancer of human. However, little knowledge is known about the homologue gene of DAP1 and its roles in the physiological process of invertebrates. In this paper, we report a novel function of DAP1 in the antivirus immunity of shrimp. A homologue gene of DAP1 was cloned from Marsupenaeus japonicus and named as Mjdap-1. The full-length of Mjdap-1 was 1761 bp with a 309 bp open reading frame that encoded 102 amino acids. Reverse transcription-PCR results showed that Mjdap-1 was expressed in all tested tissues, including hemocytes, gills, intestines, stomach, heart, hepatopancreas, testes, and ovaries. In shrimp, Mjdap-1 transcripts were up-regulated by white spot syndrome virus (WSSV) infection; Mjdap-1 knockdown decreased the virus copy in vivo and the mortality of M. japonicus to WSSV challenge. Conversely, injecting the purified recombinant MjDAP1 protein promoted the amplification of virus in shrimp. Flow cytometric assay showed, the virus infection-induced apoptosis of hemocytes was enhanced by MjDAP1 protein injection and inhibited in MjDAP1 knockdown shrimp. Furthermore, the expression of apoptosis-inducing factor (AIF) was regulated by Mjdap-1, but the caspase transcripts were not affected. Our results suggested that MjDAP1 facilitated the amplification of virus in shrimp, which may be attributed to the promotion of hemocyte apoptosis in an AIF-dependent manner. These results provided a new insight into the function of this protein that may be used for virus disease control.

  9. Apoptosis goes on a chip: advances in the microfluidic analysis of programmed cell death

    PubMed Central

    Wlodkowic, Donald; Khoshmanesh, Khashayar; Sharpe, John C; Darzynkiewicz, Zbigniew; Cooper, Jonathan Mark

    2011-01-01

    Summary Recent years have brought enormous progress in cell-based lab-on-a-chip technologies, allowing dynamic studies of cell death with an unprecedented accuracy. As interest in the microfabricated technologies for cell-based bioassays is rapidly gaining momentum, we highlight the most promising technologies that provide a new outlook for the rapid assessment of programmed and accidental cell death and are applicable in drug discovery, high-content drug screening, and personalized clinical diagnostics. PMID:21630641

  10. Cell death mechanisms of plant-derived anticancer drugs: beyond apoptosis.

    PubMed

    Gali-Muhtasib, Hala; Hmadi, Raed; Kareh, Mike; Tohme, Rita; Darwiche, Nadine

    2015-12-01

    Despite remarkable progress in the discovery and development of novel cancer therapeutics, cancer remains the second leading cause of death in the world. For many years, compounds derived from plants have been at the forefront as an important source of anticancer therapies and have played a vital role in the prevention and treatment of cancer because of their availability, and relatively low toxicity when compared with chemotherapy. More than 3000 plant species have been reported to treat cancer and about thirty plant-derived compounds have been isolated so far and have been tested in cancer clinical trials. The mechanisms of action of plant-derived anticancer drugs are numerous and most of them induce apoptotic cell death that may be intrinsic or extrinsic, and caspase and/or p53-dependent or independent mechanisms. Alternative modes of cell death by plant-derived anticancer drugs are emerging and include mainly autophagy, necrosis-like programmed cell death, mitotic catastrophe, and senescence leading to cell death. Considering that the non-apoptotic cell death mechanisms of plant-derived anticancer drugs are less reviewed than the apoptotic ones, this paper attempts to focus on such alternative cell death pathways for some representative anticancer plant natural compounds in clinical development. In particular, emphasis will be on some promising polyphenolics such as resveratrol, curcumin, and genistein; alkaloids namely berberine, noscapine, and colchicine; terpenoids such as parthenolide, triptolide, and betulinic acid; and the organosulfur compound sulforaphane. The understanding of non-apoptotic cell death mechanisms induced by these drugs would provide insights into the possibility of exploiting novel molecular pathways and targets of plant-derived compounds for future cancer therapeutics.

  11. The Janus Face of Death Receptor Signaling during Tumor Immunoediting

    PubMed Central

    O’ Reilly, Eimear; Tirincsi, Andrea; Logue, Susan E.; Szegezdi, Eva

    2016-01-01

    Cancer immune surveillance is essential for the inhibition of carcinogenesis. Malignantly transformed cells can be recognized by both the innate and adaptive immune systems through different mechanisms. Immune effector cells induce extrinsic cell death in the identified tumor cells by expressing death ligand cytokines of the tumor necrosis factor ligand family. However, some tumor cells can escape immune elimination and progress. Acquisition of resistance to the death ligand-induced apoptotic pathway can be obtained through cleavage of effector cell expressed death ligands into a poorly active form, mutations or silencing of the death receptors, or overexpression of decoy receptors and pro-survival proteins. Although the immune system is highly effective in the elimination of malignantly transformed cells, abnormal/dysfunctional death ligand signaling curbs its cytotoxicity. Moreover, DRs can also transmit pro-survival and pro-migratory signals. Consequently, dysfunctional death receptor-mediated apoptosis/necroptosis signaling does not only give a passive resistance against cell death but actively drives tumor cell motility, invasion, and contributes to consequent metastasis. This dual contribution of the death receptor signaling in both the early, elimination phase, and then in the late, escape phase of the tumor immunoediting process is discussed in this review. Death receptor agonists still hold potential for cancer therapy since they can execute the tumor-eliminating immune effector function even in the absence of activation of the immune system against the tumor. The opportunities and challenges of developing death receptor agonists into effective cancer therapeutics are also discussed. PMID:27843441

  12. Death receptor and mitochondria-mediated hepatocyte apoptosis underlies liver dysfunction in rats exposed to organic pollutants from drinking water.

    PubMed

    Yang, Guanghong; Zhou, Zhiwei; Cen, Yanli; Gui, Xiaolin; Zeng, Qibing; Ao, Yunxia; Li, Qian; Wang, Shiran; Li, Jun; Zhang, Aihua

    2015-01-01

    Persistent organic pollutants in drinking water impose a substantial risk to the health of human beings, but the evidence for liver toxic effect and the underlying mechanism is scarce. This study aimed to examine the liver toxicity and elucidate the molecular mechanism of organic pollutants in drinking water in normal human liver cell line L02 cells and rats. The data showed that organic extraction from drinking water remarkably impaired rat liver function, evident from the increase in the serum level of alanine aminotransferase, aspartate aminotransferase, and cholinesterase, and decrease in the serum level of total protein and albumin. Organic extraction dose-dependently induced apoptotic cell death in rat liver and L02 cells. Administration of rats with organic extraction promoted death receptor signaling pathway through the increase in gene and protein expression level of Fas and FasL. Treatment of rats with organic extraction also induced mitochondria-mediated apoptosis via increasing the expression level of proapoptotic protein, Bax, but decreasing the expression level of antiapoptotic protein, Bcl-2, resulting in an upregulation of cytochrome c and activation of caspase cascade at both transcriptional and post-transcriptional levels. Moreover, organic extraction enhanced rat liver glutathione S-transferases activity and reactive oxygen species generation, and upregulated aryl hydrocarbon receptor and glutathione S-transferase A1 at both transcriptional and translational levels. Collectively, the results indicate that organic extraction from drinking water impairs liver function, with the involvement of death receptor and mitochondria-mediated apoptosis in rats. The results provide evidence and molecular mechanisms for organic pollutants in drinking water-induced liver dysfunction, which may help prevent and treat organic extraction-induced liver injury.

  13. Effects of a novel β-lapachone derivative on Trypanosoma cruzi: Parasite death involving apoptosis, autophagy and necrosis.

    PubMed

    Dos Anjos, Danielle Oliveira; Sobral Alves, Eliomara Sousa; Gonçalves, Vinicius Tomaz; Fontes, Sheila Suarez; Nogueira, Mateus Lima; Suarez-Fontes, Ana Márcia; Neves da Costa, João Batista; Rios-Santos, Fabricio; Vannier-Santos, Marcos André

    2016-12-01

    Natural products comprise valuable sources for new antiparasitic drugs. Here we tested the effects of a novel β-lapachone derivative on Trypanosoma cruzi parasite survival and proliferation and used microscopy and cytometry techniques to approach the mechanism(s) underlying parasite death. The selectivity index determination indicate that the compound trypanocidal activity was over ten-fold more cytotoxic to epimastigotes than to macrophages or splenocytes. Scanning electron microscopy analysis revealed that the R72 β-lapachone derivative affected the T. cruzi morphology and surface topography. General plasma membrane waving and blebbing particularly on the cytostome region were observed in the R72-treated parasites. Transmission electron microscopy observations confirmed the surface damage at the cytostome opening vicinity. We also observed ultrastructural evidence of the autophagic mechanism termed macroautophagy. Some of the autophagosomes involved large portions of the parasite cytoplasm and their fusion/confluence may lead to necrotic parasite death. The remarkably enhanced frequency of autophagy triggering was confirmed by quantitating monodansylcadaverine labeling. Some cells displayed evidence of chromatin pycnosis and nuclear fragmentation were detected. This latter phenomenon was also indicated by DAPI staining of R72-treated cells. The apoptotis induction was suggested to take place in circa one-third of the parasites assessed by annexin V labeling measured by flow cytometry. TUNEL staining corroborated the apoptosis induction. Propidium iodide labeling indicate that at least 10% of the R72-treated parasites suffered necrosis within 24 h. The present data indicate that the β-lapachone derivative R72 selectively triggers T. cruzi cell death, involving both apoptosis and autophagy-induced necrosis.

  14. Selection of Aptamers for CED-9/Bcl-2 Family Cell Death Regulations and Their Application in Study of Apoptosis Regulation and Drug Design for Breast Cancer

    DTIC Science & Technology

    2005-07-01

    Horvitz, H. R. (1992) Nature 356, 494-499. 18 12. Hengartner , M. 0. & Horvitz, H. R. (1994) Cell 76, 665-676. 13. Finnegan, E. J. & Matzke, M. A. (2003... Apoptosis Regulation and Drug Design for Breast Cancer PRINCIPAL INVESTIGATOR: Ding Xue, Ph.D. Chonglin Yang, Ph.D. Nathan Camp CONTRACTING ORGANIZATION...Aptamers for CED-9/Bcl-2 Family Cell Death Regulations and Their Application in Study of Apoptosis Regulation and Drug Design for Breast Cancer 5b. GRANT

  15. YOPRO-1 permits cytofluorometric analysis of programmed cell death (apoptosis) without interfering with cell viability.

    PubMed

    Idziorek, T; Estaquier, J; De Bels, F; Ameisen, J C

    1995-09-25

    In the absence of cell permeabilization, the impermeant nuclear dye YOPRO-1 permits accurate analysis of apoptosis using cytofluorometry or fluorescent microscopy. Several immune cell populations were studied including dexamethasone-treated thymocytes, irradiated peripheral blood mononuclear cells and a growth factor-depleted tumor B cell line. Excellent correlation values were found with acridine orange using cytofluorometry and with eosin-hematoxylin using optical microscopy. Under fluorescent microscopy, YOPRO-1-fluorescent cells demonstrate the morphological features of cells undergoing apoptosis such as nuclear shrinkage and fragmentation. An important characteristic of the dye that differs from all other nuclear dyes previously used for the detection of apoptosis is that it does not label living cells. Cell sorting after flow cytofluorometry analysis confirmed that only the apoptotic cell population was labelled with YOPRO-1. Further studies showed that while incubation of living cells with Hoechst 33342 almost completely abrogated the capacity of T cells to proliferate in response to several stimuli, YOPRO-1 had no inhibitory effect. This new simple, rapid and reproducible use of the YOPRO-1 dye should prove useful in the analysis of apoptotic cells as well as for investigations of the functional properties of living cells in a culture containing apoptotic cells.

  16. Wingless mediated apoptosis: How cone cells direct the death of peripheral ommatidia in the developing Drosophila eye

    PubMed Central

    Kumar, Sudha R; Patel, Hina; Tomlinson, Andrew

    2016-01-01

    Morphogen gradients play pervasive roles in development, and understanding how they are established and decoded is a major goal of contemporary developmental biology. Here we examine how a Wingless (Wg) morphogen gradient patterns the peripheral specialization of the fly eye. The outermost specialization is the pigment rim; a thick band of pigment cells that circumscribes the eye and optically insulates the sides of the retina. It results from the coalescence of pigment cells that survive the death of the outermost row of developing ommatidia. We investigate here how the Wg target genes expressed in the moribund ommatidia direct the intercellular signaling, the morphogenetic movements, and ultimately the ommatidial death. A salient feature of this process is the secondary expression of the Wg morphogen elicited in the ommatidia by the primary Wg signal. We find that neither the primary nor secondary sources of Wg alone are able to promote ommatidial death, but together they suffice to drive the apoptosis. This represents an unusual gradient read-out process in which a morphogen induces its own expression in its target cells to generate a concentration spike required to push the local cellular responses to the next threshold response. PMID:26428511

  17. The Death Domain Superfamily in Intracellular Signaling of Apoptosis and Inflammation

    PubMed Central

    Park, Hyun Ho; Lo, Yu-Chih; Lin, Su-Chang; Wang, Liwei; Yang, Jin Kuk; Wu, Hao

    2010-01-01

    The death domain (DD) superfamily comprising the death domain (DD) subfamily, the death effector domain (DED) subfamily, the caspase recruitment domain (CARD) subfamily and the pyrin domains (PYD) subfamily is one of the largest domain superfamilies. By mediating homotypic interactions within each domain subfamily, these proteins play important roles in the assembly and activation of apoptotic and inflammatory complexes. In this article, we review the molecular complexes that are assembled by these proteins, the structural and biochemical features of these domains and the molecular interactions mediated by them. By analyzing the potential molecular basis for the function of these domains, we hope to provide a comprehensive understanding on the function, structure, interaction and evolution of this important family of domains. PMID:17201679

  18. Autophagy inhibition and mitochondrial remodeling join forces to amplify apoptosis in activation-induced cell death.

    PubMed

    Mauro, Corrado; Silvia, Campello

    2016-12-01

    Mitochondrial structural and functional changes and the autophagy pathway crosstalk under several stress conditions. However, their interplay under physiological cell death stimulation has been unclear. In our recent report, we show that during activation-induced cell death (AICD), the T-cell receptor (TCR)-dependent pathway that controls immune tolerance, autophagy is inhibited at an early stage. Further, we found that this inhibition is coupled with mitochondria fragmentation and cristae remodeling to unleash the apoptotic program. Last, we dissected the role of macroautophagy/autophagy versus mitophagy in the context of this physiological cell death, and bulk autophagy turned out to be able to remove dysfunctional and depolarized mitochondria. Our data suggest new possible approaches to modulate the immune function in the context of autoimmunity or immunotherapy.

  19. Cell-Centric View of Apoptosis and Apoptotic Cell Death-Inducing Antitumoral Strategies

    PubMed Central

    Apraiz, Aintzane; Boyano, Maria Dolores; Asumendi, Aintzane

    2011-01-01

    Programmed cell death and especially apoptotic cell death, occurs under physiological conditions and is also desirable under pathological circumstances. However, the more we learn about cellular signaling cascades, the less plausible it becomes to find restricted and well-limited signaling pathways. In this context, an extensive description of pathway-connections is necessary in order to point out the main regulatory molecules as well as to select the most appropriate therapeutic targets. On the other hand, irregularities in programmed cell death pathways often lead to tumor development and cancer-related mortality is projected to continue increasing despite the effort to develop more active and selective antitumoral compounds. In fact, tumor cell plasticity represents a major challenge in chemotherapy and improvement on anticancer therapies seems to rely on appropriate drug combinations. An overview of the current status regarding apoptotic pathways as well as available chemotherapeutic compounds provides a new perspective of possible future anticancer strategies. PMID:24212653

  20. The C-Kit Receptor-Mediated Signal Transduction and Tumor-Related Diseases

    PubMed Central

    Liang, Jing; Wu, Yan-Ling; Chen, Bing-Jia; Zhang, Wen; Tanaka, Yoshimasa; Sugiyama, Hiroshi

    2013-01-01

    As an important member of tyrosine kinase family, c-kit receptor causes specific expression of certain genes, regulates cell differentiation and proliferation, resists cell apoptosis, and plays a key role in tumor occurrence, development, migration and recurrence through activating the downstream signaling molecules following interaction with stem cell factor (SCF). The abnormality of SCF/c-kit signaling pathway is closely related to some certain tumors. The discovery of c-kit receptor-targeted drugs has promoted clinical-related cancer's diagnosis and treatment. In this paper, we review recent research progress on c-kit receptor-mediated signal transduction and its potential therapeutic application as a target in tumor-related diseases. PMID:23678293

  1. Noninvasive Monitoring of Pharmacodynamics and Kinetics of a Death Receptor 5 Antibody and Its Enhanced Apoptosis Induction in Sequential Application with Doxorubicin1

    PubMed Central

    Weber, Thomas G; Pöschinger, Thomas; Galbán, Stefanie; Rehemtulla, Alnawaz; Scheuer, Werner

    2013-01-01

    Induction of apoptosis plays a crucial role in the response of tumors to treatment. Thus, we investigated the pharmacodynamics and tumor saturation kinetics of a death receptor 5 antibody (anti-DR5) when combined with chemotherapeutics. For our investigations, we applied an imaging method that allows monitoring of apoptosis noninvasively in living mice. A stably transfected apoptosis reporter based on split luciferase technology facilitates to screen various chemotherapeutics and anti-DR5 on their ability to induce apoptosis in glioblastoma cells in vitro as well as in vivo. We found that doxorubicin (DOX) treatment in vitro led to significant apoptosis induction within 48 hours and to a 2.3-fold increased anti-DR5 binding to the cell surface. In contrast, cisplatin and 5-fluorouracil (5-FU) treatment altered anti-DR5 binding only marginally. Induction of apoptosis by treatment with anti-DR5 was dose- and time-dependent (both in vitro and in vivo). Simultaneous visualization of fluorescence-labeled anti-DR5 in tumor tissue and apoptosis revealed maximal apoptosis induction immediately after the compound had reached tumor site. Regarding combination therapy of anti-DR5 and DOX, we found that the sequential application of DOX before anti-DR5 resulted in synergistically enhanced apoptosis reporter activity. In striking contrast, anti-DR5 given before DOX did not lead to increased apoptosis induction. We suggest that DOX-induced recruitment of DR5 to the cell surface impacts the enhanced apoptotic effect that can be longitudinally monitored by apoptosis imaging. This study demonstrates that the combination of apoptosis and fluorescence imaging is an excellent method for optimizing dosing and treatment schedules in preclinical cancer models. PMID:23908588

  2. Porcine reproductive and respiratory syndrome virus replicates in testicular germ cells, alters spermatogenesis, and induces germ cell death by apoptosis.

    PubMed Central

    Sur, J H; Doster, A R; Christian, J S; Galeota, J A; Wills, R W; Zimmerman, J J; Osorio, F A

    1997-01-01

    Like other arteriviruses, porcine reproductive and respiratory syndrome virus (PRRSV) is shed in semen, a feature that is critical for the venereal transmission of this group of viruses. In spite of its epidemiological importance, little is known of the association of PRRSV or other arteriviruses with gonadal tissues. We experimentally infected a group of boars with PRRSV 12068-96, a virulent field strain. By combined use of in situ hybridization and immunohistochemistry, we detected infection by PRRSV in the testes of these boars. The PRRSV testicular replication in testis centers on two types of cells: (i) epithelial germ cells of the seminiferous tubules, primarily spermatids and spermatocytes, and (ii) macrophages, which are located in the interstitium of the testis. Histopathologically, hypospermatogenesis, formation of multinucleated giant cells (MGCs), and abundant germ cell depletion and death were observed. We obtained evidence that such germ cell death occurs by apoptosis, as determined by a characteristic histologic pattern and evidence of massive DNA fragmentation detected in situ (TUNEL [terminal deoxynucleotidyltransferase-mediated digoxigenin-UTP nick end labeling] assay). Simultaneously with these testicular alterations, we observed that there is a significant increase in the number of immature sperm cells (mainly MGCs, spermatids, and spermatocytes) in the ejaculates of the PRRSV-inoculated boars and that these cells are infected with PRRSV. Our results indicate that PRRSV may infect target cells other than macrophages, that these infected cells can be primarily responsible for the excretion of infectious PRRSV in semen, and that PRRSV induces apoptosis in these germ cells in vivo. PMID:9371575

  3. Novel monofunctional platinum (II) complex Mono-Pt induces apoptosis-independent autophagic cell death in human ovarian carcinoma cells, distinct from cisplatin.

    PubMed

    Guo, Wen-Jie; Zhang, Yang-Miao; Zhang, Li; Huang, Bin; Tao, Fei-Fei; Chen, Wei; Guo, Zi-Jian; Xu, Qiang; Sun, Yang

    2013-07-01

    Failure to engage apoptosis appears to be a leading mechanism of resistance to traditional platinum drugs in patients with ovarian cancer. Therefore, an alternative strategy to induce cell death is needed for the chemotherapy of this apoptosis-resistant cancer. Here we report that autophagic cell death, distinct from cisplatin-induced apoptosis, is triggered by a novel monofunctional platinum (II) complex named Mono-Pt in human ovarian carcinoma cells. Mono-Pt-induced cell death has the following features: cytoplasmic vacuolation, caspase-independent, no nuclear fragmentation or chromatin condensation, and no apoptotic bodies. These characteristics integrally indicated that Mono-Pt, rather than cisplatin, initiated a nonapoptotic cell death in Caov-3 ovarian carcinoma cells. Furthermore, incubation of the cells with Mono-Pt but not with cisplatin produced an increasing punctate distribution of microtubule-associated protein 1 light chain 3 (LC3), and an increasing ratio of LC3-II to LC3-I. Mono-Pt also caused the formation of autophagic vacuoles as revealed by monodansylcadaverine staining and transmission electron microscopy. In addition, Mono-Pt-induced cell death was significantly inhibited by the knockdown of either BECN1 or ATG7 gene expression, or by autophagy inhibitors 3-methyladenine, chloroquine and bafilomycin A 1. Moreover, the effect of Mono-Pt involved the AKT1-MTOR-RPS6KB1 pathway and MAPK1 (ERK2)/MAPK3 (ERK1) signaling, since the MTOR inhibitor rapamycin increased, while the MAPK1/3 inhibitor U0126 decreased Mono-Pt-induced autophagic cell death. Taken together, our results suggest that Mono-Pt exerts anticancer effect via autophagic cell death in apoptosis-resistant ovarian cancer. These findings lead to increased options for anticancer platinum drugs to induce cell death in cancer.

  4. Receptors mediating toxicity and their involvement in endocrine disruption.

    PubMed

    Rüegg, Joëlle; Penttinen-Damdimopoulou, Pauliina; Mäkelä, Sari; Pongratz, Ingemar; Gustafsson, Jan-Ake

    2009-01-01

    Many toxic compounds exert their harmful effects by activating of certain receptors, which in turn leads to dysregulation of transcription. Some of these receptors are so called xenosensors. They are activated by external chemicals and evoke a cascade of events that lead to the elimination of the chemical from the system. Other receptors that are modulated by toxic substances are hormone receptors, particularly the ones of the nuclear receptor family. Some environmental chemicals resemble endogenous hormones and can falsely activate these receptors, leading to undesired activity in the cell. Furthermore, excessive activation of the xenosensors can lead to disturbances of the integrity of the system as well. In this chapter, the concepts of receptor-mediated toxicity and hormone disruption are introduced. We start by describing environmental chemicals that can bind to xenosensors and nuclear hormone receptors. We then describe the receptors most commonly targeted by environmental chemicals. Finally, the mechanisms by which receptor-mediated events can disrupt the system are depicted.

  5. Multiscale Modeling of Virus Entry via Receptor-Mediated Endocytosis

    NASA Astrophysics Data System (ADS)

    Liu, Jin

    2012-11-01

    Virus infections are ubiquitous and remain major threats to human health worldwide. Viruses are intracellular parasites and must enter host cells to initiate infection. Receptor-mediated endocytosis is the most common entry pathway taken by viruses, the whole process is highly complex and dictated by various events, such as virus motions, membrane deformations, receptor diffusion and ligand-receptor reactions, occurring at multiple length and time scales. We develop a multiscale model for virus entry through receptor-mediated endocytosis. The binding of virus to cell surface is based on a mesoscale three dimensional stochastic adhesion model, the internalization (endocytosis) of virus and cellular membrane deformation is based on the discretization of Helfrich Hamiltonian in a curvilinear space using Monte Carlo method. The multiscale model is based on the combination of these two models. We will implement this model to study the herpes simplex virus entry into B78 cells and compare the model predictions with experimental measurements.

  6. Evidence for the involvement of apoptosis-inducing factor-mediated caspase-independent neuronal death in Alzheimer disease.

    PubMed

    Yu, Wenfeng; Mechawar, Naguib; Krantic, Slavica; Quirion, Rémi

    2010-05-01

    Accumulating evidence suggests the involvement of caspase-dependent and -independent mechanisms in neuronal cell death in Alzheimer disease (AD). The apoptosis-inducing factor (AIF) is a mitochondrial oxido-reductase originally characterized as a mediator of caspase-independent programmed cell death (PCD). In this postmortem study, we investigated the distribution of AIF and its possible morphological association with pathological features in the hippocampus, as well as entorhinal and medial gyrus of temporal cortices of late stage AD, dementia with Lewy bodies (DLB), and control subjects. In comparison with controls, a significant increase in neuronal AIF immunoreactivity (AIF-ir) was observed in the hippocampus and the superficial layers of entorhinal and medial gyrus of temporal cortices in AD--but not DLB--samples. AIF-ir in neuronal nuclei was also significantly more widespread in AD compared with control and DLB samples. Furthermore, AIF-ir was found to be colocalized with neurofibrillary tangles (NFTs) in AD brains. Interestingly, a significant positive correlation was seen between nuclear AIF-ir and Braak stage in CA1 of the hippocampus as well as in entorhinal and temporal cortices in AD samples. These data show for the first time: (1) the nuclear localization of AIF in the AD brain and (2) its colocalization with NFTs, suggesting a possible involvement of AIF-mediated caspase-independent PCD, at least in the late stage of this neuropathology.

  7. Betanin-Enriched Red Beetroot (Beta vulgaris L.) Extract Induces Apoptosis and Autophagic Cell Death in MCF-7 Cells.

    PubMed

    Nowacki, Laëtitia; Vigneron, Pascale; Rotellini, Laura; Cazzola, Hélène; Merlier, Franck; Prost, Elise; Ralanairina, Robert; Gadonna, Jean-Pierre; Rossi, Claire; Vayssade, Muriel

    2015-12-01

    Recent studies have pointed out the preventive role of beetroot extracts against cancers and their cytotoxic activity on cancer cells. Among many different natural compounds, these extracts contained betanin and its stereoisomer isobetanin, which belongs to the betalain group of highly bioavailable antioxidants. However, a precise identification of the molecules responsible for this tumor-inhibitory effect was still required. We isolated a betanin/isobetanin concentrate from fresh beetroots, corresponding to the highest purified betanin extract used for studying anticancer activities of these molecules. The cytotoxicity of this betanin-enriched extract was then characterized on cancer and normal cells and we highlighted the death signalling pathways involved. Betanin/isobetanin concentrate significantly decreased cancer cell proliferation and viability. Particularly in MCF-7-treated cells, the expressions of apoptosis-related proteins (Bad, TRAILR4, FAS, p53) were strongly increased and the mitochondrial membrane potential was altered, demonstrating the involvement of both intrinsic and extrinsic apoptotic pathways. Autophagosome vesicles in MCF-7-treated cells were observed, also suggesting autophagic cell death upon betanin/isobetanin treatment. Importantly, the betanin-enriched extract had no obvious effect towards normal cell lines. Our data bring new insight to consider the betanin/isobetanin mix as therapeutic anticancer compound, alone or in combination with classical chemotherapeutic drugs, especially in functional p53 tumors.

  8. The cyclomodulin Cif of Photorhabdus luminescens inhibits insect cell proliferation and triggers host cell death by apoptosis.

    PubMed

    Chavez, Carolina Varela; Jubelin, Grégory; Courties, Gabriel; Gomard, Aurélie; Ginibre, Nadège; Pages, Sylvie; Taïeb, Frédéric; Girard, Pierre-Alain; Oswald, Eric; Givaudan, Alain; Zumbihl, Robert; Escoubas, Jean-Michel

    2010-12-01

    Cycle inhibiting factors (Cif) constitute a broad family of cyclomodulins present in bacterial pathogens of invertebrates and mammals. Cif proteins are thought to be type III effectors capable of arresting the cell cycle at G(2)/M phase transition in human cell lines. We report here the first direct functional analysis of Cif(Pl), from the entomopathogenic bacterium Photorhabdus luminescens, in its insect host. The cif(Pl) gene was expressed in P. luminescens cultures in vitro. The resulting protein was released into the culture medium, unlike the well characterized type III effector LopT. During locust infection, cif(Pl) was expressed in both the hemolymph and the hematopoietic organ, but was not essential for P. luminescens virulence. Cif(Pl) inhibited proliferation of the insect cell line Sf9, by blocking the cell cycle at the G(2)/M phase transition. It also triggered host cell death by apoptosis. The integrity of the Cif(Pl) catalytic triad is essential for the cell cycle arrest and pro-apoptotic activities of this protein. These results highlight, for the first time, the dual role of Cif in the control of host cell proliferation and apoptotic death in a non-mammalian cell line.

  9. Death deflected: IL-15 inhibits TNF-alpha-mediated apoptosis in fibroblasts by TRAF2 recruitment to the IL-15Ralpha chain.

    PubMed

    Bulfone-Paus, S; Bulanova, E; Pohl, T; Budagian, V; Durkop, H; Ruckert, R; Kunzendorf, U; Paus, R; Krause, H

    1999-09-01

    Interleukin-15 (IL-15) is a potent inhibitor of several apoptosis pathways. One prominent path toward apoptosis is the ligand-induced association of TNF receptor 1 (TNFR1) with death domain adaptor proteins. Studying if and how IL-15 blocks TNFR1-mediated apoptosis in a murine fibroblast cell line (L929), we show here that IL-15 blocks TNFR1-induced apoptosis via IL-15Ralpha chain signaling. The intracellular tail of IL-15Ralpha shows sequence homologies to the TRAF2 binding motifs of CD30 and CD40. Most important, binding of IL-15 to IL-15Ralpha successfully competes with the TNFR1 complex for TRAF2 binding, which may impede assembly of key adaptor proteins to the TNFR1 complex, and induces IkappaBalpha phosphorylation. Thus, IL-15Ralpha chain stimulation is a powerful deflector of cell death very early in the apoptosis signaling cascade, while TNF-alpha and IL-15 surface as major opponents in apoptosis control.

  10. In vivo analysis of germ cell apoptosis reveals the existence of stage-specific 'social' control of germ cell death in the seminiferous epithelium.

    PubMed

    Blanco-Rodríguez, J; Martínez-García, C

    1997-12-01

    It has become clear in recent years that programmed cell death is regulated during development by signals from other cells. Nevertheless, compared to the 'social' control of cell proliferation, relatively little is known about the 'social' control of cell death in other systems. Since in a previous study we showed that induced germ cell apoptosis occurs at specific stages of the spermatogenic cycle, in this study we aimed to ascertain the existence of supracellular control of germ cell death during spermatogenesis. Therefore, the TUNEL technique has been used to analyse whether all of the different germ cell types are induced to die at these specific stages in animals injected intratesticularly with one of several inducers of apoptosis. Our findings suggest that all of the investigated agents trigger apoptosis in all the diverse progenies of germ cells existing at stages I, XII or XIV of the spermatogenic cycle. In contrast, at most other stages the number of apoptotic cells was similar to that found in control animals. These data are consistent with the existence of an intercellular control of germ cell death during spermatogenesis. We conclude that the seminiferous epithelium provides a suitable in vivo model to study the mechanisms underlying the 'social' control of apoptosis.

  11. An insight into the hepatocellular death induced by amphetamines, individually and in combination: the involvement of necrosis and apoptosis.

    PubMed

    Dias da Silva, Diana; Carmo, Helena; Lynch, Adam; Silva, Elisabete

    2013-12-01

    The liver is a vulnerable target for amphetamine toxicity, but the mechanisms involved in the drug's hepatotoxicity remain poorly understood. The purpose of the current research was to characterize the mode of death elicited by four amphetamines and to evaluate whether their combination triggered similar mechanisms in immortalized human HepG2 cells. The obtained data revealed a time- and temperature-dependent mortality of HepG2 cells exposed to 3,4-methylenedioxymethamphetamine (MDMA, ecstasy; 1.3 mM), methamphetamine (3 mM), 4-methylthioamphetamine (0.5 mM) and D-amphetamine (1.7 mM), alone or combined (1.6 mM mixture). At physiological temperature (37 °C), 24-h exposures caused HepG2 death preferentially by apoptosis, while a rise to 40.5 °C favoured necrosis. ATP levels remained unaltered when the drugs where tested at normothermia, but incubation at 40.5 °C provoked marked ATP depletion for all treatments. Further investigations on the apoptotic mechanisms triggered by the drugs (alone or combined) showed a decline in BCL-2 and BCL- XL mRNA levels, with concurrent upregulation of BAX, BIM, PUMA and BID genes. Elevation of Bax, cleaved Bid, Puma, Bak and Bim protein levels was also seen. To the best of our knowledge, Puma, Bim and Bak have never been linked with the toxicity induced by amphetamines. Time-dependent caspase-3/-7 activation, but not mitochondrial membrane potential (∆ψm) disruption, also mediated amphetamine-induced apoptosis. The cell dismantling was confirmed by poly(ADP-ribose)polymerase proteolysis. Overall, for all evaluated parameters, no relevant differences were detected between individual amphetamines and the mixture (all tested at equieffective cytotoxic concentrations), suggesting that the mode of action of the amphetamines in combination does not deviate from the mode of action of the drugs individually, when eliciting HepG2 cell death.

  12. The role of intracellular oxidation in death induction (apoptosis and necrosis) in human promonocytic cells treated with stress inducers (cadmium, heat, X-rays).

    PubMed

    Galán, A; García-Bermejo, L; Troyano, A; Vilaboa, N E; Fernández, C; de Blas, E; Aller, P

    2001-04-01

    Treatment of U-937 human promonocytic cells with the stress inducers cadmium chloride (2 h at 200 microM), heat (2 h at 42.5 C) or X-rays (20 Gy), followed by recovery, caused death by apoptosis and stimulated caspase-3 activity. In addition, all stress agents caused intracellular oxidation, as measured by peroxide and/or anion superoxide accumulation. However, while pre-incubation with antioxidants (N-acetyl-L-cysteine or butylated hydroxyanisole) inhibited the induction of apoptosis by cadmium and X-rays, it did not affect the induction by heat-shock. Pre-incubation for 24 h with the GSH-depleting agent L-buthionine-[S,R]-sulfoximine (BSO) switched the mode of death from apoptosis to necrosis in cadmium-treated cells. By contrast, BSO only caused minor modifacions in the rate of apoptosis without affecting the mode of death in heat- and X-rays-treated cells. BSO potentiated peroxide accumulation in cells treated with both cadmium and X-rays. However, while the accumulation of peroxides was stable in the case of cadmium, it was transient in the case of X-rays. Moreover, the administration of antioxidants during the recovery period sufficed to prevent necrosis and restore apoptosis in BSO plus cadmium-treated cells. Cadmium and X-rays caused a decrease in intracellular ATP levels, but the decrease was similar in both apoptotic and necrotic cells. Taken together, these results demonstrate that (i) stress inducers cause intracellular oxidation, but oxidation is not a general requirement for apoptosis; and (ii) the duration of the oxidant state seems to be critical in determining the mode of death.

  13. Magnolol-induced H460 cells death via autophagy but not apoptosis.

    PubMed

    Li, Hai-Bo; Yi, Xin; Gao, Jian-Mei; Ying, Xi-Xiang; Guan, Hong-Quan; Li, Jian-Chun

    2007-12-01

    We have reported that the protective effect of Magnolol on TBHP-induced injury in human nonsmall lung cancer H460 cells is partially via a p53 dependent mechanism. In this study, we found that Magnolol displayed a stimulatory effect at low concentrations (< or = 20 microM) whilst inhibitory effect at high concentrations (> or = 40 microM) in H460 cells. To investigate the mechanism of inducing the biphasic effect in H460 cells with Magnolol, we showed that Magnolol stimulated DNA synthesis at low concentrations and displayed an inhibition effect at high concentrations in H460 cells. More importantly, the inhibition of DNA synthesis was accompanied by the S phase cell cycle arrest and the appearance of intense intracytoplasmic vacuoles. These vacuoles can be labeled by autophagic marker monodansylcadaverin (MDC), 3-methyladenine (3-MA), an inhibitor of autophagy, was able to inhibit the occurrence of autophagy. The results of the LDH activity assay and TUNEL assay also showed that Magnolol at high concentrations inhibiting H460 cell growth was not via apoptotic pathway. Furthermore, accompanied by the occurrence of autophagy, the expression of phospho-Akt was down-regulated but PTEN significantly was up-regulated. In conclusion, Magnolol induces H460 cells death by autophagy but not apoptotic pathway. Blockade of PI3K/PTEN/Akt pathway is maybe related to Magnolol-induced autophagy. Autophagic cells death induction by Magnolol underlines the potential utility of its induction as a new cancer treatment modality.

  14. FK506 augments activation-induced programmed cell death of T lymphocytes in vivo.

    PubMed Central

    Migita, K; Eguchi, K; Kawabe, Y; Tsukada, T; Mizokami, A; Nagataki, S

    1995-01-01

    FK506 is an immunosuppressive drug that inhibits T cell receptor-mediated signal transduction. This drug can induce immunological tolerance in allograft recipients. In this study, we investigated the in vivo effects of FK506 on T cell receptor-mediated apoptosis induction. Injection of anti-CD3 antibody (Ab) in mice resulted in the elimination of CD4+ CD8+ thymocytes by DNA fragmentation. FK506 treatment significantly augmented thymic apoptosis induced by in vivo anti-CD3 Ab administration. Increased thymic apoptosis resulted in the disappearance of CD4+ CD8+ thymocytes after anti-CD3 Ab/FK506 treatment. DNA fragmentation triggered by FK506 was induced exclusively in antigen-stimulated T cells, since enhanced DNA fragmentation induced by in vivo staphylococcal enterotoxin B (SEB) injection was confirmed in SEB-reactive V beta 8+ thymocytes but not in SEB-nonreactive V beta 6+ thymocytes. In addition to thymocytes, mature peripheral T cells also die by activation-induced programmed cell death. A similar effect of FK506 on activation-induced programmed cell death was observed in SEB-activated peripheral spleen T cells. In contrast, cyclosporin A treatment did not enhance activation-induced programmed cell death of thymocytes and peripheral T cells. Apoptosis is required for the generation and maintenance of self-tolerance in the immune system. Our findings suggest that FK506-triggered apoptosis after elimination of antigen-activated T cells may represent a potential mechanism of the immunological tolerance achieved by FK506 treatment. Images PMID:7543492

  15. Activation of autophagic flux by epigallocatechin gallate mitigates TRAIL-induced tumor cell apoptosis via down-regulation of death receptors

    PubMed Central

    Park, Sang-Youel

    2016-01-01

    Epigallocatechin gallate (EGCG) is a major polyphenol in green tea. Recent studies have reported that EGCG can inhibit TRAIL-induced apoptosis and activate autophagic flux in cancer cells. However, the mechanism behind these processes is unclear. The present study found that EGCG prevents tumor cell death by antagonizing the TRAIL pathway and activating autophagy flux. Our results indicate that EGCG dose-dependently inhibits TRAIL-induced apoptosis and decreases the binding of death receptor 4 and 5 (DR4 and 5) to TRAIL. In addition, EGCG activates autophagy flux, which is involved in the inhibition of TRAIL cell death. We confirmed that the protective effect of EGCG can be reversed using genetic and pharmacological tools through re-sensitization to TRAIL. The inhibition of autophagy flux affects not only the re-sensitization of tumor cells to TRAIL, but also the restoration of death receptor proteins. This study demonstrates that EGCG inhibits TRAIL-induced apoptosis through the manipulation of autophagic flux and subsequent decrease in number of death receptors. On the basis of these results, we suggest further consideration of the use of autophagy activators such as EGCG in combination anti-tumor therapy with TRAIL. PMID:27582540

  16. Interplay between lysosomal, mitochondrial and death receptor pathways during manganese-induced apoptosis in glial cells.

    PubMed

    Gorojod, R M; Alaimo, A; Porte Alcon, S; Saravia, F; Kotler, M L

    2017-04-04

    Manganese (Mn) is an essential trace metal which plays a critical role in brain physiology by acting as a cofactor for several enzymes. However, upon overexposure, Mn preferentially accumulates within the basal ganglia leading to the development of a Parkinsonism known as Manganism. Data from our group have proved that Mn induces oxidative stress-mediated apoptosis in astrocytoma C6 cells. In the present study we described how cathepsins impact on different steps of each apoptotic cascade. Evidence obtained demonstrated that Mn generates lysosomal membrane permeabilization (LMP) and cathepsin release. Both cathepsins B (Ca-074 Me) and D (Pepstatin A) inhibitors as well as Bafilomycin A1 prevented caspases-3, -7, -8 and -9 activation, FasL upregulation, Bid cleavage, Δφm disruption and cytochrome c release. Results from in vivo studies showed that intrastriatal Mn injection increased cathepsin D levels from corpus striatum and substantia nigra pars compacta. Our results point to LMP and lysosomal cathepsins as key mediators in the apoptotic process triggered by Mn. These findings highlight the relevance of targeting the lysosomal pathway for Manganism therapy.

  17. Antigen-Specific Immune Modulation Targets mTORC1 Function To Drive Chemokine Receptor-Mediated T Cell Tolerance.

    PubMed

    Chen, Weirong; Wan, Xiaoxiao; Ukah, Tobechukwu K; Miller, Mindy M; Barik, Subhasis; Cattin-Roy, Alexis N; Zaghouani, Habib

    2016-11-01

    To contain autoimmunity, pathogenic T cells must be eliminated or diverted from reaching the target organ. Recently, we defined a novel form of T cell tolerance whereby treatment with Ag downregulates expression of the chemokine receptor CXCR3 and prevents diabetogenic Th1 cells from reaching the pancreas, leading to suppression of type 1 diabetes (T1D). This report defines the signaling events underlying Ag-induced chemokine receptor-mediated tolerance. Specifically, we show that the mammalian target of rapamycin complex 1 (mTORC1) is a major target for induction of CXCR3 downregulation and crippling of Th1 cells. Indeed, Ag administration induces upregulation of programmed death-ligand 1 on dendritic cells in a T cell-dependent manner. In return, programmed death-ligand 1 interacts with the constitutively expressed programmed death-1 on the target T cells and stimulates docking of Src homology 2 domain-containing tyrosine phosphatase 2 phosphatase to the cytoplasmic tail of programmed death-1. Active Src homology 2 domain-containing tyrosine phosphatase 2 impairs the signaling function of the PI3K/protein kinase B (AKT) pathway, leading to functional defect of mTORC1, downregulation of CXCR3 expression, and suppression of T1D. Thus, mTORC1 component of the metabolic pathway serves as a target for chemokine receptor-mediated T cell tolerance and suppression of T1D.

  18. FasL and TRAIL Induce Epidermal Apoptosis and Skin Ulceration Upon Exposure to Leishmania major

    PubMed Central

    Eidsmo, Liv; Fluur, Caroline; Rethi, Bence; Eriksson Ygberg, Sofia; Ruffin, Nicolas; De Milito, Angelo; Akuffo, Hannah; Chiodi, Francesca

    2007-01-01

    Receptor-mediated apoptosis is proposed as an important regulator of keratinocyte homeostasis in human epidermis. We have previously reported that Fas/FasL interactions in epidermis are altered during cutaneous leishmaniasis (CL) and that keratinocyte death through apoptosis may play a pathogenic role for skin ulceration. To further investigate the alterations of apoptosis during CL, a keratinocyte cell line (HaCaT) and primary human epidermal keratinocytes were incubated with supernatants from Leishmania major-infected peripheral blood mononuclear cells. An apoptosis-specific microarray was used to assess mRNA expression in HaCaT cells exposed to supernatants derived from L. major-infected cultures. Fas and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA and protein expression were significantly up-regulated, and apoptosis was detected in both HaCaT and human epidermal keratinocyte cells. The keratinocyte apoptosis was partly inhibited through blocking of Fas or FasL and even more efficiently through TRAIL neutralization. Up-regulation of Fas on keratinocytes in epidermis and the presence of FasL-expressing macrophages and T cells in dermis were previously reported by us. In this study, keratinocytes expressing TRAIL, as well as the proapoptotic receptor TRAIL-R2, were detected in skin biopsies from CL cases. We propose that activation of Fas and TRAIL apoptosis pathways, in the presence of inflammatory mediators at the site of infection, leads to tissue destruction and ulceration during CL. PMID:17200196

  19. Apoptosis-inducing factor participation in bovine macrophage Mycobacterium bovis-induced caspase-independent cell death.

    PubMed

    Vega-Manriquez, X; López-Vidal, Y; Moran, J; Adams, L G; Gutiérrez-Pabello, J A

    2007-03-01

    Mycobacterium tuberculosis complex species survive and replicate in phagosomes of the host cell. Cell death (CD) has been highlighted as one of the probable outcomes in this host-pathogen interaction. Previously, our group demonstrated macrophage apoptosis as a consequence of Mycobacterium bovis infection. In this study, we aimed to identify the contribution of apoptotic effector elements in M. bovis-induced CD. Bovine macrophages were either infected with M. bovis (multiplicity of infection, 10:1) or treated with an M. bovis cell extract (CFE). Structural changes compatible with CD were evaluated. Chromatin condensation was increased three times by the CFE. On the other hand, a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay demonstrated that levels of DNA fragmentation induced by M. bovis and CFE were 53.7% +/- 24% and 38.9% +/- 14%, respectively, whereas control cells had a basal proportion of 8.9% +/- 4.1%. Rates of DNA fragmentation were unaffected by the presence of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (z-VAD). Cells treated with 100 mug of CFE for 12 h had a fivefold decrease in the level of mitochondrial outer membrane permeabilization compared to that of untreated cells. Neither M. bovis infection nor CFE treatment induced activation of caspase 3, 8, or 9. Translocation of apoptosis-inducing factor (AIF) to the nucleus was identified in 32% +/- 3.5% and 26.3% +/- 4.9% of M. bovis-infected and CFE-treated cells, respectively. Incubation of macrophages with z-VAD prior to infection did not alter the percentage of cells showing AIF translocation. Our data suggest that M. bovis-induced CD in bovine macrophages is caspase independent with AIF participation.

  20. An anthraquinone derivative, emodin sensitizes hepatocellular carcinoma cells to TRAIL induced apoptosis through the induction of death receptors and downregulation of cell survival proteins.

    PubMed

    Subramaniam, Aruljothi; Loo, Ser Yue; Rajendran, Peramaiyan; Manu, Kanjoormana A; Perumal, Ekambaram; Li, Feng; Shanmugam, Muthu K; Siveen, Kodappully Sivaraman; Park, Joo-In; Ahn, Kwang Seok; Hui, Kam M; Kumar, Alan P; Sethi, Gautam

    2013-10-01

    Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is currently under clinical trials for cancer, however many tumor cells, including hepatocellular carcinoma (HCC) develop resistance to TRAIL-induced apoptosis. Hence, novel agents that can alleviate TRAIL-induced resistance are urgently needed. In the present report, we investigated the potential of emodin to enhance apoptosis induced by TRAIL in HCC cells. As observed by MTT cytotoxicity assay and the externalization of the membrane phospholipid phosphatidylserine, we found that emodin can significantly potentiate TRAIL-induced apoptosis in HCC cells. When investigated for the mechanism(s), we observed that emodin can downregulate the expression of various cell survival proteins, and induce the cell surface expression of both TRAIL receptors, death receptors (DR) 4 as well as 5. In addition, emodin increased the expression of C/EBP homologous protein (CHOP) in a time-dependent manner. Knockdown of CHOP by siRNA decreased the induction of emodin-induced DR5 expression and apoptosis. Emodin-induced induction of DR5 was mediated through the generation of reactive oxygen species (ROS), as N-acetylcysteine blocked the induction of DR5 and the induction of apoptosis. Also, the knockdown of X-linked inhibitor of apoptosis protein by siRNA significantly reduced the sensitization effect of emodin on TRAIL-induced apoptosis. Overall, our experimental results clearly indicate that emodin can indeed potentiate TRAIL-induced apoptosis through the downregulation of antiapoptotic proteins, increased expression of apoptotic proteins, and ROS mediated upregulation of DR in HCC cells.

  1. Inhibitor of apoptosis (IAP)-like protein lacks a baculovirus IAP repeat (BIR) domain and attenuates cell death in plant and animal systems.

    PubMed

    Kim, Woe Yeon; Lee, Sun Yong; Jung, Young Jun; Chae, Ho Byoung; Nawkar, Ganesh M; Shin, Mi Rim; Kim, Sun Young; Park, Jin Ho; Kang, Chang Ho; Chi, Yong Hun; Ahn, Il Pyung; Yun, Dae Jin; Lee, Kyun Oh; Kim, Young-Myeong; Kim, Min Gab; Lee, Sang Yeol

    2011-12-09

    A novel Arabidopsis thaliana inhibitor of apoptosis was identified by sequence homology to other known inhibitor of apoptosis (IAP) proteins. Arabidopsis IAP-like protein (AtILP) contained a C-terminal RING finger domain but lacked a baculovirus IAP repeat (BIR) domain, which is essential for anti-apoptotic activity in other IAP family members. The expression of AtILP in HeLa cells conferred resistance against tumor necrosis factor (TNF)-α/ActD-induced apoptosis through the inactivation of caspase activity. In contrast to the C-terminal RING domain of AtILP, which did not inhibit the activity of caspase-3, the N-terminal region, despite displaying no homology to known BIR domains, potently inhibited the activity of caspase-3 in vitro and blocked TNF-α/ActD-induced apoptosis. The anti-apoptotic activity of the AtILP N-terminal domain observed in plants was reproduced in an animal system. Transgenic Arabidopsis lines overexpressing AtILP exhibited anti-apoptotic activity when challenged with the fungal toxin fumonisin B1, an agent that induces apoptosis-like cell death in plants. In AtIPL transgenic plants, suppression of cell death was accompanied by inhibition of caspase activation and DNA fragmentation. Overexpression of AtILP also attenuated effector protein-induced cell death and increased the growth of an avirulent bacterial pathogen. The current results demonstrated the existence of a novel plant IAP-like protein that prevents caspase activation in Arabidopsis and showed that a plant anti-apoptosis gene functions similarly in plant and animal systems.

  2. Hibiscus sabdariffa leaf polyphenolic extract induces human melanoma cell death, apoptosis, and autophagy.

    PubMed

    Chiu, Chun-Tang; Hsuan, Shu-Wen; Lin, Hui-Hsuan; Hsu, Cheng-Chin; Chou, Fen-Pi; Chen, Jing-Hsien

    2015-03-01

    Melanoma is the least common but most fatal form of skin cancer. Previous studies have indicated that an aqueous extract of Hibiscus sabdariffa leaves possess hypoglycemic, hypolipidemic, and antioxidant effects. In this study, we want to investigate the anticancer activity of Hibiscus leaf polyphenolic (HLP) extract in melanoma cells. First, HLP was exhibited to be rich in epicatechin gallate (ECG) and other polyphenols. Apoptotic and autophagic activities of HLP and ECG were further evaluated by DAPI stain, cell-cycle analysis, and acidic vascular organelle (AVO) stain. Our results revealed that both HLP and ECG induced the caspases cleavages, Bcl-2 family proteins regulation, and Fas/FasL activation in A375 cells. In addition, we also revealed that the cells presented AVO-positive after HLP treatments. HLP could increase the expressions of autophagy-related proteins autophagy-related gene 5 (ATG5), Beclin1, and light chain 3-II (LC3-II), and induce autophagic cell death in A375 cells. These data indicated that the anticancer effect of HLP, partly contributed by ECG, in A375 cells. HLP potentially could be developed as an antimelanoma agent.

  3. Sialylation of the Fas Death Receptor by ST6Gal-I Provides Protection against Fas-mediated Apoptosis in Colon Carcinoma Cells*

    PubMed Central

    Swindall, Amanda F.; Bellis, Susan L.

    2011-01-01

    The glycosyltransferase, ST6Gal-I, adds sialic acid in an α2–6 linkage to the N-glycans of membrane and secreted glycoproteins. Up-regulation of ST6Gal-I occurs in many cancers, including colon carcinoma, and correlates with metastasis and poor prognosis. However, mechanisms by which ST6Gal-I facilitates tumor progression remain poorly understood due to limited knowledge of enzyme substrates. Herein we identify the death receptor, Fas (CD95), as an ST6Gal-I substrate, and show that α2–6 sialylation of Fas confers protection against Fas-mediated apoptosis. Intriguingly, differences in ST6Gal-I activity do not affect the function of DR4 or DR5 death receptors upon treatment with TRAIL, implicating a selective effect of ST6Gal-I on the Fas receptor. Using ST6Gal-I knockdown and forced overexpression colon carcinoma cell models, we find that α2–6 sialylation of Fas prevents apoptosis stimulated by FasL as well as the Fas-activating antibody, CH11, as evidenced by decreased activation of caspases 8 and 3. We also show that α2–6 sialylation of Fas does not alter the binding of CH11, but rather inhibits the capacity of Fas to induce apoptosis by blocking the association of FADD with Fas cytoplasmic tails, an event that initiates death-inducing signaling complex formation. Furthermore, α2–6 sialylation of Fas inhibits Fas internalization, which is required for apoptotic signaling. Although dysregulated Fas activity is a well known mechanism through which tumors evade apoptosis, the current study is the first to link Fas insensitivity to the actions of a specific sialyltransferase. This finding establishes a new paradigm by which death receptor function is impaired for the self-protection of tumors against apoptosis. PMID:21550977

  4. Apoptosis-like cell death in Leishmania donovani treated with KalsomeTM10, a new liposomal amphotericin B

    PubMed Central

    Shadab, Md.; Jha, Baijayanti; Asad, Mohammad; Deepthi, Makaraju; Kamran, Mohd.; Ali, Nahid

    2017-01-01

    Objective The present study aimed to elucidate the cell death mechanism in Leishmania donovani upon treatment with KalsomeTM10, a new liposomal amphotericin B. Methodology/Principal findings We studied morphological alterations in promastigotes through phase contrast and scanning electron microscopy. Phosphatidylserine (PS) exposure, loss of mitochondrial membrane potential and disruption of mitochondrial integrity was determined by flow cytometry using annexinV-FITC, JC-1 and mitotraker, respectively. For analysing oxidative stress, generation of H2O2 (bioluminescence kit) and mitochondrial superoxide O2− (mitosox) were measured. DNA fragmentation was evaluated using terminal deoxyribonucleotidyl transferase mediated dUTP nick-end labelling (TUNEL) and DNA laddering assay. We found that KalsomeTM10 is more effective then Ambisome against the promastigote as well as intracellular amastigote forms. The mechanistic study showed that KalsomeTM10 induced several morphological alterations in promastigotes typical of apoptosis. KalsomeTM10 treatment showed a dose- and time-dependent exposure of PS in promastigotes. Further, study on mitochondrial pathway revealed loss of mitochondrial membrane potential as well as disruption in mitochondrial integrity with depletion of intracellular pool of ATP. KalsomeTM10 treated promastigotes showed increased ROS production, diminished GSH levels and increased caspase-like activity. DNA fragmentation and cell cycle arrest was observed in KalsomeTM10 treated promastigotes. Apoptotic DNA fragmentation was also observed in KalsomeTM10 treated intracellular amastigotes. KalsomeTM10 induced generation of ROS and nitric oxide leads to the killing of the intracellular parasites. Moreover, endocytosis is indispensable for KalsomeTM10 mediated anti-leishmanial effect in host macrophage. Conclusions KalsomeTM10 induces apoptotic-like cell death in L. donovani parasites to exhibit its anti-leishmanial function. PMID:28170432

  5. Mitochondrial apoptosis is dispensable for NLRP3 inflammasome activation but non-apoptotic caspase-8 is required for inflammasome priming

    PubMed Central

    Allam, Ramanjaneyulu; Lawlor, Kate E; Yu, Eric Chi-Wang; Mildenhall, Alison L; Moujalled, Donia M; Lewis, Rowena S; Ke, Francine; Mason, Kylie D; White, Michael J; Stacey, Katryn J; Strasser, Andreas; O’Reilly, Lorraine A; Alexander, Warren; Kile, Benjamin T; Vaux, David L; Vince, James E

    2014-01-01

    A current paradigm proposes that mitochondrial damage is a critical determinant of NLRP3 inflammasome activation. Here, we genetically assess whether mitochondrial signalling represents a unified mechanism to explain how NLRP3 is activated by divergent stimuli. Neither co-deletion of the essential executioners of mitochondrial apoptosis BAK and BAX, nor removal of the mitochondrial permeability transition pore component cyclophilin D, nor loss of the mitophagy regulator Parkin, nor deficiency in MAVS affects NLRP3 inflammasome function. In contrast, caspase-8, a caspase essential for death-receptor-mediated apoptosis, is required for efficient Toll-like-receptor-induced inflammasome priming and cytokine production. Collectively, these results demonstrate that mitochondrial apoptosis is not required for NLRP3 activation, and highlight an important non-apoptotic role for caspase-8 in regulating inflammasome activation and pro-inflammatory cytokine levels. PMID:24990442

  6. Mitochondrial apoptosis is dispensable for NLRP3 inflammasome activation but non-apoptotic caspase-8 is required for inflammasome priming.

    PubMed

    Allam, Ramanjaneyulu; Lawlor, Kate E; Yu, Eric Chi-Wang; Mildenhall, Alison L; Moujalled, Donia M; Lewis, Rowena S; Ke, Francine; Mason, Kylie D; White, Michael J; Stacey, Katryn J; Strasser, Andreas; O'Reilly, Lorraine A; Alexander, Warren; Kile, Benjamin T; Vaux, David L; Vince, James E

    2014-09-01

    A current paradigm proposes that mitochondrial damage is a critical determinant of NLRP3 inflammasome activation. Here, we genetically assess whether mitochondrial signalling represents a unified mechanism to explain how NLRP3 is activated by divergent stimuli. Neither co-deletion of the essential executioners of mitochondrial apoptosis BAK and BAX, nor removal of the mitochondrial permeability transition pore component cyclophilin D, nor loss of the mitophagy regulator Parkin, nor deficiency in MAVS affects NLRP3 inflammasome function. In contrast, caspase-8, a caspase essential for death-receptor-mediated apoptosis, is required for efficient Toll-like-receptor-induced inflammasome priming and cytokine production. Collectively, these results demonstrate that mitochondrial apoptosis is not required for NLRP3 activation, and highlight an important non-apoptotic role for caspase-8 in regulating inflammasome activation and pro-inflammatory cytokine levels.

  7. Death receptor-induced apoptosis reveals a novel interplay between the chromosomal passenger complex and CENP-C during interphase.

    PubMed

    Faragher, Alison J; Sun, Xiao-Ming; Butterworth, Michael; Harper, Nick; Mulheran, Mike; Ruchaud, Sandrine; Earnshaw, William C; Cohen, Gerald M

    2007-04-01

    Despite the fact that the chromosomal passenger complex is well known to regulate kinetochore behavior in mitosis, no functional link has yet been established between the complex and kinetochore structure. In addition, remarkably little is known about how the complex targets to centromeres. Here, in a study of caspase-8 activation during death receptor-induced apoptosis in MCF-7 cells, we have found that cleaved caspase-8 rapidly translocates to the nucleus and that this translocation is correlated with loss of the centromere protein (CENP)-C, resulting in extensive disruption of centromeres. Caspase-8 activates cytoplasmic caspase-7, which is likely to be the primary caspase responsible for cleavage of CENP-C and INCENP, a key chromosomal passenger protein. Caspase-mediated cleavage of CENP-C and INCENP results in their mislocalization and the subsequent mislocalization of Aurora B kinase. Our results demonstrate that the chromosomal passenger complex is displaced from centromeres as a result of caspase activation. Furthermore, mutation of the primary caspase cleavage sites of INCENP and CENP-C and expression of noncleavable CENP-C or INCENP prevent the mislocalization of the passenger complex after caspase activation. Our studies provide the first evidence for a functional interplay between the passenger complex and CENP-C.

  8. Triclosan Demonstrates Synergic Effect with Amphotericin B and Fluconazole and Induces Apoptosis-Like Cell Death in Cryptococcus neoformans

    PubMed Central

    Movahed, Elaheh; Tan, Grace Min Yi; Munusamy, Komathy; Yeow, Tee Cian; Tay, Sun Tee; Wong, Won Fen; Looi, Chung Yeng

    2016-01-01

    Objectives: Cryptococcus neoformans is an opportunistic fungus that causes fatal meningoencephalitis especially in AIDS patients. There is an increasing need for discovery of new anti-cryptococcal drugs due to emergence of resistance cases in recent years. In this study, we aim to elucidate the antifungal effect of triclosan against C. neoformans. Methods: Minimal inhibitory concentration (MIC) of triclosan in different C. neoformans strains was first examined. The in vitro interactions between triclosan and two standard anti-fungal drugs (amphotericin B and fluconazole) were further evaluated by microdilution checkerboard assay. Mechanism of triclosan fungicidal activity was then investigated by viewing the cell morphology under transmission electron microscope. Results: We reported that triclosan potently inhibited the growth of C. neoformans. A combination of triclosan with amphotericin B or with fluconazole enhanced their fungicidal effects. Triclosan-treated C. neoformans displayed characteristics such as nuclear chromatin condensation, extensive intracellular vacuolation and mitochondrial swelling, indicating that triclosan triggered apoptosis-like cell death. Conclusion: In summary, our report suggests triclosan as an independent drug or synergent for C. neoformans treatment. PMID:27047474

  9. Efficient Drug Delivery and Induction of Apoptosis in Colorectal Tumors Using a Death Receptor 5-Targeted Nanomedicine

    PubMed Central

    Schmid, Daniela; Fay, Francois; Small, Donna M; Jaworski, Jakub; Riley, Joel S; Tegazzini, Diana; Fenning, Cathy; Jones, David S; Johnston, Patrick G; Longley, Daniel B; Scott, Christopher J

    2014-01-01

    Death Receptor 5 (DR5) is a pro-apoptotic cell-surface receptor that is a potential therapeutic target in cancer. Despite the potency of DR5-targeting agents in preclinical models, the translation of these effects into the clinic remains disappointing. Herein, we report an alternative approach to exploiting DR5 tumor expression using antibody-targeted, chemotherapy-loaded nanoparticles. We describe the development of an optimized polymer-based nanotherapeutic incorporating both a functionalized polyethylene glycol (PEG) layer and targeting antibodies to limit premature phagocytic clearance whilst enabling targeting of DR5-expressing tumor cells. Using the HCT116 colorectal cancer model, we show that following binding to DR5, the nanoparticles activate caspase 8, enhancing the anti-tumor activity of the camptothecin payload both in vitro and in vivo. Importantly, the combination of nanoparticle-induced DR5 clustering with camptothecin delivery overcomes resistance to DR5-induced apoptosis caused by loss of BAX or overexpression of anti-apoptotic FLIP. This novel approach may improve the clinical activity of DR5-targeted therapeutics while increasing tumor-specific delivery of systemically toxic chemotherapeutics. PMID:25200008

  10. Nitric oxide releasing iron oxide magnetic nanoparticles for biomedical applications: cell viability, apoptosis and cell death evaluations

    NASA Astrophysics Data System (ADS)

    de Lima, R.; de Oliveira, J. L.; Ludescher, A.; Molina, M. M.; Itri, R.; Seabra, A. B.; Haddad, P. S.

    2013-04-01

    Nitric oxide (NO) is involved in several physiological and pathophysiological processes, such as control of vascular tone and immune responses against microbes. Thus, there is great interest in the development of NO-releasing materials to carry and deliver NO for biomedical applications. Magnetic iron oxide nanoparticles have been used in important pharmacological applications, including drug-delivery. In this work, magnetic iron oxide nanoparticles were coated with thiol-containing hydrophilic ligands: mercaptosuccinic acid (MSA) and dimercaptosuccinic acid (DMSA). Free thiol groups on the surface of MSA- or DMSA- coated nanoparticles were nitrosated, leading to the formation of NO-releasing iron oxide nanoparticles. The cytotoxicity of MSA- or DMSA-coated magnetic nanoparticles (MNP) (thiolated nanoparticles) and nitrosated MSA- or nitrosated DMSA- coated MNPs (NO-releasing nanoparticles) were evaluated towards human lymphocytes. The results showed that MNP-MSA and MNP-DMSA have low cytotoxicity effects. On the other hand, NO-releasing MNPs were found to increase apoptosis and cell death compared to free NO-nanoparticles. Therefore, the cytotoxicity effects observed for NO-releasing MNPs may result in important biomedical applications, such as the treatment of tumors cells.

  11. 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

    PubMed Central

    Denning, Daniel P.; Hatch, Victoria; Horvitz, H. Robert

    2013-01-01

    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 corpses in parallel to

  12. Nuclear localized protein-1 (Nulp1) increases cell death of human osteosarcoma cells and binds the X-linked inhibitor of apoptosis protein

    SciTech Connect

    Steen, Hakan; Lindholm, Dan

    2008-02-08

    Nuclear localized protein-1 (Nulp1) is a recently identified gene expressed in mouse and human tissues particularly during embryonic development. Nulp1 belongs to the family of basic helix-loop-helix (bHLH) proteins that are important in development. The precise function of Nulp1 in cells is however not known. We observed that overexpression of Nulp1 induces a large increase in cell death of human osteosarcoma Saos2 cells with DNA fragmentation. In mouse N2A neuroblastoma cells Nulp1 affected cell proliferation and sensitized cells towards death induced by staurosporine. Staining using a novel antibody localized Nulp1 mainly to the cell nucleus and to some extent to the cytoplasm. Nulp1 binds the X-linked inhibitor of apoptosis protein (XIAP) and this interaction was increased during cell death. These results indicate that Nulp1 plays a role in cell death control and may influence tumor growth.

  13. Stronger Dopamine D1 Receptor-Mediated Neurotransmission in Dyskinesia.

    PubMed

    Farré, Daniel; Muñoz, Ana; Moreno, Estefanía; Reyes-Resina, Irene; Canet-Pons, Júlia; Dopeso-Reyes, Iria G; Rico, Alberto J; Lluís, Carme; Mallol, Josefa; Navarro, Gemma; Canela, Enric I; Cortés, Antonio; Labandeira-García, José L; Casadó, Vicent; Lanciego, José L; Franco, Rafael

    2015-12-01

    Radioligand binding assays to rat striatal dopamine D1 receptors showed that brain lateralization of the dopaminergic system were not due to changes in expression but in agonist affinity. D1 receptor-mediated striatal imbalance resulted from a significantly higher agonist affinity in the left striatum. D1 receptors heteromerize with dopamine D3 receptors, which are considered therapeutic targets for dyskinesia in parkinsonian patients. Expression of both D3 and D1-D3 receptor heteromers were increased in samples from 6-hydroxy-dopamine-hemilesioned rats rendered dyskinetic by treatment with 3, 4-dihydroxyphenyl-L-alanine (L-DOPA). Similar findings were obtained using striatal samples from primates. Radioligand binding studies in the presence of a D3 agonist led in dyskinetic, but not in lesioned or L-DOPA-treated rats, to a higher dopamine sensitivity. Upon D3-receptor activation, the affinity of agonists for binding to the right striatal D1 receptor increased. Excess dopamine coming from L-DOPA medication likely activates D3 receptors thus making right and left striatal D1 receptors equally responsive to dopamine. These results show that dyskinesia occurs concurrently with a right/left striatal balance in D1 receptor-mediated neurotransmission.

  14. Receptor-mediated control of regulatory volume decrease (RVD) and apoptotic volume decrease (AVD)

    PubMed Central

    Okada, Yasunobu; Maeno, Emi; Shimizu, Takahiro; Dezaki, Katsuya; Wang, Jun; Morishima, Shigeru

    2001-01-01

    A fundamental property of animal cells is the ability to regulate their own cell volume. Even under hypotonic stress imposed by either decreased extracellular or increased intracellular osmolarity, the cells can re-adjust their volume after transient osmotic swelling by a mechanism known as regulatory volume decrease (RVD). In most cell types, RVD is accomplished mainly by KCl efflux induced by parallel activation of K+ and Cl− channels. We have studied the molecular mechanism of RVD in a human epithelial cell line (Intestine 407). Osmotic swelling results in a significant increase in the cytosolic Ca2+ concentration and thereby activates intermediate-conductance Ca2+-dependent K+ (IK) channels. Osmotic swelling also induces ATP release from the cells to the extracellular compartment. Released ATP stimulates purinergic ATP (P2Y2) receptors, thereby inducing phospholipase C-mediated Ca2+ mobilization. Thus, RVD is facilitated by stimulation of P2Y2 receptors due to augmentation of IK channels. In contrast, stimulation of another G protein-coupled Ca2+-sensing receptor (CaR) enhances the activity of volume-sensitive outwardly rectifying Cl− channels, thereby facilitating RVD. Therefore, it is possible that Ca2+ efflux stimulated by swelling-induced and P2Y2 receptor-mediated intracellular Ca2+ mobilization activates the CaR, thereby secondarily upregulating the volume-regulatory Cl− conductance. On the other hand, the initial process towards apoptotic cell death is coupled to normotonic cell shrinkage, called apoptotic volume decrease (AVD). Stimulation of death receptors, such as TNFα receptor and Fas, induces AVD and thereafter biochemical apoptotic events in human lymphoid (U937), human epithelial (HeLa), mouse neuroblastoma × rat glioma hybrid (NG108-15) and rat phaeochromocytoma (PC12) cells. In those cells exhibiting AVD, facilitation of RVD is always observed. Both AVD induction and RVD facilitation as well as succeeding apoptotic events can be

  15. Pomegranate Juice Polyphenols Induce Macrophage Death via Apoptosis as Opposed to Necrosis Induced by Free Radical Generation: A Central Role for Oxidative Stress.

    PubMed

    Rom, Oren; Volkova, Nina; Nandi, Sukhendu; Jelinek, Raz; Aviram, Michael

    2016-08-01

    At high concentrations, polyphenols induce cell death, and the polyphenols-rich pomegranate juice (PJ), known for its antioxidative/antiatherogenic properties, can possibly affect cell death, including macrophage death involved in atherogenesis. In the present study, apoptotic/necrotic macrophage death was analyzed in J774A.1 macrophages and in peritoneal macrophages isolated from atherosclerotic apoE-/- mice treated with PJ. The effects of PJ were compared with those of the free radical generator 2, 2'-azobis (2-amidinopropane) dihydrochloride (AAPH). Both PJ and AAPH significantly increased J774A.1 macrophage death; however, flow cytometric and microscopic analyses using annexin V/propidium iodide revealed that PJ increased the early apoptosis of the macrophage dose dependently (up to 2.5-fold, P < 0.01), whereas AAPH caused dose-dependent increases in late apoptosis/necrosis (up to 12-fold, P < 0.001). Unlike PJ, AAPH-induced macrophage death was associated with increased intracellular oxidative stress (up to 7-fold, P < 0.001) and with lipid stress demonstrated by triglyceride accumulation (up to 3-fold, P < 0.01) and greater chromatic vesicle response to culture medium (up to 5-fold, P < 0.001). Accordingly, recombinant paraoxonase 1, which hydrolyzes oxidized lipids, attenuated macrophage death induced by AAPH, but not by PJ. Similar apoptotic and oxidative effects were found in macrophages from apoE-/- mice treated with PJ or AAPH. As macrophage apoptotic/necrotic death has considerable impact on atherosclerosis progression, these findings may provide novel mechanisms for the antiatherogenicity of PJ.

  16. The multiple functions of cytochrome c and their regulation in life and death decisions of the mammalian cell: from respiration to apoptosis

    PubMed Central

    Hüttemann, Maik; Pecina, Petr; Rainbolt, Matthew; Sanderson, Thomas H.; Kagan, Valerian E.; Samavati, Lobelia; Doan, Jeffrey W.; Lee, Icksoo

    2011-01-01

    Cytochrome c (Cytc) is essential in mitochondrial electron transport and intrinsic type II apoptosis. Mammalian Cytc also scavenges reactive oxygen species (ROS) under healthy conditions, produces ROS with the co-factor p66Shc, and oxidizes cardiolipin during apoptosis. The recent finding that Cytc is phosphorylated in vivo underpins a model for the pivotal role of Cytc regulation in making life and death decisions. An apoptotic sequence of events is proposed involving changes in Cytc phosphorylation, increased ROS via increased mitochondrial membrane potentials or the p66Shc pathway, the oxidation of cardiolipin by Cytc, and its release from the mitochondria. Cytc regulation in respiration and cell death is discussed in a human disease context including neurodegenerative and cardiovascular diseases, cancer, and sepsis. PMID:21296189

  17. The Amaryllidaceae isocarbostyril narciclasine induces apoptosis by activation of the death receptor and/or mitochondrial pathways in cancer cells but not in normal fibroblasts.

    PubMed

    Dumont, Patrick; Ingrassia, Laurent; Rouzeau, Sébastien; Ribaucour, Fabrice; Thomas, Stéphanie; Roland, Isabelle; Darro, Francis; Lefranc, Florence; Kiss, Robert

    2007-09-01

    Our study has shown that the Amaryllidaceae isocarbostyril narciclasine induces marked apoptosis-mediated cytotoxic effects in human cancer cells but not in normal fibroblasts by triggering the activation of the initiator caspases of the death receptor pathway (caspase-8 and caspase-10) at least in human MCF-7 breast and PC-3 prostate carcinoma cells. The formation of the Fas and death receptor 4 (DR4) death-inducing signaling complex was clearly evidenced in MCF-7 and PC-3 cancer cells. Caspase-8 was found to interact with Fas and DR4 receptors on narciclasine treatment. However, narciclasine-induced downstream apoptotic pathways in MCF-7 cells diverged from those in PC-3 cells, where caspase-8 directly activated effector caspases such as caspase-3 in the absence of any further release of mitochondrial proapoptotic effectors. In contrast, in MCF-7 cells, the apoptotic process was found to require an amplification step that is mitochondria-dependent, with Bid processing, release of cytochrome c, and caspase-9 activation. It is postulated that the high selectivity of narciclasine to cancer cells might be linked, at least in part, to this activation of the death receptor pathway. Normal human fibroblasts appear approximately 250-fold less sensitive to narciclasine, which does not induce apoptosis in these cells probably due to the absence of death receptor pathway activation.

  18. BID links ferroptosis to mitochondrial cell death pathways.

    PubMed

    Neitemeier, Sandra; Jelinek, Anja; Laino, Vincenzo; Hoffmann, Lena; Eisenbach, Ina; Eying, Roman; Ganjam, Goutham K; Dolga, Amalia M; Oppermann, Sina; Culmsee, Carsten

    2017-03-09

    Ferroptosis has been defined as an oxidative and iron-dependent pathway of regulated cell death that is distinct from caspase-dependent apoptosis and established pathways of death receptor-mediated regulated necrosis. While emerging evidence linked features of ferroptosis induced e.g. by erastin-mediated inhibition of the Xc(-) system or inhibition of glutathione peroxidase 4 (Gpx4) to an increasing number of oxidative cell death paradigms in cancer cells, neurons or kidney cells, the biochemical pathways of oxidative cell death remained largely unclear. In particular, the role of mitochondrial damage in paradigms of ferroptosis needs further investigation. In the present study, we find that erastin-induced ferroptosis in neuronal cells was accompanied by BID transactivation to mitochondria, loss of mitochondrial membrane potential, enhanced mitochondrial fragmentation and reduced ATP levels. These hallmarks of mitochondrial demise are also established features of oxytosis, a paradigm of cell death induced by Xc(-) inhibition by millimolar concentrations of glutamate. Bid knockout using CRISPR/Cas9 approaches preserved mitochondrial integrity and function, and mediated neuroprotective effects against both, ferroptosis and oxytosis. Furthermore, the BID-inhibitor BI-6c9 inhibited erastin-induced ferroptosis, and, in turn, the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 prevented mitochondrial dysfunction and cell death in the paradigm of oxytosis. These findings show that mitochondrial transactivation of BID links ferroptosis to mitochondrial damage as the final execution step in this paradigm of oxidative cell death.

  19. ERK-mediated activation of Fas apoptotic inhibitory molecule 2 (Faim2) prevents apoptosis of 661W cells in a model of detachment-induced photoreceptor cell death.

    PubMed

    Besirli, Cagri G; Zheng, Qiong-Duon; Reed, David M; Zacks, David N

    2012-01-01

    In this study, we examined the role of Fas apoptotic inhibitory molecule 2 (Faim2), an inhibitor of the Fas signaling pathway, and its regulation by stress kinase signaling during Fas-mediated apoptosis of 661W cells, an immortalized photoreceptor-like cell line Treatment of 661W cells with a Fas-activating antibody led to increased levels of Faim2. Both ERK and JNK stress kinase pathways were activated in Fas-treated 661W cells, but only the inhibition of the ERK pathway reduced the levels of Faim2. Blocking the ERK pathway using a pharmacological inhibitor increased the susceptibility of 661W cells to Fas-induced caspase activation and apoptosis. When the levels of Faim2 were reduced in 661W cells by siRNA knockdown, Fas activating antibody treatment resulted in earlier and more robust caspase activation, and increased cell death. These results demonstrate that Faim2 acts as a neuroprotectant during Fas-mediated apoptosis of 661W cells. The expression of Faim2 is triggered, at least in part, by Fas-receptor activation and subsequent ERK signaling. Our findings identify a novel protective pathway that auto-regulates Fas-induced photoreceptor apoptosis in vitro. Modulation of this pathway to increase Faim2 expression may be a potential therapeutic option to prevent photoreceptor death.

  20. Induction of Cell Death by Betulinic Acid through Induction of Apoptosis and Inhibition of Autophagic Flux in Microglia BV-2 Cells.

    PubMed

    Seo, Jeongbin; Jung, Juneyoung; Jang, Dae Sik; Kim, Joungmok; Kim, Jeong Hee

    2017-03-10

    Betulinic acid (BA), a natural pentacyclic triterpene found in many medicinal plants is known to have various biological activity including tumor suppression and anti-inflammatory effects. In this study, the cell-death induction effect of BA was investigated in BV-2 microglia cells. BA was cytotoxic to BV-2 cells with IC₅₀ of approximately 2.0 μM. Treatment of BA resulted in a dosedependent chromosomal DNA degradation, suggesting that these cells underwent apoptosis. Flow cytometric analysis further confirmed that BA-treated BV-2 cells showed hypodiploid DNA content. BA treatment triggered apoptosis by decreasing Bcl-2 levels, activation of capase-3 protease and cleavage of PARP. In addition, BA treatment induced the accumulation of p62 and the increase in conversion of LC3-I to LC3-II, which are important autophagic flux monitoring markers. The increase in LC3-II indicates that BA treatment induced autophagosome formation, however, accumulation of p62 represents that the downstream autophagy pathway is blocked. It is demonstrated that BA induced cell death of BV-2 cells by inducing apoptosis and inhibiting autophagic flux. These data may provide important new information towards understanding the mechanisms by which BA induce cell death in microglia BV-2 cells.

  1. Saving cells from ultrasound-induced apoptosis: quantification of cell death and uptake following sonication and effects of targeted calcium chelation

    PubMed Central

    Hutcheson, J.D.; Schlicher, R.K.; Hicks, H.K.; Prausnitz, M.R.

    2010-01-01

    Applications of ultrasound for non-invasive drug and gene delivery have been limited by associated cell death due to sonication. In this study, we sought to quantify the distribution of cellular bioeffects caused by low-frequency ultrasound (24 kHz) and test the hypothesis that Ca2+ chelation after sonication can shift this distribution by saving cells from death by apoptosis. Using flow cytometry, we quantitatively categorized sonicated cells among four populations: (1) cells that appear largely unaffected, (2) cells reversibly permeabilized, (3) cells rendered nonviable during sonication and (4) cells that appear to be viable shortly after sonication, but later undergo apoptosis and die. By monitoring cells for 6 h after ultrasound exposure, we found that up to 15% of intact cells fell into this final category. Those apoptotic cells initially had the highest levels of uptake of a marker compound, calcein; also had highly elevated levels of intracellular Ca2+; and contained an estimated plasma membrane wound radius of 100 – 300 nm. Finally, we showed that chelation of intracellular Ca2+ after sonication reduced apoptosis by up to 44%, thereby providing a strategy to save cells. We conclude that cells can be saved from ultrasound-induced death by appropriate selection of ultrasound conditions and Ca2+ chelation after sonication. PMID:20447754

  2. Chaetocin induces endoplasmic reticulum stress response and leads to death receptor 5-dependent apoptosis in human non-small cell lung cancer cells.

    PubMed

    Liu, Xianfang; Guo, Sen; Liu, Xiangguo; Su, Ling

    2015-11-01

    Epigenetic abnormalities are associated with non-small cell lung cancer (NSCLC) initiation and progression. Epigenetic drugs are being studied and in clinical trials. However, the molecular mechanism underlying the apoptosis by the epigenetic agents remains unclear. SUV39H1 is an important methyl-transferase for lysine 9 on histone H3 and usually related to gene transcriptional suppression, and chaetocin acts as the inhibitor of SUV39H1. We demonstrated here that chaetocin effectively suppressed the growth of multiple lung cancer cells through inducing apoptosis in a death receptor 5 (DR5)-dependent manner. Chaetocin treatment activated endoplasmic reticulum (ER) stress which gave rise to the up-regulation of ATF3 and CHOP. Furthermore, ATF3 and CHOP contributed to the induction of DR5 and subsequent apoptosis. When SUV39H1 was silenced with siRNA, the expression of ATF3, CHOP and DR5 was elevated. Thereafter, knockdown of SUV39H1 induced apoptosis in NSCLC cells. In summary, chaetocin pharmacologically inhibits the activity of SUV39H1 which provokes ER stress and results in up-regulation of ATF3 and CHOP, leading to DR5-dependent apoptosis eventually. These findings provide a novel interpretation on the anti-neoplastic activity of epigenetic drugs as a new therapeutic approach in NSCLC.

  3. Mitochondrial calcium signalling and cell death: approaches for assessing the role of mitochondrial Ca2+ uptake in apoptosis

    PubMed Central

    Hajnóczky, György; Csordás, György; Das, Sudipto; Garcia-Perez, Cecilia; Saotome, Masao; Roy, Soumya Sinha; Yi, Muqing

    2009-01-01

    Summary Local Ca2+ transfer between adjoining domains of the sarcoendoplasmic reticulum (ER/SR) and mitochondria allows ER/SR Ca2+ release to activate mitochondrial Ca2+ uptake and to evoke a matrix [Ca2+] ([Ca2+]m) rise. [Ca2+]m exerts control on several steps of energy metabolism to synchronize ATP generation with cell function. However, calcium signal propagation to the mitochondria may also ignite a cell death program through opening of the permeability transition pore (PTP). This occurs when the Ca2+ release from the ER/SR is enhanced or is coincident with sensitization of the PTP. Recent studies have shown that several pro-apoptotic factors, including members of the Bcl-2 family proteins and reactive oxygen species (ROS) regulate the Ca2+ sensitivity of both the Ca2+ release channels in the ER and the PTP in the mitochondria. To test the relevance of the mitochondrial Ca2+ accumulation in various apoptotic paradigms, methods are available for buffering of [Ca2+], for dissipation of the driving force of the mitochondrial Ca2+ uptake and for inhibition of the mitochondrial Ca2+ transport mechanisms. However, in intact cells, the efficacy and the specificity of these approaches have to be established. Here we discuss mechanisms that recruit the mitochondrial calcium signal to a pro-apoptotic cascade and the approaches available for assessment of the relevance of the mitochondrial Ca2+ handling in apoptosis. We also present a systematic evaluation of the effect of ruthenium red and Ru360, two inhibitors of mitochondrial Ca2+ uptake on cytosolic [Ca2+] and [Ca2+]m in intact cultured cells. PMID:17074387

  4. Apoptosis-like death in Leishmania donovani promastigotes induced by eugenol-rich oil of Syzygium aromaticum.

    PubMed

    Islamuddin, Mohammad; Sahal, Dinkar; Afrin, Farhat

    2014-01-01

    Leishmaniasis consists of a complex spectrum of infectious diseases with worldwide distribution of which visceral leishmaniasis or kala-azar caused by Leishmania donovani is the most devastating. In the absence of vaccines, chemotherapy remains the mainstay for the control of leishmaniasis. The drugs of choice are expensive and associated with multiple adverse side effects. Because of these limitations, the development of new antileishmanial compounds is imperative and plants offer prospects in this regard. The present work was conducted to study the antileishmanial potential of oil from Syzygium aromaticum flower buds (clove). The S. aromaticum oil was characterized by gas chromatography and GC-MS and eugenol as well as eugenyl acetate were found to be the most abundant compounds, composing 59.75 % and 29.24 %, respectively of the oil. Our findings have shown that eugenol-rich essential oil from S. aromaticum (EROSA) possesses significant activity against L. donovani, with 50 % inhibitory concentration of 21 ± 0.16 µg ml(-1) and 15.24 ± 0.14 µg ml(-1), respectively, against promastigotes and intracellular amastigotes. Alterations in cellular morphology and growth reversibility assay substantiated the leishmanicidal activity of EROSA. The leishmanicidal effect was mediated via apoptosis as confirmed by externalization of phosphatidylserine, DNA nicking by TdT-mediated dUTP nick-end labelling (TUNEL) assay, dyskinetoplastidy, cell cycle arrest at sub-G0-G1 phase, loss of mitochondrial membrane potential and reactive oxygen species generation. EROSA presented no adverse cytotoxic effects against murine macrophages even at 200 µg ml(-1). Our studies authenticate the promising antileishmanial activity of EROSA, which is mediated by programmed cell death, and, accordingly, EROSA may be a source of novel agents for the treatment of leishmaniasis.

  5. Loss of FAS/FASL signaling does not reduce apoptosis in Sharpin null mice.

    PubMed

    Potter, Christopher S; Silva, Kathleen A; Kennedy, Victoria E; Stearns, Timothy M; HogenEsch, Harm; Sundberg, John P

    2017-01-17

    Mice with mutations in SHANK-associated RH domain interactor (Sharpin) develop a hypereosinophilic auto-inflammatory disease known as chronic proliferative dermatitis. Affected mice have increased apoptosis in the keratinocytes of the skin, esophagus, and forestomach driven by extrinsic TNF receptor mediated apoptotic signaling pathways. FAS receptor signaling is an extrinsic apoptotic signaling mechanism frequently involved in inflammatory skin diseases. Compound mutations in Sharpin and Fas or Fasl were created to determine if these death domain proteins influenced the cutaneous phenotype in Sharpin null mice. Both Sharpin/Fas and Sharpin/Fasl compound mutant mice developed an auto-inflammatory phenotype similar to that seen in Sharpin null mice indicating that initiation of apoptosis by FAS signaling is likely not involved in the pathogenesis of this disease. This article is protected by copyright. All rights reserved.

  6. Mono(2-ethylhexyl) phthalate induces apoptosis in p53-silenced L02 cells via activation of both mitochondrial and death receptor pathways.

    PubMed

    Yang, Guangtao; Zhang, Wenjuan; Qin, Qizhi; Wang, Jing; Zheng, Hongyan; Xiong, Wei; Yuan, Jing

    2015-09-01

    Mono(2-ethylhexyl) phthalate (MEHP) is one of the main metabolites of di(2-ethylhexyl) phthalate. The evidence shows that DEHP may exert its toxic effects primarily via MEHP, which is 10-fold more potent than its parent compound in toxicity in vitro. MEHP-induced apoptosis is mediated by either p53-dependent or -independent pathway. However, the detailed mechanism of its toxicity remains unclear. In this study, immortalized normal human liver cell line L02 was chosen, as an in vitro model of nonmalignant liver, to elucidate the role of p53 in MEHP-induced apoptosis. The cells were treated with MEHP (6.25, 12.50, 25.00, 50.00, and 100.00 μM) for 24 and 36 h, then small interfering RNA (siRNA) was used to specifically silence p53 gene of L02 cells. The results indicated that MEHP caused oxidative DNA damage and apoptosis in L02 cells were associated with the p53 signaling pathway. Further study found that MEHP (50.00 and 100.00 μM) induced apoptosis in p53-silenced L02 cells, along with the up-regulations of Fas and FasL proteins as well as increased the Bax/Bcl-2 ratio and Caspase 3, 8, and 9 activities. Additionally, both FasL inhibitor (AF-016) and Caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp- fluoromethylketone (Z-VAD-FMK) could prevent the cell apoptosis induced by MEHP. The findings suggested that MEHP-induced apoptosis in L02 cells involving a Caspases-mediated mitochondrial signaling pathway and/or death receptor pathway. p53 was not absolutely necessary for MEHP-induced L02 cell apoptosis.

  7. Hyaluronan synthase-2 upregulation protects smpd3-deficient fibroblasts against cell death induced by nutrient deprivation, but not against apoptosis evoked by oxidized LDL

    PubMed Central

    Garoby-Salom, Sandra; Rouahi, Myriam; Mucher, Elodie; Auge, Nathalie; Salvayre, Robert; Negre-Salvayre, Anne

    2014-01-01

    The neutral type 2 sphingomyelinase (nSMase2) hydrolyzes sphingomyelin and generates ceramide, a major bioactive sphingolipid mediator, involved in growth arrest and apoptosis. The role of nSMase2 in apoptosis is debated, and apparently contradictory results have been observed on fibroblasts isolated from nSMase2-deficient fragilitas ossium (homozygous fro/fro) mice. These mice exhibit a severe neonatal dysplasia, a lack of long bone mineralization and delayed apoptosis patterns of hypertrophic chondrocytes in the growth plate. We hypothesized that apoptosis induced by nutrient deprivation, which mimics the environmental modifications of the growth plate, requires nSMase2 activation. In this study, we have compared the resistance of fro/fro fibroblasts to different death inducers (oxidized LDL, hydrogen peroxide and nutrient starvation). The data show that nSMase2-deficient fro/fro cells resist to apoptosis evoked by nutrient starvation (fetal calf serum/glucose/pyruvate-free DMEM), whereas wt fibroblasts die after 48 h incubation in this medium. In contrast, oxidized LDL and hydrogen peroxide are similarly toxic to fro/fro and wt fibroblasts, indicating that nSMase2 is not involved in the mechanism of toxicity evoked by these agents. Interestingly, wt fibroblasts treated with the SMase inhibitor GW4869 were more resistant to starvation-induced apoptosis. The resistance of fro/fro cells to starvation-induced apoptosis is associated with an increased expression of hyaluronan synthase 2 (HAS2) mRNAs and protein, which is inhibited by ceramide. In wt fibroblasts, this HAS2 rise and its protective effect did not occur, but exogenously added HA exhibited a protective effect against starvation-induced apoptosis. The protective mechanism of HAS2 involves an increased expression of the heat-shock protein Hsp72, a chaperone with antiapoptotic activity. Taken together, these results highlight the role of nSMase2 in apoptosis evoked by nutrient starvation that could

  8. Menthol inhibits 5-HT3 receptor-mediated currents.

    PubMed

    Ashoor, Abrar; Nordman, Jacob C; Veltri, Daniel; Yang, Keun-Hang Susan; Shuba, Yaroslav; Al Kury, Lina; Sadek, Bassem; Howarth, Frank C; Shehu, Amarda; Kabbani, Nadine; Oz, Murat

    2013-11-01

    The effects of alcohol monoterpene menthol, a major active ingredient of the peppermint plant, were tested on the function of human 5-hydroxytryptamine type 3 (5-HT3) receptors expressed in Xenopus laevis oocytes. 5-HT (1 μM)-evoked currents recorded by two-electrode voltage-clamp technique were reversibly inhibited by menthol in a concentration-dependent (IC50 = 163 μM) manner. The effects of menthol developed gradually, reaching a steady-state level within 10-15 minutes and did not involve G-proteins, since GTPγS activity remained unaltered and the effect of menthol was not sensitive to pertussis toxin pretreatment. The actions of menthol were not stereoselective as (-), (+), and racemic menthol inhibited 5-HT3 receptor-mediated currents to the same extent. Menthol inhibition was not altered by intracellular 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid injections and transmembrane potential changes. The maximum inhibition observed for menthol was not reversed by increasing concentrations of 5-HT. Furthermore, specific binding of the 5-HT3 antagonist [(3)H]GR65630 was not altered in the presence of menthol (up to 1 mM), indicating that menthol acts as a noncompetitive antagonist of the 5-HT3 receptor. Finally, 5-HT3 receptor-mediated currents in acutely dissociated nodose ganglion neurons were also inhibited by menthol (100 μM). These data demonstrate that menthol, at pharmacologically relevant concentrations, is an allosteric inhibitor of 5-HT3 receptors.

  9. Synergistic induction of apoptosis and caspase-independent autophagic cell death by a combination of nitroxide Tempo and heat shock in human leukemia U937 cells.

    PubMed

    Zhao, Qing-Li; Fujiwara, Yoshisada; Kondo, Takashi

    2010-10-01

    We have shown that heat stress or a superoxide dismutase mimic nitroxide, Tempo, induces apoptosis, while their combination causes nonapoptotic cell death; however, the underlying mechanism for this switch remains unclear. Here we identified for the first time that 10 mM Tempo present during heating at 44°C for 30 min rapidly induced autophagy in U937 leukemic cells in spite of Bax activation and mitochondrial outer membrane (MOM) permeabilization. This co-treatment inhibited the processing of heat-activated procaspases-2, -8, -9 and -3 into active small subunits, leading to the inhibition of caspase-dependent apoptosis, and instead caused the induction of autophagy. The inactivation of caspases, a key event, could result from oxidation of active-site-CysSH of all caspases by a prooxidant oxo-ammonium cation, an intermediate derived Tempo during dismutation of heat-induced superoxide anion. In addition, the co-treatment caused mitochondrial calcium overloads, the mitochondrial inner membrane permeabilization, profound mitochondrial dysfunction, and liberation of Beclin 1 from the Bcl-2/Beclin 1 complex, all of which contributed to induction of autophagy. These autophagic cells underwent propidium iodide-positive necrosis in a delayed fashion, leading to the complete proliferative inhibition. Remarkably, ruthenium red and BAPTA, which interfere with mitochondrial calcium uptake, facilitated autophagic necrotic death. Cyclosporin A, which binds to cyclophilin D, had a similar necrotic effect. 3-Methyladenine facilitated the necrosis of autophagic cells. In contrast, 5 mM Tempo-44°C/10 min or 44°C/30 min induced Bax-mediated MOM permeabilization and caspase-dependent apoptosis more potently than Tempo alone. Thus, Tempo is a unique thermosensitizer to synergistically induce apoptosis and autophagic cell death.

  10. Involvement of the PI3K and ERK signaling pathways in largemouth bass virus-induced apoptosis and viral replication.

    PubMed

    Huang, Xiaohong; Wang, Wei; Huang, Youhua; Xu, Liwen; Qin, Qiwei

    2014-12-01

    Increased reports demonstrated that largemouth Bass, Micropterus salmoides in natural and artificial environments were always suffered from an emerging iridovirus disease, largemouth Bass virus (LMBV). However, the underlying mechanism of LMBV pathogenesis remained largely unknown. Here, we investigated the cell signaling events involved in virus induced cell death and viral replication in vitro. We found that LMBV infection in epithelioma papulosum cyprini (EPC) cells induced typical apoptosis, evidenced by the appearance of apoptotic bodies, cytochrome c release, mitochondrial membrane permeabilization (MMP) destruction and reactive oxygen species (ROS) generation. Two initiators of apoptosis, caspase-8 and caspase-9, and the executioner of apoptosis, caspase-3, were all significantly activated with the infection time, suggested that not only mitochondrion-mediated, but also death receptor-mediated apoptosis were involved in LMBV infection. Reporter gene assay showed that the promoter activity of transcription factors including p53, NF-κB, AP-1 and cAMP response element-binding protein (CREB) were decreased during LMBV infection. After treatment with different signaling pathway inhibitors, virus production were significantly suppressed by the inhibition of phosphatidylinositol 3-kinase (PI3K) pathway and extracellular-signal-regulated kinases (ERK) signaling pathway. Furthermore, LMBV infection induced apoptosis was enhanced by PI3K inhibitor LY294002, but decreased by addition of ERK inhibitor UO126. Therefore, we speculated that apoptosis was sophisticatedly regulated by a series of cell signaling events for efficient virus propagation. Taken together, our results provided new insights into the molecular mechanism of ranavirus infection.

  11. Antigen receptor-induced B lymphocyte apoptosis mediated via a protease of the caspase family.

    PubMed

    Andjelic, S; Liou, H C

    1998-02-01

    An extensive body of data, in a variety of systems, denoted the caspase family of proteases as a key player in the execution of programmed cell death. This family consists of cysteine proteases that cleave after asparagine-containing motifs. It is well established that the caspases are essential for the apoptosis mediated by Fas (CD95) and TNF receptor p55, molecules that contain the "death domain" in the cytoplasmic tail. However, little is known about the mechanisms underlying the antigen receptor-mediated cell death in B lymphocytes, a process instrumental in negative selection of potentially autoreactive B cells. Here, we investigated the involvement of caspases in cell death triggered via the antigen receptor in B lymphocytes (BCR) by using specific inhibitors. Initially, we used a well-established cell line, CH31, which is a model of B cell tolerance, to demonstrate that these proteases indeed participate in the BCR-induced apoptotic pathway. Next, we confirmed the physiological relevance of the caspase-mediated cell death pathway in splenic B cell populations isolated ex vivo that were induced to undergo apoptosis by extensive cross-linking of their BCR. Most interestingly, our data demonstrated that caspases regulate not only the nuclear DNA fragmentation, but also the surface membrane phosphatidylserine translocation as well as the degradation of a specific nuclear substrate. Taken together, this report supports the hypothesis that regulation of the caspase family is crucial in controlling the life/death decision in B lymphocytes mediated by the antigen receptor signal transduction.

  12. Characterization of HJ-PI01 as a novel Pim-2 inhibitor that induces apoptosis and autophagic cell death in triple-negative human breast cancer

    PubMed Central

    Zhao, Yu-qian; Yin, Yi-qiong; Liu, Jie; Wang, Gui-hua; Huang, Jian; Zhu, Ling-juan; Wang, Jin-hui

    2016-01-01

    Aim: Pim-2 is a short-lived serine/threonine kinase, which plays a key role in metastasis of breast cancer through persistent activation of STAT3. Although the crystal structure of Pim-2 has been reported, but thus far no specific Pim-2-targeted compounds have been reported. In this study, we identified a novel Pim-2 inhibitor, HJ-PI01, by in silico analysis and experimental validation. Methods: The protein-protein interaction (PPI) network, chemical synthesis, molecular docking, and molecular dynamics (MD) simulations were used to design and discover the new Pim-2 inhibitor HJ-PI01. The anti-tumor effects of HJ-PI01 were evaluated in human breast MDA-MB-231, MDA-MB-468, MDA-MB-436, MCF-7 cells in vitro and in MDA-MB-231 xenograft mice, which were treated with HJ-PI01 (40 mg·kg−1·d−1, ig) with or without lienal polypeptide (50 mg·kg−1·d−1, ip) for 10 d. The apoptosis/autophage-inducing mechanisms of HJ-PI01 were elucidated using Western blots, immunoblots, flow cytometry, transmission electron microscopy and fluorescence microscopy. Results: Based on the PrePPI network, the potential partners interacting with Pim-2 in regulating apoptosis (160 protein pairs) and autophagy (47 protein pairs) were identified. Based on the structural characteristics of Pim-2, a total of 15 compounds (HJ-PI01 to HJ-P015) were synthesized, which showed moderate or remarkable anti-proliferative potency in the human breast cancer cell lines tested. The most effective compound HJ-PI01 exerted a robust inhibition on MDA-MB-231 cells compared with chlorpromazine and the pan-Pim inhibitor PI003. Molecular dynamics (MD) simulation revealed that HJ-PI01 had a good binding score with Pim-2. Moreover, HJ-PI01 (300 nmol/L) induced death receptor-dependent and mitochondrial apoptosis as well as autophagic death in MDA-MB-231 cells. In MDA-MB-231 xenograft mice, administration of HJ-PI01 remarkably inhibited the tumor growth and induced tumor cell apoptosis in vivo. Co-administration of

  13. Andrographolide Analogue Induces Apoptosis and Autophagy Mediated Cell Death in U937 Cells by Inhibition of PI3K/Akt/mTOR Pathway

    PubMed Central

    Kumar, Deepak; Das, Bimolendu; Sen, Rupashree; Kundu, Priyanka; Manna, Alak; Sarkar, Avijit; Chowdhury, Chinmay; Chatterjee, Mitali; Das, Padma

    2015-01-01

    Background Current chemotherapeutic agents based on apoptosis induction are lacking in desired efficacy. Therefore, there is continuous effort to bring about new dimension in control and gradual eradication of cancer by means of ever evolving therapeutic strategies. Various forms of PCD are being increasingly implicated in anti-cancer therapy and the complex interplay among them is vital for the ultimate fate of proliferating cells. We elaborated and illustrated the underlying mechanism of the most potent Andrographolide analogue (AG–4) mediated action that involved the induction of dual modes of cell death—apoptosis and autophagy in human leukemic U937 cells. Principal Findings AG–4 induced cytotoxicity was associated with redox imbalance and apoptosis which involved mitochondrial depolarisation, altered apoptotic protein expressions, activation of the caspase cascade leading to cell cycle arrest. Incubation with caspase inhibitor Z-VAD-fmk or Bax siRNA decreased cytotoxic efficacy of AG–4 emphasising critical roles of caspase and Bax. In addition, AG–4 induced autophagy as evident from LC3-II accumulation, increased Atg protein expressions and autophagosome formation. Pre-treatment with 3-MA or Atg 5 siRNA suppressed the cytotoxic effect of AG–4 implying the pro-death role of autophagy. Furthermore, incubation with Z-VAD-fmk or Bax siRNA subdued AG–4 induced autophagy and pre-treatment with 3-MA or Atg 5 siRNA curbed AG–4 induced apoptosis—implying that apoptosis and autophagy acted as partners in the context of AG–4 mediated action. AG–4 also inhibited PI3K/Akt/mTOR pathway. Inhibition of mTOR or Akt augmented AG–4 induced apoptosis and autophagy signifying its crucial role in its mechanism of action. Conclusions Thus, these findings prove the dual ability of AG–4 to induce apoptosis and autophagy which provide a new perspective to it as a potential molecule targeting PCD for future cancer therapeutics. PMID:26436418

  14. Screening of suppressors of bax-induced cell death identifies glycerophosphate oxidase-1 as a mediator of debcl-induced apoptosis in Drosophila.

    PubMed

    Colin, Jessie; Garibal, Julie; Clavier, Amandine; Szuplewski, Sébastien; Risler, Yanick; Milet, Cécile; Gaumer, Sébastien; Guénal, Isabelle; Mignotte, Bernard

    2015-05-01

    Members of the Bcl-2 family are key elements of the apoptotic machinery. In mammals, this multigenic family contains about twenty members, which either promote or inhibit apoptosis. We have previously shown that the mammalian pro-apoptotic Bcl-2 family member Bax is very efficient in inducing apoptosis in Drosophila, allowing the study of bax-induced cell death in a genetic animal model. We report here the results of the screening of a P[UAS]-element insertion library performed to identify gene products that modify the phenotypes induced by the expression of bax in Drosophila melanogaster. We isolated 17 putative modifiers involved in various function or process: the ubiquitin/proteasome pathway; cell growth, proliferation and death; pathfinding and cell adhesion; secretion and extracellular signaling; metabolism and oxidative stress. Most of these suppressors also inhibit debcl-induced phenotypes, suggesting that the activities of both proteins can be modulated in part by common signaling or metabolic pathways. Among these suppressors, Glycerophosphate oxidase-1 is found to participate in debcl-induced apoptosis by increasing mitochondrial reactive oxygen species accumulation.

  15. Pretreatment with paclitaxel enhances apo-2 ligand/tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis of prostate cancer cells by inducing death receptors 4 and 5 protein levels.

    PubMed

    Nimmanapalli, R; Perkins, C L; Orlando, M; O'Bryan, E; Nguyen, D; Bhalla, K N

    2001-01-15

    We have demonstrated that Apo-2 ligand (Apo-2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis of human prostate cancer PC-3, DU145, and LNCaP cells in a dose-dependent manner, with PC-3 cells displaying the greatest sensitivity to Apo-2L/TRAIL. Susceptibility of the prostate cancer cell types to Apo-2L/TRAIL-induced apoptosis did not appear to correlate with the levels of the Apo-2L/TRAIL receptors death receptor (DR) 4 (TRAIL receptor 1) or DR5 (TRAIL receptor 2), decoy receptor (DcR) 1 and DcR2, Flame-1, or the inhibitors of apoptosis proteins family of proteins. Apo-2L/TRAIL-induced apoptosis of PC-3 cells was associated with the processing of caspase-8, caspase-10, and the proapoptotic Bid protein, resulting in the cytosolic accumulation of cytochrome c as well as the processing of procaspase-9 and procaspase-3. Cotreatment with the caspase-8 inhibitor z-IETD-fmk or DR4:Fc significantly inhibited Apo-2L/TRAIL-induced apoptosis. Treatment with paclitaxel or taxotere increased DR4 and/or DR5 protein levels (up to 8-fold) without affecting the protein levels of DcR1 and DcR2, Apo-2L/TRAIL, Fas, or Fas ligand. Up-regulation of DR4 and DR5 was not preceded by the induction of their mRNA levels but was inhibited by cotreatment with cycloheximide. Importantly, sequential treatment of PC-3, DU145, and LNCaP cells with paclitaxel followed by Apo-2L/TRAIL induced significantly more apoptosis than Apo-2L/TRAIL treatment alone (P < 0.01). This was also associated with greater processing of procaspase-8 and Bid, as well as greater cytosolic accumulation of cytochrome c and the processing of caspase-3. These findings indicate that up-regulation of DR4 and DR5 protein levels by treatment with paclitaxel enhances subsequent Apo-2L/TRAIL-induced apoptosis of human prostate cancer cells.

  16. Inhibition of γ-secretase activity synergistically enhances tumour necrosis factor-related apoptosis-inducing ligand induced apoptosis in T-cell acute lymphoblastic leukemia cells via upregulation of death receptor 5

    PubMed Central

    Greene, Lisa M.; Nathwani, Seema M.; Zisterer, Daniela M.

    2016-01-01

    T-cell acute lymphoblastic leukemia (T-ALL) is a rare and aggressive hematopoietic malignancy prone to relapse and drug resistance. Half of all T-ALL patients exhibit mutations in Notch1, which leads to aberrant Notch1 associated signaling cascades. Notch1 activation is mediated by the γ-secretase cleavage of the Notch1 receptor into the active intracellular domain of Notch1 (NCID). Clinical trials of γ-secretase small molecule inhibitors (GSIs) as single agents for the treatment of T-ALL have been unsuccessful. The present study demonstrated, using immunofluorescence and western blotting, that blocking γ-secretase activity in T-ALL cells with N-[(3,5-difluorophenyl) acetyl]-L-alanyl-2-phenyl] glycine-1,1-dimethylethyl ester (DAPT) downregulated NCID and upregulated the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 5 (DR5). Upregulation of DR5 restored the sensitivity of T-ALL cells to TRAIL. Combination index revealed that the combined treatment of DAPT and TRAIL synergistically enhanced apoptosis compared with treatment with either drug alone. TRAIL combined with the clinically evaluated γ-secretase inhibitor 3-[(1r, 4s)-4-(4-chlorophenylsulfonyl)-4-(2, 5-difluorophenyl) cyclohexyl] propanoic acid (MK-0752) also significantly enhanced TRAIL-induced cell death compared with either drug alone. DAPT/TRAIL apoptotic synergy was dependent on the extrinsic apoptotic pathway and was associated with a decrease in BH3 interacting-domain death agonist and x-linked inhibitor of apoptosis. In conclusion, γ-secretase inhibition represents a potential therapeutic strategy to overcome TRAIL resistance for the treatment of T-ALL. PMID:27698877

  17. Electrochemical control of cell death by reduction-induced intrinsic apoptosis and oxidation-induced necrosis on CoCrMo alloy in vitro.

    PubMed

    Haeri, Morteza; Wӧllert, Torsten; Langford, George M; Gilbert, Jeremy L

    2012-09-01

    Electrochemical voltage shifts in metallic biomedical implants occur in-vivo due to a number of processes including mechanically assisted corrosion. These excursions may compromise the biocompatibility of metallic implants. Voltages can also be controlled to modulate cell function and fate. The in vitro effect of static voltages on the behavior of MC3T3-E1 pre-osteoblasts cultured on CoCrMo alloy (ASTM-1537) was studied to determine the range of cell viability and mode of cell death beyond the viable range. Cell viability and morphology, changes in actin cytoskeleton, adhesion complexes and nucleus, and mode of cell death (necrosis, or intrinsic or extrinsic apoptosis) were characterized at different voltages ranging from -1000 to +500 mV (Ag/AgCl). Moreover, electrochemical currents and metal ion concentrations at each voltage were measured and related to the observed responses. Results show that cathodic and anodic voltages outside the voltage viability range (-400 < V < +500) lead to primarily intrinsic apoptotic and necrotic cell death, respectively. Cell death is associated with cathodic current densities of 0.1 μA cm(-2) and anodic current densities of 10 μA cm(-2). Significant increase in metallic ions (Co, Cr, Ni, Mo) was seen at +500 mV, and -1000 mV (Cr only) compared to open circuit potential. The number and total projected area of adhesion complexes was also lower on the polarized alloy (p < 0.05). These results show that reduction reactions on CoCrMo alloys leads to apoptosis of cells on the surface and may be a relevant mode of cell death for metallic implants in-vivo.

  18. Programmed Death Ligand 1 (PD-L1)-targeted TRAIL combines PD-L1-mediated checkpoint inhibition with TRAIL-mediated apoptosis induction

    PubMed Central

    Hendriks, Djoke; He, Yuan; Koopmans, Iris; Wiersma, Valerie R.; van Ginkel, Robert J.; Samplonius, Douwe F.; Helfrich, Wijnand; Bremer, Edwin

    2016-01-01

    ABSTRACT Antibodies that block PD-L1/PD-1 immune checkpoints restore the activity of functionally-impaired antitumor T cells. These antibodies show unprecedented clinical benefit in various advanced cancers, particularly in melanoma. However, only a subset of cancer patients responds to current PD-L1/PD-1-blocking strategies, highlighting the need for further advancements in PD-L1/PD-1-based immunotherapy. Here, we report on a novel approach designed to combine PD-L1 checkpoint inhibition with the tumor-selective induction of apoptosis by TNF-related Apoptosis Inducing Ligand (TRAIL). In brief, a new bi-functional fusion protein, designated anti-PD-L1:TRAIL, was constructed comprising a PD-L1-blocking antibody fragment genetically fused to the extracellular domain of the pro-apoptotic tumoricidal protein TRAIL. Treatment of PD-L1-expressing cancer cells with anti-PD-L1:TRAIL induced PD-L1-directed TRAIL-mediated cancer cell death. Treatment of T cells with anti-PD-L1:TRAIL augmented T cell activation, as evidenced by increased proliferation, secretion of IFNγ and enhanced killing of cancer cell lines and primary patient-derived cancer cells in mixed T cell/cancer cell culture experiments. Of note, elevated levels of IFNγ further upregulated PD-L1 on cancer cells and simultaneously sensitized cancer cells to TRAIL-mediated apoptosis by anti-PD-L1:TRAIL. Additionally, anti-PD-L1:TRAIL converted immunosuppressive PD-L1-expressing myeloid cells into pro-apoptotic effector cells that triggered TRAIL-mediated cancer cell death. In conclusion, combining PD-L1 checkpoint inhibition with TRAIL-mediated induction of apoptosis using anti-PD-L1:TRAIL yields promising multi-fold and mutually reinforcing anticancer activity that may be exploited to enhance the efficacy of therapeutic PD-L1/PD-1 checkpoint inhibition. PMID:27622071

  19. Heat-modified citrus pectin induces apoptosis-like cell death and autophagy in HepG2 and A549 cancer cells.

    PubMed

    Leclere, Lionel; Fransolet, Maude; Cote, Francois; Cambier, Pierre; Arnould, Thierry; Van Cutsem, Pierre; Michiels, Carine

    2015-01-01

    Cancer is still one of the leading causes of death worldwide, and finding new treatments remains a major challenge. Previous studies showed that modified forms of pectin, a complex polysaccharide present in the primary plant cell wall, possess anticancer properties. Nevertheless, the mechanism of action of modified pectin and the pathways involved are unclear. Here, we show that citrus pectin modified by heat treatment induced cell death in HepG2 and A549 cells. The induced cell death differs from classical apoptosis because no DNA cleavage was observed. In addition, Z-VAD-fmk, a pan-caspase inhibitor, did not influence the observed cell death in HepG2 cells but appeared to be partly protective in A549 cells, indicating that heat-modified citrus pectin might induce caspase-independent cell death. An increase in the abundance of the phosphatidylethanolamine-conjugated Light Chain 3 (LC3) protein and a decrease in p62 protein abundance were observed in both cell types when incubated in the presence of heat-modified citrus pectin. These results indicate the activation of autophagy. To our knowledge, this is the first time that autophagy has been revealed in cells incubated in the presence of a modified form of pectin. This autophagy activation appears to be protective, at least for A549 cells, because its inhibition with 3-methyladenine increased the observed modified pectin-induced cytotoxicity. This study confirms the potential of modified pectin to improve chemotherapeutic cancer treatments.

  20. Protein Kinase Cβ Modulates Ligand-induced Cell Surface Death Receptor Accumulation

    PubMed Central

    Meng, Xue Wei; Heldebrant, Michael P.; Flatten, Karen S.; Loegering, David A.; Dai, Haiming; Schneider, Paula A.; Gomez, Timothy S.; Peterson, Kevin L.; Trushin, Sergey A.; Hess, Allan D.; Smith, B. Douglas; Karp, Judith E.; Billadeau, Daniel D.; Kaufmann, Scott H.

    2010-01-01

    Although treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) is known to protect a subset of cells from induction of apoptosis by death ligands such as Fas ligand and tumor necrosis factor-α-related apoptosis-inducing ligand, the mechanism of this protection is unknown. This study demonstrated that protection in short term apoptosis assays and long term proliferation assays was maximal when Jurkat or HL-60 human leukemia cells were treated with 2–5 nm PMA. Immunoblotting demonstrated that multiple PKC isoforms, including PKCα, PKCβ, PKCϵ, and PKCθ, translocated from the cytosol to a membrane-bound fraction at these PMA concentrations. When the ability of short hairpin RNA (shRNA) constructs that specifically down-regulated each of these isoforms was examined, PKCβ shRNA uniquely reversed PMA-induced protection against cell death. The PKCβ-selective small molecule inhibitor enzastaurin had a similar effect. Although mass spectrometry suggested that Fas is phosphorylated on a number of serines and threonines, mutation of these sites individually or collectively had no effect on Fas-mediated death signaling or PMA protection. Further experiments demonstrated that PMA diminished ligand-induced cell surface accumulation of Fas and DR5, and PKCβ shRNA or enzastaurin reversed this effect. Moreover, enzastaurin sensitized a variety of human tumor cell lines and clinical acute myelogenous leukemia isolates, which express abundant PKCβ, to tumor necrosis factor-α related apoptosis-inducing ligand-induced death in the absence of PMA. Collectively, these results identify a specific PKC isoform that modulates death receptor-mediated cytotoxicity as well as a small molecule inhibitor that mitigates the inhibitory effects of PKC activation on ligand-induced death receptor trafficking and cell death. PMID:19887445

  1. Bleomycin induced sensitivity to TRAIL/Apo-2L-mediated apoptosis in human seminomatous testicular cancer cells is correlated with upregulation of death receptors.

    PubMed

    Timur, Mujgan; Cort, Aysegul; Ozdemir, Evrim; Sarikcioglu, Sureyya Bilmen; Sanlioglu, Salih; Sanlioglu, Ahter Dilsad; Ozben, Tomris

    2015-01-01

    The most common solid tumor is testicular cancer among young men. Bleomycin is an antitumor antibiotic used for the therapy of testicular cancer. TRAIL is a proapoptotic cytokine that qualified as an apoptosis inducer in cancer cells. Killing cancer cells selectively via apoptosis induction is an encouraging therapeutic strategy in clinical settings. Combination of TRAIL with chemotherapeutics has been reported to enhance TRAIL-mediated apoptosis of different kinds of cancer cell lines. The molecular ground for sensitization of tumour cells to TRAIL by chemotherapeutics might involve upregulation of TRAIL-R1 (TR/1, DR4) and/or TRAIL-R2 (TR/2, DR5) receptors or activation of proapoptotic proteins including caspases. The curative potential of TRAIL to eradicate cancer cells selectively in testicular cancer has not been studied before. In this study, we investigated apoptotic effects of bleomycin, TRAIL, and their combined application in NTera-2 and NCCIT testicular cancer cell lines. We measured caspase 3 levels as an apoptosis indicator, and TRAIL receptor expressions using flow cytometry. Both NTera-2 and NCCIT cells were fairly resistant to TRAIL's apoptotic effect. Incubation of bleomycin alone caused a significant increase in caspase 3 activity in NCCIT. Combined incubation with bleomycin and TRAIL lead to elevated caspase 3 activity in Ntera-2. Exposure to 72 h of bleomycin increased TR/1, TR/2, and TR/3 cell-surface expressions in NTera-2. Elevation in TR/1 cell-surface expression was evident only at 24 h of bleomycin application in NCCIT. It can be concluded that TRAIL death receptor expressions in particular are increased in testicular cancer cells via bleomycin treatment, and TRAIL-induced apoptosis is initiated.

  2. 1-Nitropyrene (1-NP) induces apoptosis and apparently a non-apoptotic programmed cell death (paraptosis) in Hepa1c1c7 cells

    SciTech Connect

    Asare, Nana Landvik, Nina E.; Lagadic-Gossmann, Dominique; Rissel, Mary; Tekpli, Xavier; Ask, Kjetil; Lag, Marit; Holme, Jorn A.

    2008-07-15

    Mechanistic studies of nitro-PAHs (polycyclic aromatic hydrocarbons) of interest might help elucidate which chemical characteristics are most important in eliciting toxic effects. 1-Nitropyrene (1-NP) is the predominant nitrated PAH emitted in diesel exhaust. 1-NP-exposed Hepa1c1c7 cells exhibited marked changes in cellular morphology, decreased proliferation and different forms of cell death. A dramatic increase in cytoplasmic vacuolization was observed already after 6 h of exposure and the cells started to round up at 12 h. The rate of cell proliferation was markedly reduced at 24 h and apoptotic as well as propidium iodide (PI)-positive cells appeared. Electron microscopic examination revealed that the vacuolization was partly due to mitochondria swelling. The caspase inhibitor Z-VAD-FMK inhibited only the apoptotic cell death and Nec-1 (an inhibitor of necroptosis) exhibited no inhibitory effects on either cell death or vacuolization. In contrast, cycloheximide markedly reduced both the number of apoptotic and PI-positive cells as well as the cytoplasmic vacuolization, suggesting that 1-NP induced paraptotic cell death. All the MAPKs; ERK1/2, p38 and JNK, appear to be involved in the death process since marked activation was observed upon 1-NP exposure, and their inhibitors partly reduced the induced cell death. The ERK1/2 inhibitor PD 98057 completely blocked the induced vacuolization, whereas the other MAPKs inhibitors only had minor effects on this process. These findings suggest that 1-NP may cause apoptosis and paraptosis. In contrast, the corresponding amine (1-aminopyrene) elicited only minor apoptotic and necrotic cell death, and cells with characteristics typical of paraptosis were absent.

  3. Receptor-mediated toxicity of pahutoxin, a marine trunkfish surfactant.

    PubMed

    Kalmanzon, Eliahu; Rahamim, Yocheved; Barenholz, Yechezkel; Carmeli, Shmuel; Zlotkin, Eliahu

    2003-07-01

    Pahutoxin (PHN, choline chloride ester of 3-acetoxypalmitic acid) is a natural fish-killing (ichthyotoxic) agent derived from the defensive secretions of trunkfish. In spite of its obvious structural resemblance to synthetic cationic long-chain quaternary ammonium detergents, we show that PHN's action does not rely on its surfactant properties and is in fact, receptor-mediated. The above conclusion is supported by the following data: 1. Ichthyotoxicity is not related to its detergency or surfactivity, as indicated by the fact that the lethal concentration is about 1.5 orders of magnitude below its critical micelle concentration value (69 microM) and its liposomal/seawater partition coefficient is low (62-85); 2. The trunkfish is tolerant to its own pahutoxin; 3. Ichthyotoxicity occurs only upon application to the surrounding water, suggesting the existence of externally located receptors; 4. The receptor hypothesis was supported by the aid of equilibrium saturation binding assays revealing the presence of specific binding sites to PHN on the fish gill membranes; 5. The PHN tolerant trunkfish was shown to be devoid of PHN-binding sites. Some chemo-ecological, and environmental implications are discussed.

  4. Biologically bounded risk assessment for receptor-mediated nongenotoxic carcinogens.

    PubMed

    Gastel, J A; Sutter, T R

    1995-12-01

    We have developed a biologically bounded marginal effect model for use in risk assessment of human exposure to receptor-mediated nongenotoxic carcinogens. Schematically this model can be reduced to four components: CI, the absence of an observable biological response; CII, observable biochemical responses but no observable pathology; CIII, observable pathology; and CIV, both observable pathology and lethality. The inflection point in the marginal response curve between CI and CII is defined as the biologically evaluated scientifically tested no observable effect level (BESTNOEL). We demonstrate the utility of this approach by applying it to the well-studied nongenotoxic carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Using a well-developed mechanistic understanding of the initial interactions of TCDD with the cell, we justify the selection of the minimal effective dose for CYP1A1 mRNA induction as the BESTNOEL. With allowance for variation in human sensitivity to TCDD, the BESTNOEL is assigned a human liver tissue burden of approximately 0.25-25 ppt and an allowable daily intake level in the range of 15-1500 fg/kg/day. In the future, the BESTNOEL can help establish a lower boundary for acceptable extrapolation when using either statistical or biologically based attributable risk models.

  5. Visualization of Receptor-mediated Endocytosis in Yeast

    PubMed Central

    Mulholland, Jon; Konopka, James; Singer-Kruger, Birgit; Zerial, Marino; Botstein, David

    1999-01-01

    We studied the ligand-induced endocytosis of the yeast α-factor receptor Ste2p by immuno-electron microscopy. We observed and quantitated time-dependent loss of Ste2p from the plasma membrane of cells exposed to α-factor. This ligand-induced internalization of Ste2p was blocked in the well-characterized endocytosis-deficient mutant sac6Δ. We provide evidence that implicates furrow-like invaginations of the plasma membrane as the site of receptor internalization. These invaginations are distinct from the finger-like plasma membrane invaginations within actin cortical patches. Consistent with this, we show that Ste2p is not located within the cortical actin patch before and during receptor-mediated endocytosis. In wild-type cells exposed to α-factor we also observed and quantitated a time-dependent accumulation of Ste2p in intracellular, membrane-bound compartments. These compartments have a characteristic electron density but variable shape and size and are often located adjacent to the vacuole. In immuno-electron microscopy experiments these compartments labeled with antibodies directed against the rab5 homologue Ypt51p (Vps21p), the resident vacuolar protease carboxypeptidase Y, and the vacuolar H+-ATPase Vph1p. Using a new double-labeling technique we have colocalized antibodies against Ste2p and carboxypeptidase Y to this compartment, thereby identifying these compartments as prevacuolar late endosomes. PMID:10069819

  6. Purine receptor mediated actin cytoskeleton remodeling of human fibroblasts

    PubMed Central

    Goldman, Nanna; Chandler-Militello, Devin; Langevin, Helene; Nedergaard, Maiken; Takano, Takahiro

    2013-01-01

    Earlier studies have shown that activation of adenosine A1 receptors on peripheral pain fibers contributes to acupuncture-induced suppression of painful input. In addition to adenosine, acupuncture triggers the release of other purines, including ATP and ADP that may bind to purine receptors on nearby fibroblasts. We here show that purine agonists trigger increase in cytosolic Ca 2+ signaling in a cultured human fibroblasts cell line. The profile of agonist-induced Ca2+ increases indicates that the cells express functional P2yR2 and P2yR4 receptors, as well as P2yR1 and P2xR7 receptors. Unexpectedly, purine-induced Ca2+ signaling was associated with a remodeling of the actin cytoskeleton. ATP induced a transient loss in F-actin stress fiber. The changes of actin cytoskeleton occurred slowly and peaked at 10 min after agonist exposure. Inhibition of ATP-induced increases in Ca2+ by cyclopiazonic acid blocked receptor-mediated cytoskeleton remodeling. The Ca2+ ionophore failed to induce cytoskeletal remodeling despite triggering robust increases in cytosolic Ca2+. These observations indicate that purine signaling induces transient changes in fibroblast cytoarchitecture that could be related to the beneficial effects of acupuncture. PMID:23462235

  7. Hemoglobin Uptake by Paracoccidioides spp. Is Receptor-Mediated

    PubMed Central

    Bailão, Elisa Flávia Luiz Cardoso; Parente, Juliana Alves; Pigosso, Laurine Lacerda; de Castro, Kelly Pacheco; Fonseca, Fernanda Lopes; Silva-Bailão, Mirelle Garcia; Báo, Sônia Nair; Bailão, Alexandre Melo; Rodrigues, Marcio L.; Hernandez, Orville; McEwen, Juan G.; Soares, Célia Maria de Almeida

    2014-01-01

    Iron is essential for the proliferation of fungal pathogens during infection. The availability of iron is limited due to its association with host proteins. Fungal pathogens have evolved different mechanisms to acquire iron from host; however, little is known regarding how Paracoccidioides species incorporate and metabolize this ion. In this work, host iron sources that are used by Paracoccidioides spp. were investigated. Robust fungal growth in the presence of the iron-containing molecules hemin and hemoglobin was observed. Paracoccidioides spp. present hemolytic activity and have the ability to internalize a protoporphyrin ring. Using real-time PCR and nanoUPLC-MSE proteomic approaches, fungal growth in the presence of hemoglobin was shown to result in the positive regulation of transcripts that encode putative hemoglobin receptors, in addition to the induction of proteins that are required for amino acid metabolism and vacuolar protein degradation. In fact, one hemoglobin receptor ortholog, Rbt5, was identified as a surface GPI-anchored protein that recognized hemin, protoporphyrin and hemoglobin in vitro. Antisense RNA technology and Agrobacterium tumefaciens-mediated transformation were used to generate mitotically stable Pbrbt5 mutants. The knockdown strain had a lower survival inside macrophages and in mouse spleen when compared with the parental strain, which suggested that Rbt5 could act as a virulence factor. In summary, our data indicate that Paracoccidioides spp. can use hemoglobin as an iron source most likely through receptor-mediated pathways that might be relevant for pathogenic mechanisms. PMID:24831516

  8. Asialoglycoprotein receptor mediated hepatocyte targeting - strategies and applications.

    PubMed

    D'Souza, Anisha A; Devarajan, Padma V

    2015-04-10

    Hepatocyte resident afflictions continue to affect the human population unabated. The asialoglycoprotein receptor (ASGPR) is primarily expressed on hepatocytes and minimally on extra-hepatic cells. This makes it specifically attractive for receptor-mediated drug delivery with minimum concerns of toxicity. ASGPR facilitates internalization by clathrin-mediated endocytosis and exhibits high affinity for carbohydrates specifically galactose, N-acetylgalactosamine and glucose. Isomeric forms of sugar, galactose density and branching, spatial geometry and galactose linkages are key factors influencing ligand-receptor binding. Popular ligands for ASGPR mediated targeting are carbohydrate polymers, arabinogalactan and pullulan. Other ligands include galactose-bearing glycoproteins, glycopeptides and galactose modified polymers and lipids. Drug-ligand conjugates provide a viable strategy; nevertheless ligand-anchored nanocarriers provide an attractive option for ASGPR targeted delivery and are widely explored. The present review details various ligands and nanocarriers exploited for ASGPR mediated delivery of drugs to hepatocytes. Nanocarrier properties affecting ASGPR mediated uptake are discussed at length. The review also highlights the clinical relevance of ASGPR mediated targeting and applications in diagnostics. ASGPR mediated hepatocyte targeting provides great promise for improved therapy of hepatic afflictions.

  9. Cytomegalovirus cell death suppressor vMIA blocks Bax- but not Bak-mediated apoptosis by binding and sequestering Bax at mitochondria.

    PubMed

    Arnoult, Damien; Bartle, Laura M; Skaletskaya, Anna; Poncet, Delphine; Zamzami, Naoufal; Park, Peter U; Sharpe, Juanita; Youle, Richard J; Goldmacher, Victor S

    2004-05-25

    We report that the cytomegalovirus-encoded cell death suppressor vMIA binds Bax and prevents Bax-mediated mitochondrial membrane permeabilization by sequestering Bax at mitochondria in the form of a vMIA-Bax complex. vMIA mutants with a defective mitochondria-targeting domain retain their Bax-binding function but not their ability to suppress mitochondrial membrane permeabilization or cell death. vMIA does not seem to either specifically associate with Bak or suppress Bak-mediated mitochondrial membrane permeabilization. Recent evidence suggests that the contribution of Bax and Bak in the mitochondrial apoptotic signaling pathway depends on the distinct phenotypes of cells, and it appears from our data that vMIA is capable of suppressing apoptosis in cells in which this pathway is dominated by Bax, but not in cells where Bak also plays a role.

  10. Cell-permeable intrinsic cellular inhibitors of apoptosis protect and rescue intestinal epithelial cells from radiation-induced cell death

    PubMed Central

    Matsuzaki-Horibuchi, Shiori; Yasuda, Takeshi; Sakaguchi, Nagako; Yamaguchi, Yoshihiro; Akashi, Makoto

    2015-01-01

    One of the important mechanisms for gastrointestinal (GI) injury following high-dose radiation exposure is apoptosis of epithelial cells. X-linked inhibitor of apoptosis (XIAP) and cellular IAP2 (cIAP2) are intrinsic cellular inhibitors of apoptosis. In order to study the effects of exogenously added IAPs on apoptosis in intestinal epithelial cells, we constructed bacterial expression plasmids containing genes of XIAP (full-length, BIR2 domain and BIR3-RING domain with and without mutations of auto-ubiquitylation sites) and cIAP2 proteins fused to a protein-transduction domain (PTD) derived from HIV-1 Tat protein (TAT) and purified these cell-permeable recombinant proteins. When the TAT-conjugated IAPs were added to rat intestinal epithelial cells IEC6, these proteins were effectively delivered into the cells and inhibited apoptosis, even when added after irradiation. Our results suggest that PTD-mediated delivery of IAPs may have clinical potential, not only for radioprotection but also for rescuing the GI system from radiation injuries. PMID:25359904

  11. Cell-permeable intrinsic cellular inhibitors of apoptosis protect and rescue intestinal epithelial cells from radiation-induced cell death.

    PubMed

    Matsuzaki-Horibuchi, Shiori; Yasuda, Takeshi; Sakaguchi, Nagako; Yamaguchi, Yoshihiro; Akashi, Makoto

    2015-01-01

    One of the important mechanisms for gastrointestinal (GI) injury following high-dose radiation exposure is apoptosis of epithelial cells. X-linked inhibitor of apoptosis (XIAP) and cellular IAP2 (cIAP2) are intrinsic cellular inhibitors of apoptosis. In order to study the effects of exogenously added IAPs on apoptosis in intestinal epithelial cells, we constructed bacterial expression plasmids containing genes of XIAP (full-length, BIR2 domain and BIR3-RING domain with and without mutations of auto-ubiquitylation sites) and cIAP2 proteins fused to a protein-transduction domain (PTD) derived from HIV-1 Tat protein (TAT) and purified these cell-permeable recombinant proteins. When the TAT-conjugated IAPs were added to rat intestinal epithelial cells IEC6, these proteins were effectively delivered into the cells and inhibited apoptosis, even when added after irradiation. Our results suggest that PTD-mediated delivery of IAPs may have clinical potential, not only for radioprotection but also for rescuing the GI system from radiation injuries.

  12. TRAIL and DR5 promote thyroid follicular cell apoptosis in iodine excess-induced experimental autoimmune thyroiditis in NOD mice.

    PubMed

    Yu, Xiujie; Li, Lanying; Li, Qingxin; Zang, Xiaoyi; Liu, Zebing

    2011-11-01

    Death receptor-mediated apoptosis has been implicated in target organ destruction in patients with chronic autoimmune thyroiditis. Several apoptosis signaling pathways, such as Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), have been shown to be active in thyroid cells and may be involved in destructive thyroiditis. Thyroid toxicity of iodide excess has been demonstrated in animals fed with an iodide-rich diet, but its pathogenic role remains unclear. The effects of excessive iodine on TRAIL and its death receptor expression in thyroid were investigated. Experimental autoimmune thyroiditis (EAT) was induced by excessive iodine and thyroglobulin (Tg) in non-obese diabetic mice. The expression of TRAIL and its death receptor DR5 was detected by immunofluorescence staining. Following administration of excessive iodine alone, Tg, and excessive iodine combined with Tg, TRAIL-positive cells appear not only in follicular cells but also in lymphocytes infiltrated in the thyroid, whereas DR5-positive cells appear only in follicular cells. Large numbers of CD3-positive cells and a few CD22-positive cells were detected in thyroid. A great amount of follicular cells were labeled specifically by terminal deoxynucleotide transferase-mediated deoxynucleotide triphosphate nick-end labeling assay. Taken together, our results suggest that excessive iodine could induce TRAIL and DR5 abnormal expression in thyroid. TRAIL band with DR5 to promote follicular cells apoptosis thus mediate thyroid destruction in EAT.

  13. Fermented soybeans, Chungkookjang, prevent hippocampal cell death and β-cell apoptosis by decreasing pro-inflammatory cytokines in gerbils with transient artery occlusion

    PubMed Central

    Park, Sunmin; Kim, Da Sol; Moon, Bo Reum

    2015-01-01

    Since Chungkookjang, a short-term fermented soybean, is known to improve glucose metabolism and antioxidant activity, it may prevent the neurological symptoms and glucose disturbance induced by artery occlusion. We investigated the protective effects and mechanisms of traditional (TFC) and standardized Chungkookjang fermented with Bacillus licheniformis (BLFC) against ischemia/reperfusion damage in the hippocampal CA1 region and against hyperglycemia after transient cerebral ischemia in gerbils. Gerbils were subjected to either an occlusion of the bilateral common carotid arteries for 8 min to render them ischemic or a sham operation. Ischemic gerbils were fed either a 40% fat diet containing 10% of either cooked soybean (CSB), TFC, or BLFC for 28 days. Neuronal cell death and cytokine expression in the hippocampus, neurological deficit, serum cytokine levels, and glucose metabolism were measured. TFC and BLFC contained more isoflavonoid aglycones than CSB. Artery occlusion increased the expressions of IL-1β and TNF-α as well as cell death in the hippocampal CA1 region and induced severe neurological symptoms. CSB, TFC, and BLFC prevented the neuronal cell death and the symptoms such as dropped eyelid, bristling hair, reduced muscle tone and flexor reflex, and abnormal posture and walking patterns, and suppressed cytokine expressions. CSB was less effective than TFC and BLFC. Artery occlusion induced glucose intolerance due to decreased insulin secretion and β-cell mass. TFC and BLFC prevented the impairment of glucose metabolism by artery occlusion. Especially TFC and BLFC increased β-cell proliferation and suppressed the β-cell apoptosis by suppressing TNF-α and IL-1β which in turn decreased cleaved caspase-3 that caused apoptosis. In conclusion, TFC and BLFC may prevent and alleviate neuronal cell death in the hippocampal CA1 region and neurological symptoms and poststroke hyperglycemia in gerbils with artery occlusion. This might be associated with

  14. Fermented soybeans, Chungkookjang, prevent hippocampal cell death and β-cell apoptosis by decreasing pro-inflammatory cytokines in gerbils with transient artery occlusion.

    PubMed

    Park, Sunmin; Kim, Da Sol; Kang, Sunna; Moon, Bo Reum

    2016-02-01

    Since Chungkookjang, a short-term fermented soybean, is known to improve glucose metabolism and antioxidant activity, it may prevent the neurological symptoms and glucose disturbance induced by artery occlusion. We investigated the protective effects and mechanisms of traditional (TFC) and standardized Chungkookjang fermented with Bacillus licheniformis (BLFC) against ischemia/reperfusion damage in the hippocampal CA1 region and against hyperglycemia after transient cerebral ischemia in gerbils. Gerbils were subjected to either an occlusion of the bilateral common carotid arteries for 8 min to render them ischemic or a sham operation. Ischemic gerbils were fed either a 40% fat diet containing 10% of either cooked soybean (CSB), TFC, or BLFC for 28 days. Neuronal cell death and cytokine expression in the hippocampus, neurological deficit, serum cytokine levels, and glucose metabolism were measured. TFC and BLFC contained more isoflavonoid aglycones than CSB. Artery occlusion increased the expressions of IL-1β and TNF-α as well as cell death in the hippocampal CA1 region and induced severe neurological symptoms. CSB, TFC, and BLFC prevented the neuronal cell death and the symptoms such as dropped eyelid, bristling hair, reduced muscle tone and flexor reflex, and abnormal posture and walking patterns, and suppressed cytokine expressions. CSB was less effective than TFC and BLFC. Artery occlusion induced glucose intolerance due to decreased insulin secretion and β-cell mass. TFC and BLFC prevented the impairment of glucose metabolism by artery occlusion. Especially TFC and BLFC increased β-cell proliferation and suppressed the β-cell apoptosis by suppressing TNF-α and IL-1β which in turn decreased cleaved caspase-3 that caused apoptosis. In conclusion, TFC and BLFC may prevent and alleviate neuronal cell death in the hippocampal CA1 region and neurological symptoms and poststroke hyperglycemia in gerbils with artery occlusion. This might be associated with

  15. Apoptosis of Theileria-infected lymphocytes induced upon parasite death involves activation of caspases 9 and 3.

    PubMed

    Guergnon, Julien; Dessauge, Frédéric; Langsley, Gordon; Garcia, Alphonse

    2003-08-01

    The intracellular parasite Theileria parva (T. parva) can infect bovine B and T-lymphocytes. T. parva-infected cells become transformed, and they survive and proliferate independently of exogenous growth factors. In vivo the uncontrolled cellular proliferation associated with lymphocyte transformation underlies the pathogenesis of the disease called East Coast Fever. The transformed state of parasitised cells can be reversed upon elimination of the parasite by specific theilericide drugs. In this study we found that elimination of the parasite by buparvaquone induces apoptosis of transformed B and CD8(+) T-lymphocytes. Apoptosis is accompanied by the activation of caspase 9 and caspase 3 and processing of poly(ADP ribose) polymerase and is inhibited by Z-VAD a general caspase inhibitor. Based on these observations, we propose that the lack of activation of a caspase 9 > caspase 3 > poly(ADP ribose) polymerase pathway is important and protects T. parva-transformed cells from spontaneous apoptosis.

  16. Cytotoxicity of atropine to human corneal epithelial cells by inducing cell cycle arrest and mitochondrion-dependent apoptosis.

    PubMed

    Tian, Cheng-Lei; Wen, Qian; Fan, Ting-Jun

    2015-10-01

    Atropine is an anticholinergic drug for mydriasis in eye clinic, and its abuse might be cytotoxic to the cornea and result in blurred vision. However, the cytotoxicity of atropine to the cornea and its cellular and molecular mechanisms remain unknown. In this study, we investigated the cytotoxicity of atropine to corneal epithelium and its underlying mechanisms using an in vitro model of non-transfected human corneal epithelial (HCEP) cells. Our results showed that atropine, above the concentration of 0.3125 g/l (1/32 of its therapeutic dosage in eye clinic), had a dose- and time-dependent toxicity to HCEP cells by inducing morphological abnormality, cytopathic effect, viability decline, and proliferation retardation. Moreover, the proliferation-retarding effect of atropine on the cells was achieved by inducing G1/S phase arrest and downregulation of E-cadherin and β-catenin. Besides, atropine also had an apoptosis-inducing effect on the cells by inducing phosphatidylserine externalization, plasma membrane permeability elevation, DNA fragmentation and apoptotic body formation. Furthermore, atropine could also induce activations of caspase-2, -3 and -9, disruption of mitochondrial transmembrane potential, downregulation of Bcl-2 and Bcl-xL, upregulation of Bax and Bad, and upregulation of cytoplasmic cytochrome c and apoptosis-inducing factor, implying a death receptor-mediated mitochondrion-dependent pathway is most probably involved in the apoptosis of HCEP cells induced by atropine. Taken together, our results suggest that atropine has remarkable cytotoxicity to HCEP cells by inducing cell cycle arrest and death receptor-mediated mitochondrion-dependent apoptosis.

  17. Matrix metalloproteinases are involved in both type I (apoptosis) and type II (autophagy) cell death induced by sodium phenylacetate in MDA-MB-231 breast tumour cells.

    PubMed

    Augustin, Sébastien; Berard, Madeleine; Kellaf, Sabine; Peyri, Nicole; Fauvel-Lafève, Françoise; Legrand, Chantal; He, Lu; Crépin, Michel

    2009-04-01

    The effects of sodium phenylacetate (NaPa), an antitumoral molecule, on cell death and matrix metalloproteinase (MMP) activities and synthesis were investigated in two metastatic breast tumour cell lines, MDA-MB-231 and MDA-MB-435, cultured on three-dimensional type I collagen gels (3-D cultures). In both cell lines, NaPa inhibited cell proliferation and induced apoptotic cell death as measured by TUNEL assay, with an IC(30) of 20 mM and 10 mM for MDA-MB-231 and MDA-MB-435 cells, respectively. In MDA-MB-231 cells, NaPa also induced (i) an autophagic process evidenced by the appearance of autophagic vacuoles and an increased phosphatase acid activity, (ii) the formation of pseudopodia and (iii) an increase in MMP-1 and MMP-9 secretion without affecting MT1-MMP. In NaPa-treated MDA-MB-435 cells, no autophagic vacuoles were formed but F-actin depolymerisation was observed. MMP-1, MMP-9 and MT1-MMP levels were strongly enhanced in these cells but MMPs were not secreted and accumulated intracellularly. When breast cancer cells were treated with NaPa in the presence of an MMP inhibitor (GM6001), apoptotic cell death decreased and the induction of autophagic vacuoles in MDA-MB-231 cells was inhibited. Taken together, these data suggest that MMPs are involved in the autophagic cell death and/or apoptosis of breast tumour cells.

  18. Cytotoxic L-amino-acid oxidases from Amanita phalloides and Clitocybe geotropa induce caspase-dependent apoptosis

    PubMed Central

    Pišlar, A; Sabotič, J; Šlenc, J; Brzin, J; Kos, J

    2016-01-01

    L-amino-acid oxidases (LAO) purified from fungi induce cell death in various mammalian cells including human tumor cell lines. The mechanism, however, remains poorly understood. In this study, we aimed to define a precise mechanism of cell death induced in Jurkat and MCF7 cancer cell lines by ApLAO and CgLAO, LAOs isolated from Amanita phalloides and Clitocybe geotropa, respectively. Cell death induced by both LAOs is shown to be concentration- and time-dependent, with higher toxic effects in Jurkat cells. LAO activity is required for the cytotoxicity. Detailed study on Jurkat cells further demonstrated that ApLAO and CgLAO both induce the intrinsic mitochondrial pathway of apoptosis, accompanied by a time-dependent depolarization of the mitochondrial membrane through the generation of reactive oxygen species. Treatment with the LAOs resulted in an increased ratio of the expression of proapoptotic Bax to that of antiapoptotic Bcl-2, subsequently leading to the activation of caspase-9 and -3. However, the pancaspase inhibitor, Z-VAD-FMK, did not completely abolish the cell death induced by either ApLAO or CgLAO, suggesting an alternative pathway for LAO-induced apoptosis. Indeed, caspase-8 activity in ApLAO- and CgLAO-treated cells was increased. Further, Fas/FasL (Fas ligand) antagonist caused a slight reduction in toxin-induced cell death, supporting the involvement of ApLAO and CgLAO in death-receptor-mediated apoptosis. These results thus provide new evidence that ApLAO and CgLAO induce apoptosis in Jurkat cells via both the intrinsic and extrinsic pathways, although the significantly higher increase of caspase-9 over caspase-8 activity suggests that it is the intrinsic pathway that is the predominant mode of ApLAO- and CgLAO-induced apoptosis. PMID:27551514

  19. Genistein inhibition of OGD-induced brain neuron death correlates with its modulation of apoptosis, voltage-gated potassium and sodium currents and glutamate signal pathway.

    PubMed

    Ma, Xue-Ling; Zhang, Feng; Wang, Yu-Xiang; He, Cong-Cong; Tian, Kun; Wang, Hong-Gang; An, Di; Heng, Bin; Liu, Yan-Qiang

    2016-07-25

    In the present study, we established an in vitro model of hypoxic-ischemia via exposing primary neurons of newborn rats to oxygen-glucose deprivation (OGD) and observing the effects of genistein, a soybean isoflavone, on hypoxic-ischemic neuron viability, apoptosis, voltage-activated potassium (Kv) and sodium (Nav) currents, and glutamate receptor subunits. The results indicated that OGD exposure reduced the viability and increased the apoptosis of brain neurons. Meanwhile, OGD exposure caused changes in the current-voltage curves and current amplitude values of voltage-activated potassium and sodium currents; OGD exposure also decreased GluR2 expression and increased NR2 expression. However, genistein at least partially reversed the effects caused by OGD. The results suggest that hypoxic-ischemia-caused neuronal apoptosis/death is related to an increase in K(+) efflux, a decrease in Na(+) influx, a down-regulation of GluR2, and an up-regulation of NR2. Genistein may exert some neuroprotective effects via the modulation of Kv and Nav currents and the glutamate signal pathway, mediated by GluR2 and NR2.

  20. Mitomycin C potentiates TRAIL-induced apoptosis through p53-independent upregulation of death receptors: evidence for the role of c-Jun N-terminal kinase activation.

    PubMed

    Cheng, Hairong; Hong, Bo; Zhou, Lanlan; Allen, Joshua E; Tai, Guihua; Humphreys, Robin; Dicker, David T; Liu, Yingqiu Y; El-Deiry, Wafik S

    2012-09-01

    The discovery of the molecular targets of chemotherapeutic medicines and their chemical footprints can validate and improve the use of such medicines. In the present report, we investigated the effect of mitomycin C (MMC), a classical chemotherapeutic agent on cancer cell apoptosis induced by TRAIL. We found that MMC not only potentiated TRAIL-induced apoptosis in HCT116 (p53-/-) colon cancer cells but also sensitized TRAIL-resistant colon cancer cells HT-29 to the cytokine both in vitro and in vivo. MMC also augmented the pro-apoptotic effects of two TRAIL receptor agonist antibodies, mapatumumab and lexatumumab. At a mechanistic level, MMC downregulated cell survival proteins, including Bcl2, Mcl-1 and Bcl-XL, and upregulated pro-apoptotic proteins including Bax, Bim and the cell surface expression of TRAIL death receptors DR4 and DR5. Gene silencing of DR5 by short hairpin RNA reduced the apoptosis induced by combination treatment of MMC and TRAIL. Induction of DR4 and DR5 was independent of p53, Bax and Bim but was dependent on c-Jun N terminal kinase (JNK) as JNK pharmacological inhibition and siRNA abolished the induction of the TRAIL receptors by MMC.

  1. Preconditioning with low concentration NO attenuates subsequent NO-induced apoptosis in vascular smooth muscle cells via HO-1-dependent mitochondrial death pathway

    SciTech Connect

    Kwak, Hyun-Jeong; Park, Kyoung-Mi; Lee, Seahyoung; Lim, Hyun-Joung; Go, Sang-Hee; Eom, Sang-Mi; Park, Hyun-Young . E-mail: hypark65@nih.go.kr

    2006-12-01

    Nitric oxide (NO) signaling pathways are important in both the maintenance of vascular homeostasis and disease progression. Overproduction of NO has been associated with ischemia/reperfusion (I/R) injury. Growing evidences suggest that NO preconditioning has cytoprotective effects against I/R injury. However, the mechanism with which NO mediates these effects remains to be elucidated. The purpose of this study was to examine the mechanism of how NO preconditioning inhibits subsequent NO-induced apoptosis in vascular smooth muscle cells (VSMC), specifically focusing on heme oxygenase-1 (HO-1). According to our data, sodium nitroprusside (SNP) increased HO-1 expression in a concentration dependent manner. Preconditioning with low concentration SNP (0.3 mM) inhibited subsequent high concentration SNP (1.5 mM)-induced apoptosis, and this effect was reversed by the HO-1 inhibitor SnPP. Low concentration SNP-mediated protection involved p38 kinase inactivation and increased Bcl-2 expression. Furthermore, mitochondrial membrane potential was concomitantly increased with decreased expressions of Bax, Apaf-1, and activity of caspase-3, which was reversed by SnPP treatment. Our results show that low concentration SNP preconditioning suppresses subsequent high concentration SNP-induced apoptosis by inhibiting p38 kinase and mitochondrial death pathway via HO-1-dependent mechanisms in VSMC.

  2. BCL6 mediates the effects of Gastrodin on promoting M2-like macrophage polarization and protecting against oxidative stress-induced apoptosis and cell death in macrophages.

    PubMed

    Jia, Jing; Shi, Xiaojie; Jing, Xiaoqian; Li, Jianguo; Gao, Jie; Liu, Mengya; Lin, Chi-Iou; Guo, Xinzhi; Hua, Qian

    2017-03-16

    Cerebral palsy (CP) is the most common childhood disability worldwide, yet biomarkers for predicting CP are lacking. By subjecting peripheral blood samples from 62 CP patients and 30 healthy controls to Affymetrix GeneChip(®) PrimeView™ HumanGene Expression Microarray analysis, we identified the novel biomarker B-cell lymphoma 6 (BCL6) as the most upregulated gene in the CP samples. Gastrodin is a traditional Chinese medicine and bioactive compound that promotes adductor angle release, as well as gross and fine motor performance by increasing Gross Motor Function Measure-66 and Fine Motor Function Measure-45 scores. Gastrodin upregulates the mRNA expression of Mgl2 and Mrc1, M2 macrophage markers, and arginase activity, an M2 polarization indicator, in murine RAW264.7 macrophages. Moreover, these effects were blocked by BCL6 siRNA, which also abrogated the protective effects of Gastrodin against hydrogen peroxide-induced apoptosis and death in RAW264.7 cells. Our work identified BCL6 as a novel biomarker for early prediction of CP. Moreover, we demonstrated that Gastrodin not only stimulated polarization toward M2-like macrophages, which promote tissue repair, but also rescued macrophages from oxidative stress, apoptosis and death by inducing BCL6 expression. BCL6-targeted therapeutic strategies have promise for improving motor performance in CP patients.

  3. ADP and Other Metabolites Released from Acanthamoeba castellanii Lead to Human Monocytic Cell Death through Apoptosis and Stimulate the Secretion of Proinflammatory Cytokines

    PubMed Central

    Mattana, A.; Cappai, V.; Alberti, L.; Serra, C.; Fiori, P. L.; Cappuccinelli, P.

    2002-01-01

    Monocytes/macrophages are thought to be involved in Acanthamoeba infections. The aim of this work was to study whether soluble metabolites (ADP and other compounds) released by Acanthamoeba castellanii trophozoites could induce morphological and biochemical changes in human monocytic cells in vitro. We demonstrate here that ADP constitutively released in the medium by A. castellanii, interacting with specific P2y2 purinoceptors expressed on the monocytic cell membrane, caused a biphasic rise in [Ca2+]i, morphological changes characteristics of cells undergoing apoptosis, caspase-3 activation, and secretion of tumor necrosis factor alpha (TNF-α). The same results were found in monocytes exposed to purified ADP. Cell damage and TNF-α release induced by amoebic ADP were blocked by the P2y2 inhibitor suramin. Other metabolites contained in amoebic cell-free supernatants, with molecular masses of, respectively, >30 kDa and between 30 and 10 kDa, also caused morphological modifications and activation of intracellular caspase-3, characteristics of programmed cell death. Nevertheless, mechanisms by which these molecules trigger cell damage appeared to differ from that of ADP. In addition, other amoebic thermolable metabolites with molecular masses of <10 kDa caused the secretion of interleukin-1β. These findings suggest that pathogenic free-living A. castellanii by release of ADP and other metabolites lead to human monocytic cell death through apoptosis and stimulate the secretion of proinflammatory cytokines. PMID:12117953

  4. Histamine H3 receptor-mediated inhibition of noradrenaline release in the human brain.

    PubMed

    Schlicker, E; Werthwein, S; Zentner, J

    1999-01-01

    Stimulation-evoked 3H-noradrenaline release in human cerebrocortical slices was inhibited by histamine (in a manner sensitive to clobenpropit) and by imetit, suggesting H3 receptor-mediated inhibition of noradrenaline release in human brain.

  5. Dinitro-o-cresol induces apoptosis-like cell death but not alternative oxidase expression in soybean cells.

    PubMed

    Aranha, Márcia M; Matos, Ana R; Teresa Mendes, Ana; Vaz Pinto, Vera; Rodrigues, Cecília M P; Arrabaça, João D

    2007-06-01

    In plants, programmed cell death is thought to be activated during differentiation and in response to biotic and abiotic stresses. Although its mechanisms are far less clear, several morphological and biochemical features have been described in different experimental systems, including DNA laddering and cytosolic protease activation. Moreover, plant mitochondria have an alternative terminal oxidase (AOX), which is thought to be involved in protection against increased reactive oxygen species production, perhaps representing a mechanism to prevent programmed cell death. In this study, we analysed cell death induced by the herbicide dinitro-o-cresol (DNOC) in soybean (Glycine max) suspension cell cultures and evaluated biochemical and molecular events associated with programmed cell death. AOX capacity and expression were also determined. DNOC-treated cells showed fragmented nuclear DNA as assessed by an in situ assay that detects 3'-OH ends. In addition, specific colorimetric assays and immunoblot analysis revealed activation of caspase-3-like proteins and release of cytochrome c from mitochondria, respectively, confirming the apoptotic-like phenotype. Surprisingly, AOX capacity and protein levels decreased in DNOC-treated cells, suggesting no association between cell death and AOX under these experimental conditions. In conclusion, the results show that DNOC induces programmed cell death in soybean cells, suggesting that plants and animals might share similar pathways. Further, the role of AOX in cell death has not been confirmed, and may depend on the nature and intensity of stress conditions.

  6. Palladium(II) saccharinate complexes with bis(2-pyridylmethyl)amine induce cell death by apoptosis in human breast cancer cells in vitro.

    PubMed

    Ari, Ferda; Ulukaya, Engin; Sarimahmut, Mehmet; Yilmaz, Veysel T

    2013-06-01

    The outcomes of breast cancer patients are still poor although new compounds have recently been introduced into the clinic. Therefore, novel chemical approaches are required. In the present study, palladium(II) and corresponding platinum(II) complexes containing bis(2-pyridylmethyl)amine (bpma) and saccharine were synthesized and tested against human breast cancer cell lines, MCF-7 and MDA-MB-231, in vitro. Cytotoxicity was first screened by the MTT assay and the results were further confirmed by the ATP assay. The palladium complexes 1 and 3 yielded stronger cytotoxicity than the corresponding platinum complexes 2 and 4 at the same doses. The palladium complex 3 was found to be the most cytotoxic one. Therefore, a more comprehensive study was carried out with this complex only. The mode of cell death was determined morphologically under fluorescent microscope and biochemically with detection of active caspase-3 and PARP cleavage by Western blot. Changes in apoptosis-related gene expressions were measured with qPCR. It was demonstrated that complex 3 caused cell death by apoptosis determined by fluorescence imaging and Western blot. As a sign of apoptosis, PARP was cleaved in both of the cell lines. In addition, caspase-3 was cleaved in MDA-MB-231 cells while this cleavage was not observed in MCF-7. The results show that the complex 3 is a promising anti-cancer compound against breast cancer with an IC50 value of 3.9 μM for MCF-7 and 4.2 μM for MDA-MB-231 cells, which warrants further animal experiments.

  7. c-Met Inhibitor Synergizes with Tumor Necrosis Factor–Related Apoptosis-Induced Ligand to Induce Papillary Thyroid Carcinoma Cell Death

    PubMed Central

    Bu, Rong; Uddin, Shahab; Ahmed, Maqbool; Hussain, Azhar R; Alsobhi, Saif; Amin, Tarek; Al-Nuaim, Abdurahman; Al-Dayel, Fouad; Abubaker, Jehad; Bavi, Prashant; Al-Kuraya, Khawla S

    2012-01-01

    The Met receptor tyrosine kinase is overexpressed and/or activated in variety of human malignancies. Previously we have shown that c-Met is overexpressed in Middle Eastern papillary thyroid carcinoma (PTC) and significantly associated with an aggressive phenotype, but its role has not been fully elucidated in PTC. The aim of this study was to determine the functional link between the c-Met/AKT signaling pathway and death receptor 5 (DR5) in a large cohort of PTC in a tissue microarray format followed by functional studies using PTC cell lines and nude mice. Our data showed that high expressions of p-Met and DR5 were significantly associated with an aggressive phenotype of PTC and correlated with BRAF mutation. Treatment of PTC cell lines with PHA665752, an inhibitor of c-Met tyrosine kinase, inhibited cell proliferation and induced apoptosis via the mitochondrial pathway in PTC cell lines. PHA665752 treatment or expression of c-Met small interfering (si)RNA resulted in dephosphorylation of c-Met, AKT and its downstream effector molecules. Furthermore, PHA665752 treatment upregulated DR5 expression via generation of reactive oxygen species in PTC cell lines, and synergistically potentiated death receptor–induced apoptosis with tumor necrosis factor–related apoptosis-inducing ligand (TRAIL). Finally, cotreatment with PHA665752 and TRAIL caused more pronounced effects on PTC xenograft tumor growth in nude mice. Our data suggest that the c-Met/AKT pathway may be a potential target for therapeutic intervention for treatment of PTC refractory to conventionally therapeutic modalities. PMID:22113498

  8. GABAA Receptor-Mediated Activity in a Model of Cortical Dysplasia

    DTIC Science & Technology

    2012-06-29

    a model of cortical dysplasia" Name of Candidate: Joseph Abbah Doctor of Philosophy Degree June 29, 2012 DISSERTATION AND ABSTRACT APPROVED...receptor-mediated activity in a model of cortical dysplasia’ is appropriately acknowledged and, beyond brief excerpts, is with the permission of the...certifies that the use of any copyrighted material in this dissertation manuscript entitled: “GABAA receptor-mediated activity in a model of

  9. TRAIL induces apoptosis in oral squamous carcinoma cells--a crosstalk with oncogenic Ras regulated cell surface expression of death receptor 5.

    PubMed

    Chen, Jun-Jie; Mikelis, Constantinos M; Zhang, Yaqin; Gutkind, J Silvio; Zhang, Baolin

    2013-02-01

    TNF-related apoptosis inducing ligand (TRAIL) induces apoptosis through its death receptors (DRs) 4 and/or 5 expressed on the surface of target cells. The selectivity of TRAIL towards cancer cells has promoted clinical evaluation of recombinant human TRAIL (rhTRAIL) and its agonistic antibodies in treating several major human cancers including colon and non-Hodgkin's lymphoma. However, little is known about their ability in killing oral squamous cell carcinoma (OSCC) cells. In this study, we tested the apoptotic responses of a panel of seven human OSCC cell lines (HN31, HN30, HN12, HN6, HN4, Cal27, and OSCC3) to rhTRAIL and monoclonal antibodies against DR4 or DR5. We found that rhTRAIL is a potent inducer of apoptosis in most of the oral cancer cell lines tested both in vitro and in vivo. We also showed that DR5 was expressed on the surface of the tested cell lines which correlated with the cellular susceptibility to apoptosis induced by rhTRAIL and anti-DR5 antibody. By contrast, little or no DR4 was detected on the surface of OSCC3 and HN6 cells rendering cellular resistance to DR4 antibody and a reduced sensitivity to rhTRAIL. Notably, the overall TRAIL sensitivity correlated well with the levels of endogenous active Ras in the cell lines tested. Expression of a constitutively active Ras mutant (RasV12) in OSCC3 cells selectively upregulated surface expression of DR5, but not DR4, and restored TRAIL sensitivity. Our findings could have implications for the use of TRAIL receptor targeted therapies in the treatment of human OSCC tumors particularly the ones harboring constitutively active Ras mutant.

  10. Induction of aryl hydrocarbon receptor-mediated and estrogen receptor-mediated activities, and modulation of cell proliferation by dinaphthofurans.

    PubMed

    Vondrácek, Jan; Chramostová, Katerina; Plísková, Martina; Bláha, Ludek; Brack, Werner; Kozubík, Alois; Machala, Miroslav

    2004-09-01

    A group of heterocyclic aromatic compounds, dinaphthofurans (DNFs), recently have been identified as potentially significant contaminants in freshwater sediments. In the present study, a battery of in vitro assays was used for detection of toxic effects of DNFs that are potentially associated with endocrine disruption and tumor promotion. Dinaphthofurans were found to act as relatively potent inducers of aryl hydrocarbon receptor (AhR)-mediated activity in the chemical-activated luciferase reporter gene expression DR-CALUX assay. The relative AhR-inducing potencies of DNFs were similar or even higher than relative potencies of unsubstituted polycyclic aromatic hydrocarbons (PAHs), with dinaphtho[1,2-b;2'3'-d]furan being the most potent AhR agonist. Two compounds, dinaphtho[2,1-b;2'3'-d]furan and dinaphtho[1,2-b;1'2'-d]furan, induced estrogen receptor (ER)-mediated activity in the estrogen receptor-mediated CALUX (the ER-CALUX) assay. Two types of potential tumor-promoting effects of DNFs were investigated, using in vitro bioassays for detection of inhibition of gap-junctional intercellular communication and detection of a release from contact inhibition. Although the acute inhibition of gap-junctional intercellular communication was not observed, all six tested DNFs were able to release rat liver epithelial WB-F344 cells from contact inhibition at concentrations as low as 100 nM. In summary, the present study indicated that DNFs can exert multiple biological effects in vitro, including induction of the AhR-mediated activity, release of cells from contact inhibition, and induction of ER-mediated activity.

  11. Up-regulation of FLIP in cisplatin-selected HeLa cells causes cross-resistance to CD95/Fas death signalling.

    PubMed Central

    Kamarajan, Pachiyappan; Sun, Nian-Kang; Chao, Chuck C-K

    2003-01-01

    Cisplatin-selected cervix carcinoma HeLa cell lines induced less apoptosis, and weaker activation by cisplatin or Fas-activating antibody, of mitochondrial-associated caspase-9 and death receptor-mediated caspase-8 than did parental cells. Furthermore, less DISC (death-inducing signalling complex) was formed in cisplatin-selected cell lines than in parental cells. Ac-IETD-CHO (acetyl-Ile-Glu-Thr-Asp-aldehyde), which has a certain preference for inhibiting caspase-8, or Fas-antagonistic antibody, significantly inhibited cisplatin-induced apoptosis in both parental and cisplatin-selected HeLa cell lines. These results imply that cell-surface death signalling is inducible by cisplatin; that reduction of this pathway is associated with drug resistance, and that cisplatin-selected cells acquire cross-resistance to cell-surface death signalling. Sequential up-regulation of FLIP (FLICE-like inhibitory protein), but not Bcl-2, Bcl-x(L) or inhibitors of apoptosis protein (IAPs), was observed in resistant cells but not in parental cells. The inhibition of FLIP by FLIP antisense oligonucleotides promotes cisplatin and Fas-antibody-induced apoptosis. However, the modulation of apoptosis by FLIP antisense oligonucleotides in resistant cells is greater than that in parental cells. The presented data reveal that the up-regulation of FLIP may contribute to the suppression of apoptosis and thereby change cells that are resistant to cisplatin and Fas-mediated death signals. The results also show that cancer cells that have undergone long-term chemotherapy and become chemoresistant may change the FLIP level, becoming cross-resistant to death factors such as Fas. PMID:12911332

  12. Inactivation of BRCA2 in human cancer cells identifies a subset of tumors with enhanced sensitivity towards death receptormediated apoptosis

    PubMed Central

    De Toni, Enrico N.; Ziesch, Andreas; Rizzani, Antonia; Török, Helga-Paula; Hocke, Sandra; Lü, Shuai; Wang, Shao-Chun; Hucl, Tomas; Göke, Burkhard; Bruns, Christiane; Gallmeier, Eike

    2016-01-01

    Purpose DNA repair defects due to detrimental BRCA2-mutations confer increased susceptibility towards DNA interstrand-crosslinking (ICL) agents and define patient subpopulations for individualized genotype-based cancer therapy. However, due to the side effects of these drugs, there is a need to identify additional agents, which could be used alone or in combination with ICL-agents. Therefore, we investigated whether BRCA2-mutations might also increase the sensitivity towards TRAIL-receptors (TRAIL-R)-targeting compounds. Experimental design Two independent model systems were applied: a BRCA2 gene knockout and a BRCA2 gene complementation model. The effects of TRAIL-R-targeting compounds and ICL-agents on cell viability, apoptosis and cell cycle distribution were compared in BRCA2-proficient versus-deficient cancer cells in vitro. In addition, the effects of the TRAIL-R2-targeting antibody LBY135 were assessed in vivo using a murine tumor xenograft model. Results BRCA2-deficient cancer cells displayed an increased sensitivity towards TRAIL-R-targeting agents. These effects exceeded and were mechanistically distinguishable from the well-established effects of ICL-agents. In vitro, ICL-agents expectedly induced an early cell cycle arrest followed by delayed apoptosis, whereas TRAIL-R-targeting compounds caused early apoptosis without prior cell cycle arrest. In vivo, treatment with LBY135 significantly reduced the tumor growth of BRCA2-deficient cancer cells in a xenograft model. Conclusions BRCA2 mutations strongly increase the in vitro- and in vivo-sensitivity of cancer cells towards TRAIL-R-mediated apoptosis. This effect is mechanistically distinguishable from the well-established ICL-hypersensitivity of BRCA2-deficient cells. Our study thus defines a new genetic subpopulation of cancers susceptible towards TRAIL-R-targeting compounds, which could facilitate novel therapeutic approaches for patients with BRCA2-deficient tumors. PMID:26843614

  13. Sanguinarine-induced oxidative stress and apoptosis-like programmed cell death(AL-PCD) in root meristem cells of Allium cepa.

    PubMed

    Żabka, Aneta; Winnicki, Konrad; Polit, Justyna Teresa; Maszewski, Janusz

    2017-03-01

    A vast number of studies on plant cell systems clearly indicate that various biotic and abiotic stresses give rise to the uncontrolled increase in the level of reactive oxygen species (ROS). Excess concentrations of ROS result in damage to proteins, lipids, carbohydrates, and DNA, which may lead, in consequence, to the apoptotic cell death. The current study investigates the effects of sanguinarine (SAN), a natural alkaloid derived from the roots of Sanguinaria canadensis, on root apical meristem cells of Allium cepa. It is shown that SAN treatment generated large amounts of hydrogen peroxide (H2O2) and superoxide anion (O2·-). Oxidative stress induced in SAN-treated cells was correlated with DNA fragmentation, formation of micronuclei (MN), altered and 'degenerated' chromatin structures characteristic of apoptosis-like programmed cell death (AL-PCD). The experiments with SAN + MG132 (a proteasome inhibitor engaged in Topo II-mediated formation of cleavable complexes) and SAN + ascorbic acid (AA; H2O2 scavenger) seem to suggest, however, that the high level of H2O2 is not the only factor responsible for changes observed at the chromatin level and for the consequent cell death. Our findings imply that Topo II-DNA covalent complexes and 26S proteasomes are also involved in SAN-induced DNA damage.

  14. Water-Soluble Coenzyme Q10 Inhibits Nuclear Translocation of Apoptosis Inducing Factor and Cell Death Caused by Mitochondrial Complex I Inhibition

    PubMed Central

    Li, Haining; Chen, Guisheng; Ma, Wanrui; Li, Ping-An Andy

    2014-01-01

    The objectives of the study were to explore the mechanism of rotenone-induced cell damage and to examine the protective effects of water-soluble Coenzyme Q10 (CoQ10) on the toxic effects of rotenone. Murine hippocampal HT22 cells were cultured with mitochondrial complex I inhibitor rotenone. Water-soluble CoQ10 was added to the culture media 3 h prior to the rotenone incubation. Cell viability was determined by alamar blue, reactive oxygen species (ROS) production by dihydroethidine (DHE) and mitochondrial membrane potential by tetramethyl rhodamine methyl ester (TMRM). Cytochrome c, caspase-9 and apoptosis-inducing factor (AIF) were measured using Western blotting after 24 h rotenone incubation. Rotenone caused more than 50% of cell death, increased ROS production, AIF nuclear translocation and reduction in mitochondrial membrane potential, but failed to cause mitochondrial cytochrome c release and caspase-9 activation. Pretreatment with water-soluble CoQ10 enhanced cell viability, decreased ROS production, maintained mitochondrial membrane potential and prevented AIF nuclear translocation. The results suggest that rotenone activates a mitochondria-initiated, caspase-independent cell death pathway. Water-soluble CoQ10 reduces ROS accumulation, prevents the fall of mitochondrial membrane potential, and inhibits AIF translocation and subsequent cell death. PMID:25089873

  15. Apoptosis and autophagy: BIM as a mediator of tumour cell death in response to oncogene-targeted therapeutics.

    PubMed

    Gillings, Annette S; Balmanno, Kathryn; Wiggins, Ceri M; Johnson, Mark; Cook, Simon J

    2009-11-01

    The BCL-2 homology domain 3 (BH3)-only protein, B-cell lymphoma 2 interacting mediator of cell death (BIM) is a potent pro-apoptotic protein belonging to the B-cell lymphoma 2 protein family. In recent years, advances in basic biology have provided a clearer picture of how BIM kills cells and how BIM expression and activity are repressed by growth factor signalling pathways, especially the extracellular signal-regulated kinase 1/2 and protein kinase B pathways. In tumour cells these oncogene-regulated pathways are used to counter the effects of BIM, thereby promoting tumour cell survival. In parallel, a new generation of targeted therapeutics has been developed, which show remarkable specificity and efficacy in tumour cells that are addicted to particular oncogenes. It is now apparent that the expression and activation of BIM is a common response to these new therapeutics. Indeed, BIM has emerged from this marriage of basic and applied biology as an important mediator of tumour cell death in response to such drugs. The induction of BIM alone may not be sufficient for significant tumour cell death, as BIM is more likely to act in concert with other BH3-only proteins, or other death pathways, when new targeted therapeutics are used in combination with traditional chemotherapy agents. Here we discuss recent advances in understanding BIM regulation and review the role of BIM as a mediator of tumour cell death in response to novel oncogene-targeted therapeutics.

  16. Geraniin-mediated apoptosis by cleavage of focal adhesion kinase through up-regulation of Fas ligand expression in human melanoma cells.

    PubMed

    Lee, Jang-Chang; Tsai, Chih-Yen; Kao, Jung-Yie; Kao, Ming-Ching; Tsai, Shih-Chang; Chang, Chih-Shiang; Huang, Li-Jiau; Kuo, Sheng-Chu; Lin, Jen-Kun; Way, Tzong-Der

    2008-06-01

    Geraniin, a form of tannin separated from geranium, causes cell death through induction of apoptosis; however, cell death characteristics for geraniin have not yet been elucidated. Here, we investigated the mechanism of geraniin-induced apoptosis in human melanoma cells and demonstrated that geraniin was able to induce cell apoptosis in a concentration- and time-dependent manner. We also examined the signaling pathway related to geraniin-induced apoptosis. To clarify the relationship between focal adhesion kinase (FAK) and geraniin-induced apoptosis, we treated human melanoma cells with geraniin and found that this resulted dose- and time-dependent degradation in FAK. However, FAK cleavage was significantly inhibited when cells were pretreated with a selective inhibitor of caspase-3 (Ac-Asp-Glu-Val-Asp-CHO). Here, we demonstrated for the first time that geraniin triggered cell death by caspase-3-mediated cleavage of FAK. There were two possible mechanisms for activating caspase-3, mitochondria-mediated and receptor-mediated apoptosis. To confirm the geraniin-relevant signaling pathway, using immunoblot analysis we found that geraniin-induced apoptosis was associated with the up-regulation of Fas ligand expression, the activation of caspase-8, the cleavage of Bid, and the induction of cytochrome c release from mitochondria to the cytosol. Treatment with geraniin caused induction of caspase-3 activity in a dose- and time-dependent manner followed by proteolytic cleavage of poly-(ADP-ribose) polymerase, and DNA fragmentation factor 45. The geraniin-induced apoptosis may provide a pivotal mechanism for its cancer-chemopreventive action.

  17. Physangulidine A, a withanolide from Physalis angulata, perturbs the cell cycle and induces cell death by apoptosis in prostate cancer cells.

    PubMed

    Reyes-Reyes, E Merit; Jin, Zhuang; Vaisberg, Abraham J; Hammond, Gerald B; Bates, Paula J

    2013-01-25

    Recently, our group reported the discovery of three new withanolides, physangulidines A-C, from Physalis angulata. In this study, the biological effects of physangulidine A (1), which was the most active and abundant of the three new constituents, are described. It was found that 1 significantly reduces survival in clonogenic assays for two hormone-independent prostate cancer cell lines. Flow cytometry and confocal microscopy studies in DU145 human prostate cancer cells indicated that 1 induces cell cycle arrest in the G(2)/M phase and causes defective mitosis. It was determined also that 1 produces programed cell death by apoptosis, as evidenced by biochemical markers and distinct changes in cell morphology. These results imply that the antimitotic and proapoptotic effects of 1 may contribute significantly to the biological activities and potential medicinal properties of its plant of origin.

  18. A possible role of oxidative stress in the switch mechanism of the cell death mode from apoptosis to necrosis--studies on rho0 cells.

    PubMed

    Wochna, Agnieszka; Niemczyk, Edyta; Kurono, Chieko; Masaoka, Makoto; Kedzior, Jakub; Słomińska, Ewa; Lipiński, Marcin; Wakabayashi, Takashi

    2007-01-01

    Apoptosis is induced not only during morphogenesis and embryogenesis but also under various pathological conditions, especially related to oxidative stress. Apoptotic cells are phagocytized by neighboring cells while necrotic cells cause local and general reactions sometimes lethal to our bodies. Data have been accumulated to demonstrate that the switch of the cell death mode from apoptosis to necrosis does occur. However, detailed mechanisms involved in the switch mechanism remain unsolved although decreases in the intracellular level of ATP and a burst in the cellular level of reactive oxygen species (ROS) have been proposed. Recently, we have shown that the population of apoptotic cells reaches maximum in human osteosarcoma 143B cells treated for 6h with menadione (MEN) while necrotic cells become predominant at 9h of the treatment. In the present study we have attempted to clarify the role of cellular ATP in the switch mechanism using rho(0) cells derived from human osteosarcoma rho+ cells. Results are summarized as follows: (1) Apoptotic and necrotic changes in rho(0) cells are much faster than rho+ cells after the treatment with MEN. (2) Cellular level of ATP in rho(0) cells remains essentially in the same level before and after the MEN-treatment while intracellular levels of superoxide continuously increase after the MEN-treatment. (3) rho+ cells treated with MEN in the presence of antimycin A plus oligomycin show similar changes to those of MEN-treated rho(0) cells. (4) MEN-induced increases in the cellular level of superoxide are distinctly suppressed by inhibitors of NADPH oxidase. These results suggest that the intracellular level of superoxide may be a key factor directly related to the switch mechanism from apoptosis to necrosis, and that decreases in cellular level of ATP accelerate both apoptotic and necrotic changes of the cells.

  19. Role of mitochondria in the switch mechanism of the cell death mode from apoptosis to necrosis--studies on rho0 cells.

    PubMed

    Wochna, Agnieszka; Niemczyk, Edyta; Kurono, Chieko; Masaoka, Makoto; Majczak, Anna; Kedzior, Jakub; Slominska, Ewa; Lipinski, Marcin; Wakabayashi, Takashi

    2005-04-01

    Detailed mechanisms of the switch of the cell death mode from apoptosis to necrosis remain to be solved, although the intracellular level of ATP and that of free radicals have been postulated to be the major factors involved in the mechanisms. In the present study menadione (MEN)-induced cell injury processes were studied using rho0 cells derived from human osteosarcoma 143B cells and parental rho+ cells co-treated with inhibitors of electron transfer chain of mitochondria or oligomycin, an inhibitor of ATP synthesis. Treatment of rho+ cells with 100 microM MEN induced apoptosis, which reached the maximum at 6 h, and was followed by an abrupt decrease thereafter, while necrotic cells (NC) increased continuously when they were judged by Annexin V and PI double staining. On the other hand, MEN induced apoptotic and necrotic changes much faster in rho0 cells compared to rho+ cells. The frequency to find apoptotic cells (AP) in the former cells was distinctly smaller than that to find NC judged by Annexin V and PI double staining. Electron microscopically, a major population of rho0 cells treated with MEN for 6 h consisted of intermediate cells, and a small number of AP co-existed. At 9 h of the treatment intermediate cells were exclusively seen, and AP were hardly detected. When parental rho+ cells were treated with MEN in the presence of oligomycin or oligomycin plus antimycin A both apoptotic and necrotic changes of the cells were distinctly accelerated. The intracellular level of superoxide in rho0 cells continuously increased after the MEN treatment, whereas that of ATP remained distinctly low before and after the MEN treatment compared to that in rho+ cells. These data suggest that the intracellular level of superoxide may be a key factor controlling the switch from apoptosis to necrosis.

  20. Hesperidin from Citrus seed induces human hepatocellular carcinoma HepG2 cell apoptosis via both mitochondrial and death receptor pathways.

    PubMed

    Banjerdpongchai, Ratana; Wudtiwai, Benjawan; Khaw-On, Patompong; Rachakhom, Wasitta; Duangnil, Natthachai; Kongtawelert, Prachya

    2016-01-01

    Citrus seeds are full of phenolic compounds, such as flavonoids. The aims of this study were to identify the types of flavonoids in Citrus seed extracts, the cytotoxic effect, mode of cell death, and signaling pathway in human hepatic cancer HepG2 cells. The flavonoids contain anticancer, free radical scavenging, and antioxidant activities. Neohesperidin, hesperidin, and naringin, active flavanone glycosides, were identified in Citrus seed extract. The cytotoxic effect of three compounds was in a dose-dependent manner, and IC50 levels were determined. The sensitivity of human HepG2 cells was as follows: hesperidin > naringin > neohesperidin > naringenin. Hesperidin induced HepG2 cells to undergo apoptosis in a dose-dependent manner as evidenced by the externalization of phosphatidylserine and determined by annexin V-fluorescein isothiocyanate and propidium iodide staining using flow cytometry. Hesperidin did not induce the generation of reactive oxygen species, which was determined by using 2',7'-dichlorohydrofluorescein diacetate and flow cytometry method. The number of hesperidin-treated HepG2 cells with the loss of mitochondrial transmembrane potential increased concentration dependently, using 3,3'-dihexyloxacarbocyanine iodide employing flow cytometry. Caspase-9, -8, and -3 activities were activated and increased in hesperidin-treated HepG2 cells. Bcl-xL protein was downregulated whereas Bax, Bak, and tBid protein levels were upregulated after treatment with hesperidin in a dose-dependent manner. In conclusion, the bioflavanone from Citrus seeds, hesperidin, induced human HepG2 cell apoptosis via mitochondrial pathway and death receptor pathway. Citrus seed flavonoids are beneficial and can be developed as anticancer drug or food supplement, which still needs further in vivo investigation in animals and human beings.

  1. Green tea polyphenols induce cell death in breast cancer MCF-7 cells through induction of cell cycle arrest and mitochondrial-mediated apoptosis*

    PubMed Central

    Liu, Shu-min; Ou, Shi-yi; Huang, Hui-hua

    2017-01-01

    In order to study the molecular mechanisms of green tea polyphenols (GTPs) in treatment or prevention of breast cancer, the cytotoxic effects of GTPs on five human cell lines (MCF-7, A549, Hela, PC3, and HepG2 cells) were determined and the antitumor mechanisms of GTPs in MCF-7 cells were analyzed. The results showed that GTPs exhibited a broad spectrum of inhibition against the detected cancer cell lines, particularly the MCF-7 cells. Studies on the mechanisms revealed that the main modes of cell death induced by GTPs were cell cycle arrest and mitochondrial-mediated apoptosis. Flow cytometric analysis showed that GTPs mediated cell cycle arrest at both G1/M and G2/M transitions. GTP dose dependently led to apoptosis of MCF-7 cells via the mitochondrial pathways, as evidenced by induction of chromatin condensation, reduction of mitochondrial membrane potential (ΔΨ m), improvement in the generation of reactive oxygen species (ROS), induction of DNA fragmentation, and activations of caspase-3 and caspase-9 in the present paper. PMID:28124838

  2. Activation of the intrinsic cell death pathway, increased apoptosis and modulation of astrocytes in the cerebellum of diabetic rats.

    PubMed

    Lechuga-Sancho, Alfonso M; Arroba, Ana I; Frago, Laura M; Pañeda, Covadonga; García-Cáceres, Cristina; Delgado Rubín de Célix, Arancha; Argente, Jesús; Chowen, Julie A

    2006-08-01

    Poorly controlled diabetes mellitus results in structural and functional changes in many brain regions. We demonstrate that in streptozotocin-induced diabetic rats cell death is increased and proliferation decreased in the cerebellum, indicating overall cell loss. Levels of both the proform and cleaved forms of caspases 3, 6 and 9 are increased, with no change in caspases 7, 8 or 12. Colocalization of glial fibrillary acidic protein (GFAP) and cleaved caspase 3 and GFAP in TUNEL-positive cells increased in diabetic rats. Changes in GFAP levels paralleled modifications in proliferating cell nuclear antigen (PCNA), increasing at 1 week of diabetes and decreasing thereafter, and proliferating GFAP-positive cells were decreased in the cerebellum of diabetic rats. These results suggest that astrocytes are dramatically affected in the cerebellum, including an increase in cell death and a decrease in proliferation, and this could play a role in the structural and functional changes in this brain area in diabetes.

  3. Newly synthesized quinazolinone HMJ-38 suppresses angiogenetic responses and triggers human umbilical vein endothelial cell apoptosis through p53-modulated Fas/death receptor signaling

    SciTech Connect

    Chiang, Jo-Hua; Yang, Jai-Sing; Lu, Chi-Cheng; Hour, Mann-Jen; Chang, Shu-Jen; Lee, Tsung-Han; Chung, Jing-Gung

    2013-06-01

    The current study aims to investigate the antiangiogenic responses and apoptotic death of human umbilical vein endothelial cells (HUVECs) by a newly synthesized compound named 2-(3′-methoxyphenyl)-6-pyrrolidinyl-4-quinazolinone (HMJ-38). This work attempted to not only explore the effects of angiogenesis on in vivo and ex vivo studies but also hypothesize the implications for HUVECs (an ideal cell model for angiogenesis in vitro) and further undermined apoptotic experiments to verify the underlying molecular signaling by HMJ-38. Our results demonstrated that HMJ-38 significantly inhibited blood vessel growth and microvessel formation by the mouse Matrigel plug assay of angiogenesis, and the suppression of microsprouting from the rat aortic ring assay was observed after HMJ-38 exposure. In addition, HMJ-38 disrupted the tube formation and blocked the ability of HUVECs to migrate in response to VEGF. We also found that HMJ-38 triggered cell apoptosis of HUVECs in vitro. HMJ-38 concentration-dependently suppressed viability and induced apoptotic damage in HUVECs. HMJ-38-influenced HUVECs were performed by determining the oxidative stress (ROS production) and ATM/p53-modulated Fas and DR4/DR5 signals that were examined by flow cytometry, Western blotting, siRNA and real-time RT-PCR analyses, respectively. Our findings demonstrate that p53-regulated extrinsic pathway might fully contribute to HMJ-38-provoked apoptotic death in HUVECs. In view of these observations, we conclude that HMJ-38 reduces angiogenesis in vivo and ex vivo as well as induces apoptosis of HUVECs in vitro. Overall, HMJ-38 has a potent anti-neovascularization effect and could warrant being a vascular targeting agent in the future. - Highlights: • HMJ-38 suppresses angiogenic actions in vivo and ex vivo. • Inhibitions of blood vessel and microvessel formation by HMJ-38 are acted. • Cytotoxic effects of HUVECs occur by HMJ-38 challenge. • p53-modulated extrinsic pathway contributes to HMJ-38

  4. FLIP the Switch: Regulation of Apoptosis and Necroptosis by cFLIP

    PubMed Central

    Tsuchiya, Yuichi; Nakabayashi, Osamu; Nakano, Hiroyasu

    2015-01-01

    cFLIP (cellular FLICE-like inhibitory protein) is structurally related to caspase-8 but lacks proteolytic activity due to multiple amino acid substitutions of catalytically important residues. cFLIP protein is evolutionarily conserved and expressed as three functionally different isoforms in humans (cFLIPL, cFLIPS, and cFLIPR). cFLIP controls not only the classical death receptor-mediated extrinsic apoptosis pathway, but also the non-conventional pattern recognition receptor-dependent apoptotic pathway. In addition, cFLIP regulates the formation of the death receptor-independent apoptotic platform named the ripoptosome. Moreover, recent studies have revealed that cFLIP is also involved in a non-apoptotic cell death pathway known as programmed necrosis or necroptosis. These functions of cFLIP are strictly controlled in an isoform-, concentration- and tissue-specific manner, and the ubiquitin-proteasome system plays an important role in regulating the stability of cFLIP. In this review, we summarize the current scientific findings from biochemical analyses, cell biological studies, mathematical modeling, and gene-manipulated mice models to illustrate the critical role of cFLIP as a switch to determine the destiny of cells among survival, apoptosis, and necroptosis. PMID:26694384

  5. Balance between short and long isoforms of cFLIP regulates Fas-mediated apoptosis in vivo

    PubMed Central

    Ram, Daniel R.; Ilyukha, Vladimir; Volkova, Tatyana; Buzdin, Anton; Tai, Albert; Smirnova, Irina; Poltorak, Alexander

    2016-01-01

    cFLIP, an inhibitor of apoptosis, is a crucial regulator of cellular death by apoptosis and necroptosis; its importance in development is exemplified by the embryonic lethality in cFLIP–deficient animals. A homolog of caspase 8 (CASP8), cFLIP exists in two main isoforms: cFLIPL (long) and cFLIPR (short). Although both splice variants regulate death receptor (DR)-induced apoptosis by CASP8, the specific role of each isoform is poorly understood. Here, we report a previously unidentified model of resistance to Fas receptor-mediated liver failure in the wild-derived MSM strain, compared with susceptibility in C57BL/6 (B6) mice. Linkage analysis in F2 intercross (B6 x MSM) progeny identified several MSM loci controlling resistance to Fas-mediated death, including the caspase 8- and FADD-like apoptosis regulator (Cflar) locus encoding cFLIP. Furthermore, we identified a 21-bp insertion in the 3′ UTR of the fifth exon of Cflar in MSM that influences differential splicing of cFLIP mRNA. Intriguingly, we observed that MSM liver cells predominantly express the FLIPL variant, in contrast to B6 liver cells, which have higher levels of cFLIPR. In keeping with this finding, genome-wide RNA sequencing revealed a relative abundance of FLIPL transcripts in MSM hepatocytes whereas B6 liver cells had significantly more FLIPR mRNA. Importantly, we show that, in the MSM liver, CASP8 is present exclusively as its cleaved p43 product, bound to cFLIPL. Because of partial enzymatic activity of the heterodimer, it might prevent necroptosis. On the other hand, it prevents cleavage of CASP8 to p10/20 necessary for cleavage of caspase 3 and, thus, apoptosis induction. Therefore, MSM hepatocytes are predisposed for protection from DR-mediated cell death. PMID:26798068

  6. Receptor-mediated endocytosis for drug delivery in African trypanosomes: fulfilling Paul Ehrlich's vision of chemotherapy.

    PubMed

    Alsford, Sam; Field, Mark C; Horn, David

    2013-05-01

    Bloodstream-form cells of Trypanosoma brucei exhibit massively increased endocytic activity relative to the insect midgut stage, enabling rapid recycling of variant surface glycoprotein and antibody clearance from the surface. In addition, recent advances have identified a role for receptor-mediated endocytosis in the uptake of the antitrypanosomal drug, suramin, via invariant surface glycoprotein 75, and in the uptake of trypanosome lytic factor 1 via haptoglobin-haemoglobin receptor. Here, we argue that receptor-mediated endocytosis represents both a validated drug target and a promising route for the delivery of novel therapeutics into trypanosomes.

  7. Cleavage of DFNA5 by caspase-3 during apoptosis mediates progression to secondary necrotic/pyroptotic cell death

    PubMed Central

    Rogers, Corey; Fernandes-Alnemri, Teresa; Mayes, Lindsey; Alnemri, Diana; Cingolani, Gino; Alnemri, Emad S.

    2017-01-01

    Apoptosis is a genetically regulated cell suicide programme mediated by activation of the effector caspases 3, 6 and 7. If apoptotic cells are not scavenged, they progress to a lytic and inflammatory phase called secondary necrosis. The mechanism by which this occurs is unknown. Here we show that caspase-3 cleaves the GSDMD-related protein DFNA5 after Asp270 to generate a necrotic DFNA5-N fragment that targets the plasma membrane to induce secondary necrosis/pyroptosis. Cells that express DFNA5 progress to secondary necrosis, when stimulated with apoptotic triggers such as etoposide or vesicular stomatitis virus infection, but disassemble into small apoptotic bodies when DFNA5 is deleted. Our findings identify DFNA5 as a central molecule that regulates apoptotic cell disassembly and progression to secondary necrosis, and provide a molecular mechanism for secondary necrosis. Because DFNA5-induced secondary necrosis and GSDMD-induced pyroptosis are dependent on caspase activation, we propose that they are forms of programmed necrosis. PMID:28045099

  8. Resveratrol Induces Apoptosis-Like Death and Prevents In Vitro and In Vivo Virulence of Entamoeba histolytica.

    PubMed

    Pais-Morales, Jonnatan; Betanzos, Abigail; García-Rivera, Guillermina; Chávez-Munguía, Bibiana; Shibayama, Mineko; Orozco, Esther

    2016-01-01

    Entamoeba histolytica causes amoebiasis, an infection that kills 100,000 individuals each year. Metronidazole and its derivatives are currently used against this protozoan, but these drugs present adverse effects on human health. Here, we investigated the effect of resveratrol (a natural compound) on E. histolytica trophozoites viability, as well as its influence on the parasite virulence. Trophozoites growth was arrested by 72 μM resveratrol and the IC50 was determined as 220 μM at 48 h. Cells appeared smaller, rounded and in clusters, with debris-containing vacuoles and with abnormally condensed chromatin. Resveratrol triggered reactive oxygen species production. It caused lipid peroxidation and produced phosphatidylserine externalization and DNA fragmentation this latter evidenced by TUNEL assays. It also provoked an increase of intracellular Ca2+ concentration, activated calpain and decreased superoxide dismutase activity, indicating that an apoptosis-like event occurred; however, autophagy was not detected. Cytopathic activity, phagocytosis, encystment and in vivo virulence were diminished dramatically by pre-incubation of trophozoites with resveratrol, evidencing that resveratrol attenuated the trophozoite virulence in vitro. Interestingly, after the inoculation of virulent trophozoites, animals treated with the drug did not develop or developed very small abscesses. Our findings propose that resveratrol could be an alternative to contend amoebiasis.

  9. Resveratrol Induces Apoptosis-Like Death and Prevents In Vitro and In Vivo Virulence of Entamoeba histolytica

    PubMed Central

    Pais-Morales, Jonnatan; Betanzos, Abigail; García-Rivera, Guillermina; Chávez-Munguía, Bibiana; Shibayama, Mineko; Orozco, Esther

    2016-01-01

    Entamoeba histolytica causes amoebiasis, an infection that kills 100,000 individuals each year. Metronidazole and its derivatives are currently used against this protozoan, but these drugs present adverse effects on human health. Here, we investigated the effect of resveratrol (a natural compound) on E. histolytica trophozoites viability, as well as its influence on the parasite virulence. Trophozoites growth was arrested by 72 μM resveratrol and the IC50 was determined as 220 μM at 48 h. Cells appeared smaller, rounded and in clusters, with debris-containing vacuoles and with abnormally condensed chromatin. Resveratrol triggered reactive oxygen species production. It caused lipid peroxidation and produced phosphatidylserine externalization and DNA fragmentation this latter evidenced by TUNEL assays. It also provoked an increase of intracellular Ca2+ concentration, activated calpain and decreased superoxide dismutase activity, indicating that an apoptosis-like event occurred; however, autophagy was not detected. Cytopathic activity, phagocytosis, encystment and in vivo virulence were diminished dramatically by pre-incubation of trophozoites with resveratrol, evidencing that resveratrol attenuated the trophozoite virulence in vitro. Interestingly, after the inoculation of virulent trophozoites, animals treated with the drug did not develop or developed very small abscesses. Our findings propose that resveratrol could be an alternative to contend amoebiasis. PMID:26731663

  10. The regulation of p53 up-regulated modulator of apoptosis by JNK/c-Jun pathway in β-amyloid-induced neuron death.

    PubMed

    Akhter, Rumana; Sanphui, Priyankar; Das, Hrishita; Saha, Pampa; Biswas, Subhas Chandra

    2015-09-01

    Neuronal loss in selective areas of brain underlies the pathology of Alzheimer's disease (AD). Recent evidences place oligomeric β-amyloid (Aβ) central to the disease. However, mechanism of neuron death in response to Aβ remains elusive. Activation of the c-Jun N-terminal kinase (JNK) pathway and induction of the AP-1 transcription factor c-Jun are reported in AD. However, targets of JNK/c-Jun in Aβ-induced neuron death are mostly unknown. Our study shows that pro-apoptotic proteins, Bim (Bcl-2 interacting mediator of cell death) and Puma (p53 up-regulated modulator of apoptosis) are targets of c-Jun in Aβ-treated neurons. We demonstrate that the JNK/c-Jun pathway is activated, in cultures of cortical neurons following treatment with oligomeric Aβ and in AD transgenic mice, and that inhibition of this pathway by selective inhibitor blocks induction of Puma by Aβ. We also find that both JNK and p53 pathways co-operatively regulate Puma expression in Aβ-treated neurons. Moreover, we identified a novel AP1-binding site on rat puma gene which is necessary for direct binding of c-Jun with Puma promoter. Finally, we find that knocking down of c-Jun by siRNA provides significant protection from Aβ toxicity and that induction of Bim and Puma by Aβ in neurons requires c-Jun. Taken together, our results suggest that both Bim and Puma are target of c-Jun and elucidate the intricate regulation of Puma expression by JNK/c-Jun and p53 pathways in neurons upon Aβ toxicity. JNK/c-Jun pathway is shown to be activated in neurons of the Alzheimer's disease (AD) brain and plays a vital role in neuron death in AD models. However, downstream targets of c-Jun in this disease have not been thoroughly elucidated. Our study shows that two important pro-apoptotic proteins, Bim (Bcl-2 interacting mediator of cell death) and Puma (p53 up-regulated modulator of apoptosis) are targets of c-Jun in Aβ-treated neurons. We demonstrate that the JNK/c-jun pathway is activated, in cultures

  11. BARC: A Novel Apoptosis Regulator

    DTIC Science & Technology

    2004-07-01

    turnover is normally achieved through programmed cell death , also known as apoptosis. Effects in apoptosis occur in breast cancers and other types of...malignancies, making tumor cells difficult to kill by chemotherapy, hormonal therapy, and radiation. Restoring function of cell death pathways is a strategy...These findings provide new insights into cell death regulation in breast cancer.

  12. Leptospermum flavescens Constituent-LF1 Causes Cell Death through the Induction of Cell Cycle Arrest and Apoptosis in Human Lung Carcinoma Cells

    PubMed Central

    Navanesan, Suerialoasan; Abdul Wahab, Norhanom; Manickam, Sugumaran; Sim, Kae Shin

    2015-01-01

    Leptospermum flavescens Sm. (Myrtaceae), locally known as ‘Senna makki’ is a smallish tree that is widespread and recorded to naturally occur in the montane regions above 900 m a.s.l from Burma to Australia. Although the species is recorded to be used traditionally to treat various ailments, there is limited data on biological and chemical investigations of L. flavescens. The aim of the present study was to investigate and understand the ability of L. flavescens in inducing cell death in lung cancer cells. The cytotoxic potentials of the extraction yields (methanol, hexane, ethyl acetate and water extracts as wells as a semi pure fraction, LF1) were evaluated against two human non-small cell lung carcinoma cell lines (A549 and NCI-H1299) using the MTT assay. LF1 showed the greatest cytotoxic effect against both cell lines with IC50 values of 7.12 ± 0.07 and 9.62 ± 0.50 μg/ml respectively. LF1 treated cells showed a sub-G1 region in the cell cycle analysis and also caused the presence of apoptotic morphologies in cells stained with acridine orange and ethidium bromide. Treatment with LF1 manifested an apoptotic population in cells that were evaluated using the Annexin V/ propidium iodide assay. Increasing dosage of LF1 caused a rise in the presence of activated caspase-3 enzymes in treated cells. Blockage of cell cycle progression was also observed in LF1-treated cells. These findings suggest that LF1 induces apoptosis and cell cycle arrest in treated lung cancer cells. Further studies are being conducted to isolate and identify the active compound as well to better understand the mechanism involved in inducing cell death. PMID:26287817

  13. A methyl jasmonate derivative, J-7, induces apoptosis in human hepatocarcinoma Hep3B cells in vitro.

    PubMed

    Park, Cheol; Jin, Cheng-Yun; Kim, Gi-Young; Cheong, JaeHun; Jung, Jee H; Yoo, Young Hyun; Choi, Yung Hyun

    2010-10-01

    The pro-apoptotic activity of J-7, a synthetic methyl jasmonate derivative, on the Hep3B human hepatocarcinoma cell line was investigated. Treatment of Hep3B cells with J-7 resulted in growth inhibition and the induction of apoptosis as measured by trypan blue-excluding cells, MTT assay, nuclear staining, DNA fragmentation, and flow cytometry analysis. The increased apoptotic events in Hep3B cells caused by J-7 were associated with the alteration in the ratio of Bax/Bcl-2 protein expression. J-7 treatment induced the expression of death receptor-related proteins such as death receptor 5, which triggered the activation of caspase-8 and the down-regulation of the whole Bid expression. In addition, the apoptosis induction by J-7 was correlated with the activation of caspase-9 and caspase-3, down-regulation IAP family proteins such as XIAP and cIAP-1, and concomitant degradation of poly (ADP-ribose) polymerase. However, the cytotoxic and apoptotic effects induced by J-7 were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor, which demonstrates the important role that caspase-3 plays in the process. Furthermore, blocking the extracellular signal-regulated protein kinase and c-Jun N-terminal kinase pathways showed increased apoptosis and the activation of caspases in J-7-induced apoptosis. The results indicated that J-7 induces the apoptosis of Hep3B cells through a signaling cascade of death-receptor-mediated extrinsic as well as mitochondria-mediated intrinsic caspase pathways, which are associated with the activation of the mitogen-activated protein kinases signal pathway.

  14. The inhibition of voltage-gated H(+) channel (HVCN1) induces acidification of leukemic Jurkat T cells promoting cell death by apoptosis.

    PubMed

    Asuaje, Agustín; Smaldini, Paola; Martín, Pedro; Enrique, Nicolás; Orlowski, Alejandro; Aiello, Ernesto A; Gonzalez León, Carlos; Docena, Guillermo; Milesi, Verónica

    2017-02-01

    Cellular energetic deregulation is widely known to produce an overproduction of acidic species in cancer cells. This acid overload must be counterbalanced with a high rate of H(+) extrusion to maintain cell viability. In this sense, many H(+) transporters have been reported to be crucial for cell survival and proposed as antineoplastic target. By the way, voltage-gated proton channels (Hv1) mediate highly selective H(+) outward currents, capable to compensate acid burden in brief periods of time. This structure is canonically described acting as NADPH oxidase counterbalance in reactive oxygen species production. In this work, we show, for the first time in a oncohematologic cell line, that inhibition of Hv1 channels by Zn(2+) and the more selective blocker 2-(6-chloro-1H-benzimidazol-2-yl)guanidine (ClGBI) progressively decreases intracellular pH in resting conditions. This acidification is evident minutes after blockade and progresses under prolonged exposure (2, 17, and 48 h), and we firstly demonstrate that this is followed by cell death through apoptosis (annexin V binding). Altogether, these results contribute strong evidence that this channel might be a new therapeutic target in cancer.

  15. Nanomaterial-induced cell death in pulmonary and hepatic cells following exposure to three different metallic materials: The role of autophagy and apoptosis.

    PubMed

    Kermanizadeh, Ali; Jantzen, Kim; Ward, Michael B; Durhuus, Jon Ambæk; Juel Rasmussen, Lene; Loft, Steffen; Møller, Peter

    2017-03-01

    Autophagy is the catabolic process involving the sequestration of the cytoplasm within double-membrane vesicles, which fuse with lysosomes to form autolysosomes in which autophagic targets are degraded. Since most endocytic routes of nanomaterial uptake converge upon the lysosome and the possibility that autophagy induction by NMs may be an attempt by the cell to self-preserve following the external challenge, this study investigated the role of autophagy following exposure to a panel of widely used metal-based NMs with high toxicity (Ag and ZnO) or low toxicity (TiO2) in a pulmonary (A549) and hepatic (HepG2) cell line. The in vitro exposure to the Ag and ZnO NMs resulted in the induction of both apoptosis and autophagy pathways in both cell types. However, the progression of autophagy was blocked in the formation of the autolysosome, which coincided with morphologic changes in the actin cytoskeleton. This response was not observed following the exposure to low-toxicity TiO2 NMs. Overall, the results show that high toxicity NMs can cause a dysfunction in the autophagy pathway which is associated with apoptotic cell death.

  16. Death-domain associated protein-6 (DAXX) mediated apoptosis in hantavirus infection is counter-balanced by activation of interferon-stimulated nuclear transcription factors

    SciTech Connect

    Khaiboullina, Svetlana F.; Morzunov, Sergey P.; Boichuk, Sergei V.; Palotás, András; Jeor, Stephen St.; Lombardi, Vincent C.; Rizvanov, Albert A.

    2013-09-01

    Hantaviruses are negative strand RNA species that replicate predominantly in the cytoplasm. They also activate numerous cellular responses, but their involvement in nuclear processes is yet to be established. Using human umbilical vein endothelial cells (HUVECs), this study investigates the molecular finger-print of nuclear transcription factors during hantavirus infection. The viral-replication-dependent activation of pro-myelocytic leukemia protein (PML) was followed by subsequent localization in nuclear bodies (NBs). PML was also found in close proximity to activated Sp100 nuclear antigen and interferon-stimulated gene 20 kDa protein (ISG-20), but co-localization with death-domain associated protein-6 (DAXX) was not observed. These data demonstrate that hantavirus triggers PML activation and localization in NBs in the absence of DAXX-PLM-NB co-localization. The results suggest that viral infection interferes with DAXX-mediated apoptosis, and expression of interferon-activated Sp100 and ISG-20 proteins may indicate intracellular intrinsic antiviral attempts.

  17. Caffeine-Induced Premature Chromosome Condensation Results in the Apoptosis-Like Programmed Cell Death in Root Meristems of Vicia faba

    PubMed Central

    Rybaczek, Dorota; Musiałek, Marcelina Weronika; Balcerczyk, Aneta

    2015-01-01

    We have demonstrated that the activation of apoptosis-like programmed cell death (AL-PCD) was a secondary result of caffeine (CF) induced premature chromosome condensation (PCC) in hydroxyurea-synchronized Vicia faba root meristem cells. Initiation of the apoptotic-like cell degradation pathway seemed to be the result of DNA damage generated by treatment with hydroxyurea (HU) [double-stranded breaks (DSBs) mostly] and co-treatment with HU/CF [single-stranded breaks (SSBs) mainly]. A single chromosome comet assay was successfully used to study different types of DNA damage (neutral variant–DSBs versus alkaline–DSBs or SSBs). The immunocytochemical detection of H2AXS139Ph and PARP-2 were used as markers for DSBs and SSBs, respectively. Acridine orange and ethidium bromide (AO/EB) were applied for quantitative immunofluorescence measurements of dead, dying and living cells. Apoptotic-type DNA fragmentation and positive TUNEL reaction finally proved that CF triggers AL-PCD in stressed V. faba root meristem cells. In addition, the results obtained under transmission electron microscopy (TEM) further revealed apoptotic-like features at the ultrastructural level of PCC-type cells: (i) extensive vacuolization; (ii) abnormal chromatin condensation, its marginalization and concomitant degradation; (iii) formation of autophagy-like vesicles (iv) protoplast shrinkage (v) fragmentation of cell nuclei and (vi) extensive degeneration of the cells. The results obtained have been discussed with respect to the vacuolar/autolytic type of plant-specific AL-PCD. PMID:26545248

  18. BIM (BCL-2 interacting mediator of cell death) SAHB (stabilized α helix of BCL2) not always convinces BAX (BCL-2-associated X protein) for apoptosis.

    PubMed

    Verma, Sharad; Goyal, Sukriti; Tyagi, Chetna; Jamal, Salma; Singh, Aditi; Grover, Abhinav

    2016-06-01

    The interaction of BAX (BCL-2-associated X protein) with BIM (BCL-2 interacting mediator of cell death) SAHB (stabilized α helix of BCL2) directly initiates BAX-mediated mitochondrial apoptosis. This molecular dynamics study reveals that BIM SAHB forms a stable complex with BAX but it remains in a non-functional conformation. N terminal of BAX folds towards the core which has been reported exposed in the functional monomer. The α1-α2 loop, which has been reported in open conformation in functional BAX, acquires a closed conformation during the simulation. BH3/α2 remains less exposed as compared to initial structure. The hydrophobic residues of BIM accommodates in the rear pocket of BAX during the simulation. A steep decrease in radius of gyration and solvent accessible surface area (SASA) indicates the complex folding to acquire a more stable but inactive conformation. Further the covariance matrix reveals that the backbone atoms' motions favour the inactive conformation of the complex. This is the first report on the non-functional BAX-BIM SAHB complex by molecular dynamics simulation in the best of our knowledge.

  19. First description of programmed cell death10 (PDCD10) in rock bream (Oplegnathus fasciatus): Potential relations to the regulation of apoptosis by several pathogens.

    PubMed

    Kim, Ju-Won; Jeong, Ji-Min; Bae, Jin-Sol; Cho, Dong-Hee; Jung, Sung Hee; Hwang, Jee-Youn; Kwon, Mun-Gyeong; Seo, Jung Soo; Baeck, Gun-Wook; Park, Chan-Il

    2016-02-01

    In this study, we isolated and characterized programmed cell death10 (PDCD10), which is known to be related to apoptosis, from rock bream (Oplegnathus fasciatus). The full-length rock bream PDCD10 (RbPDCD10) cDNA (1459 bp) contains an open reading frame of 633 bp that encodes 210 amino acids. Furthermore, multiple alignments revealed that the six of the α-helix bundles were well conserved among the other PDCD10 sequences tested. RbPDCD10 was significantly expressed in the liver, RBC (red blood cell), gill, intestine, trunk kidney and spleen. RbPDCD10 gene expression was also examined in several tissues, including the kidney, spleen, liver, and gill, under bacterial and viral challenges. Generally, all of the examined tissues from the fish that were infected with Edwardsiella tarda and the red sea bream iridovirus (RSIV) exhibited significant up-regulations of RbPDCD10 expression compared to the controls. However, RbPDCD10 expression exhibited dramatic down-regulations in all of the examined tissues following injections of Streptococcus iniae, which is major bacterial pathogen that is responsible for mass mortality in rock bream. Our results revealed that rock bream PDCD10 may be involved in the apoptotic regulation of rock bream immune responses.

  20. ZFAT plays critical roles in peripheral T cell homeostasis and its T cell receptor-mediated response

    SciTech Connect

    Doi, Keiko; Fujimoto, Takahiro; Okamura, Tadashi; Ogawa, Masahiro; Tanaka, Yoko; Mototani, Yasumasa; Goto, Motohito; Ota, Takeharu; Matsuzaki, Hiroshi; Kuroki, Masahide; Tsunoda, Toshiyuki; Sasazuki, Takehiko; Shirasawa, Senji

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer We generated Cd4-Cre-mediated T cell-specific Zfat-deficient mice. Black-Right-Pointing-Pointer Zfat-deficiency leads to reduction in the number of the peripheral T cells. Black-Right-Pointing-Pointer Impaired T cell receptor-mediated response in Zfat-deficient peripheral T cells. Black-Right-Pointing-Pointer Decreased expression of IL-7R{alpha}, IL-2R{alpha} and IL-2 in Zfat-deficient peripheral T cells. Black-Right-Pointing-Pointer Zfat plays critical roles in peripheral T cell homeostasis. -- Abstract: ZFAT, originally identified as a candidate susceptibility gene for autoimmune thyroid disease, has been reported to be involved in apoptosis, development and primitive hematopoiesis. Zfat is highly expressed in T- and B-cells in the lymphoid tissues, however, its physiological function in the immune system remains totally unknown. Here, we generated the T cell-specific Zfat-deficient mice and demonstrated that Zfat-deficiency leads to a remarkable reduction in the number of the peripheral T cells. Intriguingly, a reduced expression of IL-7R{alpha} and the impaired responsiveness to IL-7 for the survival were observed in the Zfat-deficient T cells. Furthermore, a severe defect in proliferation and increased apoptosis in the Zfat-deficient T cells following T cell receptor (TCR) stimulation was observed with a reduced IL-2R{alpha} expression as well as a reduced IL-2 production. Thus, our findings reveal that Zfat is a critical regulator in peripheral T cell homeostasis and its TCR-mediated response.

  1. Cinnabar-Induced Subchronic Renal Injury Is Associated with Increased Apoptosis in Rats

    PubMed Central

    Wang, Ying; Wang, Dapeng; Wu, Jie; Wang, Bohan; Gao, Xianhui; Wang, Liangjun; Ma, Honglin

    2015-01-01

    The aim of this study was to explore the role of apoptosis in cinnabar-induced renal injury in rats. To test this role, rats were dosed orally with cinnabar (1 g/kg/day) for 8 weeks or 12 weeks, and the control rats were treated with 5% carboxymethylcellulose solution. Levels of urinary mercury (UHg), renal mercury (RHg), serum creatinine (SCr), and urine kidney injury molecule 1 (KIM-1) were assessed, and renal pathology was analyzed. Apoptotic cells were identified and the apoptotic index was calculated. A rat antibody array was used to analyze expression of cytokines associated with apoptosis. Results from these analyses showed that UHg, RHg, and urine KIM-1, but not SCr, levels were significantly increased in cinnabar-treated rats. Renal pathological changes in cinnabar-treated rats included vacuolization of tubular cells, formation of protein casts, infiltration of inflammatory cells, and increase in the number of apoptotic tubular cells. In comparison to the control group, expression of FasL, Fas, TNF-α, TRAIL, activin A, and adiponectin was upregulated in the cinnabar-treated group. Collectively, our results suggest that prolonged use of cinnabar results in kidney damage due to accumulation of mercury and that the underlying mechanism involves apoptosis of tubular cells via a death receptor-mediated pathway. PMID:25629042

  2. Nicotine effects on muscarinic receptor-mediated free Ca[Formula: see text] level changes in the facial nucleus following facial nerve injury.

    PubMed

    Sun, Dawei; Zhou, Rui; Dong, Anbing; Sun, Wenhai; Zhang, Hongmei; Tang, Limin

    2016-06-01

    It was suggested that muscarinic, and nicotinic receptors increase free Ca[Formula: see text] levels in the facial nerve nucleus via various channels following facial nerve injury. However, intracellular Ca[Formula: see text] overload can trigger either necrotic or apoptotic cell death. It is assumed that, following facial nerve injury, the interactions of nicotinic and muscarinic acetylcholine receptors in facial nerve nucleus may negatively regulate free Ca[Formula: see text] concentrations in the facial nerve nucleus, which provide important information for the repair and regeneration of the facial nerve. The present study investigated the regulatory effects of nicotine on muscarinic receptor-mediated free calcium ion level changes in the facial nucleus in a rat model of facial nerve injury at 7, 30, and 90 days following facial nerve injury using laser confocal microscopy. The dose-dependent regulation of nicotine on muscarinic receptor-mediated free calcium ion level changes in the facial nucleus may decrease the range of free Ca[Formula: see text] increases following facial nerve injury, which is important for nerve cell regeneration. It is concluded that the negative effects of nicotine on muscarinic receptors are related to the [Formula: see text] subtype of nicotinic receptors.

  3. Extracellular signal‑regulated kinase inhibition is required for methanol extract of Smilax china L.‑induced apoptosis through death receptor 5 in human oral mucoepidermoid carcinoma cells.

    PubMed

    Yu, Hyun-Ju; Shin, Ji-Ae; Lee, Syng-Ook; Kwon, Ki Han; Cho, Sung-Dae

    2014-02-01

    Smilax china L., a well‑known Chinese traditional medicine, has been used as an anti‑inflammatory, anti‑cancer and analgesic agent, but its role has not yet been fully elucidated in oral mucoepidermoid carcinoma (MEC). The present study focused on addressing the anticancer activity and molecular mechanism of methanol extract of Smilax china L. (MESC) in MC‑3 human oral MEC cells. The results indicated that MESC inhibited cell growth and induced apoptosis in MC‑3 cells. These observations were found to correlate with increases in truncated BH3 interacting‑domain death agonist and B‑cell lymphoma 2 (Bcl‑2) interacting mediator of cell death, but not Bcl‑2 homologous antagonist killer, Bcl‑2‑associated X protein, Bcl‑2, B‑cell lymphoma‑extra large and induced myeloid leukemia cell differentiation protein levels. MESC also damaged the mitochondrial membrane potential, cleaved caspase‑8 protein and increased death receptor 5 (DR5) protein levels by enhancing the stability of DR5 protein. Furthermore, MESC affected the phosphorylation of extracellular signal‑regulated kinase (ERK) only, and did not affect c‑Jun N‑terminal kinase or p38 phosphorylation. Co‑treatment with MESC and an ERK inhibitor (PD98059) significantly increased the expression of DR5 to induce apoptosis in MC‑3 cells. Therefore, these results suggest that MESC may induce apoptosis via the ERK pathway and may be a potential anticancer drug candidate against human oral MEC.

  4. Ethanol inhibits epileptiform activity and NMDA receptor-mediated synaptic transmission in rat amygdaloid slices

    SciTech Connect

    Gean, P.W. )

    1992-02-26

    The effect of ethanol on the epileptiform activity induced by Mg{sup ++}-free solution was studied in rat amygdalar slices using intracellular recording techniques. The spontaneous and evoked epileptiform discharges consisting of an initial burst followed by afterdischarges were observed 20-30 min after switching to Mg{sup ++}-free medium. Superfusion with ethanol reversibly reduced the duration of spontaneous and evoked bursting discharges in a concentration-dependent manner. Synaptic response mediated by N-methyl-D-aspartate (NMDA) receptor activation was isolated by application of a solution containing the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and either in Mg{sup ++}-free solution or in the presence of 50 {mu}M bicuculline. Application of ethanol reversibly suppressed the duration of NMDA receptor-mediated synaptic response. These results suggest that intoxicating concentrations of ethanol possess anticonvulsant activity through blocking the NMDA receptor-mediated synaptic excitation.

  5. Apoptosis-like cell death induced by nematocyst venom from Chrysaora helvola Brandt jellyfish and an in vitro evaluation of commonly used antidotes.

    PubMed

    Qu, Xiaosheng; Xia, Xianghua; Lai, Zefeng; Zhong, Taozheng; Li, Gang; Fan, Lanlan; Shu, Wei

    2016-02-01

    The present work investigated the in vitro cytotoxicity of nematocyst venom (NV) from Chrysaora helvola Brandt (C. helvola) jellyfish against human MCF-7 and CNE-2 tumor cell lines. Potent cytotoxicity was quantified using the MTT assay (LC50=12.07±3.13 and 1.6±0.22μg/mL (n=4), respectively). Apoptosis-like cell death was further confirmed using the LDH release assay and Annexin V/PI double staining-based flow cytometry analysis. However, only activation of caspase-4 was observed. It is possible that some caspase-independent pathways were activated by the NV treatment. Since no reference or antivenom is available, the effects of several commonly used antidotes on the cytotoxicity of NV were examined on more sensitive CNE-2 cells to determine the appropriate emergency measures for envenomation by C. helvola. The phospholipase A2 (PLA2) inhibitor para-bromophenacyl bromide (pBPB) showed no protective effect, while Mg(2+) potentiated cytotoxicity. Voltage-gated L-type Ca(2+) channel blockers (verapamil, nifedipine and felodipine) and Na-Ca(2+) exchanger inhibitor KB-R7943 also showed no effect. Assays using Ca(2+)-free culture media or the intracellular Ca(2+) chelator BAPTA also could not inhibit the cytotoxicity. Taken together, these results suggest that PLA2 and Ca(2+) are not directly involved in the cytotoxicity of NV from C. helvola. Our work also suggests caution regarding the choice for first aid for envenomation by C. helvola jellyfish.

  6. Menthol enhances phasic and tonic GABAA receptor-mediated currents in midbrain periaqueductal grey neurons

    PubMed Central

    Lau, Benjamin K; Karim, Shafinaz; Goodchild, Ann K; Vaughan, Christopher W; Drew, Geoffrey M

    2014-01-01

    Background and Purpose Menthol, a naturally occurring compound in the essential oil of mint leaves, is used for its medicinal, sensory and fragrant properties. Menthol acts via transient receptor potential (TRPM8 and TRPA1) channels and as a positive allosteric modulator of recombinant GABAA receptors. Here, we examined the actions of menthol on GABAA receptor-mediated currents in intact midbrain slices. Experimental Approach Whole-cell voltage-clamp recordings were made from periaqueductal grey (PAG) neurons in midbrain slices from rats to determine the effects of menthol on GABAA receptor-mediated phasic IPSCs and tonic currents. Key Results Menthol (150–750 μM) produced a concentration-dependent prolongation of spontaneous GABAA receptor-mediated IPSCs, but not non-NMDA receptor-mediated EPSCs throughout the PAG. Menthol actions were unaffected by TRPM8 and TRPA1 antagonists, tetrodotoxin and the benzodiazepine antagonist, flumazenil. Menthol also enhanced a tonic current, which was sensitive to the GABAA receptor antagonists, picrotoxin (100 μM), bicuculline (30 μM) and Zn2+ (100 μM), but unaffected by gabazine (10 μM) and a GABAC receptor antagonist, 1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid hydrate (TPMPA; 50 μM). In addition, menthol potentiated currents induced by the extrasynaptic GABAA receptor agonist THIP/gaboxadol (10 μM). Conclusions and Implications These results suggest that menthol positively modulates both synaptic and extrasynaptic populations of GABAA receptors in native PAG neurons. The development of agents that potentiate GABAA-mediated tonic currents and phasic IPSCs in a manner similar to menthol could provide a basis for novel GABAA-related pharmacotherapies. PMID:24460753

  7. Nanoscale imaging and mechanical analysis of Fc receptor-mediated macrophage phagocytosis against cancer cells.

    PubMed

    Li, Mi; Liu, Lianqing; Xi, Ning; Wang, Yuechao; Xiao, Xiubin; Zhang, Weijing

    2014-02-18

    Fc receptor-mediated macrophage phagocytosis against cancer cells is an important mechanism in the immune therapy of cancers. Traditional research about macrophage phagocytosis was based on optical microscopy, which cannot reveal detailed information because of the 200-nm-resolution limit. Quantitatively investigating the macrophage phagocytosis at micro- and nanoscale levels is still scarce. The advent of atomic force microscopy (AFM) offers an excellent analytical instrument for quantitatively investigating the biological processes at single-cell and single-molecule levels under native conditions. In this work, we combined AFM and fluorescence microscopy to visualize and quantify the detailed changes in cell morphology and mechanical properties during the process of Fc receptor-mediated macrophage phagocytosis against cancer cells. Lymphoma cells were discernible by fluorescence staining. Then, the dynamic process of phagocytosis was observed by time-lapse optical microscopy. Next, AFM was applied to investigate the detailed cellular behaviors during macrophage phagocytosis under the guidance of fluorescence recognition. AFM imaging revealed the distinct features in cellular ultramicrostructures for the different steps of macrophage phagocytosis. AFM cell mechanical property measurements indicated that the binding of cancer cells to macrophages could make macrophages become stiffer. The experimental results provide novel insights in understanding the Fc-receptor-mediated macrophage phagocytosis.

  8. Mannose receptor-mediated gene delivery into antigen presenting dendritic cells.

    PubMed

    Diebold, Sandra S; Plank, Christian; Cotten, Matt; Wagner, Ernst; Zenke, Martin

    2002-11-01

    Dendritic cells are professional antigen presenting cells and are unique in their ability to prime naïve T cells. Gene modification of dendritic cells is of particular interest for immunotherapy of diseases where the immune system has failed or is aberrantly regulated, such as in cancer or autoimmune disease, respectively. Dendritic cells abundantly express mannose receptor and mannose receptor-related receptors, and receptor-mediated gene transfer via mannose receptor offers a versatile tool for targeted gene delivery into these cells. Accordingly, mannose polyethylenimine DNA transfer complexes were generated and used for gene delivery into dendritic cells. Mannose receptor belongs to the group of scavenger receptors that allow dendritic cells to take up pathogenic material, which is directed for degradation and MHC class II presentation. Therefore, a limiting step of transgene expression by mannose receptor-mediated gene delivery is endosomal degradation of DNA. Several strategies have been explored to overcome this limitation including the addition of endosomolytic components to DNA transfer complexes like adenovirus particles and influenza peptides. Here, we review the current understanding of mannose receptor-mediated gene delivery into dendritic cells and discuss strategies to identify appropriate endosomolytic agents to improve DNA transfer efficacy.

  9. Slowly developing depression of N-methyl-D-aspartate receptor mediated responses in young rat hippocampi

    PubMed Central

    Dozmorov, Mikhail; Li, Rui; Xu, Hui-Ping; Jilderos, Barbro; Wigström, Holger

    2004-01-01

    Background Activation of N-methyl-D-aspartate (NMDA) type glutamate receptors is essential in triggering various forms of synaptic plasticity. A critical issue is to what extent such plasticity involves persistent changes of glutamate receptor subtypes and many prior studies have suggested a main role for alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors in mediating the effect. Our previous work in hippocampal slices revealed that, under pharmacological unblocking of NMDA receptors, both AMPA and NMDA receptor mediated responses undergo a slowly developing depression. In the present study we have further adressed this phenomenon, focusing on the contribution via NMDA receptors. Pharmacologically isolated NMDA receptor mediated excitatory postsynaptic potentials (EPSPs) were recorded for two independent synaptic pathways in CA1 area using perfusion with low Mg2+ (0.1 mM) to unblock NMDA receptors. Results Following unblocking of NMDA receptors, there was a gradual decline of NMDA receptor mediated EPSPs for 2–3 hours towards a stable level of ca. 60–70 % of the maximal size. If such an experimental session was repeated twice in the same pathway with a period of NMDA receptor blockade in between, the depression attained in the first session was still evident in the second one and no further decay occurred. The persistency of the depression was also validated by comparison between pathways. It was found that the responses of a control pathway, unstimulated in the first session of receptor unblocking, behaved as novel responses when tested in association with the depressed pathway under the second session. In similar experiments, but with AP5 present during the first session, there was no subsequent difference between NMDA EPSPs. Conclusions Our findings show that merely evoking NMDA receptor mediated responses results in a depression which is input specific, induced via NMDA receptor activation, and is maintained for several hours through

  10. Site of action of the general anesthetic propofol in muscarinic M1 receptor-mediated signal transduction.

    PubMed

    Murasaki, Osamu; Kaibara, Muneshige; Nagase, Yoshihisa; Mitarai, Sayaka; Doi, Yoshiyuki; Sumikawa, Koji; Taniyama, Kohtaro

    2003-12-01

    Although a potential target site of general anesthetics is primarily the GABA A receptor, a chloride ion channel, a previous study suggested that the intravenous general anesthetic propofol attenuates the M1 muscarinic acetylcholine receptor (M1 receptor)-mediated signal transduction. In the present study, we examined the target site of propofol in M1 receptor-mediated signal transduction. Two-electrode voltage-clamp method was used in Xenopus oocytes expressing both M1 receptors and associated G protein alpha subunits (Gqalpha). Propofol inhibited M1 receptor-mediated signal transduction in a dose-dependent manner (IC50 = 50 nM). Injection of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) into oocytes overexpressing Gqalpha was used to investigate direct effects of propofol on G protein coupled with the M1 receptor. Propofol did not affect activation of Gqalpha-mediated signal transduction with the intracellular injection of GTPgammaS. We also studied effects of propofol on l-[N-methyl-3H]scopolamine methyl chloride ([3H]NMS) binding and M1 receptor-mediated signal transduction in mammalian cells expressing M1 receptor. Propofol inhibited the M1 receptor-mediated signal transduction but did not inhibit binding of [3H]NMS. Effects of propofol on Gs- and Gi/o-coupled signal transduction were investigated, using oocytes expressing the beta2 adrenoceptor (beta2 receptor)/cystic fibrosis transmembrane conductance regulator or oocytes expressing the M2 muscarinic acetylcholine receptor (M2 receptor)/Kir3.1 (a member of G protein-gated inwardly rectifying K(+) channels). Neither beta2 receptor-mediated nor M2 receptor-mediated signal transduction was inhibited by a relatively high concentration of propofol (50 microM). These results indicate that propofol inhibits M1 receptor-mediated signal transduction by selectively disrupting interaction between the receptor and associated G protein.

  11. Identification by DNA macroarray of nur77 as a gene induced by di-n-butyltin dichloride: its role in organotin-induced apoptosis.

    PubMed

    Gennari, Alessandra; Bleumink, Rob; Viviani, Barbara; Galli, Corrado Lodovico; Marinovich, Marina; Pieters, Raymond; Corsini, Emanuela

    2002-05-15

    The thymotoxic organotin compounds di-n-butyltin dichloride (DBTC) and tri-n-butyltin chloride (TBTC) are known to induce apoptosis in vitro in rat thymocytes. They also affect macromolecular synthesis, inhibiting DNA synthesis and increasing RNA synthesis. Since these RNA molecules, likely to be involved in the initiation of the apoptotic process, have not been identified yet, the purpose of this research was to characterize by a cDNA macroarray the expression of genes involved in DBTC-induced apoptosis. We found that nur77 was rapidly transcripted in vitro following exposure of freshly isolated rat thymocytes to 3 microM DBTC. nur77 induction has also been observed in vivo after treatment of rats with apoptotic doses (60 mg/kg body wt) of DBTC. The products of nur77 are known to be involved in the apoptotic process, as nur77 is a transcription factor expressed in response to T-cell receptor-mediated apoptosis in immature T cells. Antisense oligonucleotide inhibition of nur77 expression prevented apoptosis induced by DBTC, supporting a role for nur77 in organotin-induced apoptotic cell death.

  12. Hypericin-photodynamic therapy leads to interleukin-6 secretion by HepG2 cells and their apoptosis via recruitment of BH3 interacting-domain death agonist and caspases

    PubMed Central

    Barathan, M; Mariappan, V; Shankar, E M; Abdullah, B JJ; Goh, K L; Vadivelu, J

    2013-01-01

    Photodynamic therapy (PDT) has emerged as a capable therapeutic modality for the treatment of cancer. PDT is a targeted cancer therapy that reportedly leads to tumor cell apoptosis and/or necrosis by facilitating the secretion of certain pro-inflammatory cytokines and expression of multiple apoptotic mediators in the tumor microenvironment. In addition, PDT also triggers oxidative stress that directs tumor cell killing and activation of inflammatory responses. However, the cellular and molecular mechanisms underlying the role of PDT in facilitating tumor cell apoptosis remain ambiguous. Here, we investigated the ability of PDT in association with hypericin (HY) to induce tumor cell apoptosis by facilitating the induction of reactive oxygen species (ROS) and secretion of Th1/Th2/Th17 cytokines in human hepatocellular liver carcinoma cell line (HepG2) cells. To discover if any apoptotic mediators were implicated in the enhancement of cell death of HY-PDT-treated tumor cells, selected gene profiling in response to HY-PDT treatment was implemented. Experimental results showed that interleukin (IL)-6 was significantly increased in all HY-PDT-treated cells, especially in 1 μg/ml HY-PDT, resulting in cell death. In addition, quantitative real-time PCR analysis revealed that the expression of apoptotic genes, such as BH3-interacting-domain death agonist (BID), cytochrome complex (CYT-C) and caspases (CASP3, 6, 7, 8 and 9) was remarkably higher in HY-PDT-treated HepG2 cells than the untreated HepG2 cells, entailing that tumor destruction of immune-mediated cell death occurs only in PDT-treated tumor cells. Hence, we showed that HY-PDT treatment induces apoptosis in HepG2 cells by facilitating cytotoxic ROS, and potentially recruits IL-6 and apoptosis mediators, providing additional hints for the existence of alternative mechanisms of anti-tumor immunity in hepatocellular carcinoma, which contribute to long-term suppression of tumor growth following PDT. PMID:23807226

  13. T Cell Receptor-induced Activation and Apoptosis In Cycling Human T Cells Occur throughout the Cell Cycle

    PubMed Central

    Karas, Michael; Zaks, Tal Z.; JL, Liu; LeRoith, Derek

    1999-01-01

    Previous studies have found conflicting associations between susceptibility to activation-induced cell death and the cell cycle in T cells. However, most of the studies used potentially toxic pharmacological agents for cell cycle synchronization. A panel of human melanoma tumor-reactive T cell lines, a CD8+ HER-2/neu-reactive T cell clone, and the leukemic T cell line Jurkat were separated by centrifugal elutriation. Fractions enriched for the G0–G1, S, and G2–M phases of the cell cycle were assayed for T cell receptor-mediated activation as measured by intracellular Ca2+ flux, cytolytic recognition of tumor targets, and induction of Fas ligand mRNA. Susceptibility to apoptosis induced by recombinant Fas ligand and activation-induced cell death were also studied. None of the parameters studied was specific to a certain phase of the cell cycle, leading us to conclude that in nontransformed human T cells, both activation and apoptosis through T cell receptor activation can occur in all phases of the cell cycle. PMID:10588669

  14. [Apoptosis in allergic disease].

    PubMed

    Rojas Ramos, E; Martínez Jiménez, N E; Martínez Aguilar, N E; Garfias Becerra, J

    2000-01-01

    Apoptosis (cell programmed death) it is a mechanism that implicate a physiological suicide, to keep the cellular homeostasis in big amount of tissues. Fas (APO-1; CD95) system is one of the most important cellular responsible via to induce apoptosis on different tissues. Eosinophillia on peripheral blood and tissues are the main characteristics on allergic like asthma. Eosinophil apoptosis is upper regulated in those diseases by IL-5 y GM-CSF. Corticoids, teophyllin and some macrolids have been used like apoptosis inductors on eosinophills, these could be a novel mechanism to promote a better solution on inflammatory allergic diseases.

  15. Induction of apoptosis in leukemia cell lines by new copper(II) complexes containing naphthyl groups via interaction with death receptors.

    PubMed

    Fernandes, Christiane; Horn, Adolfo; Lopes, Bruna F; Bull, Erika S; Azeredo, Nathália F B; Kanashiro, Milton M; Borges, Franz V; Bortoluzzi, Adailton J; Szpoganicz, Bruno; Pires, Anderson B; Franco, Roberto W A; Almeida, João Carlos de A; Maciel, Leide L F; Resende, Jackson A L C; Schenk, Gerhard

    2015-12-01

    The synthesis, physico-chemical characterization and cytotoxicity of four new ligands and their respective copper(II) complexes toward two human leukemia cell lines (THP-1 and U937) are reported (i.e. [(HL1)Cu(μ-Cl)2Cu(HL1)]Cl2·H2O (1), [(H2L2)Cu(μ-Cl)2Cu(H2L2)]Cl2·5H2O (2), [(HL3)Cu(μ-Cl)2Cu(HL3)]Cl2·4H2O (3), [(H2L4)Cu(μ-Cl)2Cu(H2L4)]Cl2·6H2O (4)). Ligands HL1 and HL3 contain two pyridines, amine and alcohol moieties with a naphthyl pendant unit yielding a N3O coordination metal environment. Ligands H2L2 and H2L4 have pyridine, phenol, amine and alcohol groups with a naphthyl pendant unit providing a N2O2 coordination metal environment. These compounds are likely to be dinuclear in the solid state but form mononuclear species in solution. The complexes have an antiproliferative effect against both leukemia cell lines; complex (2) exhibits higher activity than cisplatin against U937 (8.20 vs 16.25μmoldm(-3)) and a comparable one against THP-1. These human neoplastic cells are also more susceptible than peripheral blood mononuclear cells (PBMCs) toward the tested compounds. Using C57BL/6 mice an LD50 of 55mgkg(-1) was determined for complex (2), suggesting that this compound is almost four times less toxic than cisplatin (LD50=14.5mgkg(-1)). The mechanism of cell death promoted by ligand H2L2 and by complexes (2) and (4) was investigated by a range of techniques demonstrating that the apoptosis signal triggered at least by complex (2) starts from an extrinsic pathway involving the activation of caspases 4 and 8. This signal is amplified by mitochondria with the concomitant release of cytochrome c and the activation of caspase 9.

  16. Association of active caspase 8 with the mitochondrial membrane during apoptosis: potential roles in cleaving BAP31 and caspase 3 and mediating mitochondrion-endoplasmic reticulum cross talk in etoposide-induced cell death.

    PubMed

    Chandra, Dhyan; Choy, Grace; Deng, Xiaodi; Bhatia, Bobby; Daniel, Peter; Tang, Dean G

    2004-08-01

    It was recently demonstrated that during apoptosis, active caspase 9 and caspase 3 rapidly accumulate in the mitochondrion-enriched membrane fraction (D. Chandra and D. G. Tang, J. Biol. Chem.278:17408-17420, 2003). We now show that active caspase 8 also becomes associated with the membranes in apoptosis caused by multiple stimuli. In MDA-MB231 breast cancer cells treated with etoposide (VP16), active caspase 8 is detected only in the membrane fraction, which contains both mitochondria and endoplasmic reticulum (ER), as revealed by fractionation studies. Immunofluorescence microscopy, however, shows that procaspase 8 and active caspase 8 predominantly colocalize with the mitochondria. Biochemical analysis demonstrates that both procaspase 8 and active caspase 8 are localized mainly on the outer mitochondrial membrane (OMM) as integral proteins. Functional analyses with dominant-negative mutants, small interfering RNAs, peptide inhibitors, and Fas-associated death domain (FADD)- and caspase 8-deficient Jurkat T cells establish that the mitochondrion-localized active caspase 8 results mainly from the FADD-dependent and tumor necrosis factor receptor-associated death domain-dependent mechanisms and that caspase 8 activation plays a causal role in VP16-induced caspase 3 activation and cell death. Finally, we present evidence that the OMM-localized active caspase 8 can activate cytosolic caspase 3 and ER-localized BAP31. Cleavage of BAP31 leads to the generation of ER- localized, proapoptotic BAP20, which may mediate mitochondrion-ER cross talk through a Ca(2+)-dependent mechanism.

  17. Ultrasound imaging of apoptosis: high-resolution non-invasive monitoring of programmed cell death in vitro, in situ and in vivo

    PubMed Central

    Czarnota, G J; Kolios, M C; Abraham, J; Portnoy, M; Ottensmeyer, F P; Hunt, J W; Sherar, M D

    1999-01-01

    A new non-invasive method for monitoring apoptosis has been developed using high frequency (40 MHz) ultrasound imaging. Conventional ultrasound backscatter imaging techniques were used to observe apoptosis occurring in response to anticancer agents in cells in vitro, in tissues ex vivo and in live animals. The mechanism behind this ultrasonic detection was identified experimentally to be the subcellular nuclear changes, condensation followed by fragmentation, that cells undergo during apoptosis. These changes dramatically increase the high frequency ultrasound scattering efficiency of apoptotic cells over normal cells (25- to 50-fold change in intensity). The result is that areas of tissue undergoing apoptosis become much brighter in comparison to surrounding viable tissues. The results provide a framework for the possibility of using high frequency ultrasound imaging in the future to non-invasively monitor the effects of chemotherapeutic agents and other anticancer treatments in experimental animal systems and in patients. © 1999 Cancer Research Campaign PMID:10507779

  18. Lipid rafts and raft-mediated supramolecular entities in the regulation of CD95 death receptor apoptotic signaling.

    PubMed

    Gajate, Consuelo; Mollinedo, Faustino

    2015-05-01

    Membrane lipid rafts are highly ordered membrane domains enriched in cholesterol, sphingolipids and gangliosides that have the property to segregate and concentrate proteins. Lipid and protein composition of lipid rafts differs from that of the surrounding membrane, thus providing sorting platforms and hubs for signal transduction molecules, including CD95 death receptor-mediated signaling. CD95 can be recruited to rafts in a reversible way through S-palmitoylation following activation of cells with its physiological cognate ligand as well as with a wide variety of inducers, including several antitumor drugs through ligand-independent intracellular mechanisms. CD95 translocation to rafts can be modulated pharmacologically, thus becoming a target for the treatment of apoptosis-defective diseases, such as cancer. CD95-mediated signaling largely depends on protein-protein interactions, and the recruitment and concentration of CD95 and distinct downstream apoptotic molecules in membrane raft domains, forming raft-based supramolecular entities that act as hubs for apoptotic signaling molecules, favors the generation and amplification of apoptotic signals. Efficient CD95-mediated apoptosis involves CD95 and raft internalization, as well as the involvement of different subcellular organelles. In this review, we briefly summarize and discuss the involvement of lipid rafts in the regulation of CD95-mediated apoptosis that may provide a new avenue for cancer therapy.

  19. IFN Regulatory Factor 8 Sensitizes Soft Tissue Sarcoma Cells to Death Receptor-initiated Apoptosis via Repression of FLICE-like Protein Expression

    PubMed Central

    Yang, Dafeng; Wang, Suizhao; Brooks, Craig; Dong, Zheng; Schoenlein, Patricia; Kumar, Vijay; Ouyang, Xinshou; Xiong, Huabao; Lahat, Guy; Hayes-Jordan, Andrea; Lazar, Alexander; Pollock, Raphael; Lev, Dina; Liu, Kebin

    2008-01-01

    Interferon Regulatory Factor 8 (IRF8) has been shown to suppress tumor development at least partly through regulating apoptosis of tumor cells; however, the molecular mechanisms underlying IRF8 regulation of apoptosis are still not fully understood. Here, we demonstrated that disrupting IRF8 function resulted in inhibition of cytochrome C release, caspases 9 and 3 activation, and PARP cleavage in soft tissue sarcoma (STS) cells. Inhibition of the mitochondrion-dependent apoptosis signaling cascade is apparently due to blockage of caspase 8 and Bid activation. Analysis of signaling events upstream of caspsse 8 revealed that disrupting IRF8 function dramatically increases FLIP mRNA stability, resulting in increased IRF8 protein level. Furthermore, primary myeloid cells isolated from IRF8 null mice also exhibited increased FLIP protein level, suggesting that IRF8 might be a general repressor of FLIP. Nuclear IRF8 protein was absent in 92% (55/60) of human STS specimens, and 99% (59/60) human STS specimens exhibited FLIP expression, suggesting that the nuclear IRF8 protein level is inversely correlated with FLIP level in vivo. Silencing FLIP expression significantly increased human sarcoma cells to both FasL and TRAIL-induced apoptosis, and ectopic expression of IRF8 also significantly increased the sensitivity of these human sarcoma cells to FasL and TRAIL-induced apoptosis. Taken together, our data suggest that IRF8 mediates FLIP expression level to regulate apoptosis and targeting IRF8 expression is a potentially effective therapeutic strategy to sensitize apoptosis-resistant human STS to apoptosis, thereby possibly overcoming chemoresistance of STS, currently a major obstacle in human STS therapy. PMID:19155307

  20. The Membrane-Active Tri-Block Copolymer Pluronic F-68 Profoundly Rescues Rat Hippocampal Neurons from Oxygen–Glucose Deprivation-Induced Death through Early Inhibition of Apoptosis

    PubMed Central

    Shelat, Phullara B.; Plant, Leigh D.; Wang, Janice C.; Lee, Elizabeth

    2013-01-01

    Pluronic F-68, an 80% hydrophilic member of the Pluronic family of polyethylene-polypropylene-polyethylene tri-block copolymers, protects non-neuronal cells from traumatic injuries and rescues hippocampal neurons from excitotoxic and oxidative insults. F-68 interacts directly with lipid membranes and restores membrane function after direct membrane damage. Here, we demonstrate the efficacy of Pluronic F-68 in rescuing rat hippocampal neurons from apoptosis after oxygen–glucose deprivation (OGD). OGD progressively decreased neuronal survival over 48 h in a severity-dependent manner, the majority of cell death occurring after 12 h after OGD. Administration of F-68 for 48 h after OGD rescued neurons from death in a dose-dependent manner. At its optimal concentration (30 μm), F-68 rescued all neurons that would have died after the first hour after OGD. This level of rescue persisted when F-68 administration was delayed 12 h after OGD. F-68 did not alter electrophysiological parameters controlling excitability, NMDA receptor-activated currents, or NMDA-induced increases in cytosolic calcium concentrations. However, F-68 treatment prevented phosphatidylserine externalization, caspase activation, loss of mitochondrial membrane potential, and BAX translocation to mitochondria, indicating that F-68 alters apoptotic mechanisms early in the intrinsic pathway of apoptosis. The profound neuronal rescue provided by F-68 after OGD and the high level of efficacy with delayed administration indicate that Pluronic copolymers may provide a novel, membrane-targeted approach to rescuing neurons after brain ischemia. The ability of membrane-active agents to block apoptosis suggests that membranes or their lipid components play prominent roles in injury-induced apoptosis. PMID:23884935

  1. Chloride transporters and receptor-mediated endocytosis in the renal proximal tubule

    PubMed Central

    Devuyst, Olivier; Luciani, Alessandro

    2015-01-01

    Abstract The epithelial cells lining the proximal tubules of the kidney reabsorb a large amount of filtered ions and solutes owing to receptor-mediated endocytosis and polarized transport systems that reflect final cell differentiation. Dedifferentiation of proximal tubule cells and dysfunction of receptor-mediated endocytosis characterize Dent’s disease, a rare disorder caused by inactivating mutations in the CLCN5 gene that encodes the endosomal chloride–proton exchanger, ClC-5. The disease is characterized by a massive urinary loss of solutes (renal Fanconi syndrome), with severe metabolic complications and progressive renal failure. Investigations of mutations affecting the gating of ClC-5 revealed that the proximal tubule dysfunction may occur despite normal endosomal acidification. In addition to defective endocytosis, proximal tubule cells lacking ClC-5 show a trafficking defect in apical receptors and transporters, as well as lysosomal dysfunction and typical features of dedifferentiation, proliferation and oxidative stress. A similar but milder defect is observed in mouse models with defective CFTR, a chloride channel that is also expressed in the endosomes of proximal tubule cells. These data suggest a major role for endosomal chloride transport in the maintenance of epithelial differentiation and reabsorption capacity of the renal proximal tubule. Key points The reabsorptive activity of renal proximal tubule cells is mediated by receptor-mediated endocytosis and polarized transport systems that reflect final cell differentiation. Loss-of-function mutations of the endosomal chloride–proton exchanger ClC-5 (Dent’s disease) cause a major trafficking defect in proximal tubule cells, associated with lysosomal dysfunction, oxidative stress and dedifferentiation/proliferation. A similar but milder defect is associated with mutations in CFTR (cystic fibrosis transmembrane conductance regulator). Vesicular chloride transport appears to be important for

  2. Investigations of receptor-mediated phagocytosis by hormone-induced (imprinted) Tetrahymena pyriformis.

    PubMed

    Kovács, P; Sundermann, C A; Csaba, G

    1996-08-15

    Receptor-mediated endocytosis by Tetrahvmena pyriformis was studied using tetramethylrhodamine isothiocyanate-labeled concanavalin A (TRITC-Con A) with fluorescence and confocal microscopy. In the presence of insulin, or 24 h after insulin pretreatment (hormonal imprinting), the binding and uptake of TRITC-Con A increased when compared to controls, owing to the binding of TRITC-Con A to sugar oligomers of insulin receptors. Mannose inhibited the binding of Con A, thus demonstrating the specificity of binding. Histamine, a phagocytosis-promoting factor in mammals and Tetrahymena, and galactose, did not influence the uptake of TRITC-Con A.

  3. Receptor-Mediated Drug Delivery to Macrophages in Chemotherapy of Leishmaniasis

    NASA Astrophysics Data System (ADS)

    Mukhopadhyay, Amitabha; Chaudhuri, Gautam; Arora, Sunil K.; Sehgal, Shobha; Basu, Sandip K.

    1989-05-01

    Methotrexate coupled to maleylated bovine serum albumin was taken up efficiently through the ``scavenger'' receptors present on macrophages and led to selective killing of intracellular Leishmania mexicana amazonensis amastigotes in cultured hamster peritoneal macrophages. The drug conjugate was nearly 100 times as effective as free methotrexate in eliminating the intracellular parasites. Furthermore, in a model of experimental cutaneous leishmaniasis in hamsters, the drug conjugate brought about more than 90% reduction in the size of footpad lesions within 11 days. In contrast, the free drug at a similar concentration did not significantly affect lesion size. These studies demonstrate the potential of receptor-mediated drug delivery in the therapy of macrophage-associated diseases.

  4. ROS-induced toxicity: exposure of 3T3, RAW264.7, and MCF7 cells to superparamagnetic iron oxide nanoparticles results in cell death by mitochondria-dependent apoptosis

    NASA Astrophysics Data System (ADS)

    Hsieh, Hui-Chen; Chen, Chung-Ming; Hsieh, Wen-Yuan; Chen, Ching-Yun; Liu, Chia-Ching; Lin, Feng-Huei

    2015-02-01

    Superparamagnetic nanoparticles (Fe3O4, SPIO) have been used as magnetic resonance imaging enhancers for years. However, bio-safety issues concerning nanoparticles remain largely unexplored. Of particular concern is the possible cellular impact of nanoparticles during SPIO uptake and subsequent oxidative stress. SPIO causes cell death by apoptosis via a little understood mitochondrial pathway. To more closely examine this process, three kinds of cells—3T3, RAW264.7, and MCF7—were treated with SPIO coated with polyethylene glycol (SPIO-PEG) and monitored by transmission electron microscopy (TEM), using cytotoxicity evaluation, mitochondrial activity, reactive oxygen species (ROS) generation, and Annexin V assay. TEM revealed that SPIO-PEG nanoparticles surrounded the cellular endosome membrane, creating a bulge in the endosome. Compared to 3T3 cells, greater numbers of SPIO-PEG nanoparticles infiltrated the mitochondria of RAW264.7 and MCF7 cells. SPIO-PEG residency is associated with boosted ROS, with elevated levels of mitochondrial activity, and advancement of cell apoptosis. Furthermore, correlation analysis showed that a polynomial model demonstrates a better fit than a linear model in MCF7, implying that cytotoxicity may have alternative impacts on cell death at different concentrations. Thus, we believe that MCF7 cell death results from the apoptosis pathway triggered by mitochondria, and we find lower cytotoxicity in 3T3. We propose that optimal levels of SPIO-PEG nanoparticles lead to increased levels of ROS and a resulting oxidative stress environment which will kill only cancer cells while sparing normal cells. This finding has great potential for use in cancer therapies in the future.

  5. Cinnamaldehyde-induced apoptosis in human hepatoma PLC/PRF/5 cells involves the mitochondrial death pathway and is sensitive to inhibition by cyclosporin A and z-VAD-fmk.

    PubMed

    Lin, Liang-Tzung; Tai, Chen-Jei; Chang, Shun-Pang; Chen, Jin-Liang; Wu, Shu-Jing; Lin, Chun-Ching

    2013-12-01

    Cinnamaldehyde (CIN) has been shown to exert chemopreventive activity against several types of human cancer cells. We previously reported that CIN induced apoptosis of human hepatoma PLC/PRF/5 cells and this effect was associated with activation of the pro-apoptotic Bcl-2 family of proteins and the MAPK cascade. To further clarify the underlying mechanism of CIN-induced apoptosis, we examined in this study its relationship with the mitochondrial death pathway using the mitochondrial permeability transition (MPT) inhibitor, cyclosporin A (CsA), and the general caspase inhibitor, z-VAD-fmk. Results indicated that CIN-induced apoptosis involved enhanced ROS generation, disruption of mitochondrial potential, and the mitochondrial release of cytochrome c and Smac/DIABLO into the cytosol, which in turn promoted caspase-3 to its active form and the subsequent cleavage of PARP. Treatment with CIN also downregulated protein levels of the anti-apoptotic factors XIAP and Bcl-2 with concomitant accumulation of the pro-apoptotic Bax in a timedependent manner. These mitochondria-related apoptotic effects induced by CIN were however blocked by CsA and z-VAD-fmk pretreatments, which prevented cells from undergoing programmed cell death triggered by CIN. Furthermore, the increase of Bax and decrease of Bcl-2 and XIAP protein expression due to CIN treatment were also reversely modulated by the two inhibitors. Taken together, these results suggested that CIN is an apoptotic inducer that acts on the mitochondrial death pathway in PLC/PRF/5 cells and its effect could be blocked by CsA and z-VAD-fmk.

  6. Knockdown of FAM3B triggers cell apoptosis through p53-dependent pathway.

    PubMed

    Mou, Haiwei; Li, Zongmeng; Yao, Pengle; Zhuo, Shu; Luan, Wei; Deng, Bo; Qian, Lihua; Yang, Mengmei; Mei, Hong; Le, Yingying

    2013-03-01

    FAM3B, also named PANDER, is a cytokine-like protein identified in 2002. Previous studies showed that FAM3B regulates glucose and lipid metabolism through interaction with liver and endocrine pancreas. FAM3B is also expressed by other tissues but its basic function is unclear. In this study, we found that FAM3B was expressed in mouse colon, intestine, liver and lung tissues and multiple types of cell lines, including murine pancreatic β-cell (Min6), microglia (N9) and muscle cell (C2C12); human colon cancer cells (HCT8, HCT116, HT29), hepatocyte (HL-7702), hepatocellular carcinoma cell (SMMC-7721) and lung carcinoma cell (A549). Inhibition of FAM3B expression by RNA interference induced apoptotic cell death of HCT8, HCT116, A549, N9, C2C12 and Min6 cells and decreased cell viability of HL-7702 and murine primary hepatocytes. Further studies with HCT8 cells showed that knockdown of FAM3B increased the protein levels of membrane-bound Fas and Bax, reduced the expression of Bcl-2, promoted the cleavage of caspases-8, -3, -9 and PARP, and the nuclear translocation of cleaved PARP. These results suggest that FAM3B silencing activates both extrinsic and intrinsic apoptotic pathways. Mechanistic studies showed that neutralizing antibody against Fas or silencing Fas-associated death domain had no effect on, while caspase inhibitors could significantly reverse FAM3B knockdown induced apoptosis, suggesting Fas and death receptor mediated extrinsic apoptotic pathway is not involved in FAM3B silencing induced apoptosis. Further studies showed that p53 was significantly upregulated after FAM3B knockdown. Silencing p53 could almost completely reverse FAM3B knockdown induced upregulation of Bax, downregulation of Bcl-2, cleavage of caspases-8, -9, -3, and apoptotic cell death, suggesting p53-dependent pathway plays critical roles in FAM3B silencing induced apoptosis. Studies with HCT116 cells confirmed that inhibition of FAM3B expression induced apoptosis through p53-dependent

  7. Decreases in mitochondrial reactive oxygen species initiate GABAA receptor-mediated electrical suppression in anoxia-tolerant turtle neurons

    PubMed Central

    Hogg, David W; Pamenter, Matthew E; Dukoff, David J; Buck, Leslie T

    2015-01-01

    Key points Anoxia induces hyper-excitability and cell death in mammalian brain but in the western painted turtle (Chrysemys picta bellii) enhanced GABA transmission prevents injury. The mechanism responsible for increased GABA transmission is unknown; however, reactive oxygen species (ROS) generated by mitochondria may play a role because this is an oxygen-sensitive process. In this study, we show that inhibition of mitochondrial ROS production is sufficient to initiate a redox-sensitive GABA signalling cascade that suppresses pyramidal neuron action potential frequency. These results further our understanding of the turtle's unique strategy for reducing ATP consumption during anoxia and highlights a natural mechanism in which to explore therapies to protect mammalian brain from low-oxygen insults (e.g. cerebral stroke). Abstract Anoxia induces hyper-excitability and cell death in mammalian brain but in the anoxia-tolerant western painted turtle (Chrysemys picta bellii) neuronal electrical activity is suppressed (i.e. spike arrest), adenosine triphosphate (ATP) consumption is reduced, and cell death does not occur. Electrical suppression is primarily the result of enhanced γ-aminobutyric acid (GABA) transmission; however, the underlying mechanism responsible for initiating oxygen-sensitive GABAergic spike arrest is unknown. In turtle cortical pyramidal neurons there are three types of GABAA receptor-mediated currents: spontaneous inhibitory postsynaptic currents (IPSCs), giant IPSCs and tonic currents. The aim of this study was to assess the effects of reactive oxygen species (ROS) scavenging on these three currents since ROS levels naturally decrease with anoxia and may serve as a redox signal to initiate spike arrest. We found that anoxia, pharmacological ROS scavenging, or inhibition of mitochondrial ROS generation enhanced all three types of GABA currents, with tonic currents comprising ∼50% of the total current. Application of hydrogen peroxide inhibited

  8. Human agonistic TRAIL receptor antibodies Mapatumumab and Lexatumumab induce apoptosis in malignant mesothelioma and act synergistically with cisplatin

    PubMed Central

    Belyanskaya, Larisa L; Marti, Thomas M; Hopkins-Donaldson, Sally; Kurtz, Stefanie; Felley-Bosco, Emanuela; Stahel, Rolf A

    2007-01-01

    Background The incidence of malignant pleural mesothelioma (MPM) is associated with exposure to asbestos, and projections suggest that the yearly number of deaths in Western Europe due to MPM will increase until 2020. Despite progress in chemo- and in multimodality therapy, MPM remains a disease with a poor prognosis. Inducing apoptosis by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or agonistic monoclonal antibodies which target TRAIL-receptor 1 (TRAIL-R1) or TRAIL-R2 has been thought to be a promising cancer therapy. Results We have compared the sensitivity of 13 MPM cell lines or primary cultures to TRAIL and two fully human agonistic monoclonal antibodies directed to TRAIL-R1 (Mapatumumab) and TRAIL-R2 (Lexatumumab) and examined sensitization of the MPM cell lines to cisplatin-induced by the TRAIL-receptor antibodies. We found that sensitivity of MPM cells to TRAIL, Mapatumumab and Lexatumumab varies largely and is independent of TRAIL-receptor expression. TRAIL-R2 contributes more than TRAIL-R1 to death-receptor mediated apoptosis in MPM cells that express both receptors. The combination of cisplatin with Mapatumumab or Lexatumumab synergistically inhibited the cell growth and enhanced apoptotic death. Furthermore, pre-treatment with cisplatin followed by Mapatumumab or Lexatumumab resulted in significant higher cytotoxic effects as compared to the reverse sequence. Combination-induced cell growth inhibition was significantly abrogated by pre-treatment of the cells with the antioxidant N-acetylcysteine. Conclusion Our results suggest that the sequential administration of cisplatin followed by Mapatumumab or Lexatumumab deserves investigation in the treatment of patients with MPM. PMID:17953743

  9. Upregulation of DR5 receptor by the diaminothiazole DAT1 [4-amino-5-benzoyl-2-(4-methoxy phenyl amino) thiazole] triggers an independent extrinsic pathway of apoptosis in colon cancer cells with compromised pro and antiapoptotic proteins.

    PubMed

    Thomas, Sannu A; Vasudevan, Smreti; Thamkachy, Reshma; Lekshmi, Swathi U; Santhoshkumar, Thankayyan R; Rajasekharan, Kallikat N; Sengupta, Suparna

    2013-06-01

    Mitochondria mediated signalling is the more common way of apoptosis induction exhibited by many chemotherapeutic agents in cancer cells. Death receptor mediated signalling for apoptosis in many cells also requires further amplification from the mitochondrial pathway activation through tBid. Thus the potential of most chemotherapeutic agents in tumours with intrinsic apoptosis resistance due to changes in molecules involved in the mitochondrial pathway is limited. Diaminothiazoles were shown earlier to bind to tubulin thereby exhibiting cytotoxicity towards different cancer cells. We observed that the lead diaminothiazole, DAT1 [4-amino-5-benzoyl-2-(4-methoxy phenyl amino) thiazole] could induce apoptosis in the colon cancer cell line HCT116 by both pathways. However, in contrast to many other chemotherapeutic agents, DAT1 triggered apoptosis where the intrinsic pathway was blocked by changing the pro and antiapoptotic proteins. An independent extrinsic pathway activation triggered by the upregulation of DR5 receptor accounted for that. The induction of DR5 occurred in the transcriptional level and the essential role of DR5 was confirmed by the fact that siRNA downregulation of DR5 significantly reduced DAT1 induced apoptosis. HCT116 cells were earlier shown to have a type II response for apoptosis induction where extrinsic pathway was connected to the intrinsic pathway via the mediator protein tBid. Our finding thus indicates that the signalling events in the manifestation of apoptosis depend not only on the cancer cell type, but also on the inducer. Our results also place diaminothiazoles in a promising position in the treatment of tumours with compromised apoptotic factors.

  10. The miR-199-dynamin regulatory axis controls receptor-mediated endocytosis.

    PubMed

    Aranda, Juan F; Canfrán-Duque, Alberto; Goedeke, Leigh; Suárez, Yajaira; Fernández-Hernando, Carlos

    2015-09-01

    Small non-coding RNAs (microRNAs) are important regulators of gene expression that modulate many physiological processes; however, their role in regulating intracellular transport remains largely unknown. Intriguingly, we found that the dynamin (DNM) genes, a GTPase family of proteins responsible for endocytosis in eukaryotic cells, encode the conserved miR-199a and miR-199b family of miRNAs within their intronic sequences. Here, we demonstrate that miR-199a and miR-199b regulate endocytic transport by controlling the expression of important mediators of endocytosis such as clathrin heavy chain (CLTC), Rab5A, low-density lipoprotein receptor (LDLR) and caveolin-1 (Cav-1). Importantly, miR-199a-5p and miR-199b-5p overexpression markedly inhibits CLTC, Rab5A, LDLR and Cav-1 expression, thus preventing receptor-mediated endocytosis in human cell lines (Huh7 and HeLa). Of note, miR-199a-5p inhibition increases target gene expression and receptor-mediated endocytosis. Taken together, our work identifies a new mechanism by which microRNAs regulate intracellular trafficking. In particular, we demonstrate that the DNM, miR-199a-5p and miR-199b-5p genes act as a bifunctional locus that regulates endocytosis, thus adding an unexpected layer of complexity in the regulation of intracellular trafficking.

  11. Target shape dependence in a simple model of receptor-mediated endocytosis and phagocytosis.

    PubMed

    Richards, David M; Endres, Robert G

    2016-05-31

    Phagocytosis and receptor-mediated endocytosis are vitally important particle uptake mechanisms in many cell types, ranging from single-cell organisms to immune cells. In both processes, engulfment by the cell depends critically on both particle shape and orientation. However, most previous theoretical work has focused only on spherical particles and hence disregards the wide-ranging particle shapes occurring in nature, such as those of bacteria. Here, by implementing a simple model in one and two dimensions, we compare and contrast receptor-mediated endocytosis and phagocytosis for a range of biologically relevant shapes, including spheres, ellipsoids, capped cylinders, and hourglasses. We find a whole range of different engulfment behaviors with some ellipsoids engulfing faster than spheres, and that phagocytosis is able to engulf a greater range of target shapes than other types of endocytosis. Further, the 2D model can explain why some nonspherical particles engulf fastest (not at all) when presented to the membrane tip-first (lying flat). Our work reveals how some bacteria may avoid being internalized simply because of their shape, and suggests shapes for optimal drug delivery.

  12. Understanding magnetic nanoparticle osteoblast receptor-mediated endocytosis using experiments and modeling

    NASA Astrophysics Data System (ADS)

    Tran, Nhiem; Webster, Thomas J.

    2013-05-01

    Iron oxide nanoparticles are promising candidates for controlling drug delivery through an external magnetic force to treat a wide range of diseases, including osteoporosis. Previous studies have demonstrated that in the presence of hydroxyapatite coated magnetite (Fe3O4) nanoparticles, osteoblast (or bone forming cell) proliferation and long-term functions (such as calcium deposition) were significantly enhanced. Hydroxyapatite is the major inorganic component of bone. As a further attempt to understand why, in the current study, the uptake of such nanoparticles into osteoblasts was experimentally investigated and mathematically modeled. Magnetite nanoparticles were synthesized using a co-precipitation method and were coated with hydroxyapatite. A cellular uptake experiment at low temperatures indicated that receptor-mediated endocytosis contributed to the internalization of the magnetic nanoparticles into osteoblasts. A model was further developed to explain the uptake of magnetic nanoparticles into osteoblasts using receptor-mediated endocytosis. This model may explain the internalization of hydroxyapatite into osteoblasts to elevate intracellular calcium levels necessary to promote osteoblast functions to treat a wide range of orthopedic problems, including osteoporosis.

  13. Neuregulin-1-beta1 enters brain and spinal cord by receptor-mediated transport.

    PubMed

    Kastin, Abba J; Akerstrom, Victoria; Pan, Weihong

    2004-02-01

    Proteins of the neuregulin (NRG) family play important regulatory roles in neuronal survival and synaptic activity. NRG-1-beta1 has particular potential as a therapeutic agent because it enhances myelination of neurites in spinal cord explants. In this study, we determined the permeation of NRG-1-beta1 across the blood-brain and blood-spinal cord barriers (BBB and BSCB respectively). Intact radioactively labeled NRG-1-beta1 had a saturable and relatively rapid influx rate from blood to the CNS in mice. Capillary depletion studies showed that NRG-1-beta1 entered the parenchyma of the brain and spinal cord rather than being trapped in the capillaries that compose the BBB. The possible mechanism of receptor-mediated transport was shown by the ability of antibodies to erbB3 and erbB4 receptors to inhibit the influx. Lipophilicity, less important for such saturable transport mechanisms, was measured by the octanol : buffer partition coefficient and found to be low. The results indicate that NRG-1-beta1 enters spinal cord and brain by a saturable receptor-mediated mechanism, which provides the opportunity for possible therapeutic manipulation at the BBB level.

  14. Regulation and ontogeny of subtypes of muscarinic receptors and muscarinic receptor-mediated

    SciTech Connect

    Lee, W.

    1989-01-01

    The densities of total and M1 muscarinic receptors were measured using the muscarinic receptor antagonists {sup 3}H-quinuclidinyl benzilate and {sup 3}H-pirenzepine, respectively. Thus, the difference between the density of {sup 3}H-quinuclidinyl benzilate and {sup 3}H-pirenzepine binding sites represents the density of M2 sites. In addition, there is no observable change in either acetylcholine-stimulated phosphoinositide breakdown (suggested to be an M1 receptor-mediated response) or in carbachol-mediated inhibition of cyclic AMP accumulation (suggested to be an M2 receptor-mediated response) in slices of cortex+dorsal hippocampus following chronic atropine administration. In other experiments, it has been shown that the M1 and M2 receptors in rat cortex have different ontogenetic profiles. The M2 receptor is present at adult levels at birth, while the M1 receptor develops slowly from low levels at postnatal week 1 to adult levels at postnatal week 3. The expression of acetylcholine-stimulated phosphoinositide breakdown parallels the development of M1 receptors, while the development of carbachol-mediated inhibition of cyclic AMP accumulation occurs abruptly between weeks 2 and 3 postnatally.

  15. Adaptation in sound localization: from GABA(B) receptor-mediated synaptic modulation to perception.

    PubMed

    Stange, Annette; Myoga, Michael H; Lingner, Andrea; Ford, Marc C; Alexandrova, Olga; Felmy, Felix; Pecka, Michael; Siveke, Ida; Grothe, Benedikt

    2013-12-01

    Across all sensory modalities, the effect of context-dependent neural adaptation can be observed at every level, from receptors to perception. Nonetheless, it has long been assumed that the processing of interaural time differences, which is the primary cue for sound localization, is nonadaptive, as its outputs are mapped directly onto a hard-wired representation of space. Here we present evidence derived from in vitro and in vivo experiments in gerbils indicating that the coincidence-detector neurons in the medial superior olive modulate their sensitivity to interaural time differences through a rapid, GABA(B) receptor-mediated feedback mechanism. We show that this mechanism provides a gain control in the form of output normalization, which influences the neuronal population code of auditory space. Furthermore, psychophysical tests showed that the paradigm used to evoke neuronal GABA(B) receptor-mediated adaptation causes the perceptual shift in sound localization in humans that was expected on the basis of our physiological results in gerbils.

  16. Inverse Susceptibility to Oxidative Death of Lymphocytes Obtained From Alzheimer's Patients and Skin Cancer Survivors: Increased Apoptosis in Alzheimer's and Reduced Necrosis in Cancer

    PubMed Central

    Silva, Monica; Salech, Felipe; Ponce, Daniela P.; Merino, Daniela; Sinning, Mariana; Xiong, Chengjie; Roe, Catherine M.; Quest, Andrew F. G.

    2012-01-01

    A paucity of cancer in individuals with Alzheimer's disease (AD) and low rates of AD in cancer survivors has been reported in epidemiological studies. Deregulation in opposite directions of biological mechanisms, such as susceptibility to cell death, might be shared in the two disorders. We analyzed lymphocytes from AD and skin cancer patients as well as healthy controls and found significantly increased vulnerability of AD lymphocytes to H2O2-induced apoptotic death and higher resistance to death of skin cancer lymphocytes, due to reduced necrosis, as compared with healthy controls by pairwise comparisons adjusted for age and sex. H2O2-induced death in lymphocytes was caspase independent and significantly reduced by PARP-1 inhibition in all three groups. These differences in the susceptibility to cell death observed for lymphocytes from AD and skin cancer patients may be one of the mechanisms that help explain the inverse correlation detected between these diseases in epidemiological studies. PMID:22367434

  17. Antitumour effects of Phyllanthus emblica L.: induction of cancer cell apoptosis and inhibition of in vivo tumour promotion and in vitro invasion of human cancer cells.

    PubMed

    Ngamkitidechakul, C; Jaijoy, K; Hansakul, P; Soonthornchareonnon, N; Sireeratawong, S

    2010-09-01

    Phyllanthus emblica Linn. (PE) is a medicinal fruit used in many Asian traditional medicine systems for the treatment of various diseases including cancer. The present study tested the potential anticancer effects of aqueous extract of PE in four ways: (1) against cancer cell lines, (2) in vitro apoptosis, (3) mouse skin tumourigenesis and (4) in vitro invasiveness. The PE extract at 50-100 microg/mL significantly inhibited cell growth of six human cancer cell lines, A549 (lung), HepG2 (liver), HeLa (cervical), MDA-MB-231 (breast), SK-OV3 (ovarian) and SW620 (colorectal). However, the extract was not toxic against MRC5 (normal lung fibroblast). Apoptosis in HeLa cells was also observed as PE extract caused DNA fragmentation and increased activity of caspase-3/7 and caspase-8, but not caspase-9, and up-regulation of the Fas protein indicating a death receptor-mediated mechanism of apoptosis. Treatment of PE extract on mouse skin resulted in over 50% reduction of tumour numbers and volumes in animals treated with DMBA/TPA. Lastly, 25 and 50 microg/mL of PE extract inhibited invasiveness of MDA-MB-231 cells in the in vitro Matrigel invasion assay. These results suggest P. emblica exhibits anticancer activity against selected cancer cells, and warrants further study as a possible chemopreventive and antiinvasive agent.

  18. Quercetin-3-O-(2″-galloyl)-α-L-rhamnopyranoside attenuates cholesterol oxidation product-induced apoptosis by suppressing NF-κB-mediated cell death process in differentiated PC12 cells.

    PubMed

    Lee, Da Hee; Nam, Yoon Jeong; Lee, Chung Soo

    2015-08-01

    Cholesterol oxidation products are suggested to be involved in neuronal cell degeneration. We examined the preventive effect of quercetin-3-O-(2″-galloyl)-α-L-rhamnopyranoside (QGR), a quercetin derivative, on the cholesterol oxidation product-induced neuronal cell death using differentiated PC12 cells in relation to nuclear factor (NF)-κB-mediated apoptotic process. 7-Ketocholesterol and 25-hydroxycholesterol induced a decrease in the levels of BH3 interacting-domain death agonist (Bid) and B cell lymphoma 2 (Bcl-2), increase in the levels of Bcl-2-associated X protein (Bax) and p53, loss of the mitochondrial transmembrane potential, cytochrome c release, activation of caspases, and cleavage of poly(ADP-ribose) polymerase 1 (PARP-1). 7-Ketocholesterol induced increase in cytosolic and nuclear NF-κB p65, nuclear phospho-NF-κB p65, cytosolic NF-κB p50, and cytosolic phospho-IκB-α levels. The addition of QGR, N-acetyl cysteine, or Bay 11-7085 attenuated the cholesterol oxidation product-induced changes in the apoptosis-related protein levels, activation of NF-κB, formation of reactive oxygen species, depletion of glutathione (GSH), nuclear damage, and cell death. The results show that QGR may attenuate the cholesterol oxidation product-induced apoptosis in PC12 cells by suppressing the activation of the mitochondrial pathway and the caspase-8- and Bid-dependent pathways that is mediated by NF-κB activation. The preventive effect appears to be associated with the inhibitory effect on the formation of reactive oxygen species and depletion of GSH.

  19. Enzyme induction and histopathology elucidate aryl hydrocarbon receptor-mediated versus non-aryl hydrocarbon receptor-mediated effects of Aroclor 1268 in American mink (Neovison vison).

    PubMed

    Folland, William R; Newsted, John L; Fitzgerald, Scott D; Fuchsman, Phyllis C; Bradley, Patrick W; Kern, John; Kannan, Kurunthachalam; Zwiernik, Matthew J

    2016-03-01

    Polychlorinated biphenyl (PCB) concentrations reported in preferred prey and blubber of bottlenose dolphins from the Turtle-Brunswick River estuary (Georgia, USA) suggest the potential for adverse effects. However, PCBs in Turtle-Brunswick River estuary dolphins are primarily derived from Aroclor 1268, and predicting toxic effects of Aroclor 1268 is uncertain because of the mixture's unique composition and associated physiochemical characteristics. These differences suggest that toxicity benchmarks for other PCB mixtures may not be relevant to dolphins exposed to Aroclor 1268. American mink (Neovison vison) were used as a surrogate model for cetaceans to characterize mechanisms of action associated with Aroclor 1268 exposure. Mink share similarities in phylogeny and life history with cetaceans and are characteristically sensitive to PCBs, making them an attractive surrogate species for marine mammals in ecotoxicity studies. Adult female mink and a subsequent F1 generation were exposed to Aroclor 1268 through diet, and effects on enzyme induction, histopathology, thyroid hormone regulation, hematology, organ weights, and body condition index were compared to a negative control and a 3,3',4,4',5-pentachlorobiphenyl (PCB 126)-positive control. Aroclor 1268 dietary exposure concentrations ranged from 1.8 µg/g wet weight to 29 µg/g wet weight. Anemia, hypothyroidism, and hepatomegaly were observed in mink exposed to Aroclor 1268 beyond various dietary thresholds. Cytochrome P450 induction and squamous epithelial proliferation jaw lesions were low in Aroclor 1268 treatments relative to the positive control. Differences in enzyme induction and the development of squamous epithelial proliferation jaw lesions between Aroclor 1268 treatments and the positive control, coupled with effects observed in Aroclor 1268 treatments not observed in the positive control, indicate that mechanisms additional to the aryl hydrocarbon receptor-mediated pathway are associated with

  20. The biochemistry of apoptosis.

    PubMed

    Hengartner, M O

    2000-10-12

    Apoptosis--the regulated destruction of a cell--is a complicated process. The decision to die cannot be taken lightly, and the activity of many genes influence a cell's likelihood of activating its self-destruction programme. Once the decision is taken, proper execution of the apoptotic programme requires the coordinated activation and execution of multiple subprogrammes. Here I review the basic components of the death machinery, describe how they interact to regulate apoptosis in a coordinated manner, and discuss the main pathways that are used to activate cell death.

  1. D2-like dopamine receptors mediate the response to amphetamine in a mouse model of ADHD

    PubMed Central

    Fan, Xueliang; Hess, Ellen J.

    2007-01-01

    The mechanisms underlying the effects of psychostimulants in attention deficit hyperactivity disorder (ADHD) are not well understood, but indirect evidence implicates D2 dopamine receptors. Here we dissect the components of dopaminergic neurotransmission in the hyperactive mouse mutant coloboma to identify pre- and postsynaptic elements essential for the effects of amphetamine in these mice. Amphetamine treatment reduced locomotor activity in coloboma mice, but induced a robust increase in dopamine overflow suggesting that abnormal regulation of dopamine efflux does not account for the behavioral effect. However, the D2-like dopamine receptor antagonists haloperidol and raclopride, but not the D1-like dopamine receptor antagonist SCH23390, blocked the amphetamine-induced reduction in locomotor activity in coloboma mice, providing direct evidence that D2-like dopamine receptors mediate the effect of amphetamine in these mice. With the precedent established that it is possible to directly antagonize this response, this strategy should prove useful for identifying novel therapeutics in ADHD. PMID:17291774

  2. Bicarbonate contributes to GABAA receptor-mediated neuronal excitation in surgically resected human hypothalamic hamartomas.

    PubMed

    Kim, Do-Young; Fenoglio, Kristina A; Kerrigan, John F; Rho, Jong M

    2009-01-01

    The role of bicarbonate (HCO(3)(-)) in GABA(A) receptor-mediated depolarization of human hypothalamic hamartoma (HH) neurons was investigated using cellular electrophysiological and calcium imaging techniques. Activation of GABA(A) receptors with muscimol (30 microM) provoked neuronal excitation in over 70% of large (18-22 microM) HH neurons in HCO(3)(-) buffer. Subsequent perfusion of HCO(3)(-)-free HEPES buffer produced partial suppression of muscimol-induced excitation. Additionally, 53% of large HH neurons under HCO(3)(-)-free conditions exhibited reduced intracellular calcium accumulation by muscimol. These results suggest that HCO(3)(-) efflux through GABA(A) receptors on a subpopulation of large HH neurons may contribute to membrane depolarization and subsequent activation of L-type calcium channels.

  3. Using GFP--ligand fusions to measure receptor-mediated endocytosis in living cells.

    PubMed

    Medina-Kauwe, Lali K; Chen, Xinhua

    2002-01-01

    Recombinant DNA technology has enabled the production of many types of chimeric proteins containing heterologous functional domains that have served a variety of useful capacities for cell biology research. Among proteins gaining wide use as a fusion partner is Aequorea victoria green fluorescent protein (GFP). GFP has been employed by numerous groups as a reporter gene for cell transfection and as an autofluorescent tag by recombinant fusion to foreign sequences. Here we describe the use of GFP as a tag for ligands, and provide examples of how purified recombinant GFP-ligand fusion proteins may be used to detect ligand-receptor interactions, including receptor-mediated endocytosis. Both its utility and limitations are discussed.

  4. Essential role of caspase-8 in p53/p73-dependent apoptosis induced by etoposide in head and neck carcinoma cells

    PubMed Central

    2011-01-01

    Background Caspase-8 is a key upstream mediator in death receptor-mediated apoptosis and also participates in mitochondria-mediated apoptosis via cleavage of proapoptotic Bid. However, the role of caspase-8 in p53- and p73-dependent apoptosis induced by genotoxic drugs remains unclear. We recently reported that the reconstitution of procaspase-8 is sufficient for sensitizing cisplatin- but not etoposide-induced apoptosis, in chemoresistant and caspase-8 deficient HOC313 head and neck squamous cell carcinoma (HNSCC) cells. Results We show that p53/p73-dependent caspase-8 activation is required for sensitizing etoposide-induced apoptosis by utilizing HOC313 cells carrying a temperature-sensitive p53G285K mutant. Restoration of wild-type p53 function under the permissive conditions, together with etoposide treatment, led to substantial transcriptional activation of proapoptotic Noxa and PUMA, but failed to induce apoptosis. In addition to p53 restoration, caspase-8 reconstitution was needed for sensitization to etoposide-induced apoptosis, mitochondria depolarization, and cleavage of the procaspases-3, and -9. In etoposide-sensitive Ca9-22 cells carrying a temperature-insensitive mutant p53, siRNA-based p73 knockdown blocked etoposide-induced apoptosis and procaspase-8 cleavage. However, induction of p73 protein and up-regulation of Noxa and PUMA, although observed in Ca9-22 cells, were hardly detected in etoposide-treated HOC313 cells under non-permissive conditions, suggesting a contribution of p73 reduction to etoposide resistance in HOC313 cells. Finally, the caspase-9 inhibitor Ac-LEHD-CHO or caspase-9 siRNA blocked etoposide-induced caspase-8 activation, Bid cleavage, and apoptosis in both cell lines, indicating that p53/p73-dependent caspase-8 activation lies downstream of mitochondria. Conclusions we conclude that p53 and p73 can act as upstream regulators of caspase-8, and that caspase-8 is an essential mediator of the p53/p73-dependent apoptosis induced by

  5. Current injection and receptor-mediated excitation produce similar maximal firing rates in hypoglossal motoneurons.

    PubMed

    Wakefield, Hilary E; Fregosi, Ralph F; Fuglevand, Andrew J

    2016-03-01

    The maximum firing rates of motoneurons (MNs), activated in response to synaptic drive, appear to be much lower than that elicited by current injection. It could be that the decrease in input resistance associated with increased synaptic activity (but not current injection) might blunt overall changes in membrane depolarization and thereby limit spike-frequency output. To test this idea, we recorded, in the same cells, maximal firing responses to current injection and to synaptic activation. We prepared 300 μm medullary slices in neonatal rats that contained hypoglossal MNs and used whole-cell patch-clamp electrophysiology to record their maximum firing rates in response to triangular-ramp current injections and to glutamate receptor-mediated excitation. Brief pressure pulses of high-concentration glutamate led to significant depolarization, high firing rates, and temporary cessation of spiking due to spike inactivation. In the same cells, we applied current clamp protocols that approximated the time course of membrane potential change associated with glutamate application and with peak current levels large enough to cause spike inactivation. Means (SD) of maximum firing rates obtained in response to glutamate application were nearly identical to those obtained in response to ramp current injection [glutamate 47.1 ± 12.0 impulses (imp)/s, current injection 47.5 ± 11.2 imp/s], even though input resistance was 40% less during glutamate application compared with current injection. Therefore, these data suggest that the reduction in input resistance associated with receptor-mediated excitation does not, by itself, limit the maximal firing rate responses in MNs.

  6. Voltage-dependent inhibition of recombinant NMDA receptor-mediated currents by 5-hydroxytryptamine

    PubMed Central

    Kloda, Anna; Adams, David J

    2005-01-01

    The effect of 5-HT and related indolealkylamines on heteromeric recombinant NMDA receptors expressed in Xenopus oocytes was investigated using the two-electrode voltage-clamp recording technique. In the absence of external Mg2+ ions, 5-HT inhibited NMDA receptor-mediated currents in a concentration-dependent manner. The inhibitory effect of 5-HT was independent of the NR1a and NR2 subunit combination. The inhibition of glutamate-evoked currents by 5-HT was use- and voltage-dependent. The voltage sensitivity of inhibition for NR1a+NR2 subunit combinations by 5-HT was similar, exhibiting an e-fold change per ∼20 mV, indicating that 5-HT binds to a site deep within the membrane electric field. The inhibition of the open NMDA receptor by external Mg2+ and 5-HT was not additive, suggesting competition between Mg2+ and 5-HT for a binding site in the NMDA receptor channel. The concentration-dependence curves for 5-HT and 5-methoxytryptamine (5-MeOT) inhibition of NMDA receptor-mediated currents are shifted to the right in the presence of external Mg2+. The related indolealkylamines inhibited glutamate-evoked currents with the following order of inhibitory potency: 5-MeOT=5-methyltryptamine>tryptamine>7-methyltryptamine>5-HT≫tryptophan=melatonin. Taken together, these data suggest that 5-HT and related compounds can attenuate glutamate-mediated excitatory synaptic responses and may provide a basis for drug treatment of excitoxic neurodegeneration. PMID:15655527

  7. [Pathophysiologic programming of cell death].

    PubMed

    Dobryszycka, W

    1998-01-01

    In multicellular organisms homeostasis depends on a balance between cell proliferation and cell death. In this review principles of the physiology of programmed cell death (apoptosis), i.e. biochemical features, involved genes and proteolytic enzymes, are described. Alterations in apoptosis contribute to the pathogenesis of a number of human diseases, including cancer, viral infections, inflammation, hematopoietic and immunological system defects (e.g. AIDS), neurodegenerative disorders. Specific effect on regulation of apoptosis might lead to new possibilities for treatment. Methods of quantitative determinations of apoptosis are discussed.

  8. Multi-walled carbon nanotubes induce apoptosis in RAW 264.7 cell-derived osteoclasts through mitochondria-mediated death pathway.

    PubMed

    Ye, Shefang; Jiang, Yuanqin; Zhang, Honggang; Wang, Yifang; Wu, Yihui; Hou, Zhenqing; Zhang, Qiqing

    2012-03-01

    Carbon nanotubes (CNTs) have attracted great interest with respect to biomaterials, particularly for use as an implant material in bone-tissue engineering. Accordingly, the bone-tissue compatibility of CNTs and their influence on new bone formation are important issues. In the present study, we examined the effects of multi-wall carbon nanotubes (MWCNTs) on the receptor activator of nuclear factor kappaB ligand (RANKL)-supported osteoclastogenesis using a murine monocytic cell line RAW 264.7. MWCNTs significantly suppressed the differentiation of RAW 264.7 cells into osteoclasts. Treatment with MWCNTs induced apoptosis in osteoclasts as characterized by nuclear condensation, DNA fragmentation, caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage, but did not decrease the cell viability of the osteoblast-like cell line MC3T3-E1. MWCNTs also induced loss of the mitochondrial membrane potential (deltapsim) by regulating expression of Bcl-2 family proteins and caused release of cytochrome c from mitochondria to cytosol. MWCNTs-induced apoptosis in osteoclasts was inhibited both by cyclosporin A, a blocker of the mitochondrial permeability transition pore, and by DEVD-CHO, a cell-permeable inhibitor of caspase-3. The present study suggests that MWCNTs suppresse osteoclastogenesis via the inhibition of osteoclast differentiation and the induction of apoptosis in osteoclasts, rendering them promising candidate for the treatment of osteoclast-related diseases.

  9. Achyranthes aspera Root Extracts Induce Human Colon Cancer Cell (COLO-205) Death by Triggering the Mitochondrial Apoptosis Pathway and S Phase Cell Cycle Arrest

    PubMed Central

    Arora, Shagun; Tandon, Simran

    2014-01-01

    Achyranthes aspera (AA) has been used traditionally for the cure of various disorders. However, the action of root extracts of AA as anticancer agent and its cellular mechanism remain unclear. The aim was to screen the antitumor effect of ethanolic (EAA) and aqueous (AAA) root extracts on the growth of colon cancer COLO-205 cells by testing their cytotoxicity, followed by their effect on clonogenicity, migration, and induction of apoptosis. Mechanisms leading to apoptosis and cell cycle arrest were also investigated by expression studies of caspase-9, caspase-3, Bax, Bcl-2, p16, p21, and p27 genes, followed by flow cytometric analysis for cell cycle distribution. Cytotoxicity screening of AA extracts indicated greater cytotoxic activity of AAA extract against COLO-205 cells. A series of events marked by apoptosis revealed loss of cell viability, chromatin condensation, and DNA fragmentation in AAA treated cells to a greater extent. The mRNA expression levels of caspase-9, caspase-3, Bax, p16, p21, and p27 were markedly increased in the AAA treated cells, along with decreased Bcl-2 expression. The cell cycle arrest at S phase was detected by flow cytometric analysis after treatment with AAA. Overall the study signifies the aqueous extracts as a promising therapeutic candidate against cancer. PMID:25401123

  10. The essential role of p53-up-regulated modulator of apoptosis (Puma) and its regulation by FoxO3a transcription factor in β-amyloid-induced neuron death.

    PubMed

    Akhter, Rumana; Sanphui, Priyankar; Biswas, Subhas Chandra

    2014-04-11

    Neurodegeneration underlies the pathology of Alzheimer disease (AD). The molecules responsible for such neurodegeneration in AD brain are mostly unknown. Recent findings indicate that the BH3-only proteins of the Bcl-2 family play an essential role in various cell death paradigms, including neurodegeneration. Here we report that Puma (p53-up-regulated modulator of apoptosis), an important member of the BH3-only protein family, is up-regulated in neurons upon toxic β-amyloid 1-42 (Aβ(1-42)) exposure both in vitro and in vivo. Down-regulation of Puma by specific siRNA provides significant protection against neuron death induced by Aβ(1-42). We further demonstrate that the activation of p53 and inhibition of PI3K/Akt pathways induce Puma. The transcription factor FoxO3a, which is activated when PI3K/Akt signaling is inhibited, directly binds with the Puma gene and induces its expression upon exposure of neurons to oligomeric Aβ(1-42). Moreover, Puma cooperates with another BH3-only protein, Bim, which is already implicated in AD. Our results thus suggest that Puma is activated by both p53 and PI3K/Akt/FoxO3a pathways and cooperates with Bim to induce neuron death in response to Aβ(1-42).

  11. Methamphetamine induces autophagy and apoptosis in a mesencephalic dopaminergic neuronal culture model: role of cathepsin-D in methamphetamine-induced apoptotic cell death.

    PubMed

    Kanthasamy, Arthi; Anantharam, V; Ali, Syed F; Kanthasamy, A G

    2006-08-01

    Autophagy is a phylogenetically conserved process that plays a critical role in the degradation of oxidatively damaged proteins and organelle turnover. The role of oxidative stress and apoptosis in methamphetamine (METH)-induced neurotoxicity is well known; however, the potential contribution of autophagy to METH-induced oxidative damage in dopaminergic neuronal systems remains unclear. The goals of the present article were twofold: (a) to develop an in vitro dopaminergic cell culture model to study cellular and molecular mechanisms underlying METH-induced autophagy and apoptosis, and (b) to determine whether lysosomal protease cathepsin-D activation, resulting from the loss of lysosomal membrane integrity, contributes to METH-induced apoptosis. To accomplish these goals, we characterized morphological and biochemical changes in an immortalized mesencephalic dopaminergic neuronal cell line (N27 cells) following treatment with METH. Exposure of METH (2 mM) to N27 cells resulted in the appearance of cytoplasmic vacuolar structures reminiscent of autophagic vacuoles within 3 h. In order to ascertain the identity of the vacuolar structures that are formed following METH exposure, immunohistochemical staining for markers of autophagy were performed. LAMP 2, a classical marker of autophagolysosomes, revealed an extensive punctuate pattern of distribution on the vacuolar membrane surface, with exclusive localization in the cytoplasm. Additionally, using DNA fragmentation analysis we showed a dose-dependent increase in fragmented DNA in METH treated N27 cells. Since METH-induced autophagy preceded DNA fragmentation, we tested whether dysfunction of the autophagolysosomal system contributes to nuclear damage. Immunofluorescence studies with cathepsin-d demonstrated a granular pattern of staining in untreated cells, whereas an increased cathepsin- D immunoreactivity with a globular pattern of staining was observed in METH-treated cells. Nevertheless, blockade of cathepsin

  12. Synthesis and mannose receptor-mediated uptake of clustered glycomimetics by human dendritic cells: effect of charge.

    PubMed

    Angyalosi, Gerhild; Grandjean, Cyrille; Lamirand, Mélanie; Auriault, Claude; Gras-Masse, Hélène; Melnyk, Oleg

    2002-10-07

    Effect of charge and shape of multivalent lysine-based cluster glycomimetics on their mannose receptor-mediated uptake by human dendritic cells has been evaluated: The capture is strongly affected by the shape of the ligands. The effect of charge is less pronounced although positive charges on the ligands seem to favor non-specific endocytosis capture.

  13. Acrolein-Induced Oxidative Stress and Cell Death Exhibiting Features of Apoptosis in the Yeast Saccharomyces cerevisiae Deficient in SOD1.

    PubMed

    Kwolek-Mirek, Magdalena; Zadrąg-Tęcza, Renata; Bednarska, Sabina; Bartosz, Grzegorz

    2015-04-01

    The yeast Saccharomyces cerevisiae is a useful eukaryotic model to study the toxicity of acrolein, an important environmental toxin and endogenous product of lipid peroxidation. The study was aimed at elucidation of the cytotoxic effect of acrolein on the yeast deficient in SOD1, Cu, Zn-superoxide dismutase which is hypersensitive to aldehydes. Acrolein generated within the cell from its precursor allyl alcohol caused growth arrest and cell death of the yeast cells. The growth inhibition involved an increase in production of reactive oxygen species and high level of protein carbonylation. DNA condensation and fragmentation, exposition of phosphatidylserine at the cell surface as well as decreased dynamic of actin microfilaments and mitochondria disintegration point to the induction of apoptotic-type cell death besides necrotic cell death.

  14. Artepillin C (3,5-diprenyl-4-hydroxycinnamic acid) sensitizes LNCaP prostate cancer cells to TRAIL-induced apoptosis

    PubMed Central

    SZLISZKA, EWELINA; ZYDOWICZ, GRZEGORZ; MIZGALA, ELZBIETA; KROL, WOJCIECH

    2012-01-01

    Naturally occurring phenolic compounds have been shown to sensitize prostate cancer cells to tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. TRAIL is a potent stimulator of apoptosis in cancer cells and an important immune effector molecule in the surveillance and elimination of developing tumours. However, many cancer cells are resistant to TRAIL-mediated death. In this study, we aimed to determine the mechanisms by which TRAIL resistance can be overcome in prostate cancer cells by 3,5-diprenyl-4-hydroxycinnamic acid (artepillin C). Artepillin C is a bioactive component of Brazilian green propolis that possesses antitumour and chemopreventive activities. TRAIL-resistant LNCaP prostate cancer cells were treated with TRAIL and artepillin C. Cytotoxicity was measured by MTT and lactate dehydrogenase (LDH) assays. Apoptosis was detected using Annexin V-FITC staining by flow cytometry and fluorescence microscopy. Death receptor (DR) (TRAIL-R1/DR4 and TRAIL-R2/DR5) expression was analyzed using flow cytometry. Mitochondrial membrane potential (ΔΨm) was evaluated using DePsipher staining by fluorescence micro scopy. The inhibition of NF-κB (p65) activation was confirmed with the ELISA-based TransAM NF-κB kit. Caspase-8 and caspase-3 activities were determined by colorimetric protease assays. The results showed that artepillin C sensitized the TRAIL-resistant LNCaP cells by engaging the extrinsic (receptor-mediated) and intrinsic (mitochondrial) apoptotic pathways. Artepillin C increased the expression of TRAIL-R2 and decreased the activity of NF-κB. Co-treatment with TRAIL and artepillin C induced the significant activation of caspase-8 and caspase-3, as well as the disruption of ΔΨm. These findings show that prostate cancer cells can be sensitized to TRAIL-mediated immunoprevention by artepillin C and confirm the role of phenolic compounds in prostate cancer immunochemoprevention. PMID:22735465

  15. Cytotoxicity of atropine to human corneal endothelial cells by inducing mitochondrion-dependent apoptosis.

    PubMed

    Wen, Qian; Fan, Ting-Jun; Tian, Cheng-Lei

    2016-07-01

    Atropine, a widely used topical anticholinergic drug, might have adverse effects on human corneas in vivo. However, its cytotoxic effect on human corneal endothelium (HCE) and its possible mechanisms are unclear. Here, we investigated the cytotoxicity of atropine and its underlying cellular and molecular mechanisms using an in vitro model of HCE cells and verified the cytotoxicity using cat corneal endothelium (CCE) in vivo. Our results showed that atropine at concentrations above 0.3125 g/L could induce abnormal morphology and viability decline in a dose- and time-dependent manner in vitro. The cytotoxicity of atropine was proven by the induced density decrease and abnormality of morphology and ultrastructure of CCE cells in vivo. Meanwhile, atropine could also induce dose- and time-dependent elevation of plasma membrane permeability, G1 phase arrest, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation of HCE cells. Moreover, 2.5 g/L atropine could also induce caspase-2/-3/-9 activation, mitochondrial transmembrane potential disruption, downregulation of anti-apoptotic Bcl-2 and Bcl-xL, upregulation of pro-apoptotic Bax and Bad, and upregulation of cytoplasmic cytochrome c and apoptosis-inducing factor. In conclusion, atropine above 1/128 of its clinical therapeutic dosage has a dose- and time-dependent cytotoxicity to HCE cells in vitro which is confirmed by CCE cells in vivo, and its cytotoxicity is achieved by inducing HCE cell apoptosis via a death receptor-mediated mitochondrion-dependent signaling pathway. Our findings provide new insights into the cytotoxicity and apoptosis-inducing effect of atropine which should be used with great caution in eye clinic.

  16. Cytotoxicity of atropine to human corneal endothelial cells by inducing mitochondrion-dependent apoptosis

    PubMed Central

    Wen, Qian; Tian, Cheng-Lei

    2016-01-01

    Atropine, a widely used topical anticholinergic drug, might have adverse effects on human corneas in vivo. However, its cytotoxic effect on human corneal endothelium (HCE) and its possible mechanisms are unclear. Here, we investigated the cytotoxicity of atropine and its underlying cellular and molecular mechanisms using an in vitro model of HCE cells and verified the cytotoxicity using cat corneal endothelium (CCE) in vivo. Our results showed that atropine at concentrations above 0.3125 g/L could induce abnormal morphology and viability decline in a dose- and time-dependent manner in vitro. The cytotoxicity of atropine was proven by the induced density decrease and abnormality of morphology and ultrastructure of CCE cells in vivo. Meanwhile, atropine could also induce dose- and time-dependent elevation of plasma membrane permeability, G1 phase arrest, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation of HCE cells. Moreover, 2.5 g/L atropine could also induce caspase-2/-3/-9 activation, mitochondrial transmembrane potential disruption, downregulation of anti-apoptotic Bcl-2 and Bcl-xL, upregulation of pro-apoptotic Bax and Bad, and upregulation of cytoplasmic cytochrome c and apoptosis-inducing factor. In conclusion, atropine above 1/128 of its clinical therapeutic dosage has a dose- and time-dependent cytotoxicity to HCE cells in vitro which is confirmed by CCE cells in vivo, and its cytotoxicity is achieved by inducing HCE cell apoptosis via a death receptor-mediated mitochondrion-dependent signaling pathway. Our findings provide new insights into the cytotoxicity and apoptosis-inducing effect of atropine which should be used with great caution in eye clinic. PMID:27022135

  17. A Small Molecule Inhibitor Selectively Induces Apoptosis in Cells Transformed by High Risk Human Papilloma Viruses

    PubMed Central

    Lee, Min S.; Qi, Huilin; Chaniewski, Susan; Zheng, Xiaofan; Farr, Glen A.; Esposito, Kim; Harden, David; Lei, Ming; Schweizer, Liang; Friborg, Jacques; Agler, Michele; McPhee, Fiona; Gentles, Robert; Beno, Brett R.; Chupak, Lou; Mason, Stephen

    2016-01-01

    A phenotypic high-throughput cell culture screen was performed to identify compounds that prevented proliferation of the human Papilloma virus type 16 (HPV-16) transformed cell line Ca Ski. A series of quinoxaline compounds exemplified by Compound 1 was identified. Testing against a panel of cell lines demonstrated that Compound 1 selectively inhibited replication of all HPV-16, HPV-18, and HPV-31 transformed cell lines tested with 50% Inhibitory Concentration (IC50) values of 2 to 8 μM relative to IC50 values of 28 to 73 μM in HPV-negative cell lines. Treatment with Compound 1 resulted in a cascade of multiple apoptotic events, including selective activation of effector caspases 3 and 7, fragmentation of cellular DNA, and PARP (poly(ADP-ribose) polymerase) cleavage in HPV-positive cells relative to HPV-negative cells. Unregulated proliferation of HPV transformed cells is dependent on the viral oncogenes, E6 and E7. Treatment with Compound 1 resulted in a decrease in HPV E7 protein in Ca Ski cells. However, the timing of this reduction relative to other effects of compound treatment suggests that this was a consequence, rather than a cause, of the apoptotic cascade. Likewise, compound treatment resulted in no obvious effects on the E6- and E7- mediated down regulation of p53 and Rb, or their downstream effectors, p21 or PCNA. Further investigation of apoptotic signals induced by Compound 1 revealed cleavage of Caspase-8 in HPV-positive cells as early as 2 hours post-treatment, suggesting the compound initiates apoptosis through the extrinsic, death receptor-mediated, pathway of cell death. These studies provide proof of concept that cells transformed by oncogenic Papillomaviruses can be selectively induced to undergo apoptosis by compound treatment. PMID:27280728

  18. AG36 Inhibits Human Breast Cancer Cells Proliferation by Promotion of Apoptosis In vitro and In vivo

    PubMed Central

    Mu, Li-Hua; Wang, Yu-Ning; Wang, Dong-Xiao; Zhang, Jing; Liu, Li; Dong, Xian-Zhe; Hu, Yuan; Liu, Ping

    2017-01-01

    AG36 is the biotransformation product of triterpenoid saponin from Ardisia gigantifolia stapf. In this study, the antitumor activity and underlying molecular mechanisms of AG36 against human breast MCF-7, MDA-MB-231, and SK-BR-3 cancer cells were investigated. AG36 inhibited the viability of MCF-7, MDA-MB-231, and SK-BR-3 cells in a dose and time-dependent manner, with an IC50 of approximately 0.73, 18.1, and 23.4 μM at 48 h, respectively. AG36 obviously induced apoptosis and G2/M arrest of all the three breast cancer cells. Moreover, AG36 decreased the protein expression of cycle regulatory proteins cyclin B1 or cyclin D1. In MCF-7 and MDA-MB-231 cells, AG36 strongly increased the cleaved caspase-3 and -8 protein expressions, while in SK-BR-3 cells, AG36 only increased the protein expression of cleaved caspase-3. In all the three breast cancer cells, the ratio of Bax/Bcl-2 and cytosolic cytochrome c content increased significantly compared with control group. The death receptor-related proteins Fas/FasL, TNFR1, and DR5 were detected by Western blot, it showed that different breast cancer cells activated the death receptor-mediated extrinsic caspase-8 pathway through different receptors. In addition, the caspase-8 inhibitor z-IETD-fmk could significantly block AG36-triggered MCF-7 cells apoptosis. The in vivo studies showed that AG36 significantly inhibited the growth of MCF-7 xenograft tumors in BALB/c nude mice comparing with control. In conclusion, AG36 inhibited MCF-7, MDA-MB-231, and SK-BR-3 cells proliferation by the intrinsic mitochondrial and the extrinsic death receptor pathways and AG36 might be a potential breast cancer therapeutic agent. PMID:28184196

  19. Pigment epithelium-derived factor (PEDF) promotes tumor cell death by inducing macrophage membrane tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

    PubMed

    Ho, Tsung-Chuan; Chen, Show-Li; Shih, Shou-Chuan; Chang, Shing-Jyh; Yang, Su-Lin; Hsieh, Jui-Wen; Cheng, Huey-Chuan; Chen, Lee-Jen; Tsao, Yeou-Ping

    2011-10-14

    Pigment epithelium-derived factor (PEDF) is an intrinsic anti-angiogenic factor and a potential anti-tumor agent. The tumoricidal mechanism of PEDF, however, has not been fully elucidated. Here we report that PEDF induces the apoptosis of TC-1 and SK-Hep-1 tumor cells when they are cocultured with bone marrow-derived macrophages (BMDMs). This macrophage-mediated tumor killing is prevented by blockage of TNF-related apoptosis-inducing ligand (TRAIL) following treatment with the soluble TRAIL receptor. PEDF also increases the amount of membrane-bound TRAIL on cultured mouse BMDMs and on macrophages surrounding subcutaneous tumors. PEDF-induced tumor killing and TRAIL induction are abrogated by peroxisome proliferator-activated receptor γ (PPARγ) antagonists or small interfering RNAs targeting PPARγ. PEDF also induces PPARγ in BMDMs. Furthermore, the activity of the TRAIL promoter in human macrophages is increased by PEDF stimulation. Chromatin immunoprecipitation and DNA pull-down assays confirmed that endogenous PPARγ binds to a functional PPAR-response element (PPRE) in the TRAIL promoter, and mutation of this PPRE abolishes the binding of the PPARγ-RXRα heterodimer. Also, PPARγ-dependent transactivation and PPARγ-RXRα binding to this PPRE are prevented by PPARγ antagonists. Our results provide a novel mechanism for the tumoricidal activity of PEDF, which involves tumor cell killing via PPARγ-mediated TRAIL induction in macrophages.

  20. Xanthohumol induces apoptosis in cultured 40-16 human colon cancer cells by activation of the death receptor- and mitochondrial pathway.

    PubMed

    Pan, Lydia; Becker, Hans; Gerhäuser, Clarissa

    2005-09-01

    Xanthohumol (XN) is one of the major prenylflavonoids found in hop cones (Humulus lupulus L.). In this study, we investigated the cell growth inhibitory potential of XN on cultured human colon cancer cells. Cell proliferation was measured by sulforhodamine B staining. Poly(ADP-ribose)polymerase (PARP) cleavage, activation of caspases-3, -7, -8, and -9, and Bcl-2 family protein expression were detected by Western blot analyses. XN significantly reduced proliferation of the HCT 116-derived colon cancer cell line 40--16. Half-maximal inhibitory concentrations decreased from 4.1 microM after 24 h treatment to 3.6 and 2.6 microM after 48 and 72 h incubation, respectively. Treatment with 15 microM XN for 48 h and with 5 microM for 72 h led to the detection of the cleaved 89 kDa fragment of 116 kDa PARP as an indication of apoptosis induction. Concomitantly, we observed activation and cleavage of the effector caspases-3 and -7, induced by activation of the initiator caspases -8 and -9. Expression of anti-apoptotic Bcl-2 was down regulated when the cells were treated with XN for 48--72 h. We conclude that induction of apoptosis by downregulation of Bcl-2 and activation of the caspase cascade may contribute to the chemopreventive or therapeutic potential of XN.

  1. Glutathione and apoptosis

    PubMed Central

    Circu, Magdalena L.; Yee Aw, Tak

    2011-01-01

    Apoptosis or programmed cell death represents a physiologically conserved mechanism of cell death that is pivotal in normal development and tissue homeostasis in all organisms. As a key modulator of cell functions, the most abundant non-protein thiol, glutathione (GSH), has important roles in cellular defense against oxidant aggression, redox regulation of proteins thiols and maintaining redox homeostasis that is critical for proper function of cellular processes, including apoptosis. Thus, a shift in the cellular GSH-to-GSSG redox balance in favour of the oxidized species, GSSG, constitutes an important signal that could decide the fate of a cell. The current review will focus on three main areas: (1) general description of cellular apoptotic pathways, (2) cellular compartmentation of GSH and the contribution of mitochondrial GSH and redox proteins to apoptotic signalling and (3) role of redox mechanisms in the initiation and execution phases of apoptosis. PMID:18671159

  2. Methods for determining Myc-induced apoptosis.

    PubMed

    Lu, Dan; Littlewood, Trevor D

    2013-01-01

    Although many oncoproteins promote cell growth and proliferation, some also possess the potential to induce cell death by apoptosis. Deregulated expression of the myc oncogene promotes apoptosis in both cultured cells and in some tissues in vivo. Here we describe techniques to detect Myc-induced apoptosis in vitro using flow cytometry and microscopy and in vivo using immunohistochemical staining.

  3. Programmed Cell Death in Breast Cancer

    DTIC Science & Technology

    1998-10-01

    Programmed cell death , or apoptosis, is a genetically regulated process through which a cell is active in bringing about its own death for the sake...delays and inhibits the cell death response, so that the breast cancer cell lines are much less susceptible to thapsigargin-induced apoptosis than...lymphoid cell lines, an observation that parallels the differential susceptibility of breast cancer and lymphomas to chemotherapy-induced cell death in

  4. Can plant oncogenes inhibit programmed cell death? The rolB oncogene reduces apoptosis-like symptoms in transformed plant cells.

    PubMed

    Gorpenchenko, Tatiana Y; Aminin, Dmitry L; Vereshchagina, Yuliya V; Shkryl, Yuri N; Veremeichik, Galina N; Tchernoded, Galina K; Bulgakov, Victor P

    2012-09-01

    The rolB oncogene was previously identified as an important player in ROS metabolism in transformed plant cells. Numerous reports indicate a crucial role for animal oncogenes in apoptotic cell death. Whether plant oncogenes such as rolB can induce programmed cell death (PCD) in transformed plant cells is of particular importance. In this investigation, we used a single-cell assay based on confocal microscopy and fluorescent dyes capable of discriminating between apoptotic and necrotic cells. Our results indicate that the expression of rolB in plant cells was sufficient to decrease the proportion of apoptotic cells in steady-state conditions and diminish the rate of apoptotic cells during induced PCD. These data suggest that plant oncogenes, like animal oncogenes, may be involved in the processes mediating PCD.

  5. Nicotine impairs cyclooxygenase-2-dependent kinin-receptor-mediated murine airway relaxations

    SciTech Connect

    Xu, Yuan Cardell, Lars-Olaf

    2014-02-15

    Introduction: Cigarette smoke induces local inflammation and airway hyperreactivity. In asthmatics, it worsens the symptoms and increases the risk for exacerbation. The present study investigates the effects of nicotine on airway relaxations in isolated murine tracheal segments. Methods: Segments were cultured for 24 h in the presence of vehicle, nicotine (10 μM) and/or dexamethasone (1 μM). Airway relaxations were assessed in myographs after pre-contraction with carbachol (1 μM). Kinin receptors, cyclooxygenase (COX) and inflammatory mediator expressions were assessed by real-time PCR and confocal-microscopy-based immunohistochemistry. Results: The organ culture procedure markedly increased bradykinin- (selective B{sub 2} receptor agonist) and des-Arg{sup 9}-bradykinin- (selective B{sub 1} receptor agonist) induced relaxations, and slightly increased relaxation induced by isoprenaline, but not that induced by PGE{sub 2}. The kinin receptor mediated relaxations were epithelium-, COX-2- and EP2-receptor-dependent and accompanied by drastically enhanced mRNA levels of kinin receptors, as well as inflammatory mediators MCP-1 and iNOS. Increase in COX-2 and mPGES-1 was verified both at mRNA and protein levels. Nicotine selectively suppressed the organ-culture-enhanced relaxations induced by des-Arg{sup 9}-bradykinin and bradykinin, at the same time reducing mPGES-1 mRNA and protein expressions. α7-nicotinic acetylcholine receptor inhibitors α-bungarotoxin and MG624 both blocked the nicotine effects on kinin B{sub 2} receptors, but not those on B{sub 1}. Dexamethasone completely abolished kinin-induced relaxations. Conclusion: It is tempting to conclude that a local inflammatory process per se could have a bronchoprotective component by increasing COX-2 mediated airway relaxations and that nicotine could impede this safety mechanism. Dexamethasone further reduced airway inflammation together with relaxations. This might contribute to the steroid resistance seen in

  6. Histamine H4 receptor mediates eosinophil chemotaxis with cell shape change and adhesion molecule upregulation

    PubMed Central

    Ling, Ping; Ngo, Karen; Nguyen, Steven; Thurmond, Robin L; Edwards, James P; Karlsson, Lars; Fung-Leung, Wai-Ping

    2004-01-01

    During mast cell degranulation, histamine is released in large quantities. Human eosinophils were found to express histamine H4 but not H3 receptors. The possible effects of histamine on eosinophils and the receptor mediating these effects were investigated in our studies. Histamine (0.01–30 μM) induced a rapid and transient cell shape change in human eosinophils, but had no effects on neutrophils. The maximal shape change was at 0.3 μM histamine with EC50 at 19 nM. After 60 min incubation with 1 μM histamine, eosinophils were desensitized and were refractory to shape change response upon histamine restimulation. Histamine (0.01–1 μM) also enhanced the eosinophil shape change induced by other chemokines. Histamine-induced eosinophil shape change was mediated by the H4 receptor. This effect was completely inhibited by H4 receptor-specific antagonist JNJ 7777120 (IC50 0.3 μM) and H3/H4 receptor antagonist thioperamide (IC50 1.4 μM), but not by selective H1, H2 or H3 receptor antagonists. H4 receptor agonists imetit (EC50 25 nM) and clobenpropit (EC50 72 nM) could mimic histamine effect in inducing eosinophil shape change. Histamine (0.01–100 μM) induced upregulation of adhesion molecules CD11b/CD18 (Mac-1) and CD54 (ICAM-1) on eosinophils. This effect was mediated by the H4 receptor and could be blocked by H4 receptor antagonists JNJ 7777120 and thioperamide. Histamine (0.01–10 μM) induced eosinophil chemotaxis with an EC50 of 83 nM. This effect was mediated by the H4 receptor and could be blocked by H4 receptor antagonists JNJ 7777120 (IC50 86 nM) and thioperamide (IC50 519 nM). Histamine (0.5 μM) also enhanced the eosinophil shape change induced by other chemokines. In conclusion, we have demonstrated a new mechanism of eosinophil recruitment driven by mast cells via the release of histamine. Using specific histamine receptor ligands, we have provided a definitive proof that the H4 receptor mediates eosinophil chemotaxis, cell shape change and

  7. The Hha-TomB Toxin-Antitoxin System Shows Conditional Toxicity and Promotes Persister Cell Formation by Inhibiting Apoptosis-Like Death in S. Typhimurium

    PubMed Central

    Jaiswal, Sangeeta; Paul, Prajita; Padhi, Chandrashekhar; Ray, Shilpa; Ryan, Daniel; Dash, Shantoshini; Suar, Mrutyunjay

    2016-01-01

    Toxin-antitoxin (TA) modules are two component “addictive” genetic elements found on either plasmid or bacterial chromosome, sometimes on both. TA systems perform a wide range of functions like biofilm formation, persistence, programmed cell death, phage abortive infection etc. Salmonella has been reported to contain several such TA systems. However, the hemolysin expression modulating protein (Hha) and its adjacent uncharacterized hypothetical protein TomB (previously known as YbaJ), have not been listed as a TA module in Salmonella. In this study we established that Hha and TomB form a bonafide TA system where Hha serves as a toxin while TomB functions as an antitoxin. Interestingly, the toxicity of Hha was conditional causing cell death under acid stress. The antitoxin attenuated the toxicity of Hha by forming a TA complex through stable interactions. The Hha-TomB TA system was found to increase persistence and inhibit programmed cell death under antibiotic stress where a phenotypically diverse population expressing differential level of TA components was observed. Therefore we propose that Hha and TomB prevent cells from committing suicide thereby promoting persister cell formation. PMID:27910884

  8. An artemisinin-mediated ROS evolving and dual protease light-up nanocapsule for real-time imaging of lysosomal tumor cell death.

    PubMed

    Huang, Liwei; Luo, Yingping; Sun, Xian; Ju, Huangxian; Tian, Jiangwei; Yu, Bo-Yang

    2017-06-15

    Lysosomes are critical organelles for cellular homeostasis and can be used as potential targets to kill tumor cells from inside. Many photo-therapeutic methods have been developed to overproduce reactive oxygen species (ROS) to trigger lysosomal membrane permeabilization (LMP)-associated cell death pathway. However, these technologies rely on extra irradiation to activate the photosensitizers, which limits the applications in treating deep seated tumors and widespread metastatic lesions. This work reports a multifunctional nanocapsule to achieve targeted lysosomal tumor cell death without irradiation and real-time monitoring of drug effect through encapsulating artemisinin and dual protease light-up nanoprobe in a folate-functionalized liposome. The nanocapsule can be specifically uptaken by tumor cells via folate receptor-mediated endocytosis to enter lysosomes, in which artemisinin reacts with ferrous to generate ROS for LMP-associated cell death. By virtue of confocal fluorescence imaging, the artemisinin location in lysosome, ROS-triggered LMP and ultimate cell apoptosis can be visualized with the cathepsin B and caspase-3 activatable nanoprobe. Notably, the artemisinin-mediated ROS evolving for tumor therapy and real-time therapeutic monitoring were successfully implemented by living imaging in tumor-bearing mice, which broaden the nanocapsule for in vivo theranostics and may offer new opportunities for precise medicine.

  9. Tetrandrine induces cell death in SAS human oral cancer cells through caspase activation-dependent apoptosis and LC3-I and LC3-II activation-dependent autophagy.

    PubMed

    Huang, An-Cheng; Lien, Jin-Cherng; Lin, Meng-Wei; Yang, Jai-Sing; Wu, Ping-Ping; Chang, Shu-Jen; Lai, Tung-Yuan

    2013-08-01

    Numerous studies have demonstrated that autophagy is associated with cancer development. Thus, agents to induce autophagy could be employed in some cases for the treatment of cancer. Our results showed that tetrandrine significantly decreased the viability of SAS cells in a concentration- and time-dependent manner. Tetrandrine induced nuclear condensation, demonstrated by DAPI staining. The early events in apoptosis analysed by Annexin V/PI staining indicated that the percentage of cells staining positive for Annexin V was slightly increased in SAS cells with tetrandrine treatment but was much lower following bafilomycin A1 pre-treatment. Tetrandrine caused AVO and MDC induction in SAS cells in a concentration-dependent manner by fluorescence microscopy. Tetrandrine also caused LC-3 expression in SAS cells in a time-dependent manner. Our results show that tetrandrine treatment induced the levels of cleaved caspase-3 in a concentration- and time-dependent manner. Tetrandrine treatment induced the levels of LC-3 II, Atg-5, beclin-1, p-S6, p-ULK, p-mTOR, p-Akt (S473) and raptor. Tetrandrine decreased cell viability, but bafilomycin A1, 3-MA, chloroquine and NAC protected tetrandrine-treated SAS cells against decrease of cell viability. Atg-5, beclin-1 siRNA decreased tetrandrine-induced cleaved caspase-3 and cleaved PARP in SAS cells and protected tetrandrine-treated SAS cells against decrease in cell viability. Chloroquine, NAC and bafilomycin A1 also decreased tetrandrine-induced cleaved caspase-3 and cleaved PARP in SAS cells. Our results indicate the tetrandrine induces apoptosis and autophagy of SAS human cancer cells via caspase-dependent and LC3-I and LC3-II‑dependent pathways.

  10. H2 receptor-mediated facilitation and H3 receptor-mediated inhibition of noradrenaline release in the guinea-pig brain.

    PubMed

    Timm, J; Marr, I; Werthwein, S; Elz, S; Schunack, W; Schlicker, E

    1998-03-01

    , hippocampal or hypothalamic slices were used instead of cortical slices. The Ca2+-induced tritium overflow in guinea-pig cortex slices was inhibited by histamine (in the presence of ranitidine); this effect was abolished by clobenpropit. In slices superfused in the presence of clobenpropit, impromidine failed to facilitate the Ca2+-evoked tritium overflow. The electrically evoked tritium overflow in mouse brain cortex slices was inhibited by histamine by about 60% (both in the absence or presence of ranitidine). The inhibitory effect of histamine was abolished (but not reversed) by clobenpropit. In conclusion, noradrenaline release in the guinea-pig brain cortex is inhibited via presynaptic H3 receptors and facilitated via H2 receptors not located presynaptically. In the mouse brain cortex, only inhibitory H3 receptors occur. The extent of the H3 receptor-mediated effect is more marked in the mouse than in the guinea-pig brain cortex.

  11. A Boolean Network Model of Nuclear Receptor Mediated Cell Cycle Progression

    EPA Science Inventory

    Nuclear receptors (NRs) are ligand-activated transcription factors that regulate a broad range of cellular processes. Hormones, lipids and xenobiotics have been shown to activate NRs with a range of consequences on development, metabolism, oxidative stress, apoptosis, and prolif...

  12. A Boolean Network Model of Nuclear Receptor Mediated Cell Cycle Progression (S)

    EPA Science Inventory

    Nuclear receptors (NRs) are ligand-activated transcription factors that regulate a broad range of cellular processes. Hormones, lipids and xenobiotics have been shown to activate NRs with a range of consequences on development, metabolism, oxidative stress, apoptosis, and prolif...

  13. Lactate modulates the activity of primary cortical neurons through a receptor-mediated pathway.

    PubMed

    Bozzo, Luigi; Puyal, Julien; Chatton, Jean-Yves

    2013-01-01

    Lactate is increasingly described as an energy substrate of the brain. Beside this still debated metabolic role, lactate may have other effects on brain cells. Here, we describe lactate as a neuromodulator, able to influence the activity of cortical neurons. Neuronal excitability of mouse primary neurons was monitored by calcium imaging. When applied in conjunction with glucose, lactate induced a decrease in the spontaneous calcium spiking frequency of neurons. The effect was reversible and concentration dependent (IC50 ∼4.2 mM). To test whether lactate effects are dependent on energy metabolism, we applied the closely related substrate pyruvate (5 mM) or switched to different glucose concentrations (0.5 or 10 mM). None of these conditions reproduced the effect of lactate. Recently, a Gi protein-coupled receptor for lactate called HCA1 has been introduced. To test if this receptor is implicated in the observed lactate sensitivity, we incubated cells with pertussis toxin (PTX) an inhibitor of Gi-protein. PTX prevented the decrease of neuronal activity by L-lactate. Moreover 3,5-dyhydroxybenzoic acid, a specific agonist of the HCA1 receptor, mimicked the action of lactate. This study indicates that lactate operates a negative feedback on neuronal activity by a receptor-mediated mechanism, independent from its intracellular metabolism.

  14. Lactate Modulates the Activity of Primary Cortical Neurons through a Receptor-Mediated Pathway

    PubMed Central

    Bozzo, Luigi; Puyal, Julien; Chatton, Jean-Yves

    2013-01-01

    Lactate is increasingly described as an energy substrate of the brain. Beside this still debated metabolic role, lactate may have other effects on brain cells. Here, we describe lactate as a neuromodulator, able to influence the activity of cortical neurons. Neuronal excitability of mouse primary neurons was monitored by calcium imaging. When applied in conjunction with glucose, lactate induced a decrease in the spontaneous calcium spiking frequency of neurons. The effect was reversible and concentration dependent (IC50 ∼4.2 mM). To test whether lactate effects are dependent on energy metabolism, we applied the closely related substrate pyruvate (5 mM) or switched to different glucose concentrations (0.5 or 10 mM). None of these conditions reproduced the effect of lactate. Recently, a Gi protein-coupled receptor for lactate called HCA1 has been introduced. To test if this receptor is implicated in the observed lactate sensitivity, we incubated cells with pertussis toxin (PTX) an inhibitor of Gi-protein. PTX prevented the decrease of neuronal activity by L-lactate. Moreover 3,5-dyhydroxybenzoic acid, a specific agonist of the HCA1 receptor, mimicked the action of lactate. This study indicates that lactate operates a negative feedback on neuronal activity by a receptor-mediated mechanism, independent from its intracellular metabolism. PMID:23951229

  15. Substance P selectively modulates GABA(A) receptor-mediated synaptic transmission in striatal cholinergic interneurons.

    PubMed

    Govindaiah, G; Wang, Yanyan; Cox, Charles L

    2010-02-01

    Substance P (SP) is co-localized and co-released with gamma-amino butyric acid (GABA) from approximately 50% of GABAergic medium spiny neurons (MSNs) in the striatum. MSNs innervate several cellular targets including neighboring MSNs and cholinergic interneurons via collaterals. However, the functional role of SP release onto striatal interneurons is unknown. Here we examined SP-mediated actions on inhibitory synaptic transmission in cholinergic interneurons using whole-cell recordings in mouse corticostriatal slices. We found that SP selectively suppressed GABA(A) receptor-mediated inhibitory post-synaptic currents (IPSCs), but not excitatory post-synaptic currents (EPSCs) in cholinergic interneurons. In contrast, SP did not alter IPSCs in fast-spiking interneurons and MSNs. SP suppressed IPSC amplitude in a concentration-dependent and reversible manner, and the NK1 receptor antagonist RP67580 attenuated the SP-mediated suppression. In addition, RP67580 alone enhanced the evoked IPSC amplitude in cholinergic interneurons, suggesting an endogenous action of SP on regulation of inhibitory synaptic transmission. SP did not alter the paired-pulse ratio, but reduced the amplitudes of GABA(A) agonist muscimol-induced outward currents and miniature IPSCs in cholinergic interneurons, suggesting SP exerts its effects primarily at the post-synaptic site. Our results indicate that the physiological effects of SP are to enhance the activity of striatal cholinergic interneurons and provide a rationale for designing potential new antiparkinsonian agents.

  16. Hormone stimulation of androgen receptor mediates dynamic changes in DNA methylation patterns at regulatory elements

    PubMed Central

    Dhiman, Vineet K.; Attwood, Kristopher; Campbell, Moray J.; Smiraglia, Dominic J.

    2015-01-01

    DNA methylation is an epigenetic modification that contributes to stable gene silencing by interfering with the ability of transcriptional regulators to bind to DNA. Recent findings have revealed that hormone stimulation of certain nuclear receptors induces rapid, dynamic changes in DNA methylation patterns alongside transcriptional responses at a subset of target loci, over time. However, the ability of androgen receptor (AR) to dynamically regulate gene transcription is relatively under-studied and its role in the regulation of DNA methylation patterns remains to be elucidated. Here we demonstrate in normal prostate cells that hormone stimulated AR activity results in dynamic changes in the transcription rate and DNA methylation patterns at the AR target genes, TIPARP and SGK1. Time-resolved chromatin immunoprecipitation experiments on the SGK1 locus reveals dynamic recruitment of AR and RNA Polymerase II, as well as the recruitment of proteins involved in the DNA demethylation process, TET1 and TDG. Furthermore, the presence of DNA methylation at dynamic regions inhibits protein binding and transcriptional activity of SGK1. These findings establish AR activity as a contributing factor to the dynamic regulation of DNA methylation patterns at target genes in prostate biology and infer further complexity involved in nuclear receptor mediation of transcriptional regulation. PMID:26646795

  17. Dual effects of anandamide on NMDA receptor-mediated responses and neurotransmission.

    PubMed

    Hampson, A J; Bornheim, L M; Scanziani, M; Yost, C S; Gray, A T; Hansen, B M; Leonoudakis, D J; Bickler, P E

    1998-02-01

    Anandamide is an endogenous ligand of cannabinoid receptors that induces pharmacological responses in animals similar to those of cannabinoids such as delta9-tetrahydrocannabinol (THC). Typical pharmacological effects of cannabinoids include disruption of pain, memory formation, and motor coordination, systems that all depend on NMDA receptor mediated neurotransmission. We investigated whether anandamide can influence NMDA receptor activity by examining NMDA-induced calcium flux (deltaCa2+NMDA) in rat brain slices. The presence of anandamide reduced deltaCa2+NMDA and the inhibition was disrupted by cannabinoid receptor antagonist, pertussis toxin treatment, and agatoxin (a calcium channel inhibitor). Whereas these treatments prevented anandamide inhibiting deltaCa2+NMDA, they also revealed another, underlying mechanism by which anandamide influences deltaCa2+NMDA. In the presence of cannabinoid receptor antagonist, anandamide potentiated deltaCa2+NMDA in cortical, cerebellar, and hippocampal slices. Anandamide (but not THC) also augmented NMDA-stimulated currents in Xenopus oocytes expressing cloned NMDA receptors, suggesting a capacity to directly modulate NMDA receptor activity. In a similar manner, anandamide enhanced neurotransmission across NMDA receptor-dependent synapses in hippocampus in a manner that was not mimicked by THC and was unaffected by cannabinoid receptor antagonist. These data demonstrate that anandamide can modulate NMDA receptor activity in addition to its role as a cannabinoid receptor ligand.

  18. Spinal GABA receptors mediate the suppressive effect of electroacupuncture on cold allodynia in rats.

    PubMed

    Park, Jung-Hyun; Han, Jae-Bok; Kim, Sun-Kwang; Park, Jung-Hyuk; Go, Dong-Hyun; Sun, Boram; Min, Byung-Il

    2010-03-31

    This study was performed to determine whether spinal GABAergic systems mediate the relieving effects of low frequency electroacupuncture (EA) on cold allodynia in a rat tail model of neuropathic pain. For neuropathic surgery, the right superior caudal trunk was resected at the level between the S1 and S2 spinal nerves innervating the tail. Two weeks after the nerve injury, the intrathecal catheter was implanted. Five days after the catheterization, rats were intrathecally injected with gabazine (GABA(A) receptor antagonist, 0.0003, 0.001 or 0.003mug), or saclofen (GABA(B) receptor antagonist, 3, 10 or 30mug). Ten minutes after the injection, EA (2Hz) was applied to the ST36 acupoint for 30min. The cold allodynia was assessed by the tail immersion test (i.e. immersing the tail in cold (4 degrees C) water and measuring the latency of an abrupt tail movement) before and after the EA treatment. EA stimulation at ST36 significantly inhibited the cold allodynia sign, whereas EA at non-acupoint and plain acupuncture at ST36 (without electrical stimulation) did not show antiallodynic effects. Intrathecal administration of gabazine or saclofen blocked the relieving effects of ST36 EA stimulation on cold allodynia. These results suggest that spinal GABA(A) and GABA(B) receptors mediate the suppressive effect of low frequency EA on cold allodynia in the tail neuropathic rats.

  19. Cloned M1 muscarinic receptors mediate both adenylate cyclase inhibition and phosphoinositide turnover.

    PubMed Central

    Stein, R; Pinkas-Kramarski, R; Sokolovsky, M

    1988-01-01

    The rat M1 muscarinic receptor gene was cloned and expressed in a rat cell line lacking endogenous muscarinic receptors. Assignment of the cloned receptors to the M1 class was pharmacologically confirmed by their high affinity for the M1-selective muscarinic antagonist pirenzepine and low affinity for the M2-selective antagonist AF-DX-116. Guanylyl imidodiphosphate [Gpp(NH)p] converted agonist binding sites on the receptor, from high-affinity to the low-affinity state, thus indicating that the cloned receptors couple to endogenous G-proteins. The cloned receptors mediated both adenylate cyclase inhibition and phosphoinositide hydrolysis, but by different mechanisms. Pertussis toxin blocked the inhibition of adenylate cyclase (indicating coupling of the receptor to inhibitory G-protein), but did not affect phosphoinositide turnover. Furthermore, the stimulation of phosphoinositide hydrolysis was less efficient than the inhibition of adenylate cyclase. These findings demonstrate that cloned M1 receptors are capable of mediating multiple responses in the cell by coupling to different effectors, possibly to different G-proteins. Images PMID:2846274

  20. M2-like macrophages are responsible for collagen degradation through a mannose receptor-mediated pathway.

    PubMed

    Madsen, Daniel H; Leonard, Daniel; Masedunskas, Andrius; Moyer, Amanda; Jürgensen, Henrik Jessen; Peters, Diane E; Amornphimoltham, Panomwat; Selvaraj, Arul; Yamada, Susan S; Brenner, David A; Burgdorf, Sven; Engelholm, Lars H; Behrendt, Niels; Holmbeck, Kenn; Weigert, Roberto; Bugge, Thomas H

    2013-09-16

    Tissue remodeling processes critically depend on the timely removal and remodeling of preexisting collagen scaffolds. Nevertheless, many aspects related to the turnover of this abundant extracellular matrix component in vivo are still incompletely understood. We therefore took advantage of recent advances in optical imaging to develop an assay to visualize collagen turnover in situ and identify cell types and molecules involved in this process. Collagen introduced into the dermis of mice underwent cellular endocytosis in a partially matrix metalloproteinase-dependent manner and was subsequently routed to lysosomes for complete degradation. Collagen uptake was predominantly executed by a quantitatively minor population of M2-like macrophages, whereas more abundant Col1a1-expressing fibroblasts and Cx3cr1-expressing macrophages internalized collagen at lower levels. Genetic ablation of the collagen receptors mannose receptor (Mrc1) and urokinase plasminogen activator receptor-associated protein (Endo180 and Mrc2) impaired this intracellular collagen degradation pathway. This study demonstrates the importance of receptor-mediated cellular uptake to collagen turnover in vivo and identifies a key role of M2-like macrophages in this process.

  1. Muscarinic receptor-mediated inositol tetrakisphosphate response in bovine adrenal chromaffin cells

    SciTech Connect

    Sanborn, B.B.; Schneider, A.S. )

    1990-01-01

    Inositol trisphosphate (IP{sub 3}), a product of the phosphoinositide cycle, mobilizes intracellular Ca{sup 2+} in many cell types. New evidence suggests that inositol tetrakisphosphate (IP{sub 4}), an IP{sub 3} derivative, may act as another second messenger to further alter calcium homeostasis. However, the function and mechanism of action of IP{sub 4} are presently unresolved. We now report evidence of muscarinic receptor-mediated accumulation of IP{sub 4} in bovine adrenal chromaffin cells, a classic neurosecretory system in which calcium movements have been well studied. Muscarine stimulated an increase in ({sup 3}H)IP{sub 4} and ({sup 3}H)IP{sub 3} accumulation in chromaffin cells and this effect was completely blocked by atropine. ({sup 3}H)IP{sub 4} accumulation was detectable within 15 sec, increased to a maximum by 30 sec and thereafter declined. 2,3-diphosphoglycerate, an inhibitor of IP{sub 3} and IP{sub 4} hydrolysis, enhanced accumulation of these inositol polyphosphates. The results provide the first evidence of a rapid inositol tetrakisphosphate response in adrenal chromaffin cells, which should facilitate the future resolution of the relationship between IP{sub 4} and calcium homeostasis.

  2. Accumbens shell AMPA receptors mediate expression of extinguished reward seeking through interactions with basolateral amygdala.

    PubMed

    Millan, E Zayra; McNally, Gavan P

    2011-07-01

    Extinction is the reduction in drug seeking when the contingency between drug seeking behavior and the delivery of drug reward is broken. Here, we investigated a role for the nucleus accumbens shell (AcbSh). Rats were trained to respond for 4% (v/v) alcoholic beer in one context (Context A) followed by extinction in a second context (Context B). Rats were subsequently tested in the training context, A (ABA), or the extinction context, B (ABB). Pre-test injections of the glutamate AMPA receptor antagonist, NBQX (1 µg) into AcbSh had no effect on renewal of alcoholic beer seeking when rats were returned to the training context (ABA). However, NBQX increased responding when rats were tested in the extinction context (ABB). In a second experiment, rats received training, extinction, and test in the same context. Pre-test injections of NBQX (0, 0.3, and 1 µg) into the AcbSh dose-dependently attenuated expression of extinction. We also found that NBQX in the AcbSh had no effect on initial acquisition of extinction or the motivation to respond for reward as measured by break point on a progressive ratio schedule. Finally, we show that pharmacological disconnection of a basolateral amygdala (BLA) → AcbSh pathway via NBQX in AcbSh combined with reversible inactivation of the contralateral BLA attenuates expression of extinction. Together, these results suggest that AcbSh AMPA receptors mediate expression of extinguished reward seeking through glutamatergic inputs from the BLA.

  3. Optimization of stress response through the nuclear receptor-mediated cortisol signalling network

    PubMed Central

    Kolodkin, Alexey; Sahin, Nilgun; Phillips, Anna; Hood, Steve R.; Bruggeman, Frank J.; Westerhoff, Hans V.; Plant, Nick

    2013-01-01

    It is an accepted paradigm that extended stress predisposes an individual to pathophysiology. However, the biological adaptations to minimize this risk are poorly understood. Using a computational model based upon realistic kinetic parameters we are able to reproduce the interaction of the stress hormone cortisol with its two nuclear receptors, the high-affinity glucocorticoid receptor and the low-affinity pregnane X-receptor. We demonstrate that regulatory signals between these two nuclear receptors are necessary to optimize the body’s response to stress episodes, attenuating both the magnitude and duration of the biological response. In addition, we predict that the activation of pregnane X-receptor by multiple, low-affinity endobiotic ligands is necessary for the significant pregnane X-receptor-mediated transcriptional response observed following stress episodes. This integration allows responses mediated through both the high and low-affinity nuclear receptors, which we predict is an important strategy to minimize the risk of disease from chronic stress. PMID:23653204

  4. A new Kupffer cell receptor mediating plasma clearance of carcinoembryonic antigen by the rat.

    PubMed Central

    Toth, C A; Thomas, P; Broitman, S A; Zamcheck, N

    1982-01-01

    Native human carcinoembryonic antigen is rapidly removed from the circulation by the rat liver Kupffer cell after intravenous injection. The molecule is subsequently transferred to the hepatocyte in an immunologically identifiable form. Carcinoembryonic antigen has a circulatory half-life of 3.7 (+/- 0.8) min, and cellular entry is by receptor-mediated endocytosis. Non-specific fluid pinocytosis and phagocytosis can be excluded as possible mechanisms by the kinetics of clearance and failure of colloidal carbon to inhibit uptake. Substances with known affinity for the hepatic receptors for mannose, N-acetylglucosamine, fucose and galactose all fail to inhibit carcinoembryonic antigen clearance. After two cycles of the Smith degradation, carcinoembryonic antigen is still able to inhibit clearance of the native molecule. Receptor specificity is apparently not dependent on those non-reducing terminal sugars of the native molecule. Performic acid-oxidized carcinoembryonic antigen also inhibits clearance of carcinoembryonic antigen in vivo. Receptor binding is not dependent on tertiary protein conformation. Non-specific cross-reacting antigen, a glycoprotein structurally similar to carcinoembryonic antigen, is cleared by the same mechanism. PMID:6896821

  5. A new Kupffer cell receptor mediating plasma clearance of carcinoembryonic antigen by the rat.

    PubMed

    Toth, C A; Thomas, P; Broitman, S A; Zamcheck, N

    1982-05-15

    Native human carcinoembryonic antigen is rapidly removed from the circulation by the rat liver Kupffer cell after intravenous injection. The molecule is subsequently transferred to the hepatocyte in an immunologically identifiable form. Carcinoembryonic antigen has a circulatory half-life of 3.7 (+/- 0.8) min, and cellular entry is by receptor-mediated endocytosis. Non-specific fluid pinocytosis and phagocytosis can be excluded as possible mechanisms by the kinetics of clearance and failure of colloidal carbon to inhibit uptake. Substances with known affinity for the hepatic receptors for mannose, N-acetylglucosamine, fucose and galactose all fail to inhibit carcinoembryonic antigen clearance. After two cycles of the Smith degradation, carcinoembryonic antigen is still able to inhibit clearance of the native molecule. Receptor specificity is apparently not dependent on those non-reducing terminal sugars of the native molecule. Performic acid-oxidized carcinoembryonic antigen also inhibits clearance of carcinoembryonic antigen in vivo. Receptor binding is not dependent on tertiary protein conformation. Non-specific cross-reacting antigen, a glycoprotein structurally similar to carcinoembryonic antigen, is cleared by the same mechanism.

  6. Different receptors mediate the electrophysiological and growth cone responses of an identified neuron to applied dopamine.

    PubMed

    Dobson, K S; Dmetrichuk, J M; Spencer, G E

    2006-09-15

    Neurotransmitters are among the many cues that may guide developing axons toward appropriate targets in the developing nervous system. We have previously shown in the mollusk Lymnaea stagnalis that dopamine, released from an identified pre-synaptic cell, differentially affects growth cone behavior of its target and non-target cells in vitro. Here, we describe a group of non-target cells that also produce an inhibitory electrophysiological response to applied dopamine. We first determined, using pharmacological blockers, which receptors mediate this physiological response. We demonstrated that the dopaminergic electrophysiological responses of non-target cells were sensitive to a D2 receptor antagonist, as are known target cell responses. However, the non-target cell receptors were linked to different G-proteins and intracellular signaling pathways than the target cell receptors. Despite the presence of a D2-like receptor at the soma, the growth cone collapse of these non-target cells was mediated by D1-like receptors. This study shows that different dopamine receptor sub-types mediated the inhibitory physiological and growth cone responses of an identified cell type. We therefore not only provide further evidence that D2- and D1-like receptors can be present on the same neuron in invertebrates, but also show that these receptors are likely involved in very different cellular functions.

  7. LRP6 protein regulates low density lipoprotein (LDL) receptor-mediated LDL uptake.

    PubMed

    Ye, Zhi-jia; Go, Gwang-Woong; Singh, Rajvir; Liu, Wenzhong; Keramati, Ali Reza; Mani, Arya

    2012-01-06

    Genetic variations in LRP6 gene are associated with high serum LDL cholesterol levels. We have previously shown that LDL clearance in peripheral B-lymphocytes of the LRP6(R611C) mutation carriers is significantly impaired. In this study we have examined the role of wild type LRP6 (LRP6(WT)) and LRP6(R611C) in LDL receptor (LDLR)-mediated LDL uptake. LDL binding and uptake were increased when LRP6(WT) was overexpressed and modestly reduced when it was knocked down in LDLR-deficient CHO (ldlA7) cells. These findings implicated LRP6 in LDLR-independent cellular LDL binding and uptake. However, LRP6 knockdown in wild type CHO cells resulted in a much greater decline in LDL binding and uptake compared with CHO-ldlA7 cells, suggesting impaired function of the LDLR. LDLR internalization was severely diminished when LRP6 was knocked down and was restored after LRP6 was reintroduced. Further analysis revealed that LRP6(WT) forms a complex with LDLR, clathrin, and ARH and undergoes a clathrin-mediated internalization after stimulation with LDL. LDLR and LRP6 internalizations as well as LDL uptake were all impaired in CHO-k1 cells expressing LRP6(R611C). These studies identify LRP6 as a critical modulator of receptor-mediated LDL endocytosis and introduce a mechanism by which variation in LRP6 may contribute to high serum LDL levels.

  8. S-nitrosylated SHP-2 contributes to NMDA receptor-mediated excitotoxicity in acute ischemic stroke

    PubMed Central

    Shi, Zhong-Qing; Sunico, Carmen R.; McKercher, Scott R.; Cui, Jiankun; Feng, Gen-Sheng; Nakamura, Tomohiro; Lipton, Stuart A.

    2013-01-01

    Overproduction of nitric oxide (NO) can cause neuronal damage, contributing to the pathogenesis of several neurodegenerative diseases and stroke (i.e., focal cerebral ischemia). NO can mediate neurotoxic effects at least in part via protein S-nitrosylation, a reaction that covalently attaches NO to a cysteine thiol (or thiolate anion) to form an S-nitrosothiol. Recently, the tyrosine phosphatase Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2) and its downstream pathways have emerged as important mediators of cell survival. Here we report that in neurons and brain tissue NO can S-nitrosylate SHP-2 at its active site cysteine, forming S-nitrosylated SHP-2 (SNO–SHP-2). We found that NMDA exposure in vitro and transient focal cerebral ischemia in vivo resulted in increased levels of SNO–SHP-2. S-Nitrosylation of SHP-2 inhibited its phosphatase activity, blocking downstream activation of the neuroprotective physiological ERK1/2 pathway, thus increasing susceptibility to NMDA receptor-mediated excitotoxicity. These findings suggest that formation of SNO–SHP-2 represents a key chemical reaction contributing to excitotoxic damage in stroke and potentially other neurological disorders. PMID:23382182

  9. MAGI-1 acts as a scaffolding molecule for NGF receptor-mediated signaling pathway.

    PubMed

    Ito, Hidenori; Morishita, Rika; Iwamoto, Ikuko; Mizuno, Makoto; Nagata, Koh-ichi

    2013-10-01

    We have recently found that the membrane-associated guanylate kinase with inverted organization-1 (MAGI-1) was enriched in rat nervous tissues such as the glomeruli in olfactory bulb of adult rats and dorsal root entry zone in spinal cord of embryonic rats. In addition, we revealed the localization of MAGI-1 in the growth cone of the primary cultured rat dorsal root ganglion cells. These results point out the possibility that MAGI-1 is involved in the regulation of neurite extension or guidance. In this study, we attempted to reveal the physiological role(s) of MAGI-1 in neurite extension. We found that RNA interference (RNAi)-mediated knockdown of MAGI-1 caused inhibition of nerve growth factor (NGF)-induced neurite outgrowth in PC12 rat pheochromocytoma cells. To clarify the involvement of MAGI-1 in NGF-mediated signal pathway, we tried to identify binding partners for MAGI-1 and identified p75 neurotrophin receptor (p75NTR), a low affinity NGF receptor, and Shc, a phosphotyrosine-binding adaptor. These three proteins formed an immunocomplex in PC12 cells. Knockdown as well as overexpression of MAGI-1 caused suppression of NGF-stimulated activation of the Shc-ERK pathway, which is supposed to play important roles in neurite outgrowth of PC12 cells. These results indicate that MAGI-1 may act as a scaffolding molecule for NGF receptor-mediated signaling pathway.

  10. The effect of vanadate on receptor-mediated endocytosis of asialoorosomucoid in rat liver parenchymal cells

    SciTech Connect

    Kindberg, G.M.; Gudmundsen, O.; Berg, T. )

    1990-06-05

    Vanadate is a phosphate analogue that inhibits enzymes involved in phosphate release and transfer reactions. Since such reactions may play important roles in endocytosis, we studied the effects of vanadate on various steps in receptor-mediated endocytosis of asialoorosomucoid labeled with 125I-tyramine-cellobiose (125I-TC-AOM). The labeled degradation products formed from 125I-TC-AOM are trapped in the lysosomes and may therefore serve as lysosomal markers in subcellular fractionation studies. Vanadate reduced the amount of active surface asialoglycoprotein receptors approximately 70%, but had no effect on the rate of internalization and retroendocytosis of ligand. The amount of surface asialoglycoprotein receptors can be reduced by lowering the incubation temperature gradually from 37 to 15 degrees C; vanadate affected only the temperature--sensitive receptors. Vanadate inhibited degradation of 125I-TC-AOM 70-80%. Degradation was much more sensitive to vanadate than binding; half-maximal effects were seen at approximately 1 mM vanadate for binding and approximately 0.1 mM vanadate for degradation. By subcellular fractionation in sucrose and Nycodenz gradients, it was shown that vanadate completely prevented the transfer of 125I-TC-AOM from endosomes to lysosomes. Therefore, the inhibition of degradation by vanadate was indirect; in the presence of vanadate, ligand did not gain access to the lysosomes. The limited degradation in the presence of vanadate took place in a prelysosomal compartment. Vanadate did not affect cell viability and ATP content.

  11. Receptor-mediated uptake and transport of macromolecules in the human placenta.

    PubMed

    Schneider, Henning; Miller, Richard K

    2010-01-01

    The human placenta is required to be the anchor, the conduit and the controller during pregnancy. The survival of the baby and its associated placenta is dependent upon the placenta shielding the embryo/fetus from harm, e.g., autoimmune disease - thrombophilia, antiphospholipid syndrome or infections, while simultaneously providing for the passage of critical nutrients (e.g., amino acids, vitamins) and beneficial immunoglobulins. In a number of instances, the movements of macromolecules into and through the placenta can result in the passage of the intact molecules into the fetal circulation or in the case of proteins - catabolism to amino acids which are utilized by the placenta and the fetus for continued growth and development. The transfer of two such macromolecules, immunoglobulin G (IgG) and vitamin B12 (cyanocobalamin or B12), are examined as to the unique receptor-mediated transfer capability of the human placenta, its transfer specificity as related to specific receptors and the role of endogeneous placental proteins (trancobalamins) in facilitating the recognition and transport of specifically B12. Brief comparisons will be made to other animal species and the differences in specific organ transfer capabilities.

  12. Cryptococcus neoformans is internalized by receptor-mediated or 'triggered' phagocytosis, dependent on actin recruitment.

    PubMed

    Guerra, Caroline Rezende; Seabra, Sergio Henrique; de Souza, Wanderley; Rozental, Sonia

    2014-01-01

    Cryptococcosis by the encapsulated yeast Cryptococcus neoformans affects mostly immunocompromised individuals and is a frequent neurological complication in AIDS patients. Recent studies support the idea that intracellular survival of Cryptococcus yeast cells is important for the pathogenesis of cryptococcosis. However, the initial steps of Cryptococcus internalization by host cells remain poorly understood. Here, we investigate the mechanism of Cryptococcus neoformans phagocytosis by peritoneal macrophages using confocal and electron microscopy techniques, as well as flow cytometry quantification, evaluating the importance of fungal capsule production and of host cell cytoskeletal elements for fungal phagocytosis. Electron microscopy analyses revealed that capsular and acapsular strains of C. neoformans are internalized by macrophages via both 'zipper' (receptor-mediated) and 'trigger' (membrane ruffle-dependent) phagocytosis mechanisms. Actin filaments surrounded phagosomes of capsular and acapsular yeasts, and the actin depolymerizing drugs cytochalasin D and latrunculin B inhibited yeast internalization and actin recruitment to the phagosome area. In contrast, nocodazole and paclitaxel, inhibitors of microtubule dynamics decreased internalization but did not prevent actin recruitment to the site of phagocytosis. Our results show that different uptake mechanisms, dependent on both actin and tubulin dynamics occur during yeast internalization by macrophages, and that capsule production does not affect the mode of Cryptococcus uptake by host cells.

  13. Cellular mechanisms of the 5-HT7 receptor-mediated signaling

    PubMed Central

    Guseva, Daria; Wirth, Alexander; Ponimaskin, Evgeni

    2014-01-01

    Serotonin (5-hydroxytryptamine or 5-HT) is an important neurotransmitter regulating a wide range of physiological and pathological functions via activation of heterogeneously expressed 5-HT receptors. The 5-HT7 receptor is one of the most recently described members of the 5-HT receptor family. Functionally, 5-HT7 receptor is associated with a number of physiological and pathological responses, including serotonin-induced phase shifting of the circadian rhythm, control of memory as well as locomotor and exploratory activity. A large body of evidence indicates involvement of the 5-HT7 receptor in anxiety and depression, and recent studies suggest that 5-HT7 receptor can be highly relevant for the treatment of major depressive disorders. The 5-HT7 receptor is coupled to the stimulatory Gs-protein, and receptor stimulation results in activation of adenylyl cyclase (AC) leading to a rise of cAMP concentration. In addition, this receptor is coupled to the G12-protein to activate small GTPases of the Rho family. This review focuses on molecular mechanisms responsible for the 5-HT7 receptor-mediated signaling. We provide detailed overview of signaling cascades controlled and regulated by the 5-HT7 receptor and discuss the functional impact of 5-HT7 receptor for the regulation of different cellular and subcellular processes. PMID:25324743

  14. Cellular mechanisms of the 5-HT7 receptor-mediated signaling.

    PubMed

    Guseva, Daria; Wirth, Alexander; Ponimaskin, Evgeni

    2014-01-01

    Serotonin (5-hydroxytryptamine or 5-HT) is an important neurotransmitter regulating a wide range of physiological and pathological functions via activation of heterogeneously expressed 5-HT receptors. The 5-HT7 receptor is one of the most recently described members of the 5-HT receptor family. Functionally, 5-HT7 receptor is associated with a number of physiological and pathological responses, including serotonin-induced phase shifting of the circadian rhythm, control of memory as well as locomotor and exploratory activity. A large body of evidence indicates involvement of the 5-HT7 receptor in anxiety and depression, and recent studies suggest that 5-HT7 receptor can be highly relevant for the treatment of major depressive disorders. The 5-HT7 receptor is coupled to the stimulatory Gs-protein, and receptor stimulation results in activation of adenylyl cyclase (AC) leading to a rise of cAMP concentration. In addition, this receptor is coupled to the G12-protein to activate small GTPases of the Rho family. This review focuses on molecular mechanisms responsible for the 5-HT7 receptor-mediated signaling. We provide detailed overview of signaling cascades controlled and regulated by the 5-HT7 receptor and discuss the functional impact of 5-HT7 receptor for the regulation of different cellular and subcellular processes.

  15. Killing of intracellular Mycobacterium tuberculosis by receptor-mediated drug delivery

    SciTech Connect

    Majumdar, S.; Basu, S.K. )

    1991-01-01

    p-Aminosalicylic acid (PAS) conjugated to maleylated bovine serum albumin (MBSA) was taken up efficiently through high-affinity MBSA-binding sites on macrophages. Binding of the radiolabeled conjugate to cultured mouse peritoneal macrophages at 4 degrees C was competed for by MBSA but not by PAS. At 37 degrees C, the radiolabeled conjugate was rapidly degraded by the macrophages, leading to release of acid-soluble degradation products in the medium. The drug conjugate was nearly 100 times as effective as free PAS in killing the intracellular mycobacteria in mouse peritoneal macrophages infected in culture with Mycobacterium tuberculosis. The killing of intracellular mycobacteria mediated by the drug conjugate was effectively prevented by simultaneous addition of excess MBSA (100 micrograms/ml) or chloroquine (3 microM) to the medium, whereas these agents did not affect the microbicidal action of free PAS. These results suggest that (i) uptake of the PAS-MBSA conjugate was mediated by cell surface receptors on macrophages which recognize MBSA and (ii) lysosomal hydrolysis of the internalized conjugate resulted in intracellular release of a pharmacologically active form of the drug, which led to selective killing of the M. tuberculosis harbored by mouse macrophages infected in culture. This receptor-mediated modality of delivering drugs to macrophages could contribute to greater therapeutic efficacy and minimization of toxic side effects in the management of tuberculosis and other intracellular mycobacterial infections.

  16. Apoptosis and the Airway Epithelium

    PubMed Central

    White, Steven R.

    2011-01-01

    The airway epithelium functions as a barrier and front line of host defense in the lung. Apoptosis or programmed cell death can be elicited in the epithelium as a response to viral infection, exposure to allergen or to environmental toxins, or to drugs. While apoptosis can be induced via activation of death receptors on the cell surface or by disruption of mitochondrial polarity, epithelial cells compared to inflammatory cells are more resistant to apoptotic stimuli. This paper focuses on the response of airway epithelium to apoptosis in the normal state, apoptosis as a potential regulator of the number and types of epithelial cells in the airway, and the contribution of epithelial cell apoptosis in important airways diseases. PMID:22203854

  17. Suppression of apoptosis by glucocorticoids in glomerular endothelial cells: effects on proapoptotic pathways

    PubMed Central

    Meßmer, Udo K; Winkel, Gundula; Briner, Verena A; Pfeilschifter, Josef

    2000-01-01

    Tumour necrosis factor-α (TNF-α)- and lipopolysaccharide (LPS)-induced apoptosis of bovine glomerular endothelial cells is now recognized as an important part in the pathogenesis of glomerulonephritis characterized by early mitochondrial cytochrome c release, mitochondrial permeability transition, Bak protein upregulation, Bcl-XL protein downregulation and caspase-3 activation.Co-treatment of cells with 10 nM dexamethasone and TNF-α or LPS blocked roughly 90% of apoptotic cell death in glomerular endothelial cells. The action of glucocorticoids could be documented in that they prevented all apoptotic markers such as DNA laddering, DNA fragmentation measured by the diphenylamine assay as well as morphological alterations.To mechanistically elucidate the action of glucocorticoids we evaluated whether glucocorticoids elicit a time-dependent effect. For dexamethasone, to maximally inhibit DNA fragmentation a preincubation period was not required. Even if dexamethasone was supplemented 6 h following TNF-α or LPS we observed a maximal inhibitory effect.Concerning its influence on TNF-α and LPS signal transduction, we found that dexamethasone only partially prevented cytochrome-c-release as a first sign of apoptotic cell death but efficiently blocked mitochondrial permeability transition. Moreover, TNF-α- and LPS-induced Bak upregulation, Bcl-XL-downregulation, and the activation of caspase-3-like proteases, measured fluorometrically using DEVD-AMC and PARP cleavage, were efficiently blocked by dexamethasone.We postulate that glucocorticoids exert their inhibitory action upstream of the terminal death pathways but downstream of primary receptor mediated signals by blocking pro-apoptotic signals pre- and/or post cytochrome c release and mitochondrial signalling. PMID:10780973

  18. Apoptosis-like (possible quantum thermodynamic) cell death induced by Yoshixol and wood oil of Chamaecyparis obtusa (Kiso-Hinoki) on HeLa cell.

    PubMed

    Koyama, S; Tanaka, S; Yamaguchi, Y; Motoyoshya, J

    1997-05-01

    1. We report on the cytotoxic effects of neutral wood oil extracted from Chamaecyparis obtusa (Kiso-Hinoki) and of the newly synthetized substance Yoshixol (4,4-dimethyl-6-methylene-2-cyclohexen-1-one) on cultured HeLa cells. 2. The neutral wood oil produced cell death, led to the formation of granules, which were connected with fibrous networks, and reduced cell size. 3. Yoshixol caused a separation of cells, granulation, formation of high-density materials (probably apoptotic body), and reduction of cell size. 4. DNA fragmentation on the electrophoresis was observed with Yoshixol. A low-molecular-weight smear band appeared in the supernatant after treatment with the neutral wood oil. Neither treatment showed higher lactate dehydrogenase (LDH) activity in the culture medium than seen with ethanol as a control. 5. These findings suggest that both the neutral wood oil and Yoshixol have a similar cytotoxic mechanism, reducing cell size and producing a granulation (fragmentation) of eukaryotic cells. 6. Yoshixol may be a potent antitumor agent that induces apoptotic-like cell death. This possible mechanism is discussed.

  19. Triptolide induced cell death through apoptosis and autophagy in murine leukemia WEHI-3 cells in vitro and promoting immune responses in WEHI-3 generated leukemia mice in vivo.

    PubMed

    Chan, Shih-Feng; Chen, Ya-Yin; Lin, Jen-Jyh; Liao, Ching-Lung; Ko, Yang-Ching; Tang, Nou-Ying; Kuo, Chao-Lin; Liu, Kuo-Ching; Chung, Jing-Gung

    2017-02-01

    Triptolide, a traditional Chinese medicine, obtained from Tripterygium wilfordii Hook F, has anti-inflammatory, antiproliferative, and proapoptotic properties. We investigated the potential efficacy of triptolide on murine leukemia by measuring the triptolide-induced cytotoxicity in murine leukemia WEHI-3 cells in vitro. Results indicated that triptolide induced cell morphological changes and induced cytotoxic effects through G0/G1 phase arrest, induction of apoptosis. Flow cytometric assays showed that triptolide increased the production of reactive oxygen species, Ca(2+) release and mitochondrial membrane potential (ΔΨm ), and activations of caspase-8, -9, and -3. Triptolide increased protein levels of Fas, Fas-L, Bax, cytochrome c, caspase-9, Endo G, Apaf-1, PARP, caspase-3 but reduced levels of AIF, ATF6α, ATF6β, and GRP78 in WEHI-3 cells. Triptolide stimulated autophagy based on an increase in acidic vacuoles, monodansylcadaverine staining for LC-3 expression and increased protein levels of ATG 5, ATG 7, and ATG 12. The in vitro data suggest that the cytotoxic effects of triptolide may involve cross-talk between cross-interaction of apoptosis and autophagy. Normal BALB/c mice were i.p. injected with WEHI-3 cells to generate leukemia and were oral treatment with triptolide at 0, 0.02, and 0.2 mg/kg for 3 weeks then animals were weighted and blood, liver, spleen samples were collected. Results indicated that triptolide did not significantly affect the weights of animal body, spleen and liver of leukemia mice, however, triptolide significant increased the cell populations of T cells (CD3), B cells (CD19), monocytes (CD11b), and macrophage (Mac-3). Furthermore, triptolide increased the phagocytosis of macrophage from peripheral blood mononuclear cells (PBMC) but not effects from peritoneum. Triptolide promoted T and B cell proliferation at 0.02 and 0.2 mg/kg treatment when cells were pretreated with Con A and LPS stimulation, respectively; however, triptolide

  20. Neonatal Death

    MedlinePlus

    ... Home > Complications & Loss > Loss & grief > Neonatal death Neonatal death E-mail to a friend Please fill in ... cope with your baby’s death. What is neonatal death? Neonatal death is when a baby dies in ...

  1. Antitumor effects of nano-bubble hydrogen-dissolved water are enhanced by coexistent platinum colloid and the combined hyperthermia with apoptosis-like cell death.

    PubMed

    Asada, Ryoko; Kageyama, Katsuhiro; Tanaka, Hiroshi; Matsui, Hisakazu; Kimura, Masatsugu; Saitoh, Yasukazu; Miwa, Nobuhiko

    2010-12-01

    In order to erase reactive oxygen species (ROS) related with the proliferation of tumor cells by reducing activity of hydrogen, we developed functional water containing nano-bubbles (diameters: <900 nm for 71%/population) hydrogen of 1.1-1.5 ppm (the theoretical maximum: 1.6 ppm) with a reducing ability (an oxidation-reduction potential -650 mV, normal water: +100-200 mV) using a microporous-filter hydrogen-jetting device. We showed that hydrogen water erased ROS indispensable for tumor cell growth by ESR/spin trap, the redox indicator CDCFH-DA assay, and was cytotoxic to Ehrlich ascites tumor cells as assessed by WST-8 assay, crystal violet dye stain and scanning electron microscopy, after 24-h or 48-h incubation sequent to warming at 37°C or 42°C. Hydrogen water supplemented with platinum colloid (0.3 ppm Pt in 4% polyvinylpyrrolidone) had more antitumor activity than hydrogen water alone, mineral water alone (15.6%), hydrogen water plus mineral water, or platinum colloid alone as observed by decreased cell numbers, cell shrinkage and pycnosis (nuclear condensation)/karyorrhexis (nuclear fragmentation) indicative of apoptosis, together with cell deformation and disappearance of microvilli on the membrane surface. These antitumor effects were promoted by combination with hyperthermia at 42°C. Thus, the nano-bubble hydrogen water with platinum colloid is potent as an anti-tumor agent.

  2. A pp32-retinoblastoma protein complex modulates androgen receptor-mediated transcription and associates with components of the splicing machinery

    SciTech Connect

    Adegbola, Onikepe; Pasternack, Gary R. . E-mail: gpastern@jhmi.edu

    2005-08-26

    We have previously shown pp32 and the retinoblastoma protein interact. pp32 and the retinoblastoma protein are nuclear receptor transcriptional coregulators: the retinoblastoma protein is a coactivator for androgen receptor, the major regulator of prostate cancer growth, while pp32, which is highly expressed in prostate cancer, is a corepressor of the estrogen receptor. We now show pp32 increases androgen receptor-mediated transcription and the retinoblastoma protein modulates this activity. Using affinity purification and mass spectrometry, we identify members of the pp32-retinoblastoma protein complex as PSF and nonO/p54nrb, proteins implicated in coordinate regulation of nuclear receptor-mediated transcription and splicing. We show that the pp32-retinoblastoma protein complex is modulated during TPA-induced K562 differentiation. Present evidence suggests that nuclear receptors assemble multiprotein complexes to coordinately regulate transcription and mRNA processing. Our results suggest that pp32 and the retinoblastoma protein may be part of a multiprotein complex that coordinately regulates nuclear receptor-mediated transcription and mRNA processing.

  3. The Orphan Nuclear Receptor ERRγ Regulates Hepatic CB1 Receptor-Mediated Fibroblast Growth Factor 21 Gene Expression

    PubMed Central

    Jung, Yoon Seok; Lee, Ji-Min; Kim, Don-Kyu; Lee, Yong-Soo; Kim, Ki-Sun; Kim, Yong-Hoon; Kim, Jina; Lee, Myung-Shik; Lee, In-Kyu; Kim, Seong Heon; Cho, Sung Jin; Jeong, Won-Il; Lee, Chul-Ho; Harris, Robert A.; Choi, Hueng-Sik

    2016-01-01

    Background Fibroblast growth factor 21 (FGF21), a stress inducible hepatokine, is synthesized in the liver and plays important roles in glucose and lipid metabolism. However, the mechanism of hepatic cannabinoid type 1 (CB1) receptor-mediated induction of FGF21 gene expression is largely unknown. Results Activation of the hepatic CB1 receptor by arachidonyl-2’-chloroethylamide (ACEA), a CB1 receptor selective agonist, significantly increased FGF21 gene expression. Overexpression of estrogen-related receptor (ERR) γ increased FGF21 gene expression and secretion both in hepatocytes and mice, whereas knockdown of ERRγ decreased ACEA-mediated FGF21 gene expression and secretion. Moreover, ERRγ, but not ERRα and ERRβ, induced FGF21 gene promoter activity. In addition, deletion and mutation analysis of the FGF21 promoter identified a putative ERRγ-binding motif (AGGTGC, a near-consensus response element). A chromatin immunoprecipitation assay revealed direct binding of ERRγ to the FGF21 gene promoter. Finally, GSK5182, an ERRγ inverse agonist, significantly inhibited hepatic CB1 receptor-mediated FGF21 gene expression and secretion. Conclusion Based on our data, we conclude that ERRγ plays a key role in hepatic CB1 receptor-mediated induction of FGF21 gene expression and secretion. PMID:27455076

  4. Cell death and tendinopathy.

    PubMed

    Yuan, Jun; Wang, Min-Xia; Murrell, George A C

    2003-10-01

    Apoptosis and necrosis are presently recognized as the two major types of physiological and pathological cell death. Apoptosis is a tightly regulated cell deletion process that differs morphologically and biochemically from necrotic cell death. Tendinopathy is defined as a tendon injury that originates from intrinsic and extrinsic etiological factors. Excessive apoptosis has recently been described in degenerative tendon. The increased number of apoptotic tendon cells in degenerative tendon tissue could affect the rate of collagen synthesis and repair. Impaired or dysfunctional protein synthesis may lead to weaker tendon tissue and eventually increase the risk for tendon rupture. Clearly, there are many details to insert into this pathway, but there is hope that if the fine details of the pathway can be fleshed out, then strategies may be able to be developed to break the cycle at one or more points and prevent or treat tendinopathy more effectively.

  5. Overexpression of cellular repressor of E1A-stimulated genes inhibits TNF-{alpha}-induced apoptosis via NF-{kappa}B in mesenchymal stem cells

    SciTech Connect

    Peng, Cheng-Fei; Han, Ya-Ling; Jie-Deng,; Yan, Cheng-Hui; Jian-Kang,; Bo-Luan,; Jie-Li

    2011-03-25

    Research highlights: {yields} CREG protected MSCs from tumor necrosis factor-{alpha} (TNF-{alpha}) induced apoptosis. {yields} CREG inhibits the phosphorylation of I{kappa}B{alpha} and prevents the activation of NF-{kappa}B. {yields} CREG inhibits NF-{kappa}B nuclear translocation and pro-apoptosis protein transcription. {yields} CREG anti-apoptotic effect involves inhibition of the death receptor pathway. {yields} p53 is downregulated by CREG via NF-{kappa}B pathway under TNF-{alpha} stimulation. -- Abstract: Bone marrow-derived mesenchymal stem cells (MSCs) show great potential for therapeutic repair after myocardial infarction. However, poor viability of transplanted MSCs in the ischemic heart has limited their use. Cellular repressor of E1A-stimulated genes (CREG) has been identified as a potent inhibitor of apoptosis. This study therefore aimed to determine if rat bone marrow MSCs transfected with CREG-were able to effectively resist apoptosis induced by inflammatory mediators, and to demonstrate the mechanism of CREG action. Apoptosis was determined by flow cytometric and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assays. The pathways mediating these apoptotic effects were investigated by Western blotting. Overexpression of CREG markedly protected MSCs from tumor necrosis factor-{alpha} (TNF-{alpha}) induced apoptosis by 50% after 10 h, through inhibition of the death-receptor-mediated apoptotic pathway, leading to attenuation of caspase-8 and caspase-3. Moreover, CREG resisted the serine phosphorylation of I{kappa}B{alpha} and prevented the nuclear translocation of the transcription factor nuclear factor-{kappa}B (NF-{kappa}B) under TNF-{alpha} stimulation. Treatment of cells with the NF-{kappa}B inhibitor pyrrolidine dithiocarbamate (PDTC) significantly increased the transcription of pro-apoptosis proteins (p53 and Fas) by NF-{kappa}B, and attenuated the anti-apoptotic effects of CREG on MSCs. The results of this study

  6. Characterization of putative 5-HT7 receptors mediating tachycardia in the cat

    PubMed Central

    Villalón, Carlos M; Heiligers, Jan P C; Centurión, David; De Vries, Peter; Saxena, Pramod R

    1997-01-01

    , sumatriptan (30, 100 and 300 μg kg−1) and indorenate (300 and 1000 μg kg−1) or the 5-HT4 receptor (partial) agonist cisapride (300 and 1000 μg kg−1) were devoid of effects on feline heart rate per se and failed to modify significantly 5-HT-induced tachycardic responses. Based upon the above rank order of agonist potency, the failure of sumatriptan, indorenate or cisapride to produce cardioacceleration and the blockade by a series of drugs showing high affinity for the cloned 5-ht7 receptor, the present results indicate that the 5-HT receptor mediating tachycardia in the cat is operationally similar to other putative 5-HT7 receptors mediating vascular and non-vascular responses (e.g. relaxation of the rabbit femoral vein, canine external carotid and coronary arteries, rat systemic vasculature and guinea-pig ileum). Since these responses represent functional correlates of the 5-ht7 gene product, the 5-HT7 receptor appellation is reinforced. Therefore, the present experimental model, which is not complicated by the presence of other 5-HT receptors, can be utilized to characterize and develop new drugs with potential agonist and antagonist properties at functional 5-HT7 receptors. PMID:9249256

  7. P2X7 Cell Death Receptor Activation and Mitochondrial Impairment in Oxaliplatin-Induced Apoptosis and Neuronal Injury: Cellular Mechanisms and In Vivo Approach

    PubMed Central

    Massicot, France; Hache, Guillaume; David, Ludivine; Chen, Dominique; Leuxe, Charlotte; Garnier-Legrand, Laure; Rat, Patrice; Laprévote, Olivier; Coudoré, François

    2013-01-01

    Limited information is available regarding the cellular mechanisms of oxaliplatin-induced painful neuropathy during exposure of patients to this drug. We therefore determined oxidative stress in cultured cells and evaluated its occurrence in C57BL/6 mice. Using both cultured neuroblastoma (SH-SY5Y) and macrophage (RAW 264.7) cell lines and also brain tissues of oxaliplatin-treated mice, we investigated whether oxaliplatin (OXA) induces oxidative stress and apoptosis. Cultured cells were treated with 2–200 µM OXA for 24 h. The effects of pharmacological inhibitors of oxidative stress or inflammation (N-acetyl cysteine, ibuprofen, acetaminophen) were also tested. Inhibitors were added 30 min before OXA treatment and then in combination with OXA for 24 h. In SH-SY5Y cells, OXA caused a significant dose-dependent decrease in viability, a large increase in ROS and NO production, lipid peroxidation and mitochondrial impairment as assessed by a drop in mitochondrial membrane potential, which are deleterious for the cell. An increase in levels of negatively charged phospholipids such as cardiolipin but also phosphatidylserine and phosphatidylinositol, was also observed. Additionally, OXA caused concentration-dependent P2X7 receptor activation, increased chromatin condensation and caspase-3 activation associated with TNF-α and IL-6 release. The majority of these toxic effects were equally observed in Raw 264.7 which also presented high levels of PGE2. Pretreatment of SH-SY5Y cells with pharmacological inhibitors significantly reduced or blocked all the neurotoxic OXA effects. In OXA-treated mice (28 mg/kg cumulated dose) significant cold hyperalgesia and oxidative stress in the tested brain areas were shown. Our study suggests that targeting P2X7 receptor activation and mitochondrial impairment might be a potential therapeutic strategy against OXA-induced neuropathic pain. PMID:23826152

  8. P2X7 Cell Death Receptor Activation and Mitochondrial Impairment in Oxaliplatin-Induced Apoptosis and Neuronal Injury: Cellular Mechanisms and In Vivo Approach.

    PubMed

    Massicot, France; Hache, Guillaume; David, Ludivine; Chen, Dominique; Leuxe, Charlotte; Garnier-Legrand, Laure; Rat, Patrice; Laprévote, Olivier; Coudoré, François

    2013-01-01

    Limited information is available regarding the cellular mechanisms of oxaliplatin-induced painful neuropathy during exposure of patients to this drug. We therefore determined oxidative stress in cultured cells and evaluated its occurrence in C57BL/6 mice. Using both cultured neuroblastoma (SH-SY5Y) and macrophage (RAW 264.7) cell lines and also brain tissues of oxaliplatin-treated mice, we investigated whether oxaliplatin (OXA) induces oxidative stress and apoptosis. Cultured cells were treated with 2-200 µM OXA for 24 h. The effects of pharmacological inhibitors of oxidative stress or inflammation (N-acetyl cysteine, ibuprofen, acetaminophen) were also tested. Inhibitors were added 30 min before OXA treatment and then in combination with OXA for 24 h. In SH-SY5Y cells, OXA caused a significant dose-dependent decrease in viability, a large increase in ROS and NO production, lipid peroxidation and mitochondrial impairment as assessed by a drop in mitochondrial membrane potential, which are deleterious for the cell. An increase in levels of negatively charged phospholipids such as cardiolipin but also phosphatidylserine and phosphatidylinositol, was also observed. Additionally, OXA caused concentration-dependent P2X7 receptor activation, increased chromatin condensation and caspase-3 activation associated with TNF-α and IL-6 release. The majority of these toxic effects were equally observed in Raw 264.7 which also presented high levels of PGE2. Pretreatment of SH-SY5Y cells with pharmacological inhibitors significantly reduced or blocked all the neurotoxic OXA effects. In OXA-treated mice (28 mg/kg cumulated dose) significant cold hyperalgesia and oxidative stress in the tested brain areas were shown. Our study suggests that targeting P2X7 receptor activation and mitochondrial impairment might be a potential therapeutic strategy against OXA-induced neuropathic pain.

  9. Bradykinin B2-receptor-mediated modulation of membrane currents in guinea-pig cardiomyocytes

    PubMed Central

    Sakamoto, Naoya; Uemura, Hiroko; Hara, Yukio; Saito, Toshihiro; Masuda, Yoshiaki; Nakaya, Haruaki

    1998-01-01

    In order to define the electrophysiological mechanism(s) responsible for bradykinin (BK)-induced positive inotropic and chronotropic responses in isolated guinea-pig atria, effects of BK on the membrane currents were examined in isolated atrial cells using patch clamp techniques.BK (0.1–1000 nM) increased the L-type Ca2+ current (ICa), which was recorded from enzymatically-dissociated atrial myocytes by the nystatin-perforated patch method, in a concentration-dependent fashion, and the calculated EC50 value for increasing ICa was 5.2 nM. In conventional ruptured patch experiments, BK inhibited the muscarinic acetylcholine receptor-operated K+ current (IK.ACh) that was activated by the muscarinic agonist carbachol (1 μM) with an EC50 value of 0.57 nM. Both the increase in ICa and the decrease in IK.ACh were blocked by HOE140, a selective bradykinin B2 receptor antagonist.The BK-induced inhibition of IK.ACh was significantly attenuated by staurosporine and calphostin C, protein kinase C inhibitors. In addition, the IK.ACh inhibition by BK was also attenuated by the tyrosine kinase inhibitor genistein or tyrphostin but not by daidzein, an inactive analogue of genistein. However, neither protein kinase C inhibitor nor tyrosine kinase inhibitor affected the BK-induced increase in ICa.In the presence and absence of muscarinic stimulation, BK prolonged the action potential recorded from the atrial cells in the current clamp mode.We conclude that BK increases ICa and decreases IK.ACh in atrial cells, resulting in positive inotropic and chronotropic responses in atrial preparations. Protein kinase C activation, and possibly tyrosine kinase activation, may be involved in the B2-receptor-mediated IK.ACh inhibition. PMID:9786500

  10. Greater Beta-Adrenergic Receptor Mediated Vasodilation in Women Using Oral Contraceptives

    PubMed Central

    Limberg, Jacqueline K.; Peltonen, Garrett L.; Johansson, Rebecca E.; Harrell, John W.; Kellawan, Jeremy M.; Eldridge, Marlowe W.; Sebranek, Joshua J.; Walker, Benjamin J.; Schrage, William G.

    2016-01-01

    Background: β-adrenergic receptors play an important role in mitigating the pressor effects of sympathetic nervous system activity in young women. Based on recent data showing oral contraceptive use in women abolishes the relationship between muscle sympathetic nervous system activity and blood pressure, we hypothesized forearm blood flow responses to a β-adrenergic receptor agonist would be greater in young women currently using oral contraceptives (OC+, n = 13) when compared to those not using oral contraceptives (OC–, n = 10). Methods: Women (18–35 years) were studied during the early follicular phase of the menstrual cycle (days 1–5) or placebo phase of oral contraceptive use. Forearm blood flow (FBF, Doppler ultrasound) and mean arterial blood pressure (MAP, brachial arterial catheter) were measured at baseline and during graded brachial artery infusion of the β-adrenergic receptor agonist, Isoproterenol (ISO), as well as Acetylcholine (ACH, endothelium-dependent vasodilation) and Nitroprusside (NTP, endothelium-independent vasodilation). Forearm vascular conductance was calculated (FVC = FBF/MAP, ml/min/100 mmHg) and the rise in FVC from baseline during infusion quantified vasodilation (ΔFVC = FVCinfusion − FVCbaseline). Results: ISO increased FVC in both groups (p < 0.01) and ISO-mediated ΔFVC was greater in OC+ compared to OC– (Main effect of group, p = 0.02). Expressing data as FVC and FBF resulted in similar conclusions. FVC responses to both ACH and NTP were also greater in OC+ compared to OC–. Conclusions: These data are the first to demonstrate greater β-adrenergic receptor-mediated vasodilation in the forearm of women currently using oral contraceptives (placebo phase) when compared to those not using oral contraceptives (early follicular phase), and suggest oral contraceptive use influences neurovascular control. PMID:27375493

  11. P2Y6 Receptor-Mediated Proinflammatory Signaling in Human Bronchial Epithelia

    PubMed Central

    Hao, Yuan; Liang, Jocelyn F.; Chow, Alison W.; Cheung, Wing-tai; Ko, Wing-hung

    2014-01-01

    P2Y receptors are expressed in virtually all epithelia and are responsible for the control of fluid and electrolyte transport. In asthmatic inflammation, the bronchial epithelia are damaged by eosinophil-derived, highly toxic cationic proteins, such as major basic protein (MBP). Consequently, extracellular nucleotides are released into the extracellular space from airway epithelial cells, and act in an autocrine or paracrine fashion to regulate immune functions. Our data show damage to the human bronchial epithelial cell line, 16HBE14o-, by poly-L-arginine-induced UDP release into the extracellular medium. Activation of P2Y6 receptor by its natural ligand, UDP, or its specific agonist, MRS 2693, led to the production of two proinflammatory cytokines, interleukin (IL)-6 and IL-8. This may have resulted from increased IL-6 and IL-8 mRNA expression, and activation of p38 and ERK1/2 MAPK, and NF-κB pathways. Our previous study demonstrated that UDP stimulated transepithelial Cl− secretion via both Ca2+- and cAMP-dependent pathways in 16HBE14o- epithelia. This was further confirmed in this study by simultaneous imaging of Ca2+ and cAMP levels in single cells using the Fura-2 fluorescence technique and a FRET-based approach, respectively. Moreover, the P2Y6 receptor-mediated production of IL-6 and IL-8 was found to be dependent on Ca2+, but not the cAMP/PKA pathway. Together, these studies show that nucleotides released during the airway inflammatory processes will activate P2Y6 receptors, which will lead to further release of inflammatory cytokines. The secretion of cytokines and the formation of such “cytokine networks” play an important role in sustaining the airway inflammatory disease. PMID:25243587

  12. Characterization of Parameters Influencing Receptor-Mediated Endocytosis in Cultured Soybean Cells 1

    PubMed Central

    Horn, Mark A.; Heinstein, Peter F.; Low, Philip S.

    1992-01-01

    In a recent publication, we were able to demonstrate that biotin enters plant cells by receptor-mediated endocytosis and that impermeable macromolecules can be cotransported into cells by the same pathway if they are first covalently linked to biotin. In the present study, we have exploited the biotin endocytosis pathway to evaluate the variables in the cell wall and surrounding growth medium that influence the efficiency of endocytosis in plants. Under normal growth conditions, the major constraint limiting macromolecule endocytosis was found to be the size of the internalized macromolecule. Thus, a log-linear relationship with a negative slope exists between the molecular weight of the biotin-conjugated macromolecule and its rate of internalization by cultured soybean cells. This relationship, which extends from insulin (Mr approximately 5700) to immunoglobulin G (Mr approximately 160,000), is characterized by a slope of −1.04 × 105 molecules/cell/min per log Mr unit and an x intercept (no endocytosis detectable) of approximately log 160,000 daltons. Unfortunately, mild digestion with cell wall-degrading enzymes is unable to increase significantly the upper size limit of molecules that can be internalized, but uptake of lower molecular weight proteins can be enhanced by mild cell wall digestion. The optimal extracellular pH for endocytosis was found to be 4.6, i.e. near the normal pH of the cell culture medium. Furthermore, the osmotic strength at which endocytosis occurs most rapidly was observed to be isotonic to slightly hypotonic, suggesting that turgor pressure within the plant cell must not be a major determinant of endocytosis rates by cultured soybean (Glycine max) cells. Finally, cell age was found to impact significantly on the rate of macromolecule internalization, with maximal uptake rates occurring during early exponential growth and decreasing by a factor of 2 when the cells reach stationary growth phase. PMID:16668694

  13. PAC₁ receptors mediate positive chronotropic responses to PACAP-27 and VIP in isolated mouse atria.

    PubMed

    Hoover, Donald B; Girard, Beatrice M; Hoover, Jeffrey L; Parsons, Rodney L

    2013-08-05

    PACAP and VIP have prominent effects on cardiac function in several species, but little is known about their influence on the murine heart. Accordingly, we evaluated the expression of PACAP/VIP receptors in mouse heart and the response of isolated atria to peptide agonists. Quantitative PCR demonstrated that PAC₁, VPAC₁, and VPAC₂ receptor mRNAs are present throughout the mouse heart. Expression of all three receptor transcripts was low, PAC₁ being the lowest. No regional differences in expression were detected for individual receptor mRNAs after normalization to L32. Pharmacological effects of PACAP-27, VIP, and the selective PAC₁ agonist maxadilan were evaluated in isolated, spontaneously beating atria from C57BL/6 mice of either sex. Incremental additions of PACAP-27 at 1 min intervals caused a concentration-dependent tachycardia with a logEC₅₀=-9.08 ± 0.15 M (n=7) and a maximum of 96.3 ± 5.9% above baseline heart rate. VIP and maxadilan also caused tachycardia but their potencies were about two orders of magnitude less. Increasing the dosing interval to 5 min caused a leftward shift of the concentration-response curve to maxadilan but no changes in the curves for PACAP-27 or VIP. Under this condition, neither the potency nor the efficacy of maxadilan differed from those of PACAP-27. Neither PACAP-27 nor maxadilan caused tachyphylaxis, and maximal responses to maxadilan were maintained for at least 2 h. We conclude that all three VIP/PACAP family receptors are expressed by mouse cardiac tissue, but only PAC₁ receptors mediate positive chronotropic responses to PACAP-27 and VIP.

  14. cap alpha. /sub 2/-Adrenergic receptor-mediated sensitization of forskolin-stimulated cyclic AMP production

    SciTech Connect

    Jones, S.B.; Toews, M.L.; Turner, J.T.; Bylund, D.B.

    1987-03-01

    Preincubation of HT29 human colonic adenocarcinoma cells with ..cap alpha../sub 2/-adrenergic agonists resulted in a 10- to 20-fold increase in forskolin-stimulated cyclic AMP production as compared to cells preincubated without agonist. Similar results were obtained using either a (/sup 3/H)adenine prelabeling assay or a cyclic AMP radioimmunoassay to measure cyclic AMP levels. This phenomenon, which is termed sensitization, is ..cap alpha../sub 2/-adrenergic receptor-mediated and rapid in onset and reversal. Yohimbine, an ..cap alpha../sub 2/-adrenergic receptor-selective antagonist, blocked norepinephrine-induced sensitization, whereas prazosin (..cap alpha../sub 1/-adrenergic) and sotalol (..beta..-adrenergic) did not. The time for half-maximal sensitization was 5 min and the half-time for reversal was 10 min. Only a 2-fold sensitization of cyclic AMP production stimulated by vasoactive intestinal peptide was observed, indicating that sensitization is relatively selective for forskolin. Sensitization reflects an increased production of cyclic AMP and not a decreased degradation of cyclic AMP, since incubation with a phosphodiesterase inhibitor and forskolin did not mimic sensitization. Increasing the levels of cyclic AMP during the preincubation had no effect on sensitization, indicating that sensitization is not caused by decreased cyclic AMP levels during the preincubation. This rapid and dramatic sensitization of forskolin-stimulated cyclic AMP production is a previously unreported effect that can be added to the growing list of ..cap alpha../sub 2/-adrenergic responses that are not mediated by a decrease in cyclic AMP.

  15. Potentiating effect of eszopiclone on GABA(A) receptor-mediated responses in pedunculopontine neurons.

    PubMed

    Ye, Meijun; Garcia-Rill, Edgar

    2009-07-01

    The pedunculopontine nucleus (PPN) is part of the cholinergic arm of the reticular activating system, which is mostly active during waking and REM sleep. GABAergic modulation of this area appears to regulate sleep-wake cycles. Eszopiclone (ESZ), a nonbenzodiazepine hypnotic agent, appears to modulate GABAergic receptors. However, the action site of ESZ in the brain is still unresolved. We tested the hypothesis that ESZ acts by potentiating GABA(A) receptors on PPN neurons. Wholecell voltage clamp recordings were performed on PPN neurons in 7-15 day rat brainstem slices, and the potentiating effects of ESZ on the responses to the GABA(A) receptor agonist isoguvacine (IGV), and on GABA(A) receptor-mediated inhibitory post-synaptic currents (IPSCs), were determined. In the presence of tetrodotoxin, ESZ (1) increased the amplitude of the outward current induced by IGV, (2) increased its duration, and (3) enhanced the IGV-induced decrease in input resistance (Rin). The GABA(A) receptor antagonist gabazine (GBZ) blocked these effects. ESZ alone did not induce detectable currents or change Rin at a holding potential of -60 mV, but when held at 0 mV, ESZ induced an outward current in 13/21 PPN cells, an effect blocked by GBZ. ESZ also increased the amplitude (n = 18/21), duration (n = 17/21), and frequency (n = 13/15) of IPSCs. ESZ may potentiate GABA(A) inhibition in the PPN via pre- and post-synaptic modulation, which may underlie the hypnotic effects of ESZ. The differential effects of ESZ on both pre- and post-synaptic sites may partially explain why it has less significant side effects compared to other hypnotic agents.

  16. Endostatin inhibits androgen-independent prostate cancer growth by suppressing nuclear receptor-mediated oxidative stress.

    PubMed

    Lee, Joo Hyoung; Kang, Minsung; Wang, Hong; Naik, Gurudatta; Mobley, James A; Sonpavde, Guru; Garvey, W Timothy; Darley-Usmar, Victor M; Ponnazhagan, Selvarangan

    2017-04-01

    Androgen-deprivation therapy has been identified to induce oxidative stress in prostate cancer (PCa), leading to reactivation of androgen receptor (AR) signaling in a hormone-refractory manner. Thus, antioxidant therapies have gained attention as adjuvants for castration-resistant PCa. Here, we report for the first time that human endostatin (ES) prevents androgen-independent growth phenotype in PCa cells through its molecular targeting of AR and glucocorticoid receptor (GR) and downstream pro-oxidant signaling. This reversal after ES treatment significantly decreased PCa cell proliferation through down-regulation of GR and up-regulation of manganese superoxide dismutase and reduced glutathione levels. Proteome and biochemical analyses of ES-treated PCa cells further indicated a significant up-regulation of enzymes in the major reactive oxygen species (ROS) scavenging machinery, including catalase, glutathione synthetase, glutathione reductase, NADPH-cytochrome P450 reductase, biliverdin reductase, and thioredoxin reductase, resulting in a concomitant reduction of intracellular ROS. ES further augmented the antioxidant system through up-regulation of glucose influx, the pentose phosphate pathway, and NAD salvaging pathways. This shift in cancer cell redox homeostasis by ES significantly decreased the effect of protumorigenic oxidative machinery on androgen-independent PCa growth, suggesting that ES can suppress GR-induced resistant phenotype upon AR antagonism and that the dual targeting action of ES on AR and GR can be further translated to PCa therapy.-Lee, J. H., Kang, M., Wang, H., Naik, G., Mobley, J. A., Sonpavde, G., Garvey, W. T., Darley-Usmar, V. M., Ponnazhagan, S. Endostatin inhibits androgen-independent prostate cancer growth by suppressing nuclear receptor-mediated oxidative stress.

  17. Direct muscarinic and nicotinic receptor-mediated excitation of rat medial vestibular nucleus neurons in vitro

    NASA Technical Reports Server (NTRS)

    Phelan, K. D.; Gallagher, J. P.

    1992-01-01

    We have utilized intracellular recording techniques to investigate the cholinoceptivity of rat medial vestibular nucleus (MVN) neurons in a submerged brain slice preparation. Exogenous application of the mixed cholinergic agonists, acetylcholine (ACh) or carbachol (CCh), produced predominantly membrane depolarization, induction of action potential firing, and decreased input resistance. Application of the selective muscarinic receptor agonist muscarine (MUSC), or the selective nicotinic receptor agonists nicotine (NIC) or 1,1-dimethyl-4-phenylpiperazinium (DMPP) also produced membrane depolarizations. The MUSC-induced depolarization was accompanied by decreased conductance, while an increase in conductance appeared to underlie the NIC- and DMPP-induced depolarizations. The muscarinic and nicotinic receptor mediated depolarizations persisted in tetrodotoxin and/or low Ca2+/high Mg2+ containing media, suggesting direct postsynaptic receptor activation. The MUSC-induced depolarization could be reversibly blocked by the selective muscarinic-receptor antagonist, atropine, while the DMPP-induced depolarization could be reversibly suppressed by the selective ganglionic nicotinic-receptor antagonist, mecamylamine. Some neurons exhibited a transient membrane hyperpolarization during the depolarizing response to CCh or MUSC application. This transient inhibition could be reversibly blocked by the gamma-aminobutyric acid (GABA) antagonist, bicuculline, suggesting that the underlying hyperpolarization results indirectly from the endogenous release of GABA acting at GABA receptors. This study confirms the cholinoceptivity of MVN neurons and establishes that individual MVN cells possess muscarinic as well as nicotinic receptors. The data provide support for a prominent role of cholinergic mechanisms in the direct and indirect regulation of the excitability of MVN neurons.

  18. PKCɛ mediates substance P inhibition of GABAA receptors-mediated current in rat dorsal root ganglion.

    PubMed

    Li, Li; Zhao, Lei; Wang, Yang; Ma, Ke-tao; Shi, Wen-yan; Wang, Ying-zi; Si, Jun-qiang

    2015-02-01

    The mechanism underlying the modulatory effect of substance P (SP) on GABA-activated response in rat dorsal root ganglion (DRG) neurons was investigated. In freshly dissociated rat DRG neurons, whole-cell patch-clamp technique was used to record GABA-activated current and sharp electrode intracellular recording technique was used to record GABA-induced membrane depolarization. Application of GABA (1-1000 μmol/L) induced an inward current in a concentration-dependent manner in 114 out of 127 DRG neurons (89.8 %) examined with whole-cell patch-clamp recordings. Bath application of GABA (1-1000 μmol/L) evoked a depolarizing response in 236 out of 257 (91.8%) DRG neurons examined with intracellular recordings. Application of SP (0.001-1 μmol/L) suppressed the GABA-activated inward current and membrane depolarization. The inhibitory effects were concentration-dependent and could be blocked by the selective neurokinin 1 (NK1) receptors antagonist spantide but not by L659187 and SR142801 (1 μmol/L, n=7), selective antagonists of NK2 and NK3. The inhibitory effect of SP was significantly reduced by the calcium chelator BAPTA-AM, phospholipase C (PLC) inhibitor U73122, and PKC inhibitor chelerythrine, respectively. The PKA inhibitor H-89 did not affect the SP effect. Remarkably, the inhibitory effect of SP on GABA-activated current was nearly completely removed by a selective PKCε inhibitor epilon-V1-2 but not by safingol and LY333531, selective inhibitors of PKCα and PKCβ. Our results suggest that NK1 receptor mediates SP-induced inhibition of GABA-activated current and membrane depolarization by activating intracellular PLC-Ca²⁺-PKCε cascade. SP might regulate the excitability of peripheral nociceptors through inhibition of the "pre-synaptic inhibition" evoked by GABA, which may explain its role in pain and neurogenic inflammation.

  19. N-Methyl-D-Aspartate Receptor-Mediated Axonal Injury in Adult Rat Corpus Callosum

    PubMed Central

    Zhang, Jingdong; Liu, Jianuo; Fox, Howard S.; Xiong, Huangui

    2013-01-01

    Damage to white matter such as corpus callosum (CC) is a pathological characteristic in many brain disorders. Glutamate (Glut) excitotoxicity through AMPA receptors on oligodendrocyte (OL) was previously considered as a mechanism for white matter damage. Recent studies have shown that N-methyl-D-aspartate receptors (NMDARs) are expressed on myelin sheath of neonatal rat OL processes and that activation of these receptors mediated demyelization. Whether NMDARs are expressed in the adult CC and are involved in excitotoxic axonal injury remains to be determined. In this study, we demonstrate the presence of NMDARs in the adult rat CC and their distributions in myelinated nerve fibers and OL somata by means of immunocytochemical staining and Western blot. Incubation of the CC slices with Glut or NMDA induced axonal injury as revealed by analyzing amplitude of CC fiber compound action potentials (CAPs) and input–output response. Both Glut and NMDA decreased the CAP amplitude and input–output responses, suggesting an involvement of NMDARs in Glut- and NMDA-induced axonal injury. The involvement of NMDAR in Glut-induced axonal injury was further assayed by detection of β-amyloid precursor protein (β-APP) in the CC axonal fibers. Treatment of the CC slices with Glut resulted in β-APP accumulation in the CC fibers as detected by Western blot, reflecting an impairment of axonal transport function. This injurious effect of Glut on CC axonal transport was significantly blocked by MK801. Taken together, these results show that NMDARs are expressed in the adult CC and are involved in excitotoxic activity in adult CC slices in vitro. PMID:23161705

  20. Receptor-mediated cell attachment and detachment kinetics. I. Probabilistic model and analysis.

    PubMed Central

    Cozens-Roberts, C.; Lauffenburger, D. A.; Quinn, J. A.

    1990-01-01

    The kinetics of receptor-mediated cell adhesion to a ligand-coated surface play a key role in many physiological and biotechnology-related processes. We present a probabilistic model of receptor-ligand bond formation between a cell and surface to describe the probability of adhesion in a fluid shear field. Our model extends the deterministic model of Hammer and Lauffenburger (Hammer, D.A., and D.A. Lauffenburger. 1987. Biophys. J. 52:475-487) to a probabilistic framework, in which we calculate the probability that a certain number of bonds between a cell and surface exists at any given time. The probabilistic framework is used to account for deviations from ideal, deterministic behavior, inherent in chemical reactions involving relatively small numbers of reacting molecules. Two situations are investigated: first, cell attachment in the absence of fluid stress; and, second, cell detachment in the presence of fluid stress. In the attachment case, we examine the expected variance in bond formation as a function of attachment time; this also provides an initial condition for the detachment case. Focusing then on detachment, we predict transient behavior as a function of key system parameters, such as the distractive fluid force, the receptor-ligand bond affinity and rate constants, and the receptor and ligand densities. We compare the predictions of the probabilistic model with those of a deterministic model, and show how a deterministic approach can yield some inaccurate results; e.g., it cannot account for temporally continuous cell attach mentor detachment, it can underestimate the time needed for cell attachment, it can overestimate the time required for cell detachment for a given level of force, and it can overestimate the force necessary for cell detachment. PMID:2174271

  1. Fc receptor-mediated immune responses: new tools but increased complexity in HIV prevention.

    PubMed

    Vargas-Inchaustegui, Diego A; Robert-Guroff, Marjorie

    2013-07-01

    The modest success of the RV144 HIV vaccine trial in Thailand and the ensuing suggestion that a Fc-receptormediated antibody activity might have played a role in the protection observed have intensified investigations on Fcrelated immune responses. HIV neutralizing antibodies have been and continue to be the focal point of research into humoral immune protection. However, recent knowledge that their protective efficacy can be augmented by Fc-FcR interactions has increased the complexity of identifying immune correlates of protection. If anything, continued studies of both humoral and cellular immune mechanisms point to the lack of a single protective anti-HIV immune response. Here we focus on humoral immunity, analyzing the role played by Fc receptor-related responses and discussing how new knowledge of their interactions requires further investigation, but may also spur novel vaccination approaches. We initially address classical Fc-receptor mediated anti-viral mechanisms including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell mediated viral inhibition (ADCVI), and antibody-dependent cellular phagocytosis (ADCP), as well as the effector cells that mediate these functions. Next, we summarize key aspects of FcR-Fc interactions that are important for potential control of HIV/SIV such as FcR polymorphisms and post-transcriptional modifications. Finally we discuss less commonly studied non-mechanistic anti-HIV immune functions: antibody avidity and envelopespecific B cell memory. Overall, a spectrum of immune responses, reflecting the immune system's redundancy, will likely be needed to prevent HIV infection and/or disease progression. Aside from elicitation of critical immune mechanisms, a successful vaccine will need to induce mature B cell responses and long-lasting immune memory.

  2. Receptor-mediated endocytosis and endosomal acidification is impaired in proximal tubule epithelial cells of Dent disease patients.

    PubMed

    Gorvin, Caroline M; Wilmer, Martijn J; Piret, Sian E; Harding, Brian; van den Heuvel, Lambertus P; Wrong, Oliver; Jat, Parmjit S; Lippiat, Jonathan D; Levtchenko, Elena N; Thakker, Rajesh V

    2013-04-23

    Receptor-mediated endocytosis, involving megalin and cubilin, mediates renal proximal-tubular reabsorption and is decreased in Dent disease because of mutations of the chloride/proton antiporter, chloride channel-5 (CLC-5), resulting in low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. To facilitate studies of receptor-mediated endocytosis and the role of CLC-5, we established conditionally immortalized proximal-tubular epithelial cell lines (ciPTECs) from three patients with CLC-5 mutations (30:insH, R637X, and del132-241) and a normal male. Confocal microscopy using the tight junction marker zona occludens-1 (ZO-1) and end-binding protein-1 (EB-1), which is specific for the plus end of microtubules demonstrated that the ciPTECs polarized. Receptor-mediated endocytic uptake of fluorescent albumin and transferrin in 30:insH and R637X ciPTECs was significantly decreased, compared with normal ciPTECs, and could be further reduced by competition with 10-fold excess of unlabeled albumin and transferrin, whereas in the del132-241 ciPTEC, receptor-mediated endocytic uptake was abolished. Investigation of endosomal acidification by live-cell imaging of pHluorin-VAMP2 (vesicle-associated membrane protein-2), a pH-sensitive-GFP construct, revealed that the endosomal pH in normal and 30:insH ciPTECs was similar, whereas in del132-241 and R637X ciPTECs, it was significantly more alkaline, indicating defective acidification in these ciPTECs. The addition of bafilomycin-A1, a V-ATPase inhibitor, raised the pH significantly in all ciPTECs, demonstrating that the differences in acidification were not due to alterations in the V-ATPase, but instead to abnormalities of CLC-5. Thus, our studies, which have established human Dent disease ciPTECs that will facilitate studies of mechanisms in renal reabsorption, demonstrate that Dent disease-causing CLC-5 mutations have differing effects on endosomal acidification and receptor-mediated endocytosis

  3. Neurosteroid dehydroepiandrosterone interacts with nerve growth factor (NGF) receptors, preventing neuronal apoptosis.

    PubMed

    Lazaridis, Iakovos; Charalampopoulos, Ioannis; Alexaki, Vassilia-Ismini; Avlonitis, Nicolaos; Pediaditakis, Iosif; Efstathopoulos, Paschalis; Calogeropoulou, Theodora; Castanas, Elias; Gravanis, Achille

    2011-04-01

    The neurosteroid dehydroepiandrosterone (DHEA), produced by neurons and glia, affects multiple processes in the brain, including neuronal survival and neurogenesis during development and in aging. We provide evidence that DHEA interacts with pro-survival TrkA and pro-death p75(NTR) membrane receptors of neurotrophin nerve growth factor (NGF), acting as a neurotrophic factor: (1) the anti-apoptotic effects of DHEA were reversed by siRNA against TrkA or by a specific TrkA inhibitor; (2) [(3)H]-DHEA binding assays showed that it bound to membranes isolated from HEK293 cells transfected with the cDNAs of TrkA and p75(NTR) receptors (K(D): 7.4 ± 1.75 nM and 5.6 ± 0.55 nM, respectively); (3) immobilized DHEA pulled down recombinant and naturally expressed TrkA and p75(NTR) receptors; (4) DHEA induced TrkA phosphorylation and NGF receptor-mediated signaling; Shc, Akt, and ERK1/2 kinases down-stream to TrkA receptors and TRAF6, RIP2, and RhoGDI interactors of p75(NTR) receptors; and (5) DHEA rescued from apoptosis TrkA receptor positive sensory neurons of dorsal root ganglia in NGF null embryos and compensated NGF in rescuing from apoptosis NGF receptor positive sympathetic neurons of embryonic superior cervical ganglia. Phylogenetic findings on the evolution of neurotrophins, their receptors, and CYP17, the enzyme responsible for DHEA biosynthesis, combined with our data support the hypothesis that DHEA served as a phylogenetically ancient neurotrophic factor.

  4. Neurosteroid Dehydroepiandrosterone Interacts with Nerve Growth Factor (NGF) Receptors, Preventing Neuronal Apoptosis

    PubMed Central

    Alexaki, Vassilia-Ismini; Avlonitis, Nicolaos; Pediaditakis, Iosif; Efstathopoulos, Paschalis; Calogeropoulou, Theodora; Castanas, Elias; Gravanis, Achille

    2011-01-01

    The neurosteroid dehydroepiandrosterone (DHEA), produced by neurons and glia, affects multiple processes in the brain, including neuronal survival and neurogenesis during development and in aging. We provide evidence that DHEA interacts with pro-survival TrkA and pro-death p75NTR membrane receptors of neurotrophin nerve growth factor (NGF), acting as a neurotrophic factor: (1) the anti-apoptotic effects of DHEA were reversed by siRNA against TrkA or by a specific TrkA inhibitor; (2) [3H]-DHEA binding assays showed that it bound to membranes isolated from HEK293 cells transfected with the cDNAs of TrkA and p75NTR receptors (KD: 7.4±1.75 nM and 5.6±0.55 nM, respectively); (3) immobilized DHEA pulled down recombinant and naturally expressed TrkA and p75NTR receptors; (4) DHEA induced TrkA phosphorylation and NGF receptor-mediated signaling; Shc, Akt, and ERK1/2 kinases down-stream to TrkA receptors and TRAF6, RIP2, and RhoGDI interactors of p75NTR receptors; and (5) DHEA rescued from apoptosis TrkA receptor positive sensory neurons of dorsal root ganglia in NGF null embryos and compensated NGF in rescuing from apoptosis NGF receptor positive sympathetic neurons of embryonic superior cervical ganglia. Phylogenetic findings on the evolution of neurotrophins, their receptors, and CYP17, the enzyme responsible for DHEA biosynthesis, combined with our data support the hypothesis that DHEA served as a phylogenetically ancient neurotrophic factor. PMID:21541365

  5. Green Synthesis of Silver and Titanium Dioxide Nanoparticles Using Euphorbia prostrata Extract Shows Shift from Apoptosis to G0/G1 Arrest followed by Necrotic Cell Death in Leishmania donovani

    PubMed Central

    Zahir, Abdul Abduz; Chauhan, Indira Singh; Bagavan, Asokan; Kamaraj, Chinnaperumal; Elango, Gandhi; Shankar, Jai; Arjaria, Nidhi; Roopan, Selvaraj Mohana

    2015-01-01

    The aim of the present study was to synthesize silver (Ag) and titanium dioxide (TiO2) nanoparticles (NPs) using green synthesis from aqueous leaf extract of Euphorbia prostrata as antileishmanial agents and to explore the underlying molecular mechanism of induced cell death. In vitro antileishmanial activity of synthesized NPs was tested against promastigotes of Leishmania donovani by alamarBlue and propidium iodide uptake assays. Antileishmanial activity of synthesized NPs on intracellular amastigotes was assessed by Giemsa staining. The leishmanicidal effect of synthesized Ag NPs was further confirmed by DNA fragmentation assay and by cell cycle progression and transmission electron microscopy (TEM) of the treated parasites. TEM analysis of the synthesized Ag NPs showed a spherical shape with an average size of 12.82 ± 2.50 nm, and in comparison to synthesized TiO2 NPs, synthesized Ag NPs were found to be most active against Leishmania parasites after 24 h exposure, with 50% inhibitory concentrations (IC50) of 14.94 μg/ml and 3.89 μg/ml in promastigotes and intracellular amastigotes, respectively. A significant increase in G0/G1 phase of the cell cycle with a subsequent decrease in S (synthesis) and G2/M phases compared to controls was observed. The growth-inhibitory effect of synthesized Ag NPs was attributed to increased length of S phase. A decreased reactive oxygen species level was also observed, which could be responsible for the caspase-independent shift from apoptosis (G0/G1 arrest) to massive necrosis. High-molecular-weight DNA fragmentation as a positive consequence of necrotic cell death was also visualized. We also report that the unique trypanothione/trypanothione reductase (TR) system of Leishmania cells was significantly inhibited by synthesized Ag NPs. The green-synthesized Ag NPs may provide promising leads for the development of cost-effective and safer alternative treatment against visceral leishmaniasis. PMID:26033724

  6. Green Synthesis of Silver and Titanium Dioxide Nanoparticles Using Euphorbia prostrata Extract Shows Shift from Apoptosis to G0/G1 Arrest followed by Necrotic Cell Death in Leishmania donovani.

    PubMed

    Zahir, Abdul Abduz; Chauhan, Indira Singh; Bagavan, Asokan; Kamaraj, Chinnaperumal; Elango, Gandhi; Shankar, Jai; Arjaria, Nidhi; Roopan, Selvaraj Mohana; Rahuman, Abdul Abdul; Singh, Neeloo

    2015-08-01

    The aim of the present study was to synthesize silver (Ag) and titanium dioxide (TiO2) nanoparticles (NPs) using green synthesis from aqueous leaf extract of Euphorbia prostrata as antileishmanial agents and to explore the underlying molecular mechanism of induced cell death. In vitro antileishmanial activity of synthesized NPs was tested against promastigotes of Leishmania donovani by alamarBlue and propidium iodide uptake assays. Antileishmanial activity of synthesized NPs on intracellular amastigotes was assessed by Giemsa staining. The leishmanicidal effect of synthesized Ag NPs was further confirmed by DNA fragmentation assay and by cell cycle progression and transmission electron microscopy (TEM) of the treated parasites. TEM analysis of the synthesized Ag NPs showed a spherical shape with an average size of 12.82 ± 2.50 nm, and in comparison to synthesized TiO2 NPs, synthesized Ag NPs were found to be most active against Leishmania parasites after 24 h exposure, with 50% inhibitory concentrations (IC50) of 14.94 μg/ml and 3.89 μg/ml in promastigotes and intracellular amastigotes, respectively. A significant increase in G0/G1 phase of the cell cycle with a subsequent decrease in S (synthesis) and G2/M phases compared to controls was observed. The growth-inhibitory effect of synthesized Ag NPs was attributed to increased length of S phase. A decreased reactive oxygen species level was also observed, which could be responsible for the caspase-independent shift from apoptosis (G0/G1 arrest) to massive necrosis. High-molecular-weight DNA fragmentation as a positive consequence of necrotic cell death was also visualized. We also report that the unique trypanothione/trypanothione reductase (TR) system of Leishmania cells was significantly inhibited by synthesized Ag NPs. The green-synthesized Ag NPs may provide promising leads for the development of cost-effective and safer alternative treatment against visceral leishmaniasis.

  7. Bortezomib induces caspase-dependent apoptosis in Hodgkin lymphoma cell lines and is associated with reduced c-FLIP expression: a gene expression profiling study with implications for potential combination therapies.

    PubMed

    Zhao, Xiaoxian; Qiu, Wansong; Kung, Jiachun; Zhao, Xinyu; Peng, Xuejun; Yegappan, Mani; Yen-Lieberman, Belinda; Hsi, Eric D

    2008-02-01

    The Hodgkin cells and Reed-Sternberg cells (HRS) of classical Hodgkin lymphoma (CHL) are derived from germinal center B cells. The pathogenesis of CHL is unclear but constitutive activation of NFkappaB may contribute. Proteasome inhibition aimed at inhibiting NFkappaB has been shown to result in apoptosis in HRS cells. Here we investigated the effects of bortezomib, a proteasome inhibitor, in HRS cells with a combination of functional assays and gene expression profiling (GEP). Exposure of KMH2 and L428 cells to bortezomib resulted in inhibition of proliferation and induction of apoptosis. Gene expression analysis of KMH2 cells by oligonucleotide cDNA microarrays showed that a limited set of genes were differentially expressed involving several key cellular pathways including cell cycle and apoptosis. Among them, the caspase 8 inhibitor cFLIP was down-regulated and confirmed by Q-PCR. Given the evidence that cFLIP in HRS cells contribute to cells' insensitive to death receptor-mediated apoptosis, we combined bortezomib and TRAIL. This combination caused further down-regulation of cFLIP protein and increased apoptosis in CHL cells demonstrated by PARP p85 immunohistochemistry and immunoblotting. Such apoptotic effects were inhibited by caspase inhibitor z-VAD-FMK, confirming the pro-apoptotic effects of bortezomib and TRAIL are caspase-dependent. Bortezomib has no detectable effect on expression of TRAIL receptor DR4/DR5 in these two cell lines. Tissue microarray analysis of primary Hodgkin lymphomas displayed that 82% cases (95/116) expressed cFLIP in Reed-Sternberg cells. The discovery of apoptotic pathways that can be manipulated by proteasome inhibition provides rationale for the combination of bortezomib and agents such as TRAIL in CHL treatment.

  8. Isoorientin induces apoptosis and autophagy simultaneously by reactive oxygen species (ROS)-related p53, PI3K/Akt, JNK, and p38 signaling pathways in HepG2 cancer cells.

    PubMed

    Yuan, Li; Wei, Shuping; Wang, Jing; Liu, Xuebo

    2014-06-11

    Cell death is closely related to autophagy under some circumstances; however, the effect of isoorientin (ISO) on autophagy and the interplay between apoptosis and autophagy in human hepatoblastoma cancer (HepG2) cells remains poorly understood. The present study showed that ISO induced autophagy, which was correlated with the formation of autophagic vacuoles and the overexpression of Beclin-1 and LC3-II. The autophagy inhibitor 3-methyladenine (3-MA) markedly inhibited apoptosis, and the apoptosis inhibitor ZVAD-fmk also decreased ISO-induced autophagy. In addition, the PI3K/Akt inhibitor LY294002 enhanced Beclin-1, LC3-II, and poly(ADP-ribose) polymerase (PARP) cleavage levels. Also, the reactive oxygen species (ROS) inhibitor N-acetyl-L-cysteine (NAC), the JNK inhibitor SP600125, and the p38 inhibitor SB203580 efficiently downregulated the levels of these proteins. Moreover, the p53 inhibitor pifithrin-α and the nuclear factor (NF)-κB inhibitor pyrrolidinedithiocarbamic acid (PDTC) clearly suppressed Beclin-1 and LC3-II and increased cytochrome c release, caspase-3 activation, and PARP cleavage. These results demonstrated for the first time that ISO simultaneously induced apoptosis and autophagy by ROS-related p53, PI3K/Akt, JNK, and p38 signaling pathways. Furthermore, ISO-induced apoptosis by activating the Fas receptor-mediated apoptotic pathway and suppressing the p53 and PI3K/Akt-dependent NF-κB signaling pathway, with the subsequent increase in the release of cytochrome c, caspase-3 activation, and PARP cleavage.

  9. Programmed Cell Death in Breast Cancer.

    DTIC Science & Technology

    1996-10-01

    TITLE: Programmed Cell Death in Breast Cancer PRINCIPAL INVESTIGATOR: Clark W. Distelhorst, M.D. CONTRACTING ORGANIZATION: Case Western Reserve...Programmed Cell Death in Breast Cancer DAMD17-94-J-4451 6. AUTHOR(S) Clark W. Distelhorst, M.D. 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8...cell death , apoptosis, in breast cancer cells has been developed. This model is based on induction of apoptosis by the selective endoplasmic reticulum

  10. THE ROLE OF APOPTOSIS IN NEUROTOXICOLOGY

    EPA Science Inventory

    Apoptosis, a form of programmed cell death, occurs in the nervous system throughout development, but with a preponderance of cell death occurring during the prenatal and perinatal periods. Aberrant periods of increased or decreased cell death, induced by toxicants in air, water,...

  11. Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012

    PubMed Central

    Galluzzi, L; Vitale, I; Abrams, J M; Alnemri, E S; Baehrecke, E H; Blagosklonny, M V; Dawson, T M; Dawson, V L; El-Deiry, W S; Fulda, S; Gottlieb, E; Green, D R; Hengartner, M O; Kepp, O; Knight, R A; Kumar, S; Lipton, S A; Lu, X; Madeo, F; Malorni, W; Mehlen, P; Nuñez, G; Peter, M E; Piacentini, M; Rubinsztein, D C; Shi, Y; Simon, H-U; Vandenabeele, P; White, E; Yuan, J; Zhivotovsky, B; Melino, G; Kroemer, G

    2012-01-01

    In 2009, the Nomenclature Committee on Cell Death (NCCD) proposed a set of recommendations for the definition of distinct cell death morphologies and for the appropriate use of cell death-related terminology, including ‘apoptosis', ‘necrosis' and ‘mitotic catastrophe'. In view of the substantial progress in the biochemical and genetic exploration of cell death, time has come to switch from morphological to molecular definitions of cell death modalities. Here we propose a functional classification of cell death subroutines that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death and mitotic catastrophe. Moreover, we discuss the utility of expressions indicating additional cell death modalities. On the basis of the new, revised NCCD classification, cell death subroutines are defined by a series of precise, measurable biochemical features. PMID:21760595

  12. Mechanisms Involved in Virus-Induced Neural Cell Death

    DTIC Science & Technology

    2001-09-01

    We are using experimental infection with reoviruses as a model to study how viruses induce cell death (apoptosis) and cause dysregulation of the cell...and their ligand (TRAIL). Apoptosis involves both death-receptor (DR) and mitochondrial-associated cell death pathways, and leads to the early

  13. GABAA and GABAB receptor-mediated effects in guinea-pig ileum.

    PubMed

    Giotti, A; Luzzi, S; Spagnesi, S; Zilletti, L

    1983-03-01

    1 The effects of gamma-aminobutyric acid (GABA) and related substances were examined in guinea-pig ileum longitudinal muscle.2 GABA at doses ranging from 10(-7) M to 3 x 10(-6) M elicited a relaxation while at higher doses (3 x 10(-6) M - 10(-4) M), as previously described, it caused a contraction followed by relaxation.3 GABA-induced relaxation was bicuculline-insensitive, was mimicked by (-)-baclofen but not by homotaurine and muscimol. The effect of baclofen was stereospecific. GABA- and (-)-baclofen-induced relaxations were dose-dependent and their ED(50) values were similar. A specific cross-desensitization occurred between GABA and (-)-baclofen.4 The bicuculline-insensitive relaxation induced by GABA and (-)-baclofen was prevented by tetrodotoxin and hyoscine but not by phentolamine plus propranolol, naloxone or theophylline.5 In preparations in which the muscle tone was raised by histamine or prostaglandin F(2alpha), GABA and (-)-baclofen induced relaxation to the same extent as before increasing the tone. If the tone was raised by DMPP, a greater bicuculline-insensitive relaxation occurred.6 Contraction caused by GABA was bicuculline-sensitive and was mimicked by homotaurine and muscimol. Contraction was dose-dependent and muscimol was about three times more potent than GABA or homotaurine. A specific cross-desensitization occurred between the contractile effects of GABA and those of homotaurine or muscimol.7 Bicuculline competitively antagonized the contractile effects of GABA, homotaurine and muscimol and gave closely similar pA(2) values. The slope of the Schild plot for the above drugs was near 1, confirming the competitive nature of the antagonism.8 The bicuculline-sensitive contraction induced by GABA, homotaurine and muscimol was abolished by tetrodotoxin and was non-competitively antagonized by hyoscine, while it was unaffected by hexamethonium, mepyramine and methysergide.9 It is concluded that two receptors mediate the GABA effects in guinea

  14. Characterization of prejunctional 5-HT receptors mediating inhibition of sympathetic vasopressor responses in the pithed rat.

    PubMed

    Villalón, C M; Contreras, J; Ramírez-San Juan, E; Castillo, C; Perusquía, M; Terrón, J A

    1995-12-01

    1. It has recently been shown that continuous infusions of 5-hydroxytryptamine (5-HT) are able to inhibit, in a dose-dependent manner, the pressor responses induced by preganglionic (T7-T9) sympathetic stimulation in pithed rats pretreated with desipramine (50 micrograms kg-1, i.v.). This inhibitory effect, besides being significantly more pronounced at lower frequencies of stimulation (0.03-I Hz) and devoid of tachyphylaxis, is reversible after interrupting the infusions of 5-HT (up to 5.6 micrograms kg-1 min-1). In the present study we have characterized the pharmacological profile of the receptors mediating the above inhibitory effect of 5-HT. 2. The inhibition induced by 5.6 micrograms kg-1 min-1 of 5-HT on sympathetically-induced pressor responses was not blocked after i.v. treatment with physiological saline (1 ml kg-1), ritanserin (0.1 mg kg-1), MDL 72222 (0.15 mg kg-1) or tropisetron (3 mg kg-1), which did not modify the sympathetically-induced pressor responses per se, but was significantly antagonized by the 5-HT1-like and 5-HT2 receptor antagonist, methysergide (0.3 mg kg-1), which also produced a slight attenuation of the pressor responses to 0.03 and 0.1 Hz per se. 3. Unexpectedly and contrasting with methysergide, the 5-HT1-like and 5-HT2 receptor antagonists, methiothepin (0.01, 0.03 and 0.1 mg kg-1) and metergoline (1 and 3 mg kg-1), apparently failed to block the above 5-HT-induced inhibition. Nevertheless, it is noteworthy that these antagonists also blocked the electrically-induced pressor responses per se, presumably by blockade of vascular alpha 1-adrenoceptors and, indeed, this property might have masked their potential antagonism at the inhibitory 5-HT1-like receptors. 4. Consistent with the above findings, 5-carboxamidotryptamine (5-CT, a potent 5-HT1-like receptor agonist), metergoline and methysergide mimicked the inhibitory action of 5-HT with the following rank order of agonist potency: 5CT > > 5-HT > metergoline > or = methysergide. 5

  15. Adenosine A2B-receptor-mediated cyclic AMP accumulation in primary rat astrocytes.

    PubMed Central

    Peakman, M. C.; Hill, S. J.

    1994-01-01

    1. The effects of adenosine receptor agonists and antagonists on the accumulation of cyclic AMP have been investigated in primary cultures of rat astrocytes. 2. Adenosine A2-receptor stimulation caused a concentration-dependent increase in the accumulation of [3H]-cyclic AMP in cells prelabelled with [3H]-adenine. The rank order of agonist potencies was 5'-N-ethylcarboxamidoadenosine (NECA; EC50 = 1 microM) > adenosine (EC50 = 5 microM) > 2-chloroadenosine (EC50 = 20 microM) >> CGS 21680 (EC50 > 10 microM). The presence of 0.5 microM dipyridamole, an adenosine uptake blocker, had no effect on the potency of adenosine. 3. The response to 10 microM NECA was antagonized in a concentration-dependent manner by the non-selective adenosine receptor antagonists, xanthine amine congener (apparent KD = 12 nM), PD 115,199 (apparent KD = 134 nM) and 8-phenyltheophylline (apparent KD = 126 nM). However, the A1-receptor-selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine, had no significant effect on the responses to NECA or 2-chloroadenosine at concentrations up to 1 microM. 4. Stimulation of A1-receptors with the selective agonist, N6-cyclopentyladenosine, did not alter the basal accumulation of [3H]-cyclic AMP but inhibited a forskolin-mediated elevation of [3H]-cyclic AMP accumulation by a maximal value of 42%. This inhibition was fully reversed in the presence of 0.1 microM, 8-cyclopentyl-1,3-dipropylxanthine. 5. The time course for NECA-mediated [3H]-cyclic AMP accumulation was investigated. The results suggest that there is a substantial efflux of cyclic AMP from the cells in addition to the rapid and sustained elevation of intracellular cyclic AMP (5 fold over basal) which was also observed. 6. These data indicate that rat astrocytes in primary culture express an A2B-adenosine receptor coupled positively to adenylyl cyclase. Furthermore, the presence of A1-receptors negatively coupled to adenylyl cyclase appears to have no significant effect on the A2B-receptor-mediated

  16. Cannabinoid CB1 receptor mediates glucocorticoid effects on hormone secretion induced by volume and osmotic changes.

    PubMed

    Ruginsk, S G; Uchoa, E T; Elias, L L K; Antunes-Rodrigues, J

    2012-02-01

    The present study provides the first in vivo evidence that the cannabinoid CB(1) receptor mediates the effects of dexamethasone on hormone release induced by changes in circulating volume and osmolality. Male adult rats were administered with the CB(1) receptor antagonist rimonabant (10 mg/Kg, p.o.), followed or not in 1 hour by dexamethasone (1 mg/Kg, i.p.). Extracellular volume expansion (EVE, 2 mL/100 g of body weight, i.v.) was performed 2 hours after dexamethasone or vehicle treatment using either isotonic (I-EVE, 0.15 mol/L) or hypertonic (H-EVE, 0.30 mol/L) NaCl solution. Five minutes after EVE, animals were decapitated and trunk blood was collected for all plasma measurements. Rimonabant potentiated oxytocin (OT) secretion induced by H-EVE and completely reversed the inhibitory effects of dexamethasone in response to the same stimulus. These data suggest that glucocorticoid modulation of OT release is mediated by the CB(1) receptor. Although dexamethasone did not affect vasopressin (AVP) secretion induced by H-EVE, the administration of rimonabant potentiated AVP release in response to the same stimulus, supporting the hypothesis that the CB(1) receptor regulates AVP secretion independently of glucocorticoid-mediated signalling. Dexamethasone alone did not affect atrial natriuretic peptide (ANP) release stimulated by I-EVE or H-EVE. However, pretreatment with rimonabant potentiated ANP secretion induced by H-EVE, suggesting a possible role for the CB(1) receptor in the control of peripheral factors that modulate cardiovascular function. Rimonabant also reversed the inhibitory effects of dexamethasone on H-EVE-induced corticosterone secretion, reinforcing the hypothesis that the CB(1) receptor may be involved in the negative feedback exerted by glucocorticoids on the activity of the hypothalamic-pituitary-adrenal axis. Collectively, the results of the present study indicate that the CB(1) receptor modulates neurohypophyseal hormone secretion and

  17. The Impact of Hyperthermia on Receptor-Mediated Interleukin-6 Regulation in Mouse Skeletal Muscle

    PubMed Central

    Welc, Steven S.; Morse, Deborah A.; Mattingly, Alex J.; Laitano, Orlando; King, Michelle A.; Clanton, Thomas L.

    2016-01-01

    In inflammatory cells, hyperthermia inhibits lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) gene expression and protein secretion. Since hyperthermia alone stimulates IL-6 in skeletal muscle, we hypothesized that it would amplify responses to other receptor-mediated stimuli. IL-6 regulation was tested in C2C12 myotubes and in soleus during treatment with epinephrine (EPI) or LPS. In EPI-treated myotubes (100 ng/ml), 1 h exposure at 40.5°C-42°C transiently increased IL-6 mRNA compared to EPI treatment alone at 37°C. In LPS-treated myotubes (1 μg/ml), exposure to 41°C-42°C also increased IL-6 mRNA. In isolated mouse soleus, similar amplifications of IL-6 gene expression were observed in 41°C, during both low (1 ng/ml) and high dose (100 ng/ml) EPI, but only in high dose LPS (1 μg/ml). In myotubes, heat increased IL-6 secretion during EPI exposure but had no effect or inhibited secretion with LPS. In soleus there were no effects of heat on IL-6 secretion during either EPI or LPS treatment. Mechanisms for the effects of heat on IL-6 mRNA were explored using a luciferase-reporter in C2C12 myotubes. Overexpression of heat shock factor-1 (HSF-1) had no impact on IL-6 promoter activity during EPI stimulation, but elevated IL-6 promoter activity during LPS stimulation. In contrast, when the activator protein-1 (AP-1) element was mutated, responses to both LPS and EPI were suppressed in heat. Using siRNA against activating transcription factor-3 (ATF-3), a heat-stress-induced inhibitor of IL-6, no ATF-3-dependent effects were observed. The results demonstrate that, unlike inflammatory cells, hyperthermia in muscle fibers amplifies IL-6 gene expression to EPI and LPS. The effect appears to reflect differential engagement of HSF-1 and AP-1 sensitive elements on the IL-6 gene, with no evidence for involvement of ATF-3. The functional significance of increased IL-6 mRNA expression during heat may serve to overcome the well-known suppression of protein synthetic

  18. Lipoprotein lipase regulates Fc receptor-mediated phagocytosis by macrophages maintained in glucose-deficient medium.

    PubMed Central

    Yin, B; Loike, J D; Kako, Y; Weinstock, P H; Breslow, J L; Silverstein, S C; Goldberg, I J

    1997-01-01

    During periods of intense activity such as phagocytosis, macrophages are thought to derive most of their energy from glucose metabolism under both aerobic and anaerobic conditions. To determine whether fatty acids released from lipoproteins by macrophage lipoprotein lipase (LPL) could substitute for glucose as a source of energy for phagocytosis, we cultured peritoneal macrophages from normal and LPL knockout (LPL-KO) mice that had been rescued from neonatal demise by expression of human LPL via the muscle creatine kinase promoter. Normal and LPL-KO macrophages were cultured in medium containing normal (5 mM) or low (1 mM) glucose, and were tested for their capacity to phagocytose IgG-opsonized sheep erythrocytes. LPL-KO macrophages maintained in 1 and 5 mM glucose phagocytosed 67 and 79% fewer IgG-opsonized erythrocytes, respectively, than macrophages from normal mice. Addition of VLDL to LPL-expressing macrophages maintained in 1 mM glucose enhanced the macrophages' phagocytosis of IgG-opsonized erythrocytes, but did not stimulate phagocytosis by LPL-KO macrophages. Inhibition of secreted LPL with a monoclonal anti-LPL antibody or with tetrahydrolipstatin blocked the ability of VLDL to enhance phagocytosis by LPL-expressing macrophages maintained in 1 mM glucose. Addition of oleic acid significantly enhanced phagocytosis by both LPL-expressing and LPL-KO macrophages maintained in 1 mM glucose. Moreover, oleic acid stimulated phagocytosis in cells cultured in non-glucose-containing medium, and increased the intracellular stores of creatine phosphate. Inhibition of oxidative phosphorylation, but not of glycolysis, blocked the capacity of oleic acid to stimulate phagocytosis. Receptor-mediated endocytosis of acetyl LDL by macrophages from LPL-expressing and LPL-KO mice was similar whether the cells were maintained in 5 or 1 mM glucose, and was not augmented by VLDL. We postulate that fatty acids derived from macrophage LPL-catalyzed hydrolysis of triglycerides and

  19. Pharmacological characterization of prostanoid receptors mediating vasoconstriction in human umbilical vein

    PubMed Central

    Daray, Federico Manuel; Minvielle, Ana Itatí; Puppo, Soledad; Rothlin, Rodolfo Pedro

    2003-01-01

    This study was undertaken to characterize pharmacologically the prostanoid receptor subtypes mediating contraction in human umbilical vein (HUV).HUV rings were mounted in organ baths and concentration–response curves to U-46619 (TXA2 mimetic) were constructed in the absence or presence of SQ-29548 or ICI-192,605 (TP receptor antagonists). U-46619 was a potent constrictor (pEC50: 8.03). SQ-29548 and ICI-192,605 competitively antagonized responses to U-46619 with pKB values of 7.96 and 9.07, respectively.Concentration–response curves to EP receptor agonists: PGE2, misoprostol and 17-phenyl-trinor-PGE2 gave pEC50 values of 5.06, 5.25 and 5.32, respectively. Neither pEC50 nor maximum of PGE2 and 17-phenyl-trinor-PGE2 concentration–response curves were modified by the DP/EP1/EP2 receptor antagonist AH 6809 (1 μM). However, ICI-192,605 produced a concentration-dependent antagonism of the responses to all the EP receptor agonists. The pA2 estimated for ICI-192,605 against PGE2 or misoprostol were 8.91 and 9.22, respectively.Concentration–response curves to FP receptor agonists: PGF2α and fluprostenol gave pEC50 values of 6.20 and 5.82, respectively. ICI-192,605 (100 nM) was completely ineffective against PGF2α or fluprostenol. In addition, lack of antagonistic effect of AH 6809 (1 μM) against PGF2α was observed.In conclusion, the findings obtained with TP-selective agonist and antagonists provide strong evidence of the involvement of TP receptors promoting vasoconstriction in HUV. Furthermore, the action of the natural and synthetic EP receptor agonists appears to be mediated via TP receptors. On the other hand, the results employing FP receptor agonists and antagonists of different prostanoid receptors suggest the presence of FP receptors mediating vasoconstriction in this vessel. PMID:12922927

  20. Characterization of prejunctional 5-HT receptors mediating inhibition of sympathetic vasopressor responses in the pithed rat.

    PubMed Central

    Villalón, C. M.; Contreras, J.; Ramírez-San Juan, E.; Castillo, C.; Perusquía, M.; Terrón, J. A.

    1995-01-01

    1. It has recently been shown that continuous infusions of 5-hydroxytryptamine (5-HT) are able to inhibit, in a dose-dependent manner, the pressor responses induced by preganglionic (T7-T9) sympathetic stimulation in pithed rats pretreated with desipramine (50 micrograms kg-1, i.v.). This inhibitory effect, besides being significantly more pronounced at lower frequencies of stimulation (0.03-I Hz) and devoid of tachyphylaxis, is reversible after interrupting the infusions of 5-HT (up to 5.6 micrograms kg-1 min-1). In the present study we have characterized the pharmacological profile of the receptors mediating the above inhibitory effect of 5-HT. 2. The inhibition induced by 5.6 micrograms kg-1 min-1 of 5-HT on sympathetically-induced pressor responses was not blocked after i.v. treatment with physiological saline (1 ml kg-1), ritanserin (0.1 mg kg-1), MDL 72222 (0.15 mg kg-1) or tropisetron (3 mg kg-1), which did not modify the sympathetically-induced pressor responses per se, but was significantly antagonized by the 5-HT1-like and 5-HT2 receptor antagonist, methysergide (0.3 mg kg-1), which also produced a slight attenuation of the pressor responses to 0.03 and 0.1 Hz per se. 3. Unexpectedly and contrasting with methysergide, the 5-HT1-like and 5-HT2 receptor antagonists, methiothepin (0.01, 0.03 and 0.1 mg kg-1) and metergoline (1 and 3 mg kg-1), apparently failed to block the above 5-HT-induced inhibition. Nevertheless, it is noteworthy that these antagonists also blocked the electrically-induced pressor responses per se, presumably by blockade of vascular alpha 1-adrenoceptors and, indeed, this property might have masked their potential antagonism at the inhibitory 5-HT1-like receptors. 4. Consistent with the above findings, 5-carboxamidotryptamine (5-CT, a potent 5-HT1-like receptor agonist), metergoline and methysergide mimicked the inhibitory action of 5-HT with the following rank order of agonist potency: 5CT > > 5-HT > metergoline > or = methysergide. 5

  1. Pharmacological and biochemical characterization of the D-1 dopamine receptor mediating acetylcholine release in rabbit retina

    SciTech Connect

    Hensler, J.G.; Cotterell, D.J.; Dubocovich, M.L.

    1987-12-01

    Superfusion with dopamine (0.1 microM-10 mM) evokes calcium-dependent (/sup 3/H)acetylcholine release from rabbit retina labeled in vitro with (/sup 3/H)choline. This effect is antagonized by the D-1 dopamine receptor antagonist SCH 23390. Activation or blockade of D-2 dopamine, alpha-2 or beta receptors did not stimulate or attenuate the release of (/sup 3/H)acetylcholine from rabbit retina. Dopamine receptor agonists evoke the release of (/sup 3/H)acetylcholine with the following order of potency: apomorphine less than or equal to SKF(R)82526 < SKF 85174 < SKF(R)38393 less than or equal to pergolide less than or equal to dopamine (EC50 = 4.5 microM) < SKF(S)82526 less than or equal to SKF(S)38393. Dopamine receptor antagonists inhibited the dopamine-evoked release of (/sup 3/H)acetylcholine: SCH 23390 (IC50 = 1 nM) < (+)-butaclamol less than or equal to cis-flupenthixol < fluphenazine < perphenazine < trans-flupenthixol < R-sulpiride. The potencies of dopamine receptor agonists and antagonists at the dopamine receptor mediating (/sup 3/H)acetylcholine release is characteristic of the D-1 dopamine receptor. These potencies were correlated with the potencies of dopamine receptor agonists and antagonists at the D-1 dopamine receptor in rabbit retina as labeled by (/sup 3/H)SCH 23390, or as determined by adenylate cyclase activity. (/sup 3/H)SCH 23390 binding in rabbit retinal membranes was stable, saturable and reversible. Scatchard analysis of (/sup 3/H)SCH 23390 saturation data revealed a single high affinity binding site (Kd = 0.175 +/- 0.002 nM) with a maximum binding of 482 +/- 12 fmol/mg of protein. The potencies of dopamine receptor agonists to stimulate (/sup 3/H)acetylcholine release were correlated with their potencies to stimulate adenylate cyclase (r = 0.784, P less than .05, n = 7) and with their affinities at (/sup 3/H)SCH 23390 binding sites (r = 0.755, P < .05, n = 8).

  2. Peroxynitrite induces apoptosis and decline in intracellular free Mg with concomitant elevation in [Ca2+]I in rat aortic smooth muscle cells: possible roles of extracellular and intracellular magnesium ions in peroxynitrite-induced cell death.

    PubMed

    Li, Jianfeng; Li, Wenyan; Liu, Weimin; Altura, Bella T; Altura, Burton M

    2007-04-01

    The present study demonstrates that exogenous ONOO(-) can result in rapid declines in intracellular free magnesium ions ([Mg(2+)](i)) concomitant with rapid rises in intracellular free calcium ions ([Ca(2+)](i)) and, subsequently, trigger apoptosis but not necrosis in rat aortic SMCs; high [Mg(2+)] significantly attenuates ONOO(-)-induced apoptosis. ONOO(-)-induced apoptosis in vascular SMCs appears to involve activation of Ca(2+)-Mg(2+)-dependent endonucleases and caspase-3. Mg deficiency itself could not induce apoptosis in these SMCs, but it could significantly enhance ONOO(-)-induced apoptosis.

  3. Zonal differences in ethanol-induced impairments in receptor-mediated endocytosis of asialoglycoproteins in isolated rat hepatocytes

    SciTech Connect

    Casey, C.A.; Kragskow, S.L.; Sorrell, M.F.; Tuma, D.J. )

    1991-02-01

    We have shown previously that ethanol-induced defects in receptor-mediated endocytosis of asialoorosomucoid occurred as early as 1 wk after ethanol feeding. This study was undertaken as an initial attempt to establish a possible role of defective receptor-mediated endocytosis in liver injury by investigating whether differences exist in the effects of ethanol on receptor-mediated endocytosis in hepatocytes isolated from different regions of the liver. Perivenule cells, present in the distal half of the liver, are thought to be more susceptible to ethanol-induced liver injury than are the periportal cells located in the proximal half of the liver acini. For these studies, we fed male Sprague-Dawley rats for 7 days with liquid diets containing either ethanol (36% of calories) or isocaloric carbohydrate. Perivenule and periportal hepatocytes were then isolated using a digitonin-collagenase perfusion method. In control animals, cells isolated from the perivenule region bound significantly more ligand than did cells from the periportal region. Amounts of ligand internalized and degraded were also greater in perivenule than in periportal cells in these animals. After ethanol feeding, cells isolated from both the perivenule and periportal regions bound significantly less ligand than their respective controls. This impairment in surface and total binding was more pronounced in perivenule than in periportal cells. Internalization and degradation of the ligand were also more adversely affected in the centrilobular region as shown by decreases of greater than 60% in perivenule cells and by only 20% to 30% in periportal cells of ethanol-fed animals compared with controls.

  4. Enhancement of postsynaptic GABAA and extrasynaptic NMDA receptor-mediated responses in the barrel cortex of Mecp2-null mice

    PubMed Central

    Lo, Fu-Sun; Blue, Mary E.

    2015-01-01

    Rett syndrome (RTT) is a neurodevelopmental disorder that results from mutations in the X-linked gene for methyl-CpG-binding protein 2 (MECP2). The underlying cellular mechanism for the sensory deficits in patients with RTT is largely unknown. This study used the Bird mouse model of RTT to investigate sensory thalamocortical synaptic transmission in the barrel cortex of Mecp2-null mice. Electrophysiological results showed an excitation/inhibition imbalance, biased toward inhibition, due to an increase in efficacy of postsynaptic GABAA receptors rather than alterations in inhibitory network and presynaptic release properties. Enhanced inhibition impaired the transmission of tonic sensory signals from the thalamus to the somatosensory cortex. Previous morphological studies showed an upregulation of NMDA receptors in the neocortex of both RTT patients and Mecp2-null mice at early ages [Blue ME, Naidu S, Johnston MV. Ann Neurol 45: 541–545, 1999; Blue ME, Kaufmann WE, Bressler J, Eyring C, O'Driscoll C, Naidu S, Johnston MV. Anat Rec (Hoboken) 294: 1624–1634, 2011]. Although AMPA and NMDA receptor-mediated excitatory synaptic transmission was not altered in the barrel cortex of Mecp2-null mice, extrasynaptic NMDA receptor-mediated responses increased markedly. These responses were blocked by memantine, suggesting that extrasynaptic NMDA receptors play an important role in the pathogenesis of RTT. The results suggest that enhancement of postsynaptic GABAA and extrasynaptic NMDA receptor-mediated responses may underlie impaired somatosensation and that pharmacological blockade of extrasynaptic NMDA receptors may have therapeutic value for RTT. PMID:26683074

  5. Synaptic NMDA receptor-mediated currents in anterior piriform cortex are reduced in the adult fragile X mouse.

    PubMed

    Gocel, James; Larson, John

    2012-09-27

    Fragile X syndrome is a neurodevelopmental condition caused by the transcriptional silencing of the fragile X mental retardation 1 (FMR1) gene. The Fmr1 knockout (KO) mouse exhibits age-dependent deficits in long term potentiation (LTP) at association (ASSN) synapses in anterior piriform cortex (APC). To investigate the mechanisms for this, whole-cell voltage-clamp recordings of ASSN stimulation-evoked synaptic currents were made in APC of slices from adult Fmr1-KO and wild-type (WT) mice, using the competitive N-methyl-D-aspartate (NMDA) receptor antagonist, CPP, to distinguish currents mediated by NMDA and AMPA receptors. NMDA/AMPA current ratios were lower in Fmr1-KO mice than in WT mice, at ages ranging from 3-18months. Since amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs) mediated by AMPA receptors were no different in Fmr1-KO and WT mice at these ages, the results suggest that NMDA receptor-mediated currents are selectively reduced in Fmr1-KO mice. Analyses of voltage-dependence and decay kinetics of NMDA receptor-mediated currents did not reveal differences between Fmr1-KO and WT mice, suggesting that reduced NMDA currents in Fmr1-KO mice are due to fewer synaptic receptors rather than differences in receptor subunit composition. Reduced NMDA receptor signaling may help to explain the LTP deficit seen at APC ASSN synapses in Fmr1-KO mice at 6-18months of age, but does not explain normal LTP at these synapses in mice 3-6months old. Evoked currents and mEPSCs were also examined in senescent Fmr1-KO and WT mice at 24-28months of age. NMDA/AMPA ratios were similar in senescent WT and Fmr1-KO mice, due to a decrease in the ratio in the WT mice, without significant change in AMPA receptor-mediated mEPSCs.

  6. Enhancement of postsynaptic GABAA and extrasynaptic NMDA receptor-mediated responses in the barrel cortex of Mecp2-null mice.

    PubMed

    Lo, Fu-Sun; Blue, Mary E; Erzurumlu, Reha S

    2016-03-01

    Rett syndrome (RTT) is a neurodevelopmental disorder that results from mutations in the X-linked gene for methyl-CpG-binding protein 2 (MECP2). The underlying cellular mechanism for the sensory deficits in patients with RTT is largely unknown. This study used the Bird mouse model of RTT to investigate sensory thalamocortical synaptic transmission in the barrel cortex of Mecp2-null mice. Electrophysiological results showed an excitation/inhibition imbalance, biased toward inhibition, due to an increase in efficacy of postsynaptic GABAA receptors rather than alterations in inhibitory network and presynaptic release properties. Enhanced inhibition impaired the transmission of tonic sensory signals from the thalamus to the somatosensory cortex. Previous morphological studies showed an upregulation of NMDA receptors in the neocortex of both RTT patients and Mecp2-null mice at early ages [Blue ME, Naidu S, Johnston MV. Ann Neurol 45: 541-545, 1999; Blue ME, Kaufmann WE, Bressler J, Eyring C, O'Driscoll C, Naidu S, Johnston MV. Anat Rec (Hoboken) 294: 1624-1634, 2011]. Although AMPA and NMDA receptor-mediated excitatory synaptic transmission was not altered in the barrel cortex of Mecp2-null mice, extrasynaptic NMDA receptor-mediated responses increased markedly. These responses were blocked by memantine, suggesting that extrasynaptic NMDA receptors play an important role in the pathogenesis of RTT. The results suggest that enhancement of postsynaptic GABAA and extrasynaptic NMDA receptor-mediated responses may underlie impaired somatosensation and that pharmacological blockade of extrasynaptic NMDA receptors may have therapeutic value for RTT.

  7. Wnt5a promotes cancer cell invasion and proliferation by receptor-mediated endocytosis-dependent and -independent mechanisms, respectively

    PubMed Central

    Shojima, Kensaku; Sato, Akira; Hanaki, Hideaki; Tsujimoto, Ikuko; Nakamura, Masahiro; Hattori, Kazunari; Sato, Yuji; Dohi, Keiji; Hirata, Michinari; Yamamoto, Hideki; Kikuchi, Akira

    2015-01-01

    Wnt5a activates the Wnt/β-catenin-independent pathway and its overexpression is associated with tumor aggressiveness enhancing invasive activity. For this action, Wnt5a-induced receptor endocytosis with clathrin is required. Wnt5a expression was previously believed to be associated with cancer cell motility but not proliferation. Recently, it was reported that Wnt5a is also implicated in cancer cell proliferation, but the mechanism was not clear. In this study, we generated a neutralizing anti-Wnt5a monoclonal antibody (mAb5A16) to investigate the mechanism by which Wnt5a regulates cancer cell proliferation. Wnt5a stimulated both invasion and proliferation of certain types of cancer cells, including HeLaS3 cervical cancer cells and A549 lung cancer cells although Wnt5a promoted invasion but not proliferation in other cancer cells such as KKLS gastric cancer cells. mAb5A16 did not affect the binding of Wnt5a to its receptor, but it suppressed Wnt5a-induced receptor-mediated endocytosis. mAb5A16 inhibited invasion but not proliferation of HeLaS3 and A549 cells. Wnt5a activated Src family kinases (SFKs) and Wnt5a-dependent cancer cell proliferation was dependent on SFKs, yet blockade of receptor-mediated endocytosis did not affect cancer cell proliferation and SFK activity. These results suggest that Wnt5a promotes invasion and proliferation of certain types of cancer cells through receptor-mediated endocytosis-dependent and -independent mechanisms, respectively. PMID:25622531

  8. NMDA receptor-mediated excitotoxicity depends on the coactivation of synaptic and extrasynaptic receptors.

    PubMed

    Zhou, X; Hollern, D; Liao, J; Andrechek, E; Wang, H

    2013-03-28

    N-methyl-D-aspartate receptors (NMDAR) overactivation is linked to neurodegeneration. The current prevailing theory suggests that synaptic and extrasynaptic NMDAR (syn- and ex-NMDAR) impose counteracting effects on cell fate, and neuronal cell death is mainly mediated by the activation of ex-NMDAR. However, several lines of evidence implicate the limitation of this theory. Here, we demonstrate that activation of NMDAR bi-directionally regulated cell fate through stimulating pro-survival or pro-death signaling. While low-dose NMDA preferentially activated syn-NMDAR and stimulated the extracellular signal-regulated kinase ½-cAMP responsive element-binding protein-brain-derived neurotrophic factor pro-survival signaling, higher doses progressively activated increasing amount of ex-NMDAR along with syn-NMDAR and triggered cell death program. Interestingly, the activation of syn- or ex-NMDAR alone did not cause measurable cell death. Consistently, activation of syn- or ex-NMDAR alone stimulated pro-survival but not pro-death signaling. Next, we found that memantine, which was previously identified as an ex-NMDAR blocker, inhibited intracellular signaling mediated by syn- or ex-NMDAR. Simultaneous blockade of syn- and ex-NMDAR by memantine dose-dependently attenuated NMDAR-mediated death. Moreover, long- but not short-term treatment with high-dose NMDA or oxygen-glucose deprivation triggered cell death and suppressed pro-survival signaling. These data implicate that activation of syn- or ex-NMDAR alone is not neurotoxic. The degree of excitotoxicity depends on the magnitude and duration of syn- and ex-NMDAR coactivation. Finally, genome-wide examination demonstrated that the activation of syn- and ex-NMDAR lead to significant overlapping rather than counteracting transcriptional responses.

  9. Apoptosis in metanephric development

    PubMed Central

    1992-01-01

    During metanephric development, non-polarized mesenchymal cells are induced to form the epithelial structures of the nephron following interaction with extracellular matrix proteins and factors produced by the inducing tissue, ureteric bud. This induction can occur in a transfilter organ culture system where it can also be produced by heterologous cells such as the embryonic spinal cord. We found that when embryonic mesenchyme was induced in vitro and in vivo, many of the cells surrounding the new epithelium showed morphological evidence of programmed cell death (apoptosis) such as condensed nuclei, fragmented cytoplasm, and cell shrinking. A biochemical correlate of apoptosis is the transcriptional activation of a calcium-sensitive endonuclease. Indeed, DNA isolated from uninduced mesenchyme showed progressive degradation, a process that was prevented by treatment with actinomycin- D or cycloheximide and by buffering intracellular calcium. These results demonstrate that the metanephric mesenchyme is programmed for apoptosis. Incubation of mesenchyme with a heterologous inducer, embryonic spinal cord prevented this DNA degradation. To investigate the mechanism by which inducers prevented apoptosis we tested the effects of protein kinase C modulators on this process. Phorbol esters mimicked the effects of the inducer and staurosporine, an inhibitor of this protein kinase, prevented the effect of the inducer. EGF also prevented DNA degradation but did not lead to differentiation. These results demonstrate that conversion of mesenchyme to epithelial requires at least two steps, rescue of the mesenchyme from apoptosis and induction of differentiation. PMID:1447305

  10. Mechanisms of Virus-Induced Neural Cell Death

    DTIC Science & Technology

    2003-09-01

    We are using experimental infection with reoviruses to study how viruses induce cell death . (apoptosis), and the significance of apoptosis in the...pathogenesis of viral infection. We have developed one of the best-characterized experimental models for investigating and manipulating viral cell death pathways...We have shown that apoptosis is a major mechanism of reovirus-induced cell death in murine models of key human viral infections including

  11. Modulation of PKC signaling and induction of apoptosis through suppression of reactive oxygen species and tumor necrosis factor receptor 1 (TNFR1): key role of quercetin in cancer prevention.

    PubMed

    Maurya, Akhilendra Kumar; Vinayak, Manjula

    2015-11-01

    Cancer cells are characterized by increased production of reactive oxygen species (ROS) and an altered redox environment as compared to normal cells. Continuous accumulation of ROS triggers oxidative stress leading to hyper-activation of signaling pathways that promote cell proliferation, survival, and metabolic adaptation to the tumor microenvironment. Therefore, antioxidants are proposed to contribute to cancer prevention. Protein kinase C (PKC) is a crucial regulator of diverse cellular processes and contributes to cancer progression. The activation of PKC is partially dependent on ROS signaling. In the present study, cancer preventive activity of natural flavonoid quercetin is analyzed in ascite cells of Dalton's lymphoma-bearing mice. The total ROS level and activity of PKC were downregulated after quercetin treatment in lymphoma-bearing mice. Quercetin modulates the expression of almost all isozymes of classical, novel, and atypical PKC as well as downregulates the level and expression of PKCα. Further, quercetin improves apoptotic potential, as observed by the levels of caspase 3, caspase 9, PARP, PKCδ, and nuclear condensation. Additionally, quercetin reduces cell survival and promotes death receptor-mediated apoptosis via differential localization of the TNFR1 level in ascite cells. The overall result suggests the cancer preventive activity of quercetin via the induction of apoptosis and modulates PKC signaling with the reduction of oxidative stress in ascite cells of lymphoma-bearing mice.

  12. Regulation of muscarinic acetylcholine receptor-mediated synaptic responses by adenosine receptors in the rat hippocampus.

    PubMed Central

    Morton, R A; Davies, C H

    1997-01-01

    '-N-ethylcarboxamidoadenosine (CGS 21680; 0.5-1.0 microM) did not significantly affect the EPSPm. 4. The selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 0.2 microM) fully reversed the depressant effects of both adenosine (100 microM) and CADO (1 microM) on the EPSPm and the stimulus-evoked reductions in spike frequency adaptation. 5. DPCPX (0.2 microM) alone caused a small but variable mean increase in the EPSPm of 22 +/- 19% and enabled activation of an EPSPm by a previously subthreshold stimulus. In contrast, the selective adenosine kinase inhibitor 5-iodotubercidin (5-IT; 10 microM) inhibited the EPSPm by 74 +/- 10%, an effect that was reversed by DPCPX. 6. The concentration-response relationship for the depressant action of CADO on the EPSPm more closely paralleled that for its presynaptic depressant action on glutamate-mediated EPSPs than that for postsynaptic hyperpolarization. The respective mean IC50 and EC50 concentrations for these effects were 0.3, 0.8 and 3.0 microM. 7. CADO (1-5 microM) did not have a significant effect on the postsynaptic depolarization, increase in input resistance and reduction in spike frequency adaptation evoked by carbachol (0.5-3.0 microM). All these effects were abolished by atropine (1 microM). 8. These data provide good evidence for an adenosine A1 receptor-mediated inhibition of mAChR-mediated synaptic responses in hippocampal CA1 pyramidal neurones. This inhibition is mediated predominantly presynaptically, is active tonically and can be enhanced when extracellular levels of endogenous adenosine are raised. PMID:9234198

  13. 5-Carboxamidotryptamine is a selective agonist at 5-hydroxytryptamine receptors mediating vasodilatation and tachycardia in anaesthetized cats.

    PubMed Central

    Connor, H. E.; Feniuk, W.; Humphrey, P. P.; Perren, M. J.

    1986-01-01

    We have attempted to characterize the 5-hydroxytryptamine (5-HT) receptors mediating bronchoconstriction, vasodilatation, vasodepression and tachycardia in anaesthetized cats following bilateral vagosympathectomy and beta-adrenoceptor blockade with propranolol. 5-HT (1-100 micrograms/kg-1 i.v.) caused dose-related bronchoconstriction and tachycardia but variable and complex effects on diastolic blood pressure and carotid arterial vascular resistance. In contrast, 5-carboxamidotryptamine (5-CT; 0.01-1 micrograms kg-1 i.v.) caused consistent, dose-related decreases in diastolic blood pressure and carotid arterial vascular resistance and increases in heart rate. 5-CT did not cause bronchoconstriction. The 5-HT-induced bronchoconstriction was dose-dependently antagonized by methiothepin, methysergide and ketanserin (10-100 micrograms kg-1 i.v.). The highest doses used of these antagonists did not antagonize bronchoconstriction induced by prostaglandin F2 alpha. The high potency of all three antagonists indicate a 5-HT2-receptor mediated effect. The 5-HT- and 5-CT-induced tachycardia as well as the 5-CT-induced vasodepressor and carotid arterial vasodilator responses were dose-dependently antagonized by low doses of methiothepin (10-100 micrograms kg-1 i.v.) and by high doses of methysergide (100-1000 micrograms kg-1 i.v.) but were little affected by ketanserin in doses up to 1000 micrograms kg-1 i.v. These selective effects of 5-CT appear to be mediated by '5-HT1-like' receptors. PMID:2937503

  14. Receptor-mediated endocytosis of albumin by kidney proximal tubule cells is regulated by phosphatidylinositide 3-kinase.

    PubMed Central

    Brunskill, N J; Stuart, J; Tobin, A B; Walls, J; Nahorski, S

    1998-01-01

    Receptor-mediated endocytosis of albumin is an important function of the kidney proximal tubule epithelium. We have measured endocytosis of [125I]-albumin in opossum kidney cells and examined the regulation of this process by phosphatidylinositide 3-kinase (PI 3-kinase). Albumin endocytosis was inhibited by both wortmannin (IC50 6.9 nM) and LY294002 (IC50 6.5 microM) at concentrations that suggested the involvement of PI 3-kinase in its regulation. Recycling rates were unaffected. We transfected OK cells with either a wild-type p85 subunit of PI 3-kinase, or a dominant negative form of the p85 subunit (Deltap85) using the LacSwitch expression system. Transfects were screened by immunoblotting with anti-PI 3-kinase antibodies. Under basal conditions, transfects demonstrated no expression of p85 or Deltap85, but expression was briskly induced by treatment of the cells with IPTG (EC50 13.7 microM). Inhibition of PI 3-kinase activity by Deltap85 was confirmed by in vitro kinase assay of anti-phosphotyrosine immunoprecipitates from transfected cells stimulated with insulin. Expression of Deltap85 resulted in marked inhibition of albumin endocytosis, predominantly as a result of reduction of the Vmax of the transport process. Expression of p85 had no significant effect on albumin uptake. The results demonstrate that PI 3-kinase regulates an early step in the receptor-mediated endocytosis of albumin by kidney proximal tubular cells. PMID:9593770

  15. Receptor-mediated oral delivery of a bioencapsulated green fluorescent protein expressed in transgenic chloroplasts into the mouse circulatory system

    PubMed Central

    Limaye, Arati; Koya, Vijay; Samsam, Mohtashem; Daniell, Henry

    2012-01-01

    Oral delivery of biopharmaceutical proteins expressed in plant cells should reduce their cost of production, purification, processing, cold storage, transportation, and delivery. However, poor intestinal absorption of intact proteins is a major challenge. To overcome this limitation, we investigate here the concept of receptor-mediated oral delivery of chloroplast-expressed foreign proteins. Therefore, the transmucosal carrier cholera toxin B-subunit and green fluorescent protein (CTB-GFP), separated by a furin cleavage site, was expressed via the tobacco chloroplast genome. Polymerase chain reaction (PCR) and Southern blot analyses confirmed site-specific transgene integration and homoplasmy. Immunoblot analysis and ELISA confirmed expression of monomeric and pentameric forms of CTB-GFP, up to 21.3% of total soluble proteins. An in vitro furin cleavage assay confirmed integrity of the engineered furin cleavage site, and a GM1 binding assay confirmed the functionality of CTB-GFP pentamers. Following oral administration of CTB-GFP expressing leaf material to mice, GFP was