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Sample records for death receptor-mediated apoptosis

  1. H-Ras regulation of TRAIL death receptor mediated apoptosis

    PubMed Central

    Chen, Jun-Jie; Bozza, William P.; Di, Xu; Zhang, Yaqin; Hallett, William; Zhang, Baolin

    2014-01-01

    TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis through the death receptors (DRs) 4 and/or 5 expressed on the cell surface. Multiple clinical trials are underway to evaluate the antitumor activity of recombinant human TRAIL and agonistic antibodies to DR4 or DR5. However, their therapeutic potential is limited by the high frequency of cancer resistance. Here we provide evidence demonstrating the role of H-Ras in TRAIL receptor mediated apoptosis. By analyzing the genome wide mRNA expression data of the NCI60 cancer cell lines, we found that H-Ras expression was consistently upregulated in TRAIL-resistant cell lines. By contrast, no correlation was found between TRAIL sensitivity and K-Ras expression levels or their mutational profiles. Notably, H-Ras upregulation associated with a surface deficiency of TRAIL death receptors. Selective inhibition of H-Ras activity in TRAIL-resistant cells restored the surface expression of both DR4 and DR5 without changing their total protein levels. The resulting cells became highly susceptible to both TRAIL and agonistic DR5 antibody, whereas K-Ras inhibition had little or no effect on TRAIL-induced apoptosis, indicating H-Ras plays a distinct role in the regulation of TRAIL death receptors. Further studies are warranted to determine the therapeutic potential of H-Ras-specific inhibitors in combination with TRAIL receptor agonists. PMID:25026275

  2. Nortriptyline induces mitochondria and death receptor-mediated apoptosis in bladder cancer cells and inhibits bladder tumor growth in vivo.

    PubMed

    Yuan, Sheau-Yun; Cheng, Chen-Li; Ho, Hao-Chung; Wang, Shian-Shiang; Chiu, Kun-Yuan; Su, Chung-Kuang; Ou, Yen-Chuan; Lin, Chi-Chen

    2015-08-15

    Nortriptyline (NTP), an antidepressant, has antitumor effects on some human cancer cells, but its effect on human bladder cancer cells is not known. In this study, we used a cell viability assay to demonstrate that NTP is cytotoxic to human TCCSUP and mouse MBT-2 bladder cancer cells in a concentration and time-dependent manner. We also performed cell cycle analysis, annexin V and mitochondrial membrane potential assays, and Western blot analysis to show that NTP inhibits cell growth in these cells by inducing both mitochondria-mediated and death receptor-mediated apoptosis. Specifically, NTP increases the expression of Fas, FasL, FADD, Bax, Bak, and cleaved forms of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase. In addition, NTP decreases the expression of Bcl-2, Bcl-xL, BH3 interacting domain death agonist, X-linked inhibitor of apoptosis protein, and survivin. Furthermore, NTP-induced apoptosis is associated with reactive oxygen species (ROS) production, which can be reduced by antioxidants, such as N-acetyl-L-cysteine. Finally, we showed that NTP suppresses tumor growth in mice inoculated with MBT-2 cells. Collectively, our results suggest that NTP induces both intrinsic and extrinsic apoptosis in human and mouse bladder cancer cells and that it may be a clinically useful chemotherapeutic agent for bladder cancer in humans. PMID:26086857

  3. Poly(ADP-ribose) Polymerase Inhibitors Sensitize Cancer Cells to Death Receptor-mediated Apoptosis by Enhancing Death Receptor Expression*

    PubMed Central

    Meng, X. Wei; Koh, Brian D.; Zhang, Jin-San; Flatten, Karen S.; Schneider, Paula A.; Billadeau, Daniel D.; Hess, Allan D.; Smith, B. Douglas; Karp, Judith E.; Kaufmann, Scott H.

    2014-01-01

    Recombinant human tumor necrosis factor-α-related apoptosis inducing ligand (TRAIL), agonistic monoclonal antibodies to TRAIL receptors, and small molecule TRAIL receptor agonists are in various stages of preclinical and early phase clinical testing as potential anticancer drugs. Accordingly, there is substantial interest in understanding factors that affect sensitivity to these agents. In the present study we observed that the poly(ADP-ribose) polymerase (PARP) inhibitors olaparib and veliparib sensitize the myeloid leukemia cell lines ML-1 and K562, the ovarian cancer line PEO1, non-small cell lung cancer line A549, and a majority of clinical AML isolates, but not normal marrow, to TRAIL. Further analysis demonstrated that PARP inhibitor treatment results in activation of the FAS and TNFRSF10B (death receptor 5 (DR5)) promoters, increased Fas and DR5 mRNA, and elevated cell surface expression of these receptors in sensitized cells. Chromatin immunoprecipitation demonstrated enhanced binding of the transcription factor Sp1 to the TNFRSF10B promoter in the presence of PARP inhibitor. Knockdown of PARP1 or PARP2 (but not PARP3 and PARP4) not only increased expression of Fas and DR5 at the mRNA and protein level, but also recapitulated the sensitizing effects of the PARP inhibition. Conversely, Sp1 knockdown diminished the PARP inhibitor effects. In view of the fact that TRAIL is part of the armamentarium of natural killer cells, these observations identify a new facet of PARP inhibitor action while simultaneously providing the mechanistic underpinnings of a novel therapeutic combination that warrants further investigation. PMID:24895135

  4. Identification of DELE, a novel DAP3-binding protein which is crucial for death receptor-mediated apoptosis induction.

    PubMed

    Harada, Tanenobu; Iwai, Atsushi; Miyazaki, Tadaaki

    2010-10-01

    Death associated protein 3 (DAP3) is known to be a highly conserved protein, and is responsible for regulating apoptosis induced by various stimuli. To understand the molecular mechanism of how DAP3 induces apoptosis, we performed yeast two-hybrid screening, and identified a novel DAP3-binding protein termed death ligand signal enhancer (DELE). In this report, we show that DELE actually binds to DAP3 in mammalian cells. We found that the cells stably expressing DELE are susceptible to apoptosis induction by the stimulation of TNF-α and TRAIL. In addition, knockdown of DELE expression rescued the HeLa cells from apoptosis induction by these stimuli. Moreover, activation of caspase-3, caspase-8 and caspase-9 induced by stimulation of TNF-α, anti-Fas or TRAIL was significantly inhibited by the knockdown of DELE expression. These results demonstrated the biological significance of DELE for apoptosis signal mediated by death receptors. PMID:20563667

  5. Ribavirin and alpha interferon enhance death receptor-mediated apoptosis and caspase activation in human hepatoma cells.

    PubMed

    Schlosser, Stephan F; Schuler, Markus; Berg, Christoph P; Lauber, Kirsten; Schulze-Osthoff, Klaus; Schmahl, Friedrich Wilhelm; Wesselborg, Sebastian

    2003-06-01

    The molecular mechanisms underlying the clinical effects of alpha interferon (IFN) and ribavirin are not understood. Elimination of infected cells occurs in part by cytotoxic T lymphocytes (CTLs) expressing CD95 ligand and thereby attacking target cells which are positive for the death receptor CD95. Since many viruses have evolved mechanisms to inhibit apoptosis, the opposite, namely, promotion of apoptosis, could be a strategy to strengthen the host antiviral response. In the present study, we have asked whether the antiviral substances IFN and ribavirin could support CD95-mediated apoptosis by interfering with the activation of caspases, a family of proteases known for their essential role in apoptosis. HepG2 cells, stimulated with the agonistic anti-CD95 antibody, served as a minimal model to mimic the CD95 stimulation occurring during a CTL attack of target cells in vivo. Apoptosis was quantitated by flow cytometric detection of hypodiploid nuclei. Caspase activity was measured by cytofluorometry, immunocytochemistry, and immunoblot analysis. IFN and ribavirin sensitized HepG2 cells for CD95-mediated apoptosis. This effect was correlated with an increase in CD95-mediated caspase activation and enhanced cleavage of the caspase substrate poly(ADP-ribose) polymerase. Furthermore, the positive effect on CD95-mediated caspase activation by IFN and ribavirin was confirmed by immunocytochemistry for activated caspase-3 and by immunoblot detection of activated caspase-3, caspase-7, and caspase-8. Our data demonstrate that the antiviral substances IFN and ribavirin are able to sensitize for CD95-mediated apoptosis. IFN and ribavirin also enhance CD95-mediated caspase activation, which might in part be responsible for the apoptosis-promoting effect of these antiviral compounds. PMID:12760867

  6. Human papilloma virus 16 E6 RNA interference enhances cisplatin and death receptor-mediated apoptosis in human cervical carcinoma cells.

    PubMed

    Tan, Shinta; Hougardy, Brigitte M T; Meersma, Gert J; Schaap, Bessel; de Vries, Elisabeth G E; van der Zee, Ate G J; de Jong, Steven

    2012-05-01

    In cervical cancer, the p53 and retinoblastoma (pRb) tumor suppressor pathways are disrupted by the human papilloma virus (HPV) E6 and E7 oncoproteins, because E6 targets p53 and E7 targets pRb for rapid proteasome-mediated degradation. We have investigated whether E6 suppression with small interfering RNA (siRNA) restores p53 functionality and sensitizes the HPV16-positive cervical cancer cell line SiHa to apoptosis by cisplatin, irradiation, recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL), or agonistic anti-Fas antibody. E6 siRNA resulted in decreased E6 mRNA levels and enhanced p53 and p21 expression, demonstrating the restoration of p53 functionality in SiHa cells, without inducing high levels of apoptosis (<10%). Cell surface expression of the proapoptotic death receptors (DRs) DR4, DR5, and Fas was not affected by E6 suppression. E6 suppression conferred susceptibility to cisplatin-induced apoptosis but not to irradiation-, rhTRAIL-, or anti-Fas antibody-induced apoptosis. Combining cisplatin with rhTRAIL or anti-Fas antibody induced even higher apoptosis levels in E6-suppressed cells. At the molecular level, cisplatin treatment resulted in elevated p53 levels, enhanced caspase-3 activation, and reduced p21 levels in E6-suppressed cells. Cisplatin in combination with death receptor ligands enhanced caspase-8 and caspase-3 activation and reduced X-linked inhibitor-of-apoptosis protein (XIAP) levels in these cells. We showed using siRNA that the enhanced apoptosis in E6-supressed cells was related to reduced XIAP levels and not due to reduced p21 levels. In conclusion, targeting E6 or XIAP in combination with cisplatin can efficiently potentiate rhTRAIL-induced apoptosis in HPV-positive cervical cancer cells. PMID:22328720

  7. 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

  8. TRPV1 receptors mediate particulate matter-induced apoptosis.

    PubMed

    Agopyan, N; Head, J; Yu, S; Simon, S A

    2004-03-01

    Exposure to airborne particulate matter (PM) is a world-wide health problem mainly because it produces adverse cardiovascular and respiratory effects that frequently result in morbidity. Despite many years of epidemiological and basic research, the mechanisms underlying PM toxicity remain largely unknown. To understand some of these mechanisms, we measured PM-induced apoptosis and necrosis in normal human airway epithelial cells and sensory neurons from both wild-type mice and mice lacking TRPV1 receptors using Alexa Fluor 488-conjugated annexin V and propidium iodide labeling, respectively. Exposure of environmental PMs containing residual oil fly ash and ash from Mount St. Helens was found to induce apoptosis, but not necrosis, as a consequence of sustained calcium influx through TRPV1 receptors. Apoptosis was completely prevented by inhibiting TRPV1 receptors with capsazepine or by removing extracellular calcium or in sensory neurons from TRPV1(-/-) mice. Binding of either one of the PMs to the cell membrane induced a capsazepine-sensitive increase in cAMP. PM-induced apoptosis was augmented upon the inhibition of PKA. PKA inhibition on its own also induced apoptosis, thereby suggesting that this pathway may be endogenously protective against apoptosis. In summary, it was found that inhibiting TRPV1 receptors prevents PM-induced apoptosis, thereby providing a potential mechanism to reduce their toxicity. PMID:14633515

  9. Reversal of endogenous dopamine receptor silencing in pituitary cells augments receptor-mediated apoptosis.

    PubMed

    Al-Azzawi, Haneen; Yacqub-Usman, Kiren; Richardson, Alan; Hofland, Leo J; Clayton, Richard N; Farrell, William E

    2011-02-01

    Dopamine (DA)-agonist targeting of the DA D(2) receptor (D2R) in prolactinomas is the first-line treatment choice for suppression of prolactin and induction of tumor shrinkage. Resistance to DA agonists seems to be related to receptor number. Using the MMQ and GH3 pituitary cell lines, that either do or do not express D2R, respectively, we explored the epigenetic profile associated with the presence or absence of D2R in these cells lines. These studies led us to explore pharmacological strategies designed to restore receptor expression and thereby potentially augment DA agonist-mediated apoptosis. We show in GH3 cells that the D2R harbors increased CpG island-associated methylation and enrichment for histone H3K27me3. Conversely, MMQ cells and normal pituitaries show enrichment for H3K9Ac and barely detectable H3K27me3. Coculture of GH3 cells with the demethylating agent zebularine and the histone deacetylase inhibitor trichostatin A was responsible for a decrease in CpG island methylation and enrichment for the histone H3K9Ac mark. In addition, challenge of GH3 cells with zebularine alone or coculture with both agents led to expression of endogenous D2R in these cells. Induced expression D2R in GH3 cells was associated with a significant increase in apoptosis indices to challenge with either DA or bromocriptine. Specificity of a receptor-mediated response was established in coincubations with specific D2R antagonist and siRNA approaches in GH3 cell and D2R expressing MMQ cell lines. These studies point to the potential efficacy of combined treatment with epigenetic drugs and DA agonists for the medical management of different pituitary tumor subtypes, resistant to conventional therapies. PMID:21177832

  10. Death-Defining Immune Responses After Apoptosis

    PubMed Central

    Campisi, L.; Cummings, R. J.; Blander, J. Magarian

    2014-01-01

    Apoptosis is a programmed form of cell death whereby characteristic internal cellular dismantling is accompanied by the preservation of plasma membrane integrity. Maintaining this order during apoptosis prevents the release of cellular contents and ensures a noninflammatory death. Here, we consider examples of apoptosis in different contexts and discuss how the same form of cell death could have different immunological consequences. Multiple parameters such as cell death as a result of microbial infection, the nature of the inflammatory microenvironment, the type of responding phagocytic cells and the genetic background of the host organism all differentially influence the immunological consequences of apoptosis. PMID:24903539

  11. Mineralocorticoid receptor mediated liposomal delivery system for targeted induction of apoptosis in cancer cells.

    PubMed

    Sharma, Priyanka; Banerjee, Rajkumar; Narayan, Kumar Pranav

    2016-02-01

    Mineralocorticoid receptors (MRs) are nuclear hormone receptors that are ubiquitously present in all cell types and are known to mediate distinct physiological functions like regulating Na(+) and K(+) balance and water excretion. MRs are linked to cell proliferation and can be exploited for the targeted control of cell mass in cancer. The present study is aimed towards extending the concept of using MR ligand spironolactone for selective delivery of genes in cancer cells. The lipoplex (SP) has shown MR mediated targeted transfections as indicated by receptor down-regulation studies using MR antagonists and siRNA. SP-targeted delivery of genes resulted in apoptosis in cell-specific manner while free drug was found to be cytotoxic irrespective of the cancerous or non-cancerous nature. In conclusion, this study presents MR as a target for efficiently delivering anticancer genes and thereby treating cancer through MR-mediated pathway. PMID:26620075

  12. Optogenetic apoptosis: light-triggered cell death.

    PubMed

    Hughes, Robert M; Freeman, David J; Lamb, Kelsey N; Pollet, Rebecca M; Smith, Weston J; Lawrence, David S

    2015-10-01

    An optogenetic Bax has been designed that facilitates light-induced apoptosis. We demonstrate that mitochondrial recruitment of a genetically encoded light-responsive Bax results in the release of mitochondrial proteins, downstream caspase-3 cleavage, changes in cellular morphology, and ultimately cell death. Mutagenesis of a key phosphorylatable residue or modification of the C-terminus mitigates background (dark) levels of apoptosis that result from Bax overexpression. The mechanism of optogenetic Bax-mediated apoptosis was explored using a series of small molecules known to interfere with various steps in programmed cell death. Optogenetic Bax appears to form a mitochondrial apoptosis-induced channel analogous to that of endogenous Bax. PMID:26418181

  13. Androgen receptor silences thioredoxin-interacting protein and competitively inhibits glucocorticoid receptor-mediated apoptosis in pancreatic β-Cells.

    PubMed

    Harada, Naoki; Katsuki, Takahiro; Takahashi, Yuji; Masuda, Tatsuya; Yoshinaga, Mariko; Adachi, Tetsuya; Izawa, Takeshi; Kuwamura, Mitsuru; Nakano, Yoshihisa; Yamaji, Ryoichi; Inui, Hiroshi

    2015-06-01

    Androgen receptor (AR) is known to bind to the same cis-element that glucocorticoid receptor (GR) binds to. However, the effects of androgen signaling on glucocorticoid signaling have not yet been elucidated. Here, we investigated the effects of testosterone on dexamethasone (DEX, a synthetic glucocorticoid)-induced apoptosis of pancreatic β-cells, which might be involved in the pathogenesis of type 2 diabetes mellitus in males. We used INS-1 #6 cells, which were isolated from the INS-1 pancreatic β-cell line and which express high levels of AR. Testosterone and dihydrotestosterone inhibited apoptosis induced by DEX in INS-1 #6 cells. AR knockdown and the AR antagonist hydroxyflutamide each diminished the anti-apoptotic effects of testosterone. AR was localized in the nucleus of both INS-1 #6 cells and pancreatic β-cells of male rats. Induction of thioredoxin-interacting protein (TXNIP) is known to cause pro-apoptotic effects in β-cells. Testosterone suppressed the DEX-induced increase of TXNIP at the transcriptional level. A Chromatin immunoprecipitation assays showed that both AR and GR competitively bound to the TXNIP promoter in ligand-dependent manners. Recombinant DNA-binding domain of AR bound to the same cis-element of the TXNIP promoter that GR binds to. Our results show that AR and GR competitively bind to the same cis-element of TXNIP promoter as a silencer and enhancer, respectively. These results indicate that androgen signaling functionally competes with glucocorticoid signaling in pancreatic β-cell apoptosis. PMID:25639671

  14. Cardiomyocytes undergo apoptosis in human immunodeficiency virus cardiomyopathy through mitochondrion- and death receptor-controlled pathways

    PubMed Central

    Twu, Cheryl; Liu, Nancy Q.; Popik, Waldemar; Bukrinsky, Michael; Sayre, James; Roberts, Jaclyn; Rania, Shammas; Bramhandam, Vishnu; Roos, Kenneth P.; MacLellan, W. Robb; Fiala, Milan

    2002-01-01

    We investigated 18 AIDS hearts (5 with and 13 without cardiomyopathy) by using immunocytochemistry and computerized image analysis regarding the roles of HIV-1 proteins and tumor necrosis factor ligands in HIV cardiomyopathy (HIVCM). HIVCM and cardiomyocyte apoptosis were significantly related to each other and to the expression by inflammatory cells of gp120 and tumor necrosis factor-α. In HIVCM heart, active caspase 9, a component of the mitochondrion-controlled apoptotic pathway, and the elements of the death receptor-mediated pathway, tumor necrosis factor-α and Fas ligand, were expressed strongly on macrophages and weakly on cardiomyocytes. HIVCM showed significantly greater macrophage infiltration and cardiomyocyte apoptosis rate compared with non-HIVCM. HIV-1 entered cultured neonatal rat ventricular myocytes by macropinocytosis but did not replicate. HIV-1- or gp120-induced apoptosis of rat myocytes through a mitochondrion-controlled pathway, which was inhibited by heparin, AOP-RANTES, or pertussis toxin, suggesting that cardiomyocyte apoptosis is induced by signaling through chemokine receptors. In conclusion, in patients with HIVCM, cardiomyocytes die through both mitochondrion- and death receptor-controlled apoptotic pathways. PMID:12379743

  15. B cell receptor-mediated apoptosis of human lymphocytes is associated with a new regulatory pathway of Bim isoform expression.

    PubMed

    Mouhamad, Shahul; Besnault, Laurence; Auffredou, Marie Thérèse; Leprince, Corinne; Bourgeade, Marie Françoise; Leca, Gérald; Vazquez, Aimé

    2004-02-15

    Studies in Bim-deficient mice have shown that the proapoptotic molecule Bim plays a key role in the control of B cell homeostasis and activation. However, the role of Bim in human B lymphocyte apoptosis is unknown. We show in this study that, depending on the degree of cross-linking, B cell receptors can mediate both Bim-dependent and apparent Bim-independent apoptotic pathways. Cross-linked anti-mu Ab-mediated activation induces an original pathway governing the expression of the various Bim isoforms. This new pathway involves the following three sequential steps: 1) extracellular signal-regulated kinase-dependent phosphorylation of the BimEL isoform, which is produced in large amounts in healthy B cells; 2) proteasome-mediated degradation of phosphorylated BimEL; and 3) increased expression of the shorter apoptotic isoforms BimL and BimS. PMID:14764673

  16. JNK (c-Jun N-terminal kinase) and p38 activation in receptor-mediated and chemically-induced apoptosis of T-cells: differential requirements for caspase activation.

    PubMed Central

    MacFarlane, M; Cohen, G M; Dickens, M

    2000-01-01

    Activation of the stress-activated mitogen-activated protein kinases (MAP kinases), c-Jun N-terminal kinase (JNK) and p38, is necessary for the induction of apoptosis in neuronal cells; however, in other cell types their involvement may be stimulus-dependent. In the present study we investigate the activation of JNK and p38 in a single non-neuronal cell type, undergoing receptor-mediated (tumour necrosis factor-related apoptosis-inducing ligand and CD95) or chemically-induced (lactacystin) apoptosis. In Jurkat T-cells, receptor-mediated and chemically-induced apoptosis resulted in a time-dependent activation of the initiator caspases-8 and -9, respectively. Both types of stimuli resulted in a significant activation of JNK and p38, which closely paralleled the time-dependent induction of apoptosis. The caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (z-VAD.FMK) inhibited receptor-mediated apoptosis and suppressed JNK and p38 activation. In contrast, inhibition of lactacystin-induced apoptosis with z-VAD.FMK, as assessed by phosphatidylserine exposure and poly(ADP-ribose) polymerase cleavage, did not inhibit activation of JNK or p38, demonstrating that during chemically-induced apoptosis, activation of JNK and p38 is independent of effector caspases. The role of p38 in apoptosis was assessed using the specific p38 inhibitor, SB203580. No effect on the induction of apoptosis or caspase activation was observed, although activation of mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK-2), an immediate downstream target of p38, was inhibited. Therefore neither p38 activation nor activation of MAPKAPK-2 is critical for induction of either receptor- or chemically-induced apoptosis. Thus, within a single cell type, (1) the mechanism of p38 and JNK activation during apoptosis is stimulus-dependent and (2) activation of the p38 pathway is not required for caspase activation or apoptosis, assessed by phosphatidylserine exposure, but

  17. [Strategy of probe selection for studying mRNAs that participate in receptor-mediated apoptosis signaling].

    PubMed

    Solntsev, L A; Starikova, V D; Sakharnov, N A; Knyazev, D I; Utkin, O V

    2015-01-01

    Death receptors (DRs) and the participants of DR-mediated signaling are characterized by a large number of mRNA isoforms generated by alternative splicing. Due to their high labor intensity and high cost, conventional methods (RT-PCR and RT-PCR in real time) are ineffective when the simultaneous detection of a plurality of mRNA isoforms is needed. In this regard, the use of DNA biochips is has prospective applications in analyzing the expression of many genes simultaneously. In this paper, we suggest an optimal strategy of probes selection aimed at detecting the maximum number of mRNA splice variants generated by major participants of DR-signaling. The objects of the study were 185 genes that form 1134 mRNA isoforms. As a result, a biochip design was developed that enables the detection of 499 mRNA isoforms (44% of total mRNA splice variants). The proposed strategy combines a high degree of modularity, the use of modern high-performance computers, and broad opportunities for setting up the selection criteria in accordance with the objectives of the study. PMID:26107906

  18. 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

  19. 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

  20. 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…

  1. Cancer cell death by design: apoptosis, autophagy and glioma virotherapy.

    PubMed

    Tyler, Matthew A; Ulasov, Ilya V; Lesniak, Maciej S

    2009-08-01

    Autophagy has been defined as a mechanism by which oncolytic adenoviruses mediate cell killing in some cancers, including malignant glioma. Until recently, however, adenovirus replication was regarded as a process that induced classical apoptosis in the infected cell. We have assessed the method of conditionally replicating adenovirus (CRAd) death in a model of malignant glioma, considering both autophagy and apoptosis as possible mechanisms of virally-induced cell death. Our initial investigations indicated that autophagy was the predominant system in CRAd-induced cell death in glioma. This appeared to be the case in vitro; however, further investigation in vivo shows that CRAds are capable of inducing both apoptotic and autophagic cell death. In this punctum, we summarize our latest research to uncover the method of oncolytic adenovirus-induced cell death in malignant glioma. Elucidating the relationship between autophagy and apoptosis in glioma virotherapy has significant implications for the design of optimal viral vectors. PMID:19430207

  2. Morphological and cytochemical determination of cell death by apoptosis

    PubMed Central

    Sobel, Burton E.; Budd, Ralph C.

    2007-01-01

    Several modes of cell death are now recognized, including necrosis, apoptosis, and autophagy. Oftentimes the distinctions between these various modes may not be apparent, although the precise mode may be physiologically important. Accordingly, it is often desirable to be able to classify the mode of cell death. Apoptosis was originally defined by structural alterations in cells observable by transmitted light and electron microscopy. Today, a wide variety of imaging and cytochemical techniques are available for the investigation of apoptosis. This review will highlight many of these methods, and provide a critique on the advantages and disadvantages associated with them for the specific identification of apoptotic cells in culture and tissues. PMID:18000678

  3. Solamargine triggers hepatoma cell death through apoptosis

    PubMed Central

    XIE, XIAODONG; ZHU, HAITAO; YANG, HUIJIAN; HUANG, WENSI; WU, YINGYING; WANG, YING; LUO, YANLING; WANG, DONGQING; SHAO, GENBAO

    2015-01-01

    Solamargine (SM), a steroidal alkaloid glycoside extracted from the traditional Chinese herb Solanum incanum, has been evidenced to inhibit the growth and induce apoptosis in a number of human cancer cell lines. In the present study, the anticancer effect of SM and underlying molecular mechanism of SM-induced apoptosis were investigated on the human hepatocellular carcinoma cells, SMMC7721 and HepG2. The proliferation effects of SM on the SMMC7721 and HepG2 cell lines were evaluated using MTT and colony formation assays. In addition, the percentage of apoptosis was measured using an Annexin V/propidium iodide staining method and the cell cycle distribution mediated by SM was analyzed using flow cytometry. The expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), caspase-3, caspase-9, proliferating cell nuclear antigen (pcna) and Ki67 proteins were examined to further demonstrate the proliferate and apoptosis effects of SM on the hepatoma cells. The results indicated that SM effectively inhibited hepatoma cell proliferation and promoted apoptosis. SM resulted in cell cycle arrest at the G2/M phase in the two cell lines. In addition, SM downregulated the levels of proliferation-associated (Ki67 and pcna) and anti-apoptotic (Bcl-2) proteins, and promoted the activity of apoptosis-associated proteins (Bax, caspase-3 and caspase-9). Therefore, the activation of the Bcl-2/Bax and caspase signaling pathways may be involved in the SM-induced apoptosis of hepatoma cells. PMID:26170994

  4. 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

  5. Do all programmed cell deaths occur via apoptosis?

    PubMed Central

    Schwartz, L M; Smith, S W; Jones, M E; Osborne, B A

    1993-01-01

    During development, large numbers of cells die by a nonpathological process referred to as programmed cell death. In many tissues, dying cells display similar changes in morphology and chromosomal DNA organization, which has been termed apoptosis. Apoptosis is such a widely documented phenomenon that many authors have assumed all programmed cell deaths occur by this process. Two well-characterized model systems for programmed cell death are (i) the death of T cells during negative selection in the mouse thymus and (ii) the loss of intersegmental muscles of the moth Manduca sexta at the end of metamorphosis. In this report we compare the patterns of cell death displayed by T cells and the intersegmental muscles and find that they differ in terms of cell-surface morphology, nuclear ultrastructure, DNA fragmentation, and polyubiquitin gene expression. Unlike the T cells, which are known to die via apoptosis, we find that the intersegmental muscles display few of the features that characterize apoptosis. These data suggest that more than one cell death mechanism is used during development. Images PMID:8430112

  6. [Glutamate receptor-mediated retinal neuronal injury in experimental glaucoma].

    PubMed

    Wang, Zhong-Feng; Yang, Xiong-Li

    2016-08-25

    Glaucoma, the second leading cause of blindness, is a neurodegenerative disease characterized by optic nerve degeneration related to apoptotic death of retinal ganglion cells (RGCs). In the pathogenesis of RGC death following the onset of glaucoma, functional changes of glutamate receptors are commonly regarded as important risk factors. During the past several years, we have explored the mechanisms underlying RGC apoptosis and retinal Müller cell reactivation (gliosis) in a rat chronic ocular hypertension (COH) model. We demonstrated that elevated intraocular pressure in COH rats may induce changes of various signaling pathways, which are involved in RGC apoptosis by modulating glutamate NMDA and AMPA receptors. Moreover, we also demonstrated that over-activation of group I metabotropic glutamate receptors (mGluR I) by excessive extracellular glutamate in COH rats could contribute to Müller cell gliosis by suppressing Kir4.1 channels. In this review, incorporating our results, we discuss glutamate receptor- mediated RGC apoptosis and Müller cell gliosis in experimental glaucoma. PMID:27546508

  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. B-cell-activating factor inhibits CD20-mediated and B-cell receptor-mediated apoptosis in human B cells

    PubMed Central

    Saito, Yohei; Miyagawa, Yoshitaka; Onda, Keiko; Nakajima, Hideki; Sato, Ban; Horiuchi, Yasuomi; Okita, Hajime; Katagiri, Yohko U; Saito, Masahiro; Shimizu, Toshiaki; Fujimoto, Junichiro; Kiyokawa, Nobutaka

    2008-01-01

    B-cell-activating factor (BAFF) is a survival and maturation factor for B cells belonging to the tumour necrosis factor superfamily. Among three identified functional receptors, the BAFF receptor (BAFF-R) is thought to be responsible for the effect of BAFF on B cells though details of how remain unclear. We determined that a hairy-cell leukaemia line, MLMA, expressed a relatively high level of BAFF-R and was susceptible to apoptosis mediated by either CD20 or B-cell antigen receptor (BCR). Using MLMA cells as an in vitro model of mature B cells, we found that treatment with BAFF could inhibit apoptosis mediated by both CD20 and BCR. We also observed, using immunoblot analysis and microarray analysis, that BAFF treatment induced activation of nuclear factor-κB2 following elevation of the expression level of Bcl-2, which may be involved in the molecular mechanism of BAFF-mediated inhibition of apoptosis. Interestingly, BAFF treatment was also found to induce the expression of a series of genes, such as that for CD40, related to cell survival, suggesting the involvement of a multiple mechanism in the BAFF-mediated anti-apoptotic effect. MLMA cells should provide a model for investigating the molecular basis of the effect of BAFF on B cells in vitro and will help to elucidate how B cells survive in the immune system in which BAFF-mediated signalling is involved. PMID:18540961

  9. Apoptosis and Beyond: Cytometry in Studies of Programmed Cell Death

    PubMed Central

    Wlodkowic, Donald; Telford, William; Skommer, Joanna; Darzynkiewicz, Zbigniew

    2012-01-01

    A cell undergoing apoptosis demonstrates multitude of characteristic morphological and biochemical features, which vary depending on the inducer of apoptosis, cell type and the “time window” at which the process of apoptosis is observed. Because the gross majority of apoptotic hallmarks can be revealed by flow and image cytometry, the cytometric methods become a technology of choice in diverse studies of cellular demise. Variety of cytometric methods designed to identify apoptotic cells, detect particular events of apoptosis and probe mechanisms associated with this mode of cell death have been developed during the past two decades. In the present review, we outline commonly used methods that are based on the assessment of mitochondrial transmembrane potential, activation of caspases, DNA fragmentation, and plasma membrane alterations. We also present novel developments in the field such as the use of cyanine SYTO and TO-PRO family of probes. Strategies of selecting the optimal multiparameter approaches, as well as potential difficulties in the experimental procedures, are thoroughly summarized. PMID:21722800

  10. Apoptosis like cell death in Raillietina echinobothrida induced by resveratrol.

    PubMed

    Giri, Bikash Ranjan; Roy, Bishnupada

    2015-08-01

    Northeast India is geographically nestled as one of the biodiversity hotspots, rich in traditionally used medicinal plants. Resveratrol, a naturally occurring phytoalexin found in berries, peanuts, grapes, red wine and also in numerous anthelmintic plants, has attracted wide interest because of its diverse pharmacological characteristics. Recently, anthelmintic potential of the compound is established. The present study was carried out to understand the possible mechanism of action of resveratrol on poultry tapeworm Raillietina echinobothrida. Resveratrol showed excellent cestocidal activity in a dose dependent manner as revealed through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. The progressive ultrastructural alterations followed by complete disruption of nuclear membrane, chromosomal condensation and in situ DNA fragmentation confirm the occurrence of apoptosis like cell death. Increased pro-apoptotic caspase activity and significant decreases in mitochondrial membrane potential in R. echinobothrida exposed to resveratrol confirm the involvement of mitochondria in the process of apoptosis. PMID:26267101

  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. β-Adrenergic Receptor-Mediated Transactivation Of Epidermal Growth Factor Receptor Decreases Cardiomyocyte Apoptosis Through Differential Subcellular Activation of ERK1/2 and Akt

    PubMed Central

    Grisanti, Laurel A.; Talarico, Jennifer A.; Carter, Rhonda L.; Yu, Justine E.; Repas, Ashley A.; Radcliffe, Scott W.; Tang, Hoang-ai; Makarewich, Catherine A.; Houser, Steven R.; Tilley, Douglas G.

    2014-01-01

    Rationale β-adrenergic receptor (βAR)-mediated transactivation of epidermal growth factor receptor (EGFR) has been shown to relay pro-survival effects via unknown mechanisms. Objective We hypothesized that acute βAR-mediated EGFR transactivation in the heart promotes differential subcellular activation of ERK1/2 and Akt, promoting cell survival through modulation of apoptosis. Methods and Results C57BL/6 mice underwent acute i.p. injection with isoproterenol (ISO) ± AG 1478 (EGFR antagonist) to assess the impact of βAR-mediated EGFR transactivation on phosphorylation of ERK1/2 (P-ERK1/2) and Akt (P-Akt) in distinct cardiac subcellular fractions. Increased P-ERK1/2 and P-Akt were observed in cytosolic, plasma membrane and nuclear fractions following ISO stimulation. Whereas the P-ERK1/2 response was EGFR-sensitive in all fractions, the P-Akt response was EGFR-sensitive only in the plasma membrane and nucleus, results confirmed in primary rat neonatal cardiomyocytes (RNCM). βAR-mediated EGFR-transactivation also decreased apoptosis in serum-depleted RNCM, as measured via TUNEL as well as caspase 3 activity/cleavage, which were sensitive to inhibition of either ERK1/2 (PD184352) or Akt (LY-294002) signaling. Caspase 3 activity/cleavage was also sensitive to inhibition of transcription, which, with an increase in nuclear P-ERK1/2 and P-Akt in response to ISO, suggested that βAR-mediated EGFR transactivation may regulate apoptotic gene transcription. An Apoptosis PCR Array identified tnfsf10 (TRAIL) to be altered by ISO in an EGFR-sensitive manner, results confirmed via RT-PCR and ELISA measurement of both membrane-bound and soluble cardiomyocyte TRAIL levels. Conclusions βAR-mediated EGFR transactivation induces differential subcellular activation of ERK1/2 and Akt leading to increased cell survival through the modulation of caspase 3 activity and apoptotic gene expression in cardiomyocytes. PMID:24566221

  13. A Gammaherpesvirus Bcl-2 Ortholog Blocks B Cell Receptor-Mediated Apoptosis and Promotes the Survival of Developing B Cells In Vivo

    PubMed Central

    Coleman, Carrie B.; McGraw, Jennifer E.; Feldman, Emily R.; Roth, Alexa N.; Keyes, Lisa R.; Grau, Katrina R.; Cochran, Stephanie L.; Waldschmidt, Thomas J.; Liang, Chengyu; Forrest, J. Craig; Tibbetts, Scott A.

    2014-01-01

    Gammaherpesviruses such as Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8) establish lifelong latency in their hosts and are associated with the development of several types of malignancies, including a subset of B cell lymphomas. These viruses are thought to co-opt the process of B cell differentiation to latently infect a fraction of circulating memory B cells, resulting in the establishment of a stable latency setpoint. However, little is known about how this infected memory B cell compartment is maintained throughout the life of the host. We have previously demonstrated that immature and transitional B cells are long-term latency reservoirs for murine gammaherpesvirus 68 (MHV68), suggesting that infection of developing B cells contributes to the maintenance of lifelong latency. During hematopoiesis, immature and transitional B cells are subject to B cell receptor (BCR)-mediated negative selection, which results in the clonal deletion of autoreactive B cells. Interestingly, numerous gammaherpesviruses encode homologs of the anti-apoptotic protein Bcl-2, suggesting that virus inhibition of apoptosis could subvert clonal deletion. To test this, we quantified latency establishment in mice inoculated with MHV68 vBcl-2 mutants. vBcl-2 mutant viruses displayed a marked decrease in the frequency of immature and transitional B cells harboring viral genome, but this attenuation could be rescued by increased host Bcl-2 expression. Conversely, vBcl-2 mutant virus latency in early B cells and mature B cells, which are not targets of negative selection, was remarkably similar to wild-type virus. Finally, in vivo depletion of developing B cells during chronic infection resulted in decreased mature B cell latency, demonstrating a key role for developing B cells in the maintenance of lifelong latency. Collectively, these findings support a model in which gammaherpesvirus latency in circulating mature B cells is sustained in part through the

  14. Activation of innate antiviral immune response via double-stranded RNA-dependent RLR receptor-mediated necroptosis

    PubMed Central

    Wang, Wei; Wang, Wei-Hua; Azadzoi, Kazem M.; Su, Ning; Dai, Peng; Sun, Jianbin; Wang, Qin; Liang, Ping; Zhang, Wentao; Lei, Xiaoying; Yan, Zhen; Yang, Jing-Hua

    2016-01-01

    Viruses induce double-stranded RNA (dsRNA) in the host cells. The mammalian system has developed dsRNA-dependent recognition receptors such as RLRs that recognize the long stretches of dsRNA as PAMPs to activate interferon-mediated antiviral pathways and apoptosis in severe infection. Here we report an efficient antiviral immune response through dsRNA-dependent RLR receptor-mediated necroptosis against infections from different classes of viruses. We demonstrated that virus-infected A549 cells were efficiently killed in the presence of a chimeric RLR receptor, dsCARE. It measurably suppressed the interferon antiviral pathway but promoted IL-1β production. Canonical cell death analysis by morphologic assessment, phosphatidylserine exposure, caspase cleavage and chemical inhibition excluded the involvement of apoptosis and consistently suggested RLR receptor-mediated necroptosis as the underlying mechanism of infected cell death. The necroptotic pathway was augmented by the formation of RIP1-RIP3 necrosome, recruitment of MLKL protein and the activation of cathepsin D. Contributing roles of RIP1 and RIP3 were confirmed by gene knockdown. Furthermore, the necroptosis inhibitor necrostatin-1 but not the pan-caspase inhibitor zVAD impeded dsCARE-dependent infected cell death. Our data provides compelling evidence that the chimeric RLR receptor shifts the common interferon antiviral responses of infected cells to necroptosis and leads to rapid death of the virus-infected cells. This mechanism could be targeted as an efficient antiviral strategy. PMID:26935990

  15. 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.

  16. 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. PMID:12615182

  17. Receptor-mediated mitophagy.

    PubMed

    Yamaguchi, Osamu; Murakawa, Tomokazu; Nishida, Kazuhiko; Otsu, Kinya

    2016-06-01

    Mitochondria are essential organelles that supply ATP through oxidative phosphorylation to maintain cellular homeostasis. Extrinsic or intrinsic agents can impair mitochondria, and these impaired mitochondria can generate reactive oxygen species (ROS) as byproducts, inducing cellular damage and cell death. The quality control of mitochondria is essential for the maintenance of normal cellular functions, particularly in cardiomyocytes, because they are terminally differentiated. Accumulation of damaged mitochondria is characteristic of various diseases, including heart failure, neurodegenerative disease, and aging-related diseases. Mitochondria are generally degraded through autophagy, an intracellular degradation system that is conserved from yeast to mammals. Autophagy is thought to be a nonselective degradation process in which cytoplasmic proteins and organelles are engulfed by isolation membrane to form autophagosomes in eukaryotic cells. However, recent studies have described the process of selective autophagy, which targets specific proteins or organelles such as mitochondria. Mitochondria-specific autophagy is called mitophagy. Dysregulation of mitophagy is implicated in the development of chronic diseases including neurodegenerative diseases, metabolic diseases, and heart failure. In this review, we discuss recent progress in research on mitophagy receptors. PMID:27021519

  18. Excitotoxic neuronal death in the immature brain is an apoptosis-necrosis morphological continuum.

    PubMed

    Portera-Cailliau, C; Price, D L; Martin, L J

    1997-02-01

    Glutamate-induced excitotoxicity is a clinically relevant degenerative process that causes selective neuronal death by mechanisms that remain unclear. Cell death is usually classified as apoptotic or necrotic based on biochemical and morphological criteria. Excitotoxic lesions in the adult rat striatum result in neuronal death associated with apoptotic DNA laddering despite a necrotic appearance of neurons ultrastructurally. This suggests that apoptosis and necrosis may not be mutually exclusive modes of cell death. Here, we characterized normal developmental cell death in the newborn rat brain with respect to DNA fragmentation patterns and ultrastructural morphology to establish a standard for apoptosis in the nervous system, and we concluded that it is essentially indistinguishable from apoptosis described in other tissues. We then investigated whether brain maturity could influence the morphology of neuronal death in vivo in the excitotoxically lesioned newborn rat forebrain. Kainic acid induced DNA laddering and death of neurons exhibiting a variety of morphologies, ranging from necrosis to apoptosis. In neurons that were dying by apoptosis, morphologic changes were characterized by a highly ordered sequence of organelle abnormalities, with swelling of endoplasmic reticulum and Golgi vesiculation preceding most nuclear changes and mitochondrial disruption. We concluded that brain maturity influences the morphologic phenotype of neurodegeneration and that excitotoxic neuronal death in the immature brain is not a uniform event but, rather, a continuum of apoptotic, necrotic, and overlapping morphologies. This excitotoxic paradigm might prove useful for analyzing the mechanisms that govern cell death under physiological and pathological conditions. PMID:9120055

  19. Mitophagy switches cell death from apoptosis to necrosis in NSCLC cells treated with oncolytic measles virus.

    PubMed

    Xia, Mao; Meng, Gang; Jiang, Aiqin; Chen, Aiping; Dahlhaus, Meike; Gonzalez, Patrick; Beltinger, Christian; Wei, Jiwu

    2014-06-15

    Although apoptotic phenomena have been observed in malignant cells infected by measles virus vaccine strain Edmonston B (MV-Edm), the precise oncolytic mechanisms are poorly defined. In this study we found that MV-Edm induced autophagy and sequestosome 1-mediated mitophagy leading to decreased cytochrome c release, which blocked the pro-apoptotic cascade in non-small cell lung cancer cells (NSCLCs). The decrease of apoptosis by mitophagy favored viral replication. Persistent viral replication sustained by autophagy ultimately resulted in necrotic cell death due to ATP depletion. Importantly, when autophagy was impaired in NSCLCs MV-Edm-induced cell death was significantly abrogated despite of increased apoptosis. Taken together, our results define a novel oncolytic mechanism by which mitophagy switches cell death from apoptosis to more efficient necrosis in NSCLCs following MV-Edm infection. This provides a foundation for future improvement of oncolytic virotherapy or antiviral therapy. PMID:25004098

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

    PubMed

    Peng, Xi; Yu, Zhengqiang; Liang, Na; Chi, Xiaofeng; Li, Xiaochong; Jiang, Min; Fang, Jing; Cui, Hengmin; Lai, Weimin; Zhou, Yi; Zhou, Shan

    2016-03-15

    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

  1. 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

  2. Host cell death due to enteropathogenic Escherichia coli has features of apoptosis.

    PubMed

    Crane, J K; Majumdar, S; Pickhardt, D F

    1999-05-01

    Enteropathogenic Escherichia coli (EPEC) is a cause of prolonged watery diarrhea in children in developing countries. The ability of EPEC to kill host cells was investigated in vitro in assays using two human cultured cell lines, HeLa (cervical) and T84 (colonic). EPEC killed epithelial cells as assessed by permeability to the vital dyes trypan blue and propidium iodide. In addition, EPEC triggered changes in the host cell, suggesting apoptosis as the mode of death; such changes included early expression of phosphatidylserine on the host cell surface and internucleosomal cleavage of host cell DNA. Genistein, an inhibitor of tyrosine kinases, and wortmannin, an inhibitor of host phosphatidylinositol 3-kinase, markedly increased EPEC-induced cell death and enhanced the features of apoptosis. EPEC-induced cell death was contact dependent and required adherence of live bacteria to the host cell. A quantitative assay for EPEC-induced cell death was developed by using the propidium iodide uptake method adapted to a fluorescence plate reader. With EPEC, the rate and extent of host cell death were less that what has been reported for Salmonella, Shigella, and Yersinia, three other genera of enteric bacteria known to cause apoptosis. However, rapid apoptosis of the host cell may not favor the pathogenic strategy of EPEC, a mucosa-adhering, noninvasive pathogen. PMID:10225923

  3. Somatostatin receptor subtype 2 sensitizes human pancreatic cancer cells to death ligand-induced apoptosis.

    PubMed

    Guillermet, Julie; Saint-Laurent, Nathalie; Rochaix, Philippe; Cuvillier, Olivier; Levade, Thierry; Schally, Andrew V; Pradayrol, Lucien; Buscail, Louis; Susini, Christiane; Bousquet, Corinne

    2003-01-01

    Somatostatin receptor subtype 2 (sst2) gene expression is lost in 90% of human pancreatic adenocarcinomas. We previously demonstrated that stable sst2 transfection of human pancreatic BxPC-3 cells, which do not endogenously express sst2, inhibits cell proliferation, tumorigenicity, and metastasis. These sst2 effects occur as a consequence of an autocrine sst2-dependent loop, whereby sst2 induces expression of its own ligand, somatostatin. Here we investigated whether sst2 induces apoptosis in sst2-transfected BxPC-3 cells. Expression of sst2 induced a 4.4- +/- 0.05-fold stimulation of apoptosis in BxPC-3 through the activation of tyrosine phosphatase SHP-1. sst2 also sensitized these cells to apoptosis induced by tumor necrosis factor alpha (TNFalpha), enhancing it 4.1- +/- 1.5-fold. Apoptosis in BxPC-3 cells mediated by TNF-related apoptosis-inducing ligand (TRAIL) and CD95L was likewise increased 2.3- +/- 0.5-fold and 7.4- +/- 2.5-fold, respectively. sst2-dependent activation and cell sensitization to death ligand-induced apoptosis involved activation of the executioner caspases, key factors in both death ligand- or mitochondria-mediated apoptosis. sst2 affected both pathways: first, by up-regulating expression of TRAIL and TNFalpha receptors, DR4 and TNFRI, respectively, and sensitizing the cells to death ligand-induced initiator capase-8 activation, and, second, by down-regulating expression of the antiapoptotic mitochondrial Bcl-2 protein. These results are of interest for the clinical management of chemoresistant pancreatic adenocarcinoma by using a combined gene therapy based on the cotransfer of genes for both the sst2 and a nontoxic death ligand. PMID:12490654

  4. Molecular mechanisms of hepatic apoptosis

    PubMed Central

    Wang, K

    2014-01-01

    Apoptosis is a prominent feature of liver diseases. Causative factors such as alcohol, viruses, toxic bile acids, fatty acids, drugs, and immune response, can induce apoptotic cell death via membrane receptors and intracellular stress. Apoptotic signaling network, including membrane death receptor-mediated cascade, reactive oxygen species (ROS) generation, endoplasmic reticulum (ER) stress, lysosomal permeabilization, and mitochondrial dysfunction, is intermixed each other, but one mechanism may dominate at a particular stage. Mechanisms of hepatic apoptosis are complicated by multiple signaling pathways. The progression of liver disease is affected by the balance between apoptotic and antiapoptotic capabilities. Therapeutic options of liver injury are impacted by the clear understanding toward mechanisms of hepatic apoptosis. PMID:24434519

  5. Different death stimuli evoke apoptosis via multiple pathways in retinal pigment epithelial cells.

    PubMed

    Ferrington, Deborah A; Tran, Tina N; Lew, Kathleen L; Van Remmen, Holly; Gregerson, Dale S

    2006-09-01

    Loss of retinal pigment epithelial (RPE) cells via apoptosis plays a prominent role in several retinal degenerative diseases, such as age-related macular degeneration, and with light damage. Strategies for preservation of vision that would interrupt the apoptotic cascade require understanding the molecular events associated with apoptosis. This study investigated the susceptibility of RPE to caspase-dependent and -independent apoptotic pathways when challenged with different stimuli, including oxidants, anti-Fas antibody, and activated cytotoxic T lymphocytes (CTLs). These experiments used novel RPE cell lines developed from wildtype and heterozygous mice with reduced levels of either Mn superoxide dismutatse (SOD) or CuZnSOD. Peroxide and 4-hydroxynonenal induced apoptosis through both caspase-independent and -dependent pathways, respectively. With both oxidants, translocation of apoptosis inducing factor into the nucleus was observed. Cells containing reduced levels of CuZnSOD were the most susceptible to oxidant-induced cell death. Targeted killing by CTLs and activation of the Fas death receptor induced caspase-dependent apoptosis. These results show stimulus-specific activation of either the caspase-dependent or -independent pathway. Since cultured RPE express the protein components required for different apoptotic pathways, they provide a good model system for studying molecular events associated with multiple signals that lead to cell death. PMID:16682026

  6. Selenium Compounds, Apoptosis and Other Types of Cell Death: An Overview for Cancer Therapy

    PubMed Central

    Sanmartín, Carmen; Plano, Daniel; Sharma, Arun K.; Palop, Juan Antonio

    2012-01-01

    Selenium (Se) is an essential trace element involved in different physiological functions of the human body and plays a role in cancer prevention and treatment. Induction of apoptosis is considered an important cellular event that can account for the cancer preventive effects of Se. The mechanisms of Se-induced apoptosis are associated with the chemical forms of Se and their metabolism as well as the type of cancer studied. So, some selenocompounds, such as SeO2 involve the activation of caspase-3 while sodium selenite induces apoptosis in the absence of the activation of caspases. Modulation of mitochondrial functions has been reported to play a key role in the regulation of apoptosis and also to be one of the targets of Se compounds. Other mechanisms for apoptosis induction are the modulation of glutathione and reactive oxygen species levels, which may function as intracellular messengers to regulate signaling pathways, or the regulation of kinase, among others. Emerging evidence indicates the overlaps between the apoptosis and other types of cell death such as autophagy. In this review we report different processes of cell death induced by Se compounds in cancer treatment and prevention. PMID:22949823

  7. Leishmania donovani: intracellular ATP level regulates apoptosis-like death in luteolin induced dyskinetoplastid cells.

    PubMed

    Sen, Nilkantha; Das, Benu Brata; Ganguly, Agneyo; Banerjee, Bijoylaxhmi; Sen, Tanusree; Majumder, Hemanta K

    2006-11-01

    Leishmaniasis presents a spectrum of diseases ranging from benign cutaneous lesions to the often-fatal visceralizing form. Luteolin, a dietary flavone induces apoptosis-like death in both promastigote and amastigote forms of Leishmania, the causative agent of the diseases. Here, we have elucidated the mechanism of action of luteolin by analyzing the mitochondrial and cytosolic changes associated with apoptosis-like death of leishmanial cells. In Leishmania donovani, treatment with luteolin induces the loss of both maxicircles and minicircles which resulted in the formation of dyskinetoplastid cells. The loss of mitochondrial DNA causes reduction in the activities of complex I, II, III, and IV of electron transport chain. However, the mitochondrial ATPase activity of complex V remains almost unaltered during treatment with luteolin but the sensitivity to oligomycin is lost. The inactivation of ETC complex is associated with decrease in mitochondrial as well as glycolytic ATP production, which is responsible for depolarization of Deltapsi(m) and alteration in mitochondrial structure. This event is followed by the release of cytochrome c from mitochondria in mt-DNA depleted leishmanial cells and causes an activation of caspase like proteases. Collectively our results provide the first insight into the mechanistic pathway of apoptosis-like death where inhibition of glycolytic ATP production is an essential event responsible for depolarization of Deltapsi(m) in mt-DNA depleted cells to propagate apoptosis-like death in leishmanial cells. PMID:16707127

  8. Nerve cell death induced in vivo by kainic acid and quinolinic acid does not involve apoptosis.

    PubMed

    Ignatowicz, E; Vezzani, A M; Rizzi, M; D'Incalci, M

    1991-11-01

    We investigated whether in vivo excitotoxicity was mediated by a mechanism of programmed cell death called apoptosis. Neurotoxic doses of kainic acid (1.2 nmol) and quinolinic acid (120 nmol) were unilaterally injected in the dorsal hippocampus of anesthetized rats. Eight or 16 h later the animals were killed and DNA was extracted from the injected hippocampi. DNA from mouse thymocytes exposed to methylprednisolone (10(-5) M for 6 h at 37 degrees C) was used as a positive control of apoptotic cells. No typical 'ladder' of DNA fragments (multimers of approximately 200 Kb) which characterizes apoptosis was seen in hippocampal cells after toxic doses of kainic or quinolinic acid, as assessed by agarose gel electrophoresis. This suggests that hippocampal nerve cell death induced in vivo by the excitotoxins is not mediated by apoptosis. PMID:1839770

  9. Anacardic acid induces apoptosis-like cell death in the rice blast fungus Magnaporthe oryzae.

    PubMed

    Muzaffar, Suhail; Bose, Chinchu; Banerji, Ashok; Nair, Bipin G; Chattoo, Bharat B

    2016-01-01

    Anacardic acid (6-pentadecylsalicylic acid), extracted from cashew nut shell liquid, is a natural phenolic lipid well known for its strong antibacterial, antioxidant, and anticancer activities. Its effect has been well studied in bacterial and mammalian systems but remains largely unexplored in fungi. The present study identifies antifungal, cytotoxic, and antioxidant activities of anacardic acid in the rice blast fungus Magnaporthe oryzae. It was found that anacardic acid causes inhibition of conidial germination and mycelial growth in this ascomycetous fungus. Phosphatidylserine externalization, chromatin condensation, DNA degradation, and loss of mitochondrial membrane potential suggest that growth inhibition of fungus is mainly caused by apoptosis-like cell death. Broad-spectrum caspase inhibitor Z-VAD-FMK treatment indicated that anacardic acid induces caspase-independent apoptosis in M. oryzae. Expression of a predicted ortholog of apoptosis-inducing factor (AIF) was upregulated during the process of apoptosis, suggesting the possibility of mitochondria dependent apoptosis via activation of apoptosis-inducing factor. Anacardic acid treatment leads to decrease in reactive oxygen species rather than increase in reactive oxygen species (ROS) accumulation normally observed during apoptosis, confirming the antioxidant properties of anacardic acid as suggested by earlier reports. Our study also shows that anacardic acid renders the fungus highly sensitive to DNA damaging agents like ethyl methanesulfonate (EMS). Treatment of rice leaves with anacardic acid prevents M. oryzae from infecting the plant without affecting the leaf, suggesting that anacardic acid can be an effective antifungal agent. PMID:26381667

  10. Novel Benzo[a]quinolizidine Analogs Induce Cancer Cell Death through Paraptosis and Apoptosis.

    PubMed

    Zheng, Hongbo; Dong, Yiwen; Li, Lin; Sun, Bin; Liu, Lei; Yuan, Huiqing; Lou, Hongxiang

    2016-05-26

    Paraptosis is nonapoptotic cell death characterized by massive endoplasmic reticulum (ER)- or mitochondria-derived vacuoles. Induction of paraptosis offers significant advantages for the treatment of chemotherapy-resistant tumors compared with anticancer drugs that rely on apoptosis. Because some natural alkaloids induce paraptotic cell death, a novel series of benzo[a]quinolizidine derivatives were synthesized, and their antiproliferative activity and ability to induce cytoplasmic vacuolation were analyzed. Structural optimization led to the identification of the potent compound 22b, which inhibited cancer cell proliferation in vitro and in vivo and profoundly facilitated paraptosis-like cell death and induced caspase-dependent apoptosis. Further investigation revealed that 22b-mediated vacuolation originated from persistent ER stress and upregulation of LC3B. Paraptosis induced by benzo[a]quinolizidine derivatives thus represents an alternative strategy for cancer chemotherapy. PMID:27077446

  11. 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

  12. Apoptosis after gamma irradiation. Is it an important cell death modality?

    PubMed Central

    Siles, E.; Villalobos, M.; Jones, L.; Guerrero, R.; Eady, J. J.; Valenzuela, M. T.; Núñez, M. I.; McMillan, T. J.; Ruiz de Almodóvar, J. M.

    1998-01-01

    Apoptosis and necrosis are two different forms of cell death that can be induced by cytotoxic stress, such as ionizing radiation. We have studied the importance of apoptotic death induced after treatment with 6 Gy of gamma-irradiation in a panel of eight human tumour cell lines of different radiosensitivities. Three different techniques based on the detection of DNA fragmentation have been used, a qualitative one--DNA ladder formation --and two quantitative approaches--in situ tailing and comet assay. No statistically significant relationship between the two quantitative assays was found (r= 0.327, P = 0.159) so these methods seem to show different aspects of the process of cell death. The presence of the DNA ladder related well to the end-labelling method in that the least amount of end labelling was seen in samples in which necrotic degradation rather than apoptotic ladders were seen. However, as the results obtained by the comet assay are not in agreement with the DNA ladder experiments, we suggest that the distinction between the degraded DNA produced by apoptosis and necrosis may be difficult by this technique. Finally, although apoptosis has been proposed to be dependent on p53 functionality, and this may explain differences in cellular radiosensitivity, no statistically significant relationship was found between these parameters and apoptosis in the eight cell lines studied. PMID:9862569

  13. miR-25 Targets TRAIL Death Receptor-4 and Promotes Apoptosis Resistance in Cholangiocarcinoma

    PubMed Central

    Razumilava, Nataliya; Bronk, Steve F.; Smoot, Rory L.; Fingas, Christian D.; Werneburg, Nathan W.; Roberts, Lewis R.; Mott, Justin L.

    2011-01-01

    It has been established that microRNA expression and function contribute to phenotypic features of malignant cells, including resistance to apoptosis. While targets and functional roles for a number of microRNAs have been described in cholangiocarcinoma, many additional microRNAs dysregulated in this tumor have not been assigned functional roles. In this study, we identify elevated miR-25 expression in malignant cholangiocarcinoma cell lines as well as patient samples. In cultured cells, treatment with the Smoothened inhibitor, cyclopamine, reduced miR-25 expression, suggesting Hedgehog signaling stimulates miR-25 production. Functionally, miR-25 was shown to protect cells against TNF-Related Apoptosis-Inducing Ligand (TRAIL)-induced apoptosis. Correspondingly, antagonism of miR-25 in culture sensitized cells to apoptotic death. Computational analysis identified the TRAIL Death Receptor-4 (DR4) as a potential novel miR-25 target, and this prediction was confirmed by immunoblot, cell staining, and reporter assays. Conclusion These data implicate elevated miR-25 levels in the control of tumor cell apoptosis in cholangiocarcinoma. The identification of the novel miR-25 target DR4 provides a mechanism by which miR-25 contributes to evasion of TRIAL-induced cholangiocarcinoma apoptosis. PMID:21953056

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

    PubMed Central

    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. PMID:18076767

  15. Hyperthermia restores apoptosis induced by death receptors through aggregation-induced c-FLIP cytosolic depletion.

    PubMed

    Morlé, A; Garrido, C; Micheau, O

    2015-01-01

    TRAIL is involved in immune tumor surveillance and is considered a promising anti-cancer agent owing to its limited side effects on healthy cells. However, some cancer cells display resistance, or become resistant to TRAIL-induced cell death. Hyperthermia can enhance sensitivity to TRAIL-induced cell death in various resistant cancer cell lines, including lung, breast, colon or prostate carcinomas. Mild heat shock treatment has been proposed to restore Fas ligand or TRAIL-induced apoptosis through c-FLIP degradation or the mitochondrial pathway. We demonstrate here that neither the mitochondria nor c-FLIP degradation are required for TRAIL-induced cell death restoration during hyperthermia. Our data provide evidence that insolubilization of c-FLIP, alone, is sufficient to enhance apoptosis induced by death receptors. Hyperthermia induced c-FLIP depletion from the cytosolic fraction, without apparent degradation, thereby preventing c-FLIP recruitment to the TRAIL DISC and allowing efficient caspase-8 cleavage and apoptosis. Hyperthermia-induced c-FLIP depletion was independent of c-FLIP DED2 FL chain assembly motif or ubiquitination-mediated c-FLIP degradation, as assessed using c-FLIP point mutants on lysine 167 and 195 or threonine 166, a phosphorylation site known to regulate ubiquitination of c-FLIP. Rather, c-FLIP depletion was associated with aggregation, because addition of glycerol not only prevented the loss of c-FLIP from the cytosol but also enabled c-FLIP recruitment within the TRAIL DISC, thus inhibiting TRAIL-induced apoptosis during hyperthermia. Altogether our results demonstrate that c-FLIP is a thermosensitive protein whose targeting by hyperthermia allows restoration of apoptosis induced by TNF ligands, including TRAIL. Our findings suggest that combining TRAIL agonists with whole-body or localized hyperthermia may be an interesting approach in cancer therapy. PMID:25675293

  16. Evidence of early involvement of apoptosis inducing factor-induced neuronal death in Alzheimer brain

    PubMed Central

    Lee, Ji-Hye; Cheon, Young-Hee; Woo, Ran-Sook; Song, Dae-Yong; Moon, Cheil

    2012-01-01

    Apoptosis inducing factor (AIF) has been proposed to act as a putative reactive oxygen species scavenger in mitochondria. When apoptotic cell death is triggered, AIF translocates to the nucleus, where it leads to nuclear chromatin condensation and large-scale DNA fragmentation which result in caspase-independent neuronal death. We performed this study to investigate the possibility that, in addition to caspase-dependent neuronal death, AIF induced neuronal death could be a cause of neuronal death in Alzheimer's disease (AD). We have found that AIF immunoreactivity was increased in the hippocampal pyramidal neurons in the Alzheimer brains compared to those of healthy, age-matched control brains. Nuclear AIF immunoreactivity was detected in the apoptotic pyramidal CA1 neurons at the early stage of AD and CA2 at the advanced stage. Nuclear AIF positive neurons were also observed in the amygdala and cholinergic neurons of the basal forebrain (BFCN) from the early stages of AD. The results of this study imply that AIF-induced apoptosis may contribute to neuronal death within the hippocampus, amygdala, and BFCN in early of AD. PMID:22536549

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

    PubMed

    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

  18. 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

  19. Stressed to Death: Targeting Endoplasmic Reticulum Stress Response Induced Apoptosis in Gliomas

    PubMed Central

    Johnson, Guyla G.; White, Misti C.; Grimaldi, Maurizio

    2012-01-01

    Glial tumors are the main primary adult brain tumor. Even with the most advanced treatments, which include stereotactic microscope aided surgical resection, internal and external radiation therapy and local and systemic chemotherapy, median survival time for patients diagnosed with these malignancies is about 12 months. We explore here the possibility that the endoplasmic reticulum stress response (ERSR) could be a possible target to develop chemotherapeutic agents to induce toxicity in glioma cells. ERSR has the dual capacity of activating repair and/or cytotoxic mechanisms. ERSR is triggered by the accumulation of unfolded proteins in the ER. The presence of unfolded proteins in the ER regulates, via a complex biochemical cascade, the upregulation of molecular chaperones, inhibition of protein synthesis, and an increase of proteasome mediated unfolded protein degradation. ERSR in particular conditions can also contribute to cell death via activation of programmed cell death. Apoptosis activation during ERSR is usually caused by the activation of one or a combination of three biochemical cascades. Induction of these pathways ultimately leads to caspase 3 activation culminating in apoptosis. Glioma cells are in a condition of constant low grade ERSR, which possibly contributes to their resistance to treatment protocols. It is conceivable that small molecules that interact with this phenomenon ultimately could be used to modulate the system to activate apoptosis and cause gliotoxicity. We will discuss here ERSR biochemically relevant features to death mechanisms and already identified small molecules that by modulating ERSR are able to activate glioma cell death. PMID:21348829

  20. Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor.

    PubMed

    Yu, Seong-Woon; Wang, Hongmin; Poitras, Marc F; Coombs, Carmen; Bowers, William J; Federoff, Howard J; Poirier, Guy G; Dawson, Ted M; Dawson, Valina L

    2002-07-12

    Poly(ADP-ribose) polymerase-1 (PARP-1) protects the genome by functioning in the DNA damage surveillance network. PARP-1 is also a mediator of cell death after ischemia-reperfusion injury, glutamate excitotoxicity, and various inflammatory processes. We show that PARP-1 activation is required for translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus and that AIF is necessary for PARP-1-dependent cell death. N-methyl-N'-nitro-N-nitrosoguanidine, H2O2, and N-methyl-d-aspartate induce AIF translocation and cell death, which is prevented by PARP inhibitors or genetic knockout of PARP-1, but is caspase independent. Microinjection of an antibody to AIF protects against PARP-1-dependent cytotoxicity. These data support a model in which PARP-1 activation signals AIF release from mitochondria, resulting in a caspase-independent pathway of programmed cell death. PMID:12114629

  1. Apoptosis-promoted tumorigenesis: gamma-irradiation-induced thymic lymphomagenesis requires Puma-driven leukocyte death.

    PubMed

    Michalak, Ewa M; Vandenberg, Cassandra J; Delbridge, Alex R D; Wu, Li; Scott, Clare L; Adams, Jerry M; Strasser, Andreas

    2010-08-01

    Although tumor development requires impaired apoptosis, we describe a novel paradigm of apoptosis-dependent tumorigenesis. Because DNA damage triggers apoptosis through p53-mediated induction of BH3-only proteins Puma and Noxa, we explored their roles in gamma-radiation-induced thymic lymphomagenesis. Surprisingly, whereas Noxa loss accelerated it, Puma loss ablated tumorigenesis. Tumor suppression by Puma deficiency reflected its protection of leukocytes from gamma-irradiation-induced death, because their glucocorticoid-mediated decimation in Puma-deficient mice activated cycling of stem/progenitor cells and restored thymic lymphomagenesis. Our demonstration that cycles of cell attrition and repopulation by stem/progenitor cells can drive tumorigenesis has parallels in human cancers, such as therapy-induced malignancies. PMID:20679396

  2. Diffusion-weighted MRI for imaging cell death after cytotoxic or apoptosis-inducing therapy

    PubMed Central

    Papaevangelou, E; Almeida, G S; Jamin, Y; Robinson, S P; deSouza, N M

    2015-01-01

    Background: Non-invasive serial imaging is desirable to detect processes such as necrotic and apoptotic cell death in cancer patients undergoing treatment. This study investigated the use of diffusion-weighted (DW-) magnetic resonance imaging (MRI) for imaging cell death induced by either a cytotoxic drug (irinotecan), or the apoptosis-inducing agent birinapant, in human tumour xenografts in vivo. Methods: Nude mice bearing human SW620 colon carcinoma xenografts were treated with vehicle, irinotecan (50 mg kg−1) or birinapant (30 mg kg−1) for up to 5 days. DW-MRI was performed prior to and on days 1, 3 and 5 during treatment. Assessment of tumour apoptosis and necrosis ex vivo was used to validate the imaging findings. Results: Both irinotecan and birinapant induced significant tumour growth delay. Irinotecan induced a small increase in the tumour apparent diffusion coefficient (ADC) after 1 day, with a 20 and 30% increase at days 3 and 5 respectively. ADC was unchanged in the vehicle- and birinapant-treated tumours despite a growth delay in the latter. Histological analysis showed that irinotecan increased necrosis at days 3 and 5, and induced apoptosis after 1 day, compared with vehicle. Birinapant induced apoptosis after day 3, but had no effect on tumour necrosis. Conclusions: Tumour ADC changes after irinotecan treatment were associated with the induction of a mixture of necrotic and apoptotic cell death, whereas induction of apoptosis alone with birinapant was not sufficient to induce changes in tissue microstructure that were detectable with DW-MRI. ADC is a useful non-invasive biomarker for early detection of response to cytotoxic drugs, but false negatives may arise while detecting apoptotic response to birinapant. PMID:25880014

  3. 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

  4. 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

  5. The interplay between human herpes simplex virus infection and the apoptosis and necroptosis cell death pathways.

    PubMed

    Yu, Xiaoliang; He, Sudan

    2016-01-01

    Human herpes simplex virus (HSV) is a ubiquitous human pathogen that establishes a lifelong latent infection and is associated with mucocutaneous lesions. In multicellular organisms, cell death is a crucial host defense mechanism that eliminates pathogen-infected cells. Apoptosis is a well-defined form of programmed cell death executed by a group of cysteine proteases, called caspases. Studies have shown that HSV has evolved strategies to counteract caspase activation and apoptosis by encoding anti-apoptotic viral proteins such as gD, gJ, Us3, LAT, and the ribonucleotide reductase large subunit (R1). Recently, necroptosis has been identified as a regulated form of necrosis that can be invoked in the absence of caspase activity. Receptor-interacting kinase 3 (RIP3 or RIPK3) has emerged as a central signaling molecule in necroptosis; it is activated via interaction with other RIP homotypic interaction motif (RHIM)-containing proteins such as RIP1 (or RIPK1). There is increasing evidence that HSV R1 manipulates necroptosis via the RHIM-dependent inactivation or activation ofRIP3 in a species-specific manner. This review summarizes the current understanding of the interplay between HSV infection and cell death pathways, with an emphasis on apoptosis and necroptosis. PMID:27154074

  6. Apoptosis, necrosis and necroptosis: cell death regulation in the intestinal epithelium.

    PubMed

    Günther, Claudia; Neumann, Helmut; Neurath, Markus F; Becker, Christoph

    2013-07-01

    Intestinal epithelial cells (IEC) are organised as a single cell layer which covers the intestine. Their primary task is to absorb nutrients present in the intestinal lumen. However, IEC also play an important role in the immune defence of our body by building a barrier that separates the bowel wall from potentially hazardous bacteria present in the gut lumen. The life cycle of IEC is determined by the time span in which cells migrate from their place of origin at the crypt base to the villus tip, from where they are shed into the lumen. Cell death in the intestinal epithelium has to be tightly regulated and irregularities might cause pathologies. Excessive cell death has been associated with chronic inflammation as seen in patients with Crohn's disease and ulcerative colitis. While until recently apoptosis was discussed as being essential for epithelial turnover and tissue homeostasis in the intestinal epithelium, recent data using gene deficient mice have challenged this concept. Moreover, an apoptosis-independent mode of programmed cell death, termed necroptosis, has been identified and described in the intestinal epithelium. The following article reviews previous studies on cell death regulation in IEC and a potential role of necroptosis for gut homeostasis. PMID:22689519

  7. MiR-133b Targets Antiapoptotic Genes and Enhances Death Receptor-Induced Apoptosis

    PubMed Central

    Bild, Matthias; Jung, Ulrike; Müller, Henrik; Arntzen, Magnus Ø.; Piso, Chloe; Stephan, Carsten; Thiede, Bernd; Mollenkopf, Hans-Joachim; Jung, Klaus; Kaufmann, Stefan H. E.; Schreiber, Jörg

    2012-01-01

    Despite the importance of microRNAs (miRs) for regulation of the delicate balance between cell proliferation and death, evidence for their specific involvement during death receptor (DR)-mediated apoptosis is scarce. Transfection with miR-133b rendered resistant HeLa cells sensitive to tumor necrosis factor-alpha (TNFα)-induced cell death. Similarly, miR-133b caused exacerbated proapoptotic responses to TNF-related apoptosis-inducing ligand (TRAIL) or an activating antibody to Fas/CD95. Comprehensive analysis, encompassing global RNA or protein expression profiling performed by microarray experiments and pulsed stable isotope labeling with amino acids in cell culture (pSILAC), led to the discovery of the antiapoptotic protein Fas apoptosis inhibitory molecule (FAIM) as immediate miR-133b target. Moreover, miR-133b impaired the expression of the detoxifying protein glutathione-S-transferase pi (GSTP1). Expression of miR-133b in tumor specimens of prostate cancer patients was significantly downregulated in 75% of the cases, when compared with matched healthy tissue. Furthermore, introduction of synthetic miR-133b into an ex-vivo model of prostate cancer resulted in impaired proliferation and cellular metabolic activity. PC3 cells were also sensitized to apoptotic stimuli after transfection with miR-133b similar to HeLa cells. These data reveal the ability of a single miR to influence major apoptosis pathways, suggesting an essential role for this molecule during cellular transformation, tumorigenesis and tissue homeostasis. PMID:22532850

  8. 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

  9. 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.

  10. APP Overexpression Causes Aβ-Independent Neuronal Death through Intrinsic Apoptosis Pathway.

    PubMed

    Cheng, Ning; Jiao, Song; Gumaste, Ankita; Bai, Li; Belluscio, Leonardo

    2016-01-01

    Accumulation of amyloid-β (Aβ) peptide in the brain is a central hallmark of Alzheimer's disease (AD) and is thought to be the cause of the observed neurodegeneration. Many animal models have been generated that overproduce Aβ yet do not exhibit clear neuronal loss, questioning this Aβ hypothesis. We previously developed an in vivo mouse model that expresses a humanized amyloid precursor protein (hAPP) in olfactory sensory neurons (OSNs) showing robust apoptosis and olfactory dysfunction by 3 weeks of age, which is consistent with early OSN loss and smell deficits, as observed in AD patients. Here we show, by deleting the β-site APP cleaving enzyme 1 (BACE1) in two distinct transgenic mouse models, that hAPP-induced apoptosis of OSNs is Aβ independent and remains cell autonomous. In addition, we reveal that the intrinsic apoptosis pathway is responsible for hAPP-induced OSN death, as marked by mitochondrial damage and caspase-9 activation. Given that hAPP expression causes OSN apoptosis despite the absence of BACE1, we propose that Aβ is not the sole cause of hAPP-induced neurodegeneration and that the early loss of olfactory function in AD may be based on a cell-autonomous mechanism, which could mark an early phase of AD, prior to Aβ accumulation. Thus, the olfactory system could serve as an important new platform to study the development of AD, providing unique insight for both early diagnosis and intervention. PMID:27517085

  11. APP Overexpression Causes Aβ-Independent Neuronal Death through Intrinsic Apoptosis Pathway

    PubMed Central

    Cheng, Ning; Jiao, Song; Gumaste, Ankita; Bai, Li

    2016-01-01

    Abstract Accumulation of amyloid-β (Aβ) peptide in the brain is a central hallmark of Alzheimer’s disease (AD) and is thought to be the cause of the observed neurodegeneration. Many animal models have been generated that overproduce Aβ yet do not exhibit clear neuronal loss, questioning this Aβ hypothesis. We previously developed an in vivo mouse model that expresses a humanized amyloid precursor protein (hAPP) in olfactory sensory neurons (OSNs) showing robust apoptosis and olfactory dysfunction by 3 weeks of age, which is consistent with early OSN loss and smell deficits, as observed in AD patients. Here we show, by deleting the β-site APP cleaving enzyme 1 (BACE1) in two distinct transgenic mouse models, that hAPP-induced apoptosis of OSNs is Aβ independent and remains cell autonomous. In addition, we reveal that the intrinsic apoptosis pathway is responsible for hAPP-induced OSN death, as marked by mitochondrial damage and caspase-9 activation. Given that hAPP expression causes OSN apoptosis despite the absence of BACE1, we propose that Aβ is not the sole cause of hAPP-induced neurodegeneration and that the early loss of olfactory function in AD may be based on a cell-autonomous mechanism, which could mark an early phase of AD, prior to Aβ accumulation. Thus, the olfactory system could serve as an important new platform to study the development of AD, providing unique insight for both early diagnosis and intervention. PMID:27517085

  12. Proteasome inhibitors prevent cytochrome c release during apoptosis but not in excitotoxic death of cerebellar granule neurons.

    PubMed

    Bobba, Antonella; Canu, Nadia; Atlante, Anna; Petragallo, Vito; Calissano, Pietro; Marra, Ersilia

    2002-03-27

    In order to find out whether and how proteasomes participate in the processes leading cerebellar granule cells to death either in necrosis, due to glutamate neurotoxicity, or in apoptosis, due to K(+) shift, we measured the three proteasome activities by using specific fluorescent probes and investigated the effect of several proteasome inhibitors, including MG132, on the cytochrome c release taking place in the early phase of both apoptosis and necrosis. We show that differently from apoptosis, the early phase of necrosis does not require proteasome activation. Inhibition of proteasome activity can prevent cytochrome c release in cerebellar granule cells undergoing apoptosis, thus improving cell survival, but not necrosis. These findings show that proteasomes play an important role in the early phase of apoptosis but not that of necrosis, and that these two types of cell death differ from each other in their mechanism of cytochrome c release. PMID:11943185

  13. Mode of cell death after acetaminophen overdose in mice: apoptosis or oncotic necrosis?

    PubMed

    Gujral, Jaspreet S; Knight, Tamara R; Farhood, Anwar; Bajt, Mary Lynn; Jaeschke, Hartmut

    2002-06-01

    Acetaminophen (AAP) overdose can cause severe liver injury and liver failure in experimental animals and humans. Recently, several authors proposed that apoptosis might be a major mechanism of cell death after AAP treatment. To address this controversial issue, we evaluated a detailed time course of liver injury after AAP (300 mg/kg) in fasted C3Heb/FeJ mice. Apoptotic hepatocytes were quantified in H&E-stained liver sections using morphologic criteria (cell shrinkage, chromatin condensation and margination, and apoptotic bodies). The number of apoptotic hepatocytes remained at baseline (0.2 +/- 0.1 cells/10 high-power fields [HPF]) up to 2 h after AAP administration. However, between 3 and 24 h, apoptotic cell death increased significantly, e.g., 6.3 +/- 0.8 cells/10 HPF at 6 h. Despite the increase in the number of hepatocytes meeting the morphological criteria of apoptosis, this cell fraction remained well below 1% of all parenchymal cells. No evidence for caspase-3 processing or increase in enzyme activity was detected at any time. These results were compared to the overall percent of necrotic cells in liver sections. Confluent areas of centrilobular necrosis were estimated to involve 40-60% of all hepatocytes between 3 and 24 h after AAP administration. These numbers correlated with the increase in plasma alanine aminotransferase activities, which reached a peak level of 5900 +/- 1350 U/l at 24 h. A similar result was obtained with higher doses of AAP and with the use of fed animals. Thus, oncotic necrosis and not apoptosis is the principal mechanism of liver-cell death after AAP overdose in vivo. PMID:12011492

  14. Apoptosis-inducing factor substitutes for caspase executioners in NMDA-triggered excitotoxic neuronal death.

    PubMed

    Wang, Hongmin; Yu, Seong-Woon; Koh, David W; Lew, Jasmine; Coombs, Carmen; Bowers, William; Federoff, Howard J; Poirier, Guy G; Dawson, Ted M; Dawson, Valina L

    2004-12-01

    The profound neuroprotection observed in poly(ADP-ribose) polymerase-1 (PARP-1) null mice to ischemic and excitotoxic injury positions PARP-1 as a major mediator of neuronal cell death. We report here that apoptosis-inducing factor (AIF) mediates PARP-1-dependent glutamate excitotoxicity in a caspase-independent manner after translocation from the mitochondria to the nucleus. In primary murine cortical cultures, neurotoxic NMDA exposure triggers AIF translocation, mitochondrial membrane depolarization, and phosphatidyl serine exposure on the cell surface, which precedes cytochrome c release and caspase activation. NMDA neurotoxicity is not affected by broad-spectrum caspase inhibitors, but it is prevented by Bcl-2 overexpression and a neutralizing antibody to AIF. These results link PARP-1 activation with AIF translocation in NMDA-triggered excitotoxic neuronal death and provide a paradigm in which AIF can substitute for caspase executioners. PMID:15574746

  15. Smac mimetic primes apoptosis-resistant acute myeloid leukaemia cells for cytarabine-induced cell death by triggering necroptosis.

    PubMed

    Chromik, Joerg; Safferthal, Charlotta; Serve, Hubert; Fulda, Simone

    2014-03-01

    The prognosis for patients with acute myeloid leukaemia (AML) is still poor, thus calling for novel treatment strategies. Here, we report that the small-molecule Smac mimetic BV6, which antagonizes Inhibitor of Apoptosis (IAP) proteins, acts in concert with cytarabine (AraC) to trigger cell death in AML cells in a highly synergistic manner (combination index 0.02-0.27). Similarly, BV6 cooperates with AraC to trigger cell death in primary AML samples, underscoring the clinical relevance of our findings. Molecular studies reveal that the TNFα-blocking antibody Enbrel significantly reduces BV6/AraC-induced cell death, demonstrating that an autocrine/paracrine TNFα loop mediates cell death. Furthermore, BV6 and AraC synergize to induce loss of mitochondrial membrane potential, caspase activation and DNA fragmentation, consistent with apoptotic cell death. Nevertheless, the caspase inhibitor zVAD.fmk fails to protect against BV6/AraC-induced cell death. Intriguingly, this cell death upon caspase inhibition is significantly reduced by pharmacological inhibition of two key components of necroptosis signaling, i.e. by RIP1 kinase inhibitor Necrostatin-1 or MLKL inhibitor NSA. Thus, BV6 sensitizes AML cells to AraC-induced cell death and overcomes apoptosis resistance by triggering necroptosis as alternative form of cell death. These findings have important implications for Smac mimetic-based strategies to bypass apoptosis resistance of AML. PMID:24184825

  16. 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

  17. Sorafenib induces autophagic cell death and apoptosis in hepatic stellate cell through the JNK and Akt signaling pathways.

    PubMed

    Hao, Huiyao; Zhang, Di; Shi, Junli; Wang, Yan; Chen, Lei; Guo, Yongze; Ma, Junji; Jiang, Xiaoyu; Jiang, Huiqing

    2016-03-01

    Increasing hepatic stellate cell (HSC) death is an attractive approach for limiting liver fibrosis. We investigated the molecular mechanisms underlying the effects of sorafenib on HSCs. LX2 cells were incubated with sorafenib and a variety of inhibitors of apoptosis, autophagy, and necrosis. Electron microscopy was used to observe autophagosomes. Inhibitors and siRNA were used to examine the role of the Akt/mTOR/p70S6K and JNK pathways. Ultrastructural analysis revealed that rat HSCs treated with 5 μmol/l sorafenib accumulated residual digested material and empty or autophagic vacuoles. Incubating LX2 cells with lysosomal protease inhibitors increased the accumulation of LC3-II, indicating that sorafenib enhances autophagic flux in HSCs. Autophagy may precede apoptosis. Lower concentrations of sorafenib and a shorter treatment time resulted in the dominance of autophagic cell death over apoptosis. Further analysis showed that Beclin 1 is inactivated by the caspases induced by sorafenib during apoptosis. Inhibition of autophagy in LX2 cells using 3-methyladenine treatment or siRNA-mediated knockdown of Atg5 resulted in a marked increase in apoptosis. Finally, sorafenib induced programmed cell death by attenuation and activation of Akt/mTOR/p70S6K and JNK signaling. Sorafenib-induced cell death is mediated by both autophagy and apoptosis. Elucidation of the signaling pathways activated by sorafenib could potentially lead to novel antifibrosis therapies for chronic liver diseases. PMID:26629768

  18. 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

  19. Why do cells die in HIV infection? Potential mechanisms inducing programmed cell death/apoptosis.

    PubMed

    Del Llano, A M; Lavergne, J A

    1994-06-01

    This work reviews the suggested mechanisms which result in programmed cell death in human HIV infection. Here we present state-of-the-art scientific information related to the newly rediscovered phenomenon of Apoptosis, and to its biological relevance in the pathogenesis of HIV disease. General features of this phenomenon are reviewed, as well as available evidence for its occurrence and possible role in AIDS pathogenesis. A complex series of cellular and molecular events leading to cellular apoptosis are also reviewed and discussed. They include events which take place at the cell membrane level and those which occur at the intramembrane level and cytoplasmic locations, which result from the immunological activation of affected cells. Cellular events which follow and occur within the mitochondrial space and at the nuclear level are also discussed. The biological significance of all these phenomena is summarized in a theoretical scheme, which attempts to integrate all cellular events leading a primed cell into its HIV-induced programmed death. PMID:7938404

  20. 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. PMID:27005397

  1. 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. PMID:25958165

  2. Autophagic Cell Death and Apoptosis Jointly Mediate Cisatracurium Besylate-Induced Cell Injury

    PubMed Central

    Zhuang, Haixia; Tian, Weili; Li, Wen; Zhang, Xingli; Wang, Jingjing; Yang, Yue; Liu, Xin; Xia, Zhengyuan; Feng, Du; Zhang, Liangqing

    2016-01-01

    Cisatracurium besylate is an ideal non-depolarizing muscle relaxant which is widely used in clinical application. However, some studies have suggested that cisatracurium besylate can affect cell proliferation. Moreover, its specific mechanism of action remains unclear. Here, we found that the number of GFP-LC3 (green fluoresent protein-light chain 3) positive autophagosomes and the rate of mitochondria fracture both increased significantly in drug-treated GFP-LC3 and MitoDsRed stable HeLa cells. Moreover, cisatracurium promoted the co-localization of LC3 and mitochondria and induced formation of autolysosomes. Levels of mitochondrial proteins decreased, which were reversed by the lysosome inhibitor Bafinomycin A1. Similar results with evidence of dose-dependent effects were found in both HeLa and Human Umbilical Vein Endothelial Cells (HUVECs). Cisatracurium lowered HUVEC viability to 0.16 (OD490) at 100 µM and to 0.05 (OD490) after 48 h in vitro; it increased the cell death rate to 56% at 100 µM and to 60% after 24 h in a concentration- and time-dependent manner (p < 0.01). Cell proliferation decreased significantly by four fold in Atg5 WT (wildtype) MEF (mouse embryonic fibroblast) (p < 0.01) but was unaffected in Atg5 KO (Knockout) MEF, even upon treatment with a high dose of cisatracurium. Cisatracurium induced significant increase in cell death of wild-type MEFs even in the presence of the apoptosis inhibitor zVAD. Thus, we conclude that activation of both the autophagic cell death and cell apoptosis pathways contributes to cisatracurium-mediated cell injury. PMID:27058536

  3. Apoptosis and cell proliferation in the mouse model of embryonic death induced by Tritrichomonas foetus infection.

    PubMed

    Woudwyk, Mariana A; Zanuzzi, Carolina N; Nishida, Fabián; Gimeno, Eduardo J; Soto, Pedro; Monteavaro, Cristina E; Barbeito, Claudio G

    2015-09-01

    Bovine tritrichomonosis is a sexually transmitted disease caused by the protozoon Tritrichomonas foetus and characterised by embryonic-death and abortion. During pregnancy, the processes of cell proliferation and death play a crucial role for blastocyst implantation and the subsequent maintenance of early pregnancy, and their misbalance may lead to the abortion. In this study, we aimed to investigate whether cell proliferation and death may be altered during tritrichomonosis. For this purpose, we used pregnant BALB/c mice as an alternative experimental animal model that has successfully reproduced the infection. We analysed the immunohistochemical expression of active caspase-3 and proliferating cell nuclear (PCNA) antigens in the endometrium of infected mice. We found an increase in the number of caspase-3 positive cells in infected mice that were not pregnant at the necropsy. Besides, the number of positive proliferating cells increased in the uterine luminal epithelium of infected animals killed at 5-7 days post coitum (dpc). Pregnant infected mice killed at 8-11 dpc showed higher proliferation than control animals. We suggest that the cytopathic effect induced by T. foetus in the uteri of infected mice may induce the apoptosis of the epithelial cells and, as a result, promote a compensatory proliferative response. The information described here will be helpful to further study the pathogenesis of the bovine tritrichomonosis. PMID:26028409

  4. Facile synthesis of autophagonizer and evaluation of its activity to induce autophagic cell death in apoptosis-defective cell line.

    PubMed

    Nguyen, Jennifer; Chen, Luxi; Kumar, Dhiraj; Lee, Jiyong

    2016-10-01

    Some cancer cells are resistant to apoptosis, rendering them irresponsive towards apoptosis-inducing chemotherapy drugs. Another mode of action to kill these apoptosis-defective cells is essential and autophagy, a dynamic process that degrades cytoplasmic contents for cellular maintenance, has been considered as one of the alternate routes. A small molecule inducer of autophagy, autophagonizer was reported to induce cell death through a novel process that is independent of extrinsic apoptosis and the normal signaling pathways of autophagy. Here, we describe an efficient synthetic procedure for the autophagonizer. The newly synthesized autophagonizer (DK-1-49) resulted in an accumulation of autophagy-associated LC3-II and enhanced levels of autophagosomes and acidic vacuoles. Furthermore, cell viability was inhibited by autophagic cell death in not only human cancer cells but also Bax/Bak double-knockout cells. These findings highlight that intrinsic apoptosis is not also involved in the induction of cellular death by the autophagonizer suggesting the autophagonizer is a promising candidate for anticancer therapeutics for cancer cells that are resistant to apoptosis-inducing chemotherapy. PMID:27597252

  5. Differential apoptosis-like cell death in amastigote and trypomastigote forms from Trypanosoma cruzi-infected heart cells in vitro.

    PubMed

    De Souza, E M; Nefertiti, A S G; Bailly, C; Lansiaux, A; Soeiro, M N C

    2010-07-01

    Apoptosis, type-I of programmed cell death (PCD-I), is not restricted to multicellular organisms since many apoptotic features have been described in different trypanosomatids, including Trypanosoma cruzi. Our present aim was to monitor, by different morphological markers, the occurrence of apoptosis-like death in amastigotes and trypomastigotes of T.cruzi (Y strain) during the infection of heart culture cells. We documented the differential occurrence of PCD-I in amastigotes and trypomastigotes, with distinct death rates noticed between these two parasite-distinct forms. Fluorescence microscopy and flow cytometry analysis using different hall markers of apoptosis (phosphatidylserine exposure, collapse of mitochondrial membrane potential and DNA fragmentation) showed that amastigotes present higher levels of apoptosis-like cell death as compared to trypomastigotes. It is possible that the higher levels of PCD-I in these highly multiplicative forms may contribute to the control of the parasite burden within the host cells. On the other hand, the apoptosis-like occurrence in the infective but non-proliferative stage of the parasite (trypomastigotes) may play a role in parasite evasion mechanisms as suggested for other parasites. PMID:20495825

  6. MicroRNA-351 Regulates Two-Types of Cell Death, Necrosis and Apoptosis, Induced by 5-fluoro-2'-deoxyuridine.

    PubMed

    Sato, Akira; Omi, Takuya; Yamamoto, Akihiro; Satake, Akito; Hiramoto, Akiko; Masutani, Mitsuko; Tanuma, Sei-ichi; Wataya, Yusuke; Kim, Hye-Sook

    2016-01-01

    Cell-death can be necrosis and apoptosis. We are investigating the mechanisms regulating the cell death that occurs on treatment of mouse cancer cell-line FM3A with antitumor 5-fluoro-2'-deoxyuridine (FUdR): necrosis occurs for the original clone F28-7, and apoptosis for its variant F28-7-A. Here we report that a microRNA (miR-351) regulates the cell death pattern. The miR-351 is expressed strongly in F28-7-A but only weakly in F28-7. Induction of a higher expression of miR-351 in F28-7 by transfecting an miRNA mimic into F28-7 resulted in a change of the death mode; necrosis to apoptosis. Furthermore, transfection of an miR-351 inhibitor into F28-7-A resulted in the morphology change, apoptosis to necrosis, in this death-by-FUdR. Possible mechanism involving lamin B1 in this miR-351's regulatory action is discussed. PMID:27071035

  7. MicroRNA-351 Regulates Two-Types of Cell Death, Necrosis and Apoptosis, Induced by 5-fluoro-2'-deoxyuridine

    PubMed Central

    Omi, Takuya; Yamamoto, Akihiro; Satake, Akito; Hiramoto, Akiko; Masutani, Mitsuko; Tanuma, Sei-ichi; Wataya, Yusuke; Kim, Hye-Sook

    2016-01-01

    Cell-death can be necrosis and apoptosis. We are investigating the mechanisms regulating the cell death that occurs on treatment of mouse cancer cell-line FM3A with antitumor 5-fluoro-2'-deoxyuridine (FUdR): necrosis occurs for the original clone F28-7, and apoptosis for its variant F28-7-A. Here we report that a microRNA (miR-351) regulates the cell death pattern. The miR-351 is expressed strongly in F28-7-A but only weakly in F28-7. Induction of a higher expression of miR-351 in F28-7 by transfecting an miRNA mimic into F28-7 resulted in a change of the death mode; necrosis to apoptosis. Furthermore, transfection of an miR-351 inhibitor into F28-7-A resulted in the morphology change, apoptosis to necrosis, in this death-by-FUdR. Possible mechanism involving lamin B1 in this miR-351’s regulatory action is discussed. PMID:27071035

  8. Targeting the Apoptosis Pathway in Hematologic Malignancies

    PubMed Central

    Zaman, Shadia; Wang, Rui; Gandhi, Varsha

    2014-01-01

    Apoptosis is a cell death program that is well-orchestrated for normal tissue homeostasis and for removal of damaged, old, or infected cells. It is regulated by intrinsic and extrinsic pathways. The intrinsic pathway responds to signals such as ultraviolet radiation or DNA damage and activates “executioner” caspases through a mitochondria-dependent pathway. The extrinsic pathway is activated by death signals induced, for example, by an infection that activates the immune system or receptor-mediated pathways. The extrinsic pathway signals also cascade down to executioner caspases that cleave target proteins and lead to cell death. Strict control of cellular apoptosis is important for the hematopoietic system as it has a high turnover rate. However, the apoptosis program is often deregulated in hematologic malignancies leading to the accumulation of malignant cells. Therefore, apoptosis pathways have been identified for development of anticancer therapeutics. We review here the proteins that have been targeted for anticancer drug development in hematologic malignancies. These include BCL-2 family proteins, death ligands and receptors, inhibitor of apoptosis family proteins, and caspases. Except for caspase activators, drugs that target each of these classes of proteins have advanced into clinical trials. PMID:24295132

  9. 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. PMID:25916905

  10. An Antimicrobial Peptidomimetic Induces Mucorales Cell Death through Mitochondria-Mediated Apoptosis

    PubMed Central

    Barbu, E. Magda; Shirazi, Fazal; McGrath, Danielle M.; Albert, Nathaniel; Sidman, Richard L.; Pasqualini, Renata; Arap, Wadih; Kontoyiannis, Dimitrios P.

    2013-01-01

    The incidence of mucormycosis has dramatically increased in immunocompromised patients. Moreover, the array of cellular targets whose inhibition results in fungal cell death is rather limited. Mitochondria have been mechanistically identified as central regulators of detoxification and virulence in fungi. Our group has previously designed and developed a proteolytically-resistant peptidomimetic motif D(KLAKLAK)2 with pleiotropic action ranging from targeted (i.e., ligand-directed) activity against cancer and obesity to non-targeted activity against antibiotic resistant gram-negative rods. Here we evaluated whether this non-targeted peptidomimetic motif is active against Mucorales. We show that D(KLAKLAK)2 has marked fungicidal action, inhibits germination, and reduces hyphal viability. We have also observed cellular changes characteristic of apoptosis in D(KLAKLAK)2-treated Mucorales cells. Moreover, the fungicidal activity was directly correlated with vacuolar injury, mitochondrial swelling and mitochondrial membrane depolarization, intracellular reactive oxygen species accumulation (ROS), and increased caspase-like enzymatic activity. Finally, these apoptotic features were prevented by the addition of the ROS scavenger N-acetyl-cysteine indicating mechanistic pathway specificity. Together, these findings indicate that D(KLAKLAK)2 makes Mucorales exquisitely susceptible via mitochondrial injury-induced apoptosis. This prototype may serve as a candidate drug for the development of translational applications against mucormycosis and perhaps other fungal infections. PMID:24098573

  11. The alkylphospholipid edelfosine shows activity against Strongyloides venezuelensis and induces apoptosis-like cell death.

    PubMed

    Legarda-Ceballos, Ana L; Rojas-Caraballo, Jose; López-Abán, Julio; Ruano, Ana Lucía; Yepes, Edward; Gajate, Consuelo; Mollinedo, Faustino; Muro, Antonio

    2016-10-01

    Strongyloidiasis is widely distributed in the tropical and subtropical areas. Ivermectin is the drug of choice for the treatment. However, the concerns about relying treatment on a single drug make identification of new molecules a priority. Alkylphospholipid analogues, including edelfosine, are a group of synthetic compounds that have shown activity against some parasites. The objective was to assess the in vitro and in vivo activity of edelfosine, miltefosine, perifosine against Strongyloides venezuelensis. Moreover, apoptosis-like mechanism in larvae after treatment was studied. Edelfosine displayed the highest activity and the best selectivity index (LD50=49.6 ± 5.4μM, SI=1.1) compared to miltefosine or perifosine. Third stage larvae after culture with edelfosine were not able to develop an infection in mice. Treatment of mice with edelfosine showed reduction of 47% in parasitic females allocated in the gut. Moreover, DNA fragmentation was observed by TUNEL staining in larvae treated with edelfosine. These results suggest that edelfosine could be an effective drug against strongyloidiasis, probably through induction of apoptosis-like cell death. PMID:27394030

  12. An antimicrobial peptidomimetic induces Mucorales cell death through mitochondria-mediated apoptosis.

    PubMed

    Barbu, E Magda; Shirazi, Fazal; McGrath, Danielle M; Albert, Nathaniel; Sidman, Richard L; Pasqualini, Renata; Arap, Wadih; Kontoyiannis, Dimitrios P

    2013-01-01

    The incidence of mucormycosis has dramatically increased in immunocompromised patients. Moreover, the array of cellular targets whose inhibition results in fungal cell death is rather limited. Mitochondria have been mechanistically identified as central regulators of detoxification and virulence in fungi. Our group has previously designed and developed a proteolytically-resistant peptidomimetic motif D(KLAKLAK)2 with pleiotropic action ranging from targeted (i.e., ligand-directed) activity against cancer and obesity to non-targeted activity against antibiotic resistant gram-negative rods. Here we evaluated whether this non-targeted peptidomimetic motif is active against Mucorales. We show that D(KLAKLAK)2 has marked fungicidal action, inhibits germination, and reduces hyphal viability. We have also observed cellular changes characteristic of apoptosis in D(KLAKLAK)2-treated Mucorales cells. Moreover, the fungicidal activity was directly correlated with vacuolar injury, mitochondrial swelling and mitochondrial membrane depolarization, intracellular reactive oxygen species accumulation (ROS), and increased caspase-like enzymatic activity. Finally, these apoptotic features were prevented by the addition of the ROS scavenger N-acetyl-cysteine indicating mechanistic pathway specificity. Together, these findings indicate that D(KLAKLAK)2 makes Mucorales exquisitely susceptible via mitochondrial injury-induced apoptosis. This prototype may serve as a candidate drug for the development of translational applications against mucormycosis and perhaps other fungal infections. PMID:24098573

  13. Dehydroabietic Acid Derivative QC4 Induces Gastric Cancer Cell Death via Oncosis and Apoptosis

    PubMed Central

    Luo, Dongjun; Ni, Qing; Ji, Anlai; Gu, Wen; Wu, Junhua

    2016-01-01

    Aim. QC4 is the derivative of rosin's main components dehydroabietic acid (DHA). We investigated the cytotoxic effect of QC4 on gastric cancer cells and revealed the mechanisms beneath the induction of cell death. Methods. The cytotoxic effect of QC4 on gastric cancer cells was evaluated by CCK-8 assay and flow cytometry. The underlying mechanisms were tested by administration of cell death related inhibitors and detection of apoptotic and oncosis related proteins. Cytomembrane integrity and organelles damage were confirmed by lactate dehydrogenase (LDH) leakage assay, mitochondrial function test, and cytosolic free Ca2+ concentration detection. Results. QC4 inhibited cell proliferation dose- and time-dependently and destroyed cell membrane integrity, activated calpain-1 autolysis, and induced apoptotic protein cleavage in gastric cancer cells. The detection of decreased ATP and mitochondrial membrane potential, ROS accumulation, and cytosolic free Ca2+ elevation confirmed organelles damage in QC4-treated gastric cancer cells. Conclusions. DHA derivative QC4 induced the damage of cytomembrane and organelles which finally lead to oncosis and apoptosis in gastric cancer cells. Therefore, as a derivative of plant derived small molecule DHA, QC4 might become a promising agent in gastric cancer therapy. PMID:27057539

  14. Induction of apoptosis-like cell death by coelomocyte extracts from Eisenia andrei earthworms.

    PubMed

    Mácsik, Levente László; Somogyi, Ildikó; Opper, Balázs; Bovári-Biri, Judit; Pollák, Edit; Molnár, László; Németh, Péter; Engelmann, Péter

    2015-10-01

    Earthworm's innate immunity is maintained by cellular and humoral components. Our objective was to characterize the cytotoxicity leading to target cell death caused by earthworm coelomocytes. Coelomocyte lysates induced strong cytotoxicity in tumor cell lines. Transmission electron microscopy revealed cell membrane and intracellular damage in cells treated with coelomocyte lysates. Using TUNEL-assay, within 5 min of incubation we detected DNA fragmentation. Moreover, we found phosphatidylserine translocation in target cell-membranes. Furthermore, we detected dose-dependent Ca(2+) influx and decrease of mitochondrial membrane potential in coelomocyte lysate-treated cells. Interestingly, caspase 3/8 activation was undetectable in exposed tumor cells. One such cytotoxic molecule, lysenin identified in earthworms binds to sphingomyelin and causes target cell lysis in vertebrates. Pretreatment with our anti-lysenin monoclonal antibody rescued the majority but not all target cells from coelomocyte induced death. These data suggest that, not only lysenin but also other factors participate in the caspase-independent apoptosis induced by coelomocytes. PMID:26049811

  15. Apoptosis-Like Cell Death Induction and Aberrant Fibroblast Properties in Human Incisional Hernia Fascia

    PubMed Central

    Diaz, Ramon; Quiles, Maria T.; Guillem-Marti, Jordi; Lopez-Cano, Manuel; Huguet, Pere; Ramon-y-Cajal, Santiago; Reventos, Jaume; Armengol, Manel; Arbos, Maria A.

    2011-01-01

    Incisional hernia often occurs following laparotomy and can be a source of serious problems. Although there is evidence that a biological cause may underlie its development, the mechanistic link between the local tissue microenvironment and tissue rupture is lacking. In this study, we used matched tissue-based and in vitro primary cell culture systems to examine the possible involvement of fascia fibroblasts in incisional hernia pathogenesis. Fascia biopsies were collected at surgery from incisional hernia patients and non-incisional hernia controls. Tissue samples were analyzed by histology and immunoblotting methods. Fascia primary fibroblast cultures were assessed at morphological, ultrastructural, and functional levels. We document tissue and fibroblast loss coupled to caspase-3 activation and induction of apoptosis-like cell-death mechanisms in incisional hernia fascia. Alterations in cytoskeleton organization and solubility were also observed. Incisional hernia fibroblasts showed a consistent phenotype throughout early passages in vitro, which was characterized by significantly enhanced cell proliferation and migration, reduced adhesion, and altered cytoskeleton properties, as compared to non-incisional hernia fibroblasts. Moreover, incisional hernia fibroblasts displayed morphological and ultrastructural alterations compatible with autophagic processes or lysosomal dysfunction, together with enhanced sensitivity to proapoptotic challenges. Overall, these data suggest an ongoing complex interplay of cell death induction, aberrant fibroblast function, and tissue loss in incisional hernia fascia, which may significantly contribute to altered matrix maintenance and tissue rupture in vivo. PMID:21641387

  16. Apoptosis-like cell death induced by Salmonella in Acanthamoeba rhysodes.

    PubMed

    Feng, Ye; Hsiao, Yi-Hsing; Chen, Hsiu-Ling; Chu, Chishih; Tang, Petrus; Chiu, Cheng-Hsun

    2009-08-01

    Free-living amoebae act as environmental hosts of several intracellular pathogens. We examined the interaction between Acanthamoeba rhysodes and Salmonella, a human intracellular pathogen. There was no difference among three different serovars of Salmonella in terms of their growth within A. rhysodes over time. The number of intracellular bacteria increased at 6 h post-infection, and the viability of A. rhysodes was significantly reduced at 24 h post-infection. Amoebic cell death was characterized by TUNEL and Annexin V assay, without DNA ladder identified, indicating an apoptosis-like cell death in Salmonella-infected A. rhysodes. Global gene expression screening between intracellular and extracellular Salmonella by microarray and quantitative PCR showed that genes from Salmonella pathogenicity islands and virulence plasmid were up-regulated within A. rhysodes. The phase-dependent expression pattern suggests their distinct roles in the pathogenesis. A. rhysodes and Salmonella provide a model to study transient symbiosis between bacterial pathogens and protozoa in an aquatic ecosystem. PMID:19446019

  17. Apoptosis Inducing Factor Binding Protein PGAM5 Triggers Mitophagic Cell Death That Is Inhibited by the Ubiquitin Ligase Activity of X-Linked Inhibitor of Apoptosis.

    PubMed

    Lenhausen, Audrey M; Wilkinson, Amanda S; Lewis, Eric M; Dailey, Kaitlin M; Scott, Andrew J; Khan, Shahzeb; Wilkinson, John C

    2016-06-14

    Apoptosis inducing factor (AIF) plays a well-defined role in controlling cell death but is also a critical factor for maintaining mitochondrial energy homeostasis; how these dueling activities are balanced has remained largely elusive. To identify new AIF binding partners that may define the continuum of AIF cellular regulation, a biochemical screen was performed that identified the mitochondrial phosphoglycerate mutase 5 (PGAM5) as an AIF associated factor. AIF binds both the short and long isoforms of PGAM5 and can reduce the ability of PGAM5 to control antioxidant responses. Transient overexpression of either PGAM5 isoform triggers caspase activation and cell death, and while AIF could reduce this caspase activation neither AIF expression nor caspase activity is required for PGAM5-mediated death. PGAM5 toxicity morphologically and biochemically resembles mitophagic cell death and is inhibited by the AIF binding protein X-linked inhibitor of apoptosis (XIAP) in a manner that depends on the ubiquitin ligase activity of XIAP. The phosphatase activity of PGAM5 was not required for cell death, and comparison of phosphatase activity between short and long PGAM5 isoforms suggested that only the long isoform is catalytically competent. This property correlated with an increased ability of PGAM5L to form dimers and/or higher order oligomers in intact cells compared to PGAM5S. Overall this study identifies an AIF/PGAM5/XIAP axis that can regulate PGAM5 activities related to the antioxidant response and mitophagy. PMID:27218139

  18. TRAIL Recombinant Adenovirus Triggers Robust Apoptosis in Multidrug-Resistant HL-60/Vinc Cells Preferentially Through Death Receptor DR5

    PubMed Central

    Wu, Ching-Huang; Kao, Ching-Hai

    2008-01-01

    Abstract Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic because of its highly selective apoptosis-inducing action on neoplastic versus normal cells. However, some cancer cells express resistance to recombinant soluble TRAIL. To overcome this problem, we used a TRAIL adenovirus (Ad5/35-TRAIL) to induce apoptosis in a drug-sensitive and multidrug-resistant variant of HL-60 leukemia cells and determined the molecular mechanisms of Ad5/35-TRAIL-induced apoptosis. Ad5/35-TRAIL did not induce apoptosis in normal human lymphocytes, but caused massive apoptosis in acute myelocytic leukemia cells. It triggered more efficient apoptosis in drug-resistant HL-60/Vinc cells than in HL-60 cells. Treating the cells with anti-DR4 and anti-DR5 neutralizing antibodies (particularly anti-DR5) reduced, whereas anti-DcR1 antibody enhanced, the apoptosis triggered by Ad5/35-TRAIL. Whereas Ad5/35-TRAIL induced apoptosis in both cell lines through activation of caspase-3 and caspase-10, known to link the cell death receptor pathway to the mitochondrial pathway, it triggered increased mitochondrial membrane potential change (Δψm) only in HL-60/Vinc cells. Ad5/35-TRAIL also increased the production of reactive oxygen species, which play an important role in apoptosis. Therefore, using Ad5/35-TRAIL may be an effective therapeutic strategy for eliminating TRAIL-resistant malignant cells and these studies may provide clues to treat and eradicate acute myelocytic leukemias. PMID:18476767

  19. 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. PMID:20150916

  20. 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

  1. Mechanism of cell death during warm hepatic ischemia-reperfusion in rats: apoptosis or necrosis?

    PubMed

    Gujral, J S; Bucci, T J; Farhood, A; Jaeschke, H

    2001-02-01

    Reperfusion injury can cause liver dysfunction after cold storage and warm ischemia. Recently it has been suggested that more than 50% of hepatocytes and sinusoidal endothelial cells (SEC) are undergoing apoptosis during the first 24 hours of reperfusion. The aim of our study was to quantify apoptotic and necrotic hepatocytes and apoptotic SEC after 60 or 120 minutes of warm, partial no-flow ischemia and 0 to 24 hours reperfusion in male SD rats. Apoptotic cells were identified by TUNEL assay in combination with morphological criteria. After 60 minutes of ischemia and 1 hour of reperfusion there was a significant increase of apoptotic hepatocytes (0.7 +/- 0.1% vs. 0.3 +/- 0.1% in controls) and SEC (1.5 +/- 0.6% vs. 0.3 +/- 0.1% in controls). The number of apoptotic SEC and hepatocytes was not different from controls at 6 hours or 24 hours of reperfusion. In contrast, the number of necrotic hepatocytes was quantified as 12 +/- 2% at 1 hour, 34 +/- 6% at 6 hours, and 57 +/- 11% at 24 hours. These results correlated with the increase in plasma ALT levels at these time points. Longer (120 min) ischemia times did not affect the number of apoptotic cells but increased hepatocellular necrosis to 58 +/- 4% at 6 hours reperfusion. No significant increase in caspase-3 activity and processing was detectable in any of these livers. Moreover, the caspase inhibitor Z-Asp-cmk (2 mg/kg IV) had no significant effect on reperfusion injury. Our results suggest that only a small minority of SEC and hepatocytes undergo apoptosis after 60 to 120 minutes of warm ischemia followed by 0 to 24 hours of reperfusion. Oncotic necrosis appears to be the principal mechanism of cell death for both cell types. PMID:11172341

  2. Targeting inhibitor of apoptosis proteins by Smac mimetic elicits cell death in poor prognostic subgroups of chronic lymphocytic leukemia.

    PubMed

    Opel, Daniela; Schnaiter, Andrea; Dodier, Dagmar; Jovanovic, Marjana; Gerhardinger, Andreas; Idler, Irina; Mertens, Daniel; Bullinger, Lars; Stilgenbauer, Stephan; Fulda, Simone

    2015-12-15

    Inhibitor of apoptosis (IAP) proteins are highly expressed in chronic lymphocytic leukemia (CLL) cells and contribute to evasion of cell death and poor therapeutic response. Here, we report that Smac mimetic BV6 dose-dependently induces cell death in 28 of 51 (54%) investigated CLL samples, while B-cells from healthy donors are largely unaffected. Importantly, BV6 is significantly more effective in prognostic unfavorable cases with, e.g., non-mutated VH status and TP53 mutation than samples with unknown or favorable prognosis. The majority of cases with 17p deletion (10/12) and Fludarabine refractory cases respond to BV6, indicating that BV6 acts independently of p53. BV6 also triggers cell death under survival conditions mimicking the microenvironment, e.g., by adding CD40 ligand or conditioned medium. Gene expression profiling identifies cell death, NF-κB and redox signaling among the top pathways regulated by BV6 not only in CLL but also in core-binding factor (CBF) acute myeloid leukemia (AML). Consistently, BV6 stimulates production of reactive oxygen species (ROS), which are contributing to BV6-induced cell death, since antioxidants reduce cell death. While BV6 causes degradation of cellular inhibitor of apoptosis (cIAP)1 and cIAP2 and nuclear factor-kappaB (NF-κB) pathway activation in primary CLL samples, BV6 induces cell death independently of caspase activity, receptor-interacting protein (RIP)1 activity or tumor necrosis factor (TNF)α, as zVAD.fmk, necrostatin-1 or TNFα-blocking antibody Enbrel fail to inhibit cell death. Together, these novel insights into BV6-regulated cell death in CLL have important implications for developing new therapeutic strategies to overcome cell death resistance especially in poor prognostic CLL subgroups. PMID:26096065

  3. High glucose-induced apoptosis in human coronary artery endothelial cells involves up-regulation of death receptors

    PubMed Central

    2011-01-01

    Background High glucose can induce apoptosis in vascular endothelial cells, which may contribute to the development of vascular complications in diabetes. We evaluated the role of the death receptor pathway of apoptotic signaling in high glucose-induced apoptosis in human coronary artery endothelial cells (HCAECs). Methods HCAECs were treated with media containing 5.6, 11.1, and 16.7 mM of glucose for 24 h in the presence or absence of tumor necrosis factor (TNF)-α. For detection of apoptosis, DNA fragmentation assay was used. HCAEC expression of death receptors were analyzed by the PCR and flow cytometry methods. Also, using immunohistochemical techniques, coronary expression of death receptors was assessed in streptozotocin-nicotinamide-induced type 2 diabetic mice. Results Exposure of HCAECs to high glucose resulted in a significant increase in TNF-R1 and Fas expression, compared with normal glucose. High glucose increased TNF-α production by HCAECs and exogenous TNF-α up-regulated TNF-R1 and Fas expression in HCAECs. High glucose-induced up-regulation of TNF-R1 and Fas expression was undetectable in the presence of TNF-α. Treatment with TNF-R1 neutralizing peptides significantly inhibited high glucose-induced endothelial cell apoptosis. Type 2 diabetic mice displayed appreciable expression of TNF-R1 and Fas in coronary vessels. Conclusions In association with increased TNF-α levels, the death receptors, TNF-R1 and Fas, are up-regulated in HCAECs under high glucose conditions, which could in turn play a role in high glucose-induced endothelial cell apoptosis. PMID:21816064

  4. Ginsenoside Rh2 induces apoptosis and paraptosis-like cell death in colorectal cancer cells through activation of p53.

    PubMed

    Li, Binghui; Zhao, Jiong; Wang, Chong-Zhi; Searle, Jennifer; He, Tong-Chuan; Yuan, Chun-Su; Du, Wei

    2011-02-28

    Ginsenosides are the main bioactive components in American ginseng, a commonly used herb. In this study, we showed that the ginsenoside Rh2 exhibited significantly more potent cell death activity than the ginsenoside Rg3 in HCT116 and SW480 colorectal cancer cells. Cell death induced by Rh2 is mediated in part by the caspase-dependent apoptosis and in part by the caspase-independent paraptosis, a type of cell death that is characterized by the accumulation of cytoplasmic vacuoles. Treatment of cells with Rh2 activated the p53 pathway and significantly increased the levels of the pro-apoptotic regulator, Bax, while decreasing the levels of anti-apoptosis regulator Bcl-2. Removal of p53 significantly blocked Rh2-induced cell death as well as vacuole formation, suggesting that both types of cell death induced by Rh2 are mediated by p53 activity. Furthermore, we show that Rh2 increased ROS levels and activated the NF-κB survival pathway. Blockage of ROS by NAC or catalase inhibited the activation of NF-κB signaling and enhanced Rh2-induced cell death, suggesting that the anti-cancer effect of Rh2 can be enhanced by antioxidants. PMID:21194832

  5. 7-Hydroxydehydronuciferine induces human melanoma death via triggering autophagy and apoptosis.

    PubMed

    Wu, Pei-Fang; Chiu, Chien-Chih; Chen, Chung-Yi; Wang, Hui-Min David

    2015-12-01

    Melanoma is the deadliest cancer. We identified 7-hydroxydehydronuciferine (7-HDNF) isolated from the leaves of Nelumbo nucifera Gaertn cv. Rosa-plena to be a bio-active agent that antagonizes melanoma tumor growth in mice xenograft model in vivo. Cell proliferation assay demonstrated strong anticancer effects of 7-HDNF to exhibit a dose-dependent behaviour and displayed minor cytotoxicities on normal human skin cells, including epidermal keratinocytes and melanocytes, and dermal fibroblasts. With acridine orange (AO) staining and flow analysis, we found 7-HDNF induced the formation of intracellular vacuoles and the augmentation of acidic vesicular organelles (AVO). The apoptotic cell death ratio was measured via two-dimensional flow cytometry by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double stained to confirm the cellular membrane asymmetry lost. One-dimensional flow cytometric analysis showed 7-HDNF increased the cellular arrest in cell cycle at the G2/M phase. Through Western blot examinations, protein expressions were discovered to verify autophagy and apoptosis response mechanisms sharing the associated pathways. Finally, 7-HDNF presented a high-quality antimigratory activity in wound-healing assay. Overall, 7-HDNF presented high-quality anticancer bio-functions and inhibited melanoma tumor growth in vivo and in vitro. PMID:26174122

  6. Gedunin inactivates the co-chaperone p23 protein causing cancer cell death by apoptosis.

    PubMed

    Patwardhan, Chaitanya A; Fauq, Abdul; Peterson, Laura B; Miller, Charles; Blagg, Brian S J; Chadli, Ahmed

    2013-03-01

    Pharmacological inhibition of Hsp90 is an exciting option for cancer therapy. The clinical efficacy of Hsp90 inhibitors is, however, less than expected. Binding of the co-chaperone p23 to Hsp90 and induced overexpression of anti-apoptotic proteins Hsp70 and Hsp27 are thought to contribute to this outcome. Herein, we report that the natural product gedunin may provide a new alternative to inactivate the Hsp90 machine. We show that gedunin directly binds to p23 and inactivates it, without overexpression of Hsp27 and relatively modest induction of Hsp70. Using molecular docking and mutational analysis, we mapped the gedunin-binding site on p23. Functional analysis shows that gedunin inhibits the p23 chaperoning activity, blocks its cellular interaction with Hsp90, and interferes with p23-mediated gene regulation. Cell treatment with gedunin leads to cancer cell death by apoptosis through inactivation of p23 and activation of caspase 7, which cleaves p23 at the C terminus. These results provide important insight into the molecular mechanism of action of this promising lead compound. PMID:23355466

  7. Cytotoxic hydrogen bridged ruthenium quinaldamide complexes showing induced cancer cell death by apoptosis.

    PubMed

    Lord, Rianne M; Allison, Simon J; Rafferty, Karen; Ghandhi, Laura; Pask, Christopher M; McGowan, Patrick C

    2016-08-16

    This report presents the first known p-cymene ruthenium quinaldamide complexes which are stabilised by a hydrogen-bridging atom, [{(p-cym)Ru(II)X(N,N)}{H(+)}{(N,N)XRu(II)(p-cym)}][PF6] (N,N = functionalised quinaldamide and X = Cl or Br). These complexes are formed by a reaction of [p-cymRu(μ-X)2]2 with a functionalised quinaldamide ligand. When filtered over NH4PF6, and under aerobic conditions the equilibrium of NH4PF6 ⇔ NH3 + HPF6 enables incorporation of HPF6 and the stabilisation of two monomeric ruthenium complexes by a bridging H(+), which are counter-balanced by a PF6 counterion. X-ray crystallographic analysis is presented for six new structures with OO distances of 2.420(4)-2.448(15) Å, which is significant for strong hydrogen bonds. Chemosensitivity studies against HCT116, A2780 and cisplatin-resistant A2780cis human cancer cells showed the ruthenium complexes with a bromide ancillary ligand to be more potent than those with a chloride ligand. The 4'-fluoro compounds show a reduction in potency for both chloride and bromide complexes against all cell lines, but an increase in selectivity towards cancer cells compared to non-cancer ARPE-19 cells, with a selectivity index >1. Mechanistic studies showed a clear correlation between IC50 values and induction of cell death by apoptosis. PMID:27417660

  8. 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.

  9. Autophagy and Apoptosis Have a Role in the Survival or Death of Stallion Spermatozoa during Conservation in Refrigeration

    PubMed Central

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

  10. The role of MAPK and FAS death receptor pathways in testicular germ cell apoptosis induced by lead.

    PubMed

    Dong, Shuying; Liang, Duoping; An, Na; Jia, Li; Shan, Yujuan; Chen, Chao; Sun, Kuo; Niu, Fei; Li, Huiyan; Fu, Songbin

    2009-09-01

    The aim of the present study is to investigate gene expression involved in the signal pathway of MAPK and death signal receptor pathway of FAS in lead-induced apoptosis of testicular germ cells. First, cell viabilities were determined by MTT assay. Second, using single cell gel-electrophoresis test (comet assay) and TUNEL staining technique, apoptotic rate and cell apoptosis localization of testicular germ cells were measured in mice treated with 0.15%, 0.3%, and 0.6% lead, respectively. Third, the immunolocalization of K-ras, c-fos, Fas, and active caspase-3 proteins was determined by immunohistochemistry. Finally, changes in the translational levels of K-ras, c-fos, Fas, and active caspase-3 were further detected by western blot analysis. Our results showed that lead could significantly induce testicular germ cell apoptosis in a dose-dependent manner (P<0.01). The mechanisms were closely related to the increased expressions of K-ras, c-fos, Fas, and active caspase-3 in apoptotic germ cells. In conclusion, K-ras/c-fos and Fas/caspase-3 death signaling receptor pathways were involved in the lead-induced apoptosis of the testicular germ cells in mice. PMID:19727529

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

    PubMed

    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

  12. 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

  13. Parthenolide enhances sensitivity of colorectal cancer cells to TRAIL by inducing death receptor 5 and promotes TRAIL-induced apoptosis.

    PubMed

    Kim, Se-Lim; Liu, Yu-Chuan; Park, Young Ran; Seo, Seung Young; Kim, Seong Hun; Kim, In Hee; Lee, Seung Ok; Lee, Soo Teik; Kim, Dae-Ghon; Kim, Sang-Wook

    2015-03-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent. Recombinant human TRAIL has been evaluated in clinical trials, however, various malignant tumors are resistant to TRAIL. Parthenolide (PT) has recently been demonstrated as a highly effective anticancer agent and has been suggested to be used for combination therapy with other anticancer agents. In this study, we investigate the molecular mechanisms by which PT sensitizes colorectal cancer (CRC) cells to TRAIL-induced apoptosis. HT-29 (TRAIL-resistant) and HCT116 (TRAIL-sensitive) cells were treated with PT and/or TRAIL. The results demonstrated that combined treatment induced apoptosis which was determined using MTT, cell cycle analysis, Annexin V assay and Hoechst 33258 staining. Interestingly, we confirmed that HCT116 cells have much higher death receptor (DR) 5 than HT-29 cells and PT upregulates DR5 protein level and surface expression in both cell lines. Apoptosis through the mitochondrial pathway was confirmed by detecting regulation of Bcl-2 family members, p53 cytochrome C release, and caspase cascades. These results suggest that PT sensitizes TRAIL-induced apoptosis via upregulation of DR5 and mitochondria-dependent pathway. Combination treatment using PT and TRAIL may offer an effective strategy to overcome TRAIL resistance of certain CRC cells. PMID:25502339

  14. Disruption of cellular homeostasis induces organelle stress and triggers apoptosis like cell-death pathways in malaria parasite

    PubMed Central

    Rathore, S; Datta, G; Kaur, I; Malhotra, P; Mohmmed, A

    2015-01-01

    A regulated protein turnover machinery in the cell is essential for effective cellular homeostasis; any interference with this system induces cellular stress and alters the normal functioning of proteins important for cell survival. In this study, we show that persistent cellular stress and organelle dysfunction because of disruption of cellular homeostasis in human malaria parasite Plasmodium falciparum, leads to apoptosis-like cell death. Quantitative global proteomic analysis of the stressed parasites before onset of cell death, showed upregulation of a number of proteins involved in cellular homeostasis; protein network analyses identified upregulated metabolic pathways that may be associated with stress tolerance and pro-survival mechanism. However, persistent stress on parasites cause structural abnormalities in endoplasmic reticulum and mitochondria, subsequently a cascade of reactions are initiated in parasites including rise in cytosolic calcium levels, loss of mitochondrial membrane potential and activation of VAD-FMK-binding proteases. We further show that activation of VAD-FMK-binding proteases in the parasites leads to degradation of phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease), a known target of metacaspases, as well as degradation of other components of spliceosomal complex. Loss of spliceosomal machinery impairs the mRNA splicing, leading to accumulation of unprocessed RNAs in the parasite and thus dysregulate vital cellular functions, which in turn leads to execution of apoptosis-like cell death. Our results establish one of the possible mechanisms of instigation of cell death by organelle stress in Plasmodium. PMID:26136076

  15. Arboviruses and apoptosis: the role of cell death in determining vector competence.

    PubMed

    Clem, Rollie J

    2016-05-01

    A relatively small number of mosquito species transmit arboviruses such as dengue, yellow fever, chikungunya and West Nile viruses to hundreds of millions of people each year, yet we still lack a thorough understanding of the molecular factors that determine vector competence. Apoptosis has been shown to be an important factor in determining the outcome of virus infection for many viruses. However, until recently, it was not clear whether apoptosis plays a role in determining the outcome of arbovirus infections in mosquitoes. Recent work has begun to shed light on the roles of apoptosis in this important process. PMID:26872460

  16. Parthenolide induces apoptosis by activating the mitochondrial and death receptor pathways and inhibits FAK-mediated cell invasion.

    PubMed

    Kwak, Sang Won; Park, Eon Sub; Lee, Chung Soo

    2014-01-01

    The natural product parthenolide induces apoptosis in cancer cells. However, the mechanism of apoptosis in ovarian cancer cells exposed to parthenolide is not clear. In addition, it is unclear whether parthenolide-induced apoptosis is mediated by the formation of reactive oxygen species and the depletion of GSH contents, and the effect of parthenolide on the invasion and migration of human epithelial ovarian cancer cells has not been studied. Therefore, we investigated the effects of parthenolide exposure on apoptosis, cell adhesion, and migration using the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. The results suggest that parthenolide may induce apoptotic cell death in ovarian carcinoma cell lines by activating the mitochondrial pathway and the caspase-8- and Bid-dependent pathways. The apoptotic effect of parthenolide appears to be mediated by the formation of reactive oxygen species and the depletion of GSH. Parthenolide inhibited fetal bovine serum-induced cell adhesion and migration of OVCAR-3 cells, possibly through the suppression the focal adhesion kinase-dependent activation of cytoskeletal-associated components. Therefore, parthenolide might be beneficial in the treatment of epithelial ovarian adenocarcinoma and combination therapy. PMID:24065392

  17. 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.

  18. Induction of apoptosis by tumor suppressor FHIT via death receptor signaling pathway in human lung cancer cells.

    PubMed

    Deng, Wu-Guo; Nishizaki, Masahiko; Fang, Bingliang; Roth, Jack A; Ji, Lin

    2007-04-20

    FHIT is a novel tumor suppressor gene located at human chromosome 3p14.2. Restoration of wild-type FHIT in 3p14.2-deficient human lung cancer cells inhibits cell growth and induces apoptosis. In this study, we analyzed potential upstream/downstream molecular targets of the FHIT protein and found that FHIT specifically targeted and regulated death receptor (DR) genes in human non-small-cell lung cancer (NSCLC) cells. Exogenous expression of FHIT by a recombinant adenoviral vector (Ad)-mediated gene transfer upregulated expression of DR genes. Treatment with a recombinant TRAIL protein, a DR-specific ligand, in Ad-FHIT-transduced NSCLC cells considerably enhanced FHIT-induced apoptosis, further demonstrating the involvement of DRs in FHIT-induced apoptosis. Moreover, we also found that FHIT targeted downstream of the DR-mediated signaling pathway. FHIT overexpression disrupted mitochondrial membrane integrity and activated multiple pro-apoptotic proteins in NSCLC cell. These results suggest that FHIT induces apoptosis through a sequential activation of DR-mediated pro-apoptotic signaling pathways in human NSCLC cells. PMID:17328863

  19. 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. PMID:26984266

  20. Ursolic Acid, a Pentacyclin Triterpene, Potentiates TRAIL-induced Apoptosis through p53-independent Up-regulation of Death Receptors

    PubMed Central

    Prasad, Sahdeo; Yadav, Vivek R.; Kannappan, Ramaswamy; Aggarwal, Bharat B.

    2011-01-01

    Discovery of the molecular targets of traditional medicine and its chemical footprints can validate the use of such medicine. In the present report, we investigated the effect of ursolic acid (UA), a pentacyclic triterpenoid found in rosemary and holy basil, on apoptosis induced by TRAIL. We found that UA potentiated TRAIL-induced apoptosis in cancer cells. In addition, UA also sensitized TRAIL-resistant cancer cells to the cytokine. When we investigated the mechanism, we found that UA down-regulated cell survival proteins and induced the cell surface expression of both TRAIL receptors, death receptors 4 and 5 (DR4 and -5). Induction of receptors by UA occurred independently of cell type. Gene silencing of either receptor by small interfering RNA reduced the apoptosis induced by UA and the effect of TRAIL. In addition, UA also decreased the expression of decoy receptor 2 (DcR2) but not DcR1. Induction of DRs was independent of p53 because UA induced DR4 and DR5 in HCT116 p53−/− cells. Induction of DRs, however, was dependent on JNK because UA induced JNK, and its pharmacologic inhibition abolished the induction of the receptors. The down-regulation of survival proteins and up-regulation of the DRs required reactive oxygen species (ROS) because UA induced ROS, and its quenching abolished the effect of the terpene. Also, potentiation of TRAIL-induced apoptosis by UA was significantly reduced by both ROS quenchers and JNK inhibitor. In addition, UA was also found to induce the expression of DRs, down-regulate cell survival proteins, and activate JNK in orthotopically implanted human colorectal cancer in a nude mouse model. Overall, our results showed that UA potentiates TRAIL-induced apoptosis through activation of ROS and JNK-mediated up-regulation of DRs and down-regulation of DcR2 and cell survival proteins. PMID:21156789

  1. Paclitaxel promotes a caspase 8-mediated apoptosis via death effector domain association with microtubules

    PubMed Central

    Mielgo, Ainhoa; Torres, Vicente A.; Clair, Kiran; Barbero, Simone; Stupack, Dwayne G.

    2009-01-01

    Microtubule-perturbing drugs have become front line chemotherapeutics, inducing cell cycle crisis as a major mechanism of action. However, these agents exhibit pleiotropic effects on cells, and can induce apoptosis via other means. Paclitaxel, a microtubule-stabilizing agent, induces a caspase-dependent apoptosis, though the precise mechanism(s) remain unclear. Here, we used genetic approaches to evaluate the role of caspase 8 in paclitaxel-mediated apoptosis. We observed that caspase 8-expressing cells are more sensitive to paclitaxel than caspase 8-deficient cells. Mechanistically, caspase 8 was found associated with microtubules, and this interaction increased following paclitaxel-treatment. The prodomains (DEDs) of caspase 8 were sufficient for interaction with microtubules, but the caspase 8 holoprotein was required for apoptosis. DED-only forms of caspase 8 were found in both primary and tumor cell lines, associating with perinuclear microtubules and the centrosome. Microtubule-association, and paclitaxel-sensitivity, depends upon a critical lysine (K156) within a microtubule-binding motif (KLD) in DED-b of caspase 8. The results reveal an unexpected pathway of apoptosis mediated by caspase 8. PMID:19668227

  2. Gefitinib upregulates death receptor 5 expression to mediate rmhTRAIL-induced apoptosis in Gefitinib-sensitive NSCLC cell line

    PubMed Central

    Yan, Dong; Ge, Yang; Deng, Haiteng; Chen, Wenming; An, Guangyu

    2015-01-01

    Background Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in tumor cells, but when used alone, it is not effective in the treatment of TRAIL-resistant tumors. Some studies have shown that gefitinib interacts with recombinant mutant human TRAIL (rmhTRAIL) to induce high levels of apoptosis in gefitinib-responsive bladder cancer cell lines; however, the molecular mechanisms underlying the anticancer effects are not fully understood. Several reports have shown that the death receptor 5 (DR5) plays an important role in sensitizing cancer cells to apoptosis induced by TRAIL. Therefore, we investigated the effects of the combination of drugs and the expression of the DR5 to analyze the growth of a gefitinib-responsive non-small cell lung cancer cell line PC9, which was treated with rmhTRAIL and gefitinib individually or in combination. Methods Human PC9 non-small cell lung cancer cells harboring an epidermal growth factor receptor mutation were used as a model for the identification of the therapeutic effects of gefitinib alone or in combination with rmhTRAIL, and cytotoxicity was assessed by MTT assays. Cell cycle and apoptosis were investigated using flow cytometry. Moreover, the effects of drugs on DR5, BAX, FLIP, and cleaved-caspase3 proteins expressions were analyzed using Western blot analyses. Finally, quantitative polymerase chain reaction analysis was carried out to assess whether rmhTRAIL and gefitinib modulate the expression of genes related to drug activity. Results Gefitinib and rmhTRAIL synergistically interact to inhibit cell proliferation, and apoptosis assessment demonstrated that associations of drug increased the apoptotic index. rmhTRAIL when used alone downregulated DR5 and upregulated BAX, FLIP, and cleaved-caspase3 proteins expressions. However, results obtained in Western blot analyses demonstrated that the combined treatment-induced cell apoptosis was achieved involving upregulated DR5, cleaved-caspase3, and

  3. Synergistic Myeloma Cell Death via Novel Intracellular Activation of Caspase-10-Dependent Apoptosis by Carfilzomib and Selinexor.

    PubMed

    Rosebeck, Shaun; Alonge, Mattina M; Kandarpa, Malathi; Mayampurath, Anoop; Volchenboum, Samuel L; Jasielec, Jagoda; Dytfeld, Dominik; Maxwell, Sean P; Kraftson, Stephanie J; McCauley, Dilara; Shacham, Sharon; Kauffman, Michael; Jakubowiak, Andrzej J

    2016-01-01

    Exportin1 (XPO1; also known as chromosome maintenance region 1, or CRM1) controls nucleo-cytoplasmic transport of most tumor suppressors and is overexpressed in many cancers, including multiple myeloma, functionally impairing tumor suppressive function via target mislocalization. Selective inhibitor of nuclear export (SINE) compounds block XPO1-mediated nuclear escape by disrupting cargo protein binding, leading to retention of tumor suppressors, induction of cancer cell death, and sensitization to other drugs. Combined treatment with the clinical stage SINE compound selinexor and the irreversible proteasome inhibitor (PI) carfilzomib induced synergistic cell death of myeloma cell lines and primary plasma cells derived from relapsing/refractory myeloma patients and completely impaired the growth of myeloma cell line-derived tumors in mice. Investigating the details of SINE/PI-induced cell death revealed (i) reduced Bcl-2 expression and cleavage and inactivation of Akt, two prosurvival regulators of apoptosis and autophagy; (ii) intracellular membrane-associated aggregation of active caspases, which depended on caspase-10 protease activity; and (iii) novel association of caspase-10 and autophagy-associated proteins p62 and LC3 II, which may prime activation of the caspase cascade. Overall, our findings provide novel mechanistic rationale behind the potent cell death induced by combining selinexor with carfilzomib and support their use in the treatment of relapsed/refractory myeloma and potentially other cancers. PMID:26637366

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

    PubMed

    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

  5. 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

  6. 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

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

    PubMed

    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

  8. Ewing's sarcoma family tumors are sensitive to tumor necrosis factor-related apoptosis-inducing ligand and express death receptor 4 and death receptor 5.

    PubMed

    Mitsiades, N; Poulaki, V; Mitsiades, C; Tsokos, M

    2001-03-15

    In this study, we investigated the sensitivity of Ewing's sarcoma family tumors (ESFTs) of children and adolescents to the tumor necrosis factor-related apoptosis-inducing Ligand (TRAIL). TRAIL binds to death receptors (DRs) DR4, DR5, DcR1, and DcR2. Either DR4 or DR5 can induce apoptosis, whereas DcR1 and DcR2 are considered inhibitory receptors. Nine of 10 ESFT cell lines, including several that were Fas resistant, underwent apoptosis with TRAIL through activation of caspase-10, capase-8 (FLICE), caspase-3, and caspase-9. In contrast to the Fas signaling pathway, caspase-10, but not caspase-8 or the Fas-associated death domain-containing molecule, was recruited to the TRAIL receptor-associated signaling complex. We found that 9 of 10 ESFT cell lines expressed both DR4 and DR5 by Western blotting, whereas the TRAIL-resistant line expressed only DR4. However, DR4 was absent from the cell surface in the resistant and two additional lines (three of five tested lines), suggesting that it may have been nonfunctional. On the contrary, DR5 was located on the cell surface in all four sensitive lines tested, being absent only from the cell surface of the resistant line that was also DR5-negative by Western blotting. In agreement with these findings, the resistance of the line was overcome by restoration of DR5 levels by transfection. Levels of DcR1 and DcR2 or levels of the FLICE-inhibitory protein (FLIP) did not correlate with TRAIL resistance, and protein synthesis inhibition did not sensitize the TRAIL-resistant line to TRAIL. Because these data suggested that sensitivity of ESFTs to TRAIL was mainly based on the presence of DR4/DR5, we investigated the presence of these receptors in 32 ESFT tissue sections by immunohistochemistry. We found that 23 of 32 tumor tissues (72%) expressed both receptors, 8 of 32 (25%) expressed one receptor only, and 1 was negative for both. Our finding of wide expression of DR4/DR5 in ESFT in vivo, in combination with their high sensitivity

  9. Block of Death-Receptor Apoptosis Protects Mouse Cytomegalovirus from Macrophages and Is a Determinant of Virulence in Immunodeficient Hosts

    PubMed Central

    Ebermann, Linda; Ruzsics, Zsolt; Guzmán, Carlos A.; van Rooijen, Nico; Casalegno-Garduño, Rosaely; Koszinowski, Ulrich; Čičin-Šain, Luka

    2012-01-01

    The inhibition of death-receptor apoptosis is a conserved viral function. The murine cytomegalovirus (MCMV) gene M36 is a sequence and functional homologue of the human cytomegalovirus gene UL36, and it encodes an inhibitor of apoptosis that binds to caspase-8, blocks downstream signaling and thus contributes to viral fitness in macrophages and in vivo. Here we show a direct link between the inability of mutants lacking the M36 gene (ΔM36) to inhibit apoptosis, poor viral growth in macrophage cell cultures and viral in vivo fitness and virulence. ΔM36 grew poorly in RAG1 knockout mice and in RAG/IL-2-receptor common gamma chain double knockout mice (RAGγC−/−), but the depletion of macrophages in either mouse strain rescued the growth of ΔM36 to almost wild-type levels. This was consistent with the observation that activated macrophages were sufficient to impair ΔM36 growth in vitro. Namely, spiking fibroblast cell cultures with activated macrophages had a suppressive effect on ΔM36 growth, which could be reverted by z-VAD-fmk, a chemical apoptosis inhibitor. TNFα from activated macrophages synergized with IFNγ in target cells to inhibit ΔM36 growth. Hence, our data show that poor ΔM36 growth in macrophages does not reflect a defect in tropism, but rather a defect in the suppression of antiviral mediators secreted by macrophages. To the best of our knowledge, this shows for the first time an immune evasion mechanism that protects MCMV selectively from the antiviral activity of macrophages, and thus critically contributes to viral pathogenicity in the immunocompromised host devoid of the adaptive immune system. PMID:23271968

  10. Cannabidiol induces programmed cell death in breast cancer cells by coordinating the cross-talk between apoptosis and autophagy.

    PubMed

    Shrivastava, Ashutosh; Kuzontkoski, Paula M; Groopman, Jerome E; Prasad, Anil

    2011-07-01

    Cannabidiol (CBD), a major nonpsychoactive constituent of cannabis, is considered an antineoplastic agent on the basis of its in vitro and in vivo activity against tumor cells. However, the exact molecular mechanism through which CBD mediates this activity is yet to be elucidated. Here, we have shown CBD-induced cell death of breast cancer cells, independent of cannabinoid and vallinoid receptor activation. Electron microscopy revealed morphologies consistent with the coexistence of autophagy and apoptosis. Western blot analysis confirmed these findings. We showed that CBD induces endoplasmic reticulum stress and, subsequently, inhibits AKT and mTOR signaling as shown by decreased levels of phosphorylated mTOR and 4EBP1, and cyclin D1. Analyzing further the cross-talk between the autophagic and apoptotic signaling pathways, we found that beclin1 plays a central role in the induction of CBD-mediated apoptosis in MDA-MB-231 breast cancer cells. Although CBD enhances the interaction between beclin1 and Vps34, it inhibits the association between beclin1 and Bcl-2. In addition, we showed that CBD reduces mitochondrial membrane potential, triggers the translocation of BID to the mitochondria, the release of cytochrome c to the cytosol, and, ultimately, the activation of the intrinsic apoptotic pathway in breast cancer cells. CBD increased the generation of reactive oxygen species (ROS), and ROS inhibition blocked the induction of apoptosis and autophagy. Our study revealed an intricate interplay between apoptosis and autophagy in CBD-treated breast cancer cells and highlighted the value of continued investigation into the potential use of CBD as an antineoplastic agent. PMID:21566064

  11. Targeted gene delivery via N-acetylglucosamine receptor mediated endocytosis.

    PubMed

    Singh, Bijay; Maharjan, Sushila; Kim, You-Kyoung; Jiang, Tai; Islam, Mohammad Ariful; Kang, Sang-Kee; Cho, Myung-Haing; Choi, Yun-Jaie; Cho, Chong-Su

    2014-11-01

    Receptor-mediated endocytosis is a promising approach of gene delivery into the target cells via receptor-ligand interaction. Vimentins at the cell surface are recently known to bind N-acetylglucosamine (GlcNAc) residue, therefore, the cell surfaces of vimentin-expressing cells could be targeted by using the GlcNAc residue as a specific ligand for receptor-mediated gene delivery. Here, we have developed polymeric gene delivery vectors, based on poly(ethylene oxide)(PEO) and poly(aspartamide), namely poly[(aspartamide)(diethylenetriamine)]-b-[PEO-(GlcNAc)] (PADPG) and poly[(aspartamide)(diethylenetriamine)]-b-[PEO] (PADP) to elucidate the efficiency of GlcNAc ligand for gene delivery through receptor mediated endocytosis. To determine the efficiency of these polymeric vectors for specific gene delivery, the DNA condensation ability of PADPG and PADP and the subsequent formation of polymeric nanoparticles were confirmed by gel retardation assay and transmission electron microscopy respectively. Both PADPG and PADP had lower cytotoxicity than polyethylenimine 25 K (PEI 25 K). However, their transfection efficiency was comparatively lower than PEI 25 K due to hydrophilic property of PEO in the vectors. To observe the stability of polymeric nanoparticles, the transfection of PADPG and PADP was carried out in the presence of serum. Favorably, the interfering effect of serum on the transfection efficiency of PADPG and PADP was also very low. Finally, when the cell specificity of these polymeric vectors was investigated, PADPG had high gene transfection in vimentin-expressing cells than vimentin-deficiency cells. The high transfection efficiency of PADPG was attributed to the GlcNAc in the polymeric vector which interact specifically with vimentin in the cells for the receptor-mediated endocytosis. The competitive inhibition assay further proved the receptor-mediated endocytosis of PADPG. Thus, this study demonstrates that conjugation of GlcNAc is an effective and rational

  12. 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. PMID:26362468

  13. 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. PMID:26316710

  14. Quercetin induced apoptosis in association with death receptors and fludarabine in cells isolated from chronic lymphocytic leukaemia patients

    PubMed Central

    Russo, M; Spagnuolo, C; Volpe, S; Mupo, A; Tedesco, I; Russo, G-L

    2010-01-01

    Background: Quercetin is a flavonoid naturally present in food and beverages belonging to the large class of phytochemicals with potential anti-cancer properties. Here, we investigated the ability of quercetin to sensitise primary cells from chronic lymphocytic leukaemia (CLL) to death receptor (DR) agonists, recombinant TNF-related-apoptosis-inducing ligand (rTRAIL) and anti-CD95, and to fludarabine, a widely used chemotherapeutic drug against CLL. Methods: Peripheral white blood cells were isolated from patients and incubated with medium containing 50 ng ml anti-CD95 agonist antibody; 10 ng ml recombinant TRAIL; 10–25 μM quercetin and 3.5–14 μM fludarabine. Neutral Red assay was used to measure cell viability, where as apoptosis was assessed by determining caspase-3 activity, exposure to Annexin V and PARP fragmentation. Results: Quercetin significantly enhanced anti-CD95- and rTRAIL-induced cell death as shown by decreased cell viability, increased caspase-3 and -9 activities, and positivity to Annexin V. In addition, association of quercetin with fludarabine increases the apoptotic response in CLL cells of about two-fold compared with quercetin monotreatment. Conclusion: This work shows that resistance to DR- and fludarabine-induced cell death in leukaemic cells isolated from CLL patients can be ameliorated or bypassed by the combined treatment with quercetin. Considering the low toxicity of the molecule, our study results are in favour of a potential use of quercetin in adjuvant chemotherapy in combination with other drugs. PMID:20648016

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

    PubMed Central

    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. PMID:26316710

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

    PubMed

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

    2015-11-15

    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. Secretory prostate apoptosis response (Par)-4 sensitizes multicellular spheroids (MCS) of glioblastoma multiforme cells to tamoxifen-induced cell death

    PubMed Central

    Jagtap, Jayashree C.; Parveen, D.; Shah, Reecha D.; Desai, Aarti; Bhosale, Dipali; Chugh, Ashish; Ranade, Deepak; Karnik, Swapnil; Khedkar, Bhushan; Mathur, Aaishwarya; Natesh, Kumar; Chandrika, Goparaju; Shastry, Padma

    2014-01-01

    Glioblastoma multiforme (GBM) is the most malignant form of brain tumor and is associated with resistance to conventional therapy and poor patient survival. Prostate apoptosis response (Par)-4, a tumor suppressor, is expressed as both an intracellular and secretory/extracellular protein. Though secretory Par-4 induces apoptosis in cancer cells, its potential in drug-resistant tumors remains to be fully explored. Multicellular spheroids (MCS) of cancer cells often acquire multi-drug resistance and serve as ideal experimental models. We investigated the role of Par-4 in Tamoxifen (TAM)-induced cell death in MCS of human cell lines and primary cultures of GBM tumors. TCGA and REMBRANT data analysis revealed that low levels of Par-4 correlated with low survival period (21.85 ± 19.30 days) in GBM but not in astrocytomas (59.13 ± 47.26 days) and oligodendrogliomas (58.04 ± 59.80 days) suggesting low PAWR expression as a predictive risk factor in GBM. Consistently, MCS of human cell lines and primary cultures displayed low Par-4 expression, high level of chemo-resistance genes and were resistant to TAM-induced cytotoxicity. In monolayer cells, TAM-induced cytotoxicity was associated with enhanced expression of Par-4 and was alleviated by silencing of Par-4 using specific siRNA. TAM effectively induced secretory Par-4 in conditioned medium (CM) of cells cultured as monolayer but not in MCS. Moreover, MCS were rendered sensitive to TAM-induced cell death by exposure to conditioned medium (CM)-containing Par-4 (derived from TAM-treated monolayer cells). Also TAM reduced the expression of Akt and PKCζ in GBM cells cultured as monolayer but not in MCS. Importantly, combination of TAM with inhibitors to PI3K inhibitor (LY294002) or PKCζ resulted in secretion of Par-4 and cell death in MCS. Since membrane GRP78 is overexpressed in most cancer cells but not normal cells, and secretory Par-4 induces apoptosis by binding to membrane GRP78, secretory Par-4 is an

  18. 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

  19. Genistein promotes cell death of ethanol-stressed HeLa cells through the continuation of apoptosis or secondary necrosis

    PubMed Central

    2013-01-01

    Background Apoptosis is a major target and treatment effect of multiple chemotherapeutical agents in cancer. A soybean isoflavone, genistein, is a well-studied chemopreventive agent and has been reported to potentiate the anticancer effect of some chemotherapeutics. However, its mechanistic basis of chemo-enhancement effect remains to be fully elucidated. Methods Apoptotic features of low concentration stressed cancer cells were studied by microscopic method, western blot, immunostaining and annexin V/PI assay. Genistein’s effects on unstressed cells and recovering cells were investigated using MTT cell viability assay and LDH cytotoxicity assay. Quantitative real-time PCR was employed to analyze the possible gene targets involved in the recovery and genistein’s effect. Results Low-concentration ethanol stressed cancer cells showed apoptotic features and could recover after stress removal. In stressed cells, genistein at sub-toxic dosage promoted the cell death. Quantitative real-time PCR revealed the up-regulation of anti-apoptotic genes MDM2 and XIAP during the recovery process in HeLa cells, and genistein treatment suppressed their expression. The application of genistein, MDM2 inhibitor and XIAP inhibitor to the recovering HeLa cells caused persistent caspase activity and enhanced cell death. Flow cytometry study indicated that genistein treatment could lead to persistent phosphatidylserine (PS) externalization and necrotic events in the recovering HeLa cells. Caspase activity inhibition shifted the major effect of genistein to necrosis. Conclusions These results suggested two possible mechanisms through which genistein promoted cell death in stressed cancer cells. Genistein could maintain the existing apoptotic signal to enhance apoptotic cell death. It could also disrupt the recovering process in caspase-independent manner, which lead to necrotic events. These effects may be related to the enhanced antitumor effect of chemotherapeutic drugs when they were

  20. Synergistic chemopreventive effects of curcumin and berberine on human breast cancer cells through induction of apoptosis and autophagic cell death

    PubMed Central

    Wang, Kai; Zhang, Chao; Bao, Jiaolin; Jia, Xuejing; Liang, Yeer; Wang, Xiaotong; Chen, Meiwan; Su, Huanxing; Li, Peng; Wan, Jian-Bo; He, Chengwei

    2016-01-01

    Curcumin (CUR) and berberine (BBR) are renowned natural compounds that exhibit potent anticancer activities through distinct molecular mechanisms. However, the anticancer capacity of either CUR or BBR is limited. This prompted us to investigate the chemopreventive potential of co-treatment of CUR and BBR against breast cancers. The results showed that CUR and BBR in combination synergistically inhibited the growth of both MCF-7 and MDA-MB-231 breast cancer cells than the compounds used alone. Further study confirmed that synergistic anti-breast cancer activities of co-treatment of these two compounds was through inducing more apoptosis and autophagic cell death (ACD). The co-treatment-induced apoptosis was caspase-dependent and through activating ERK pathways. Our data also demonstrated that co-treatment of CUR and BBR strongly up-regulated phosphorylation of JNK and Beclin1, and decreased phosphorylated Bcl-2. Inhibition of JNK by SP600125 markedly decreased LC3-II and Beclin1, restored phosphorylated Bcl-2, and reduced the cytotoxicity induced by the two compounds in combination. These results strongly suggested that JNK/Bcl-2/Beclin1 pathway played a key role in the induction of ACD in breast cancer cells by co-treatment of CUR and BBR. This study provides an insight into the potential application of curcumin and berberine in combination for the chemoprevention and treatment of breast cancers. PMID:27263652

  1. Synergistic chemopreventive effects of curcumin and berberine on human breast cancer cells through induction of apoptosis and autophagic cell death.

    PubMed

    Wang, Kai; Zhang, Chao; Bao, Jiaolin; Jia, Xuejing; Liang, Yeer; Wang, Xiaotong; Chen, Meiwan; Su, Huanxing; Li, Peng; Wan, Jian-Bo; He, Chengwei

    2016-01-01

    Curcumin (CUR) and berberine (BBR) are renowned natural compounds that exhibit potent anticancer activities through distinct molecular mechanisms. However, the anticancer capacity of either CUR or BBR is limited. This prompted us to investigate the chemopreventive potential of co-treatment of CUR and BBR against breast cancers. The results showed that CUR and BBR in combination synergistically inhibited the growth of both MCF-7 and MDA-MB-231 breast cancer cells than the compounds used alone. Further study confirmed that synergistic anti-breast cancer activities of co-treatment of these two compounds was through inducing more apoptosis and autophagic cell death (ACD). The co-treatment-induced apoptosis was caspase-dependent and through activating ERK pathways. Our data also demonstrated that co-treatment of CUR and BBR strongly up-regulated phosphorylation of JNK and Beclin1, and decreased phosphorylated Bcl-2. Inhibition of JNK by SP600125 markedly decreased LC3-II and Beclin1, restored phosphorylated Bcl-2, and reduced the cytotoxicity induced by the two compounds in combination. These results strongly suggested that JNK/Bcl-2/Beclin1 pathway played a key role in the induction of ACD in breast cancer cells by co-treatment of CUR and BBR. This study provides an insight into the potential application of curcumin and berberine in combination for the chemoprevention and treatment of breast cancers. PMID:27263652

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

    PubMed Central

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

    2013-01-01

    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 A1. 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. PMID:23580233

  3. Comparative analyses of lysophosphatidic acid receptor-mediated signaling.

    PubMed

    Fukushima, Nobuyuki; Ishii, Shoichi; Tsujiuchi, Toshifumi; Kagawa, Nao; Katoh, Kazutaka

    2015-06-01

    Lysophosphatidic acid (LPA) is a bioactive lipid mediator that activates G protein-coupled LPA receptors to exert fundamental cellular functions. Six LPA receptor genes have been identified in vertebrates and are classified into two subfamilies, the endothelial differentiation genes (edg) and the non-edg family. Studies using genetically engineered mice, frogs, and zebrafish have demonstrated that LPA receptor-mediated signaling has biological, developmental, and pathophysiological functions. Computational analyses have also identified several amino acids (aa) critical for LPA recognition by human LPA receptors. This review focuses on the evolutionary aspects of LPA receptor-mediated signaling by comparing the aa sequences of vertebrate LPA receptors and LPA-producing enzymes; it also summarizes the LPA receptor-dependent effects commonly observed in mouse, frog, and fish. PMID:25732591

  4. 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.

  5. Caffeic acid phenethyl ester enhances TRAIL-mediated apoptosis via CHOP-induced death receptor 5 upregulation in hepatocarcinoma Hep3B cells.

    PubMed

    Dilshara, Matharage Gayani; Jayasooriya, Rajapaksha Gedara Prasad Tharanga; Park, Sang Rul; Choi, Yung-Hyun; Choi, Il-Whan; Kim, Gi-Young

    2016-07-01

    Caffeic acid phenethyl ester (CAPE) exhibits various pharmaceutical properties, including anti-bacterial, anti-inflammatory, anti-viral, anti-cancer, and anti-oxidative activity. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been a promising anti-cancer agent that preferentially induces cancer cell apoptosis with negligible cytotoxicity toward normal cells. Therefore, the present study investigated whether CAPE promotes TRAIL-mediated cytotoxicity in hepatocarcinoma Hep3B cells. The present study demonstrated that CAPE sensitized TRAIL-mediated cell death in Hep3B carcinoma cells. The percentages of the apoptotic cells and annexin-V(+) cells significantly increased in combined treatment with CAPE and TRAIL (CAPE/TRAIL). Treatment with pancaspase inhibitor, z-VAD-fmk, attenuated CAPE/TRAIL-induced apoptosis, suggesting that the combined treatment triggers caspase-dependent apoptosis. Additionally, we found that CAPE stimulated the expression of death receptor 5 (DR5) and treatment with DR5/Fc chimera protein significantly blocked CAPE/TRAIL-induced apoptosis, which indicates that CAPE/TRAIL stimulated apoptosis through the binding of TRAIL to DR5. Moreover, expression of transcription factor C/EBP homologous protein (CHOP) markedly increased in response to CAPE and transient knockdown of CHOP abolished CAPE/TRAIL-mediated apoptosis. These results suggest that CHOP is a key regulator in CAPE/TRAIL-mediated apoptosis. Taken together, the present study found that CAPE significantly enhanced TRAIL-mediated apoptosis in Hep3B carcinoma cells and suggested that CAPE has promising potential in chemoprevention of hepatocellular carcinomas. PMID:27260301

  6. FK-16 Derived from the Anticancer Peptide LL-37 Induces Caspase-Independent Apoptosis and Autophagic Cell Death in Colon Cancer Cells

    PubMed Central

    Ren, Shun X.; Shen, Jin; Cheng, Alfred S. L.; Lu, Lan; Chan, Ruby L. Y.; Li, Zhi J.; Wang, Xiao J.; Wong, Clover C. M.; Zhang, Lin; Ng, Simon S. M.; Chan, Franky L.; Chan, Francis K. L.; Yu, Jun; Sung, Joseph J. Y.; Wu, William K. K.; Cho, Chi H.

    2013-01-01

    Host immune peptides, including cathelicidins, have been reported to possess anticancer properties. We previously reported that LL-37, the only cathelicidin in humans, suppresses the development of colon cancer. In this study, the potential anticancer effect of FK-16, a fragment of LL-37 corresponding to residues 17 to 32, on cultured colon cancer cells was evaluated. FK-16 induced a unique pattern of cell death, marked by concurrent activation of caspase-independent apoptosis and autophagy. The former was mediated by the nuclear translocation of AIF and EndoG whereas the latter was characterized by enhanced expression of LC3-I/II, Atg5 and Atg7 and increased formation of LC3-positive autophagosomes. Knockdown of Atg5 or Atg7 attenuated the cytotoxicity of FK-16, indicating FK-16-induced autophagy was pro-death in nature. Mechanistically, FK-16 activated nuclear p53 to upregulate Bax and downregulate Bcl-2. Knockdown of p53, genetic ablation of Bax, or overexpression of Bcl-2 reversed FK-16-induced apoptosis and autophagy. Importantly, abolition of AIF/EndoG-dependent apoptosis enhanced FK-16-induced autophagy while abolition of autophagy augmented FK-16-induced AIF−/EndoG-dependent apoptosis. Collectively, FK-16 induces caspase-independent apoptosis and autophagy through the common p53-Bcl-2/Bax cascade in colon cancer cells. Our study also uncovered previously unknown reciprocal regulation between these two cell death pathways. PMID:23700428

  7. ATG7 deficiency suppresses apoptosis and cell death induced by lysosomal photodamage

    PubMed Central

    Kessel, David H.; Price, Michael; Reiners, Jr., John J.

    2012-01-01

    Photodynamic therapy (PDT) involves photosensitizing agents that, in the presence of oxygen and light, initiate formation of cytotoxic reactive oxygen species (ROS). PDT commonly induces both apoptosis and autophagy. Previous studies with murine hepatoma 1c1c7 cells indicated that loss of autophagy-related protein 7 (ATG7) inhibited autophagy and enhanced the cytotoxicity of photosensitizers that mediate photodamage to mitochondria or the endoplasmic reticulum. In this study, we examined two photosensitizing agents that target lysosomes: the chlorin NPe6 and the palladium bacteriopheophorbide WST11. Irradiation of wild-type 1c1c7 cultures loaded with either photosensitizer induced apoptosis and autophagy, with a blockage of autophagic flux. An ATG7- or ATG5-deficiency suppressed the induction of autophagy in PDT protocols using either photosensitizer. Whereas ATG5-deficient cells were quantitatively similar to wild-type cultures in their response to NPe6 and WST11 PDT, an ATG7-deficiency suppressed the apoptotic response (as monitored by analyses of chromatin condensation and procaspase-3/7 activation) and increased the LD50 light dose by > 5-fold (as monitored by colony-forming assays). An ATG7-deficiency did not prevent immediate lysosomal photodamage, as indicated by loss of the lysosomal pH gradient. However, unlike wild-type and ATG5-deficient cells, the lysosomes of ATG7-deficient cells recovered this gradient within 4 h of irradiation, and never underwent permeabilization (monitored as release of endocytosed 10-kDa dextran polymers). We propose that the efficacy of lysosomal photosensitizers is in part due to both promotion of autophagic stress and suppression of autophagic prosurvival functions. In addition, an effect of ATG7 unrelated to autophagy appears to modulate lysosomal photodamage. PMID:22889762

  8. Dual PI-3 kinase/mTOR inhibition impairs autophagy flux and induces cell death independent of apoptosis and necroptosis

    PubMed Central

    Button, Robert W.; Vincent, Joseph H.; Strang, Conor J.; Luo, Shouqing

    2016-01-01

    The PI-3 kinase (PI-3K)/mTOR pathway is critical for cell growth and proliferation. Strategies of antagonising this signaling have proven to be detrimental to cell survival. This observation, coupled with the fact many tumours show enhanced growth signaling, has caused dual inhibitors of PI-3K and mTOR to be implicated in cancer treatment, and have thus been studied across various tumour models. Since PI-3K (class-I)/mTOR pathway negatively regulates autophagy, dual inhibitors of PI-3K/mTOR are currently believed to be autophagy activators. However, our present data show that the dual PI-3K/mTOR inhibition (DKI) potently suppresses autophagic flux. We further confirm that inhibition of Vps34/PI3KC3, the class-III PI-3K, causes the blockade to autophagosome-lysosome fusion. Our data suggest that DKI induces cell death independently of apoptosis and necroptosis, whereas autophagy perturbation by DKI may contribute to cell death. Given that autophagy is critical in cellular homeostasis, our study not only clarifies the role of a dual PI-3K/mTOR inhibitor in autophagy, but also suggests that its autophagy inhibition needs to be considered if such an agent is used in cancer chemotherapy. PMID:26814436

  9. 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. PMID:26463240

  10. BDE-99 congener induces cell death by apoptosis of human hepatoblastoma cell line - HepG2.

    PubMed

    Souza, A O; Pereira, L C; Oliveira, D P; Dorta, D J

    2013-03-01

    Polybrominated Diphenyl Ethers (PBDEs) are an important class of flame retardants with a wide range of toxic effects on biotic and abiotic systems. The toxic mechanisms of PBDEs are still not completely understood because there are several different congeners with different chemical and biological characteristics. BDE-99 is one of these, widely found in the environment and biological samples, showing evidence of neurotoxic and endocrine disruption activities, but with little information about its action mechanism described in the current literature. This work investigated the effects of BDE-99 on the HepG2 cell line in order to clarify its toxic mechanism, using concentrations of 0.5-25 μM (24 and 48 h). Our results showed that BDE-99 could cause cell death in the higher concentrations, its activity being related to a decrease in mitochondrial membrane potential and an accumulation of ROS. It was also shown that BDE-99 induced the exposure of phosphatidylserine, caspases 3 and 9 activation and DNA fragmentation in HepG2 cells, without causing the release of LDH. Thus it was shown that BDE-99 could cause HepG2 cell death by apoptosis, suggesting its toxicity to the human liver. PMID:23124135

  11. 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. PMID:9568531

  12. D,L-Sulforaphane-induced cell death in human prostate cancer cells is regulated by inhibitor of apoptosis family proteins and Apaf-1.

    PubMed

    Choi, Sunga; Lew, Karen L; Xiao, Hui; Herman-Antosiewicz, Anna; Xiao, Dong; Brown, Charles K; Singh, Shivendra V

    2007-01-01

    D,L-Sulforaphane (SFN), a synthetic analogue of cruciferous vegetable-derived isomer l-SFN, suppresses proliferation of cancer cells by causing apoptosis but the mechanism of cell death is not fully understood. We used LNCaP (wild-type p53) and PC-3 (p53 deficient) human prostate cancer cells to gain further insights into the mechanism of SFN-induced apoptosis. The LNCaP cell line was relatively more sensitive to SFN-induced apoptosis compared with PC-3. The SFN treatment caused stabilization of p53 protein in LNCaP cells, but SFN-mediated apoptosis was not attenuated by knockdown of p53 protein. Instead, the differential sensitivity of these cells to SFN-induced apoptosis correlated with difference in kinetics of Bax conformational change. Ectopic expression of Bcl-2 failed to confer protection against SFN-induced cell death in LNCaP cells. Treatment of PC-3 cells with SFN resulted in a marked decrease in the levels of inhibitor of apoptosis (IAP) family proteins (cIAP1, cIAP2 and XIAP), which was accompanied by inhibition of nuclear translocation of p65-nuclear factor kappaB (NFkappaB). The effect of SFN on levels of IAP family proteins as well as transcriptional activity of NFkappaB was biphasic in LNCaP cells. The SFN-treated LNCaP and PC-3 cells exhibited a marked increase in protein level of Apaf-1, which was accompanied by an increase in transcriptional activity of E2F1. The SFN-induced apoptosis in both cell lines was significantly attenuated by Apaf-1 protein knockdown. In conclusion, the present study reveals a complex signaling mechanism involving Bax activation, downregulation of IAP family proteins and Apaf-1 induction in regulation of SFN-induced cell death. PMID:16920735

  13. 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

  14. Programmed Death 1 (PD-1) is involved in the development of proliferative diabetic retinopathy by mediating activation-induced apoptosis

    PubMed Central

    Fang, Mengyuan; Meng, Qianli; Wang, Liya; Zhao, Zhaoxia; Zhang, Liang; Kuang, Jian; Cui, Ying; Mai, Liping; Zhu, Jiening

    2015-01-01

    Purpose Recent studies revealed that immunological mechanisms play a prominent role in the pathogenesis of proliferative diabetic retinopathy (PDR). Given the importance of the immune response in PDR and the significance of the programmed death 1 (PD-1) pathway as an immune regulatory pathway, the aim of this study is to determine the expression and functional characteristics of the PD-1 pathway in peripheral blood lymphocytes from patients with PDR. Methods Peripheral blood lymphocytes were obtained from patients with PDR, age-matched patients with diabetes mellitus and no diabetic retinopathy (DM-NDR), and controls. The mRNA expression of PD-1 and its ligands were determined using real-time PCR. The frequencies of PD-1 and its ligands, activation-induced apoptosis, IFN-γ, and IL-4 were determined by flow cytometry. Results The PD-1 mRNA expression markedly decreased, while the frequency of PD-1+ cells increased in the PDR group compared with the DM-NDR and control groups. The expression of PD-ligand 1 (PD-L1) mRNA and PD-L1+ cells in the PDR group was lower than that in the other two groups. In the PDR group, the frequency of Annexin V+PI- and Annexin V+PI-PD-1+ cells increased, while the frequency of Annexin V+PI-PD-L1+ cells decreased. Although their expression was upregulated, the ratio of PD-1+ IFN-γ+ to PD-1+IL-4+ cells in the PDR group was not significantly different to that in the DM-NDR and control groups. Conclusions These results suggest that PD-1 is involved in the development of PDR by mediating activation-induced apoptosis. PMID:26321864

  15. Cortex Moutan Induces Bladder Cancer Cell Death via Apoptosis and Retards Tumor Growth in Mouse Bladders.

    PubMed

    Lin, Mei-Yi; Lee, Ying-Ray; Chiang, Su-Yin; Li, Yi-Zhen; Chen, Yueh-Sheng; Hsu, Cheng-Da; Liu, Yi-Wen

    2013-01-01

    Cortex Moutan is the root bark of Paeonia suffruticosa Andr. It is the herbal medicine widely used in Traditional Chinese Medicine for the treatment of blood-heat and blood-stasis syndrome. Furthermore, it has been reported that Cortex Moutan has anticancer effect. In this study, the Cortex Moutan extract was evaluated in bladder cancer therapy in vitro and in vivo. Cortex Moutan extract reduces cell viability with IC50 between 1~2 mg/ml in bladder cancer cells, and it has lower cytotoxicity in normal urotheliums. It arrests cells in G1 and S phase and causes phosphatidylserine expression in the outside of cell membrane. It induces caspase-8 and caspase-3 activation and poly(ADP-ribose) polymerase degradation. The pan caspase inhibitor z-VAD-fmk reverses Cortex Moutan-induced cell death. Cortex Moutan also inhibits cell invasion activity in 5637 cells. In mouse orthotopic bladder cancer model, intravesical application of Cortex Moutan decreases the bladder tumor size without altering the blood biochemical parameters. In summary, these results demonstrate the antiproliferation and anti-invasion properties of Cortex Moutan in bladder cancer cells and its antibladder tumor effect in vivo. Cortex Moutan may provide an alternative therapeutic strategy for the intravesical therapy of superficial bladder cancer. PMID:24282433

  16. 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. PMID:25694272

  17. Sensitization for death receptor- or drug-induced apoptosis by re-expression of caspase-8 through demethylation or gene transfer.

    PubMed

    Fulda, S; Küfer, M U; Meyer, E; van Valen, F; Dockhorn-Dworniczak, B; Debatin, K M

    2001-09-13

    Resistance of tumors to treatment with cytotoxic drugs, irradiation or immunotherapy may be due to disrupted apoptosis programs. Here, we report in a variety of different tumor cells including Ewing tumor, neuroblastoma, malignant brain tumors and melanoma that caspase-8 expression acts as a key determinant of sensitivity for apoptosis induced by death-inducing ligands or cytotoxic drugs. In tumor cell lines resistant to TRAIL, anti-CD95 or TNFalpha, caspase-8 protein and mRNA expression was decreased or absent without caspase-8 gene loss. Methylation-specific PCR revealed hypermethylation of caspase-8 regulatory sequences in cells with impaired caspase-8 expression. Treatment with the demethylation agent 5-Aza-2'-deoxycytidine (5-dAzaC) reversed hypermethylation of caspase-8 resulting in restoration of caspase-8 expression and recruitment and activation of caspase-8 at the CD95 DISC upon receptor cross-linking thereby sensitizing for death receptor-, and importantly, also for drug-induced apoptosis. Inhibition of caspase-8 activity also inhibited apoptosis sensitization by 5-dAzaC. Similar to demethylation, introduction of caspase-8 by gene transfer sensitized for apoptosis induction. Hypermethylation of caspase-8 was linked to reduced caspase-8 expression in different tumor cell lines in vitro and, most importantly, also in primary tumor samples. Thus, these findings indicate that re-expression of caspase-8, e.g. by demethylation or caspase-8 gene transfer, might be an effective strategy to restore sensitivity for chemotherapy- or death receptor-induced apoptosis in various tumors in vivo. PMID:11593392

  18. 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. PMID:23700228

  19. TAK1 Is Essential for Osteoclast Differentiation and Is an Important Modulator of Cell Death by Apoptosis and Necroptosis

    PubMed Central

    Lai, YunJu; Xie, Min; Schneider, Michael D.

    2013-01-01

    Transforming growth factor β (TGF-β)-activated kinase 1 (TAK1), a mitogen-activated protein 3 (MAP3) kinase, plays an essential role in inflammation by activating the IκB kinase (IKK)/nuclear factor κB (NF-κB) and stress kinase (p38 and c-Jun N-terminal kinase [JNK]) pathways in response to many stimuli. The tumor necrosis factor (TNF) superfamily member receptor activator of NF-κB ligand (RANKL) regulates osteoclastogenesis through its receptor, RANK, and the signaling adaptor TRAF6. Because TAK1 activation is mediated through TRAF6 in the interleukin 1 receptor (IL-1R) and toll-like receptor (TLR) pathways, we sought to investigate the consequence of TAK1 deletion in RANKL-mediated osteoclastogenesis. We generated macrophage colony-stimulating factor (M-CSF)-derived monocytes from the bone marrow of mice with TAK1 deletion in the myeloid lineage. Unexpectedly, TAK1-deficient monocytes in culture died rapidly but could be rescued by retroviral expression of TAK1, inhibition of receptor-interacting protein 1 (RIP1) kinase activity with necrostatin-1, or simultaneous genetic deletion of TNF receptor 1 (TNFR1). Further investigation using TAK1-deficient mouse embryonic fibroblasts revealed that TNF-α-induced cell death was abrogated by the simultaneous inhibition of caspases and knockdown of RIP3, suggesting that TAK1 is an important modulator of both apoptosis and necroptosis. Moreover, TAK1-deficient monocytes rescued from programmed cell death did not form mature osteoclasts in response to RANKL, indicating that TAK1 is indispensable to RANKL-induced osteoclastogenesis. To our knowledge, we are the first to report that mice in which TAK1 has been conditionally deleted in osteoclasts develop osteopetrosis. PMID:23166301

  20. Death Receptor-Induced Apoptosis Signalling Regulation by Ezrin Is Cell Type Dependent and Occurs in a DISC-Independent Manner in Colon Cancer Cells

    PubMed Central

    Iessi, Elisabetta; Zischler, Luciana; Etringer, Aurélie; Bergeret, Marion; Morlé, Aymeric; Jacquemin, Guillaume; Morizot, Alexandre; Shirley, Sarah; Lalaoui, Najoua; Elifio-Esposito, Selene L.; Fais, Stefano; Garrido, Carmen; Solary, Eric; Micheau, Olivier

    2015-01-01

    Ezrin belongs to the ERM (ezrin-radixin-moesin) protein family and has been demonstrated to regulate early steps of Fas receptor signalling in lymphoid cells, but its contribution to TRAIL-induced cell death regulation in adherent cancer cells remains unknown. In this study we report that regulation of FasL and TRAIL-induced cell death by ezrin is cell type dependant. Ezrin is a positive regulator of apoptosis in T-lymphoma cell line Jurkat, but a negative regulator in colon cancer cells. Using ezrin phosphorylation or actin-binding mutants, we provide evidence that negative regulation of death receptor-induced apoptosis by ezrin occurs in a cytoskeleton- and DISC-independent manner, in colon cancer cells. Remarkably, inhibition of apoptosis induced by these ligands was found to be tightly associated with regulation of ezrin phosphorylation on serine 66, the tumor suppressor gene WWOX and activation of PKA. Deficiency in WWOX expression in the liver cancer SK-HEP1 or the pancreatic Mia PaCa-2 cell lines as well as WWOX silencing or modulation of PKA activation by pharmacological regulators, in the colon cancer cell line SW480, abrogated regulation of TRAIL signalling by ezrin. Altogether our results show that death receptor pro-apoptotic signalling regulation by ezrin can occur downstream of the DISC in colon cancer cells. PMID:26010871

  1. 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

  2. Gedunin Inactivates the Co-chaperone p23 Protein Causing Cancer Cell Death by Apoptosis*♦

    PubMed Central

    Patwardhan, Chaitanya A.; Fauq, Abdul; Peterson, Laura B.; Miller, Charles; Blagg, Brian S. J.; Chadli, Ahmed

    2013-01-01

    Pharmacological inhibition of Hsp90 is an exciting option for cancer therapy. The clinical efficacy of Hsp90 inhibitors is, however, less than expected. Binding of the co-chaperone p23 to Hsp90 and induced overexpression of anti-apoptotic proteins Hsp70 and Hsp27 are thought to contribute to this outcome. Herein, we report that the natural product gedunin may provide a new alternative to inactivate the Hsp90 machine. We show that gedunin directly binds to p23 and inactivates it, without overexpression of Hsp27 and relatively modest induction of Hsp70. Using molecular docking and mutational analysis, we mapped the gedunin-binding site on p23. Functional analysis shows that gedunin inhibits the p23 chaperoning activity, blocks its cellular interaction with Hsp90, and interferes with p23-mediated gene regulation. Cell treatment with gedunin leads to cancer cell death by apoptosis through inactivation of p23 and activation of caspase 7, which cleaves p23 at the C terminus. These results provide important insight into the molecular mechanism of action of this promising lead compound. PMID:23355466

  3. The nematocysts venom of Chrysaora helvola Brandt leads to apoptosis-like cell death accompanied by uncoupling of oxidative phosphorylation.

    PubMed

    Qu, Xiaosheng; Fan, LanLan; Zhong, Taozheng; Li, Gang; Xia, Xianghua; Long, Hairong; Huang, Danna; Shu, Wei

    2016-02-01

    The present work investigated the effects of the nematocysts venom (NV) from the Chrysaora helvola Brandt (C. helvola) jellyfish on the human nasopharyngeal carcinoma cell line, CNE-2. The medium lethal concentration (LC50), quantified by MTT assays, was 1.7 ± 0.53 μg/mL (n = 5). An atypical apoptosis-like cell death was confirmed by LDH release assay and Annexin V-FITC/PI staining-based flow cytometry. Interestingly, activation of caspase-4 other than caspase-3, -8, -9 and -1 was observed. Moreover, the NV stimuli caused a time-dependent loss of mitochondrial membrane potential (ΔΨm) as was an intracellular ROS burst. These results indicated that there was uncoupling of oxidative phosphorylation (UOP). An examination of the intracellular pH value by a pH-sensitive fluorescent probe, BCECF, suggested that the UOP was due to the time-dependent increase in the intracellular pH. This is the first report that jellyfish venom can induce UOP. PMID:26718259

  4. 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

  5. Receptor-mediated mitophagy in yeast and mammalian systems.

    PubMed

    Liu, Lei; Sakakibara, Kaori; Chen, Quan; Okamoto, Koji

    2014-07-01

    Mitophagy, or mitochondria autophagy, plays a critical role in selective removal of damaged or unwanted mitochondria. Several protein receptors, including Atg32 in yeast, NIX/BNIP3L, BNIP3 and FUNDC1 in mammalian systems, directly act in mitophagy. Atg32 interacts with Atg8 and Atg11 on the surface of mitochondria, promoting core Atg protein assembly for mitophagy. NIX/BNIP3L, BNIP3 and FUNDC1 also have a classic motif to directly bind LC3 (Atg8 homolog in mammals) for activation of mitophagy. Recent studies have shown that receptor-mediated mitophagy is regulated by reversible protein phosphorylation. Casein kinase 2 (CK2) phosphorylates Atg32 and activates mitophagy in yeast. In contrast, in mammalian cells Src kinase and CK2 phosphorylate FUNDC1 to prevent mitophagy. Notably, in response to hypoxia and FCCP treatment, the mitochondrial phosphatase PGAM5 dephosphorylates FUNDC1 to activate mitophagy. Here, we mainly focus on recent advances in our understanding of the molecular mechanisms underlying the activation of receptor-mediated mitophagy and the implications of this catabolic process in health and disease. PMID:24903109

  6. 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.

  7. The androgen receptor mediates antiapoptotic function in myometrial cells.

    PubMed

    Li, H; Li, Y; Morin, D; Plymate, S; Lye, S; Dong, X

    2014-01-01

    During pregnancy, myometrial phenotype is programmed into three characteristic stages referred to as the early proliferative, the midterm hypertrophic, and the late contractile stage. Increased myometrial growth in the early and midterm of pregnancy involves a complex process of cell proliferation, antiapoptosis and differentiation. We have previously demonstrated that the androgen receptor (AR) is required for myometrial cell proliferation by modulating IGF-1 signaling during early pregnancy. Here, we report that AR also exerts its antiapoptotic function in human myometrial cells. Enhanced AR expression protects, whereas AR silencing sensitizes myometrial cells to both intrinsic and extrinsic apoptotic stimuli. AR agonist inhibits, whereas AR antagonist induces myometrial cells to undergo apoptotic cell death. Gene microarray analysis confirms that the central functions of AR in myometrial cells are to regulate cell cycling and apoptosis through three major gene groups involving the epidermal growth factor (EGF) signaling, RNA splicing and DNA repair processes. AR mediates its antiapoptotic function through two distinct pathways. In the receptor-dependent pathway, AR is required for the expression of several protein factors within the EGF signaling pathway. Through the PI3K/Akt pathway, AR enhances the expression of the antiapoptotic protein Mcl-1. In the ligand-dependent pathway, AR agonist triggers the activation of Src kinase, which in turn phosphorylates STAT3 to increase Mcl-1 expression. We conclude from these results that the AR signaling exerts antiapoptotic function in myometrial cells, further supporting its key role in programming of myometrial phenotype. PMID:25032861

  8. Inflammasome priming increases retinal pigment epithelial cell susceptibility to lipofuscin phototoxicity by changing the cell death mechanism from apoptosis to pyroptosis.

    PubMed

    Brandstetter, Carolina; Patt, Joshua; Holz, Frank G; Krohne, Tim U

    2016-08-01

    Progressive death of retinal pigment epithelium (RPE) cells is a hallmark of age-related macular degeneration (AMD), the leading cause of blindness in all developed countries. Photooxidative damage and activation of the NLRP3 inflammasome have been suggested as contributing factors to this process. We investigated the effects of inflammasome activation on oxidative damage-induced RPE cell death. In primary human RPE cells and ARPE-19 cells, lipofuscin accumulated following incubation with oxidatively modified photoreceptor outer segments. Oxidative stress was induced by blue light irradiation (dominant wavelength: 448nm, irradiance: 0.8mW/cm(2), duration: 3 to 6h) of lipofuscin-loaded cells and resulted in cell death by apoptosis. Prior inflammasome priming by IL-1α or complement activation product C5a altered the cell death mechanism to pyroptosis and resulted in a significant increase of the phototoxic effect. Following IL-1α priming, viability 24h after irradiation was reduced in primary RPE cells and ARPE-19 cells from 65.3% and 56.7% to 22.6% (p=0.003) and 5.1% (p=0.0002), respectively. Inflammasome-mediated IL-1β release occurred only in association with pyroptotic cell lysis. Inflammasome priming by conditioned media of pyroptotic cells likewise increased cell death. Suppression of inflammasome activation by inhibition of caspase-1 or cathepsins B and L significantly reduced cell death in primed cells. In summary, inflammasome priming by IL-1α, C5a, or conditioned media of pyroptotic cells increases RPE cell susceptibility to photooxidative damage-mediated cell death and changes the mechanism of induced cell death from apoptosis to pyroptosis. This process may contribute to RPE degeneration in AMD and provide new targets for intervention. PMID:27240191

  9. 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. PMID:27010808

  10. The Amaryllidaceae Isocarbostyril Narciclasine Induces Apoptosis By Activation of the Death Receptor and/or Mitochondrial Pathways in Cancer Cells But Not in Normal Fibroblasts1

    PubMed Central

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

    2007-01-01

    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. PMID:17898872

  11. 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

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

    PubMed

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

    2001-07-15

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

  13. Puma cooperates with Bim, the rate-limiting BH3-only protein in cell death during lymphocyte development, in apoptosis induction

    PubMed Central

    Erlacher, Miriam; Labi, Verena; Manzl, Claudia; Böck, Günther; Tzankov, Alexandar; Häcker, Georg; Michalak, Ewa; Strasser, Andreas; Villunger, Andreas

    2006-01-01

    The physiological role of B cell lymphoma 2 (Bcl-2) homology 3–only proteins has been investigated in mice lacking the individual genes identifying rate-limiting roles for Bim (Bcl-2–interacting mediator of cell death) and Puma (p53–up-regulated modulator of apoptosis) in apoptosis induction. The loss of Bim protects lymphocytes from apoptosis induced by cytokine deprivation and deregulated Ca++ flux and interferes with the deletion of autoreactive lymphocytes and the shutdown of immune responses. In contrast, Puma is considered the key mediator of p53-induced apoptosis. To investigate the hypothesis that Bim and Puma have overlapping functions, we generated mice lacking both genes and found that bim−/−/puma−/− animals develop multiple postnatal defects that are not observed in the single knockout mice. Most strikingly, hyperplasia of lymphatic organs is comparable with that observed in mice overexpressing Bcl-2 in all hemopoietic cells exceeding the hyperplasia observed in bim−/− mice. Bim and Puma also have clearly overlapping functions in p53-dependent and -independent apoptosis. Their combined loss promotes spontaneous tumorigenesis, causing the malignancies observed in Bcl-2 transgenic mice, but does not exacerbate the autoimmunity observed in the absence of Bim. PMID:17178918

  14. 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. PMID:24161990

  15. 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. PMID:24706461

  16. 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

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

    PubMed Central

    Clavier, Amandine; Szuplewski, Sébastien; Risler, Yanick; Milet, Cécile; Gaumer, Sébastien; Guénal, Isabelle; Mignotte, Bernard

    2015-01-01

    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. PMID:26124923

  18. Apaf-1 and caspase-9 are required for cytokine withdrawal-induced apoptosis of mast cells but dispensable for their functional and clonogenic death.

    PubMed

    Marsden, Vanessa S; Kaufmann, Thomas; O'reilly, Lorraine A; Adams, Jerry M; Strasser, Andreas

    2006-03-01

    Cytokines promote survival of mast cells by inhibiting apoptotic pathways regulated by the Bcl-2 protein family. We previously showed that lymphocyte apoptosis can proceed via a Bcl-2-inhibitable pathway independent of the canonical initiator caspase, caspase-9, and its adaptor, Apaf-1. Here we report that mast cells lacking caspase-9 or Apaf-1 are refractory to apoptosis after cytotoxic insults but still lose effector function and ability to proliferate. In response to cytokine deprivation or DNA damage, fetal liver-derived mast cells lacking Apaf-1 or caspase-9 failed to undergo apoptosis. Nevertheless, the cytokine-starved cells were not functionally alive, because, unlike those overexpressing Bcl-2, they could not degranulate on Fcepsilon receptor stimulation or resume proliferation on re-addition of cytokine. Furthermore, mast cells lacking Apaf-1 or caspase-9 had no survival advantage over wild-type counterparts in vivo. These results indicate that the Apaf-1/caspase-9-independent apoptotic pathway observed in lymphocytes is ineffective in cytokine-deprived mast cells. However, although Apaf-1 and caspase-9 are essential for mast cell apoptosis, neither is required for the functional or clonogenic death of the cells, which may be due to mitochondrial dysfunction. PMID:16291596

  19. Lovastatin enhances adenovirus-mediated TRAIL induced apoptosis by depleting cholesterol of lipid rafts and affecting CAR and death receptor expression of prostate cancer cells.

    PubMed

    Liu, Youhong; Chen, Lin; Gong, Zhicheng; Shen, Liangfang; Kao, Chinghai; Hock, Janet M; Sun, Lunquan; Li, Xiong

    2015-02-20

    Oncolytic adenovirus and apoptosis inducer TRAIL are promising cancer therapies. Their antitumor efficacy, when used as single agents, is limited. Oncolytic adenoviruses have low infection activity, and cancer cells develop resistance to TRAIL-induced apoptosis. Here, we explored combining prostate-restricted replication competent adenovirus-mediated TRAIL (PRRA-TRAIL) with lovastatin, a commonly used cholesterol-lowering drug, as a potential therapy for advanced prostate cancer (PCa). Lovastatin significantly enhanced the efficacy of PRRA-TRAIL by promoting the in vivo tumor suppression, and the in vitro cell killing and apoptosis induction, via integration of multiple molecular mechanisms. Lovastatin enhanced PRRA replication and virus-delivered transgene expression by increasing the expression levels of CAR and integrins, which are critical for adenovirus 5 binding and internalization. Lovastatin enhanced TRAIL-induced apoptosis by increasing death receptor DR4 expression. These multiple effects of lovastatin on CAR, integrins and DR4 expression were closely associated with cholesterol-depletion in lipid rafts. These studies, for the first time, show correlations between cholesterol/lipid rafts, oncolytic adenovirus infection efficiency and the antitumor efficacy of TRAIL at the cellular level. This work enhances our understanding of the molecular mechanisms that support use of lovastatin, in combination with PRRA-TRAIL, as a candidate strategy to treat human refractory prostate cancer in the future. PMID:25605010

  20. Programmed cell death 4 (PDCD4) mediates the sensitivity of gastric cancer cells to TRAIL-induced apoptosis by down-regulation of FLIP expression.

    PubMed

    Wang, Weiqiang; Zhao, Jingjing; Wang, Hongbin; Sun, Yonggang; Peng, Zhihong; Zhou, Gang; Fan, Lilin; Wang, Xingwei; Yang, Shiming; Wang, Rongquan; Fang, Dianchun

    2010-09-10

    Tumor necrosis factor-related apoptosis induced ligand (TRAIL) is an important apoptosis inducer in a variety of tumor cells. In the present study, we determined the underlying molecular mechanisms by which certain gastric cancer cells are resistant to TRAIL. We first detected expression of programmed cell death 4 (PDCD4) in three gastric cancer cell lines and identified its association with the sensitivity of gastric cancer cells to TRAIL. We then stably transfected PDCD4 cDNA or shRNA into these gastric cell lines. Our data showed that restoration of PDCD4 expression induced TRAIL sensitivity, whereas knockdown of PDCD4 expression reduced the sensitivity of these tumor cells to TRAIL treatment. PDCD4 was able to suppress expression of FLICE-inhibiting protein (FLIP), a negative regulator of apoptosis. Knockdown of FLIP expression using FLIP shRNA had similar effects as those of restored PDCD4 expression. Furthermore, the proteasome inhibitor MG132 was able to inhibit expression of FLIP mRNA and protein and upregulate the sensitivity of these cells to TRAIL treatment. Taken together, the results from the current study demonstrated that PDCD4 plays an important role in mediating the sensitivity of gastric cancer cells to TRAIL-induced apoptosis through FLIP suppression. Therefore, the proteasome inhibitor MG132 should be further evaluated for combination therapy with TRAIL. PMID:20595005

  1. Glycogen synthase kinase-3β antagonizes ROS-induced hepatocellular carcinoma cell death through suppression of the apoptosis signal-regulating kinase 1.

    PubMed

    Zhang, Na; Liu, Lu; Dou, Yueying; Song, Danqing; Deng, Hongbin

    2016-07-01

    Glycogen synthase kinase-3β (GSK-3β), a multifunctional kinase, is an important regulator of cancer cell survival. Apoptosis signal-regulating kinase 1 (ASK1) is also a key factor for controlling several cellular events including the cell cycle, senescence, and apoptosis, in response to reactive oxygen species (ROS). The role of GSK-3β regulating the activity and protein level of ASK1 in the cancer cells remains largely unexplored. In this study, we showed that GSK-3β inhibits ROS-induced hepatocellular carcinoma cell death by suppressing ASK1. We first found that ectopic expression of GSK-3β suppressed hydrogen peroxide (H2O2)-induced cell death in HepG2 cells and knockdown of endogenous GSK-3β expression exhibited opposite effects. Moreover, GSK-3β expression clearly inhibited H2O2-induced phosphorylation of ASK1 in HepG2 cells, in association with a decrease in ASK1 protein level. Further exploration revealed that GSK-3β induced ubiquitination and proteasome-dependent degradation of ASK1 via inhibition of ubiquitin-specific protease USP9X. Our results thus suggest that GSK-3β is a key factor involved in ASK1 activation and ROS-induced cell death. PMID:27221474

  2. Exposure of insect midgut cells to Sambucus nigra L. agglutinins I and II causes cell death via caspase-dependent apoptosis.

    PubMed

    Shahidi-Noghabi, Shahnaz; Van Damme, Els J M; Iga, Masatoshi; Smagghe, Guy

    2010-09-01

    Sambucus nigra agglutinins I and II, further referred to as SNA-I and SNA-II, are two ricin-related lectins from elderberry. SNA-I is a chimeric lectin composed of an A-chain with enzymatic activity and a B-chain with carbohydrate-binding activity, and therefore belongs to the group of type 2 ribosome-inactivating proteins. In contrast, SNA-II consists only of carbohydrate-binding B-chains. The physiological effect of SNA-I was tested on different insect cell lines (midgut, ovary, fat body, embryo). In sensitive midgut CF-203 cells, SNA-I induced cell death with typical characteristics such as cell shrinkage, plasma membrane blebbing, nuclear condensation and DNA fragmentation. The effect was dose-dependent with 50% death of 4-day-exposed cells at 3nM. SNA-I exposure induced caspase-3 like activities, suggesting that SNA-I can induce the apoptotic pathway. Interestingly, the hololectin SNA-II also induced apoptosis in CF-203 cells at similar doses with the same physiological events. SNA-I and SNA-II both induced caspase-dependent apoptosis at low concentrations (nM order), leading to typical symptoms of cell death in sensitive cells. This effect seems independent from the catalytic activity of the A-chain, but depends on the carbohydrate-binding B-chain. PMID:20230823

  3. 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. PMID:22704843

  4. 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. PMID:25794149

  5. Heat-Modified Citrus Pectin Induces Apoptosis-Like Cell Death and Autophagy in HepG2 and A549 Cancer Cells

    PubMed Central

    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. PMID:25794149

  6. Poly (ADP-ribose) (PAR) Binding to Apoptosis-Inducing Factor Is Critical For PAR Polymerase-1-Dependent Cell Death (Parthanatos)

    PubMed Central

    Wang, Yingfei; Kim, No Soo; Haince, Jean-Francois; Kang, HoChul; David, Karen K.; Andrabi, Shaida A.; Poirier, Guy G.; Dawson, Valina L.; Dawson, Ted M.

    2011-01-01

    The mitochondrial protein apoptosis-inducing factor (AIF) plays a pivotal role in poly(ADP-ribose) polymerase-1 (PARP-1)-mediated cell death (parthanatos), during which it is released from the mitochondria and translocates to the nucleus. Here, we show that AIF is a high affinity poly(ADP-ribose) (PAR)–binding protein and that PAR binding to AIF is required for parthanatos both in vitro and in vivo. AIF bound PAR at a site distinct from AIF’s DNA binding site and this interaction triggered AIF release from the cytosolic side of the mitochondrial outer membrane. Mutation of the PAR binding site in AIF did not affect its NADH oxidase activity, its ability to bind FAD or DNA, or its ability to induce nuclear condensation. However, this AIF mutant was not released from mitochondria and did not translocate to the nucleus or mediate cell death following PARP-1 activation. These results suggest a mechanism for PARP-1 to initiate AIF-mediated cell death and indicate that AIF’s bioenergetic cell survival-promoting functions are separate from its effects as a mitochondrially-derived death effector. Interference with the PAR-AIF interaction or PAR signaling may provide unique opportunities for preventing cell death following activation of PARP-1. PMID:21467298

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

    PubMed

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

    2016-08-01

    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

  8. Functional Consequences for Apoptosis by Transcription Elongation Regulator 1 (TCERG1)-Mediated Bcl-x and Fas/CD95 Alternative Splicing

    PubMed Central

    Montes, Marta; Coiras, Mayte; Becerra, Soraya; Moreno-Castro, Cristina; Mateos, Elena; Majuelos, Jara; Oliver, F. Javier; Hernández-Munain, Cristina; Alcamí, José; Suñé, Carlos

    2015-01-01

    Here, we present evidence for a specific role of the splicing-related factor TCERG1 in regulating apoptosis in live cells by modulating the alternative splicing of the apoptotic genes Bcl-x and Fas. We show that TCERG1 modulates Bcl-x alternative splicing during apoptosis and its activity in Bcl-x alternative splicing correlates with the induction of apoptosis, as determined by assessing dead cells, sub-G1-phase cells, annexin-V binding, cell viability, and cleavage of caspase-3 and PARP-1. Furthermore, the effect of TCERG1 on apoptosis involved changes in mitochondrial membrane permeabilization. We also found that depletion of TCERG1 reduces the expression of the activated form of the pro-apoptotic mitochondrial membrane protein Bak, which remains inactive by heterodimerizing with Bcl-xL, preventing the initial step of cytochrome c release in Bak-mediated mitochondrial apoptosis. In addition, we provide evidence that TCERG1 also participates in the death receptor-mediated apoptosis pathway. Interestingly, TCERG1 also modulates Fas/CD95 alternative splicing. We propose that TCERG1 sensitizes a cell to apoptotic agents, thus promoting apoptosis by regulating the alternative splicing of both the Bcl-x and Fas/CD95 genes. Our findings may provide a new link between the control of alternative splicing and the molecular events leading to apoptosis. PMID:26462236

  9. 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

  10. Receptor-mediated endocytosis and brain delivery of therapeutic biologics.

    PubMed

    Xiao, Guangqing; Gan, Liang-Shang

    2013-01-01

    Transport of macromolecules across the blood-brain-barrier (BBB) requires both specific and nonspecific interactions between macromolecules and proteins/receptors expressed on the luminal and/or the abluminal surfaces of the brain capillary endothelial cells. Endocytosis and transcytosis play important roles in the distribution of macromolecules. Due to the tight junction of BBB, brain delivery of traditional therapeutic proteins with large molecular weight is generally not possible. There are multiple pathways through which macromolecules can be taken up into cells through both specific and nonspecific interactions with proteins/receptors on the cell surface. This review is focused on the current knowledge of receptor-mediated endocytosis/transcytosis and brain delivery using the Angiopep-2-conjugated system and the molecular Trojan horses. In addition, the role of neonatal Fc receptor (FcRn) in regulating the efflux of Immunoglobulin G (IgG) from brain to blood, and approaches to improve the pharmacokinetics of therapeutic biologics by generating Fc fusion proteins, and increasing the pH dependent binding affinity between Fc and FcRn, are discussed. PMID:23840214

  11. Cerebellar vermis H₂ receptors mediate fear memory consolidation in mice.

    PubMed

    Gianlorenço, A C L; Riboldi, A M; Silva-Marques, B; Mattioli, R

    2015-02-01

    Histaminergic fibers are present in the molecular and granular layers of the cerebellum and have a high density in the vermis and flocullus. Evidence supports that the cerebellar histaminergic system is involved in memory consolidation. Our recent study showed that histamine injections facilitate the retention of an inhibitory avoidance task, which was abolished by pretreatment with an H2 receptor antagonist. In the present study, we investigated the effects of intracerebellar post training injections of H1 and H2 receptor antagonists as well as the selective H2 receptor agonist on fear memory consolidation. The cerebellar vermi of male mice were implanted with guide cannulae, and after three days of recovery, the inhibitory avoidance test was performed. Immediately after a training session, animals received a microinjection of the following histaminergic drugs: experiment 1, saline or chlorpheniramine (0.016, 0.052 or 0.16 nmol); experiment 2, saline or ranitidine (0.57, 2.85 or 5.07 nmol); and experiment 3, saline or dimaprit (1, 2 or 4 nmol). Twenty-four hours later, a retention test was performed. The data were analyzed using one-way analysis of variance (ANOVA) and Duncan's tests. Animals microinjected with chlorpheniramine did not show any behavioral effects at the doses that we used. Intra-cerebellar injection of the H2 receptor antagonist ranitidine inhibited, while the selective H2 receptor agonist dimaprit facilitated, memory consolidation, suggesting that H2 receptors mediate memory consolidation in the inhibitory avoidance task in mice. PMID:25524412

  12. Receptor-Mediated Endocytosis and Brain Delivery of Therapeutic Biologics

    PubMed Central

    Xiao, Guangqing

    2013-01-01

    Transport of macromolecules across the blood-brain-barrier (BBB) requires both specific and nonspecific interactions between macromolecules and proteins/receptors expressed on the luminal and/or the abluminal surfaces of the brain capillary endothelial cells. Endocytosis and transcytosis play important roles in the distribution of macromolecules. Due to the tight junction of BBB, brain delivery of traditional therapeutic proteins with large molecular weight is generally not possible. There are multiple pathways through which macromolecules can be taken up into cells through both specific and nonspecific interactions with proteins/receptors on the cell surface. This review is focused on the current knowledge of receptor-mediated endocytosis/transcytosis and brain delivery using the Angiopep-2-conjugated system and the molecular Trojan horses. In addition, the role of neonatal Fc receptor (FcRn) in regulating the efflux of Immunoglobulin G (IgG) from brain to blood, and approaches to improve the pharmacokinetics of therapeutic biologics by generating Fc fusion proteins, and increasing the pH dependent binding affinity between Fc and FcRn, are discussed. PMID:23840214

  13. 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.

  14. 1-Nitropyrene (1-NP) induces apoptosis and apparently a non-apoptotic programmed cell death (paraptosis) in Hepa1c1c7 cells.

    PubMed

    Asare, Nana; Landvik, Nina E; Lagadic-Gossmann, Dominique; Rissel, Mary; Tekpli, Xavier; Ask, Kjetil; Låg, Marit; Holme, Jørn 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. PMID:18417179

  15. Induction of hepatoma carcinoma cell apoptosis through activation of the JNK-nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-ROS self-driven death signal circuit.

    PubMed

    Zeng, Ke-Wu; Song, Fang-Jiao; Wang, Ying-Hong; Li, Ning; Yu, Qian; Liao, Li-Xi; Jiang, Yong; Tu, Peng-Fei

    2014-10-28

    As an efficient method for inducing tumor cell apoptosis, ROS can be constantly formed and accumulated in NADPH oxidase overactivated-cells, resulting in further mitochondrial membrane damage and mitochondria-dependent apoptosis. In addition, JNK mitogen-activated protein kinase (JNK MAPK) signal also acts as a vital candidate pathway for inducing tumor cell apoptosis by targeting mitochondrial death pathway. However, the relationship between NADPH oxidase-ROS and JNK MAPK signal still remains unclear. Here, we discovered a novel self-driven signal circuit between NADPH oxidase-ROS and JNK MAPK, which was induced by a cytotoxic steroidal saponin (ASC) in hepatoma carcinoma cells. NADPH oxidase-dependent ROS production was markedly activated by ASC and directly led to JNK MAPK activation. Moreover, antioxidant, NADPH oxidase inhibitor and specific knock-out for p47 subunit of NADPH oxidase could effectively block NADPH oxidase-ROS-dependent JNK activation, suggesting that NADPH oxidase is an upstream regulator of JNK MAPK. Conversely, a specific JNK inhibitor could inhibit ASC-induced NADPH oxidase activation and down-regulate ROS levels as well, indicating that JNK might also regulate NADPH oxidase activity to some extent. These observations indicate that NADPH oxidase and JNK MAPK activate each other as a signal circuit. Furthermore, drug pretreatment experiments with ASC showed this signal circuit operated continuously via a self-driven mode and finally induced apoptosis in hepatoma carcinoma cells. Taken together, we provide a proof for inducing hepatoma carcinoma cell apoptosis by activating the JNK-NADPH oxidase-ROS-dependent self-driven signal circuit pathway. PMID:25064608

  16. 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. PMID:25173558

  17. Human parvovirus B19 non-structural protein (NS1) induces apoptosis through mitochondria cell death pathway in COS-7 cells.

    PubMed

    Hsu, Tsai-Ching; Wu, Wen-Jun; Chen, Meng-Chi; Tsay, Gregory J

    2004-01-01

    Human parvovirus B19 has been found in various tissues in addition to erythroid lineage cells, and non-structural protein (NS1) is reported to induce cytotoxicity and apoptosis in erythroid lineage cells, but the mechanism in non-permissive cells is still unclear. To address this issue, we have constructed the NS1 gene in a cytomegalovirus episomal vector, pEGFP-C1 and transfected it into monkey epithelial cells, COS-7. EGFP-NS1 expression in transfected cells was monitored and assessed by fluorescence microscopy, RT-PCR and Western blot. The flow cytometric analysis showed that the NS1-transfected cells were arrested at G1 phase by paclitaxel treatment and there was increased apoptosis. The expression of p53, an important molecule in apoptosis and cell cycle regulation, and its downstream cell cycle kinase inhibitors p16(INK4) and p21(WAF1/CIP1) were up-regulated in the NS1-transfected cells. Also, increased expression of the pro-apoptotic Bcl-2 members Bax, Bad and activation of caspase 3 and caspase 9, but not the activation of caspase 8 or Fas were detected in the NS1-transfected cells. p53-induced Bax expression and subsequent activation of caspase 9 is probably the apoptotic pathway in NS1-transfected cells since activation of the caspase 9 was suppressed by the p53 inhibitor and apoptosis was significantly inhibited by the caspase 9 inhibitor. Our results suggest that the cell death of the NS1-transfected cells is associated with mitochondria related apoptosis. These findings might provide alternative information for further study and characterization of B19 NS1 protein in B19 non-permissive cells. PMID:15370668

  18. Silencing of fas, fas-associated via death domain, or caspase 3 differentially affects lung inflammation, apoptosis, and development of trauma-induced septic acute lung injury.

    PubMed

    Messer, Mirko Philipp; Kellermann, Philipp; Weber, Sascha Jörn; Hohmann, Christoph; Denk, Stephanie; Klohs, Bettina; Schultze, Anke; Braumüller, Sonja; Huber-Lang, Markus Stefan; Perl, Mario

    2013-01-01

    Activation of Fas signaling is a potentially important pathophysiological mechanism in the development of septic acute lung injury (ALI). However, so far the optimal targets within this signaling cascade remain elusive. Thus, we tested the hypothesis that in vivo gene silencing of Fas, Fas-associated via death domain (FADD), or caspase 3 by intratracheal administration of small interfering RNA would ameliorate ALI in a clinically relevant double-hit mouse model of trauma induced septic lung injury. Male C57Bl/6 mice received small interfering (Fas, FADD, caspase 3) or control RNA 24 h before and 12 h after blunt chest trauma or sham procedures. Polymicrobial sepsis was induced by cecal ligation and puncture 24 h after chest trauma. Twelve or 24 h later, lung tissue, plasma, and bronchoalveolar lavage fluid were harvested. During ALI, lung apoptosis (active caspase 3 Western blotting, TUNEL staining) was substantially increased when compared with sham. Silencing of caspase 3 or FADD both markedly reduced pulmonary apoptosis. Fas- and FADD-small interfering RNA administration substantially decreased lung cytokine concentration, whereas caspase 3 silencing did not reduce lung inflammation. In addition, Fas silencing markedly decreased lung neutrophil infiltration. Interestingly, only in response to caspase 3 silencing, ALI-induced lung epithelial barrier dysfunction was substantially improved, and histological appearance was beneficially affected. Taken together, downstream inhibition of lung apoptosis via caspase 3 silencing proved to be superior in mitigating ALI when compared with upstream inhibition of apoptosis via Fas or FADD silencing, even in the presence of additional anti-inflammatory effects. This indicates a major pathophysiological role of lung apoptosis and suggests the importance of other than Fas-driven apoptotic pathways in trauma-induced septic ALI. PMID:23247118

  19. Signaling through C/EBP homologous protein and death receptor 5 and calpain activation differentially regulate THP-1 cell maturation-dependent apoptosis induced by Shiga toxin type 1.

    PubMed

    Lee, Moo-Seung; Cherla, Rama P; Lentz, Erin K; Leyva-Illades, Dinorah; Tesh, Vernon L

    2010-08-01

    Shiga toxins (Stxs) induce apoptosis via activation of the intrinsic and extrinsic pathways in many cell types. Toxin-mediated activation of the endoplasmic reticulum (ER) stress response was shown to be instrumental in initiating apoptosis in THP-1 myeloid leukemia cells. THP-1 cells responded to Shiga toxin type 1 (Stx1) in a cell maturation-dependent manner, undergoing rapid apoptosis in the undifferentiated state but reduced and delayed apoptosis in differentiated cells. The onset of apoptosis was associated with calpain activation and changes in expression of C/EBP homologous protein (CHOP), Bcl-2 family members, and death receptor 5 (DR5). Ligation of DR5 by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) activates the extrinsic pathway of apoptosis. We show here that expression of TRAIL and DR5 is increased by Stx1 treatment. Addition of exogenous TRAIL enhances, and anti-TRAIL antibodies inhibit, Stx1-induced apoptosis of THP-1 cells. Silencing of CHOP or DR5 expression selectively prevented caspase activation, loss of mitochondrial membrane potential, and Stx1-induced apoptosis of macrophage-like THP-1 cells. In contrast, the rapid kinetics of apoptosis induction in monocytic THP-1 cells correlated with rates of calpain cleavage. The results suggest that CHOP-DR5 signaling and calpain activation differentially contribute to cell maturation-dependent Stx1-induced apoptosis. Inhibition of these signaling pathways may protect cells from Stx cytotoxicity. PMID:20515924

  20. 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. PMID:25359904

  1. Polyamines modulate the roscovitine-induced cell death switch decision autophagy vs. apoptosis in MCF-7 and MDA-MB-231 breast cancer cells.

    PubMed

    Arisan, Elif Damla; Akkoç, Yunus; Akyüz, Kaan Gencer; Kerman, Ezgi Melek; Obakan, Pinar; Çoker-Gürkan, Ajda; Palavan Ünsal, Narçin

    2015-06-01

    Current clinical strategies against breast cancer mainly involve the use of anti‑hormonal agents to decrease estrogen production; however, development of resistance is a major problem. The resistance phenotype depends on the modulation of cell‑cycle regulatory proteins, cyclins and cyclin‑dependent kinases. Roscovitine, a selective inhibitor of cyclin‑dependent kinases, shows high therapeutic potential by causing cell‑cycle arrest in various cancer types. Autophagy is a type of cell death characterized by the enzymatic degradation of macromolecules and organelles in double‑ or multi‑membrane autophagic vesicles. This process has important physiological functions, including the degradation of misfolded proteins and organelle turnover. Recently, the switch between autophagy and apoptosis has been proposed to constitute an important regulator of cell death in response to chemotherapeutic drugs. The process is regulated by several proteins, such as the proteins of the Atg family, essential for the initial formation of the autophagosome, and PI3K, important at the early stages of autophagic vesicle formation. Polyamines (PAs) are small aliphatic amines that play major roles in a number of eukaryotic processes, including cell proliferation. The PA levels are regulated by ornithine decarboxylase (ODC), the rate‑limiting enzyme in PA biosynthesis. In this study, we aimed to investigate the role of PAs in roscovitine‑induced autophagic/apoptotic cell death in estrogen receptor‑positive MCF‑7 and estrogen receptor‑negative MDA‑MB‑231 breast cancer cells. We show that MDA‑MB‑231 cells are more resistant to roscovitine than MCF‑7 cells. This difference was related to the regulation of autophagic key molecules in MDA‑MB‑231 cells. In addition, we found that exogenous PAs have a role in the cell death decision between roscovitine‑induced apoptosis or autophagy in MCF‑7 and MDA‑MB‑231 breast cancer cells. PMID:25650699

  2. 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

  3. 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

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

    PubMed

    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

  5. Bombesin receptor-mediated imaging and cytotoxicity: review and current status

    PubMed Central

    Sancho, Veronica; Di Florio, Alessia; Moody, Terry W.; Jensen, Robert T.

    2010-01-01

    The three mammalian bombesin (Bn) receptors (gastrin-releasing peptide [GRP] receptor, neuromedin B [NMB] receptor, BRS-3) are one of the classes of G protein-coupled receptors that are most frequently over-express/ectopically expressed by common, important malignancies. Because of the clinical success of somatostatin receptor-mediated imaging and cytotoxicity with neuroendocrine tumors, there is now increasing interest in pursuing a similar approach with Bn receptors. In the last few years then have been more than 200 studies in this area. In the present paper, the in vitro and in vivo results, as well as results of human studies from many of these studies are reviewed and the current state of Bn receptor-mediated imaging or cytotoxicity is discussed. Both Bn receptor-mediated imaging studies as well as Bn receptor-mediated tumoral cytotoxic studies using radioactive and non-radioactive Bn-based ligands are covered. PMID:21034419

  6. Targeting receptor-mediated endocytotic pathways with nanoparticles: rationale and advances

    PubMed Central

    Xu, Shi; Olenyuk, Bogdan Z.; Okamoto, Curtis T.; Hamm-Alvarez, Sarah F.

    2012-01-01

    Targeting of drugs and their carrier systems by using receptor-mediated endocytotic pathways was in its nascent stages 25 years ago. In the intervening years, an explosion of knowledge focused on design and synthesis of nanoparticulate delivery systems as well as elucidation of the cellular complexity of what was previously-termed receptor-mediated endocytosis has now created a situation when it has become possible to design and test the feasibility of delivery of highly specific nanoparticle drug carriers to specific cells and tissue. This review outlines the mechanisms governing the major modes of receptor-mediated endocytosis used in drug delivery and highlights recent approaches using these as targets for in vivo drug delivery of nanoparticles. The review also discusses some of the inherent complexity associated with the simple shift from a ligand-drug conjugate versus a ligand-nanoparticle conjugate, in terms of ligand valency and its relationship to the mode of receptor-mediated internalization. PMID:23026636

  7. Dual roles of plasmalemmal chloride channels in induction of cell death.

    PubMed

    Okada, Yasunobu; Maeno, Emi; Shimizu, Takahiro; Manabe, Kenichi; Mori, Shin-Ichiro; Nabekura, Takashi

    2004-06-01

    Even under anisotonic conditions, most cells can regulate their volume by mechanisms called regulatory volume decrease (RVD) and increase (RVI) after osmotic swelling or shrinkage, respectively. In contrast, the initial processes of necrosis and apoptosis are associated with persistent swelling and shrinkage. Necrotic volume increase (NVI) is initiated by uptake of osmolytes, such as Na+, Cl- and lactate, under conditions of injury, hypoxia, ischaemia, acidosis or lactacidosis. Persistence of NVI is caused by dysfunction of RVD due to impairment of volume-sensitive Cl- channels under conditions of ATP deficiency or lactacidosis. Both lactacidosis-induced RVD dysfunction and necrotic cell death are prevented by pretreatment of cells with the vacuolating cytotoxin-A (VacA) toxin protein purified from Helicobacter pylori, which forms a lactacidosis-resistant anion channel. Apoptotic volume decrease (AVD) is triggered by activation of K+ and Cl- conductances following stimulation with a mitochondrion-mediated or death receptor-mediated apoptosis inducer. Apoptotic cell death can be prevented by blocking the Cl- channels but not the K+-Cl- cotransporters. Thus, the volume regulatory anion channel plays, unless impaired, a cell-rescuing role in the necrotic process by ensuring RVD after swelling induced by necrotic insults, whereas normotonic activation of the anion channel plays a cell-killing role in the apoptotic process by triggering AVD following stimulation with apoptosis inducers. PMID:15103464

  8. 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. PMID:27238724

  9. 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.

  10. Apoptosis-induced cell death due to oleanolic acid in HaCaT keratinocyte cells--a proof-of-principle approach for chemopreventive drug development.

    PubMed

    George, V Cijo; Kumar, D R Naveen; Suresh, P K; Kumar, R Ashok

    2012-01-01

    Oleanolic acid (OA) is a naturally occurring triterpenoid in food materials and is a component of the leaves and roots of Olea europaea, Viscum album L., Aralia chinensis L. and more than 120 other plant species. There are several reports validating its antitumor activity against different cancer cells apart from its hepatoprotective activity. However, antitumor activity against skin cancer has not been studied well thus far. Hence the present study of effects of OA against HaCaT (immortalized keratinocyte) cells--a cell-based epithelial model system for toxicity/ethnopharmacology-based studies--was conducted. Radical scavenging activity (DPPH·) and FRAP were determined spectrophotometrically. Proliferation was assessed by XTT assay at 24, 48 and 72 hrs with exposure to various concentrations (12.5-200 μM) of OA. Apoptotic induction potential of OA was demonstrated using a cellular DNA fragmentation ELISA method. Morphological studies were also carried out to elucidate its antitumor potential. The results revealed that OA induces apoptosis by altering cellular morphology as well as DNA integrity in HaCaT cells in a dose-dependent manner, with comparatively low cytotoxicity. The moderate toxicity observed in HaCaT cells, with induction of apoptosis, possibly suggests greater involvement of programmed-cell death-mediated mechanisms. We conclude that OA has relatively low toxicity and has the potential to induce apoptosis in HaCaT cells and hence provides a substantial and sound scientific basis for further validation studies. PMID:22901164

  11. Research of ALA combined with HpD-PDT which induced s180 ascitic tumor cells, death or apoptosis on cytology

    NASA Astrophysics Data System (ADS)

    Zhu, Jing; Yan, Min; Zhang, Hui-Guo; Li, Enling; Luo, Hongyu

    2005-07-01

    To ascertain the adequate dosage of ALA combined with HpD-PDT which induced tumor cell death or apoptosis on cytology. And to study the different effect of ALA-PDT and HPD-PDT used only. Rat ascitic tumor cells(S180) were randomly divided into several groups and incubated with ALA(20μg/ml 、40μg/ml、80μg/ml 、160μg/ml)、HPD(2.5μg/ml、5μg/ml、10μg/ml)and their combination dosages. 630nm light (total output 2W) was delivered to tumor cells at a constant fluence rate: 200mw/cm2 and a constant irradiated time period: 20 minutes. We set 3 groups (no photosensitizers or no irradiation or neither) to be the control groups. We used inversion microscopy to observe the morphological change of tumor cells and flow cytometry technology to detect the death or apoptosis of tumor cells during the experiment. ..

  12. Dopamine receptor-mediated regulation of neuronal "clock" gene expression.

    PubMed

    Imbesi, M; Yildiz, S; Dirim Arslan, A; Sharma, R; Manev, H; Uz, T

    2009-01-23

    Using a transgenic mice model (i.e. "clock" knockouts), clock transcription factors have been suggested as critical regulators of dopaminergic behaviors induced by drugs of abuse. Moreover, it has been shown that systemic administration of psychostimulants, such as cocaine and methamphetamine regulates the striatal expression of clock genes. However, it is not known whether dopamine receptors mediate these regulatory effects of psychostimulants at the cellular level. Primary striatal neurons in culture express dopamine receptors as well as clock genes and have been successfully used in studying dopamine receptor functioning. Therefore, we investigated the role of dopamine receptors on neuronal clock gene expression in this model using specific receptor agonists. We found an inhibitory effect on the expression of mClock and mPer1 genes with the D2-class (i.e. D2/D3) receptor agonist quinpirole. We also found a generalized stimulatory effect on the expression of clock genes mPer1, mClock, mNPAS2 (neuronal PAS domain protein 2), and mBmal1 with the D1-class (i.e. D1) receptor agonist SKF38393. Further, we tested whether systemic administration of dopamine receptor agonists causes similar changes in striatal clock gene expression in vivo. We found quinpirole-induced alterations in mPER1 protein levels in the mouse striatum (i.e. rhythm shift). Collectively, our results indicate that the dopamine receptor system may mediate psychostimulant-induced changes in clock gene expression. Using striatal neurons in culture as a model, further research is needed to better understand how dopamine signaling modulates the expression dynamics of clock genes (i.e. intracellular signaling pathways) and thereby influences neuronal gene expression, neuronal transmission, and brain functioning. PMID:19017537

  13. 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. PMID:23601820

  14. Excessive Ovarian Production of Nerve Growth Factor Elicits Granulosa Cell Apoptosis by Setting in Motion a Tumor Necrosis Factor alpha/Stathmin$Mediated Death Signaling Pathway

    PubMed Central

    Garcia-Rudaz, Cecilia; Dorfman, Mauricio; Nagalla, Srinivasa; Svechnikov, Konstantin; Söder, Olle; Ojeda, Sergio R.; Dissen, Gregory A.

    2012-01-01

    Excessive nerve growth factor (NGF) production by the ovary, achieved via a transgenic approach, results in arrested antral follicle growth, reduced ovulatory capacity, and a predisposition to cyst formation in response to mildly elevated LH levels. Two salient features in these mutant mice (termed 17NF) are an elevated production of 17-alpha hydroxyprogesterone (17-OHP4), testosterone (T4) and estradiol (E2) in response to gonadotropins, and an increased frequency of granulosa cell (GC) apoptosis. Here we show that the increase in steroidal response is associated with enhanced expression of Cyp17a1, Hsd17b, and Cyp19a1, which encode the enzymes catalyzing the synthesis of 17-OHP4, T4 and E2, respectively. Using a proteomic approach, we identified stathmin (STMN1), as a protein that is overproduced in 17NF ovaries. In its phosphorylated state, STMN1 mediates a cell death signal initiated by tumor necrosis factor alpha (TNF). STMN1 is expressed in GCs and excessive NGF increases its abundance as well as that of its forms phosphorylated at serine (Ser) 16, 25 and 38. TNF synthesis is also increased in 17NF ovaries, and this change is abolished by blocking neurotrophic tyrosine kinase (NTRK) receptors. Inhibiting TNF actions in vivo by administering a soluble TNF receptor prevented the increase in total and phosphorylated STMN1 production, as well as GC apoptosis in NGF-overproducing ovaries. These results indicate that an excess of NGF in the ovary promotes steroidogenesis by enhancing the expression of enzyme genes involved in 17-OHP4, T4 and E2 synthesis, and causes GC apoptosis by activating a TNF/STMN1-mediated cell death pathway. PMID:21646391

  15. Osteopontin-stimulated apoptosis in cardiac myocytes involves oxidative stress and mitochondrial death pathway: role of a pro-apoptotic protein BIK.

    PubMed

    Dalal, Suman; Zha, Qinqin; Singh, Mahipal; Singh, Krishna

    2016-07-01

    Increased osteopontin (OPN) expression in the heart, specifically in myocytes, associates with increased myocyte apoptosis and myocardial dysfunction. Recently, we provided evidence that OPN interacts with CD44 receptor, and induces myocyte apoptosis via the involvement of endoplasmic reticulum stress and mitochondrial death pathways. Here we tested the hypothesis that OPN induces oxidative stress in myocytes and the heart via the involvement of mitochondria and NADPH oxidase-4 (NOX-4). Treatment of adult rat ventricular myocytes (ARVMs) with OPN (20 nM) increased oxidative stress as analyzed by protein carbonylation, and intracellular reactive oxygen species (ROS) levels as analyzed by ROS detection kit and dichlorohydrofluorescein diacetate staining. Pretreatment with NAC (antioxidant), apocynin (NOX inhibitor), MnTBAP (superoxide dismutase mimetic), and mitochondrial KATP channel blockers (glibenclamide and 5-hydroxydecanoate) decreased OPN-stimulated ROS production, cytosolic cytochrome c levels, and apoptosis. OPN increased NOX-4 expression, while decreasing SOD-2 expression. OPN decreased mitochondrial membrane potential as measured by JC-1 staining, and induced mitochondrial abnormalities including swelling and reorganization of cristae as observed using transmission electron microscopy. OPN increased expression of BIK, a pro-apoptotic protein involved in reorganization of mitochondrial cristae. Expression of dominant-negative BIK decreased OPN-stimulated apoptosis. In vivo, OPN expression in cardiac myocyte-specific manner associated with increased protein carbonylation, and expression of NOX-4 and BIK. Thus, OPN induces oxidative stress via the involvement of mitochondria and NOX-4. It may affect mitochondrial morphology and integrity, at least in part, via the involvement of BIK. PMID:27262843

  16. Identification of a molecular signaling network that regulates a cellular necrotic cell death pathway by a genome wide siRNA screen

    PubMed Central

    Hitomi, Junichi; Christofferson, Dana E.; Ng, Aylwin; Yao, Jianhua; Degterev, Alexei; Xavier, Ramnik J.; Yuan, Junying

    2009-01-01

    Stimulation of death receptors by agonists such as FasL and TNFα activates apoptotic cell death in apoptotic competent conditions or a type of necrotic cell death dependent on RIP1 kinase, termed necroptosis, in apoptotic deficient conditions. In a genome-wide siRNA screen for regulators of necroptosis, we identify a set of 432 genes that regulate necroptosis, a subset of 32 genes that act downstream and/or as regulators of RIP1 kinase, 32 genes required for death receptor mediated apoptosis, and 7 genes involved in both necroptosis and apoptosis. We show that the expression of subsets of the 432 genes are enriched in the immune and nervous systems, and cellular sensitivity to necroptosis is regulated by an extensive signaling network mediating innate immunity. Interestingly, Bmf, a BH3-only Bcl-2 family member, is required for death receptor-induced necroptosis. Our study defines a cellular signaling network that regulates necroptosis and the molecular bifurcation that controls apoptosis and necroptosis. PMID:19109899

  17. 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

  18. Apoptosis of Candida albicans during the Interaction with Murine Macrophages: Proteomics and Cell-Death Marker Monitoring.

    PubMed

    Cabezón, Virginia; Vialás, Vital; Gil-Bona, Ana; Reales-Calderón, Jose A; Martínez-Gomariz, Montserrat; Gutiérrez-Blázquez, Dolores; Monteoliva, Lucía; Molero, Gloria; Ramsdale, Mark; Gil, Concha

    2016-05-01

    Macrophages may induce fungal apoptosis to fight against C. albicans, as previously hypothesized by our group. To confirm this hypothesis, we analyzed proteins from C. albicans cells after 3 h of interaction with macrophages using two quantitative proteomic approaches. A total of 51 and 97 proteins were identified as differentially expressed by DIGE and iTRAQ, respectively. The proteins identified and quantified were different, with only seven in common, but classified in the same functional categories. The analyses of their functions indicated that an increase in the metabolism of amino acids and purine nucleotides were taking place, while the glycolysis and translation levels dropped after 3 h of interaction. Also, the response to oxidative stress and protein translation were reduced. In addition, seven substrates of metacaspase (Mca1) were identified (Cdc48, Fba1, Gpm1, Pmm1, Rct1, Ssb1, and Tal1) as decreased in abundance, plus 12 proteins previously described as related to apoptosis. Besides, the monitoring of apoptotic markers along 24 h of interaction (caspase-like activity, TUNEL assay, and the measurement of ROS and cell examination by transmission electron microscopy) revealed that apoptotic processes took place for 30% of the fungal cells, thus supporting the proteomic results and the hypothesis of macrophages killing C. albicans by apoptosis. PMID:27048922

  19. Brazilian Red Propolis Induces Apoptosis-Like Cell Death and Decreases Migration Potential in Bladder Cancer Cells

    PubMed Central

    Begnini, Karine Rech; Moura de Leon, Priscila Marques; Thurow, Helena; Schultze, Eduarda; Campos, Vinicius Farias; Borsuk, Sibele; Dellagostin, Odir Antônio; Savegnago, Lucielli; Moura, Sidnei; Padilha, Francine F.; Pêgas Henriques, João Antonio; Seixas, Fabiana Kömmling

    2014-01-01

    Natural products continue to be an invaluable resource of anticancer drug discovery in recent years. Propolis is known for its biological activities such as antimicrobial and antitumor effects. This study assessed the effects of Brazilian red propolis (BRP) on apoptosis and migration potential in human bladder cancer cells. The effect of BRP ethanolic extract (25, 50, and 100 μg/mL) on 5637 cells was determined by MTT, LIVE/DEAD, and migration (scratch assay) assays. Apoptosis induction was investigated through flow cytometry and gene expression profile was investigated by qRT-PCR. Results showed cytotoxicity on MTT and LIVE/DEAD assays, with IC50 values of 95 μg/mL in 24 h of treatment. Cellular migration of 5637 cells was significantly inhibited through lower doses of BRP ethanolic extract (25 and 50 μg/mL). Flow cytometry analyses showed that BRP induced cytotoxicity through apoptosis-like mechanisms in 5637 cells and qRT-PCR revealed increased levels of Bax/Bcl-2 ratio, p53, AIF, and antioxidant enzymes genes. Data suggest that BRP may be a potential source of drugs to bladder cancer treatment. PMID:25530785

  20. Rapamycin, an mTOR inhibitor, induced apoptosis via independent mitochondrial and death receptor pathway in retinoblastoma Y79 cell

    PubMed Central

    Wang, Yan-Dong; Su, Yong-Jing; Li, Jian-Ying; Yao, Xiang-Chao; Liang, Guang-Jiang

    2015-01-01

    Rapamycin is helpful in the treatment of certain cancers by inhibiting mTOR (mammalian target of rapamycin) pathway. Here, rapamycin mediated apoptosis were investigated in human retinoblastoma Y79 cells. The MTT assay showed that the IC50 value of rapamycin against Y79 cells was 0.136 ± 0.032 μmol/L. Flow cytometry analysis indicated that the percentage of apoptotic cells was increased from 2.16 ± 0.41% to 12.24 ± 3.10%, 20.16 ± 4.22%, and 31.32 ± 5.78% after 0.1, 0.2, and 0.4 μmol/L rapamycin or without rapamycin treatment for 48 hours. Flow cytometry analysis showed that rapamycin induced mitochondrial membrane potential (∆Ψm) collapse in Y79 cells in a concentration-dependent manner. Western blot assay showed that rapamycin led to release of cytochrome c from mitochondrial membranes to cytosol. Further Western blot assays showed that rapamycin induced activation of caspase-9 and caspase-8 and the cleavage of caspase-3. Rapamycin induced cleavages of caspase-3 and apoptosis was inhibited by both Z-LETD-FMK and Z-IETD-FMK treatment. Together, all these results illustrated that rapamycin induced apoptosis in human retinoblastoma Y79 cells involvement of both intrinsic and extrinsic pathways. PMID:26379864

  1. NMDA receptors mediate calcium accumulation in myelin during chemical ischaemia.

    PubMed

    Micu, I; Jiang, Q; Coderre, E; Ridsdale, A; Zhang, L; Woulfe, J; Yin, X; Trapp, B D; McRory, J E; Rehak, R; Zamponi, G W; Wang, W; Stys, P K

    2006-02-23

    Central nervous system myelin is a specialized structure produced by oligodendrocytes that ensheaths axons, allowing rapid and efficient saltatory conduction of action potentials. Many disorders promote damage to and eventual loss of the myelin sheath, which often results in significant neurological morbidity. However, little is known about the fundamental mechanisms that initiate myelin damage, with the assumption being that its fate follows that of the parent oligodendrocyte. Here we show that NMDA (N-methyl-d-aspartate) glutamate receptors mediate Ca2+ accumulation in central myelin in response to chemical ischaemia in vitro. Using two-photon microscopy, we imaged fluorescence of the Ca2+ indicator X-rhod-1 loaded into oligodendrocytes and the cytoplasmic compartment of the myelin sheath in adult rat optic nerves. The AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)/kainate receptor antagonist NBQX completely blocked the ischaemic Ca2+ increase in oligodendroglial cell bodies, but only modestly reduced the Ca2+ increase in myelin. In contrast, the Ca2+ increase in myelin was abolished by broad-spectrum NMDA receptor antagonists (MK-801, 7-chlorokynurenic acid, d-AP5), but not by more selective blockers of NR2A and NR2B subunit-containing receptors (NVP-AAM077 and ifenprodil). In vitro ischaemia causes ultrastructural damage to both axon cylinders and myelin. NMDA receptor antagonism greatly reduced the damage to myelin. NR1, NR2 and NR3 subunits were detected in myelin by immunohistochemistry and immunoprecipitation, indicating that all necessary subunits are present for the formation of functional NMDA receptors. Our data show that the mature myelin sheath can respond independently to injurious stimuli. Given that axons are known to release glutamate, our finding that the Ca2+ increase was mediated in large part by activation of myelinic NMDA receptors suggests a new mechanism of axo-myelinic signalling. Such a mechanism may represent a

  2. Water-soluble coenzyme q10 inhibits nuclear translocation of apoptosis inducing factor and cell death caused by mitochondrial complex I inhibition.

    PubMed

    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

  3. Cleavage of bid may amplify caspase-8-induced neuronal death following focally evoked limbic seizures.

    PubMed

    Henshall, D C; Bonislawski, D P; Skradski, S L; Lan, J Q; Meller, R; Simon, R P

    2001-08-01

    The mechanism by which seizures induce neuronal death is not completely understood. Caspase-8 is a key initiator of apoptosis via extrinsic, death receptor-mediated pathways; we therefore investigated its role in mediating seizure-induced neuronal death evoked by unilateral kainic acid injection into the amygdala of the rat, terminated after 40 min by diazepam. We demonstrate that cleaved (p18) caspase-8 was detectable immediately following seizure termination coincident with an increase in cleavage of the substrate Ile-Glu-Thr-Asp (IETD)-p-nitroanilide and the appearance of cleaved (p15) Bid. Expression of Fas and FADD, components of death receptor signaling, was increased following seizures. In vivo intracerebroventricular z-IETD-fluoromethyl ketone administration significantly reduced seizure-induced activities of caspases 8, 9, and 3 as well as reducing Bid and caspase-9 cleavage, cytochrome c release, DNA fragmentation, and neuronal death. These data suggest that intervention in caspase-8 and/or death receptor signaling may confer protection on the brain from the injurious effects of seizures. PMID:11493022

  4. Follicle-stimulating Hormone Regulates Pro-apoptotic Protein Bcl-2-interacting Mediator of Cell Death-Extra Long (BimEL)-induced Porcine Granulosa Cell Apoptosis*

    PubMed Central

    Wang, Xian-Long; Wu, Yi; Tan, Lu-Bin; Tian, Zhen; Liu, Jing-Hao; Zhu, De-Sheng; Zeng, Shen-Ming

    2012-01-01

    The pro-apoptotic protein Bim (B-cell lymphoma-2 (Bcl-2)-interacting modulator of cell death) has recently been identified and shown to promote cell death in response to several stimuli. In this report, we investigated the role of Bim in porcine follicular atresia. Initially, Bim cDNA was cloned and characterized from porcine ovarian tissue. Porcine Bim had three alternative splicing variants (Bim-extra long, Bim-long, and Bim-short), all containing the consensus Bcl-2 homology 3 domain. We then found the Bim-extra long (BimEL) protein, the most abundant isoform of Bim, was strongly expressed and co-localized with apoptotic (TUNEL-positive) granulosa cells from porcine atretic follicles. Furthermore, overexpression of BimEL triggered apoptosis in granulosa cells. In primary granulosa cell cultures under basal conditions, we observed that BimEL expression was dampened by treatment with follicle-stimulating hormone (FSH). The role of the PI3K/Akt pathway in the regulation of repression was clarified by the use of the PI3K inhibitor, LY294002, and by transfection with Akt siRNA. Forkhead Box Protein O3a (FoxO3a), a well defined transcriptional activator of Bim, was phosphorylated at Ser-253 and inactivated after FSH stimulation. Also, FSH abolished FoxO3a nuclear accumulation in response to LY294002. Finally, chromatin immunoprecipitation assays demonstrated that FoxO3a directly bound and activated the bim promoter. Taken together, we conclude that BimEL induces porcine granulosa cell apoptosis during follicular atresia, and its expression is regulated by FSH via the PI3K/Akt/FoxO3a pathway. PMID:22235114

  5. 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. PMID:26194866

  6. Small molecule inhibitor YM155-mediated activation of death receptor 5 is crucial for chemotherapy-induced apoptosis in pancreatic carcinoma.

    PubMed

    Zhao, Xiangxuan; Puszyk, William M; Lu, Zaiming; Ostrov, David A; George, Thomas J; Robertson, Keith D; Liu, Chen

    2015-01-01

    Despite much effort, pancreatic cancer survival rates are still dismally low. Novel therapeutics may hold the key to improving survival. YM155 is a small molecule inhibitor that has shown antitumor activity in a number of cancers by reducing the expression of survivin. The aim of our study is to understand the mechanisms by which YM155 functions in pancreatic cancer cells. We established the antitumor effect of YM155 with in vitro studies in cultured cells, and in vivo studies using a mouse xenograft model. Our data demonstrated that YM155 reduced the expression of survivin; however, downregulation of survivin itself is insufficient to induce apoptosis in pancreatic cancer cells. We showed for the first time that treatment with YM155 increased death receptor 5 (DR5) expression in pancreatic cancer cells. We found that YM155 induced apoptosis by broad-spectrum inhibition of IAP family member proteins (e.g., CIAP1/2 and FLIP) and induced proapoptotic Bak protein upregulation and activation; the antitumor effect of YM155 treatment with either the DR5 agonist lexatumumab or gemcitabine on pancreatic cancer cells was synergistic. Our data also revealed that YM155 inhibits tumor growth in vivo, without apparent toxicity to the noncancerous human pancreatic ductal epithelial cell line. Together, these findings suggest that YM155 could be a novel therapeutic agent for pancreatic cancer. PMID:25344582

  7. Overcoming resistance to TRAIL-induced apoptosis in solid tumor cells by simultaneously targeting death receptors, c-FLIP and IAPs

    PubMed Central

    HUANG, YING; YANG, XIANG; XU, TIANRUI; KONG, QINGHONG; ZHANG, YAPING; SHEN, YUEHAI; WEI, YUNLIN; WANG, GUANLIN; CHANG, KWEN-JEN

    2016-01-01

    The discovery of the TRAIL protein and its death receptors DR4/5 changed the horizon of cancer research because TRAIL specifically kills cancer cells. However, the validity of TRAIL-based cancer therapies has yet to be established, as most cancer cells are TRAIL-resistant. In this report, we demonstrate that TRAIL-resistance of many cancer cell lines can be overcome after siRNA- or rocaglamide-mediated downregulation of c-FLIP expression and simultaneous inhibition of IAPs activity using AT406, a pan-antagonist of IAPs. Combined triple actions of the TRAIL, the IAPs inhibitor, AT406, and the c-FLIP expression inhibitor, rocaglamide (ART), markedly improve TRAIL-induced apoptotic effects in most solid cancer cell lines through the activation of an extrinsic apoptosis pathway. Furthermore, this ART combination does not harm normal cells. Among the 18 TRAIL-resistant cancer cell lines used, 15 cell lines become sensitive or highly sensitive to ART, and two out of three glioma cell lines exhibit high resistance to ART treatment due to very low levels of procaspase-8. This study provides a rationale for the development of TRAIL-induced apoptosis-based cancer therapies. PMID:27210546

  8. Overcoming resistance to TRAIL-induced apoptosis in solid tumor cells by simultaneously targeting death receptors, c-FLIP and IAPs.

    PubMed

    Huang, Ying; Yang, Xiang; Xu, Tianrui; Kong, Qinghong; Zhang, Yaping; Shen, Yuehai; Wei, Yunlin; Wang, Guanlin; Chang, Kwen-Jen

    2016-07-01

    The discovery of the TRAIL protein and its death receptors DR4/5 changed the horizon of cancer research because TRAIL specifically kills cancer cells. However, the validity of TRAIL-based cancer therapies has yet to be established, as most cancer cells are TRAIL-resistant. In this report, we demonstrate that TRAIL-resistance of many cancer cell lines can be overcome after siRNA- or rocaglamide-mediated downregulation of c-FLIP expression and simultaneous inhibition of IAPs activity using AT406, a pan-antagonist of IAPs. Combined triple actions of the TRAIL, the IAPs inhibitor, AT406, and the c-FLIP expression inhibitor, rocaglamide (ART), markedly improve TRAIL-induced apoptotic effects in most solid cancer cell lines through the activation of an extrinsic apoptosis pathway. Furthermore, this ART combination does not harm normal cells. Among the 18 TRAIL-resistant cancer cell lines used, 15 cell lines become sensitive or highly sensitive to ART, and two out of three glioma cell lines exhibit high resistance to ART treatment due to very low levels of procaspase-8. This study provides a rationale for the development of TRAIL-induced apoptosis-based cancer therapies. PMID:27210546

  9. Synergism of ursolic acid derivative US597 with 2-deoxy-D-glucose to preferentially induce tumor cell death by dual-targeting of apoptosis and glycolysis.

    PubMed

    Wang, Jichuang; Jiang, Zhou; Xiang, Liping; Li, Yuanfang; Ou, Minrui; Yang, Xiang; Shao, Jingwei; Lu, Yusheng; Lin, Lifeng; Chen, Jianzhong; Dai, Yun; Jia, Lee

    2014-01-01

    Ursolic acid (UA) is a naturally bioactive product that exhibits potential anticancer effects. The relatively safe and effective molecule intrigued us to explore a way to further improve its anti-cancer activity and tumor-targeting specificity. In the present study, a series of structural modifications of UA was achieved, which resulted in significant increase in growth inhibition on various cancer cell lines with minimal effects on normal cells. The leading molecule US597 (UA-4) caused depolarization of mitochondrial membrane potential, cell arrest in G0/G1 phase and apoptosis/necrosis in a dose-dependent manner. Structural docking suggested that the carbon chains of the modified UA derivatives compete strongly with glucose for binding to glucokinase, the key glycolysis enzyme presumably active in cancer cells. The combination of 2-deoxy-D-glucose (2-DG) and UA-4 induced cell cycle arrest in G2/M phase, promoted caspase-dependent cell death, reduced hexokinase activity, aggravated depletion of intracellular ATP, decreased lactate production and synergistically inhibited cancer cell growth in vitro (HepG2) and in vivo (H22). Collectively, our findings suggest that the structural modification enhances efficacy and selectivity of UA, and the combination of UA-4 with 2-DG produces synergistic inhibition on hepatoma cell proliferation by dual targeting of apoptosis and glycolysis. PMID:25833312

  10. 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

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

    PubMed

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

    2016-02-01

    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

  12. 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

  13. Role of Endoplasmic Reticulum Stress in α-TEA Mediated TRAIL/DR5 Death Receptor Dependent Apoptosis

    PubMed Central

    Li, Jing; Park, Sook-Kyung; Sanders, Bob G.; Kline, Kimberly

    2010-01-01

    Background α-TEA (RRR-α-tocopherol ether-linked acetic acid analog), a derivative of RRR-α-tocopherol (vitamin E) exhibits anticancer actions in vitro and in vivo in variety of cancer types. The objective of this study was to obtain additional insights into the mechanisms involved in α-TEA induced apoptosis in human breast cancer cells. Methodology/Principal Findings α-TEA induces endoplasmic reticulum (ER) stress as indicated by increased expression of CCAAT/enhancer binding protein homologous protein (CHOP) as well as by enhanced expression or activation of specific markers of ER stress such as glucose regulated protein (GRP78), phosphorylated alpha subunit of eukaryotic initiation factor 2 (peIF-2α), and spliced XBP-1 mRNA. Knockdown studies using siRNAs to TRAIL, DR5, JNK and CHOP as well as chemical inhibitors of ER stress and caspase-8 showed that: i) α-TEA activation of DR5/caspase-8 induces an ER stress mediated JNK/CHOP/DR5 positive amplification loop; ii) α-TEA downregulation of c-FLIP (L) protein levels is mediated by JNK/CHOP/DR5 loop via a JNK dependent Itch E3 ligase ubiquitination that further serves to enhance the JNK/CHOP/DR5 amplification loop by preventing c-FLIP's inhibition of caspase-8; and (iii) α-TEA downregulation of Bcl-2 is mediated by the ER stress dependent JNK/CHOP/DR5 signaling. Conclusion Taken together, ER stress plays an important role in α-TEA induced apoptosis by enhancing DR5/caspase-8 pro-apoptotic signaling and suppressing anti-apoptotic factors c-FLIP and Bcl-2 via ER stress mediated JNK/CHOP/DR5/caspase-8 signaling. PMID:20686688

  14. Plumbagin, a plant-derived naphthoquinone metabolite induces mitochondria mediated apoptosis-like cell death in Leishmania donovani: an ultrastructural and physiological study.

    PubMed

    Awasthi, Bhanu Priya; Kathuria, Manoj; Pant, Garima; Kumari, Neema; Mitra, Kalyan

    2016-08-01

    Naphthoquinones are known to exhibit a broad range of biological activities against microbes, cancer and parasitic diseases and have been widely used in Indian traditional medicine. Plumbagin is a plant-derived naphthoquinone metabolite (5-hydroxy-2-methyl-1,4-naphthoquinone) reported to inhibit trypanothione reductase, the principal enzyme and a validated drug target involved in detoxification of oxidative stress in Leishmania. Here, we report the mechanistic aspects of cell death induced by plumbagin including physiological effects in the promastigote form and ultrastructural alterations in both promastigote and amastigote forms of Leishmania donovani which till now remained largely unknown. Our observations show that oxidative stress induced by plumbagin resulted in depolarization of the mitochondrial membrane, depletion in ATP levels, elevation of cytosolic calcium, increase in caspase 3/7-like protease activity and lipid peroxidation in promastigotes. Apoptosis-like cell death induction post plumbagin treatment was confirmed by biochemical assays like Annexin V/FITC staining, TUNEL as well as morphological and ultrastructural studies. These findings collectively highlight the mode of action and importance of oxidative stress inducing agents in effectively killing both forms of the Leishmania parasite and opens up the possibility of exploring plumbagin and its derivatives as promising candidates in the chemotherapy of Leishmaniasis. PMID:27315817

  15. The pentachlorophenol metabolite tetrachlorohydroquinone induces massive ROS and prolonged p-ERK expression in splenocytes, leading to inhibition of apoptosis and necrotic cell death.

    PubMed

    Chen, Hsiu-Min; Zhu, Ben-Zhan; Chen, Rong-Jane; Wang, Bour-Jr; Wang, Ying-Jan

    2014-01-01

    Pentachlorophenol (PCP) has been used extensively as a biocide and a wood preservative and has been reported to be immunosuppressive in rodents and humans. Tetrachlorohydroquinone (TCHQ) is a major metabolite of PCP. TCHQ has been identified as the main cause of PCP-induced genotoxicity due to reactive oxidant stress (ROS). However, the precise mechanisms associated with the immunotoxic effects of PCP and TCHQ remain unclear. The aim of this study was to examine the effects of PCP and TCHQ on the induction of ROS and injury to primary mouse splenocytes. Our results shown that TCHQ was more toxic than PCP and that a high dose of TCHQ led to necrotic cell death of the splenocytes through induction of massive and sudden ROS and prolonged ROS-triggered ERK activation. Inhibition of ROS production by N-acetyl-cysteine (NAC) partially restored the mitochondrial membrane potential, inhibited ERK activity, elevated caspase-3 activity and PARP cleavage, and, eventually, switched the TCHQ-induced necrosis to apoptosis. We suggest that prolonged ERK activation is essential for TCHQ-induced necrosis, and that ROS play a pivotal role in the different TCHQ-induced cell death mechanisms. PMID:24586814

  16. Restoration of TRAIL-induced apoptosis in a caspase-8-deficient neuroblastoma cell line by stable re-expression of caspase-8.

    PubMed

    Mühlethaler-Mottet, Annick; Balmas, Katia; Auderset, Katya; Joseph, Jean-Marc; Gross, Nicole

    2003-12-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in most tumor cells, a process sometimes potentiated by chemotherapeutic drugs or cycloheximide (CHX). Childhood neuroblastoma (NB) is a clinically and biologically heterogeneous neoplasm whose behavior can be explained by differential regulation of apoptosis. The non-invasive S-type NB cell lines are sensitive to TRAIL, whereas the invasive N-type NB cell lines are resistant. We have reported the silencing of caspase-8 expression in N-type cells as a possible mechanism of death receptor-mediated resistance to apoptosis in NB. The recently observed deregulation of caspase-10 in these cells prompted us to investigate the particular contribution of caspase-8 silencing in the resistance to TRAIL in N-type cells. Stable caspase-8 expression was therefore restored in the IGR-N91 cell line by retroviral infection. The IGR-N91-C8 cells became sensitive to TRAIL-mediated apoptosis, whereas the control vector-infected IGR-N91-M cells remained resistant. Interestingly, the apoptotic response to TRAIL was enhanced by co-treatment of SH-EP and IGR-N91-C8 cells with CHX or with sub-toxic concentration of doxorubicin (DOX) in a caspase-dependent manner, as cells could be protected from death by specific caspase-8 or pan-caspase inhibitors. CHX or DOX was shown to enhance TRAIL-induced caspase-8 activation and loss of mitochondrial transmembrane potential. In conclusion, restoration of active caspase-8 expression in caspase-8- and caspase-10-deficient IGR-N-91 cell line is necessary and sufficient to fully restore TRAIL-mediated cell death. Moreover, DOX and CHX were able to sensitize NB cell lines to TRAIL-induced apoptosis in a caspase-8-dependent manner by engaging death receptor and mitochondrial signaling pathways. PMID:15033719

  17. Downregulation of Peptidylprolyl isomerase A promotes cell death and enhances doxorubicin-induced apoptosis in hepatocellular carcinoma.

    PubMed

    Cheng, Shaobing; Luo, Mengchao; Ding, Chaofeng; Peng, Chuanhui; Lv, Zhen; Tong, Rongliang; Xiao, Heng; Xie, Haiyang; Zhou, Lin; Wu, Jian; Zheng, Shusen

    2016-10-10

    Peptidylprolyl isomerase A (PPIA) is a peptidyl-prolyl cis-trans isomerase that is known to play a critical role in the development of many human cancers. However, the precise biological function of PPIA in hepatocellular carcinoma (HCC) remains largely unclear. In this study, lentiviral overexpression vectors and small interfering RNA knockdown methods were employed to investigate the biological effects of PPIA in HCC. PPIA levels in HCC tissues and peritumoral tissues were detected by real-time Polymerase Chain Reaction (RT-PCR), Western blotting, and immunohistochemistry. Our results indicate that PPIA levels were significantly higher in the HCC tissues compared to the matched peritumoral tissues. Moreover, PPIA expression was significantly associated with tumor size in these tissues. Interestingly, serum PPIA (sPPIA) levels were significantly higher in healthy controls compared to the HCC patients. Knockdown or overexpression of PPIA was shown to downregulate and upregulate cell growth, respectively. Moreover, PPIA siRNA knockdown appears to promote doxorubicin-induced apoptosis in HCC cells, altering the expression of downstream apoptotic factors. In summary, our results indicate that PPIA may play a pivotal role in HCC by regulating cell growth and could serve as a novel marker and therapeutic molecular target for HCC patients. PMID:27397650

  18. 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

  19. 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. PMID:26731663

  20. Stool-fermented Plantago ovata husk induces apoptosis in colorectal cancer cells independently of molecular phenotype.

    PubMed

    Sohn, Vanessa R; Giros, Anna; Xicola, Rosa M; Fluvià, Lourdes; Grzybowski, Mike; Anguera, Anna; Llor, Xavier

    2012-06-01

    Several studies have suggested that the partially fermentable fibre Plantago ovata husk (PO) may have a protective effect on colorectal cancer (CRC). We studied the potentially pro-apoptotic effect of PO and the implicated mechanisms in CRC cells with different molecular phenotypes (Caco-2, HCT116, LoVo, HT-29, SW480) after PO anaerobic fermentation with colonic bacteria as it occurs in the human colon. The fermentation products of PO induced apoptosis in all primary tumour and metastatic cell lines, independent of p53, adenomatous polyposis coli, β-catenin or cyclo-oxygenase-2 status. Apoptosis was caspase-dependent and both intrinsic and extrinsic pathways were implicated. The intrinsic pathway was activated through a shift in the balance towards a pro-apoptotic environment with an up-regulation of B-cell lymphoma protein 2 homologous antagonist killer (BAK) and a down-regulation of B-cell lymphoma-extra large (Bcl-xL) seen in HCT116 and LoVo cells. This resulted in mitochondrial membrane depolarisation, increased expression of caspase activators second mitochondria-derived activator of caspases (Smac)/Diablo, death effector apoptosis-inducing factor, apoptosome member apoptotic protease activating factor 1 and down-regulation of inhibitors of apoptosis Survivin and X-linked inhibitor of apoptosis in most cells. The extrinsic pathway was activated presumably through the up-regulation of death receptor (DR5). Some important differences were seen between primary tumour and metastatic CRC cells. Thus, metastatic PO-treated LoVo cells had a remarkable up-regulation of TNF-α ligand along with death-inducing signalling complex components receptor interacting protein and TNF-α receptor 1-associated death domain protein. The extrinsic pathway modulator FCICE-inhibitory protein (FLIP), an inhibitor of both spontaneous death ligand-independent and death receptor-mediated apoptosis, was significantly down-regulated after PO treatment in all primary tumour cells, but not

  1. OSTEOCYTE APOPTOSIS

    PubMed Central

    Jilka, Robert L.; Noble, Brendon; Weinstein, Robert S.

    2012-01-01

    Apoptotic death of osteocytes was recognized over 15 years ago, but its significance for bone homeostasis has remained elusive. A new paradigm has emerged that invokes osteocyte apoptosis as a critical event in the recruitment of osteoclasts to a specific site in response to skeletal unloading, fatigue damage, estrogen deficiency and perhaps in other states where bone must be removed. This is accomplished by yet to be defined signals emanating from dying osteocytes, which stimulate neighboring viable osteocytes to produce osteoclastogenic cytokines. The osteocyte apoptosis caused by chronic glucocorticoid administration does not increase osteoclasts; however, it does negatively impact maintenance of bone hydration, vascularity, and strength. PMID:23238124

  2. Tetrandrine and caffeine modulated cell cycle and increased glioma cell death via caspase-dependent and caspase-independent apoptosis pathways.

    PubMed

    Chen, Jin-Cherng; Hwang, Juen-Haur; Chiu, Wen-Hsuan; Chan, Yin-Ching

    2014-01-01

    Viability, cell cycle distribution, and expressions of eukaryotic translation initiation factor-2α (eIF-2α), cyclin D1, poly(ADP-ribose) polymerase 1 (PARP-1), and apoptosis-inducing factor (AIF) of RT-2 glioma cells were assayed under treatment of tetrandrine and caffeine for 48 h. The results showed that cell viability decreased significantly under treatment with tetrandrine (5 μM) alone or under combined treatment with tetrandrine (5 μM) and caffeine (0.5 or 1 mM). The ratio of RT-2 cells at sub G1 and G0/G1 stages increased significantly during combined treatment of tetrandrine (5 μM) and caffeine (0.5, 1 mM). The ratio of phospharylated eIF-2α to dephospharylated eIF-2α increased, whereas cyclin D1 decreased significantly under combined treatment of tetrandrine (5 μM) and caffeine (1 mM). The cleaved PARP-1 to PARP-1 ratio was elevated significantly under treatment of 5 μM tetrandrine alone, and combined treatment of 5 μM tetrandrine and caffeine (0.5, 1 mM). The expression levels of AIF increased significantly under treatment of 5 μM tetrandrine alone or 1 mM caffeine alone, and combined treatment of 5 μM tetrandrine and caffeine (0.5, 1 mM). In conclusion, tetrandrine and caffeine could induce glioma cell death possibly via increasing eIF-2α phospharylation, decreasing cyclin-D1 expression, and increasing caspase-dependent and -independent apoptosis pathways. PMID:24738643

  3. P2X7 receptors mediate deleterious renal epithelial-fibroblast cross talk.

    PubMed

    Ponnusamy, Murugavel; Ma, Li; Gong, Rujun; Pang, Maoyin; Chin, Y Eugene; Zhuang, Shougang

    2011-01-01

    Peritubular fibroblasts in the kidney are the major erythropoietin-producing cells and also contribute to renal repair following acute kidney injury (AKI). Although few fibroblasts were observed in the interstitium adjacent to damaged tubular epithelium in the early phase of AKI, the underlying mechanism by which their numbers were reduced remains unknown. In this study, we tested the hypothesis that damaged renal epithelial cells directly induce renal interstitial fibroblast death by releasing intracellular ATP and activating purinergic signaling. Exposure of a cultured rat renal interstitial fibroblast cell line (NRK-49F) to necrotic renal proximal tubular cells (RPTC) lysate or supernatant induced NRK-49F cell death by apoptosis and necrosis. Depletion of ATP with apyrase or inhibition of the P2X purinergic receptor with pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid blocked the deleterious effect of necrotic RPTC supernatant. The P2X7 receptor, an ATP-sensitive purinergic receptor, was not detected in cultured NRK-49F cells but was inducible by necrotic RPTC supernatant. Treatment with A438079, a highly selective P2X7 receptor inhibitor, or knockdown of the P2X7 receptor with small interference RNA diminished renal fibroblast death induced by necrotic RPTC supernatant. Conversely, overexpression of the P2X7 receptor potentiated this response. Collectively, these findings provide strong evidence that damaged renal epithelial cells can directly induce the death of renal interstitial fibroblasts by ATP activation of the P2X7 receptor. PMID:20861083

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

    PubMed

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

    2016-05-27

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

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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.

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

    PubMed

    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

  11. BI-1 enhances Fas-induced cell death through a Na+/H+-associated mechanism

    PubMed Central

    Lee, Geum-Hwa; Kim, Hyung-Ryong; Chae, Han-Jung

    2014-01-01

    The role of Bax inhibitor-1 (BI-1) in the protective mechanism against apoptotic stimuli has been studied; however, as little is known about its role in death receptor-mediated cell death, this study was designed to investigate the effect of BI-1 on Fas-induced cell death, and the underlying mechanisms. HT1080 adenocarcinoma cells were cultured in high concentration of glucose media and transfected with vector alone (Neo cells) or BI-1-vector (BI-1 cells), and treated with Fas. In cell viability, apoptosis, and caspase-3 analyses, the BI-1 cells showed enhanced sensitivity to Fas. Fas significantly decreased cytosolic pH in BI-1 cells, compared with Neo cells, and this decrease correlated with BI-1 oligomerization, mitochondrial Ca2+ accumulation, and significant inhibition of sodium-hydrogen exchanger (NHE) activity. Compared with Neo cells, a single treatment of BI-1 cells with the NHE inhibitor EIPA or siRNA against NHE significantly increased cell death, which suggests that the viability of BI-1 cells is affected by the maintenance of intracellular pH homeostasis through NHE. [BMB Reports 2014; 47(7): 393-398] PMID:24314142

  12. Crude saponins from Platycodon grandiflorum induce apoptotic cell death in RC-58T/h/SA#4 prostate cancer cells through the activation of caspase cascades and apoptosis-inducing factor.

    PubMed

    Lee, Ju-Hye; Oh, Eun-Kyoung; Cho, Hyun-Dong; Kim, Jae-Yong; Lee, Mi-Kyung; Seo, Kwon-Il

    2013-04-01

    Saponins are a major active component of Platycodon grandiflorum (P. grandiflorum) and are known to induce apoptosis in metastatic prostate cancer cell lines. However, thus far, no research has been conducted on the anticancer activity of saponins in RC-58T/h/SA#4 primary prostate cancer cells. In this study, we show that the treatment of prostate cancer cells with saponins extracted from P. grandiflorum (SPG) inhibits cell proliferation in a dose-dependent manner. SPG significantly induced apoptotic cell death, resulting in an increase in the sub-G1 apoptotic cell population, apoptotic DNA fragmentation and morphological changes. Pre-treatment with a caspase inhibitor modestly attenuated the SPG-induced increase in the sub-G1 cell population, suggesting that caspases play a role in SPG-induced apoptosis. Moreover, SPG-induced apoptosis was associated with changes in caspase activity, the upregulation of the apoptotic protein, Bax and the downregulation of the anti-apoptotic protein, Bcl-2. Furthermore, the caspase-independent mitochondrial apoptosis factor, apoptosis-inducing factor (AIF) was upregulated following SPG treatment. These findings indicate that SPG exerts its anticancer effects on RC-58T/h/SA#4 primary prostate cancer cells through mitochondrial caspase-dependent and -independent apoptotic pathways. PMID:23443329

  13. 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. PMID:26538054

  14. [The origin of eukaryotic cells and origination of apoptosis].

    PubMed

    Galitskiĭ, V A

    2005-01-01

    The unified conception of the origin of eukaryotic cells has been proposed. In the author's opinion, evolutionary transformation of prokaryotic cell into eukaryotic cell took place 3.3-1.4 billion years ago and involved the next four stages: 1) the appearance of intracellular membranes due to prokaryotic cell plasmalemma invaginating into its cytoplasm; 2) the cell nucleus formation by the double sheet of intracellular membrane surrounding and sequestrating genetic material of the cell; 3) the appearance of cytoskeleton in parallel with mitotic spindle formation and gradual transition from prokaryotic way of cell division to mitosis; 4) the establishment of symbiosis between the evolving nucleated cell and prokaryotic microorganicsms that subsequently transform into mitochondria and chloroplasts. Apoptosis of cells of the present day multicellular eukaryotic organisms is supposed to be an evolutionary altered response of mitochondrian predecessors to the influence of factors, which are able to damage eukaryotic host cell. The initial biological significance of this reaction pertained to attempts of endosymbionts to leave the host cell as soon as possible, if the probability of its irreversible injury was very high, and by this to escape from their death. It is possible that numerous proteins, known as sensors or transducers of proapoptotic signals in Bcl-2--p53-dependent apoptotic pathway, were initially encoded by mitochondrial genome, whereas antiapoptotic factors and also components of receptor-mediated and granzyme B perforin dependent apoptotic pathways have cellular origin. PMID:16706173

  15. Ilimaquinone induces death receptor expression and sensitizes human colon cancer cells to TRAIL-induced apoptosis through activation of ROS-ERK/p38 MAPK-CHOP signaling pathways.

    PubMed

    Do, Minh Truong; Na, MinKyun; Kim, Hyung Gyun; Khanal, Tilak; Choi, Jae Ho; Jin, Sun Woo; Oh, Seok Hoon; Hwang, In Hyun; Chung, Young Chul; Kim, Hee Suk; Jeong, Tae Cheon; Jeong, Hye Gwang

    2014-09-01

    TRAIL induces apoptosis in a variety of tumor cells. However, development of resistance to TRAIL is a major obstacle to more effective cancer treatment. Therefore, novel pharmacological agents that enhance sensitivity to TRAIL are necessary. In the present study, we investigated the molecular mechanisms by which ilimaquinone isolated from a sea sponge sensitizes human colon cancer cells to TRAIL. Ilimaquinone pretreatment significantly enhanced TRAIL-induced apoptosis in HCT 116 cells and sensitized colon cancer cells to TRAIL-induced apoptosis through increased caspase-8, -3 activation, PARP cleavage, and DNA damage. Ilimaquinone also reduced the cell survival proteins Bcl2 and Bcl-xL, while strongly up-regulating death receptor (DR) 4 and DR5 expression. Induction of DR4 and DR5 by ilimaquinone was mediated through up-regulation of CCAAT/enhancer-binding protein homologous protein (CHOP). The up-regulation of CHOP, DR4 and DR5 expression was mediated through activation of extracellular-signal regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways. Finally, the generation of ROS was required for CHOP and DR5 up-regulation by ilimaquinone. These results demonstrate that ilimaquinone enhanced the sensitivity of human colon cancer cells to TRAIL-induced apoptosis through ROS-ERK/p38 MAPK-CHOP-mediated up-regulation of DR4 and DR5 expression, suggesting that ilimaquinone could be developed into an adjuvant chemotherapeutic drug. PMID:24930757

  16. Cysteine cathepsins are not critical for TRAIL- and CD95-induced apoptosis in several human cancer cell lines.

    PubMed

    Špes, Aleš; Sobotic, Barbara; Turk, Vito; Turk, Boris

    2012-12-01

    prevented apoptosis and its progression nor the mitochondrial and lysosomal membrane permeabilization associated with this type of cell death. Consequently, cathepsin release into the cytosol was also not prevented. Together, these data indicate that cysteine cathepsins are not required for the TRAIL- and CD95-mediated apoptosis in various human cancer cell lines. This does not, however, rule out that lysosomes and cysteine cathepsins are involved in the amplification, but not in the initiation, of death receptor-mediated apoptosis in certain cell lines or under different stimulation conditions than the ones employed here. PMID:23667901

  17. Arsenic trioxide (As(2)O(3)) induces apoptosis and necrosis mediated cell death through mitochondrial membrane potential damage and elevated production of reactive oxygen species in PLHC-1 fish cell line.

    PubMed

    Selvaraj, Vellaisamy; Armistead, Mindy Yeager; Cohenford, Menashi; Murray, Elizabeth

    2013-01-01

    Several environmental pollutants, including metals can induce toxicological effect on aquatic animal species. Most studies to understand the toxicity of arsenic compounds were performed in mammalian cells; however, the study of the arsenic toxicity to the aquatic animals' species, including fish, is limited. So the objective of this study was first to investigate the effects of As(2)O(3) induced toxicity particularly on apoptosis and necrosis mediated cell death in fish cell PLHC-1 as compared to the mechanism of toxicity from known mammalian cell lines, secondly to relate in vitro effects in fish to those demonstrated by in vivo systems. To conduct this study, PLHC-1 cells were exposed to various concentrations of As(2)O(3) (0-100 μM) for 10, 20 and 40 h. The results indicate that As(2)O(3) exposure promoted apoptotic and necrotic mediated cell death in a concentration and time dependent manner. Cell death (apoptotic and necrotic) induced by As(2)O(3) was further confirmed by changes in various phases of cell cycle, DNA fragmentation (necro- comet and apo-comet) in the comet assay, alteration in mitochondrial membrane potential and formation of increased reactive oxygen species (ROS). Apoptotic mediated cell death was confirmed further by observing the increased caspase-3 activity and elevated expression of p53, cytochrome c and Bax proteins levels in the same experimental conditions. PLHC-1 cells were shown to be a good model for evaluating biochemical/cytotoxic effects following exposure to various reference chemicals and environmental contaminants. In vitro data obtained from this study provides a comprehensive approach for the elucidating the actual molecular mechanism for As(2)O(3) induced toxicity particularly apoptosis and necrosis mediated cell death in PLHC-1 cell line. PMID:23121984

  18. Tc-99m-galactosyl-neoglycoalbumin: in vivo characterization of receptor-mediated binding to hepatocyctes

    SciTech Connect

    Vera, D.R.; Krohn, K.A.; Stadalnik, R.C.; Scheibe, P.O.

    1984-04-01

    The biodistribution and kinetics of a receptor-binding hepatic radiopharmaceutical, Tc-99m-galactosyl-neoglycoalbumin (Tc-NGA), were investigated using mammalian and avian models. The radiopharmaceutical exhibited four significant features associated with receptor-mediated binding at the hepatocyte membrane in mammals: (a) high tissue specificity, (b) high molecular specificity, (c) affinity-dependent uptake, and (d) dose-dependent uptake. Diminished hepatic uptake by the avian model illustrated low nonspecific binding. The kinetic sensitivity to ligand-receptor affinity and stoichiometry illustrated the principal feature of receptor-binding radiopharmaceuticals, namely, quantitative assessment of tissue function based upon the biochemical interaction of a ligand and its specific receptor.

  19. 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.

  20. [Molecular physiology of receptor mediated endocytosis and its role in overcoming multidrug resistance].

    PubMed

    Severin, E S; Posypanova, G A

    2011-06-01

    Receptor-mediated endocytosis plays important role in the selective uptake of proteins at the plasma membrane of eukaryotic cells. Endocytosis regulates many processes of cell signalling by controlling the number of functional receptors on the cell surface. The article reviews the mechanism of clathrin-dependent endocytosis and the possibility of using this phenomenon for the targeted delivery of drugs. Use of certain proteins as targeting component of drug delivery systems can significantly improve the selectivity of this drug, as well as to overcome the multidrug resistance of cells resulting from the activity of the ABC-transporters. PMID:21874867

  1. 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

  2. Prevention of neonatal oxygen-induced brain damage by reduction of intrinsic apoptosis

    PubMed Central

    Sifringer, M; Bendix, I; Börner, C; Endesfelder, S; von Haefen, C; Kalb, A; Holifanjaniaina, S; Prager, S; Schlager, G W; Keller, M; Jacotot, E; Felderhoff-Mueser, U

    2012-01-01

    Within the last decade, it became clear that oxygen contributes to the pathogenesis of neonatal brain damage, leading to neurocognitive impairment of prematurely born infants in later life. Recently, we have identified a critical role for receptor-mediated neuronal apoptosis in the immature rodent brain. However, the contribution of the intrinsic apoptotic pathway accompanied by activation of caspase-2 under hyperoxic conditions in the neonatal brain still remains elusive. Inhibition of caspases appears a promising strategy for neuroprotection. In order to assess the influence of specific caspases on the developing brain, we applied a recently developed pentapeptide-based group II caspase inhibitor (5-(2,6-difluoro-phenoxy)-3(R,S)-(2(S)-(2(S)-(3-methoxycarbonyl-2(S)-(3-methyl-2(S)-((quinoline-2-carbonyl)-amino)-butyrylamino)propionylamino)3-methylbutyrylamino)propionylamino)-4-oxo-pentanoic acid methyl ester; TRP601). Here, we report that elevated oxygen (hyperoxia) triggers a marked increase in active caspase-2 expression, resulting in an initiation of the intrinsic apoptotic pathway with upregulation of key proteins, namely, cytochrome c, apoptosis protease-activating factor-1, and the caspase-independent protein apoptosis-inducing factor, whereas BH3-interacting domain death agonist and the anti-apoptotic protein B-cell lymphoma-2 are downregulated. These results coincide with an upregulation of caspase-3 activity and marked neurodegeneration. However, single treatment with TRP601 at the beginning of hyperoxia reversed the detrimental effects in this model. Hyperoxia-mediated neurodegeneration is supported by intrinsic apoptosis, suggesting that the development of highly selective caspase inhibitors will represent a potential useful therapeutic strategy in prematurely born infants. PMID:22237207

  3. Receptor-mediated binding and uptake of GnRH agonist and antagonist by pituitary cells

    SciTech Connect

    Jennes, L.; Stumpf, W.E.; Conn, P.M.

    1984-01-01

    The intracellular pathway of an enzyme resistant GnRH agonist (D- Lys6 -GnRH) conjugated to ferritin or to colloidal gold was followed in cultured pituitary cells. After an initial uniform distribution over the cell surface of gonadotropes, the electrondense marker was internalized, either individually or in small groups. After longer incubation times, the marker appeared in the lysosomal compartment and the Golgi apparatus, where it could be found in the vesicular as well as cisternal portion. In addition, the receptor-mediated endocytosis of the GnRH antagonist D-p-Glu1-D-Phe2-D-Trp3-D- Lys6 -GnRH was studied by light and electron microscopic autoradiography after 30 and 60 min of incubation to ensure uptake. At both time points, in in vitro as well as in vivo studies, silver grains were localized over cytoplasmic organelles of castration cells, including dilated endoplasmic reticulum, lysosomes, and clear vesicles. No consistent association with cell nuclei, mitochondria, or secretory vesicles could be observed. The results suggest that both agonist and antagonist are binding selectively to the plasma membrane of gonadotropes and subsequently are taken up via receptor-mediated endocytosis for degradation or possible action on synthetic processes.

  4. Target shape dependence in a simple model of receptor-mediated endocytosis and phagocytosis

    PubMed Central

    Richards, David M.; Endres, Robert G.

    2016-01-01

    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. PMID:27185939

  5. Receptor-mediated tumor targeting with radiopeptides. Part 1. General principles and methods.

    PubMed

    Eberle, Alex N; Mild, Gabriele

    2009-01-01

    Radiolabeled peptides have become important tools for preclinical cancer research, and in nuclear oncology they serve as diagnostic and more recently also as therapeutic agents. In the latter application, radiolabeled peptides represent a distinct sector of the molecular targeting approach, which in many areas of therapy implements the old "magic bullet" concept by specifically directing the therapeutic agent to the site of action. Although in the past few years the development of receptor-mediated targeting for therapy has been confined to some radiolabeled antibodies and to somatostatin/SRIF, research into an increasing number of radiolabeled peptides and their receptors expressed by different tumors will soon lead to a wider use of peptide radiopharmaceuticals. In a consecutive series of six reviews we present a comprehensive overview of the literature on receptor-mediated tumor targeting with the different radiopeptides currently studied. Part 1 summarizes the concepts and methods of radiopeptide targeting, the selection of radioisotopes, chelators, the criteria of peptide ligand development and some general aspects of diagnostic and therapeutic application of peptide radiopharmaceuticals. PMID:19519167

  6. Interleukin 1 amplifies receptor-mediated activation of phospholipase A2 in 3T3 fibroblasts.

    PubMed Central

    Burch, R M; Connor, J R; Axelrod, J

    1988-01-01

    Human recombinant interleukin 1 alpha (IL-1 alpha) and IL-1 beta stimulated prostaglandin E2 synthesis in 3T3 fibroblasts in a time- and concentration-dependent manner. Enhanced prostaglandin E2 synthesis after IL-1 treatment was apparent by 1 hr and continued to increase for at least 2 days. Half-maximal stimulation occurred at 0.5 pM IL-1 alpha or IL-1 beta, and both interleukins were equally effective, with maximal stimulation occurring in response to 5-10 pM IL-1. In contrast to IL-1, bradykinin stimulation of prostaglandin E2 synthesis is rapid; its effect is maximal by 5 min. In cells that had been pretreated with IL-1 for 24 hr, prostaglandin E2 synthesis in response to bradykinin was amplified more than 10-fold. IL-1 also amplified the receptor-mediated formation of prostaglandin E2 by bombesin and thrombin. The lymphokine did not affect bradykinin receptor number or affinity. IL-1 treatment induced phospholipase A2 and cyclooxygenase but not phospholipase C or prostaglandin E isomerase. It also enhanced bradykinin-stimulated GTPase activity, suggesting possible induction of the GTP-binding regulatory protein coupled to the bradykinin receptor. Thus, IL-1 enhanced receptor-mediated release of prostaglandin E2 in response to bradykinin, bombesin, and thrombin by increasing the cellular levels of phospholipase A2, cyclooxygenase, and GTP-binding regulatory protein(s). PMID:2901097

  7. Opioid receptors mediate direct predictive fear learning: Evidence from one-trial blocking

    PubMed Central

    Cole, Sindy; McNally, Gavan P.

    2007-01-01

    Pavlovian fear learning depends on predictive error, so that fear learning occurs when the actual outcome of a conditioning trial exceeds the expected outcome. Previous research has shown that opioid receptors, including μ-opioid receptors in the ventrolateral quadrant of the midbrain periaqueductal gray (vlPAG), mediate such predictive fear learning. Four experiments reported here used a within-subject one-trial blocking design to study whether opioid receptors mediate a direct or indirect action of predictive error on Pavlovian association formation. In Stage I, rats were trained to fear conditioned stimulus (CS) A by pairing it with shock. In Stage II, CSA and CSB were co-presented once and co-terminated with shock. Two novel stimuli, CSC and CSD, were also co-presented once and co-terminated with shock in Stage II. The results showed one-trial blocking of fear learning (Experiment 1) as well as one-trial unblocking of fear learning when Stage II training employed a higher intensity footshock than was used in Stage I (Experiment 2). Systemic administrations of the opioid receptor antagonist naloxone (Experiment 3) or intra-vlPAG administrations of the selective μ-opioid receptor antagonist CTAP (Experiment 4) prior to Stage II training prevented one-trial blocking. These results show that opioid receptors mediate the direct actions of predictive error on Pavlovian association formation. PMID:17404385

  8. 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.

  9. Heterogeneity and probabilistic binding contributions to receptor-mediated cell detachment kinetics.

    PubMed Central

    Saterbak, A; Kuo, S C; Lauffenburger, D A

    1993-01-01

    Biospecific cell adhesion is mediated by receptor-ligand bonds. Early theoretical work presented a deterministic analysis of receptor-mediated cell attachment and detachment for a homogeneous cell population. However, initial comparison of a deterministic framework to experimental detachment profiles of model "cells" (antibody-coated latex beads) did not show qualitative or quantitative agreement (Cozens-Roberts, C., D.A. Lauffenburger, and J.A. Quinn. 1990. Biophys. J. 58:857-872). Hence, we determine the contributions of population heterogeneity and probabilistic binding to the detachment behavior of this experimental system which was designed to minimize experimental and theoretical complications. This work also corrects an error in the numerical solution of the probabilistic model of receptor-mediated cell attachment and detachment developed previously (Cozens-Roberts, C., D.A. Lauffenburger, and J.A. Quinn. 1990. Biophys J. 58:841-856). Measurement of the population distribution of the number of receptors per bead has enabled us to explicitly consider the effect of receptor number heterogeneity within the cell-surface contact area. A deterministic framework that incorporates receptor number heterogeneity qualitatively and quantitatively accounts in large part for the model cell detachment data. Using measured and estimated parameter values for the model cell system, we estimate that about 90% of the observed kinetic detachment behavior originates from heterogeneity effects, while about 10% is due to probabilistic binding effects. In general, these relative contributions may differ for other systems. PMID:8396454

  10. 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. PMID:24141311

  11. 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.

  12. Cytotoxic and apoptosis-inducing activities of limonoids from the seeds of Azadirachta indica (neem).

    PubMed

    Kikuchi, Takashi; Ishii, Koichi; Noto, Taisuke; Takahashi, Akitomo; Tabata, Keiichi; Suzuki, Takashi; Akihisa, Toshihiro

    2011-04-25

    Thirty-five limonoids, including 15 of the azadiradione type (1-15), five of the gedunin type (16-20), four of the azadirachtin type (21-24), nine of the nimbin type (25-33), and two degraded limonoids (34, 35), isolated from Azadirachta indica seed extracts, were evaluated for their cytotoxic activities against five human cancer cell lines. Seven compounds (3, 6, 7, 16, 18, 28, and 29) exhibited cytotoxic activity against one or more cell lines. Among these compounds, 7-deacetyl-7-benzoylepoxyazadiradione (7), 7-deacetyl-7-benzoylgeduin (18), and 28-deoxonimbolide (28) exhibited potent cytotoxic activity against HL60 leukemia cells with IC(50) values in the range 2.7-3.1 μM. Compounds 7, 18, and 28 induced early apoptosis in HL60 cells, observed by flow cytometry. Western blot analysis showed that compounds 7, 18, and 28 activated caspases-3, -8, and -9 in HL60 cells. This suggested that compounds 7, 18, and 28 induced apoptotic cell death in HL60 cells via both the mitochondrial- and the death receptor-mediated pathways. Futhermore, compound 7 was shown to possess high selective cytotoxicity for leukemia cells since it exhibited only weak cytotoxicity against a normal lymphocyte cell line (RPMI 1788). PMID:21381696

  13. Presynaptic adenosine A2A receptors dampen cannabinoid CB1 receptor-mediated inhibition of corticostriatal glutamatergic transmission

    PubMed Central

    Ferreira, S G; Gonçalves, F Q; Marques, J M; Tomé, Â R; Rodrigues, R J; Nunes-Correia, I; Ledent, C; Harkany, T; Venance, L; Cunha, R A; Köfalvi, A

    2015-01-01

    Background and Purpose Both cannabinoid CB1 and adenosine A2A receptors (CB1 receptors and A2A receptors) control synaptic transmission at corticostriatal synapses, with great therapeutic importance for neurological and psychiatric disorders. A postsynaptic CB1−A2A receptor interaction has already been elucidated, but the presynaptic A2A receptor-mediated control of presynaptic neuromodulation by CB1 receptors remains to be defined. Because the corticostriatal terminals provide the major input to the basal ganglia, understanding the interactive nature of converging neuromodulation on them will provide us with novel powerful tools to understand the physiology of corticostriatal synaptic transmission and interpret changes associated with pathological conditions. Experimental Approach Pharmacological manipulation of CB1 and A2A receptors was carried out in brain nerve terminals isolated from rats and mice, using flow synaptometry, immunoprecipitation, radioligand binding, ATP and glutamate release measurement. Whole-cell patch-clamp recordings were made in horizontal corticostriatal slices. Key Results Flow synaptometry showed that A2A receptors were extensively co-localized with CB1 receptor-immunopositive corticostriatal terminals and A2A receptors co-immunoprecipitated CB1 receptors in these purified terminals. A2A receptor activation decreased CB1 receptor radioligand binding and decreased the CB1 receptor-mediated inhibition of high-K+-evoked glutamate release in corticostriatal terminals. Accordingly, A2A receptor activation prevented CB1 receptor-mediated paired-pulse facilitation and attenuated the CB1 receptor-mediated inhibition of synaptic transmission in glutamatergic synapses of corticostriatal slices. Conclusions and Implications Activation of presynaptic A2A receptors dampened CB1 receptor-mediated inhibition of corticostriatal terminals. This constitutes a thus far unrecognized mechanism to modulate the potent CB1 receptor-mediated presynaptic

  14. 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

  15. EP4 prostanoid receptor-mediated vasodilatation of human middle cerebral arteries

    PubMed Central

    Davis, Richard J; Murdoch, Colin E; Ali, Mozam; Purbrick, Stuart; Ravid, Rivka; Baxter, Gordon S; Tilford, Nick; Sheldrick, Robert L G; Clark, Kenneth L; Coleman, Robert A

    2004-01-01

    Dilatation of the cerebral vasculature is recognised to be involved in the pathophysiology of migraine. Furthermore, elevated levels of prostaglandin E2 (PGE2) occur in the blood, plasma and saliva of migraineurs during an attack, suggestive of a contributory role. In the present study, we have characterised the prostanoid receptors involved in the relaxation and contraction of human middle cerebral arteries in vitro. In the presence of indomethacin (3 μM) and the TP receptor antagonist GR32191 (1 μM), PGE2 was found to relax phenylephrine precontracted cerebral arterial rings in a concentration-dependent manner (mean pEC50 8.0±0.1, n=5). Establishment of a rank order of potency using the EP4>EP2 agonist 11-deoxy PGE1, and the EP2>EP4 agonist PGE1-OH (mean pEC50 of 7.6±0.1 (n=6) and 6.4±0.1 (n=4), respectively), suggested the presence of functional EP4 receptors. Furthermore, the selective EP2 receptor agonist butaprost at concentrations <1 μM failed to relax the tissues. Blockade of EP4 receptors with the EP4 receptor antagonists AH23848 and EP4A caused significant rightward displacements in PGE2 concentration–response curves, exhibiting pA2 and pKB values of 5.7±0.1, n=3, and 8.4, n=3, respectively. The IP receptor agonists iloprost and cicaprost relaxed phenylephrine precontracted cerebral arterial rings (mean pEC50 values 8.3±0.1 (n=4) and 8.1±0.1 (n=9), respectively). In contrast, the DP and FP receptor agonists PGD2 and PGF2α failed to cause appreciable relaxation or contraction at concentrations of up to 30 μM. In the absence of phenylephrine contraction and GR32191, the TP receptor agonist U46619 caused concentration-dependent contraction of cerebral artery (mean pEC50 7.4±0.3, n=3). These data demonstrate the presence of prostanoid EP4 receptors mediating PGE2 vasodilatation of human middle cerebral artery. IP receptors mediating relaxation and TP receptors mediating contraction were also functionally demonstrated. PMID:14744815

  16. AMPA receptor desensitization is the determinant of AMPA receptor mediated excitotoxicity in purified retinal ganglion cells.

    PubMed

    Park, Yong H; Mueller, Brett H; McGrady, Nolan R; Ma, Hai-Ying; Yorio, Thomas

    2015-03-01

    The ionotropic glutamate receptors (iGLuR) have been hypothesized to play a role in neuronal pathogenesis by mediating excitotoxic death. Previous studies on iGluR in the retina have focused on two broad classes of receptors: NMDA and non-NMDA receptors including the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) and kainate receptor. In this study, we examined the role of receptor desensitization on the specific excitotoxic effects of AMPAR activation on primary retinal ganglion cells (RGCs). Purified rat RGCs were isolated from postnatal day 4-7 Sprague-Dawley rats. Calcium imaging was used to identify the functionality of the AMPARs and selectivity of the s-AMPA agonist. Phosphorylated CREB and ERK1/2 expression were performed following s-AMPA treatment. s-AMPA excitotoxicity was determined by JC-1 mitochondrial membrane depolarization assay, caspase 3/7 luciferase activity assay, immunoblot analysis for α-fodrin, and Live (calcein AM)/Dead (ethidium homodimer-1) assay. RGC cultures of 98% purity, lacking Iba1 and GFAP expression were used for the present studies. Isolated prenatal RGCs expressed calcium permeable AMPAR and s-AMPA (100 μM) treatment of cultured RGCs significantly increased phosphorylation of CREB but not that of ERK1/2. A prolonged (6 h) AMPAR activation in purified RGCs using s-AMPA (100 μM) did not depolarize the RGC mitochondrial membrane potential. In addition, treatment of cultured RGCs with s-AMPA, both in the presence and absence of trophic factors (BDNF and CNTF), did not increase caspase 3/7 activities or the cleavage of α-fodrin (neuronal apoptosis marker), as compared to untreated controls. Lastly, a significant increase in cell survival of RGCs was observed after s-AMPA treatment as compared to control untreated RGCs. However, preventing the desensitization of AMPAR with the treatment with either kainic acid (100 μM) or the combination of s-AMPA and cyclothiazide (50 μM) significantly reduced cell

  17. Zyflamend Sensitizes Tumor Cells to TRAIL-Induced Apoptosis Through Up-Regulation of Death Receptors and Down-Regulation of Survival Proteins: Role of ROS-Dependent CCAAT/Enhancer-Binding Protein-Homologous Protein Pathway

    PubMed Central

    Kim, Ji Hye; Park, Byoungduck; Gupta, Subash C.; Kannappan, Ramaswamy; Sung, Bokyung

    2012-01-01

    Abstract Aim: TNF (tumor necrosis factor)-related apoptosis-inducing ligand (TRAIL), is a selective killer of tumor cells, although its potential is limited by the development of resistance. In this article, we investigated whether the polyherbal preparation Zyflamend® can sensitize tumor cells to TRAIL. Results: We found that Zyflamend potentiated TRAIL-induced apoptosis in human cancer cells. Zyflamend manifested its effects through several mechanisms. First, it down-regulated the expression of cell survival proteins known to be linked to resistance to TRAIL. Second, Zyflamend up-regulated the expression of pro-apoptotic protein, Bax. Third, Zyflamend up-regulated the expression of death receptors (DRs) for TRAIL. Up-regulation of DRs was critical as gene-silencing of these receptors significantly reduced the effect of Zyflamend on TRAIL-induced apoptosis. The up-regulation of DRs was dependent on CCAAT/enhancer-binding protein-homologous protein (CHOP), as Zyflamend induced CHOP, its gene-silencing abolished the induction of receptors, and mutation of the CHOP binding site on DR5 promoter abolished Zyflamend-mediated DR5 transactivation. Zyflamend mediated its effects through reactive oxygen species (ROS), as ROS quenching reduced its effect. Further, Zyflamend induced DR5 and CHOP and down-regulated the expression of cell survival proteins in nude mice bearing human pancreatic cancer cells. Innovation: Zyflamend can sensitize tumor cells to TRAIL through modulation of multiple cell signaling mechanisms that are linked to ROS. Conclusion: Zyflamend potentiates TRAIL-induced apoptosis through the ROS-CHOP-mediated up-regulation of DRs, increase in pro-apoptotic protein and down-regulation of cell survival proteins. Antioxid. Redox Signal. 16, 413–427. PMID:22004570

  18. 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

  19. Receptor-mediated entry of diphtheria toxin into monkey kidney (Vero) cells: electron microscopic evaluation.

    PubMed Central

    Morris, R E; Gerstein, A S; Bonventre, P F; Saelinger, C B

    1985-01-01

    To express toxicity in living cells, diphtheria toxin (DT) must cross a membrane barrier and reach its target in the cytosol. Here we examine the entry of DT into the toxin-sensitive monkey kidney (Vero) cells. Using electron microscopy we directly demonstrated for the first time that DT is internalized by receptor-mediated endocytosis, i.e., via clathrin-coated pits, and enters the endosomal system. Methylamine, which is known to protect cells from DT, stopped the movement of toxin to coated areas of the cell membrane. In the presence of amine, prebound biotinyl-DT was internalized, but toxicity was inhibited. Biochemical evidence revealed that methylamine maintained toxin molecules at a site accessible to neutralization by antitoxin. The data suggest that DT entering Vero cells in the presence of methylamine is sequestered within the cell and does not express toxicity. Images PMID:4066029

  20. Administration of pyrene lipids by receptor-mediated endocytosis and their degradation in skin fibroblasts

    SciTech Connect

    Agmon, V.; Dinur, T.; Cherbu, S.; Dagan, A.; Gatt, S. )

    1991-10-01

    Sphingomyelin and seven glycosphingolipids were labeled with the fluorescent probe pyrene and administered into cultured fibroblasts by receptor-mediated endocytosis. For this purpose pyrene sphingomyelin or mixtures of pyrene glycolipid and unlabeled sphingomyelin were dispersed as small, unilamellar liposomes. Apolipoprotein E was then added and the receptor for this ligand on the cell surface was utilized for uptake of the liposomes and their transport to the lysosomes, where the respective pyrene lipids were degraded. Following incubation with each of the respective pyrene lipids, only the administered compound and the pyrene ceramide were present; intermediate hydrolysis products were not detected. This indicated that, in skin fibroblasts, the lysosomal ceramidase was limiting and controlled the rate of total degradation of the pyrene sphingolipids.

  1. Transferrin protein nanospheres: a nanoplatform for receptor-mediated cancer cell labeling and gene delivery

    NASA Astrophysics Data System (ADS)

    McDonald, Michael A.; Spurlin, Tighe A.; Tona, Alessandro; Elliott, John T.; Halter, Michael; Plant, Anne L.

    2010-02-01

    This paper presents preliminary results on the use of transferrin protein nanospheres (TfpNS) for targeting cancer cells in vitro. Protein nanospheres represent an easily prepared and modifiable nanoplatform for receptor-specific targeting, molecular imaging and gene delivery. Rhodamine B isothiocyanate conjugated TfpNS (RBITC-TfpNS) show significantly enhanced uptake in vitro in SK-MEL-28 human malignant melanoma cells known to overexpress transferrin receptors compared to controls. RBITCTfpNS labeling of the cancer cells is due to transferrin receptor-mediated uptake, as demonstrated by competitive inhibition with native transferrin. Initial fluorescence microscopy studies indicate GFP plasmid can be transfected into melanoma cells via GFP plasmid encapsulated by TfpNS.

  2. Targeting receptor-mediated transport for delivery of biologics across the blood-brain barrier.

    PubMed

    Lajoie, Jason M; Shusta, Eric V

    2015-01-01

    Biologics are an emerging class of medicines with substantial promise to treat neurological disorders such as Alzheimer's disease, stroke, and multiple sclerosis. However, the blood-brain barrier (BBB) presents a formidable obstacle that appreciably limits brain uptake and hence the therapeutic potential of biologics following intravenous administration. One promising strategy for overcoming the BBB to deliver biologics is the targeting of endogenous receptor-mediated transport (RMT) systems that employ vesicular trafficking to transport ligands across the BBB endothelium. If a biologic is modified with an appropriate targeting ligand, it can gain improved access to the brain via RMT. Various RMT-targeting strategies have been developed over the past 20 years, and this review explores exciting recent advances, emphasizing studies that show brain targeting in vivo. PMID:25340933

  3. Receptor-mediated uptake of low density lipoprotein stimulates bile acid synthesis by cultured rat hepatocytes

    SciTech Connect

    Junker, L.H.; Davis, R.A. )

    1989-12-01

    The cellular mechanisms responsible for the lipoprotein-mediated stimulation of bile acid synthesis in cultured rat hepatocytes were investigated. Adding 280 micrograms/ml of cholesterol in the form of human or rat low density lipoprotein (LDL) to the culture medium increased bile acid synthesis by 1.8- and 1.6-fold, respectively. As a result of the uptake of LDL, the synthesis of (14C)cholesterol from (2-14C)acetate was decreased and cellular cholesteryl ester mass was increased. Further studies demonstrated that rat apoE-free LDL and apoE-rich high density lipoprotein (HDL) both stimulated bile acid synthesis 1.5-fold, as well as inhibited the formation of (14C)cholesterol from (2-14C)acetate. Reductive methylation of LDL blocked the inhibition of cholesterol synthesis, as well as the stimulation of bile acid synthesis, suggesting that these processes require receptor-mediated uptake. To identify the receptors responsible, competitive binding studies using 125I-labeled apoE-free LDL and 125I-labeled apoE-rich HDL were performed. Both apoE-free LDL and apoE-rich HDL displayed an equal ability to compete for binding of the other, suggesting that a receptor or a group of receptors that recognizes both apolipoproteins is involved. Additional studies show that hepatocytes from cholestyramine-treated rats displayed 2.2- and 3.4-fold increases in the binding of apoE-free LDL and apoE-rich HDL, respectively. These data show for the first time that receptor-mediated uptake of LDL by the liver is intimately linked to processes activating bile acid synthesis.

  4. Potentiation of NMDA receptor-mediated transmission in striatal cholinergic interneurons.

    PubMed

    Oswald, Manfred J; Schulz, Jan M; Kelsch, Wolfgang; Oorschot, Dorothy E; Reynolds, John N J

    2015-01-01

    Pauses in the tonic firing of striatal cholinergic interneurons (CINs) emerge during reward-related learning in response to conditioning of a neutral cue. We have previously reported that augmenting the postsynaptic response to cortical afferents in CINs is coupled to the emergence of a cell-intrinsic afterhyperpolarization (AHP) underlying pauses in tonic activity. Here we investigated in a bihemispheric rat-brain slice preparation the mechanisms of synaptic plasticity of excitatory afferents to CINs and the association with changes in the AHP. We found that high frequency stimulation (HFS) of commissural corticostriatal afferents from the contralateral hemisphere induced a robust long-term depression (LTD) of postsynaptic potentials (PSP) in CINs. Depression of the PSP of smaller magnitude and duration was observed in response to HFS of the ipsilateral white matter or cerebral cortex. In Mg(2+)-free solution HFS induced NMDA receptor-dependent potentiation of the PSP, evident in both the maximal slope and amplitude of the PSP. The increase in maximal slope corroborates previous findings, and was blocked by antagonism of either D1-like dopamine receptors with SCH23390 or D2-like dopamine receptors with sulpiride during HFS in Mg(2+)-free solution. Potentiation of the slower PSP amplitude component was due to augmentation of the NMDA receptor-mediated potential as this was completely reversed on subsequent application of the NMDA receptor antagonist AP5. HFS similarly potentiated NMDA receptor currents isolated by blockade of AMPA/kainate receptors with CNQX. The plasticity-induced increase in the slow PSP component was directly associated with an increase in the subsequent AHP. Thus plasticity of cortical afferent synapses is ideally suited to influence the cue-induced firing dynamics of CINs, particularly through potentiation of NMDA receptor-mediated synaptic transmission. PMID:25914618

  5. Withanolide E sensitizes renal carcinoma cells to TRAIL-induced apoptosis by increasing cFLIP degradation

    PubMed Central

    Henrich, C J; Brooks, A D; Erickson, K L; Thomas, C L; Bokesch, H R; Tewary, P; Thompson, C R; Pompei, R J; Gustafson, K R; McMahon, J B; Sayers, T J

    2015-01-01

    Withanolide E, a steroidal lactone from Physalis peruviana, was found to be highly active for sensitizing renal carcinoma cells and a number of other human cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Withanolide E, the most potent and least toxic of five TRAIL-sensitizing withanolides identified, enhanced death receptor-mediated apoptotic signaling by a rapid decline in the levels of cFLIP proteins. Other mechanisms by which TRAIL sensitizers have been reported to work: generation of reactive oxygen species (ROS), changes in pro-and antiapoptotic protein expression, death receptor upregulation, activation of intrinsic (mitochondrial) apoptotic pathways, ER stress, and proteasomal inhibition proved to be irrelevant to withanolide E activity. Loss of cFLIP proteins was not due to changes in expression, but rather destabilization and/or aggregation, suggesting impairment of chaperone proteins leading to degradation. Indeed, withanolide E treatment altered the stability of a number of HSP90 client proteins, but with greater apparent specificity than the well-known HSP90 inhibitor geldanamycin. As cFLIP has been reported to be an HSP90 client, this provides a potentially novel mechanism for sensitizing cells to TRAIL. Sensitization of human renal carcinoma cells to TRAIL-induced apoptosis by withanolide E and its lack of toxicity were confirmed in animal studies. Owing to its novel activity, withanolide E is a promising reagent for the analysis of mechanisms of TRAIL resistance, for understanding HSP90 function, and for further therapeutic development. In marked contrast to bortezomib, among the best currently available TRAIL sensitizers, withanolide E's more specific mechanism of action suggests minimal toxic side effects. PMID:25719250

  6. Withanolide E sensitizes renal carcinoma cells to TRAIL-induced apoptosis by increasing cFLIP degradation.

    PubMed

    Henrich, C J; Brooks, A D; Erickson, K L; Thomas, C L; Bokesch, H R; Tewary, P; Thompson, C R; Pompei, R J; Gustafson, K R; McMahon, J B; Sayers, T J

    2015-01-01

    Withanolide E, a steroidal lactone from Physalis peruviana, was found to be highly active for sensitizing renal carcinoma cells and a number of other human cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Withanolide E, the most potent and least toxic of five TRAIL-sensitizing withanolides identified, enhanced death receptor-mediated apoptotic signaling by a rapid decline in the levels of cFLIP proteins. Other mechanisms by which TRAIL sensitizers have been reported to work: generation of reactive oxygen species (ROS), changes in pro-and antiapoptotic protein expression, death receptor upregulation, activation of intrinsic (mitochondrial) apoptotic pathways, ER stress, and proteasomal inhibition proved to be irrelevant to withanolide E activity. Loss of cFLIP proteins was not due to changes in expression, but rather destabilization and/or aggregation, suggesting impairment of chaperone proteins leading to degradation. Indeed, withanolide E treatment altered the stability of a number of HSP90 client proteins, but with greater apparent specificity than the well-known HSP90 inhibitor geldanamycin. As cFLIP has been reported to be an HSP90 client, this provides a potentially novel mechanism for sensitizing cells to TRAIL. Sensitization of human renal carcinoma cells to TRAIL-induced apoptosis by withanolide E and its lack of toxicity were confirmed in animal studies. Owing to its novel activity, withanolide E is a promising reagent for the analysis of mechanisms of TRAIL resistance, for understanding HSP90 function, and for further therapeutic development. In marked contrast to bortezomib, among the best currently available TRAIL sensitizers, withanolide E's more specific mechanism of action suggests minimal toxic side effects. PMID:25719250

  7. 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

  8. The inhibition of apoptosis by melatonin in VSC4.1 motoneurons exposed to oxidative stress, glutamate excitotoxicity, or TNF-α toxicity involves membrane melatonin receptors

    PubMed Central

    Das, Arabinda; McDowell, Misty; Pava, Matthew J; Smith, Joshua A.; Reiter, Russel J.; Woodward, John J.; Varma, Abhay K.; Ray, Swapan K.; Banik, Naren L.

    2009-01-01

    Loss of motoneurons may underlie some of the deficits in motor function associated with CNS injuries and diseases. We tested whether melatonin, a potent antioxidant and free radical scavenger, would prevent motoneuron apoptosis following exposure to toxins and whether this neuroprotection is mediated by melatonin receptors. Exposure of VSC4.1 motoneurons to either 50 μM H2O2, 25 μM glutamate (LGA), or 50 ng/ml tumor necrosis factor-alpha (TNF-α) for 24 h caused significant increases in apoptosis, as determined by Wright staining and ApopTag assay. Analyses of mRNA and proteins showed increased expression and activities of stress kinases and cysteine proteases and loss of mitochondrial membrane potential during apoptosis. These insults also caused increases in intracellular free [Ca2+] and activities of calpain and caspases. Cells exposed to stress stimuli for 15 min were then treated with 200 nM melatonin. Post-treatment of cells with melatonin attenuated production of reactive oxygen species (ROS) and phosphorylation of p38, MAPK, and JNK1, prevented cell death, and maintained whole-cell membrane potential, indicating functional neuroprotection. Melatonin receptors (MT1 and MT2) were upregulated following treatment with melatonin. To confirm the involvement of MT1 and MT2 in providing neuroprotection, cells were post-treated (20 min) with 10 μM luzindole (melatonin receptor antagonist). Luzindole significantly attenuated melatonin-induced neuroprotection, suggesting that melatonin worked, at least in part, via its receptors to prevent VSC4.1 motoneuron apoptosis. Results suggest that neuroprotection rendered by melatonin to motoneurons is receptor mediated and melatonin may be an effective neuroprotective agent to attenuate motoneuron death in CNS injuries and diseases. PMID:20082663

  9. A Small Molecule Inhibitor Selectively Induces Apoptosis in Cells Transformed by High Risk Human Papilloma Viruses.

    PubMed

    Sheaffer, Amy K; 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

  10. 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

  11. Sustained adenosine exposure causes lung endothelial apoptosis: a possible contributor to cigarette smoke-induced endothelial apoptosis and lung injury

    PubMed Central

    Sakhatskyy, Pavlo; Newton, Julie; Shamirian, Paul; Hsiao, Vivian; Curren, Sean; Gabino Miranda, Gustavo Andres; Pedroza, Mesias; Blackburn, Michael R.; Rounds, Sharon

    2013-01-01

    Pulmonary endothelial cell (EC) apoptosis has been implicated in the pathogenesis of emphysema. Cigarette smoke (CS) causes lung EC apoptosis and emphysema. In this study, we show that CS exposure increased lung tissue adenosine levels in mice, an effect associated with increased lung EC apoptosis and the development of emphysema. Adenosine has a protective effect against apoptosis via adenosine receptor-mediated signaling. However, sustained elevated adenosine increases alveolar cell apoptosis in adenosine deaminase-deficient mice. We established an in vitro model of sustained adenosine exposure by incubating lung EC with adenosine in the presence of an adenosine deaminase inhibitor, deoxycoformicin. We demonstrated that sustained adenosine exposure caused lung EC apoptosis via nucleoside transporter-facilitated intracellular adenosine uptake, subsequent activation of p38 and JNK in mitochondria, and ultimately mitochondrial defects and activation of the mitochondria-mediated intrinsic pathway of apoptosis. Our results suggest that sustained elevated adenosine may contribute to CS-induced lung EC apoptosis and emphysema. Our data also reconcile the paradoxical effects of adenosine on apoptosis, demonstrating that prolonged exposure causes apoptosis via nucleoside transporter-mediated intracellular adenosine signaling, whereas acute exposure protects against apoptosis via activation of adenosine receptors. Inhibition of adenosine uptake may become a new therapeutic target in treatment of CS-induced lung diseases. PMID:23316066

  12. Apoptosis in Anthracycline Cardiomyopathy

    PubMed Central

    Shi, Jianjian; Abdelwahid, Eltyeb; Wei, Lei

    2011-01-01

    Apoptosis is a tightly regulated physiologic process of programmed cell death that occurs in both normal and pathologic tissues. Numerous in vitro or in vivo studies have indicated that cardiomyocyte death through apoptosis and necrosis is a primary contributor to the progression of anthracycline-induced cardiomyopathy. There are now several pieces of evidence to suggest that activation of intrinsic and extrinsic apoptotic pathways contribute to anthracycline-induced apoptosis in the heart. Novel strategies were developed to address a wide variety of cardiotoxic mechanisms and apoptotic pathways by which anthracycline influences cardiac structure and function. Anthracycline-induced apoptosis provides a very valid representation of cardiotoxicity in the heart, an argument which has implications for the most appropriate animal models of damaged heart plus diverse pharmacological effects. In this review we describe various aspects of the current understanding of apoptotic cell death triggered by anthracycline. Differences in the sensitivity to anthracycline-induced apoptosis between young and adult hearts are also discussed. PMID:22212952

  13. The combination of irreversible EGFR TKIs and SAHA induces apoptosis and autophagy-mediated cell death to overcome acquired resistance in EGFR T790M-mutated lung cancer.

    PubMed

    Lee, Tae-Gul; Jeong, Eun-Hui; Kim, Seo Yun; Kim, Hye-Ryoun; Kim, Cheol Hyeon

    2015-06-01

    To overcome T790M-mediated acquired resistance of lung cancer cells to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs), second generation TKIs such as BIBW2992 (afatinib) and third generation TKIs including WZ4002 have been developed. However, clinical data on their efficacy in treating T790M mutant tumors are lacking. Histone deacetylase (HDAC) inhibitors have been reported to arrest cell growth and to lead to differentiation and apoptosis of various cancer cells, both in vitro and in vivo. In the present study, we assessed whether the combination of suberoylanilide hydroxamic acid (SAHA, vorinostat), a potent HDAC inhibitor, and BIBW2992 or WZ4002 could overcome EGFR TKI resistance associated with T790M mutation in lung cancer cells. While treatment with BIBW2992 or WZ4002 alone slightly reduced the viability of PC-9G and H1975 cells, which possess T790M mutation, combining them with SAHA resulted in significantly decreased cell viability through the activation of the apoptotic pathway. This combination also enhanced autophagy occurrence and inhibition of autophagy significantly reduced the apoptosis induced by the combination treatment, showing that autophagy is required for the enhanced apoptosis. Caspase-independent autophagic cell death was also induced by the combination treatment with SAHA and either BIBW2992 or WZ4002. Finally, the combined treatment with SAHA and either BIBW2992 or WZ4002 showed an enhanced anti-tumor effect on xenografts of H1975 cells in vivo. In conclusion, the combination of new generation EGFR TKIs and SAHA may be a new strategy to overcome the acquired resistance to EGFR TKIs in T790M mutant lung cancer. PMID:25382705

  14. The human programmed cell death-2 (PDCD2) gene is a target of BCL6 repression: Implications for a role of BCL6 in the down-regulation of apoptosis

    PubMed Central

    Baron, Beverly W.; Anastasi, John; Thirman, Michael J.; Furukawa, Yoichi; Fears, Scott; Kim, David C.; Simone, Federico; Birkenbach, Mark; Montag, Anthony; Sadhu, Annamma; Zeleznik-Le, Nancy; McKeithan, Timothy W.

    2002-01-01

    BCL6, a gene on chromosome 3 band q27, encodes a Kruppel-type zinc finger transcriptional repressor. Rearrangements of this gene are frequent in various kinds of lymphomas, particularly of the large-cell B-cell type. The BCL6 nuclear phosphoprotein is expressed in a variety of tissues and is up-regulated particularly in lymph node germinal centers. The zinc fingers of BCL6 bind DNA in a sequence-specific manner. To identify targets of the BCL6 repressive effects, we used a VP16-BCL6 fusion protein containing the zinc fingers but devoid of the repressor domains to compete with the binding of endogenous BCL6 in a transiently transfected B-cell line and then performed subtractive hybridization by using a method to selectively amplify sequences that are differentially expressed. We found that the programmed cell death-2 (PDCD2) gene is a target of BCL6 repression. This gene is the human homolog of Rp8, a rat gene associated with programmed cell death in thymocytes. Immunohistochemistry reveals the anticipated inverse relationship between BCL6 and PDCD2 expression in human tonsil. PDCD2 is detectable in cells of the germinal center in areas where there is less BCL6 expression as well as in the mantle zone, where there is little or no BCL6 expression. These results raise the possibility that BCL6 may regulate apoptosis by means of its repressive effects on PDCD2. BCL6 deregulation may lead to persistent down-regulation of PDCD2, reduced apoptosis, and, as a consequence, accumulation of BCL6-containing lymphoma cells. PMID:11854457

  15. Induction of indoleamine 2,3-dioxygenase (IDO) enzymatic activity contributes to interferon-gamma induced apoptosis and death receptor 5 expression in human non-small cell lung cancer cells.

    PubMed

    Chung, Ting Wen; Tan, Kok-Tong; Chan, Hong-Lin; Lai, Ming-Derg; Yen, Meng-Chi; Li, Yi-Ron; Lin, Sheng Hao; Lin, Chi-Chen

    2014-01-01

    Interferon-gamma (IFN-γ) has been used to treat various malignant tumors. However, the molecular mechanisms underlying the direct anti-proliferative activity of IFN-γ are poorly understood. In the present study, we examined the in vitro antitumor activity of IFN-γ on two human non-small-cell lung carcinoma (NSCLC) cell lines, H322M and H226. Our findings indicated that IFN-γ treatment caused a time-dependent reduction in cell viability and induced apoptosis through a FADD-mediated caspase-8/tBid/mitochondria-dependent pathway in both cell lines. Notably, we also postulated that IFN-γ increased indoleamine 2,3-dioxygenase (IDO) expression and enzymatic activity in H322M and H226 cells. In addition, inhibition of IDO activity by the IDO inhibitor 1-MT or tryptophan significantly reduced IFN-γ-induced apoptosis and death receptor 5 (DR5) expression, which suggests that IDO enzymatic activity plays an important role in the anti-NSCLC cancer effect of IFN-γ. These results provide new mechanistic insights into interferon-γ antitumor activity and further support IFN-γ as a potential therapeutic adjuvant for the treatment of NCSLC. PMID:25292102

  16. Quinovic acid glycosides purified fraction from Uncaria tomentosa induces cell death by apoptosis in the T24 human bladder cancer cell line.

    PubMed

    Dietrich, Fabrícia; Kaiser, Samuel; Rockenbach, Liliana; Figueiró, Fabrício; Bergamin, Letícia Scussel; da Cunha, Fernanda Monte; Morrone, Fernanda Bueno; Ortega, George González; Battastini, Ana Maria Oliveira

    2014-05-01

    Bladder cancer is the second most prevalent malignancy in the genitourinary tract and remains a therapeutic challenge. In the search for new treatments, researchers have attempted to find compounds with low toxicity. With this goal in mind, Uncaria tomentosa is noteworthy because the bark and root of this species are widely used in traditional medicine and in adjuvant therapy for the treatment of numerous diseases. The objective of this study was to investigate the antitumor effect of one purified bioactive fraction of U.tomentosa bark on cell proliferation in two human bladder cancer cell lines, T24 and RT4. Quinovic acid glycosides purified fraction (QAPF) of U.tomentosa decreased the growth and viability of both T24 and RT4 cell lines. In T24 cells, QAPF induced apoptosis by activating caspase-3 and NF-κB. Further study showed that this fraction does not induce cell cycle arrest and does not alter PTEN and ERK levels. In conclusion, we demonstrated that QAPF of U.tomentosa has a potent inhibitory effect on the growth of human bladder cancer cell lines by inducing apoptosis through modulation of NF-κB, and we suggest that QAPF may become a potential therapeutic agent for the prevention and/or treatment of this cancer. PMID:24607820

  17. 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

  18. Apoptosis in colorectal cancer.

    PubMed

    Stoian, M; State, N; Stoica, V; Radulian, G

    2014-06-15

    Apoptosis is an inborn process that has been preserved during evolution; it allows the cells to systematically inactivate, destroy and dispose of their own components thus leading to their death. This programme can be activated by both intra and extracellular mechanisms. The intracellular components involve a genetically defined development programme while the extracellular aspects regard endogenous proteins, cytokines and hormones as well as xenobiotics, radiations, oxidative stress and hypoxia. The ability of a cell to enter apoptosis as a response to a "death" signal depends on its proliferative status, the position in the cell cycle and also on the controlled expression of those genes that have the capacity of promoting and inhibiting cell death. The fine regulation of these parameters needs to be maintained in order to ensure the physiological environment required for the induction of apoptosis. Any malfunction in any of the steps of controlled cellular death can lead to dysfunctions and, as a consequence, to different pathological conditions. The importance of apoptosis lies in its active nature and in the potential of controlling biological systems. PMID:25408720

  19. [Apoptosis and its biomedical significance].

    PubMed

    Ortega-Camarillo, C; Díaz-Flores, M; Avalos-Rodríguez, A; Vergara-Onofre, M; Rosales-Torres, A M

    2001-01-01

    Cell death can occur through apoptotic or necrotic death pathways. Membrane disruption leads to inflammation, a typical feature of necrosis. Apoptosis constitutes a genetically controlled physiologic process of cell removal. It is characterized by cell shrinkage, chromatin condensation, and DNA cleavage. Apoptotic cells are rapidly recognized and engulfed by phagocytes thus inhibiting an inflammatory response following necrosis. Apoptosis has been proposed as a basic event to protect tissue homeostasis. This paper analyzes the genetic, biochemical, and morphologic characteristics related to apoptosis, as well as its relationship to certain illnesses. PMID:11766462

  20. PTEN Overexpression Cooperates With Lithium to Reduce the Malignancy and to Increase Cell Death by Apoptosis via PI3K/Akt Suppression in Colorectal Cancer Cells.

    PubMed

    de Araujo, Wallace Martins; Robbs, Bruno Kaufmann; Bastos, Lilian G; de Souza, Waldemir F; Vidal, Flávia C B; Viola, João P B; Morgado-Diaz, Jose A

    2016-02-01

    Lithium is a well-established non-competitive inhibitor of glycogen synthase kinase-3β (GSK-3β), a kinase that is involved in several cellular processes related to cancer progression. GSK-3β is regulated upstream by PI3K/Akt, which is negatively modulated by PTEN. The role that lithium plays in cancer is controversial because lithium can activate or inhibit survival signaling pathways depending on the cell type. In this study, we analyzed the mechanisms by which lithium can modulate events related to colorectal cancer (CRC) progression and evaluated the role that survival signaling pathways such as PI3K/Akt and PTEN play in this context. We show that the administration of lithium decreased the proliferative potential of CRC cells in a GSK-3β-independent manner but induced the accumulation of cells in G2/M phase. Furthermore, high doses of lithium increased apoptosis, which was accompanied by decreased proteins levels of Akt and PTEN. Then, cells that were induced to overexpress PTEN were treated with lithium; we observed that low doses of lithium strongly increased apoptosis. Additionally, PTEN overexpression reduced proliferation, but this effect was minor compared with that in cells treated with lithium alone. Furthermore, we demonstrated that PTEN overexpression and lithium treatment separately reduced cell migration, colony formation, and invasion, and these effects were enhanced when lithium treatment and PTEN overexpression were combined. In conclusion, our findings indicate that PTEN overexpression and lithium treatment cooperate to reduce the malignancy of CRC cells and highlight lithium and PTEN as potential candidates for studies to identify new therapeutic approaches for CRC treatment. PMID:26224641

  1. Toll Receptor-Mediated Hippo Signaling Controls Innate Immunity in Drosophila.

    PubMed

    Liu, Bo; Zheng, Yonggang; Yin, Feng; Yu, Jianzhong; Silverman, Neal; Pan, Duojia

    2016-01-28

    The Hippo signaling pathway functions through Yorkie to control tissue growth and homeostasis. How this pathway regulates non-developmental processes remains largely unexplored. Here, we report an essential role for Hippo signaling in innate immunity whereby Yorkie directly regulates the transcription of the Drosophila IκB homolog, Cactus, in Toll receptor-mediated antimicrobial response. Loss of Hippo pathway tumor suppressors or activation of Yorkie in fat bodies, the Drosophila immune organ, leads to elevated cactus mRNA levels, decreased expression of antimicrobial peptides, and vulnerability to infection by Gram-positive bacteria. Furthermore, Gram-positive bacteria acutely activate Hippo-Yorkie signaling in fat bodies via the Toll-Myd88-Pelle cascade through Pelle-mediated phosphorylation and degradation of the Cka subunit of the Hippo-inhibitory STRIPAK PP2A complex. Our studies elucidate a Toll-mediated Hippo signaling pathway in antimicrobial response, highlight the importance of regulating IκB/Cactus transcription in innate immunity, and identify Gram-positive bacteria as extracellular stimuli of Hippo signaling under physiological settings. PMID:26824654

  2. Receptor-Mediated Entry of Pristine Octahedral DNA Nanocages in Mammalian Cells.

    PubMed

    Vindigni, Giulia; Raniolo, Sofia; Ottaviani, Alessio; Falconi, Mattia; Franch, Oskar; Knudsen, Birgitta R; Desideri, Alessandro; Biocca, Silvia

    2016-06-28

    DNA offers excellent programming properties for the generation of nanometer-scaled polyhedral structures with a broad variety of potential applications. Translation to biomedical applications requires improving stability in biological fluids, efficient and selective cell binding, and/or internalization of the assembled DNA nanostructures. Here, we report an investigation on the selective mechanism of cellular uptake of pristine DNA nanocages in cells expressing the receptor "oxidized low-density lipoprotein receptor-1" (LOX-1), a scavenger receptor associated with cardiovascular diseases and, more recently, identified as a tumor marker. For this purpose a truncated octahedral DNA nanocage functionalized with a single biotin molecule, which allows DNA cage detection through the biotin-streptavidin assays, was constructed. The results indicate that DNA nanocages are stable in biological fluids, including human serum, and are selectively bound and very efficiently internalized in vesicles only in LOX-1-expressing cells. The amount of internalized cages is 30 times higher in LOX-1-expressing cells than in normal fibroblasts, indicating that the receptor-mediated uptake of pristine DNA nanocages can be pursued for a selective cellular internalization. These results open the route for a therapeutic use of pristine DNA cages targeting LOX-1-overexpressing tumor cells. PMID:27214742

  3. Delivery of liposomes into cultured KB cells via folate receptor-mediated endocytosis.

    PubMed

    Lee, R J; Low, P S

    1994-02-01

    Folic acid was covalently conjugated to 66-nm liposomes via spacers of various lengths in an attempt to target the liposomes to KB cells expressing folate receptors. Spacers of short and intermediate lengths were unable to mediate association of folate-conjugated liposomes with receptor-bearing cells, however, use of a 250 A polyethyleneglycol spacer (PEG, M(r) approximately 3350) permitted avid uptake of the liposomes at approximately 2.5 x 10(5) sites/cell. The binding of folate-PEG liposomes to KB cells could be competitively inhibited by excess free folate or by antiserum against the folate receptor, demonstrating the interaction is mediated by the cell surface folate-binding protein. Following binding, cell-associated folate-PEG liposomes were internalized by folate-receptor-mediated endocytosis at 37 degrees C but not at 4 degrees C. These folate-PEG liposomes show potential for delivering large quantities of low molecular weight compounds nondestructively into folate receptor-bearing cells. PMID:8106354

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. Receptor-mediated endocytosis of lysozyme in renal proximal tubules of the frog Rana temporaria.

    PubMed

    Seliverstova, E V; Prutskova, N P

    2015-01-01

    The mechanism of protein reabsorption in the kidney of lower vertebrates remains insufficiently investigated in spite of raising interest to the amphibian and fish kidneys as a useful model for physiological and pathophysiological examinations. In the present study, we examined the renal tubular uptake and the internalization rote of lysozyme after its intravenous injection in the wintering frog Rana temporaria using immunohisto- and immunocytochemistry and specific markers for some endocytic compartments. The distinct expression of megalin and cubilin in the proximal tubule cells of lysozyme-injected frogs was revealed whereas kidney tissue of control animals showed no positive immunoreactivity. Lysozyme was detected in the apical endocytic compartment of the tubular cells and colocalized with clathrin 10 min after injection. After 20 min, lysozyme was located in the subapical compartment negative to clathrin (endosomes), and intracellular trafficking of lysozyme was coincided with the distribution of megalin and cubilin. However, internalized protein was retained in the endosomes and did not reach lysosomes within 30 min after treatment that may indicate the inhibition of intracellular trafficking in hibernating frogs. For the first time, we provided the evidence that lysozyme is filtered through the glomeruli and absorbed by receptor-mediated clathrin-dependent endocytosis in the frog proximal tubule cells. Thus, the protein uptake in the amphibian mesonephros is mediated by megalin and cubilin that confirms a critical role of endocytic receptors in the renal reabsorption of proteins in amphibians as in mammals. PMID:26150156

  9. Co-receptors are dispensable for tethering receptor-mediated phagocytosis of apoptotic cells.

    PubMed

    Park, B; Lee, J; Moon, H; Lee, G; Lee, D-H; Cho, J Hoon; Park, D

    2015-01-01

    During efferocytosis, phagocytic cells recognize dying cells by receptors binding to ligands specifically exposed on apoptotic cells. Multiple phagocytic receptors and some of their signaling pathways have been identified. However, the downstream pathways of tethering receptors that secure apoptotic cells remain elusive. It is generally assumed that tethering receptors induce signaling to mediate engulfment via interacting with co-receptors or other engulfment receptors located nearby. However, it is poorly understood whether co-receptors for tethering receptors exist during efferocytosis, and, if they do, whether they are indispensable for this process. Here, we address this issue using glycophosphatidylinositol (GPI)-anchored annexin A5 (Anxa5-GPI), an artificial tethering receptor without a putative co-receptor. Phagocytes expressing Anxa5-GPI exhibited enhanced binding of apoptotic cells, resulting in promoted ingestion of apoptotic cells in a phosphatidylserine-dependent manner. Anxa5-GPI-induced phagocytosis of apoptotic cells relied on the known cytoskeletal engulfment machinery but partially depended on the Elmo-Dock-Rac module or the integrin pathway. In addition, Anxa5-GPI-mediated efferocytosis provoked anti-inflammatory responses. Taken together, our work suggests that co-receptors are dispensable for tethering receptor-induced efferocytosis and that tethering receptors mediate the engulfment of apoptotic cells through multiple engulfment signaling pathways. PMID:26018733

  10. Evidence for 5-HT7 receptors mediating relaxation of human colonic circular smooth muscle

    PubMed Central

    Prins, Nicolaas H; Briejer, Michel R; Van Bergen, Patrick J E; Akkermans, Louis M A; Schuurkes, Jan A J

    1999-01-01

    5-HT4 receptors mediate relaxation of human colon circular muscle. However, after 5-HT4 receptor blockade (SB 204070 10 nM), 5-HT still induced a relaxation (pEC50 6.3). 5-HT4 receptors were sufficiently blocked, as the curves to 5-HT obtained in the presence of 10 and 100 nM SB 204070 were indistinguishable. This 5-HT-induced relaxation was tetrodotoxin-insensitive, indicative of a smooth muscle relaxant 5-HT receptor. This, and the rank order of potency (5-CT=5-MeOT=5-HT) suggested involvement of 5-HT1 or 5-HT7 receptors. Mesulergine, a 5-HT7 receptor antagonist at nanomolar concentrations, and a 5-HT1 receptor antagonist at micromolar concentrations, competitively antagonized the 5-HT-induced relaxation (pKB 8.3) and antagonized the relaxation to 5-CT. Methysergide antagonized the 5-HT-induced relaxation (pA2 7.6). It is concluded that the profile of the smooth muscle inhibitory 5-HT receptor resembles that of the 5-HT7 receptor. These data provide the first evidence for functional human 5-HT7 receptors. PMID:10556917

  11. 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.

  12. Effects of chronic ethanol administration on receptor mediated endocytosis of asialoorosomucoid (ASOR) in isolated rat hepatocytes

    SciTech Connect

    Casey, C.A.; Kragskow, S.L.; Sorrell, M.F.; Tuma, D.J.

    1986-05-01

    The authors have previously shown that acute and chronic ethanol administration decreases hepatic glycoprotein secretion and membrane biogenesis. The present study was undertaken to determine the effects of chronic ethanol feeding on receptor-mediated endocytosis using the endocytosis of ASOR as a model system. Rats were fed either rat chow ad lib or pair-fed with Lieber-DeCarli diet (ethanol or isocaloric glucose as 36% of total calories) for 5 to 7 weeks. Binding of /sup 125/I ASOR to isolated hepatocytes was studied at 0-4/sup 0/C. Internalization (cell-associated acid precipitable radioactivity) and degradation (acid soluble radioactivity) were determined at 37/sup 0/C for periods up to 240 min. Results were expressed as pmoles ASOR bound, degraded or internalized/10/sup 6/ cells. In ethanol-fed rats the number of pmoles ASOR bound/10/sup 6/ cells was decreased by 40-50% (p< 0.01) as compared to pair-fed and chow-fed animals. Rates of degradation and internalization of the ligand were also 50-70% lower (p< 0.01) in chronic ethanol-treated animals. No significant differences were observed for either binding or internalization of ASOR between chow-fed and pair-fed animals. These results indicate that chronic ethanol feeding decreases internalization and degradation of ASOR in rat hepatocytes.

  13. Progesterone stimulates respiration through a central nervous system steroid receptor-mediated mechanism in cat.

    PubMed Central

    Bayliss, D A; Millhorn, D E; Gallman, E A; Cidlowski, J A

    1987-01-01

    We have examined the effect on respiration of the steroid hormone progesterone, administered either intravenously or directly into the medulla oblongata in anesthetized and paralyzed male and female cats. The carotid sinus and vagus nerves were cut, and end-tidal PCO2 and temperature were kept constant with servo-controllers. Phrenic nerve activity was used to quantitate central respiratory activity. Repeated doses of progesterone (from 0.1 to 2.0 micrograms/kg, cumulative) caused a sustained (greater than 45 min) facilitation of phrenic nerve activity in female and male cats; however, the response was much more variable in females. Progesterone injected into the region of nucleus tractus solitarii, a respiratory-related area in the medulla oblongata, also caused a prolonged stimulation of respiration. Progesterone administration at high concentration by both routes also caused a substantial hypotension. Identical i.v. doses of other classes of steroid hormones (17 beta-estradiol, testosterone, and cortisol) did not elicit the same respiratory effect. Pretreatment with RU 486, a progesterone-receptor antagonist, blocked the facilitatory effect of progesterone. We conclude that progesterone acts centrally through a steroid receptor-mediated mechanism to facilitate respiration. PMID:3478727

  14. Plasma clearance of rat bikunin: evidence for receptor-mediated uptake.

    PubMed Central

    Sjöberg, E M; Blom, A; Larsson, B S; Alston-Smith, J; Sjöquist, M; Fries, E

    1995-01-01

    Bikunin is a chondroitin sulphate-containing protease inhibitor with a molecular mass of 25 kDa. It is secreted into the blood by hepatocytes, and recent observations indicate that it may have an extravascular function. Here we have studied the plasma clearance of bikunin in rats and mice. On intravenous injection, radiolabelled bikunin was found to have a half-life of 10 min; in rats with ligated renal arteries, the clearance time was twice as long, implying that the kidneys account for half the uptake. As judged by gel filtration, the size of bikunin is similar to that of albumin. Autoradiographic analysis of kidneys removed 2 min after the injection of radiolabelled bikunin indicated that, despite its size, bikunin is cleared by glomerular filtration. On ligation of the renal arteries, the plasma concentration of bikunin increased linearly to at least four times normal. This finding shows that the non-renal uptake system is saturated and therefore presumably receptor-mediated. Most of the non-renal uptake of injected bikunin was found to occur in non-visceral tissues such as the skin. Analysis of skin samples by autoradiography after injection of radiolabelled bikunin suggested that bikunin had been transferred from the plasma to the interstitial space. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:8948446

  15. Co-receptors are dispensable for tethering receptor-mediated phagocytosis of apoptotic cells

    PubMed Central

    Park, B; Lee, J; Moon, H; Lee, G; Lee, D-H; Hoon Cho, J; Park, D

    2015-01-01

    During efferocytosis, phagocytic cells recognize dying cells by receptors binding to ligands specifically exposed on apoptotic cells. Multiple phagocytic receptors and some of their signaling pathways have been identified. However, the downstream pathways of tethering receptors that secure apoptotic cells remain elusive. It is generally assumed that tethering receptors induce signaling to mediate engulfment via interacting with co-receptors or other engulfment receptors located nearby. However, it is poorly understood whether co-receptors for tethering receptors exist during efferocytosis, and, if they do, whether they are indispensable for this process. Here, we address this issue using glycophosphatidylinositol (GPI)-anchored annexin A5 (Anxa5-GPI), an artificial tethering receptor without a putative co-receptor. Phagocytes expressing Anxa5-GPI exhibited enhanced binding of apoptotic cells, resulting in promoted ingestion of apoptotic cells in a phosphatidylserine-dependent manner. Anxa5-GPI-induced phagocytosis of apoptotic cells relied on the known cytoskeletal engulfment machinery but partially depended on the Elmo-Dock-Rac module or the integrin pathway. In addition, Anxa5-GPI-mediated efferocytosis provoked anti-inflammatory responses. Taken together, our work suggests that co-receptors are dispensable for tethering receptor-induced efferocytosis and that tethering receptors mediate the engulfment of apoptotic cells through multiple engulfment signaling pathways. PMID:26018733

  16. Tonic GABAA Receptor-Mediated Inhibition in the Rat Dorsal Motor Nucleus of the Vagus

    PubMed Central

    Gao, Hong

    2010-01-01

    Type A γ-aminobutyric acid (GABAA) receptors expressed in the dorsal motor nucleus of vagus (DMV) critically regulate the activity of vagal motor neurons and, by inference, the gastrointestinal (GI) tract. Two types of GABAA receptor-mediated inhibition have been identified in the brain, represented by phasic (Iphasic) and tonic (Itonic) inhibitory currents. The hypothesis that Itonic regulates neuron activity was tested in the DMV using whole cell patch-clamp recordings in transverse brain stem slices from rats. An Itonic was present in a subset of DMV neurons, which was determined to be mediated by different receptors than those mediating fast, synaptic currents. Preapplication of tetrodotoxin significantly decreased the resting Itonic amplitude in DMV neurons, suggesting that most of the current was due to action potential (AP)–dependent GABA release. Blocking GABA transport enhanced Itonic and multiple GABA transporters cooperated to regulate Itonic. The Itonic was composed of both a gabazine-insensitive component that was nearly saturated under basal conditions and a gabazine-sensitive component that was activated when extracellular GABA concentration was elevated. Perfusion of THIP (10 μM) significantly increased Itonic amplitude without increasing Iphasic amplitude. The Itonic played a major role in determining the overall excitability of DMV neurons by contributing to resting membrane potential and AP frequency. Our results indicate that Itonic contributes to DMV neuron membrane potential and activity and is thus an important regulator of vagally mediated GI function. PMID:20018836

  17. Peptides in Receptor-Mediated Radiotherapy: From Design to the Clinical Application in Cancers

    PubMed Central

    Lozza, Catherine; Navarro-Teulon, Isabelle; Pèlegrin, André; Pouget, Jean-Pierre; Vivès, Eric

    2013-01-01

    Short peptides can show high affinity for specific receptors overexpressed on tumor cells. Some of these are already used in cancerology as diagnostic tools and others are in clinical trials for therapeutic applications. Therefore, peptides exhibit great potential as a diagnostic tool but also as an alternative or an additional antitumoral approach upon the covalent attachment of a therapeutic moiety such as a radionuclide or a cytotoxic drug. The chemistry offers flexibility to graft onto the targeting-peptide either fluorine or iodine directly, or metallic radionuclides through appropriate chelating agent. Since short peptides are straightforward to synthesize, there is an opportunity to further improve existing peptides or to design new ones for clinical applications. However, several considerations have to be taken into account to optimize the recognition properties of the targeting-peptide to its receptor, to improve its stability in the biological fluids and its residence in the body, or to increase its overall therapeutic effect. In this review, we highlight the different aspects which need to be considered for the development of an efficient peptide receptor-mediated radionuclide therapy in different neoplasms. PMID:24093086

  18. Pharmacological characterization of the receptors mediating vasoactive intestinal peptide-induced vasodilation in rat aorta

    SciTech Connect

    Turner, J.T.; Bylund, D.B.

    1986-03-01

    Vasoactive intestinal peptide (VIP)-contain nerve fibers associated with blood vessels are widely distributed, both in the central nervous system and in the periphery. VIP has been shown to be a potent vasodilator in a variety of vascular preparations. The authors have evaluated VIP, the VIP fragment 10-28, and several related peptides including PHI-27, PHM-27 and secretin in terms of their potencies in (1) stimulating the synthesis of cyclic AMP, using the method of Shimizu, in aortic rings; and (2) reversing norepinephrine induced contraction in aortic rings. The authors results indicate that VIP is the most potent of the peptides in both experimental protocols and that the rank order potencies of the various peptides are consistent between the two parameters measured. The authors are currently conducting radioligand binding studies with (/sup 125/I)VIP to further characterize the receptors involved. Additionally, the authors experiments in rat aorta indicate that the presence of the endothelial layer is not required for VIP receptor mediated effects to occur. A potential role for synthetic compounds with high specificity for the VIP receptor in treating hypertension is suggested.

  19. Receptor-mediated adhesion phenomena. Model studies with the Radical-Flow Detachment Assay.

    PubMed Central

    Cozens-Roberts, C; Quinn, J A; Lauffenberger, D A

    1990-01-01

    Receptor-mediated cell adhesion phenomena play a vital role in many physiological and biotechnology-related processes. To investigate the physical and chemical factors that influence the cell/surface interaction, we have used a radial flow device, a so-called Radial-Flow Detachment Assay (RFDA). The RFDA allows us to make direct observations of the detachment process under specified experimental conditions. In results reported here, we have studied the detachment of receptor-coated latex beads (prototype cells) from ligand-coated glass surfaces. The receptors and ligands used in this work are complementary antibodies. The beads enable us to examine several aspects of the adhesion process with particles having uniform properties that can be varied systematically. Advantages of the RFDA are many, especially direct observation of cell detachment over a range of shear stresses with quantitative measurement of the adhesive force. We focus our studies on the effects of ligand and receptor densities, along with the influence of pH and ionic strength of the medium. These data are analyzed with a mathematical model based on the theoretical framework of Bell, G. I. (1978. Science [Wash. DC]. 200:618-627) and Hammer, D. A. and D. A. Lauffenburger (1987. Biophys. J. 52:475-487). We demonstrate experimental validation of a theoretical expression for the critical shear stress for particle detachment, and show that it is consistent with reasonable estimates for the receptor-ligand bond affinity. Images FIGURE 2 PMID:2166596

  20. LRP6 Protein Regulates Low Density Lipoprotein (LDL) Receptor-mediated LDL Uptake*

    PubMed Central

    Ye, Zhi-jia; Go, Gwang-Woong; Singh, Rajvir; Liu, Wenzhong; Keramati, Ali Reza; Mani, Arya

    2012-01-01

    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 LRP6R611C mutation carriers is significantly impaired. In this study we have examined the role of wild type LRP6 (LRP6WT) and LRP6R611C in LDL receptor (LDLR)-mediated LDL uptake. LDL binding and uptake were increased when LRP6WT 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 LRP6WT 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 LRP6R611C. 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. PMID:22128165

  1. Evidence that 5-HT1D receptors mediate inhibition of sympathetic ganglionic transmission in anaesthetized cats.

    PubMed Central

    Jones, J. F.; Martin, G. R.; Ramage, A. G.

    1995-01-01

    In anaesthetized cats, 5-carboxamidotryptamine (5-CT) or 5-hydroxytryptamine (5-HT) (0.3-300 micrograms kg-1,i.v.) inhibited the postganglionic compound action potential evoked by preganglionic electrical stimulation (0.5 Hz) with a similar potency in the stellate and splanchnic ganglia. In the 5-HT experiments transmission thorough the inferior mesenteric ganglia was also recorded. The maximal inhibitory effect of 5-HT was greater on the stellate and splanchnic ganglia (60 +/- 4 and 52 +/- 5%) than on the inferior mesenteric (15 +/- 2%). The effects of 5-HT were unaffected by pretreatment with antagonists (1 mg kg-1;i.v.) for 5-HT2 (BW501C67), 5-HT1A (WAY-100635) and 5-HT3 receptors (ondansetron). However, responses to both 5-HT and 5-CT were attenuated significantly by GR127935 (1 mg kg-1) except the responses to 5-HT at the inferior mesenteric ganglia. These results are consistent with the involvement of 5-HT1D receptors mediating inhibition of sympathetic ganglionic transmission in vivo. PMID:8528548

  2. Progesterone stimulates respiration through a central nervous system steroid receptor-mediated mechanism in cat.

    PubMed

    Bayliss, D A; Millhorn, D E; Gallman, E A; Cidlowski, J A

    1987-11-01

    We have examined the effect on respiration of the steroid hormone progesterone, administered either intravenously or directly into the medulla oblongata in anesthetized and paralyzed male and female cats. The carotid sinus and vagus nerves were cut, and end-tidal PCO2 and temperature were kept constant with servo-controllers. Phrenic nerve activity was used to quantitate central respiratory activity. Repeated doses of progesterone (from 0.1 to 2.0 micrograms/kg, cumulative) caused a sustained (greater than 45 min) facilitation of phrenic nerve activity in female and male cats; however, the response was much more variable in females. Progesterone injected into the region of nucleus tractus solitarii, a respiratory-related area in the medulla oblongata, also caused a prolonged stimulation of respiration. Progesterone administration at high concentration by both routes also caused a substantial hypotension. Identical i.v. doses of other classes of steroid hormones (17 beta-estradiol, testosterone, and cortisol) did not elicit the same respiratory effect. Pretreatment with RU 486, a progesterone-receptor antagonist, blocked the facilitatory effect of progesterone. We conclude that progesterone acts centrally through a steroid receptor-mediated mechanism to facilitate respiration. PMID:3478727

  3. Receptor-Mediated Endocytosis of Lysozyme in Renal Proximal Tubules of the Frog Rana Temporaria

    PubMed Central

    Seliverstova, E.V.

    2015-01-01

    The mechanism of protein reabsorption in the kidney of lower vertebrates remains insufficiently investigated in spite of raising interest to the amphibian and fish kidneys as a useful model for physiological and pathophysiological examinations. In the present study, we examined the renal tubular uptake and the internalization rote of lysozyme after its intravenous injection in the wintering frog Rana temporaria using immunohisto- and immunocytochemistry and specific markers for some endocytic compartments. The distinct expression of megalin and cubilin in the proximal tubule cells of lysozyme-injected frogs was revealed whereas kidney tissue of control animals showed no positive immunoreactivity. Lysozyme was detected in the apical endocytic compartment of the tubular cells and colocalized with clathrin 10 min after injection. After 20 min, lysozyme was located in the subapical compartment negative to clathrin (endo-somes), and intracellular trafficking of lysozyme was coincided with the distribution of megalin and cubilin. However, internalized protein was retained in the endosomes and did not reach lysosomes within 30 min after treatment that may indicate the inhibition of intra-cellular trafficking in hibernating frogs. For the first time, we provided the evidence that lysozyme is filtered through the glomeruli and absorbed by receptor-mediated clathrin-dependent endocytosis in the frog proximal tubule cells. Thus, the protein uptake in the amphibian mesonephros is mediated by megalin and cubilin that confirms a critical role of endocytic receptors in the renal reabsorption of proteins in amphibians as in mammals. PMID:26150156

  4. Coupling of mitochondrial import and export translocases by receptor-mediated supercomplex formation.

    PubMed

    Qiu, Jian; Wenz, Lena-Sophie; Zerbes, Ralf M; Oeljeklaus, Silke; Bohnert, Maria; Stroud, David A; Wirth, Christophe; Ellenrieder, Lars; Thornton, Nicolas; Kutik, Stephan; Wiese, Sebastian; Schulze-Specking, Agnes; Zufall, Nicole; Chacinska, Agnieszka; Guiard, Bernard; Hunte, Carola; Warscheid, Bettina; van der Laan, Martin; Pfanner, Nikolaus; Wiedemann, Nils; Becker, Thomas

    2013-08-01

    The mitochondrial outer membrane harbors two protein translocases that are essential for cell viability: the translocase of the outer mitochondrial membrane (TOM) and the sorting and assembly machinery (SAM). The precursors of β-barrel proteins use both translocases-TOM for import to the intermembrane space and SAM for export into the outer membrane. It is unknown if the translocases cooperate and where the β-barrel of newly imported proteins is formed. We established a position-specific assay for monitoring β-barrel formation in vivo and in organello and demonstrated that the β-barrel was formed and membrane inserted while the precursor was bound to SAM. β-barrel formation was inhibited by SAM mutants and, unexpectedly, by mutants of the central import receptor, Tom22. We show that the cytosolic domain of Tom22 links TOM and SAM into a supercomplex, facilitating precursor transfer on the intermembrane space side. Our study reveals receptor-mediated coupling of import and export translocases as a means of precursor channeling. PMID:23911324

  5. 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...

  6. 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...

  7. Neuroprotective effects of pramipexole against tunicamycin-induced cell death in PC12 cells.

    PubMed

    Nakayama, Hitoshi; Zhao, Jing; Ei-Fakhrany, Amany; Isosaki, Minoru; Satoh, Hiroyasu; Kyotani, Yoji; Yoshizumi, Masanori

    2009-12-01

    1. Pramipexole (PPX), a dopamine D2 and D3 receptor agonist, exerts neuroprotective effects via both dopamine receptor-mediated and non-dopaminergic mechanisms. In the present study, we demonstrate that PPX reduces the toxicity of tunicamycin, a typical endoplasmic reticulum (ER) stressor, in PC12h cells, a subline of PC12 cells. 2. The PC12h cells were treated with 300 micromol / L PPX in the presence of 0.5 micromol / L tunicamycin for 24 h. The neuroprotective effects of PPX against tunicamycin-induced cell death were evaluated using 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release assays, Hoechst 33258 staining and western blot analysis. 3. Tunicamycin (0.2, 0.3 and 0.5 microg / mL) dose-dependently decreased MTT activity and increased LDH release from PC12h cells. Treatment with 300 micromol / L PPX rescued the tunicamycin-induced decrease in cell viability. 4. Spiperone (10 micromol / L), a dopamine D2 and D4 receptor antagonist, had no effect on PPX neuroprotection against tunicamycin in these cells. Marker proteins of ER stress and apoptosis are known to be upregulated by tunicamycin, but we detected no significant effects of PPX on these factors. 5. In conclusion, we speculate that a combination of several mechanisms may be involved in PPX-induced neuroprotection. PMID:19515063

  8. 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

  9. Silent NMDA receptor-mediated synapses are developmentally regulated in the dorsal horn of the rat spinal cord.

    PubMed

    Baba, H; Doubell, T P; Moore, K A; Woolf, C J

    2000-02-01

    In vitro whole cell patch-clamp recording techniques were utilized to study silent pure-N-methyl-D-aspartate (NMDA) receptor-mediated synaptic responses in lamina II (substantia gelatinosa, SG) and lamina III of the spinal dorsal horn. To clarify whether these synapses are present in the adult and contribute to neuropathic pain, transverse lumbar spinal cord slices were prepared from neonatal, naive adult and adult sciatic nerve transected rats. In neonatal rats, pure-NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) were elicited in SG neurons either by focal intraspinal stimulation (n = 15 of 20 neurons) or focal stimulation of the dorsal root (n = 2 of 7 neurons). In contrast, in slices from naive adult rats, no silent pure-NMDA EPSCs were recorded in SG neurons following focal intraspinal stimulation (n = 27), and only one pure-NMDA EPSC was observed in lamina III (n = 23). Furthermore, in rats with chronic sciatic nerve transection, pure-NMDA EPSCs were elicited by focal intraspinal stimulation in only 2 of 45 SG neurons. Although a large increase in Abeta fiber evoked mixed alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and NMDA receptor-mediated synapses was detected after sciatic nerve injury, Abeta fiber-mediated pure-NMDA EPSCs were not evoked in SG neurons by dorsal root stimulation. Pure-NMDA receptor-mediated EPSCs are therefore a transient, developmentally regulated phenomenon, and, although they may have a role in synaptic refinement in the immature dorsal horn, they are unlikely to be involved in receptive field plasticity in the adult. PMID:10669507

  10. Hyperoside and rutin of Nelumbo nucifera induce mitochondrial apoptosis through a caspase-dependent mechanism in HT-29 human colon cancer cells

    PubMed Central

    GUON, TAE EUN; CHUNG, HA SOOK

    2016-01-01

    The present study demonstrates the mechanism of 2 flavonol glycosides, hyperoside and rutin, in the induction of apoptosis in HT-29 human colon cancer cells through the bioactivity-guided fractionation and isolation method. The chemical structure of hyperoside and rutin, isolated from the roots of Nelumbo nucifera, were established using extensive 1- and 2-dimensional nuclear magnetic resonance experiments and absolute high resolution fast-atom bombardment mass spectrometry, ultraviolet-visible and Fourier transform infrared spectroscopy spectral analytical methods. The treatment of HT-29 colon cancer cells with hyperoside and rutin significantly decreased cell viability in a dose-dependent manner. The concomitant activation of the mitochondria-dependent apoptotic pathway of hyperoside and rutin occurred via modulation of Bcl-2-associated X protein and B-cell lymphoma 2 expression, resulting in the activation of cleaved caspases-3, −8 and −9 and cleaved poly-(ADP-ribose) polymerase. The findings of the present study indicate that hyperoside and rutin induce apoptosis in HT-29 human colon cancer cells, and that this phenomenon is mediated via the death receptor-mediated and mitochondria-mediated apoptotic pathways. These results suggest that hyperoside and rutin may be useful in the development of a colon cancer therapy protocol. PMID:27073499

  11. 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

  12. Electrophysiological actions of phenytoin on N-methyl-D-aspartate receptor-mediated responses in rat hippocampus in vitro.

    PubMed Central

    Laffling, A. J.; Scherr, P.; McGivern, J. G.; Patmore, L.; Sheridan, R. D.

    1995-01-01

    1. The effects of the anticonvulsant, phenytoin, have been examined on N-methyl-D-aspartate (NMDA) receptor-mediated population spikes in the CA1 region of the rat hippocampus in vitro. 2. The 'conventional' (AMPA receptor-mediated) CA1 population spike, evoked by electrical stimulation of the Schaffer collateral/commissural pathway, was abolished by 5 min treatment with 5 x 10(-6) M 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), after which superfusion with a nominally Mg(2+)-free Krebs solution (containing 5 x 10(-6) M CNQX) led to the appearance of an epileptiform population spike which was fully developed by 30-40 min. 3. The epileptiform population spike was abolished by the non-competitive NMDA antagonist, dizocilpine (1 x 10(-6) M, 20-30 min) and inhibited by the competitive NMDA receptor antagonist, D-CPP (IC50 for reducing the amplitude of the first spike in the train = 8.3 x 10(-7) M), demonstrating that the response was mediated by activation of NMDA receptors and validating its use as an assay for antagonists acting at the NMDA receptor/channel complex. 4. Phenytoin (0.1, 0.3 and 1 x 10(-4) M applied cumulatively for 30 min at each concentration) failed to inhibit the NMDA receptor-mediated epileptiform population response (n = 7 slices).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7647985

  13. The BCL2L1 and PGAM5 axis defines hypoxia-induced receptor-mediated mitophagy

    PubMed Central

    Wu, Hao; Xue, Danfeng; Chen, Guo; Han, Zhe; Huang, Li; Zhu, Chongzhuo; Wang, Xiaohui; Jin, Haijing; Wang, Jun; Zhu, Yushan; Liu, Lei; Chen, Quan

    2014-01-01

    Receptor-mediated mitophagy is one of the major mechanisms of mitochondrial quality control essential for cell survival. We previously have identified FUNDC1 as a mitophagy receptor for selectively removing damaged mitochondria in mammalian systems. A critical unanswered question is how receptor-mediated mitophagy is regulated in response to cellular and environmental cues. Here, we report the striking finding that BCL2L1/Bcl-xL, but not BCL2, suppresses mitophagy mediated by FUNDC1 through its BH3 domain. Mechanistically, we demonstrate that BCL2L1, but not BCL2, interacts with and inhibits PGAM5, a mitochondrially localized phosphatase, to prevent the dephosphorylation of FUNDC1 at serine 13 (Ser13), which activates hypoxia-induced mitophagy. Our results showed that the BCL2L1-PGAM5-FUNDC1 axis is critical for receptor-mediated mitophagy in response to hypoxia and that BCL2L1 possesses unique functions distinct from BCL2. PMID:25126723

  14. 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.

  15. Regulation of the death-associated protein kinase 1 expression and autophagy via ATF6 requires apoptosis signal-regulating kinase 1.

    PubMed

    Gade, Padmaja; Manjegowda, Srikanta B; Nallar, Shreeram C; Maachani, Uday B; Cross, Alan S; Kalvakolanu, Dhananjaya V

    2014-11-01

    The death-associated protein kinase 1 (DAPK1) is an important regulator of cell death and autophagy. Recently, we have identified that ATF6, an endoplasmic reticulum-resident transcription factor, in association with the transcription factor CEBP-β, regulates the gamma interferon (IFN-γ)-induced expression of Dapk1 (P. Gade et al., Proc. Natl. Acad. Sci. U. S. A. 109:10316-10321, 2012, doi.org/10.1073/pnas.1119273109). IFN-γ-induced proteolytic processing of ATF6 and phosphorylation of C/EBP-β were essential for the formation of a novel transcriptional complex that regulates DAPK1. Here, we report that IFN-γ activates the ASK1-MKK3/MKK6-p38 mitogen-activated protein kinase (MAPK) pathway for controlling the activity of ATF6. The terminal enzyme in this pathway, p38 MAPK, phosphorylates a critical threonine residue in ATF6 upstream of its DNA binding domain. ATF6 mutants defective for p38 MAPK phosphorylation fail to undergo proteolytic processing in the Golgi apparatus and drive IFN-γ-induced gene expression and autophagy. We also show that mice lacking Ask1 are highly susceptible to lethal bacterial infection owing to defective autophagy. Together, these results identify a novel host defense pathway controlled by IFN-γ signaling. PMID:25135476

  16. 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

  17. Tantalizing Thanatos: unexpected links in death pathways.

    PubMed

    Cohen, Isabelle; Castedo, Maria; Kroemer, Guido

    2002-07-01

    Cell death is most frequently the result of apoptosis, an event that is often controlled by mitochondrial membrane permeabilization (MMP). Recent data reveal unexpected functional links between apoptosis and autophagic cell death, in the sense that MMP can trigger autophagy of damaged mitochondria. Conversely, one of the major signal-transducing molecules involved in the activation of autophagy during apoptosis--the so-called DAP kinase--can induce cell death through MMP. Connections are also emerging between apoptosis, autophagy, replicative senescence and cancer-specific metabolic changes. PMID:12185842

  18. 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. PMID:21042740

  19. 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

  20. 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.

  1. Scavenger receptors mediate the role of SUMO and Ftz-f1 in Drosophila steroidogenesis.

    PubMed

    Talamillo, Ana; Herboso, Leire; Pirone, Lucia; Pérez, Coralia; González, Monika; Sánchez, Jonatan; Mayor, Ugo; Lopitz-Otsoa, Fernando; Rodriguez, Manuel S; Sutherland, James D; Barrio, Rosa

    2013-04-01

    SUMOylation participates in ecdysteroid biosynthesis at the onset of metamorphosis in Drosophila melanogaster. Silencing the Drosophila SUMO homologue smt3 in the prothoracic gland leads to reduced lipid content, low ecdysone titers, and a block in the larval-pupal transition. Here we show that the SR-BI family of Scavenger Receptors mediates SUMO functions. Reduced levels of Snmp1 compromise lipid uptake in the prothoracic gland. In addition, overexpression of Snmp1 is able to recover lipid droplet levels in the smt3 knockdown prothoracic gland cells. Snmp1 expression depends on Ftz-f1 (an NR5A-type orphan nuclear receptor), the expression of which, in turn, depends on SUMO. Furthermore, we show by in vitro and in vivo experiments that Ftz-f1 is SUMOylated. RNAi-mediated knockdown of ftz-f1 phenocopies that of smt3 at the larval to pupal transition, thus Ftz-f1 is an interesting candidate to mediate some of the functions of SUMO at the onset of metamorphosis. Additionally, we demonstrate that the role of SUMOylation, Ftz-f1, and the Scavenger Receptors in lipid capture and mobilization is conserved in other steroidogenic tissues such as the follicle cells of the ovary. smt3 knockdown, as well as ftz-f1 or Scavenger knockdown, depleted the lipid content of the follicle cells, which could be rescued by Snmp1 overexpression. Therefore, our data provide new insights into the regulation of metamorphosis via lipid homeostasis, showing that Drosophila Smt3, Ftz-f1, and SR-BIs are part of a general mechanism for uptake of lipids such as cholesterol, required during development in steroidogenic tissues. PMID:23637637

  2. Receptor-mediated mechanism for the transport of prolactin from blood to cerebrospinal fluid

    SciTech Connect

    Walsh, R.J.; Slaby, F.J.; Posner, B.I.

    1987-05-01

    Prolactin (PRL) interacts with areas of the central nervous system which reside behind the blood-brain barrier. While vascular PRL does not cross this barrier, it is readily accessible to the cerebrospinal fluid (CSF) from which it may gain access to the PRL-responsive areas of the brain. Studies were undertaken to characterize the mechanism responsible for the translocation of PRL from blood to CSF. Rats were given external jugular vein injections of (/sup 125/-I)iodo-PRL in the presence or absence of an excess of unlabeled ovine PRL (oPRL), human GH, bovine GH, or porcine insulin. CSF and choroid plexus were removed 60 min later. CSF samples were electrophoresed on sodium dodecyl sulfate-polyacrylamide slab gels and resultant autoradiographs were analyzed with quantitative microdensitometry. The data revealed that unlabeled lactogenic hormones, viz. oPRL and human GH, caused a statistically significant inhibition of (/sup 125/I)iodo-PRL transport from blood to CSF. In contrast, nonlactogenic hormones, viz bovine GH and insulin, had no effect on (/sup 125/I)iodo-PRL transport into the CSF. An identical pattern of competition was observed in the binding of hormone to the choroid plexus. Furthermore, vascular injections of (/sup 125/I)iodo-PRL administered with a range of concentrations of unlabeled oPRL revealed a dose-response inhibition in the transport of (/sup 125/I)iodo-PRL from blood to CSF. The study demonstrates that PRL enters the CSF by a specific, PRL receptor-mediated transport mechanism. The data is consistent with the hypothesis that the transport mechanism resides at the choroid plexus. The existence of this transport mechanism reflects the importance of the cerebroventricular system in PRL-brain interactions.

  3. Receptor-mediated uptake of labeled transferrin by embryonic chicken dorsal root ganglion neurons in culture.

    PubMed

    Markelonis, G J; Oh, T H; Park, L P; Azari, P; Max, S R

    1985-01-01

    Transferrin is a growth-promoting plasma protein which is known to occur within developing neurons. Since little information exists on the process by which transferrin is internalized by neurons, we studied this process using dissociated embryonic chicken dorsal root ganglion neurons in culture. Cultured dorsal root ganglion neurons were incubated in the presence of 3.75 nM (125)I-transferrin at 37°C, the cultures were extensively washed, the neurons were solubilized in a Triton-containing buffer and internalized (125)I-transferrin was quantified with a gamma counter. (125)I-transferrin was internalized in a linear fashion for at least 60 min, and this uptake was abolished by the presence of 1.25 μM unlabeled transferrin. No competition for the uptake of (125)I-transferrin was observed in the presence of 1.25 μM ovalbumin, cytochrome c, hemoglobin, insulin, horseradish peroxidase, aldolase or the carboxyl-terminal fragment ('half-site') of transferrin. By contrast, uptake was inhibited by approximately 50% in the presence of the ammo-terminal fragment ('half-site') of transferrin (1.25 μM) or in the presence of concanavalin A (1.25 μM). The binding of transferrin conjugated to fluorescein isothiocyanate to neurons at 4°C and its subsequent internalization at 37°C was demonstrated by fluorescence microscopy of unfixed cells following incubation of the neurons in the presence of the fluorescently labeled protein. Furthermore, the transferrin receptors were visualized immunocytochemically on the surface membranes of dorsal root ganglion neurons using rabbit antibodies directed against transferrin receptors from chicken reticulocytes. From these data, we conclude that transferrin is internalized by neurons via receptor-mediated endocytosis, and suggest that this protein may serve an important role in the development and survival of dorsal root ganglion neurons. PMID:24874753

  4. Renal opiate receptor mediation of renin secretion to renal nerve stimulation in the dog.

    PubMed

    Koyama, S; Hosomi, H

    1986-06-01

    The present study was designed to evaluate renal opiate receptor mediation of the renin secretion response to electrical stimulation of the renal nerves in the pentobarbital sodium-anesthetized dog by use of the opiate agonist leucine-enkephalin (Leu-enk) and the opiate antagonist naloxone. In all animals studied, left kidneys were pump perfused at a constant renal blood flow. Renal perfusion pressure (RPP) and glomerular filtration rate (GFR) were unaltered at a stimulation frequency of 1.0 Hz; however, renin secretion rate (RSR) increased significantly in the nontreated group. High-frequency renal nerve stimulation (10 Hz) increased RPP and decreased GFR. RSR at the high-frequency stimulation was significantly augmented in the nontreated group. Renal arterial infusion of either Leu-enk (25 micrograms X kg-1 X min-1) or naloxone (7 micrograms X kg-1 X min-1) did not alter base-line levels of renal hemodynamics and RSR and did not produce significant changes in these variables even when renal nerves were stimulated at the low frequency; however, Leu-enk inhibited RPP and RSR responses to the high-frequency stimulation, and naloxone augmented these responses. Phentolamine (13 micrograms X kg-1 X min-1) prevented renal hemodynamic responses to the renal nerve stimulation, whereas RSR responses to the stimulation were unaffected. Propranolol (8 micrograms X kg-1 X min-1) resulted in decreases in RSR at the renal nerve stimulation despite the presence of changes in renal hemodynamics similar to the other groups. The results indicate that intrarenal opiate receptors may participate in inhibiting renal secretion of renin mediated by the renal nerves when renal vasoconstriction and reduction of GFR occurred at the high-frequency stimulation. PMID:3013030

  5. β2 Adrenergic receptors mediate important electrophysiological effects in human ventricular myocardium

    PubMed Central

    Lowe, M; Rowland, E; Brown, M; Grace, A

    2001-01-01

    OBJECTIVE—To define the effects of β2 adrenergic receptor stimulation on ventricular repolarisation in vivo.
DESIGN—Prospective study.
SETTING—Tertiary referral centre.
PATIENTS—85 patients with coronary artery disease and 22 normal controls.
INTERVENTIONS—Intravenous and intracoronary salbutamol (a β2 adrenergic receptor selective agonist; 10-30 µg/min and 1-10 µg/min), and intravenous isoprenaline (a mixed β1/β2 adrenergic receptor agonist; 1-5 µg/min), infused during fixed atrial pacing.
MAIN OUTCOME MEASURES—QT intervals, QT dispersion, monophasic action potential duration.
RESULTS—In patients with coronary artery disease, salbutamol decreased QTonset and QTpeak but increased QTend duration; QTonset-QTpeak and QTpeak-QTend intervals increased, resulting in T wave prolongation (mean (SEM): 201 (2) ms to 233 (2) ms; p < 0.01). There was a large increase in dispersion of QTonset, QTpeak, and QTend which was more pronounced in patients with coronary artery disease—for example, QTend dispersion: 50 (2) ms baseline v 98 (4) ms salbutamol (controls), and 70 (1) ms baseline v 108 (3) ms salbutamol (coronary artery disease); p < 0.001. Similar responses were obtained with isoprenaline. Monophasic action potential duration at 90% repolarisation shortened during intracoronary infusion of salbutamol, from 278 (4.1) ms to 257 (3.8) ms (p < 0.05).
CONCLUSIONS—β2 adrenergic receptors mediate important electrophysiological effects in human ventricular myocardium. The increase in dispersion of repolarisation provides a mechanism whereby catecholamines acting through this receptor subtype may trigger ventricular arrhythmias.


Keywords: β2 adrenergic receptors; ventricular repolarisation; QT dispersion; salbutamol; isoprenaline PMID:11410561

  6. Scavenger Receptors Mediate the Role of SUMO and Ftz-f1 in Drosophila Steroidogenesis

    PubMed Central

    Talamillo, Ana; Herboso, Leire; Pirone, Lucia; Pérez, Coralia; González, Monika; Sánchez, Jonatan; Mayor, Ugo; Lopitz-Otsoa, Fernando; Rodriguez, Manuel S.; Sutherland, James D.; Barrio, Rosa

    2013-01-01

    SUMOylation participates in ecdysteroid biosynthesis at the onset of metamorphosis in Drosophila melanogaster. Silencing the Drosophila SUMO homologue smt3 in the prothoracic gland leads to reduced lipid content, low ecdysone titers, and a block in the larval–pupal transition. Here we show that the SR-BI family of Scavenger Receptors mediates SUMO functions. Reduced levels of Snmp1 compromise lipid uptake in the prothoracic gland. In addition, overexpression of Snmp1 is able to recover lipid droplet levels in the smt3 knockdown prothoracic gland cells. Snmp1 expression depends on Ftz-f1 (an NR5A-type orphan nuclear receptor), the expression of which, in turn, depends on SUMO. Furthermore, we show by in vitro and in vivo experiments that Ftz-f1 is SUMOylated. RNAi–mediated knockdown of ftz-f1 phenocopies that of smt3 at the larval to pupal transition, thus Ftz-f1 is an interesting candidate to mediate some of the functions of SUMO at the onset of metamorphosis. Additionally, we demonstrate that the role of SUMOylation, Ftz-f1, and the Scavenger Receptors in lipid capture and mobilization is conserved in other steroidogenic tissues such as the follicle cells of the ovary. smt3 knockdown, as well as ftz-f1 or Scavenger knockdown, depleted the lipid content of the follicle cells, which could be rescued by Snmp1 overexpression. Therefore, our data provide new insights into the regulation of metamorphosis via lipid homeostasis, showing that Drosophila Smt3, Ftz-f1, and SR-BIs are part of a general mechanism for uptake of lipids such as cholesterol, required during development in steroidogenic tissues. PMID:23637637

  7. 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

  8. Self-Assembly into Nanoparticles Is Essential for Receptor Mediated Uptake of Therapeutic Antisense Oligonucleotides.

    PubMed

    Ezzat, Kariem; Aoki, Yoshitsugu; Koo, Taeyoung; McClorey, Graham; Benner, Leif; Coenen-Stass, Anna; O'Donovan, Liz; Lehto, Taavi; Garcia-Guerra, Antonio; Nordin, Joel; Saleh, Amer F; Behlke, Mark; Morris, John; Goyenvalle, Aurelie; Dugovic, Branislav; Leumann, Christian; Gordon, Siamon; Gait, Michael J; El-Andaloussi, Samir; Wood, Matthew J A

    2015-07-01

    Antisense oligonucleotides (ASOs) have the potential to revolutionize medicine due to their ability to manipulate gene function for therapeutic purposes. ASOs are chemically modified and/or incorporated within nanoparticles to enhance their stability and cellular uptake, however, a major challenge is the poor understanding of their uptake mechanisms, which would facilitate improved ASO designs with enhanced activity and reduced toxicity. Here, we study the uptake mechanism of three therapeutically relevant ASOs (peptide-conjugated phosphorodiamidate morpholino (PPMO), 2'Omethyl phosphorothioate (2'OMe), and phosphorothioated tricyclo DNA (tcDNA) that have been optimized to induce exon skipping in models of Duchenne muscular dystrophy (DMD). We show that PPMO and tcDNA have high propensity to spontaneously self-assemble into nanoparticles. PPMO forms micelles of defined size and their net charge (zeta potential) is dependent on the medium and concentration. In biomimetic conditions and at low concentrations, PPMO obtains net negative charge and its uptake is mediated by class A scavenger receptor subtypes (SCARAs) as shown by competitive inhibition and RNAi silencing experiments in vitro. In vivo, the activity of PPMO was significantly decreased in SCARA1 knockout mice compared to wild-type animals. Additionally, we show that SCARA1 is involved in the uptake of tcDNA and 2'OMe as shown by competitive inhibition and colocalization experiments. Surface plasmon resonance binding analysis to SCARA1 demonstrated that PPMO and tcDNA have higher binding profiles to the receptor compared to 2'OMe. These results demonstrate receptor-mediated uptake for a range of therapeutic ASO chemistries, a mechanism that is dependent on their self-assembly into nanoparticles. PMID:26042553

  9. 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

  10. Evidence for 5-HT1-like receptor-mediated vasoconstriction in human pulmonary artery.

    PubMed Central

    MacLean, M. R.; Clayton, R. A.; Templeton, A. G.; Morecroft, I.

    1996-01-01

    1. The 5-hydroxytryptamine (5-HT) receptors mediating contraction of human isolated pulmonary artery rings were investigated. Responses to the agonists 5-carboximidotryptamine (5-CT, non-selective 5-HT1 agonist), sumatriptan (5-HT1D-like receptor agonist), 5-HT and 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT, 5-HT1A receptor agonist) were studied. Responses to 5-HT and sumatriptan in the presence of the antagonists, methiothepin (non-selective 5-HT1+2-receptor antagonist), ketanserin (5-HT2A receptor antagonist) and the novel antagonist, GR55562 (5-HT1D receptor antagonist) were also studied. 2. All agonists contracted human pulmonary artery ring preparations in the following order of potency 5-CT > 5-HT = sumatriptan > 8-OH-DPAT. Maximum responses to 5-HT, 5-CT and sumatriptan were not significantly different. 3. Methiothepin 1 nM and 10 nM, but not 0.1 nM reduced the maximum contractile responses to 5-HT but did not alter tissue sensitivity to 5-HT. Methiothepin 0.1 nM, 1 nM and 10 nM had a similar effect on responses to sumatriptan. 4. The 5-HT2A receptor antagonist ketanserin (10 nM, 100 nM and 1 microM) also reduced the maximum contractile response to both 5-HT and sumatriptan without affecting tissue sensitivity to these agonists. 5. The novel 5-HT1D receptor antagonist, GR55562, inhibited responses to 5-HT and sumatriptan in a true competitive fashion. 6. The results suggest that the human pulmonary artery has a functional population of 5-HT1D-like receptors which are involved in the contractile response to 5-HT. PMID:8886409

  11. Characterization of muscarinic receptors mediating relaxation and contraction in the rat iris dilator muscle.

    PubMed Central

    Masuda, Y; Yamahara, N S; Tanaka, M; Ryang, S; Kawai, T; Imaizumi, Y; Watanabe, M

    1995-01-01

    1. The characteristics of muscarinic receptors mediating relaxation and/or contraction in the rat iris dilator muscle were examined. 2. Relaxation was induced in a dilator muscle by application of acetylcholine (ACh) at low doses (3 microM or less) and contraction was induced by high doses. Methacholine and carbachol also showed biphasic effects similar to those of ACh; in contrast, bethanechol, arecoline, pilocarpine and McN-A-343 induced mainly relaxation but no substantial contraction. 3. After parasympathetic denervation by ciliary ganglionectomy, the relaxant response to muscarinic agonists disappeared upon nerve stimulation. Application of McN-A-343 and pilocarpine induced only small contractions in denervated dilator muscles, indicating that these are partial agonists for contraction. 4. pA2 values of pirenzepine, methoctramine, AF-DX 116, himbacine, and 4-DAMP for antagonism to pilocarpine-induced relaxation in normal dilator muscles and those for antagonism to ACh-induced contraction in denervated dilator muscles were determined. The pA2 values for antagonism to relaxation of all these antagonists were most similar to those for M3-type muscarinic receptors. 5. Although pA2 values for contraction of these antagonists, except for methoctramine, were very close to those for relaxation, contraction was not significantly antagonized by methoctramine. Contraction might be mediated by M3-like receptors which have a very low affinity for methoctramine. 6. In conclusion, ACh-induced biphasic responses in rat iris dilator muscles were clearly distinguished from each other by specific muscarinic agonists and parasympathetic denervation, whereas muscarinic receptors could not be subclassified according to the pA2 values of 5 specific antagonists only. PMID:7539696

  12. Regulation of muscarinic acetylcholine receptor-mediated synaptic responses by GABAB receptors in the rat hippocampus

    PubMed Central

    Morton, Robin A; Manuel, Nick A; Bulters, Diederick O; Cobb, Stuart R; Davies, Ceri H

    2001-01-01

    Both GABAB and muscarinic acetylcholine receptors (mAChRs) influence hippocampal-dependent mnemonic processing. Here the possibility of a direct interaction between GABAB receptors and mAChR-mediated synaptic responses has been studied using intracellular recording in rat hippocampal slices. The GABAB receptor agonist(−)-baclofen (5–10 μm) depressed an atropine-sensitive slow EPSP (EPSPM) and occluded the GABAB-receptor-mediated IPSP (IPSPB) which preceded it. These inhibitory effects were accompanied by postsynaptic hyperpolarization (9 ± 2 mV) and a reduction in cell input resistance (12 ± 3 %). The selective GABAB receptor antagonist CGP 55845A (1 μm) fully reversed the depressant effects of (−)-baclofen (5–10 μm) such that in the combined presence of (−)-baclofen and CGP 55845A the EPSPM was 134 ± 21 % of control. (−)-Baclofen (5–10 μm) caused a small (28 ± 11 %) inhibition of carbachol-induced (3.0 μm) postsynaptic depolarizations and increases in input resistance. CGP 55845A (1 μm) alone caused an increase in the amplitude of the EPSPM (253 ± 74 % of control) and blocked the IPSPB that preceded it. In contrast, the selective GABA uptake inhibitor NNC 05–0711 (10 μm) increased the amplitude of the IPSPB by 141 ± 38 % and depressed the amplitude of the EPSPM by 58 ± 10 %. This inhibition was abolished by CGP 55845A (1 μm). Taken together these data provide good evidence that synaptically released GABA activates GABAB receptors that inhibit mAChR-mediated EPSPs in hippocampal CA1 pyramidal neurones. The mechanism of inhibition may involve both pre- and postsynaptic elements. PMID:11559773

  13. Role of Calpain in Apoptosis

    PubMed Central

    Momeni, Hamid Reza

    2011-01-01

    Apoptosis, a form of programmed cell death that occurs under physiological as well as pathological conditions, is characterized by morphological and biochemical features. While the importance of caspases in apoptosis is established, several noncaspase proteases (Ca2+-dependent proteases) such as calpain may play a role in the execution of apoptosis. The calpain family consists of two major isoforms, calpain I and calpain II which require µM and mM Ca2+ concentrations to initiate their activity. An increase in intracellular Ca2+ level is thought to trigger a cascade of biochemical processes including calpain activation. Once activated, calpains degrade membrane, cytoplasmic and nuclear substrates, leading to the breakdown of cellular architecture and finally apoptosis. The activation of calpain has been implicated in neuronal apoptosis following spinal cord injuries and neurodegenerative diseases. This review focuses on calpain with an emphasis on its key role in the proteolysis of cellular protein substrates following apoptosis. PMID:23507938

  14. TNF-like weak inducer of apoptosis promotes blood brain barrier disruption and increases neuronal cell death in MRL/lpr mice.

    PubMed

    Wen, Jing; Doerner, Jessica; Weidenheim, Karen; Xia, Yumin; Stock, Ariel; Michaelson, Jennifer S; Baruch, Kuti; Deczkowska, Aleksandra; Gulinello, Maria; Schwartz, Michal; Burkly, Linda C; Putterman, Chaim

    2015-06-01

    Neuropsychiatric disease is one of the most common manifestations of human systemic lupus erythematosus, but the mechanisms remain poorly understood. In human brain microvascular endothelial cells in vitro, TNF-like weak inducer of apoptosis (TWEAK) decreases tight junction ZO-1 expression and increases the permeability of monolayer cell cultures. Furthermore, knockout (KO) of the TWEAK receptor, Fn14, in the MRL/lpr lupus mouse strain markedly attenuates neuropsychiatric disease, as demonstrated by significant reductions in depressive-like behavior and improved cognitive function. The purpose of the present study was to determine the mechanisms by which TWEAK signaling is instrumental in the pathogenesis of neuropsychiatric lupus (NPSLE). Evaluating brain sections of MRL/lpr Fn14WT and Fn14KO mice, we found that Fn14KO mice displayed significantly decreased cellular infiltrates in the choroid plexus. To evaluate the integrity of the blood brain barrier (BBB) in MRL/lpr mice, Western blot for fibronectin, qPCR for iNOS, and immunohistochemical staining for VCAM-1/ICAM-1 were performed. We found preserved BBB permeability in MRL/lpr Fn14KO mice, attributable to reduced brain expression of VCAM-1/ICAM-1 and iNOS. Additionally, administration of Fc-TWEAK intravenously directly increased the leakage of a tracer (dextran-FITC) into brain tissue. Furthermore, MRL/lpr Fn14KO mice displayed reduced antibody (IgG) and complement (C3, C6, and C4a) deposition in the brain. Finally, we found that MRL/lpr Fn14KO mice manifested reduced neuron degeneration and hippocampal gliosis. Our studies indicate that TWEAK/Fn14 interactions play an important role in the pathogenesis of NPSLE by increasing the accumulation of inflammatory cells in the choroid plexus, disrupting BBB integrity, and increasing neuronal damage, suggesting a novel target for therapy in this disease. PMID:25911200

  15. TNF-like weak inducer of apoptosis promotes blood brain barrier disruption and increases neuronal cell death in MRL/lpr mice

    PubMed Central

    Wen, Jing; Doerner, Jessica; Weidenheim, Karen; Xia, Yumin; Stock, Ariel; Michaelson, Jennifer S.; Baruch, Kuti; Deczkowska, Aleksandra; Gulinello, Maria; Schwartz, Michal; Burkly, Linda C.; Putterman, Chaim

    2015-01-01

    Neuropsychiatric disease is one of the most common manifestations of human systemic lupus erythematosus, but the mechanisms remain poorly understood. In human brain microvascular endothelial cells in vitro, TNF-like weak inducer of apoptosis (TWEAK) decreases tight junction ZO-1 expression and increases the permeability of monolayer cell cultures. Furthermore, knockout (KO) of the TWEAK receptor, Fn14, in the MRL/lpr lupus mouse strain markedly attenuates neuropsychiatric disease, as demonstrated by significant reductions in depressive-like behavior and improved cognitive function. The purpose of the present study was to determine the mechanisms by which TWEAK signaling is instrumental in the pathogenesis of neuropsychiatric lupus (NPSLE). Evaluating brain sections of MRL/lpr Fn14WT and Fn14KO mice, we found that Fn14KO mice displayed significantly decreased cellular infiltrates in the choroid plexus. To evaluate the integrity of the blood brain barrier (BBB) in MRL/lpr mice, Western blot for fibronectin, qPCR for iNOS, and immunohistochemical staining for VCAM-1/ICAM-1 were performed. We found preserved BBB permeability in MRL/lpr Fn14KO mice, attributable to reduced brain expression of VCAM-1/ICAM-1 and iNOS. Additionally, administration of Fc-TWEAK intravenously directly increased the leakage of a tracer (dextran-FITC) into brain tissue. Furthermore, MRL/lpr Fn14KO mice displayed reduced antibody (IgG) and complement (C3, C6, and C4a) deposition in the brain. Finally, we found that MRL/lpr Fn14KO mice manifested reduced neuron degeneration and hippocampal gliosis. Our studies indicate that TWEAK/Fn14 interactions play an important role in the pathogenesis of NPSLE by increasing the accumulation of inflammatory cells in the choroid plexus, disrupting BBB integrity, and increasing neuronal damage, suggesting a novel target for therapy in this disease. PMID:25911200

  16. [Apoptosis during embryo development].

    PubMed

    Jezek, Davor; Kozina, Viviana

    2009-10-01

    The development of human embryo includes two essential processes, i.e., rapid mitotic activity of cells and gradual differentiation of tissues and organs. The latter process is very often characterized by extensive migration of cells from their site of origin to the site of definitive location, inductive action of the neighboring germ layers and programmed cell death (apoptosis). This paper describes examples of proliferative and apoptotic processes during the development of human embryo. The development of trilaminar germ disk, skin, gonads, central and peripheral nerve system as well as limbs provides instructive examples of how apoptosis regulates the development and differentiation of cells. PMID:19999545

  17. Transient elevations of cytosolic free calcium retard subsequent apoptosis in neutrophils in vitro.

    PubMed Central

    Whyte, M K; Hardwick, S J; Meagher, L C; Savill, J S; Haslett, C

    1993-01-01

    Elevation of cytosolic calcium ([Ca2+]i) has been reported to induce apoptosis in a number of cell types. However, in the neutrophil, which undergoes apoptosis constitutively during aging in vitro, activation by inflammatory mediators elevates [Ca2+]i and prolongs lifespan via inhibition of apoptosis. To examine this paradox, we investigated the effects of modulation of [Ca2+]i upon apoptosis of neutrophils in vitro. Calcium ionophores (A23187, ionomycin) retarded apoptosis in neutrophil populations after 20 h (P < 0.001). Conversely, intracellular Ca(2+)-chelation, using bis-(o-aminophenoxy)-N,N,N'N'-tetraacetic acid (BAPTA) acetoxymethyl ester (AM) promoted apoptosis (P < 0.02). W-7 (an inhibitor of calmodulin) also promoted apoptosis (P < 0.05). Measurements of [Ca2+]i, using fura-2, showed (a) increased apoptosis in neutrophil populations was not associated with elevated [Ca2+]i, (b) neutrophils cultured with ionophore at concentrations inhibiting apoptosis exhibited transient (< 1 h) elevations of [Ca2+]i, to levels previously reported with receptor-mediated stimuli, and (c) BAPTA was able to prevent the elevation of [Ca2+]i and the inhibition of apoptosis produced by ionophore. Modulation of apoptosis occurred without alterations in intracellular pH. Thus, in the neutrophil, unlike lymphoid cells, elevation of [Ca2+]i exerts an inhibitory effect upon apoptosis. Furthermore, these data suggest that transient elevation of [Ca2+]i elicits signaling events leading to prolonged inhibition of apoptosis. Images PMID:8392090

  18. 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

  19. 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. PMID:26033724

  20. Induction of intrinsic and extrinsic apoptosis through oxidative stress in drug-resistant cancer by a newly synthesized Schiff base copper chelate.

    PubMed

    Banerjee, Kaushik; Basu, Soumya; Das, Satyajit; Sinha, Abhinaba; Biswas, Manas Kumar; Choudhuri, Soumitra Kumar

    2016-04-01

    Multidrug resistance (MDR) in cancer represents a variety of strategies employed by tumor cells to evade the beneficial cytotoxic effects of structurally different anticancer drugs and thus confers impediments to the successful treatment of cancers. Efflux of drugs by MDR protein-1, functional P-glycoprotein and elevated level of reduced glutathione confer resistance to cell death or apoptosis and thus provide a possible therapeutic target for overcoming MDR in cancer. Previously, we reported that a Schiff base ligand, potassium-N-(2-hydroxy 3-methoxy-benzaldehyde)-alaninate (PHMBA) overcomes MDR in both in vivo and in vitro by targeting intrinsic apoptotic/necrotic pathway through induction of reactive oxygen species (ROS). The present study describes the synthesis and spectroscopic characterization of a copper chelate of Schiff base, viz., copper (II)-N-(2-hydroxy-3-methoxy-benzaldehyde)-alaninate (CuPHMBA) and the underlying mechanism of cell death induced by CuPHMBA in vitro. CuPHMBA kills both the drug-resistant and sensitive cell types irrespective of their drug resistance phenotype. The cell death induced by CuPHMBA follows apoptotic pathway and moreover, the cell death is associated with intrinsic mitochondrial and extrinsic receptor-mediated pathways. Oxidative stress plays a pivotal role in the process as proved by the fact that antioxidant enzyme; polyethylene glycol conjugated-catalase completely blocked CuPHMBA-induced ROS generation and abrogated cell death. To summarize, the present work provides a compelling rationale for the future clinical use of CuPHMBA, a redox active copper chelate in the treatment of cancer patients, irrespective of their drug-resistance status. PMID:26733073

  1. 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,...

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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).

  7. Reactivation of apolipoprotein II gene transcription by cycloheximide reveals two steps in the deactivation of estrogen receptor-mediated transcription.

    PubMed

    Sensel, M G; Binder, R; Lazier, C B; Williams, D L

    1994-03-01

    In this report, we describe apolipoprotein II (apoII) gene expression in cell lines derived by stable expression of the chicken estrogen receptor in LMH chicken hepatoma cells. In cell lines expressing high levels of receptor (LMH/2A), apoII gene expression is increased by estrogen 300-fold compared with levels in the receptor-deficient parent LMH line. LMH/2A cells show apoII mRNA induction and turnover kinetics similar to those in chicken liver. Inhibition of protein synthesis with cycloheximide (CHX) or puromycin following estrogen withdrawal superinduces apoII mRNA without affecting apoII mRNA stability. Superinduction is due to an estrogen-independent reactivation of apoII gene transcription. The apoII gene can be reactivated by CHX for up to 24 h following hormone withdrawal, suggesting that the gene is in a repressed yet transcriptionally competent state. These results reveal two distinct events necessary for termination of estrogen receptor-mediated transcription. The first event, removal of hormone, is sufficient to stop transcription when translation is ongoing. The second event is revealed by the CHX-induced superinduction of apoII mRNA following hormone withdrawal. This superinduction suggests that deactivation of estrogen receptor-mediated transcription requires a labile protein. Furthermore, reactivation of apoII gene expression by CHX and estrogen is additive, suggesting that estrogen is unable to overcome repression completely. Thus, a labile protein may act to repress estrogen receptor-mediated transcription of the apoII gene. PMID:8114707

  8. 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. PMID:26683074

  9. 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.

  10. 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

  11. Inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase reduce receptor-mediated endocytosis in opossum kidney cells.

    PubMed

    Sidaway, James E; Davidson, Robert G; McTaggart, Fergus; Orton, Terry C; Scott, Robert C; Smith, Graham J; Brunskill, Nigel J

    2004-09-01

    Renal proximal tubule cells are responsible for the reabsorption of proteins that are present in the tubular lumen. This occurs by receptor-mediated endocytosis, a process that has a requirement for some GTP-binding proteins. Statins are inhibitors of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase used for the therapeutic reduction of cholesterol-containing plasma lipoproteins. However, they can also reduce intracellular levels of isoprenoid pyrophosphates that are derived from the product of the enzyme, mevalonate, and are required for the prenylation and normal function of GTP-binding proteins. The hypothesis that inhibition of HMG-CoA reductase in renal proximal tubule cells could reduce receptor mediated-endocytosis was therefore tested. Five different statins inhibited the uptake of FITC-labeled albumin by the proximal tubule-derived opossum kidney cell line in a dose-dependent manner and in the absence of cytotoxicity. The reduction in albumin uptake was related to the degree of inhibition of HMG-CoA reductase. Simvastatin (e.g., statin) inhibited receptor-mediated endocytosis of both FITC-albumin and FITC-beta(2)-microglobulin to similar extents but without altering the binding of albumin to the cell surface. The effect on albumin endocytosis was prevented by mevalonate and by the isoprenoid geranylgeranyl pyrophosphate but not by cholesterol. Finally, evidence that the inhibitory effect of statins on endocytosis of proteins may be caused by reduced prenylation and thereby decreased function of one or more GTP-binding proteins is provided. These data establish the possibility in principle that inhibition of HMG-CoA reductase by statins in proximal tubule cells may reduce tubular protein reabsorption. PMID:15339975

  12. Effects of particle size and ligand density on the kinetics of receptor-mediated endocytosis of nanoparticles

    NASA Astrophysics Data System (ADS)

    Yuan, Hongyan; Zhang, Sulin

    2010-01-01

    We elucidate, from thermodynamic arguments, the governing factors of receptor-mediated endocytosis of nanoparticles (NPs). We show that the endocytic energetics specifies a minimal particle size and a minimal ligand density below which endocytosis is not possible. Due to the entropic penalty involved in ligand-receptor binding, endocytosis may occur with a large fraction of ligands unbound with receptors. Our analyses suggest that the endocytic time depends interrelatedly on the particle size and ligand density. There exists an optimal condition at which the endocytic time minimizes. These findings may provide valuable guidance to the rational designs of NP-based biomarkers and anticancer bioagents.

  13. Effects of rasagiline, its metabolite aminoindan and selegiline on glutamate receptor mediated signalling in the rat hippocampus slice in vitro

    PubMed Central

    2011-01-01

    Background Rasagiline, a new drug developed to treat Parkinson's disease, is known to inhibit monoamine oxidase B. However, its metabolite R-(-)-aminoindan does not show this kind of activity. The present series of in vitro experiments using the rat hippocampal slice preparation deals with effects of both compounds on the pyramidal cell response after electric stimulation of the Schaffer Collaterals in comparison to selegiline, another MAO B inhibitor. Method Stimulation of the Schaffer Collaterals by single stimuli (SS) or theta burst stimulation (TBS) resulted in stable responses of pyramidal cells measured as population spike amplitude (about 1 mV under control SS conditions or about 2 mV after TBS). Results During the first series, this response was attenuated in the presence of rasagiline and aminoindan-to a lesser degree of selegiline-in a concentration dependent manner (5-50 μM) after single stimuli as well as under TBS. During oxygen/glucose deprivation for 10 min the amplitude of the population spike breaks down by 75%. The presence of rasagiline and aminoindan, but rarely the presence of selegiline, prevented this break down. Following glutamate receptor mediated enhancements of neuronal transmission in a second series of experiments very clear differences could be observed in comparison to the action of selegiline: NMDA receptor, AMPA receptor as well as metabotropic glutamate receptor mediated increases of transmission were concentration dependently (0,3 - 2 μM) antagonized by rasagiline and aminoindan, but not by selegiline. On the opposite, only selegiline attenuated kainate receptor mediated increases of excitability. Thus, both monoamino oxidase (MAO) B inhibitors show attenuation of glutamatergic transmission in the hippocampus but interfere with different receptor mediated excitatory modulations at low concentrations. Conclusions Since aminoindan does not induce MAO B inhibition, these effects must be regarded as being independent from MAO B

  14. Proximal tubule sphingosine kinase-1 has a critical role in A1 adenosine receptor-mediated renal protection from ischemia

    PubMed Central

    Park, Sang Won; Kim, Mihwa; Kim, Joo Yun; Brown, Kevin M.; Haase, Volker H.; D’Agati, Vivette D.; Lee, H. Thomas

    2012-01-01

    Renal ischemia reperfusion injury is a major cause of acute kidney injury. We previously found that renal A1 adenosine receptor (A1AR) activation attenuated multiple cell death pathways including necrosis, apoptosis and inflammation. Here, we tested whether induction of cytoprotective sphingosine kinase (SK)-1 and sphingosine-1 phosphate (S1P) synthesis might be the mechanism of protection. A selective A1AR agonist (CCPA) increased the synthesis of S1P and selectively induced SK-1 in mouse kidney and HK-2 cells. This agonist failed to protect SK1-knockout but protected SK2-knockout mice against renal ischemia reperfusion injury indicating a critical role of SK1 in A1AR-mediated renal protection. Inhibition of SK prevented A1AR-mediated defense against necrosis and apoptosis in HK-2 cells. A selective S1P1R antagonist (W146) and global in vivo gene knockdown of S1P1Rs with small interfering RNA completely abolished the renal protection provided by CCPA. Mice selectively deficient in renal proximal tubule S1P1Rs (S1P1Rflox/flox PEPCKCre/−) were not protected against renal ischemia reperfusion injury by CCPA. Mechanistically, CCPA increased nuclear translocation of hypoxia inducible factor-1α in HK-2 cells and selective hypoxia inducible factor-1α inhibition blocked A1AR-mediated induction of SK1. Thus, proximal tubule SK-1 has a critical role in A1AR-mediated protection against renal ischemia reperfusion injury. PMID:22695326

  15. Dopamine D2 Receptor-Mediated Regulation of Pancreatic β Cell Mass.

    PubMed

    Sakano, Daisuke; Choi, Sungik; Kataoka, Masateru; Shiraki, Nobuaki; Uesugi, Motonari; Kume, Kazuhiko; Kume, Shoen

    2016-07-12

    Understanding the molecular mechanisms that regulate β cell mass and proliferation is important for the treatment of diabetes. Here, we identified domperidone (DPD), a dopamine D2 receptor (DRD2) antagonist that enhances β cell mass. Over time, islet β cell loss occurs in dissociation cultures, and this was inhibited by DPD. DPD increased proliferation and decreased apoptosis of β cells through increasing intracellular cAMP. DPD prevented β cell dedifferentiation, which together highly contributed to the increased β cell mass. DRD2 knockdown phenocopied the effects of domperidone and increased the number of β cells. Drd2 overexpression sensitized the dopamine responsiveness of β cells and increased apoptosis. Further analysis revealed that the adenosine agonist 5'-N-ethylcarboxamidoadenosine, a previously identified promoter of β cell proliferation, acted with DPD to increase the number of β cells. In humans, dopamine also modulates β cell mass through DRD2 and exerts an inhibitory effect on adenosine signaling. PMID:27373926

  16. Mechanisms of methicillin-resistant Staphylococcus aureus pneumonia-induced intestinal epithelial apoptosis.

    PubMed

    Perrone, Erin E; Jung, Enjae; Breed, Elise; Dominguez, Jessica A; Liang, Zhe; Clark, Andrew T; Dunne, W Michael; Burd, Eileen M; Coopersmith, Craig M

    2012-07-01

    Methicillin-resistant Staphylococcus aureus (MRSA) pneumonia-induced sepsis is a common cause of morbidity in the intensive care unit. Although pneumonia is initiated in the lungs, extrapulmonary manifestations occur commonly. In light of the key role the intestine plays in the pathophysiology of sepsis, we sought to determine whether MRSA pneumonia induces intestinal injury. FVB/N mice were subjected to MRSA or sham pneumonia and killed 24 h later. Septic animals had a marked increase in intestinal epithelial apoptosis by both hematoxylin-eosin and active caspase 3 staining. Methicillin-resistant S. aureus-induced intestinal apoptosis was associated with an increase in the expression of the proapoptotic proteins Bid and Bax and the antiapoptotic protein Bcl-xL in the mitochondrial pathway. In the receptor-mediated pathway, MRSA pneumonia induced an increase in Fas ligand but decreased protein levels of Fas, FADD, pFADD, TNF-R1, and TRADD. To assess the functional significance of these changes, MRSA pneumonia was induced in mice with genetic manipulations in proteins in either the mitochondrial or receptor-mediated pathways. Both Bid-/- mice and animals with intestine-specific overexpression of Bcl-2 had decreased intestinal apoptosis compared with wild-type animals. In contrast, Fas ligand-/- mice had no alterations in apoptosis. To determine if these findings were organism-specific, similar experiments were performed in mice subjected to Pseudomonas aeruginosa pneumonia. Pseudomonas aeruginosa induced gut apoptosis, but unlike MRSA, this was associated with increased Bcl-2 and TNF-R1 and decreased Fas. Methicillin-resistant S. aureus pneumonia thus induces organism-specific changes in intestinal apoptosis via changes in both the mitochondrial and receptor-mediated pathways, although the former may be more functionally significant. PMID:22592747

  17. Mechanisms of methicillin-resistant Staphylococcus aureus pneumonia-induced intestinal epithelial apoptosis

    PubMed Central

    Perrone, Erin E.; Jung, Enjae; Breed, Elise; Dominguez, Jessica A.; Liang, Zhe; Clark, Andrew T.; Dunne, W. Michael; Burd, Eileen M.; Coopersmith, Craig M.

    2012-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) pneumonia-induced sepsis is a common cause of morbidity in the intensive care unit. Although pneumonia is initiated in the lungs, extrapulmonary manifestations occur commonly. In light of the key role the intestine plays in the pathophysiology of sepsis, we sought to determine whether MRSA pneumonia induces intestinal injury. FVB/N mice were subjected to MRSA or sham pneumonia and sacrificed 24 hours later. Septic animals had a marked increase in intestinal epithelial apoptosis by both H&E and active caspase-3 staining. MRSA-induced intestinal apoptosis was associated with an increase in the expression of the pro-apoptotic proteins Bid and Bax and the anti-apoptotic protein Bcl-xL in the mitochondrial pathway. In the receptor-mediated pathway, MRSA pneumonia induced an increase in Fas-ligand but decreased protein levels of Fas, FADD, pFADD, TNF-R1 and TRADD. To assess the functional significance of these changes, MRSA pneumonia was induced in mice with genetic manipulations in proteins in either the mitochondrial or receptor-mediated pathways. Both Bid−/− mice and animals with intestine specific overexpression of Bcl-2 had decreased intestinal apoptosis compared to wild type animals. In contrast, Fas-ligand−/− mice had no alterations in apoptosis. To determine if these findings were organism-specific, similar experiments were performed in mice subjected to Pseudomonas aeruginosa pneumonia. P. aeruginosa induced gut apoptosis, but unlike MRSA, this was associated with increased Bcl-2 and TNF-R1 and decreased Fas. MRSA pneumonia thus induces organism-specific changes in intestinal apoptosis via changes in both the mitochondrial and receptor-mediated pathways although the former may be more functionally significant. PMID:22592747

  18. Techniques to Distinguish Apoptosis from Necroptosis.

    PubMed

    Feoktistova, Maria; Wallberg, Fredrik; Tenev, Tencho; Geserick, Peter; Leverkus, Martin; Meier, Pascal

    2016-04-01

    The processes by which cells die are as tightly regulated as those that govern cell growth and proliferation. Recent studies of the molecular pathways that regulate and execute cell death have uncovered a plethora of signaling cascades that lead to distinct modes of cell death, including "apoptosis," "necrosis," "autophagic cell death," and "mitotic catastrophe." Cells can readily switch from one form of death to another; therefore, it is vital to have the ability to monitor the form of death that cells are undergoing. A number of techniques are available that allow the detection of cell death and when combined with either knockdown approaches or inhibitors of specific signaling pathways, such as caspase or RIP kinase pathways, they allow the rapid dissection of divergent cell death pathways. However, techniques that reveal the end point of cell death cannot reconstruct the sequence of events that have led to death; therefore, they need to be complemented with methods that can distinguish all forms of cell death. Apoptotic cells frequently undergo secondary necrosis under in vitro culture conditions; therefore, novel methods relying on high-throughput time-lapse fluorescence video microscopy are necessary to provide temporal resolution to cell death events. Further, visualizing the assembly of multiprotein signaling hubs that can execute apoptosis or necroptosis helps to explore the underlying processes. Here we introduce a suite of techniques that reliably distinguish necrosis from apoptosis and secondary necrosis, and that enable investigation of signaling platforms capable of instructing apoptosis or necroptosis. PMID:27037077

  19. Receptor-Mediated Endocytosis of Two-Dimensional Nanomaterials Undergoes Flat Vesiculation and Occurs by Revolution and Self-Rotation.

    PubMed

    Mao, Jian; Chen, Pengyu; Liang, Junshi; Guo, Ruohai; Yan, Li-Tang

    2016-01-26

    Two-dimensional nanomaterials, such as graphene and transitional metal dichalcogenide nanosheets, are promising materials for the development of antimicrobial surfaces and the nanocarriers for intracellular therapy. Understanding cell interaction with these emerging materials is an urgently important issue to promoting their wide applications. Experimental studies suggest that two-dimensional nanomaterials enter cells mainly through receptor-mediated endocytosis. However, the detailed molecular mechanisms and kinetic pathways of such processes remain unknown. Here, we combine computer simulations and theoretical derivation of the energy within the system to show that the receptor-mediated transport of two-dimensional nanomaterials, such as graphene nanosheet across model lipid membrane, experiences a flat vesiculation event governed by the receptor density and membrane tension. The graphene nanosheet is found to undergo revolution relative to the membrane and, particularly, unique self-rotation around its normal during membrane wrapping. We derive explicit expressions for the formation of the flat vesiculation, which reveals that the flat vesiculation event can be fundamentally dominated by a dimensionless parameter and a defined relationship determined by complicated energy contributions. The mechanism offers an essential understanding on the cellular internalization and cytotoxicity of the emerging two-dimensional nanomaterials. PMID:26741298

  20. Receptor-mediated endocytosis of albumin by kidney proximal tubule cells is regulated by phosphatidylinositide 3-kinase.

    PubMed

    Brunskill, N J; Stuart, J; Tobin, A B; Walls, J; Nahorski, S

    1998-05-15

    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

  1. Downregulation of Plk1 Expression By Receptor-Mediated Uptake of Antisense Oligonucleotide-Loaded Nanoparticles1

    PubMed Central

    Spänkuch, Birgit; Steinhauser, Isabel; Wartlick, Heidrun; Kurunci-Csacsko, Elisabeth; Strebhardt, Klaus I; Langer, Klaus

    2008-01-01

    Human serum albumin (HSA) nanoparticles represent a promising tool for targeted drug delivery to tumor cells. The coupling of the antibody trastuzumab to nanoparticles uses the capability of human epidermal growth factor receptor 2 (HER2)-positive cells to incorporate agents linked to HER2. In our present study, we developed targeted nanoparticles loaded with antisense oligonucleotides (ASOs) against polo-like kinase 1 (Plk1). We evaluated the receptor-mediated uptake into HER2-positive and -negative breast cancer and murine cell lines. We performed quantitative real-time PCR and Western blot analyses to monitor the impact on Plk1 expression in HER2-positive breast cancer cells. Antibody-conjugated nanoparticles showed a specific targeting to HER2-overexpressing cells with cellular uptake by receptor-mediated endocytosis and a release into HER2-positive BT-474 cells. We observed a significant reduction of Plk1 mRNA and protein expression and increased activation of Caspase 3/7. Thus, this is the first report about ASO-loaded HSA nanoparticles, where an impact on gene expression could be observed. The data provide the basis for the further development of carrier systems for Plk1-specific ASOs to reduce off-target effects evoked by systemically administered ASOs and to achieve a better penetration into primary and metastatic target cells. Treatment of tumors using trastuzumab-conjugated ASO-loaded HSA nanoparticles could be a promising approach to reach this goal. PMID:18320067

  2. Androgen receptor-mediated regulation of adrenocortical activity in the sand rat, Psammomys obesus.

    PubMed

    Benmouloud, Abdelouafi; Amirat, Zaina; Khammar, Farida; Patchev, Alexandre V; Exbrayat, Jean M; Almeida, Osborne F X

    2014-12-01

    The wild sand rat, Psammomys obesus, displays seasonal variations in adrenocortical activity that parallel those of testicular activity, indicating functional cross-talk between the hypothalamo-pituitary-adrenal and hypothalamo-pituitary-gonadal axes. In the present study, we examined androgen receptor (AR)-mediated actions of testicular steroids in the regulation of adrenocortical function in the sand rat. Specifically, we examined the expression of AR in the adrenal cortex, as well as adrenal apoptosis in male sand rats that had been surgically castrated or castrated and supplemented with testosterone; biochemical indices of adrenocortical function and hormone profiles were also measured. Orchiectomy was followed by an increase in adrenocorticotropic hormone secretion from the anterior pituitary and subsequently, increased adrenocortical activity; the latter was evidenced by orchiectomy-induced increases in the adrenal content of cholesterol and lipids as well as adrenal hypertrophy (seen as an elevation of the RNA/DNA ratio). Further, androgen deprivation respectively up- and downregulated the incidence of apoptosis within the glucocorticoid-producing zona fasciculata and sex steroid-producing zona reticularis. Interestingly, orchiectomy resulted in increased expression of AR in the zona fasciculata. All of the orchiectomy-induced cellular and biochemical responses were reversible after testosterone substitution therapy. Together, these data suggest that adrenocortical activity in the sand rat is seasonally modulated by testicular androgens that act through AR located in the adrenal cortex itself. PMID:25179180

  3. Honokiol enhances paclitaxel efficacy in multi-drug resistant human cancer model through the induction of apoptosis.

    PubMed

    Wang, Xu; Beitler, Jonathan J; Wang, Hong; Lee, Michael J; Huang, Wen; Koenig, Lydia; Nannapaneni, Sreenivas; Amin, A R M Ruhul; Bonner, Michael; Shin, Hyung Ju C; Chen, Zhuo Georgia; Arbiser, Jack L; Shin, Dong M

    2014-01-01

    Resistance to chemotherapy remains a major obstacle in cancer therapy. This study aimed to evaluate the molecular mechanism and efficacy of honokiol in inducing apoptosis and enhancing paclitaxel chemotherapy in pre-clinical multi-drug resistant (MDR) cancer models, including lineage-derived human MDR (KB-8-5, KB-C1, KB-V1) and their parental drug sensitive KB-3-1 cancer cell lines. In vitro analyses demonstrated that honokiol effectively inhibited proliferation in KB-3-1 cells and the MDR derivatives (IC50 ranging 3.35 ± 0.13 µg/ml to 2.77 ± 0.22 µg/ml), despite their significant differences in response to paclitaxel (IC50 ranging 1.66 ± 0.09 ng/ml to 6560.9 ± 439.52 ng/ml). Honokiol induced mitochondria-dependent and death receptor-mediated apoptosis in MDR KB cells, which was associated with inhibition of EGFR-STAT3 signaling and downregulation of STAT3 target genes. Combined treatment with honokiol and paclitaxel synergistically augmented cytotoxicity in MDR KB cells, compared with treatment with either agent alone in vitro. Importantly, the combined treatment significantly inhibited in vivo growth of KB-8-5 tumors in a subcutaneous model. Tumor tissues from the combination group displayed a significant inhibition of Ki-67 expression and an increase in TUNEL-positive cells compared with the control group. These results suggest that targeting multidrug resistance using honokiol in combination with chemotherapy drugs may provide novel therapeutic opportunities. PMID:24586249

  4. CHCHD2 connects mitochondrial metabolism to apoptosis

    PubMed Central

    Liu, Yong; Zhang, Yanping

    2015-01-01

    As the powerhouse of cells and gatekeeper for apoptosis, mitochondria control life and death. CHCHD2, a mitochondrial protein previously known to regulate metabolism, has recently been identified as an apoptosis inhibitor. New data suggest a model in which CHCHD2 performs a prosurvival function by acting as both a reactive oxygen species scavenger and BCL-XL activator. PMID:27308501

  5. Beyond Apoptosis in Lupus

    PubMed Central

    Colonna, Lucrezia; Lood, Christian; Elkon, Keith B.

    2014-01-01

    Purpose of review Systemic lupus erythematosus (SLE) is characterized by autoantibodies directed against nuclear autoantigens normally concealed from immune recognition in healthy individuals. Here we summarize recently identified mechanisms of abnormal cell death leading to exposure and aberrant processing of nucleoprotein self antigens, and discuss their role in the SLE pathogenesis. Recent findings During the past few years, the unveiling of several new forms of cell death has expanded our understanding beyond the simple view of “apoptotic” versus “necrotic” cell death. SLE patients show abnormalities in cell death at several levels, including increased rates of apoptosis, necrosis, and autophagy, as well as reduced clearance of dying cells. These abnormalities lead to an increased autoantigen burden and also antigen modifications, such as nucleic acid oxidation that increase the inflammatory properties of self antigens. Recent investigations have highlighted the role of opsonins in determining the immunogenic versus tolerogenic characteristics of self antigens. Summary Dysregulation of different forms of programmed cell death contributes to increased exposure, availability, and immunogenic characteristic of intracellular self antigens, which all participate in development of lupus autoimmunity. As our understanding of abnormalities of cell death in SLE advances, potential therapeutic opportunities await human implementation. PMID:25036095

  6. Mechanism-Based Tumor-Targeting Drug Delivery System. Validation of Efficient Vitamin Receptor-Mediated Endocytosis and Drug Release

    SciTech Connect

    Chen, S.; Wong, S.; Zhao, X.; Chen, J.; Chen, J.; Kuznetsova, L.; Ojima, I.

    2010-05-01

    An efficient mechanism-based tumor-targeting drug delivery system, based on tumor-specific vitamin-receptor mediated endocytosis, has been developed. The tumor-targeting drug delivery system is a conjugate of a tumor-targeting molecule (biotin: vitamin H or vitamin B-7), a mechanism-based self-immolative linker and a second-generation taxoid (SB-T-1214) as the cytotoxic agent. This conjugate (1) is designed to be (i) specific to the vitamin receptors overexpressed on tumor cell surface and (ii) internalized efficiently through receptor-mediated endocytosis, followed by smooth drug release via glutathione-triggered self-immolation of the linker. In order to monitor and validate the sequence of events hypothesized, i.e., receptor-mediated endocytosis of the conjugate, drug release, and drug-binding to the target protein (microtubules), three fluorescent/fluorogenic molecular probes (2, 3, and 4) were designed and synthesized. The actual occurrence of these processes was unambiguously confirmed by means of confocal fluorescence microscopy (CFM) and flow cytometry using L1210FR leukemia cells, overexpressing biotin receptors. The molecular probe 4, bearing the taxoid linked to fluorescein, was also used to examine the cell specificity (i.e., efficacy of receptor-based cell targeting) for three cell lines, L1210FR (biotin receptors overexpressed), L1210 (biotin receptors not overexpressed), and WI38 (normal human lung fibroblast, biotin receptor negative). As anticipated, the molecular probe 4 exhibited high specificity only to L1210FR. To confirm the direct correlation between the cell-specific drug delivery and anticancer activity of the probe 4, its cytotoxicity against these three cell lines was also examined. The results clearly showed a good correlation between the two methods. In the same manner, excellent cell-specific cytotoxicity of the conjugate 1 (without fluorescein attachment to the taxoid) against the same three cell lines was confirmed. This mechanism

  7. Valerian inhibits rat hepatocarcinogenesis by activating GABA(A) receptor-mediated signaling.

    PubMed

    Kakehashi, Anna; Kato, Ayumi; Ishii, Naomi; Wei, Min; Morimura, Keiichirou; Fukushima, Shoji; Wanibuchi, Hideki

    2014-01-01

    Valerian is widely used as a traditional medicine to improve the quality of sleep due to interaction of several active components with the γ-aminobutyric acid (GABA) A receptor (GABA(A)R) system. Recently, activation of GABA signaling in stem cells has been reported to suppress cell cycle progression in vivo. Furthermore, possible inhibitory effects of GABA(A)R agonists on hepatocarcinogenesis have been reported. The present study was performed to investigate modulating effects of Valerian on hepatocarcinogenesis using a medium-term rat liver bioassay. Male F344 rats were treated with one of the most powerful Valerian species (Valeriana sitchensis) at doses of 0, 50, 500 and 5000 ppm in their drinking water after initiation of hepatocarcinogenesis with diethylnitrosamine (DEN). Formation of glutathione S-transferase placental form positive (GST-P(+)) foci was significantly inhibited by Valerian at all applied doses compared with DEN initiation control rats. Generation of 8-hydroxy-2'-deoxyguanosine in the rat liver was significantly suppressed by all doses of Valerian, likely due to suppression of Nrf2, CYP7A1 and induction of catalase expression. Cell proliferation was significantly inhibited, while apoptosis was induced in areas of GST-P(+) foci of Valerian groups associated with suppression of c-myc, Mafb, cyclin D1 and induction of p21(Waf1/Cip1), p53 and Bax mRNA expression. Interestingly, expression of the GABA(A)R alpha 1 subunit was observed in GST-P(+) foci of DEN control rats, with significant elevation associated with Valerian treatment. These results indicate that Valerian exhibits inhibitory effects on rat hepatocarcinogenesis by inhibiting oxidative DNA damage, suppressing cell proliferation and inducing apoptosis in GST-P(+) foci by activating GABA(A)R-mediated signaling. PMID:25419570

  8. Valerian Inhibits Rat Hepatocarcinogenesis by Activating GABA(A) Receptor-Mediated Signaling

    PubMed Central

    Kakehashi, Anna; Kato, Ayumi; Ishii, Naomi; Wei, Min; Morimura, Keiichirou; Fukushima, Shoji; Wanibuchi, Hideki

    2014-01-01

    Valerian is widely used as a traditional medicine to improve the quality of sleep due to interaction of several active components with the γ-aminobutyric acid (GABA) A receptor (GABA(A)R) system. Recently, activation of GABA signaling in stem cells has been reported to suppress cell cycle progression in vivo. Furthermore, possible inhibitory effects of GABA(A)R agonists on hepatocarcinogenesis have been reported. The present study was performed to investigate modulating effects of Valerian on hepatocarcinogenesis using a medium-term rat liver bioassay. Male F344 rats were treated with one of the most powerful Valerian species (Valeriana sitchensis) at doses of 0, 50, 500 and 5000 ppm in their drinking water after initiation of hepatocarcinogenesis with diethylnitrosamine (DEN). Formation of glutathione S-transferase placental form positive (GST-P+) foci was significantly inhibited by Valerian at all applied doses compared with DEN initiation control rats. Generation of 8-hydroxy-2′-deoxyguanosine in the rat liver was significantly suppressed by all doses of Valerian, likely due to suppression of Nrf2, CYP7A1 and induction of catalase expression. Cell proliferation was significantly inhibited, while apoptosis was induced in areas of GST-P+ foci of Valerian groups associated with suppression of c-myc, Mafb, cyclin D1 and induction of p21Waf1/Cip1, p53 and Bax mRNA expression. Interestingly, expression of the GABA(A)R alpha 1 subunit was observed in GST-P+ foci of DEN control rats, with significant elevation associated with Valerian treatment. These results indicate that Valerian exhibits inhibitory effects on rat hepatocarcinogenesis by inhibiting oxidative DNA damage, suppressing cell proliferation and inducing apoptosis in GST-P+ foci by activating GABA(A)R-mediated signaling. PMID:25419570

  9. Fc receptor-mediated phagocytosis, superoxide production and calcium signaling of beta 2 integrin-deficient bovine neutrophils.

    PubMed

    Nagahata, H; Sawada, C; Higuchi, H; Teraoka, H; Yamaguchi, M

    1997-01-01

    Fc receptor for immunoglobulin G-mediated phagocytosis, superoxide production and intracellular calcium ([Ca2+]i) signaling of complement receptor type 3 (CR3)-deficient neutrophils from a heifer with leukocyte adhesion deficiency (BLAD) were compared to those of control heifers. The mean phagocytic activity of IgG-coated yeasts and aggregated bovine IgG (Agg-IgG)-induced superoxide production of CR3-deficient neutrophils were 10% and 77.9%, respectively, of those of control neutrophils. The [Ca2+]i signals in CR3-deficient neutrophils stimulated with Agg-IgG or concanavalin A were different with mean peak [Ca2+]i concentrations of 78% and 41.9%, respectively, of those of control neutrophils. These findings suggest that Fc receptor-mediated neutrophil functions are closely dependent on the presence of CR3 (CD11b/CD18) on the neutrophil cell surfaces. PMID:9343828

  10. The influence of receptor-mediated interactions on reaction-diffusion mechanisms of cellular self-organisation.

    PubMed

    Klika, Václav; Baker, Ruth E; Headon, Denis; Gaffney, Eamonn A

    2012-04-01

    Understanding the mechanisms governing and regulating self-organisation in the developing embryo is a key challenge that has puzzled and fascinated scientists for decades. Since its conception in 1952 the Turing model has been a paradigm for pattern formation, motivating numerous theoretical and experimental studies, though its verification at the molecular level in biological systems has remained elusive. In this work, we consider the influence of receptor-mediated dynamics within the framework of Turing models, showing how non-diffusing species impact the conditions for the emergence of self-organisation. We illustrate our results within the framework of hair follicle pre-patterning, showing how receptor interaction structures can be constrained by the requirement for patterning, without the need for detailed knowledge of the network dynamics. Finally, in the light of our results, we discuss the ability of such systems to pattern outside the classical limits of the Turing model, and the inherent dangers involved in model reduction. PMID:22072186

  11. Multi-functionalized hyaluronic acid nanogels crosslinked with carbon dots as dual receptor-mediated targeting tumor theranostics.

    PubMed

    Jia, Xu; Han, Yu; Pei, Mingliang; Zhao, Xubo; Tian, Kun; Zhou, Tingting; Liu, Peng

    2016-11-01

    Hyaluronic acid (HA)-based theranostic nanogels were designed for the tumor diagnosis and chemotherapy, by crosslinking the folate-terminated poly(ethylene glycol) modified hyaluronic acid (FA-PEG-HA) with carbon dots (CDs) for the first time. Due to the extraordinary fluorescence property of the integrated CDs, the theranostic nanogels could be used for the real-time and noninvasive location tracking to cancer cells. HA could load Doxorubicin (DOX) via electrostatic interaction with a drug-loading capacity (DLC) of 32.5%. The nanogels possessed an ideal release of DOX in the weak acid environment, while it was restrained in the neutral media, demonstrating the pH-responsive controlled release behavior. The cytotoxicity and cellular uptake results clearly illustrated that most DOX was released and accumulated in the cell nuclei and killed the cancer cells efficaciously, due to their dual receptor-mediated targeting characteristics. PMID:27516286

  12. Apoptosis in the vasculature: mechanisms and functional importance

    PubMed Central

    Mallat, Ziad; Tedgui, Alain

    2000-01-01

    Apoptotic death has now been recognized in a number of common and threatening vascular diseases, including atherosclerosis. Interest in apoptosis research relates to the fact that apoptosis, in contrast to oncosis, is a highly regulated process of cell death which raises the hope for the development of specific therapeutic strategies to alter disease progression. This review summarizes the mechanisms involved in vascular endothelial and smooth muscle cell survival/apoptosis, and the potential roles of apoptotic death in atherosclerosis and restenosis. The potential effects of modulation of apoptosis in these diseases are also discussed. PMID:10882378

  13. Estrous cycle regulation of extrasynaptic δ-containing GABA(A) receptor-mediated tonic inhibition and limbic epileptogenesis.

    PubMed

    Wu, Xin; Gangisetty, Omkaram; Carver, Chase Matthew; Reddy, Doodipala Samba

    2013-07-01

    The ovarian cycle affects susceptibility to behavioral and neurologic conditions. The molecular mechanisms underlying these changes are poorly understood. Deficits in cyclical fluctuations in steroid hormones and receptor plasticity play a central role in physiologic and pathophysiologic menstrual conditions. It has been suggested that synaptic GABA(A) receptors mediate phasic inhibition in the hippocampus and extrasynaptic receptors mediate tonic inhibition in the dentate gyrus. Here we report a novel role of extrasynaptic δ-containing GABA(A) receptors as crucial mediators of the estrous cycle-related changes in neuronal excitability in mice, with hippocampus subfield specificity. In molecular and immunofluorescence studies, a significant increase occurred in δ-subunit, but not α4- and γ2-subunits, in the dentate gyrus during diestrus. However, δ-subunit upregulation was not evident in the CA1 region. The δ-subunit expression was undiminished by age and ovariectomy and in mice lacking progesterone receptors, but it was significantly reduced by finasteride, a neurosteroid synthesis inhibitor. Electrophysiologic studies confirmed greater potentiation of GABA currents by progesterone-derived neurosteroid allopregnanolone in dissociated dentate gyrus granule cells in diestrus than in CA1 pyramidal cells. The baseline conductance and allopregnanolone potentiation of tonic currents in dentate granule cells from hippocampal slices were higher than in CA1 pyramidal cells. In behavioral studies, susceptibility to hippocampus kindling epileptogenesis was lower in mice during diestrus. These results demonstrate the estrous cycle-related plasticity of neurosteroid-sensitive, δ-containing GABA(A) receptors that mediate tonic inhibition and seizure susceptibility. These findings may provide novel insight on molecular cascades of menstrual disorders like catamenial epilepsy, premenstrual syndrome, and migraine. PMID:23667248

  14. Agonist-induced desensitization of histamine H1 receptor-mediated inositol phospholipid hydrolysis in human umbilical vein endothelial cells.

    PubMed Central

    McCreath, G; Hall, I P; Hill, S J

    1994-01-01

    1. The regulation of histamine-induced [3H]-inositol phosphate formation was studied in human cultured umbilical vein endothelial cells (HUVEC). 2. Histamine (EC50 4.8 microM) produced a 12.7 fold increase in [3H]-inositol phosphate formation over basal levels. Prior exposure to 0.1 mM histamine (2 h) produced a 78% reduction in the response to subsequent histamine (0.1 mM) challenge. The IC50 for this histamine-induced desensitization was 0.9 microM. 3. The inositol phosphate response to histamine (0.1 mM) was inhibited by phorbol dibutyrate (IC50 40 nM; maximal reduction 64%). This effect was antagonized by both staurosporine (100 nM) and Ro 31-8220 (10 microM). However, the histamine-induced desensitization of the H1-receptor-mediated inositol phosphate response was insensitive to the protein kinase inhibitors, staurosporine, Ro 31-8220, K252a and KN62. 4. Prior exposure to sodium nitroprusside (100 microM), forskolin (10 microM) or dibutyryl cyclic AMP (1 mM) had no effect upon histamine-induced [3H]-inositol phosphate formation. 5. NaF (20 mM) and thrombin (EC50 0.4 u ml-1) also induced inositol phosphate formation in HUVEC. Histamine pretreatment (0.1 mM, 10-120 min) failed to modify the inositol phosphate response to a subsequent NaF or thrombin challenge. 6. We conclude that the desensitization of histamine H1-receptor-mediated [3H]-inositol phosphate formation occurs at the level of the receptor and involves a mechanism independent of activation of protein kinase A, G, or C, or calcium calmodulin-dependent protein kinase II. PMID:7858873

  15. Halothane inhibits the cholinergic-receptor-mediated influx of calcium in primary culture of bovine adrenal medulla cells

    SciTech Connect

    Yashima, N.; Wada, A.; Izumi, F.

    1986-04-01

    Adrenal medulla cells are cholinoceptive cells. Stimulation of the acetylcholine receptor causes the influx of Ca to the cells, and Ca acts as the coupler of the stimulus-secretion coupling. In this study, the authors investigated the effects of halothane on the receptor-mediated influx of /sup 45/Ca using cultured bovine adrenal medulla cells. Halothane at clinical concentrations (0.5-2%) inhibited the influx of /sup 45/Ca caused by carbachol, with simultaneous inhibition of catecholamine secretion. The influx of /sup 45/Ca and the secretion of catecholamines caused by K depolarization were inhibited by a large concentration of Mg, which competes with Ca at Ca channels, but not inhibited by halothane. Inhibition of the /sup 45/Ca influx by halothane was not overcome by increase in the carbachol concentration. Inhibition of the /sup 45/Ca influx by halothane was examined in comparison with that caused by a large concentration of Mg by the application of Scatchard analysis as the function of the external Ca concentration. Halothane decreased the maximal influx of /sup 45/Ca without altering the apparent kinetic constant of Ca to Ca channels. On the contrary, a large concentration of Mg increased the apparent kinetic constant without altering the maximal influx of /sup 45/Ca. Based on these findings, the authors suggest that inhibition of the /sup 45/Ca influx by halothane was not due to the direct competitive inhibition of Ca channels, nor to the competitive antagonism of agonist-receptor interaction. As a possibility, halothane seems to inhibit the receptor-mediated activation of Ca channels through the interference of coupling between the receptor and Ca channels.

  16. Biomarkers of Chondrocyte Apoptosis and Autophagy in Osteoarthritis

    PubMed Central

    Musumeci, Giuseppe; Castrogiovanni, Paola; Trovato, Francesca Maria; Weinberg, Annelie Martina; Al-Wasiyah, Mohammad K.; Alqahtani, Mohammed H.; Mobasheri, Ali

    2015-01-01

    Cell death with morphological and molecular features of apoptosis has been detected in osteoarthritic (OA) cartilage, which suggests a key role for chondrocyte death/survival in the pathogenesis of OA. Identification of biomarkers of chondrocyte apoptosis may facilitate the development of novel therapies that may eliminate the cause or, at least, slow down the degenerative processes in OA. The aim of this review was to explore the molecular markers and signals that induce chondrocyte apoptosis in OA. A literature search was conducted in PubMed, Scopus, Web of Science and Google Scholar using the keywords chondrocyte death, apoptosis, osteoarthritis, autophagy and biomarker. Several molecules considered to be markers of chondrocyte apoptosis will be discussed in this brief review. Molecular markers and signalling pathways associated with chondroycte apoptosis may turn out to be therapeutic targets in OA and approaches aimed at neutralizing apoptosis-inducing molecules may at least delay the progression of cartilage degeneration in OA. PMID:26334269

  17. Role of NMDA Receptor-Mediated Glutamatergic Signaling in Chronic and Acute Neuropathologies

    PubMed Central

    2016-01-01

    N-Methyl-D-aspartate receptors (NMDARs) have two opposing roles in the brain. On the one hand, NMDARs control critical events in the formation and development of synaptic organization and synaptic plasticity. On the other hand, the overactivation of NMDARs can promote neuronal death in neuropathological conditions. Ca2+ influx acts as a primary modulator after NMDAR channel activation. An imbalance in Ca2+ homeostasis is associated with several neurological diseases including schizophrenia, Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. These chronic conditions have a lengthy progression depending on internal and external factors. External factors such as acute episodes of brain damage are associated with an earlier onset of several of these chronic mental conditions. Here, we will review some of the current evidence of how traumatic brain injury can hasten the onset of several neurological conditions, focusing on the role of NMDAR distribution and the functional consequences in calcium homeostasis associated with synaptic dysfunction and neuronal death present in this group of chronic diseases.

  18. Just So Stories about the Evolution of Apoptosis.

    PubMed

    Green, Douglas R; Fitzgerald, Patrick

    2016-07-11

    Apoptosis is a form of active cell death engaged by developmental cues as well as many different cellular stresses in which the dying cell essentially 'packages' itself for removal. The process of apoptotic cell death, as defined at the molecular level, is unique to the Metazoa (animals). Yet active cell death exists in non-animal organisms, and in some cases molecules involved in such death show some sequence similarities to those involved in apoptosis, leading to extensive speculation regarding the evolution of apoptosis. Here, we examine such speculation from the perspective of the functional properties of molecules of the mitochondrial apoptotic cell death pathway. We suggest scenarios for the evolution of one pathway of apoptosis, the mitochondrial pathway, and consider how they might be tested. We conclude with a 'Just So Story' of how the mitochondrial pathway of apoptosis might have evolved during eukaryotic evolution. PMID:27404257

  19. TRIM32 promotes neural differentiation through retinoic acid receptor-mediated transcription.

    PubMed

    Sato, Tomonobu; Okumura, Fumihiko; Kano, Satoshi; Kondo, Takeshi; Ariga, Tadashi; Hatakeyama, Shigetsugu

    2011-10-15

    Retinoic acid (RA), a metabolite of vitamin A, plays versatile roles in development, differentiation, cell cycles and regulation of apoptosis by regulating gene transcription through nuclear receptor activation. Ubiquitinylation, which is one of the post-translational modifications, appears to be involved in the transcriptional activity of intranuclear receptors including retinoic acid receptor α (RARα). Mutations in the tripartite motif-containing protein 32 gene (TRIM32; also known as E3 ubiquitin-protein ligase) have been reported to be responsible for limb-girdle muscular dystrophy type 2H in humans, and its encoded protein has been shown to interact with several other important proteins. In this study, we found that TRIM32 interacts with RARα and enhances its transcriptional activity in the presence of RA. We also found that overexpression of TRIM32 in mouse neuroblastoma cells and embryonal carcinoma cells promoted stability of RARα, resulting in enhancement of neural differentiation. These findings suggest that TRIM32 functions as one of the co-activators for RARα-mediated transcription, and thereby TRIM32 is a potential therapeutic target for developmental disorders and RA-dependent leukemias. PMID:21984809

  20. P2Y6 Receptor-Mediated Microglial Phagocytosis in Radiation-Induced Brain Injury.

    PubMed

    Xu, Yongteng; Hu, Weihan; Liu, Yimin; Xu, Pengfei; Li, Zichen; Wu, Rong; Shi, Xiaolei; Tang, Yamei

    2016-08-01

    Microglia are the resident immune cells and the professional phagocytic cells of the CNS, showing a multitude of cellular responses after activation. However, how microglial phagocytosis changes and whether it is involved in radiation-induced brain injury remain unknown. In the current study, we found that microglia were activated and microglial phagocytosis was increased by radiation exposure both in cultured microglia in vitro and in mice in vivo. Radiation increased the protein expression of the purinergic receptor P2Y6 receptor (P2Y6R) located on microglia. The selective P2Y6 receptor antagonist MRS2578 suppressed microglial phagocytosis after radiation exposure. Inhibition of microglial phagocytosis increased inhibitory factor Nogo-A and exacerbated radiation-induced neuronal apoptosis and demyelination. We also found that the levels of protein expression for phosphorylated Ras-related C3 botulinum toxin substrate 1 (Rac1) and myosin light chain kinase (MLCK) were elevated, indicating that radiation exposure activated Rac1 and MLCK. The Rac1 inhibitor NSC23766 suppressed expression of MLCK, indicating that the Rac1-MLCK pathway was involved in microglial phagocytosis. Taken together, these findings suggest that the P2Y6 receptor plays a critical role in mediating microglial phagocytosis in radiation-induced brain injury, which might be a potential strategy for therapeutic intervention to alleviate radiation-induced brain injury. PMID:26099306

  1. An AIF orthologue regulates apoptosis in yeast

    PubMed Central

    Wissing, Silke; Ludovico, Paula; Herker, Eva; Büttner, Sabrina; Engelhardt, Silvia M.; Decker, Thorsten; Link, Alexander; Proksch, Astrid; Rodrigues, Fernando; Corte-Real, Manuela; Fröhlich, Kai-Uwe; Manns, Joachim; Candé, Céline; Sigrist, Stephan J.; Kroemer, Guido; Madeo, Frank

    2004-01-01

    Apoptosis-inducing factor (AIF), a key regulator of cell death, is essential for normal mammalian development and participates in pathological apoptosis. The proapoptotic nature of AIF and its mode of action are controversial. Here, we show that the yeast AIF homologue Ynr074cp controls yeast apoptosis. Similar to mammalian AIF, Ynr074cp is located in mitochondria and translocates to the nucleus of yeast cells in response to apoptotic stimuli. Purified Ynr074cp degrades yeast nuclei and plasmid DNA. YNR074C disruption rescues yeast cells from oxygen stress and delays age-induced apoptosis. Conversely, overexpression of Ynr074cp strongly stimulates apoptotic cell death induced by hydrogen peroxide and this effect is attenuated by disruption of cyclophilin A or the yeast caspase YCA1. We conclude that Ynr074cp is a cell death effector in yeast and rename it AIF-1 (Aif1p, gene AIF1). PMID:15381687

  2. Neurotrophin signalling pathways regulating neuronal apoptosis.

    PubMed

    Miller, F D; Kaplan, D R

    2001-07-01

    Recent evidence indicates that naturally occurring neuronal death in mammals is regulated by the interplay between receptor-mediated prosurvival and proapoptotic signals. The neurotrophins, a family of growth factors best known for their positive effects on neuronal biology, have now been shown to mediate both positive and negative survival signals, by signalling through the Trk and p75 neurotrophin receptors, respectively. The mechanisms whereby these two neurotrophin receptors interact to determine neuronal survival have been difficult to decipher, largely because both can signal independently or coincidentally, depending upon the cell or developmental context. Nonetheless, the past several years have seen significant advances in our understanding of this receptor signalling system. In this review, we focus on the proapoptotic actions of the p75 neurotrophin receptor (p75NTR), and on the interplay between Trk and p75NTR that determines neuronal survival. PMID:11529497

  3. JNK Signaling in Apoptosis

    PubMed Central

    Dhanasekaran, Danny N.; Reddy, E. Premkumar

    2011-01-01

    Jun N-terminal kinases or JNKs play a critical role in death receptor-initiated extrinsic as well as mitochondrial intrinsic apoptotic pathways. JNKs activate apoptotic signaling by the upregulation pro-apoptotic genes via the transactivation of specific transcription factors or by directly modulating the activities of mitochondrial pro- and anti-apoptotic proteins through distinct phosphorylation events. This review analyzes our present understanding of the role of JNK in apoptotic signaling and the various mechanisms by which JNK promotes apoptosis PMID:18931691

  4. Apoptosis and Molecular Targeting Therapy in Cancer

    PubMed Central

    Hassan, Mohamed; Watari, Hidemichi; AbuAlmaaty, Ali; Ohba, Yusuke; Sakuragi, Noriaki

    2014-01-01

    Apoptosis is the programmed cell death which maintains the healthy survival/death balance in metazoan cells. Defect in apoptosis can cause cancer or autoimmunity, while enhanced apoptosis may cause degenerative diseases. The apoptotic signals contribute into safeguarding the genomic integrity while defective apoptosis may promote carcinogenesis. The apoptotic signals are complicated and they are regulated at several levels. The signals of carcinogenesis modulate the central control points of the apoptotic pathways, including inhibitor of apoptosis (IAP) proteins and FLICE-inhibitory protein (c-FLIP). The tumor cells may use some of several molecular mechanisms to suppress apoptosis and acquire resistance to apoptotic agents, for example, by the expression of antiapoptotic proteins such as Bcl-2 or by the downregulation or mutation of proapoptotic proteins such as BAX. In this review, we provide the main regulatory molecules that govern the main basic mechanisms, extrinsic and intrinsic, of apoptosis in normal cells. We discuss how carcinogenesis could be developed via defective apoptotic pathways or their convergence. We listed some molecules which could be targeted to stimulate apoptosis in different cancers. Together, we briefly discuss the development of some promising cancer treatment strategies which target apoptotic inhibitors including Bcl-2 family proteins, IAPs, and c-FLIP for apoptosis induction. PMID:25013758

  5. Cot Deaths.

    ERIC Educational Resources Information Center

    Tyrrell, Shelagh

    1985-01-01

    Addresses the tragedy of crib deaths, giving particular attention to causes, prevention, and medical research on Sudden Infant Death Syndrome (SIDS). Gives anecdotal accounts of coping strategies used by parents and families of SIDS infants. (DT)

  6. Understanding Death.

    ERIC Educational Resources Information Center

    Heath, Charles P.

    1986-01-01

    Bibliotherapy can help children prepare for and understand the death of a loved one. An annotated bibliography lists references with age level information on attitudes toward death and deaths of a father, friend, grandparent, mother, pet, and sibling. (Author/CL)

  7. Tissue plasminogen activator inhibits NMDA-receptor-mediated increases in calcium levels in cultured hippocampal neurons

    PubMed Central

    Robinson, Samuel D.; Lee, Tet Woo; Christie, David L.; Birch, Nigel P.

    2015-01-01

    NMDA receptors (NMDARs) play a critical role in neurotransmission, acting as essential mediators of many forms of synaptic plasticity, and also modulating aspects of development, synaptic transmission and cell death. NMDAR-induced responses are dependent on a range of factors including subunit composition and receptor location. Tissue-type plasminogen activator (tPA) is a serine protease that has been reported to interact with NMDARs and modulate NMDAR activity. In this study we report that tPA inhibits NMDAR-mediated changes in intracellular calcium levels in cultures of primary hippocampal neurons stimulated by low (5 μM) but not high (50 μM) concentrations of NMDA. tPA also inhibited changes in calcium levels stimulated by presynaptic release of glutamate following treatment with bicucculine/4-aminopyridine (4-AP). Inhibition was dependent on the proteolytic activity of tPA but was unaffected by α2-antiplasmin, an inhibitor of the tPA substrate plasmin, and receptor-associated protein (RAP), a pan-ligand blocker of the low-density lipoprotein receptor, two proteins previously reported to modulate NMDAR activity. These findings suggest that tPA can modulate changes in intracellular calcium levels in a subset of NMDARs expressed in cultured embryonic hippocampal neurons through a mechanism that involves the proteolytic activity of tPA and synaptic NMDARs. PMID:26500501

  8. The trk family of receptors mediates nerve growth factor and neurotrophin-3 effects in melanocytes.

    PubMed Central

    Yaar, M; Eller, M S; DiBenedetto, P; Reenstra, W R; Zhai, S; McQuaid, T; Archambault, M; Gilchrest, B A

    1994-01-01

    We have recently shown that (a) human melanocytes express the p75 nerve growth factor (NGF) receptor in vitro; (b) that melanocyte dendricity and migration, among other behaviors, are regulated at least in part by NGF; and (c) that cultured human epidermal keratinocytes produce NGF. We now report that melanocyte stimulation with phorbol 12-tetra decanoate 13-acetate (TPA), previously reported to induce p75 NGF receptor, also induces trk in melanocytes, and TPA effect is further potentiated by the presence of keratinocytes in culture. Moreover, trk in melanocytes becomes phosphorylated within minutes after NGF stimulation. As well, cultures of dermal fibroblasts express neurotrophin-3 (NT-3) mRNA; NT-3 mRNA levels in cultured fibroblasts are modulated by mitogenic stimulation, UV irradiation, and exposure to melanocyte-conditioned medium. Moreover, melanocytes constitutively express low levels of trk-C, and its expression is downregulated after TPA stimulation. NT-3 supplementation to cultured melanocytes maintained in Medium 199 alone prevents cell death. These combined data suggest that melanocyte behavior in human skin may be influenced by neurotrophic factors, possibly of keratinocyte and fibroblast origin, which act through high affinity receptors. Images PMID:7929831

  9. Central role for hydrogen peroxide in P2Y1 ADP receptor-mediated cellular responses in vascular endothelium

    PubMed Central

    Kalwa, Hermann; Sartoretto, Juliano L.; Martinelli, Roberta; Romero, Natalia; Steinhorn, Benjamin S.; Tao, Ming; Ozaki, C. Keith; Carman, Christopher V.; Michel, Thomas

    2014-01-01

    ADP activates a family of cell surface receptors that modulate signaling pathways in a broad range of cells. ADP receptor antagonists are widely used to treat cardiovascular disease states. These studies identify a critical role for the stable reactive oxygen species hydrogen peroxide (H2O2) in mediating cellular responses activated by the G protein-coupled P2Y1 receptor for ADP. We found that ADP-dependent phosphorylation of key endothelial signaling proteins—including endothelial nitric oxide synthase, AMP-activated protein kinase, and the actin-binding MARCKS protein—was blocked by preincubation with PEG-catalase, which degrades H2O2. ADP treatment promoted the H2O2-dependent phosphorylation of c-Abl, a nonreceptor tyrosine kinase that modulates the actin cytoskeleton. Cellular imaging experiments using fluorescence resonance energy transfer-based biosensors revealed that ADP-stimulated activation of the cytoskeleton-associated small GTPase Rac1 was independent of H2O2. However, Rac1-dependent activation of AMP-activated protein kinase, the signaling phospholipid phosphatidylinositol-(4, 5)-bisphosphate, and the c-Abl–interacting protein CrkII are mediated by H2O2. We transfected endothelial cells with differentially targeted HyPer2 H2O2 biosensors and found that ADP promoted a marked increase in H2O2 levels in the cytosol and caveolae, and a smaller increase in mitochondria. We performed a screen for P2Y1 receptor-mediated receptor tyrosine kinase transactivation and discovered that ADP transactivates Fms-like tyrosine kinase 3 (Flt3), a receptor tyrosine kinase expressed in these cells. Our observation that P2Y1 receptor-mediated responses involve Flt3 transactivation may identify a unique mechanism whereby cancer chemotherapy with receptor tyrosine kinase inhibitors promotes vascular dysfunction. Taken together, these findings establish a critical role for endogenous H2O2 in control of ADP-mediated signaling responses in the vascular wall. PMID:24550450

  10. Genetically designed biomolecular capping system for mesoporous silica nanoparticles enables receptor-mediated cell uptake and controlled drug release

    NASA Astrophysics Data System (ADS)

    Datz, Stefan; Argyo, Christian; Gattner, Michael; Weiss, Veronika; Brunner, Korbinian; Bretzler, Johanna; von Schirnding, Constantin; Torrano, Adriano A.; Spada, Fabio; Vrabel, Milan; Engelke, Hanna; Bräuchle, Christoph; Carell, Thomas; Bein, Thomas

    2016-04-01

    Effective and controlled drug delivery systems with on-demand release and targeting abilities have received enormous attention for biomedical applications. Here, we describe a novel enzyme-based cap system for mesoporous silica nanoparticles (MSNs) that is directly combined with a targeting ligand via bio-orthogonal click chemistry. The capping system is based on the pH-responsive binding of an aryl-sulfonamide-functionalized MSN and the enzyme carbonic anhydrase (CA). An unnatural amino acid (UAA) containing a norbornene moiety was genetically incorporated into CA. This UAA allowed for the site-specific bio-orthogonal attachment of even very sensitive targeting ligands such as folic acid and anandamide. This leads to specific receptor-mediated cell and stem cell uptake. We demonstrate the successful delivery and release of the chemotherapeutic agent Actinomycin D to KB cells. This novel nanocarrier concept provides a promising platform for the development of precisely controllable and highly modular theranostic systems.Effective and controlled drug delivery systems with on-demand release and targeting abilities have received enormous attention for biomedical applications. Here, we describe a novel enzyme-based cap system for mesoporous silica nanoparticles (MSNs) that is directly combined with a targeting ligand via bio-orthogonal click chemistry. The capping system is based on the pH-responsive binding of an aryl-sulfonamide-functionalized MSN and the enzyme carbonic anhydrase (CA). An unnatural amino acid (UAA) containing a norbornene moiety was genetically incorporated into CA. This UAA allowed for the site-specific bio-orthogonal attachment of even very sensitive targeting ligands such as folic acid and anandamide. This leads to specific receptor-mediated cell and stem cell uptake. We demonstrate the successful delivery and release of the chemotherapeutic agent Actinomycin D to KB cells. This novel nanocarrier concept provides a promising platform for the

  11. Prolactin receptor-mediated internalization of imaging agents detects epithelial ovarian cancer

    NASA Astrophysics Data System (ADS)

    Sundaram, Karthik M.

    Epithelial ovarian cancer (EOC) has the highest mortality rate of all gynecologic malignant tumors. Diagnosis of epithelial ovarian cancer (EOC) presents two main challenges. The first challenge is detecting low volume (< 1 g) and early stage (≤ stage II) masses to prevent rapid progression to late stages and ultimately death. The second challenge is differentiating malignant from benign tissue to avoid costly and invasive surgeries (19.5 surgeries are required to find 1 cancer even with multiple screenings). First-line diagnostic tests such as ultrasound and serum marker tests (e.g. CA-125) aid in diagnosis but they lack the sensitivity and specificity required to overcome both challenges. Magnetic resonance imaging (MRI), a second-line diagnostic aided by gadolinium based contrast agents (CAs), offers higher resolution pictures for classifying indeterminate ovarian masses. But as currently practiced, MRI still lacks the sensitivity and specificity required to alter patient outcomes. In this work we develop a new paradigm for EOC diagnosis that targets the prolactin receptor (PRLR) - a cell surface tyrosine kinase receptor that is over-expressed in moderate to high levels on > 98% of epithelial ovarian cancers. Upon binding of native ligands to PRLR, the ligand:PRLR complex is internalized by cells. By conjugating gadolinium-chelates, molecules normally used as contrast agents diagnostically, to human placental lactogen (hPL), a native ligand of PRLR, we show that MRI becomes highly sensitive and specific for detecting PRLR (+) tumors in a nude mouse model of EOC. We further establish the adaptability of this approach for fluorescence-based imaging techniques using an hPL conjugated Cy5.5 dye. We conclude that molecular imaging of PRLR with hPL-conjugated imaging agents can address the current challenges that limit EOC diagnosis.

  12. Apoptosis pathways and their therapeutic exploitation in pancreatic cancer.

    PubMed

    Fulda, Simone

    2009-07-01

    Resistance to apoptosis (programmed cell death) is a characteristic feature of human malignancies including pancreatic cancer, which is one of the leading causes of cancer deaths in the western world. Defects in this intrinsic cell death program can contribute to the multistep process of tumorigenesis, because too little cell death can disturb tissue homeostasis. Further, blockade of apoptosis pathways can cause treatment failure, because intact apoptosis signalling cascades largely mediate therapy-induced cytotoxicity. The elucidation of apoptosis pathways in pancreatic carcinoma over the last decade has resulted in the identification of various molecular defects. How apoptosis pathways can be exploited for the treatment of pancreatic cancer will be discussed in this review. PMID:19382915

  13. Apoptosis pathways and their therapeutic exploitation in pancreatic cancer

    PubMed Central

    Fulda, Simone

    2009-01-01

    Resistance to apoptosis (programmed cell death) is a characteristic feature of human malignancies including pancreatic cancer, which is one of the leading causes of cancer deaths in the western world. Defects in this intrinsic cell death program can contribute to the multistep process of tumorigenesis, because too little cell death can disturb tissue homeostasis. Further, blockade of apoptosis pathways can cause treatment failure, because intact apoptosis signalling cascades largely mediate therapy-induced cytotoxicity. The elucidation of apoptosis pathways in pancreatic carcinoma over the last decade has resulted in the identification of various molecular defects. How apoptosis pathways can be exploited for the treatment of pancreatic cancer will be discussed in this review. PMID:19382915

  14. Receptor interconversion model of hormone action. 3. Estrogen receptor mediated repression of reporter gene activity in A431 cells.

    PubMed

    Nag, A; Park, I; Krust, A; Smith, R G

    1990-03-20

    The chicken estrogen receptor exists in three interconvertible forms, two of which bind estradiol with high affinity and one which lacks the capacity to bind estradiol. Interconversion is regulated by reactions involving ATP/Mg2+. By cotransfecting into A431 cells estrogen receptor cDNA in an expression vector together with the pA2 (-821/-87) tk-CAT vitellogenin construct, we demonstrate that constitutive expression of chloramphenicol acetyltransferase (CAT) activity can be regulated either by selection of ligand or by modifying phosphorylation reactions in the recipient cells. In the presence of estrogen receptors, constitutive expression of CAT activity is inhibited in three situations: (i) in the absence of an estrogenic ligand; (ii) in the presence of an anti-estrogen; and (iii) in the presence of an estrogenic ligand together with 12-O-tetradecanoylphorbol 13-acetate (TPA). Estrogen receptor mediated repression of constitutive CAT activity is not observed with the pA2 (-331/-87) tk-CAT construct, indicating that DNA sequences required for repression are located between -821 and -331 base pairs upstream of the transcription initiation site. PMID:2346742

  15. Clathrin and AP2 Are Required for Phagocytic Receptor-Mediated Apoptotic Cell Clearance in Caenorhabditis elegans

    PubMed Central

    Liu, Xuezhao; Zhang, Yuanya; Liang, Jingjing; Qi, Xiaying; Du, Hongwei; Zou, Wei; Chen, Lianwan; Chai, Yongping; Ou, Guangshuo; Miao, Long; Wang, Yingchun; Yang, Chonglin

    2013-01-01

    Clathrin and the multi-subunit adaptor protein complex AP2 are central players in clathrin-mediated endocytosis by which the cell selectively internalizes surface materials. Here, we report the essential role of clathrin and AP2 in phagocytosis of apoptotic cells. In Caenorhabditis elegans, depletion of the clathrin heavy chain CHC-1 and individual components of AP2 led to a significant accumulation of germ cell corpses, which resulted from defects in both cell corpse engulfment and phagosome maturation required for corpse removal. CHC-1 and AP2 components associate with phagosomes in an inter-dependent manner. Importantly, we found that the phagocytic receptor CED-1 interacts with the α subunit of AP2, while the CED-6/Gulp adaptor forms a complex with both CHC-1 and the AP2 complex, which likely mediates the rearrangement of the actin cytoskeleton required for cell corpse engulfment triggered by the CED-1 signaling pathway. In addition, CHC-1 and AP2 promote the phagosomal association of LST-4/Snx9/18/33 and DYN-1/dynamin by forming a complex with them, thereby facilitating the maturation of phagosomes necessary for corpse degradation. These findings reveal a non-classical role of clathrin and AP2 and establish them as indispensable regulators in phagocytic receptor-mediated apoptotic cell clearance. PMID:23696751

  16. The positive feedback action of vasopressin on its own release from rat septal tissue in vitro is receptor-mediated.

    PubMed

    Landgraf, R; Ramirez, A D; Ramirez, V D

    1991-04-01

    The effect of arginine vasopressin (AVP) on its own septal release was evaluated using an in vitro superfusion procedure. As compared to basal release from septal fragments, pulses of synthetic AVP (15 pg/5 min) resulted in a 25-fold augmented release of endogenous AVP, indicating a positive feedback action. Both the basal and stimulated AVP release were significantly increased by 60 mM potassium and markedly reduced by omission of calcium. Preincubation of the septal fragments with the V2/V1 AVP receptor antagonist d(CH2)5 [D-Tyr (Et)2,Val4]AVP resulted in a dose-dependent inhibition of the positive feedback action of AVP which was nearly completely blocked at doses between 1.25 and 5 ng per 100 microliters incubation medium. As compared to this effect, the V1 antagonist d(CH2)5 Tyr (Me)2 AVP as well as oxytocin were significantly less potent. The results suggest that the positive feedback action of AVP on its own release from septal fragments is potassium-stimulated, calcium-dependent and mainly V2 receptor-mediated. The physiological significance of this phenomenon remains to be shown. PMID:1830507

  17. α(5)GABA(A) receptors mediate primary afferent fiber tonic excitability in the turtle spinal cord.

    PubMed

    Loeza-Alcocer, Emanuel; Canto-Bustos, Martha; Aguilar, Justo; González-Ramírez, Ricardo; Felix, Ricardo; Delgado-Lezama, Rodolfo

    2013-11-01

    γ-Amino butyric acid (GABA) plays a key role in the regulation of central nervous system by activating synaptic and extrasynaptic GABAA receptors. It is acknowledged that extrasynaptic GABAA receptors located in the soma, dendrites, and axons may be activated tonically by low extracellular GABA concentrations. The activation of these receptors produces a persistent conductance that can hyperpolarize or depolarize nerve cells depending on the Cl(-) equilibrium potential. In an in vitro preparation of the turtle spinal cord we show that extrasynaptic α5GABAA receptors mediate the tonic state of excitability of primary afferents independently of the phasic primary afferent depolarization mediated by synaptic GABAA receptors. Blockade of α5GABAA receptors with the inverse agonist L-655,708 depressed the dorsal root reflex (DRR) without affecting the phasic increase in excitability of primary afferents. Using RT-PCR and Western blotting, we corroborated the presence of the mRNA and the α5GABAA protein in the dorsal root ganglia of the turtle spinal cord. The receptors were localized in primary afferents in dorsal root, dorsal root ganglia, and peripheral nerve terminals using immunoconfocal microscopy. Considering the implications of the DRR in neurogenic inflammation, α5GABAA receptors may serve as potential pharmacological targets for the treatment of pain. PMID:23966669

  18. Fatty acyl specificity of the receptor-mediated release of polyunsaturated fatty acids from vascular endothelial cells

    SciTech Connect

    Rosenthal, M.D.

    1987-05-01

    Histamine and bradykinin appear to exhibit the same fatty acid specificity as thrombin. Incubation of human umbilical vein endothelial cells with 10 ..mu..M histamine for 10 min in buffered saline containing 50 ..mu..M fat-free albumin stimulates the release of previously incorporated (/sup 14/C)arachidonate but not (/sup 14/C)22:4(n-6) or (/sup 14/C)20:3(n-6). Similarly calf pulmonary artery endothelial cells release (/sup 14/C)arachidonate but not (/sup 14/C)22:4(n-6) in response to either bradykinin (1 /sup +/g/ml) or histamine (10..mu..M). In both types of endothelial cells, the calcium ionophore A23187 (10 ..mu..M) exhibits the same pattern of fatty acyl specificity as the receptor-mediated agonists. By contrast, mellitin (2-4 ..mu..g/ml) stimulates the release of free 22:4(n-6) and oleate in addition to arachidonate; release of 22:4(n-6) is 30-70% that of arachidonate. These results suggest that histamine, bradykinin and thrombin stimulate a common calcium-dependent fatty acyl-specific phospholipase activity.

  19. Interrogating the Role of Receptor-Mediated Mechanisms: Biological Fate of Peptide-Functionalized Radiolabeled Gold Nanoparticles in Tumor Mice.

    PubMed

    Silva, Francisco; Zambre, Ajit; Campello, Maria Paula Cabral; Gano, Lurdes; Santos, Isabel; Ferraria, Ana Maria; Ferreira, Maria João; Singh, Amolak; Upendran, Anandhi; Paulo, António; Kannan, Raghuraman

    2016-04-20

    To get a better insight on the transport mechanism of peptide-conjugated nanoparticles to tumors, we performed in vivo biological studies of bombesin (BBN) peptide functionalized gold nanoparticles (AuNPs) in human prostate tumor bearing mice. Initially, we sought to compare AuNPs with thiol derivatives of acyclic and macrocyclic chelators of DTPA and DOTA types. The DTPA derivatives were unable to provide a stable coordination of (67)Ga, and therefore, the functionalization with the BBN analogues was pursued for the DOTA-containing AuNPs. The DOTA-coated AuNPs were functionalized with BBN[7-14] using a unidentate cysteine group or a bidentate thioctic group to attach the peptide. AuNPs functionalized with thioctic-BBN displayed the highest in vitro cellular internalization (≈ 25%, 15 min) in gastrin releasing peptide (GRP) receptor expressing cancer cells. However, these results fail to translate to in vivo tumor uptake. Biodistribution studies following intravenous (IV) and intraperitoneal (IP) administration of nanoconjugates in tumor bearing mice indicated that the presence of BBN influences to some degree the biological profile of the nanoconstructs. For IV administration, the receptor-mediated pathway appears to be outweighed by the EPR effect. By contrast, in IP administration, it is reasoned that the GRPr-mediated mechanism plays a role in pancreas uptake. PMID:27003101

  20. Leukotriene D4 receptor-mediated hydrolysis of phosphoinositide and mobilization of calcium in sheep tracheal smooth muscle cells

    SciTech Connect

    Mong, S.; Miller, J.; Wu, H.L.; Crooke, S.T.

    1988-02-01

    A sheep tracheal smooth muscle primary culture cell system was developed to characterize leukotriene D4 (LTD4) receptor-mediated biochemical and pharmacological effects. (/sup 3/H)LTD4 binding to the enriched plasma membrane receptor was specific, stereoselective and saturable. LTE4 and high affinity receptor antagonists bound to the receptors with a rank-order potency that was expected from previous smooth muscle contraction studies. In the (/sup 3/H)myoinositol labeled cells, LTD4 and LTE4 induced phosphoinositide hydrolysis. The biosynthesis of (/sup 3/H)inositol-trisphosphate was rapid and the induction of biosynthesis of (/sup 3/H)inositol-monophosphate by LTs was stereoselective and specific and was inhibited specifically by a receptor antagonist, SKF 104353. In the fura-2 loaded smooth muscle cells, LTD4 and LTE4 induced transient intracellular Ca++ mobilization. The fura-2/Ca++ transient was stereoselective and specific and was inhibited by receptor antagonist, SKF 104353. These results suggest that the cultured sheep tracheal smooth muscle cells have plasma membrane receptors for LTD4. These receptors were coupled to a phospholipase C that, when activated by agonists, induced hydrolysis of inositol containing phospholipids. The hydrolysis products, e.g. diacylglycerol and inositol-trisphosphate, may serve as intracellular messengers that trigger or contribute to the contractile effect in sheep tracheal smooth muscle.

  1. The overexpressed human 46-kDa mannose 6-phosphate receptor mediates endocytosis and sorting of. beta. -glucuronidase

    SciTech Connect

    Watanabe, H.; Grubb, J.H.; Sly, W.S. )

    1990-10-01

    The authors studied the function of the human small (46-kDa) mannose 6-phosphate receptor (SMPR) in transfected mouse L cells that do not express the larger insulin-like growth factor II/mannose 6-phosphate receptor. Cells overexpressing human SMPR were studied for enzyme binding to cell surface receptors, for binding to intracellular receptors in permeabilized cells, and for receptor-mediated endocytosis of recombinant human {beta}-glucuronidase. Specific binding to human SMPR in permeabilized cells showed a pH optimum between pH 6.0 and pH 6.5. Binding was significant in the present of EDTA but was enhanced by added divalent cations. Up to 2.3{percent} of the total functional receptor could be detected on the cell surface by enzyme binding. They present experiments showing that at very high levels of overexpression, and at pH 6.5, human SMPR mediated the endocytosis of {beta}-glucuronidase. At pH 7.5, the rate of endocytosis was only 14{percent} the rate seen at pH 6.5. Cells overexpressing human SMPR also showed reduced secretion of newly synthesized {beta}-glucuronidase when compared to cells transfected with vector only, suggesting that overexpressed human SMPR can participate in sorting of newly synthesized {beta}-glucuronidase and partially correct the sorting defect in mouse L cells that do not express the insulin-like growth factor II/mannose 6-phosphate receptor.

  2. Cryptococcus neoformans Is Internalized by Receptor-Mediated or ‘Triggered’ Phagocytosis, Dependent on Actin Recruitment

    PubMed Central

    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. PMID:24586631

  3. Receptor-mediated cell attachment and detachment kinetics. II. Experimental model studies with the radial-flow detachment assay.

    PubMed Central

    Cozens-Roberts, C; Quinn, J A; Lauffenburger, D A

    1990-01-01

    Quantitative information regarding the kinetics of receptor-mediated cell adhesion to a ligand-coated surface are crucial for understanding the role of certain key parameters in many physiological and biotechnology-related processes. Here, we use the probabilistic attachment and detachment models developed in the preceding paper to interpret transient data from well-defined experiments. These data are obtained with a simple model cell system that consists of receptor-coated latex beads (prototype cells) and a Radial-Flow Detachment Assay (RFDA) using a ligand-coated glass disc. The receptors and ligands used in this work are complementary antibodies. The beads enable us to examine transient behavior with particles that possess fairly uniform properties that can be varied systematically, and the RFDA is designed for direct observation of adhesion to the ligand-coated glass surface over a range of shear stresses. Our experiments focus on the effects of surface shear stress, receptor density, and ligand density. These data provide a crucial test of the probabilistic framework. We show that these data can be explained with the probabilistic analyses, whereas they cannot be readily interpreted on the basis of a deterministic analysis. In addition, we examine transient data on cell adhesion reported from other assays, demonstrating the consistency of these data with the predictions of the probabilistic models. Images FIGURE 2 PMID:2174272

  4. Receptor-mediated activation of a plant Ca2+-permeable ion channel involved in pathogen defense

    PubMed Central

    Zimmermann, Sabine; Nürnberger, Thorsten; Frachisse, Jean-Marie; Wirtz, Wolfgang; Guern, Jean; Hedrich, Rainer; Scheel, Dierk

    1997-01-01

    Pathogen recognition at the plant cell surface typically results in the initiation of a multicomponent defense response. Transient influx of Ca2+ across the plasma membrane is postulated to be part of the signaling chain leading to pathogen resistance. Patch-clamp analysis of parsley protoplasts revealed a novel Ca2+-permeable, La3+-sensitive plasma membrane ion channel of large conductance (309 pS in 240 mM CaCl2). At an extracellular Ca2+ concentration of 1 mM, which is representative of the plant cell apoplast, unitary channel conductance was determined to be 80 pS. This ion channel (LEAC, for large conductance elicitor-activated ion channel) is reversibly activated upon treatment of parsley protoplasts with an oligopeptide elicitor derived from a cell wall protein of Phytophthora sojae. Structural features of the elicitor found previously to be essential for receptor binding, induction of defense-related gene expression, and phytoalexin formation are identical to those required for activation of LEAC. Thus, receptor-mediated stimulation of this channel appears to be causally involved in the signaling cascade triggering pathogen defense in parsley. PMID:11038609

  5. B cells from patients with systemic lupus erythematosus display abnormal antigen receptor-mediated early signal transduction events.

    PubMed Central

    Liossis, S N; Kovacs, B; Dennis, G; Kammer, G M; Tsokos, G C

    1996-01-01

    To understand the molecular mechanisms that are responsible for the B cell overactivity that is observed in patients with SLE, we have conducted experiments in which the surface immunoglobulin (sIg)-mediated early cell signaling events were studied. The anti-sIgM-mediated free intracytoplasmic calcium ([Ca2+]i) responses were significantly higher in SLE B cells compared with responses of normal individuals and to those of patients with other systemic autoimmune rheumatic diseases. The anti-IgD mAb induced [Ca2+]i responses were also higher in lupus B cells than in controls. The magnitude of anti-sIgM-mediated Ca2+ release from intracellular stores was also increased in B cells from SLE patients compared with normal controls. The amount of inositol phosphate metabolites produced upon crosslinking of sIgM was slightly higher in patients with lupus than in normal controls, although the difference was not statistically significant. In contrast, the degree of anti-sIgM-induced protein tyrosine phosphorylation was obviously increased in lupus patients. Our study demonstrates clearly for the first time that SLE B cells exhibit aberrant early signal transduction events, including augmented calcium responses after crosslinking of the B cell receptor and increased antigen-receptor-mediated phosphorylation of protein tyrosine residues. Because the above abnormalities did not correlate with disease activity or treatment status, we propose that they may have pathogenic significance. PMID:8958217

  6. [Morphological and biochemical criteria for cell death].

    PubMed

    Chernikov, V P; Belousova, T A; Kakturskiĭ, L V

    2010-01-01

    The state-of-the-art of classifications of and criteria for cell death in the light of the 2009 recommendations of the Nomenclature Committee on Cell Death is presented as a lecture. Motivation is given for the necessity of using the unified criteria in the description of cell death and more than one study in its verification. The major structural and biochemical signs of four typical types of cell death--apoptosis, autophagia, keratinization, and necrosis are compared. Data are given on the major atypical forms of cell death--mitotic catastrophe, anoikis, exitotoxicity, Wallerian degeneration, paraptosis, pyroptosis, pyronecrosis, and entosis. PMID:20734836

  7. Cell death proteomics database: consolidating proteomics data on cell death.

    PubMed

    Arntzen, Magnus Ø; Bull, Vibeke H; Thiede, Bernd

    2013-05-01

    Programmed cell death is a ubiquitous process of utmost importance for the development and maintenance of multicellular organisms. More than 10 different types of programmed cell death forms have been discovered. Several proteomics analyses have been performed to gain insight in proteins involved in the different forms of programmed cell death. To consolidate these studies, we have developed the cell death proteomics (CDP) database, which comprehends data from apoptosis, autophagy, cytotoxic granule-mediated cell death, excitotoxicity, mitotic catastrophe, paraptosis, pyroptosis, and Wallerian degeneration. The CDP database is available as a web-based database to compare protein identifications and quantitative information across different experimental setups. The proteomics data of 73 publications were integrated and unified with protein annotations from UniProt-KB and gene ontology (GO). Currently, more than 6,500 records of more than 3,700 proteins are included in the CDP. Comparing apoptosis and autophagy using overrepresentation analysis of GO terms, the majority of enriched processes were found in both, but also some clear differences were perceived. Furthermore, the analysis revealed differences and similarities of the proteome between autophagosomal and overall autophagy. The CDP database represents a useful tool to consolidate data from proteome analyses of programmed cell death and is available at http://celldeathproteomics.uio.no. PMID:23537399

  8. Apoptosis deregulation in myeloproliferative neoplasms

    PubMed Central

    Tognon, Raquel; Nunes, Natália de Souza; de Castro, Fabíola Attié

    2013-01-01

    ABSTRACT Philadelphia-chromosome negative chronic myeloproliferative neoplasms are clonal hematologic diseases characterized by hematopoietic progenitor independence from or hypersensitivity to cytokines. The cellular and molecular mechanisms involved in the pathophysiology of myeloproliferative neoplasms have not yet been fully clarified. Pathophysiologic findings relevant for myeloproliferative neoplasms are associated with genetic alterations, such as, somatic mutation in the gene that codifies JAK-2 (JAK V617F). Deregulation of the process of programmed cellular death, called apoptosis, seems to participate in the pathogenesis of these disorders. It is known that expression deregulation of pro- and anti-apoptotic genes promotes cell resistance to apoptosis, culminating with the accumulation of myeloid cells and establishing neoplasms. This review will focus on the alterations in apoptosis regulation in myeloproliferative neoplasms, and the importance of a better understanding of this mechanism for the development of new therapies for these diseases. PMID:24488400

  9. Monitoring apoptosis in real time.

    PubMed

    Green, Allan M; Steinmetz, Neil D

    2002-01-01

    Many therapeutically active anticancer treatments exert their effect by the induction of apoptosis and necrosis. Serial biopsies in breast cancer patients have suggested that response to therapy correlates with early posttreatment increases in tumor apoptotic index. Radiolabeled technetium Tc 99m-recombinant human (rh) annexin V provides a noninvasive technique for imaging treatment-induced cell death. Annexin V is a naturally occurring human protein that binds avidly to membrane-associated phosphatidylserine (PS). PS is normally found only on the inner leaflet of the cell membrane double layer, but it is actively transported to the outer layer as an early event in apoptosis and becomes available for annexin binding. Annexin also gains access to PS as a result of the membrane fragmentation associated with necrosis. In vitro studies of apoptosis using fluorescein annexin have shown good correlation with assessments of apoptosis documented by nuclear DNA degradation and caspase activation. In vivo localization of intravenously administered Tc 99m-annexin V has been demonstrated in numerous preclinical models of apoptosis, including anti-Fas-mediated hepatic apoptosis, rejection of allogeneic heterotopic cardiac allografts, cyclophosphamide treatment of murine lymphoma, cyclophosphamide-induced apoptosis in bone marrow, and leukocyte apoptosis associated with abscess formation. Scintigraphic studies in humans using Tc 99m-rh annexin V have demonstrated the feasibility of imaging cell death in acute myocardial infarction, in tumors with a high apoptotic index, and in response to anti-tumor chemotherapy of non-small cell lung cancer, small-cell lung cancer, breast cancer, lymphoma, and sarcoma. Increased localization of Tc 99m-rh annexin V within 1 to 3 days of chemotherapy has been noted in some, but not all, subjects with these tumors. To date, most subjects showing increased Tc 99m-rh annexin V uptake after the first course of chemotherapy have shown objective

  10. [The comeback of mitochondria in Drosophila apoptosis].

    PubMed

    Clavier, Amandine; Rincheval-Arnold, Aurore; Mignotte, Bernard; Guénal, Isabelle

    2016-05-01

    The role of the mitochondrion in mammalian cell apoptosis has been established since the mid-1990s. However, the importance of this organelle in non-mammalian apoptosis has long been regarded as minor, notably because of the absence of a crucial role for cytochrome c in caspase activation. Recent results indicate that the control of caspase activation and apoptosis in Drosophila cell death occurs at the mitochondrial level. Numerous proteins that appear key for Drosophila apoptosis regulation constitutively or transiently bind to mitochondria. They participate in the cell death process at different levels such as degradation of an IAP caspase inhibitor, production of mitochondrial reactive oxygen species or stimulation of the mitochondrial fission machinery. The aim of this review is to take stock of these events that might have their counterpart in humans. PMID:27225920

  11. Nicotinic acetylcholine receptor-mediated GABAergic inputs to cholinergic interneurons in the striosomes and the matrix compartments of the mouse striatum.

    PubMed

    Inoue, Ritsuko; Suzuki, Takeo; Nishimura, Kinya; Miura, Masami

    2016-06-01

    The striatum consists of two neurochemically distinct compartments: the striosomes (or patches) and the extrastriosomal matrix. Although striatal neurons are strongly innervated by intrinsic cholinergic interneurons, acetylcholinesterase is expressed more abundantly in the matrix than in the striosomes. At present, little is known about the different cholinergic functions of the striatal compartments. In this study, we examined gamma-aminobutyric acidergic (GABAergic) inputs to cholinergic interneurons in both compartments. We found that nicotinic receptor-mediated GABAergic responses were evoked more frequently in the matrix than in the striosomes. Furthermore, a single action potential of cholinergic neurons induced nicotinic receptor-mediated GABAergic inputs to the cholinergic neurons themselves, suggesting mutual connections that shape the temporal firing pattern of cholinergic neurons. The nicotinic receptor-mediated GABAergic responses were attenuated by continuous application of acetylcholine or the acetylcholinesterase inhibitor eserine and were enhanced by desformylflustrabromine, a positive allosteric modulator of the α4β2 subunit containing a nicotinic receptor. These results suggest that the nicotinic impact on the GABAergic responses are not uniform despite the massive and continuous cholinergic innervation. It has been reported that differential activation of neurons in the striosomes and the matrix produce a repetitive behavior called stereotypy. Drugs acting on α4β2 nicotinic receptors might provide potential tools for moderating the imbalanced activities between the compartments. PMID:26808315

  12. Regulation of rat cortical 5-hydroxytryptamine2A-receptor mediated electrophysiological responses by repeated daily treatment with electroconvulsive shock or imipramine

    PubMed Central

    Marek, Gerard J.

    2008-01-01

    Down-regulation of 5-hydroxytryptamine2A (5-HT2A) receptors has been a consistent effect induced by most antidepressant drugs. In contrast, electroconvulsive shock (ECS) up-regulates the number of 5-HT2A receptor binding sites. However, the effects of antidepressants on 5-HT2A receptor-mediated responses on identified cells of the cerebral cortex has not been examined. The purpose of the present study was to compare the effects of the tricyclic antidepressant imipramine and ECS on 5-HT2A receptor-mediated electrophysiological responses involving glutamatergic and GABAergic neurotransmission in the rat medial prefrontal cortex (mPFC) and piriform cortex, respectively. The electrophysiological effects of activating 5-HT2A receptors was consistent with 5-HT2A receptor binding regulation for imipramine and ECS except for the mPFC where chronic ECS decreased the potency of 5-HT at a 5-HT2A receptor-mediated response. These findings are consistent with the general hypothesis that chronic antidepressant treatments shift the balance of serotonergic neurotransmission towards inhibitory effects in the cortex. PMID:18294819

  13. Anti-Inflammatory Benefits of Antibiotic-Induced Neutrophil Apoptosis: Tulathromycin Induces Caspase-3-Dependent Neutrophil Programmed Cell Death and Inhibits NF-κB Signaling and CXCL8 Transcription▿

    PubMed Central

    Fischer, Carrie D.; Beatty, Jennifer K.; Zvaigzne, Cheryl G.; Morck, Douglas W.; Lucas, Merlyn J.; Buret, A. G.

    2011-01-01

    Clearance of apoptotic neutrophils is a central feature of the resolution of inflammation. Findings indicate that immuno-modulation and induction of neutrophil apoptosis by macrolide antibiotics generate anti-inflammatory benefits via mechanisms that remain obscure. Tulathromycin (TUL), a new antimicrobial agent for bovine respiratory disease, offers superior clinical efficacy for reasons not fully understood. The aim of this study was to identify the immuno-modulating effects of tulathromycin and, in this process, to establish tulathromycin as a new model for characterizing the novel anti-inflammatory properties of antibiotics. Bronchoalveolar lavage specimens were collected from Holstein calves 3 and 24 h postinfection, challenged intratracheally with live Mannheimia haemolytica (2 × 107 CFU), and treated with vehicle or tulathromycin (2.5 mg/kg body weight). Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining and enzyme-linked immunosorbent assay (ELISA) revealed that tulathromycin treatment significantly increased leukocyte apoptosis and reduced levels of proinflammatory leukotriene B4 in M. haemolytica-challenged calves. In vitro, tulathromycin concentration dependently induced apoptosis in freshly isolated bovine neutrophils from healthy steers in a capase-3-dependent manner but failed to induce apoptosis in bovine fibroblasts, epithelial cells, and endothelial cells, as well as freshly isolated bovine blood monocytes and monocyte-derived macrophages. The proapoptotic effects of TUL were also, in part, drug specific; equimolar concentrations of penicillin G, oxytetracycline, and ceftiofur failed to cause apoptosis in bovine neutrophils. In addition, tulathromycin significantly reduced levels of phosphorylated IκBα, nuclear translocation of NF-κB p65, and mRNA levels of proinflammatory interleukin-8 in lipopolysaccharide (LPS)-stimulated bovine neutrophils. The findings illustrate novel mechanisms through which

  14. Novel Levamisole Derivative Induces Extrinsic Pathway of Apoptosis in Cancer Cells and Inhibits Tumor Progression in Mice

    PubMed Central

    Hegde, Mahesh; Karki, Subhas S.; Thomas, Elizabeth; Kumar, Sujeet; Panjamurthy, Kuppusamy; Ranganatha, Somasagara R.; Rangappa, Kanchugarakoppal S.; Choudhary, Bibha; Raghavan, Sathees C.

    2012-01-01

    Background Levamisole, an imidazo(2,1-b)thiazole derivative, has been reported to be a potential antitumor agent. In the present study, we have investigated the mechanism of action of one of the recently identified analogues, 4a (2-benzyl-6-(4′-fluorophenyl)-5-thiocyanato-imidazo[2,1-b][1], [3], [4]thiadiazole). Materials and Methods ROS production and expression of various apoptotic proteins were measured following 4a treatment in leukemia cell lines. Tumor animal models were used to evaluate the effect of 4a in comparison with Levamisole on progression of breast adenocarcinoma and survival. Immunohistochemistry and western blotting studies were performed to understand the mechanism of 4a action both ex vivo and in vivo. Results We have determined the IC50 value of 4a in many leukemic and breast cancer cell lines and found CEM cells most sensitive (IC50 5 µM). Results showed that 4a treatment leads to the accumulation of ROS. Western blot analysis showed upregulation of pro-apoptotic proteins t-BID and BAX, upon treatment with 4a. Besides, dose-dependent activation of p53 along with FAS, FAS-L, and cleavage of CASPASE-8 suggest that it induces death receptor mediated apoptotic pathway in CEM cells. More importantly, we observed a reduction in tumor growth and significant increase in survival upon oral administration of 4a (20 mg/kg, six doses) in mice. In comparison, 4a was found to be more potent than its parental analogue Levamisole based on both ex vivo and in vivo studies. Further, immunohistochemistry and western blotting studies indicate that 4a treatment led to abrogation of tumor cell proliferation and activation of apoptosis by the extrinsic pathway even in animal models. Conclusion Thus, our results suggest that 4a could be used as a potent chemotherapeutic agent. PMID:22970136

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

    PubMed

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

    2013-01-01

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

  16. Silencing of Pokemon Enhances Caspase-Dependent Apoptosis via Fas- and Mitochondria-Mediated Pathways in Hepatocellular Carcinoma Cells

    PubMed Central

    Lin, Bi-Yun; Shi, Ying; Liu, Yun-Peng; Liu, Jing-Jing; Guleng, Bayasi; Ren, Jian-Lin

    2013-01-01

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

  17. Practicing death.

    PubMed

    Avny, Ohad; Alon, Aya

    2016-07-01

    This narrative describes the struggle of a primary care physician contending with the challenge of remaining committed to his patient's care despite a sense of burnout in relation to an intense period of patient deaths. The story presents two patient deaths and the physician's reflections on how he handled both cases. PMID:26899633

  18. PDT: death pathways

    NASA Astrophysics Data System (ADS)

    Kessel, David

    2007-02-01

    Cellular targets of photodynamic therapy include mitochondria, lysosomes, the endoplasmic reticulum (ER) and the plasma membrane. PDT can evoke necrosis, autophagy and apoptosis, or combinations of these, depending on the PDT dose, the site(s) of photodamage and the cellular phenotype. It has been established that loss of viability occurs even when the apoptotic program is inhibited. Studies assessing effects of ER or mitochondrial photodamage, involving loss of Bcl-2 function, indicate that low-dose PDT elicited a rapid autophagic response in L1210 cells. This was attributed to the ability of autophagy to recycle photodamaged organelles, and there was partial protection from loss of viability. This effect was not observed in L1210/Atg7, where autophagy was silenced. At higher PDT doses, apoptotic cells were observed within 60 min in both cell lines, but more so in L1210. The ability of L1210 cells to undergo autophagy did not offer protection from cell death at the higher PDT dose. Previous studies had indicated that autophagy can contribute to cell death, since L1210 cells that do not undergo an initial apoptotic response often contain multiple autophagic vacuoles 24 hr later. With L1210/Atg7, apoptosis alone may account for the loss of viability at an LD 90 PDT dose.

  19. N-methyl-D-aspartate receptor-mediated mitochondrial Ca(2+) overload in acute excitotoxic motor neuron death: a mechanism distinct from chronic neurotoxicity after Ca(2+) influx.

    PubMed

    Urushitani, M; Nakamizo, T; Inoue, R; Sawada, H; Kihara, T; Honda, K; Akaike, A; Shimohama, S

    2001-03-01

    Mitochondrial uptake of Ca(2+) has recently been found to play an important role in glutamate-induced neurotoxicity (GNT) as well as in the activation of Ca(2+)-dependent molecules, such as calmodulin and neuronal nitric oxide synthase (nNOS), in the cytoplasm. Prolonged exposure to glutamate injures motor neurons predominantly through the activation of Ca(2+)/calmodulin-nNOS, as previously reported, and is, in part, associated with the pathogenesis of amyotrophic lateral sclerosis (ALS). In the present study, we investigated how mitochondrial uptake of Ca(2+) is involved in GNT in spinal motor neurons. Acute excitotoxicity induced by exposure to 0.5 mM glutamate for 5 min was found in both motor and nonmotor neurons in cultured spinal cords from rat embryos and was dependent on extracellular Ca(2+) and on N-methyl-D-aspartate (NMDA) receptor activation. Mitochondrial uncouplers markedly blocked acute excitotoxicity, and membrane-permeable superoxide dismutase mimics attenuated acute excitotoxicity induced by glutamate and NMDA but not by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) or kainate. Fluorimetric analysis showed that mitochondrial Ca(2+) was elevated promptly with subsequent accumulation of reactive oxygen species (ROS) in the mitochondria. An NMDA receptor antagonist and a mitochondrial uncoupler eliminated the increase in fluorescence of mitochondrial Ca(2+) and ROS indicators. These data indicate that acute excitotoxicity in spinal neurons is mediated by mitochondrial Ca(2+) overload and ROS generation through the activation of NMDA receptors. This mechanism is different from that of chronic GNT. PMID:11223912

  20. Sensitization of vascular smooth muscle cell to TNF-{alpha}-mediated death in the presence of palmitate

    SciTech Connect

    Rho, Mun-Chual; Ah Lee, Kyeong; Mi Kim, Sun; Sik Lee, Chang; Jeong Jang, Min; Kook Kim, Young; Sun Lee, Hyun; Hyun Choi, Yung; Yong Rhim, Byung; Kim, Koanhoi . E-mail: koanhoi@pusan.ac.kr

    2007-05-01

    Saturated free fatty acids (FFAs), including palmitate, can activate the intrinsic death pathway in cells. However, the relationship between FFAs and receptor-mediated death pathway is still unknown. In this study, we have investigated whether FFAs are able to trigger receptor-mediated death. In addition, to clarify the mechanisms responsible for the activation, we examined the biochemical changes in dying vascular smooth muscle cell (VSMC) and the effects of various molecules to the receptor-mediated VSMC death. Tumor necrosis factor (TNF)-{alpha}-mediated VSMC death occurred in the presence of sub-cytotoxic concentration of palmitate as determined by assessing viability and DNA degradation, while the cytokine did not influence VSMC viability in the presence of oleate. The VSMC death was inhibited by the gene transfer of a dominant-negative Fas-associated death domain-containing protein and the baculovirus p35, but not by the bcl-xL or the c-Jun N-terminal kinase (JNK) binding domain of JNK-interacting protein-1, in tests utilizing recombinant adenoviruses. The VSMC death was also inhibited by a neutralizing anti-TNF receptor 1 antibody, the caspase inhibitor z-VAD, and the cathepsin B inhibitor CA074, a finding indicative of the role of both caspases and cathepsin B in this process. Consistent with this finding, caspase-3 activation and an increase in cytosolic cathepsin B activity were detected in the dying VSMC. Palmitate inhibited an increase of TNF-{alpha}-mediated nuclear factor kappa B (NF-{kappa}B) activity, the survival pathway activated by the cytokine, by hindering the translocation of the NF-{kappa}B subunit of p65 from the cytosol into the nucleus. The gene transfer of inhibitor of NF-{kappa}B predisposed VSMC to palmitate-induced cell death. To the best of our knowledge, this study is the first report to demonstrate the activation of TNF-{alpha}-mediated cell death in the presence of palmitate. The current study proposes that FFAs would take part in

  1. THE PROS AND CONS OF APOPTOSIS ASSAYS FOR USE IN THE STUDY OF CELLS, TISSUES AND ORGANS

    EPA Science Inventory

    Abstract
    Programmed cell death or apoptosis occurs in many tissues during normal development and in the normal homeostasis of adult tissues. Apoptosis also plays a significant role in abnormal development and disease. Increased interest in apoptosis and cell death in general...

  2. Reduction of spinal glycine receptor-mediated miniature inhibitory postsynaptic currents in streptozotocin-induced diabetic neuropathic pain.

    PubMed

    Chiu, Yu-Chi; Liao, Wen-Tzu; Liu, Chia-Kai; Wu, Chih-Hsien; Lin, Chung-Ren

    2016-01-12

    Diabetic neuropathic pain (DNP) is a common clinical problem, and the mechanisms underlying the onset and progression of this complication are poorly understood. The present study examined the glycine receptors (GlyR) in the control of synaptic input to dorsal horn neurons in diabetes. Male Sprague-Dawley rats with or without streptozotocin (STZ) intraperitoneal injections were used. Tactile sensitivities were assessed by measuring paw withdrawal thresholds to von Frey filaments for four weeks. The extent of GlyR-mediated inhibition controlling primary afferent-evoked excitation in dorsal horn neurons was examined by using the whole cell patch clamp recording technique in isolated adult rat spinal cord slices. The content of the spinal dorsal horn glycine levels was measured by microdialysis. An intrathecal glycine agonist injection was used to test whether mimicking endogenous glycine-receptor-mediated inhibition reduces DNP. We found that persistent hyperglycemia induced by the administration of STZ caused a decrease in the paw withdrawal latency to mechanical stimuli. The miniature inhibitory post-synaptic current (mIPSC) rise, decay kinetics and mean GlyR-mediated mIPSC amplitude were not affected in DNP. The mean frequency of GlyR-mediated mIPSC of lamina I neurons from DNP rats was, however, significantly reduced when compared with neurons from control rats. Principal passive and active membrane properties and the firing patterns of spinal lamina I neurons were not changed in DNP rats. Spinal microdialysis rats had a significantly decreased glycine level following its initial elevation. The intrathecal administration of glycine diminished tactile pain hypersensitivity in DNP rats. In conclusion, these results indicate that long-lasting hyperglycemia induced by STZ injections leads to a reduced glycinergic inhibitory control of spinal lamina I neurons through a presynaptic mechanism. PMID:26598022

  3. Immunomodulatory parasites and toll-like receptor-mediated tumour necrosis factor alpha responsiveness in wild mammals

    PubMed Central

    Jackson, Joseph A; Friberg, Ida M; Bolch, Luke; Lowe, Ann; Ralli, Catriona; Harris, Philip D; Behnke, Jerzy M; Bradley, Janette E

    2009-01-01

    Background Immunological analyses of wild populations can increase our understanding of how vertebrate immune systems respond to 'natural' levels of exposure to diverse infections. A major recent advance in immunology has been the recognition of the central role of phylogenetically conserved toll-like receptors in triggering innate immunity and the subsequent recruitment of adaptive response programmes. We studied the cross-sectional associations between individual levels of systemic toll-like receptor-mediated tumour necrosis factor alpha responsiveness and macro- and microparasite infections in a natural wood mouse (Apodemus sylvaticus) population. Results Amongst a diverse group of macroparasites, only levels of the nematode Heligmosomoides polygyrus and the louse Polyplax serrata were correlated (negatively) with innate immune responsiveness (measured by splenocyte tumour necrosis factor alpha responses to a panel of toll-like receptor agonists). Polyplax serrata infection explained a strikingly high proportion of the total variation in innate responses. Contrastingly, faecal oocyst count in microparasitic Eimeria spp. was positively associated with innate immune responsiveness, most significantly for the endosomal receptors TLR7 and TLR9. Conclusion Analogy with relevant laboratory models suggests the underlying causality for the observed patterns may be parasite-driven immunomodulatory effects on the host. A subset of immunomodulatory parasite species could thus have a key role in structuring other infections in natural vertebrate populations by affecting the 'upstream' innate mediators, like toll-like receptors, that are important in initiating immunity. Furthermore, the magnitude of the present result suggests that populations free from immunosuppressive parasites may exist at 'unnaturally' elevated levels of innate immune activation, perhaps leading to an increased risk of immunopathology. PMID:19386086

  4. Dexamethasone modulates TCR zeta chain expression and antigen receptor-mediated early signaling events in human T lymphocytes.

    PubMed

    Nambiar, M P; Enyedy, E J; Fisher, C U; Warke, V G; Juang, Y T; Tsokos, G C

    2001-02-25

    Dexamethasone is a potent anti-inflammatory and immunosupressive agent that has complex, yet incompletely defined, effects on the immune response. Here, we explored the effect of dexamethasone on the expression of TCR zeta chain and TCR/CD3-induced early signaling events in human T lymphocytes. Immunoblotting studies using TCR zeta chain specific mAb showed a dose-dependent biphasic effect of dexamethasone on TCR zeta chain expression, that is, it was increased when cells were incubated with 10 nM, whereas the expression was decreased when incubated with 100 nM dexamethasone. The dose-dependent biphasic effect of dexamethsone on the TCR zeta chain expression was also revealed by FACS analysis of permeabilized cells. Time course studies showed that upregulation of the TCR zeta chain at 10 nM dexamethasone reached maximum levels at 24 h and remained elevated up to 48 h. Other subunits of the TCR/CD3 complex were minimally affected under these conditions. The increased expression of the TCR zeta chain following treatment with 10 nM dexamethasone correlated with increased anti-CD3 antibody-induced tyrosine phosphorylation of the TCR zeta chain and downstream signaling intermediate ZAP-70 and PLC gamma with faster kinetics. Similarly, the induction of TCR zeta chain expression at 10 nM dexamethasone correlated with increased and more sustained TCR/CD3-mediated [Ca(2+)](i) response. Reporter gene assays using TCR zeta chain promoter-driven luciferase gene constructs in Jurkat cells showed that treatment with 10 nM dexamethasone increased TCR zeta chain promoter activity and that the region between -160 and +58 was responsible for the observed effect. These results suggest that dexamethasone primarily acts at the transcriptional level and differentially modulates TCR zeta chain expression and antigen receptor-mediated early signaling events in human peripheral T lymphocytes. PMID:11277620

  5. Testin, a novel binding partner of the calcium-sensing receptor, enhances receptor-mediated Rho-kinase signalling

    SciTech Connect

    Magno, Aaron L.; Ingley, Evan; Brown, Suzanne J.; Conigrave, Arthur D.; Ratajczak, Thomas; Ward, Bryan K.

    2011-09-09

    Highlights: {yields} A yeast two-hybrid screen revealed testin bound to the calcium-sensing receptor. {yields} The second zinc finger of LIM domain 1 of testin is critical for interaction. {yields} Testin bound to a region of the receptor tail important for cell signalling. {yields} Testin and receptor interaction was confirmed in mammalian (HEK293) cells. {yields} Overexpression of testin enhanced receptor-mediated Rho signalling in HEK293 cells. -- Abstract: The calcium-sensing receptor (CaR) plays an integral role in calcium homeostasis and the regulation of other cellular functions including cell proliferation and cytoskeletal organisation. The multifunctional nature of the CaR is manifested through ligand-dependent stimulation of different signalling pathways that are also regulated by partner binding proteins. Following a yeast two-hybrid library screen using the intracellular tail of the CaR as bait, we identified several novel binding partners including the focal adhesion protein, testin. Testin has not previously been shown to interact with cell surface receptors. The sites of interaction between the CaR and testin were mapped to the membrane proximal region of the receptor tail and the second zinc-finger of LIM domain 1 of testin, the integrity of which was found to be critical for the CaR-testin interaction. The CaR-testin association was confirmed in HEK293 cells by coimmunoprecipitation and confocal microscopy studies. Ectopic expression of testin in HEK293 cells stably expressing the CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling. These results suggest an interplay between the CaR and testin in the regulation of CaR-mediated Rho signalling with possible effects on the cytoskeleton.

  6. Receptor-Mediated Recognition and Uptake of Iron from Human Transferrin by Staphylococcus aureus and Staphylococcus epidermidis

    PubMed Central

    Modun, Belinda; Evans, Robert W.; Joannou, Christopher L.; Williams, Paul

    1998-01-01

    Staphylococcus aureus and Staphylococcus epidermidis both recognize and bind the human iron-transporting glycoprotein, transferrin, via a 42-kDa cell surface protein receptor. In an iron-deficient medium, staphylococcal growth can be promoted by the addition of human diferric transferrin but not human apotransferrin. To determine whether the staphylococcal transferrin receptor is involved in the removal of iron from transferrin, we employed 6 M urea–polyacrylamide gel electrophoresis, which separates human transferrin into four forms (diferric, monoferric N-lobe, and monoferric C-lobe transferrin and apotransferrin). S. aureus and S. epidermidis but not Staphylococcus saprophyticus (which lacks the transferrin receptor) converted diferric human transferrin into its apotransferrin form within 30 min. During conversion, iron was removed sequentially from the N lobe and then from the C lobe. Metabolic poisons such as sodium azide and nigericin inhibited the release of iron from human transferrin, indicating that it is an energy-requiring process. To demonstrate that this process is receptor rather than siderophore mediated, we incubated (i) washed staphylococcal cells and (ii) the staphylococcal siderophore, staphyloferrin A, with porcine transferrin, a transferrin species which does not bind to the staphylococcal receptor. While staphyloferrin A removed iron from both human and porcine transferrins, neither S. aureus nor S. epidermidis cells could promote the release of iron from porcine transferrin. In competition binding assays, both native and recombinant N-lobe fragments of human transferrin as well as a naturally occurring human transferrin variant with a mutation in the C-lobe blocked binding of 125I-labelled transferrin. Furthermore, the staphylococci removed iron efficiently from the iron-loaded N-lobe fragment of human transferrin. These data demonstrate that the staphylococci efficiently remove iron from transferrin via a receptor-mediated process and

  7. Protein kinases A and C regulate receptor-mediated increases in cAMP in rabbit erythrocytes

    PubMed Central

    Sridharan, Meera; Bowles, Elizabeth A.; Stephenson, Alan H.; Ellsworth, Mary L.; Sprague, Randy S.

    2010-01-01

    Activation of the β-adrenergic receptor (β-AR) or the prostacyclin receptor (IPR) results in increases in cAMP and ATP release from erythrocytes. cAMP levels depend on a balance between synthesis via adenylyl cyclase and hydrolysis by phosphodiesterases (PDEs). Previously, we reported that cAMP increases associated with activation of the β-AR and IPR in rabbit and human erythrocytes are tightly regulated by distinct PDEs (1). Importantly, inhibitors of these PDEs potentiated both increases in cAMP and ATP release. It has been shown that increases in protein kinase (PK) activity can activate PDE3 and PDE4. Both PKA and PKC are present in the erythrocyte and can phosphorylate and activate these PDEs. Here we investigate the hypothesis that PKA regulates PDE activity associated with the β-AR and both PKA and PKC regulate the PDE activity associated with the IPR in rabbit erythrocytes. Pretreatment of erythrocytes with the PKA inhibitor, H89 (10 μM), in the presence of the PDE4 inhibitor, rolipram (10 μM), augmented isoproterenol (1 μM)-induced cAMP increases. In contrast, in the presence of the PDE3 inhibitor, cilostazol (10 μM), pretreatment of erythrocytes with either H89 (1 μM) or two chemically dissimilar inhibitors of PKC, calphostin C (1 μM) or GFX109203X (1 μM), potentiated iloprost (1 μM)-induced cAMP increases. Furthermore, pretreatment of erythrocytes with both H89 and GFX109203X in the presence of cilostazol augmented the iloprost-induced increases in cAMP to a greater extent than either PK inhibitor individually. These results support the hypothesis that PDEs associated with receptor-mediated increases in cAMP in rabbit erythrocytes are regulated by kinases specific to the receptor's signaling pathway. PMID:20008267

  8. The Role of cGMP on Adenosine A1 Receptor-mediated Inhibition of Synaptic Transmission at the Hippocampus

    PubMed Central

    Pinto, Isa; Serpa, André; Sebastião, Ana M.; Cascalheira, José F.

    2016-01-01

    Both adenosine A1 receptor and cGMP inhibit synaptic transmission at the hippocampus and recently it was found that A1 receptor increased cGMP levels in hippocampus, but the role of cGMP on A1 receptor-mediated inhibition of synaptic transmission remains to be established. In the present work we investigated if blocking the NOS/sGC/cGMP/PKG pathway using nitric oxide synthase (NOS), protein kinase G (PKG), and soluble guanylyl cyclase (sGC) inhibitors modify the A1 receptor effect on synaptic transmission. Neurotransmission was evaluated by measuring the slope of field excitatory postsynaptic potentials (fEPSPs) evoked by electrical stimulation at hippocampal slices. N6-cyclopentyladenosine (CPA, 15 nM), a selective A1 receptor agonist, reversibly decreased the fEPSPs by 54 ± 5%. Incubation of the slices with an inhibitor of NOS (L-NAME, 200 μM) decreased the CPA effect on fEPSPs by 57 ± 9% in female rats. In males, ODQ (10 μM), an sGC inhibitor, decreased the CPA inhibitory effect on fEPSPs by 23 ± 6%, but only when adenosine deaminase (ADA,1 U/ml) was present; similar results were found in females, where ODQ decreased CPA-induced inhibition of fEPSP slope by 23 ± 7%. In male rats, the presence of the PKG inhibitor (KT5823, 1 nM) decreased the CPA effect by 45.0 ± 9%; similar results were obtained in females, where KT5823 caused a 32 ± 9% decrease on the CPA effect. In conclusion, the results suggest that the inhibitory action of adenosine A1 receptors on synaptic transmission at hippocampus is, in part, mediated by the NOS/sGC/cGMP/PKG pathway. PMID:27148059

  9. Asialoglycoprotein receptor mediates the toxic effects of an asialofetuin-diphtheria toxin fragment A conjugate on cultured rat hepatocytes

    SciTech Connect

    Cawley, D.B.; Simpson, D.L.; Herschman, H.R.

    1981-06-01

    We have constructed a toxic hybrid protein that is recognized by asialoglycoprotein (ASGP) receptors of cultured rat hepatocytes. The conjugate consists of fragment A of diphtheria toxin (DTA) linked by a disulfide bond to asialofetuin (ASF). This conjugate is highly toxic, inhibiting protein synthesis in primary rat hepatocytes at concentrations as low as 10 pM. The ASF-DTA conjugate was 600 and 1800 times as toxic as diphtheria toxin and DTA, respectively, on primary rat hepatocytes. The ASGP receptor recognizes galactose-terminated proteins. We tested a series of glycoproteins for their ability to block the action of the ASF-DTA conjugate. Fetuin and orosomucoid, two glycoproteins with terminal sialic acid on their oligosaccharide chains, did not block the action of the conjugate. Their galactose-terminated asialo derivatives, ASF and asialoorosomucoid, as expected, did block the action of the conjugate. The N-acetylglucosaminyl-terminated derivative (asialoagalactoorosomucoid) had no appreciable effect on the activity of the conjugate. We tested the ASF-DTA conjugate on six cell types; except for primary rat hepatocytes, none of them were affected by a high concentration (10 nM) of ASF-DTA conjugate. A fetuin-DTA conjugate was less toxic by a factor of 300 than the ASF-DTA conjugate and exerted its effects primarily through non-receptor-mediated mechanisms. The highly toxic ASF-DTA conjugate is cell-type specific, and its action is mediated by a well-characterized receptor, whose mechanism of receptor-ligand internalization has been extensively investigated.

  10. Two types of functionally different GABAA receptors mediate GABA modulation of cholinergic transmission in cat terminal ileum.

    PubMed

    Radomirov, R; Pencheva, N

    1995-08-01

    1. The effects of GABA (1 microM-2 mM) on longitudinally or circularly oriented organ bath preparations of cat terminal ileum consisted of a relaxation phase with an inhibition of the rhythmic spontaneous phasic contractions, followed by a phase of contractions characterized by an elevation in basal tone and an increase in amplitude of the spontaneous phasic contractions. 2. Muscimol (100 microM), but not baclofen (100 microM), mimicked the relaxation phase of the response to applied GABA (100 microM) in all tissue preparations. In addition, muscimol induced a phase of contractile activity in the circular muscle layer whilst baclofen exerted a 'GABA-like' contractile effect on the longitudinal muscle layer. Bicuculline (30 microM) or picrotoxinin (30 microM) antagonized the GABA- or muscimol-induced relaxations in all preparations and decreased the GABA- but not the baclofen-induced contractions of the longitudinal muscle layer. 3. Tetrodotoxin (0.5 microM) or atropine (0.1 microM) prevented the bicuculline-sensitive phases of the GABA or muscimol effects on both muscle layers but not the contractile effect of baclofen on the longitudinal muscle layer. 4. The bicuculline-sensitive phases of the GABA effect on both muscle layers were almost completely eliminated by 1 nM pirenzepine. At this concentration pirenzepine did not affect the electrically-evoked cholinergic twitch contractions or contractile responses to applied acetylcholine of both muscle layers. 5. During electrically-evoked cholinergic twitch contractions of both muscle layers, GABA (100 microM) had an inhibitory effect. The inhibition occurred in the presence of pirenzepine (1 nM) but not of bicuculline (30 microM). 6. It is suggested that two types of functionally different bicuculline-sensitive GABAA receptors mediate an exitatory presynaptic and an inhibitory prejunctional action of GABA on the cholinergic transmission in cat terminal ileum. PMID:8576270

  11. Blockade of thromboxane/endoperoxide receptor-mediated responses in the pulmonary vascular bed of the cat by sulotroban.

    PubMed

    Nossaman, B D; McMahon, T J; Ragheb, M S; Ibrahim, I N; Babycos, C R; Hood, J S; Kadowitz, P J

    1992-03-17

    The effects of sulotroban (BM13.177; SK & F 95587), a thromboxane (TX) A2/endoperoxide (PGH2) receptor blocking agent on responses to the TXA2/PGH2 mimics, U46619 and U44069, were investigated in the pulmonary vascular bed of the intact-chest cat under constant flow conditions. Injections of U46619 and U44069 directly into the perfused lobar artery caused dose-related increases in lobar arterial pressure without altering left atrial pressure. Following administration of sulotroban in a dose of 5 mg/kg i.v., dose-response curves for U46619 and U44069 were shifted to the right in a parallel manner. The duration of the blocking effect of sulotroban was investigated, and responses to U46619 returned to approximately 50% of control in 120 min and were not significantly different from control 240 min after administration of the receptor antagonist. Sulotroban was without significant effect on responses to prostaglandin (PG) D2 or F2 alpha or serotonin, histamine, norepinephrine, angiotensin II or BAY K8644, an agent which enhances calcium entry. Sulotroban was without effect on responses to endothelin (ET)-1, sarafotoxin (S) 6a or S6c and platelet-activating factor (PAF). Sulotroban did not alter baseline vascular pressures in the cat and responses to the PG and TXA2/PGH2 precursor, arachidonic acid, were reduced. The present data show that sulotroban selectively blocks TXA2/PGH2 receptor-mediated responses in a competitive and reversible manner in the pulmonary vascular bed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1379928

  12. Anandamide, an endogenous cannabimimetic eicosanoid, binds to the cloned human cannabinoid receptor and stimulates receptor-mediated signal transduction.

    PubMed Central

    Felder, C C; Briley, E M; Axelrod, J; Simpson, J T; Mackie, K; Devane, W A