Multiple programmed cell death pathways are involved in N-methyl-N-nitrosourea-induced photoreceptor degeneration.

PubMed

Reisenhofer, Miriam; Balmer, Jasmin; Zulliger, Rahel; Enzmann, Volker

2015-05-01

To identify programmed cell death (PCD) pathways involved in N-methyl-N-nitrosourea (MNU)-induced photoreceptor (PR) degeneration. Adult C57BL/6 mice received a single MNU i.p. injection (60 mg/kg bodyweight), and were observed over a period of 7 days. Degeneration was visualized by H&E overview staining and electron microscopy. PR cell death was measured by quantifying TUNEL-positive cells in the outer nuclear layer (ONL). Activity measurements of key PCD enzymes (calpain, caspases) were used to identify the involved cell death pathways. Furthermore, the expression level of C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78), key players in endoplasmic reticulum (ER) stress-induced apoptosis, was analyzed using quantitative real-time PCR. A decrease in ONL thickness and the appearance of apoptotic PR nuclei could be detected beginning 3 days post-injection (PI). This was accompanied by an increase of TUNEL-positive cells. Significant upregulation of activated caspases (3, 9, 12) was found at different time periods after MNU injection. Additionally, several other players of nonconventional PCD pathways were also upregulated. Consequently, calpain activity increased in the ONL, with a maximum on day 7 PI and an upregulation of CHOP and GRP78 expression beginning on day 1 PI was found. The data indicate that regular apoptosis is the major cause of MNU-induced PR cell death. However, alternative PCD pathways, including ER stress and calpain activation, are also involved. Knowledge about the mechanisms involved in this mouse model of PR degeneration could facilitate the design of putative combinatory therapeutic approaches.

Nitric Oxide and Protein S-Nitrosylation Are Integral to Hydrogen Peroxide-Induced Leaf Cell Death in Rice1[W][OA

PubMed Central

Lin, Aihong; Wang, Yiqin; Tang, Jiuyou; Xue, Peng; Li, Chunlai; Liu, Linchuan; Hu, Bin; Yang, Fuquan; Loake, Gary J.; Chu, Chengcai

2012-01-01

Nitric oxide (NO) is a key redox-active, small molecule involved in various aspects of plant growth and development. Here, we report the identification of an NO accumulation mutant, nitric oxide excess1 (noe1), in rice (Oryza sativa), the isolation of the corresponding gene, and the analysis of its role in NO-mediated leaf cell death. Map-based cloning revealed that NOE1 encoded a rice catalase, OsCATC. Furthermore, noe1 resulted in an increase of hydrogen peroxide (H2O2) in the leaves, which consequently promoted NO production via the activation of nitrate reductase. The removal of excess NO reduced cell death in both leaves and suspension cultures derived from noe1 plants, implicating NO as an important endogenous mediator of H2O2-induced leaf cell death. Reduction of intracellular S-nitrosothiol (SNO) levels, generated by overexpression of rice S-nitrosoglutathione reductase gene (GSNOR1), which regulates global levels of protein S-nitrosylation, alleviated leaf cell death in noe1 plants. Thus, S-nitrosylation was also involved in light-dependent leaf cell death in noe1. Utilizing the biotin-switch assay, nanoliquid chromatography, and tandem mass spectrometry, S-nitrosylated proteins were identified in both wild-type and noe1 plants. NO targets identified only in noe1 plants included glyceraldehyde 3-phosphate dehydrogenase and thioredoxin, which have been reported to be involved in S-nitrosylation-regulated cell death in animals. Collectively, our data suggest that both NO and SNOs are important mediators in the process of H2O2-induced leaf cell death in rice. PMID:22106097

Autophagy regulates death of retinal pigment epithelium cells in age-related macular degeneration.

PubMed

Kaarniranta, Kai; Tokarz, Paulina; Koskela, Ali; Paterno, Jussi; Blasiak, Janusz

2017-04-01

Age-related macular degeneration (AMD) is an eye disease underlined by the degradation of retinal pigment epithelium (RPE) cells, photoreceptors, and choriocapillares, but the exact mechanism of cell death in AMD is not completely clear. This mechanism is important for prevention of and therapeutic intervention in AMD, which is a hardly curable disease. Present reports suggest that both apoptosis and pyroptosis (cell death dependent on caspase-1) as well as necroptosis (regulated necrosis dependent on the proteins RIPK3 and MLKL, caspase-independent) can be involved in the AMD-related death of RPE cells. Autophagy, a cellular clearing system, plays an important role in AMD pathogenesis, and this role is closely associated with the activation of the NLRP3 inflammasome, a central event for advanced AMD. Autophagy can play a role in apoptosis, pyroptosis, and necroptosis, but its contribution to AMD-specific cell death is not completely clear. Autophagy can be involved in the regulation of proteins important for cellular antioxidative defense, including Nrf2, which can interact with p62/SQSTM, a protein essential for autophagy. As oxidative stress is implicated in AMD pathogenesis, autophagy can contribute to this disease by deregulation of cellular defense against the stress. However, these and other interactions do not explain the mechanisms of RPE cell death in AMD. In this review, we present basic mechanisms of autophagy and its involvement in AMD pathogenesis and try to show a regulatory role of autophagy in RPE cell death. This can result in considering the genes and proteins of autophagy as molecular targets in AMD prevention and therapy.

On the role of phosphatidylinositol 3-kinase, protein kinase b/Akt, and glycogen synthase kinase-3β in photodynamic injury of crayfish neurons and glial cells.

PubMed

Komandirov, Maxim A; Knyazeva, Evgeniya A; Fedorenko, Yulia P; Rudkovskii, Mikhail V; Stetsurin, Denis A; Uzdensky, Anatoly B

2011-10-01

Photodynamic treatment that causes intense oxidative stress and cell death is currently used in neurooncology. However, along with tumor cells, it may damage healthy neurons and glia. To study the involvement of signaling processes in photodynamic injury or protection of neurons and glia, we used crayfish mechanoreceptor consisting of a single neuron surrounded by glial cells. It was photosensitized with alumophthalocyanine Photosens. Application of specific inhibitors showed that phosphatidylinositol 3-kinase did not participate in photoinduced death of neurons and glia. Akt was involved in photoinduced necrosis but not in apoptosis of neurons and glia. Glycogen synthase kinase-3β participated in photoinduced apoptosis of glial cells and in necrosis of neurons. Therefore, phosphatidylinositol 3-kinase/protein kinase Akt/glycogen synthase kinase-3β pathway was not involved as a whole in photodynamic injury of crayfish neurons and glia but its components, Akt and glycogen synthase kinase-3β, independently and cell specifically regulated death of neurons and glial cells. According to these data, necrosis in this system was a controlled but not a non-regulated cell death mode. The obtained results may be used for the search of pharmacological agents selectively modulating death and survival of normal neurons and glial cells during photodynamic therapy of brain tumors.

Sticholysin II-mediated cytotoxicity involves the activation of regulated intracellular responses that anticipates cell death.

PubMed

Soto, Carmen; Bergado, Gretchen; Blanco, Rancés; Griñán, Tania; Rodríguez, Hermis; Ros, Uris; Pazos, Fabiola; Lanio, María Eliana; Hernández, Ana María; Álvarez, Carlos

2018-05-01

Sticholysin II (StII) is a pore-forming toxin of biomedical interest that belongs to the actinoporin protein family. Sticholysins are currently under examination as an active immunomodulating component of a vaccinal platform against tumoral cells and as a key element of a nucleic acids delivery system to cell cytosol. These proteins form pores in the plasma membrane leading to ion imbalance and cell lysis. However, the intracellular mechanisms triggered by actinoporins upon binding to membranes and its consequences for cell death are barely understood. Here, we have examined the cytotoxicity and intracellular responses induced by StII upon binding to human B-cell lymphoma Raji in vitro. StII cytotoxicity involves a functional actin cytoskeleton, induces cellular swelling, lysis and the concomitant release of cytosol content. In addition, StII induces calcium release mainly from the Endoplasmic Reticulum, activates Mitogen-Activated Protein Kinase ERK and impairs mitochondrial membrane potential. Furthermore, StII stimulates the expression of receptor interacting protein kinase 1 (RIP1), normally related to different forms of regulated cell death such as apoptosis and necroptosis. In correspondence, necrostatin-1, an inhibitor of this kinase, reduces StII cytotoxicity. However, the mechanism of cell death activated by StII does not involve caspases activation, typical molecular features of apoptosis and pyroptosis. Our results suggest that, beyond pore-formation and cell lysis, StII-induced cytotoxicity could involve other regulated intracellular mechanisms connected to RIP1-MEK1/2 -ERK1/2- pathways. This opens new perspectives and challenges the general point of view that these toxins induce a completely unregulated mechanism of necrotic cell death. This study contributes to a better understanding of the molecular mechanisms involved in toxin-cell interaction and the implications for cell functioning, with connotation for the exploitations of these toxins in clinical settings. Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Photodynamic therapy: the role of paraptosis

NASA Astrophysics Data System (ADS)

Kessel, David; Cho, Won-Jin; Kim, Hyeong-Reh

2018-02-01

Apoptosis is a pathway to cell death frequently observed after photodynamic therapy (PDT). Sub-cellular photodamage to mitochondria, lysosomes, the ER, or combinations of these targets, can lead to apoptotic death. We have recently investigated another pathway to cell death after PDT termed `paraptosis'. This is characterized by extensive cytoplasmic vacuolization, does not involve caspase activation or nuclear fragmentation, requires a brief interval of continued protein synthesis and appears to derive from ER stress. Determinants and further characteristics of PDT-derived paraptosis are explored in the A549 non small-cell lung cancer cell line and in cells derived from head and neck cancer tissues. We provide evidence that ER photodamage and JNK pathway activation are involved in PDT-mediated paraptosis.

MCP-1 causes cardiomyoblast death via autophagy resulting from ER stress caused by oxidative stress generated by inducing a novel zinc-finger protein, MCPIP.

PubMed

Younce, Craig W; Kolattukudy, Pappachan E

2010-01-27

MCP-1 (monocyte chemotactic protein-1) plays a critical role in the development of heart failure that is known to involve apoptosis. How MCP-1 contributes to cell death involved in the development of heart disease is not understood. In the present study we show that MCP-1 causes death in cardiac myoblasts, H9c2 cells, by inducing oxidative stress which causes ER stress leading to autophagy via a novel zinc-finger protein, MCPIP (MCP-1-induced protein). MCPIP expression caused cell death, and knockdown of MCPIP attenuated MCP-1-induced cell death. It caused induction of iNOS (inducible NO synthase), translocation of the NADPH oxidase subunit phox47 from the cytoplasm to the membrane, production of ROS (reactive oxygen species), and induction of ER (endoplasmic reticulum) stress markers HSP40 (heat-shock protein 40), PDI (protein disulfide-isomerase), GRP78 (guanine-nucleotide-releasing protein 78) and IRE1alpha (inositol-requiring enzyme 1alpha). It also caused autophagy, as indicated by beclin-1 induction, cleavage of LC3 (microtubule-associated protein 1 light chain 3) and autophagolysosome formation, and apoptosis, as indicated by caspase 3 activation and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) assay. Inhibitors of oxidative stress, including CeO2 nanoparticles, inhibited ROS formation, ER stress, autophagy and cell death. Specific inhibitors of ER stress inhibited autophagy and cell death as did knockdown of the ER stress signalling protein IRE1. Knockdown of beclin-1 and autophagy inhibitors prevented cell death. This cell death involved caspase 2 and caspase 12, as specific inhibitors of these caspases prevented MCPIP-induced cell death. Microarray analysis showed that MCPIP expression caused induction of a variety of genes known to be involved in cell death. MCPIP caused activation of JNK (c-Jun N-terminal kinase) and p38 and induction of p53 and PUMA (p53 up-regulated modulator of apoptosis). Taken together, these results suggest that MCPIP induces ROS/RNS (reactive nitrogen species) production that causes ER stress which leads to autophagy and apoptosis through caspase 2/12 and IRE1alpha-JNK/p38-p53-PUMA pathway. These results provide the first molecular insights into the mechanism by which elevated MCP-1 levels associated with chronic inflammation may contribute to the development of heart failure.

Cell Death Pathways and Phthalocyanine as an Efficient Agent for Photodynamic Cancer Therapy

PubMed Central

Mfouo-Tynga, Ivan; Abrahamse, Heidi

2015-01-01

The mechanisms of cell death can be predetermined (programmed) or not and categorized into apoptotic, autophagic and necrotic pathways. The process of Hayflick limits completes the execution of death-related mechanisms. Reactive oxygen species (ROS) are associated with oxidative stress and subsequent cytodamage by oxidizing and degrading cell components. ROS are also involved in immune responses, where they stabilize and activate both hypoxia-inducible factors and phagocytic effectors. ROS production and presence enhance cytodamage and photodynamic-induced cell death. Photodynamic cancer therapy (PDT) uses non-toxic chemotherapeutic agents, photosensitizer (PS), to initiate a light-dependent and ROS-related cell death. Phthalocyanines (PCs) are third generation and stable PSs with improved photochemical abilities. They are effective inducers of cell death in various neoplastic models. The metallated PCs localize in critical cellular organelles and are better inducers of cell death than other previous generation PSs as they favor mainly apoptotic cell death events. PMID:25955645

Cell life and death in the anterior pituitary gland: role of oestrogens.

PubMed

Seilicovich, A

2010-07-01

Apoptotic processes play an important role in the maintenance of cell numbers in the anterior pituitary gland during physiological endocrine events. In this review, we summarise the regulation of apoptosis of anterior pituitary cells, particularly lactotrophs, somatotrophs and gonadotrophs, and analyse the possible mechanisms involved in oestrogen-induced apoptosis in anterior pituitary cells. Oestrogens exert apoptotic actions in several cell types and act as modulators of pituitary cell renewal, sensitising cells to both mitogenic and apoptotic signals. Local synthesis of growth factors and cytokines induced by oestradiol as well as changes in phenotypic features that enhance the responsiveness of anterior pituitary cells to pro-apoptotic factors may account for cyclical apoptotic activity in anterior pituitary cells during the oestrous cycle. Considering that tissue homeostasis results from a balance between cell proliferation and death and that mechanisms involved in apoptosis are tightly regulated, defects in cell death processes could have a considerable physiopathological impact.

Mitochondrial fission proteins regulate programmed cell death in yeast.

PubMed

Fannjiang, Yihru; Cheng, Wen-Chih; Lee, Sarah J; Qi, Bing; Pevsner, Jonathan; McCaffery, J Michael; Hill, R Blake; Basañez, Gorka; Hardwick, J Marie

2004-11-15

The possibility that single-cell organisms undergo programmed cell death has been questioned in part because they lack several key components of the mammalian cell death machinery. However, yeast encode a homolog of human Drp1, a mitochondrial fission protein that was shown previously to promote mammalian cell death and the excessive mitochondrial fragmentation characteristic of apoptotic mammalian cells. In support of a primordial origin of programmed cell death involving mitochondria, we found that the Saccharomyces cerevisiae homolog of human Drp1, Dnm1, promotes mitochondrial fragmentation/degradation and cell death following treatment with several death stimuli. Two Dnm1-interacting factors also regulate yeast cell death. The WD40 repeat protein Mdv1/Net2 promotes cell death, consistent with its role in mitochondrial fission. In contrast to its fission function in healthy cells, Fis1 unexpectedly inhibits Dnm1-mediated mitochondrial fission and cysteine protease-dependent cell death in yeast. Furthermore, the ability of yeast Fis1 to inhibit mitochondrial fission and cell death can be functionally replaced by human Bcl-2 and Bcl-xL. Together, these findings indicate that yeast and mammalian cells have a conserved programmed death pathway regulated by a common molecular component, Drp1/Dnm1, that is inhibited by a Bcl-2-like function.

Necrotic enlargement of cone photoreceptor cells and the release of high-mobility group box-1 in retinitis pigmentosa

PubMed Central

Murakami, Y; Ikeda, Y; Nakatake, S; Tachibana, T; Fujiwara, K; Yoshida, N; Notomi, S; Nakao, S; Hisatomi, T; Miller, J W; Vavvas, DG; Sonoda, KH; Ishibashi, T

2015-01-01

Retinitis pigmentosa (RP) refers to a group of inherited retinal degenerations resulting form rod and cone photoreceptor cell death. The rod cell death due to deleterious genetic mutations has been shown to occur mainly through apoptosis, whereas the mechanisms and features of the secondary cone cell death have not been fully elucidated. Our previous study showed that the cone cell death in rd10 mice, an animal model of RP, involves necrotic features and is partly mediated by the receptor interacting protein kinase. However, the relevancy of necrotic cone cell death in human RP patients remains unknown. In the present study, we showed that dying cone cells in rd10 mice exhibited cellular enlargement, along with necrotic changes such as cellular swelling and mitochondrial rupture. In human eyes, live imaging of cone cells by adaptive optics scanning laser ophthalmoscopy revealed significantly increased percentages of enlarged cone cells in the RP patients compared with the control subjects. The vitreous of the RP patients contained significantly higher levels of high-mobility group box-1, which is released extracellularly associated with necrotic cell death. These findings suggest that necrotic enlargement of cone cells is involved in the process of cone degeneration, and that necrosis may be a novel target to prevent or delay the loss of cone-mediated central vision in RP. PMID:27551484

Non-conventional apoptotic response to ionising radiation mediated by N-methyl D-aspartate receptors in immature neuronal cells

PubMed Central

SAMARI, NADA; DE SAINT-GEORGES, LOUIS; PANI, GIUSEPPE; BAATOUT, SARAH; LEYNS, LUC; BENOTMANE, MOHAMMED ABDERRAFI

2013-01-01

During cortical development, N-methyl D-aspartate (NMDA) receptors are highly involved in neuronal maturation and synapse establishment. Their implication in the phenomenon of excitotoxicity has been extensively described in several neurodegenerative diseases due to the permissive entry of Ca2+ ions and massive accumulation in the intracellular compartment, which is highly toxic to cells. Ionising radiation is also a source of stress to the cells, particularly immature neurons. Their capacity to induce cell death has been described for various cell types either by directly damaging the DNA or indirectly through the generation of reactive oxygen species responsible for the activation of a battery of stress response effectors leading in certain cases, to cell death. In this study, in order to determine whether a link exists between NMDA receptors-mediated excitotoxicity and radiation-induced cell death, we evaluated radiation-induced cell death in vitro and in vivo in maturing neurons during the fetal period. Cell death induction was assessed by TUNEL, caspase-3 activity and DNA ladder assays, with or without the administration of dizocilpine (MK-801), a non-competitive NMDA receptor antagonist which blocks neuronal Ca2+ influx. To further investigate the possible involvement of Ca2+-dependent enzyme activation, known to occur at high Ca2+ concentrations, we examined the protective effect of a calpain inhibitor on cell death induced by radiation. Doses ranging from 0.2 to 0.6 Gy of X-rays elicited a clear apoptotic response that was prevented by the injection of dizocilpine (MK-801) or calpain inhibitor. These data demonstrate the involvement of NMDA receptors in radiation-induced neuronal death by the activation of downstream effectors, including calpain-related pathways. An increased apoptotic process elicited by radiation, occurring independently of the normal developmental scheme, may eliminate post-mitotic but immature neuronal cells and deeply impair the establishment of the neuronal network, which in the case of cortical development is critical for cognitive capacities. PMID:23338045

Control of non-apoptotic nurse cell death by engulfment genes in Drosophila.

PubMed

Timmons, Allison K; Mondragon, Albert A; Meehan, Tracy L; McCall, Kimberly

2017-04-03

Programmed cell death occurs as a normal part of oocyte development in Drosophila. For each egg that is formed, 15 germline-derived nurse cells transfer their cytoplasmic contents into the oocyte and die. Disruption of apoptosis or autophagy only partially inhibits the death of the nurse cells, indicating that other mechanisms significantly contribute to nurse cell death. Recently, we demonstrated that the surrounding stretch follicle cells non-autonomously promote nurse cell death during late oogenesis and that phagocytosis genes including draper, ced-12, and the JNK pathway are crucial for this process. When phagocytosis genes are inhibited in the follicle cells, events specifically associated with death of the nurse cells are impaired. Death of the nurse cells is not completely blocked in draper mutants, suggesting that other engulfment receptors are involved. Indeed, we found that the integrin subunit, αPS3, is enriched on stretch follicle cells during late oogenesis and is required for elimination of the nurse cells. Moreover, double mutant analysis revealed that integrins act in parallel to draper. Death of nurse cells in the Drosophila ovary is a unique example of programmed cell death that is both non-apoptotic and non-cell autonomously controlled.

Ion channels involved in cell volume regulation: effects on migration, proliferation, and programmed cell death in non adherent EAT cells and adherent ELA cells.

PubMed

Hoffmann, Else Kay

2011-01-01

This mini review outlines studies of cell volume regulation in two closely related mammalian cell lines: nonadherent Ehrlich ascites tumour cells (EATC) and adherent Ehrlich Lettre ascites (ELA) cells. Focus is on the regulatory volume decrease (RVD) that occurs after cell swelling, the volume regulatory ion channels involved, and the mechanisms (cellular signalling pathways) that regulate these channels. Finally, I shall also briefly review current investigations in these two cell lines that focuses on how changes in cell volume can regulate cell functions such as cell migration, proliferation, and programmed cell death. Copyright © 2011 S. Karger AG, Basel.

Network analysis of genes involved in the enhancement of hyperthermia sensitivity by the knockdown of BAG3 in human oral squamous cell carcinoma cells.

PubMed

Yunoki, Tatsuya; Tabuchi, Yoshiaki; Hayashi, Atsushi; Kondo, Takashi

2016-07-01

BCL2-associated athanogene 3 (BAG3), a co-chaperone of the heat shock 70 kDa protein (HSPA) family of proteins, is a cytoprotective protein that acts against various stresses, including heat stress. The aim of the present study was to identify gene networks involved in the enhancement of hyperthermia (HT) sensitivity by the knockdown (KD) of BAG3 in human oral squamous cell carcinoma (OSCC) cells. Although a marked elevation in the protein expression of BAG3 was detected in human the OSCC HSC-3 cells exposed to HT at 44˚C for 90 min, its expression was almost completely suppressed in the cells transfected with small interfering RNA against BAG3 (siBAG) under normal and HT conditions. The silencing of BAG3 also enhanced the cell death that was increased in the HSC-3 cells by exposure to HT. Global gene expression analysis revealed many genes that were differentially expressed by >2-fold in the cells exposed to HT and transfected with siBAG. Moreover, Ingenuity® pathways analysis demonstrated two unique gene networks, designated as Pro-cell death and Anti-cell death, which were obtained from upregulated genes and were mainly associated with the biological functions of induction and the prevention of cell death, respectively. Of note, the expression levels of genes in the Pro-cell death and Anti-cell death gene networks were significantly elevated and reduced in the HT + BAG3-KD group compared to those in the HT control group, respectively. These results provide further insight into the molecular mechanisms involved in the enhancement of HT sensitivity by the silencing of BAG3 in human OSCC cells.

Colourful death: six-parameter classification of cell death by flow cytometry--dead cells tell tales.

PubMed

Munoz, Luis E; Maueröder, Christian; Chaurio, Ricardo; Berens, Christian; Herrmann, Martin; Janko, Christina

2013-08-01

The response of the immune system against dying and dead cells strongly depends on the cell death phenotype. Beside other forms of cell death, two clearly distinct populations, early apoptotic and secondary necrotic cells, have been shown to induce anti-inflammation/tolerance and inflammation/immune priming, respectively. Cytofluorometry is a powerful technique to detect morphological and phenotypical changes occurring during cell death. Here, we describe a new technique using AnnexinA5, propidiumiodide, DiIC1(5) and Hoechst 33342 to sub-classify populations of apoptotic and/or necrotic cells. The method allows the fast and reliable identification of several different phases and pathways of cell death by analysing the following cell death associated changes in a single tube: cellular granularity and shrinkage, phosphatidylserine exposure, ion selectivity of the plasma membrane, mitochondrial membrane potential, and DNA content. The clear characterisation of cell death is of major importance for instance in immunization studies, in experimental therapeutic settings, and in the exploration of cell-death associated diseases. It also enables the analysis of immunological properties of distinct populations of dying cells and the pathways involved in this process.

Taxifolin synergizes Andrographolide-induced cell death by attenuation of autophagy and augmentation of caspase dependent and independent cell death in HeLa cells

PubMed Central

Alzaharna, Mazen; Alqouqa, Iyad; Cheung, Hon-Yeung

2017-01-01

Andrographolide (Andro) has emerged recently as a potential and effective anticancer agent with induction of apoptosis in some cancer cell lines while induction of G2/M arrest with weak apoptosis in others. Few studies have proved that Andro is also effective in combination therapy. The flavonoid Taxifolin (Taxi) has showed anti-oxidant and antiproliferative effects against different cancer cells. Therefore, the present study investigated the cytotoxic effects of Andro alone or in combination with Taxi on HeLa cells. The combination of Andro with Taxi was synergistic at all tested concentrations and combination ratios. Andro alone induced caspase-dependent apoptosis which was enhanced by the combination with Taxi and attenuated partly by using Z-Vad-Fmk. Andro induced a protective reactive oxygen species (ROS)-dependent autophagy which was attenuated by Taxi. The activation of p53 was involved in Andro-induced autophagy where the use of Taxi or pifithrin-α (PFT-α) decreased it while the activation of JNK was involved in the cell death of HeLa cells but not in the induction of autophagy. The mitochondrial outer-membrane permeabilization (MOMP) plays an important role in Andro-induced cell death in HeLa cells. Andro alone increased the MOMP which was further increased in the case of combination. This led to the increase in AIF and cytochrome c release from mitochondria which consequently increased caspase-dependent and independent cell death. In conclusion, Andro induced a protective autophagy in HeLa cells which was reduced by Taxi and the cell death was increased by increasing the MOMP and subsequently the caspase-dependent and independent cell death. PMID:28182713

Violacein Induces Death of Resistant Leukaemia Cells via Kinome Reprogramming, Endoplasmic Reticulum Stress and Golgi Apparatus Collapse

PubMed Central

Queiroz, Karla C. S.; Milani, Renato; Ruela-de-Sousa, Roberta R.; Fuhler, Gwenny M.; Justo, Giselle Z.; Zambuzzi, Willian F.; Duran, Nelson; Diks, Sander H.; Spek, C. Arnold; Ferreira, Carmen V.; Peppelenbosch, Maikel P.

2012-01-01

It is now generally recognised that different modes of programmed cell death (PCD) are intimately linked to the cancerous process. However, the mechanism of PCD involved in cancer chemoprevention is much less clear and may be different between types of chemopreventive agents and tumour cell types involved. Therefore, from a pharmacological view, it is crucial during the earlier steps of drug development to define the cellular specificity of the candidate as well as its capacity to bypass dysfunctional tumoral signalling pathways providing insensitivity to death stimuli. Studying the cytotoxic effects of violacein, an antibiotic dihydro-indolone synthesised by an Amazon river Chromobacterium, we observed that death induced in CD34+/c-Kit+/P-glycoprotein+/MRP1+ TF1 leukaemia progenitor cells is not mediated by apoptosis and/or autophagy, since biomarkers of both types of cell death were not significantly affected by this compound. To clarify the working mechanism of violacein, we performed kinome profiling using peptide arrays to yield comprehensive descriptions of cellular kinase activities. Pro-death activity of violacein is actually carried out by inhibition of calpain and DAPK1 and activation of PKA, AKT and PDK, followed by structural changes caused by endoplasmic reticulum stress and Golgi apparatus collapse, leading to cellular demise. Our results demonstrate that violacein induces kinome reprogramming, overcoming death signaling dysfunctions of intrinsically resistant human leukaemia cells. PMID:23071514

Endoplasmic reticulum-resident E3 ubiquitin ligase Hrd1 controls B-cell immunity through degradation of the death receptor CD95/Fas

PubMed Central

Kong, Sinyi; Yang, Yi; Xu, Yuanming; Wang, Yajun; Zhang, Yusi; Melo-Cardenas, Johanna; Xu, Xiangping; Gao, Beixue; Thorp, Edward B.; Zhang, Donna D.; Zhang, Bin; Song, Jianxun; Zhang, Kezhong; Zhang, Jianning; Zhang, Jinping; Li, Huabin; Fang, Deyu

2016-01-01

Humoral immunity involves multiple checkpoints during B-cell development, maturation, and activation. The cell death receptor CD95/Fas-mediated apoptosis plays a critical role in eliminating the unwanted activation of B cells by self-reactive antigens and in maintaining B-cell homeostasis through activation-induced B-cell death (AICD). The molecular mechanisms controlling AICD remain largely undefined. Herein, we show that the E3 ubiquitin ligase Hrd1 protected B cells from activation-induced cell death by degrading the death receptor Fas. Hrd1-null B cells exhibited high Fas expression during activation and rapidly underwent Fas-mediated apoptosis, which could be largely inhibited by FasL neutralization. Fas mutation in Hrd1 KO mice abrogated the increase in B-cell AICD. We identified Hrd1 as the first E3 ubiquitin ligase of the death receptor Fas and Hrd1-mediated Fas destruction as a molecular mechanism in regulating B-cell immunity. PMID:27573825

Endoplasmic reticulum-resident E3 ubiquitin ligase Hrd1 controls B-cell immunity through degradation of the death receptor CD95/Fas.

PubMed

Kong, Sinyi; Yang, Yi; Xu, Yuanming; Wang, Yajun; Zhang, Yusi; Melo-Cardenas, Johanna; Xu, Xiangping; Gao, Beixue; Thorp, Edward B; Zhang, Donna D; Zhang, Bin; Song, Jianxun; Zhang, Kezhong; Zhang, Jianning; Zhang, Jinping; Li, Huabin; Fang, Deyu

2016-09-13

Humoral immunity involves multiple checkpoints during B-cell development, maturation, and activation. The cell death receptor CD95/Fas-mediated apoptosis plays a critical role in eliminating the unwanted activation of B cells by self-reactive antigens and in maintaining B-cell homeostasis through activation-induced B-cell death (AICD). The molecular mechanisms controlling AICD remain largely undefined. Herein, we show that the E3 ubiquitin ligase Hrd1 protected B cells from activation-induced cell death by degrading the death receptor Fas. Hrd1-null B cells exhibited high Fas expression during activation and rapidly underwent Fas-mediated apoptosis, which could be largely inhibited by FasL neutralization. Fas mutation in Hrd1 KO mice abrogated the increase in B-cell AICD. We identified Hrd1 as the first E3 ubiquitin ligase of the death receptor Fas and Hrd1-mediated Fas destruction as a molecular mechanism in regulating B-cell immunity.

24(S)-Hydroxycholesterol induces RIPK1-dependent but MLKL-independent cell death in the absence of caspase-8.

PubMed

Vo, Diep-Khanh Ho; Urano, Yasuomi; Takabe, Wakako; Saito, Yoshiro; Noguchi, Noriko

2015-07-01

24(S)-Hydroxycholesterol (24S-OHC), which is enzymatically produced in the brain, is known to play an important role in maintaining brain cholesterol homeostasis. We have previously reported that 24S-OHC induces a type of non-apoptotic programmed necrosis in neuronal cells expressing little caspase-8. Necroptosis has been characterized as a type of programmed necrosis in which activation of receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like (MLKL) is involved in the signaling pathway. In the present study, we investigated the involvement of these three proteins in 24S-OHC-induced cell death. We found that RIPK1 but neither RIPK3 nor MLKL was expressed in human neuroblastoma SH-SY5Y cells, while all three proteins were expressed in human T lymphoma caspase-8-deficient Jurkat (Jurkat(Cas8-/-)) cells. In Jurkat(Cas8-/-) cells, tumor necrosis factor α (TNFα)-induced cell death was significantly suppressed by treatment with respective inhibitors of RIPK1, RIPK3, and MLKL. In contrast, only RIPK1 inhibitor showed significant suppression of 24S-OHC-induced cell death, and even this was less prominent than was observed in TNFα-induced cell death. In Jurkat(Cas8-/-) cells, knockdown of either RIPK1 or RIPK3 caused moderate but significant suppression of 24S-OHC-induced cell death, but no such effect was observed as a result of knockdown of MLKL. Collectively, these results suggest that, for both SH-SY5Y cells and Jurkat(Cas8-/-) cells, 24S-OHC-induced cell death is dependent on RIPK1 but not on MLKL. We therefore conclude that, in the absence of caspase-8 activity, 24S-OHC induces a necroptosis-like cell death which is RIPK1-dependent but MLKL-independent. Copyright © 2015 Elsevier Inc. All rights reserved.

Regulatory mechanism of the flavoprotein Tah18-dependent nitric oxide synthesis and cell death in yeast.

PubMed

Yoshikawa, Yuki; Nasuno, Ryo; Kawahara, Nobuhiro; Nishimura, Akira; Watanabe, Daisuke; Takagi, Hiroshi

2016-07-01

Nitric oxide (NO) is a ubiquitous signaling molecule involved in the regulation of a large number of cellular functions. The regulatory mechanism of NO generation in unicellular eukaryotic yeast cells is poorly understood due to the lack of mammalian and bacterial NO synthase (NOS) orthologues, even though yeast produces NO under oxidative stress conditions. Recently, we reported that the flavoprotein Tah18, which was previously shown to transfer electrons to the iron-sulfur cluster protein Dre2, is involved in NOS-like activity in the yeast Saccharomyces cerevisiae. On the other hand, Tah18 was reported to promote apoptotic cell death after exposure to hydrogen peroxide (H2O2). Here, we showed that NOS-like activity requiring Tah18 induced cell death upon treatment with H2O2. Our experimental results also indicate that Tah18-dependent NO production and cell death are suppressed by enhancement of the interaction between Tah18 and its molecular partner Dre2. Our findings indicate that the Tah18-Dre2 complex regulates cell death as a molecular switch via Tah18-dependent NOS-like activity in response to environmental changes. Copyright © 2016 Elsevier Inc. All rights reserved.

Apoptosis-inducing factor (Aif1) mediates anacardic acid-induced apoptosis in Saccharomyces cerevisiae.

PubMed

Muzaffar, Suhail; Chattoo, Bharat B

2017-03-01

Anacardic acid is a medicinal phytochemical that inhibits proliferation of fungal as well as several types of cancer cells. It induces apoptotic cell death in various cell types, but very little is known about the mechanism involved in the process. Here, we used budding yeast Saccharomyces cerevisiae as a model to study the involvement of some key elements of apoptosis in the anacardic acid-induced cell death. Plasma membrane constriction, chromatin condensation, DNA degradation, and externalization of phosphatidylserine (PS) indicated that anacardic acid induces apoptotic cell death in S. cerevisiae. However, the exogenous addition of broad-spectrum caspase inhibitor Z-VAD-FMK or deletion of the yeast caspase Yca1 showed that the anacardic acid-induced cell death is caspase independent. Apoptosis-inducing factor (AIF1) deletion mutant was resistant to the anacardic acid-induced cell death, suggesting a key role of Aif1. Overexpression of Aif1 made cells highly susceptible to anacardic acid, further confirming that Aif1 mediates anacardic acid-induced apoptosis. Interestingly, instead of the increase in the intracellular reactive oxygen species (ROS) normally observed during apoptosis, anacardic acid caused a decrease in the intracellular ROS levels. Quantitative real-time PCR analysis showed downregulation of the BIR1 survivin mRNA expression during the anacardic acid-induced apoptosis.

The deaths of a cell: how language and metaphor influence the science of cell death.

PubMed

Reynolds, Andrew S

2014-12-01

Multicellular development and tissue maintenance involve the regular elimination of damaged and healthy cells. The science of this genetically regulated cell death is particularly rich in metaphors: 'programmed cell death' or 'cell suicide' is considered an 'altruistic' act on the part of a cell for the benefit of the organism as a whole. It is also considered a form of 'social control' exerted by the body/organism over its component cells. This paper analyzes the various functions of these metaphors and critical discussion about them within the scientific community. Bodies such as the Nomenclature Committee on Cell Death (NCCD) have been charged with bringing order to the language of cell death to facilitate scientific progress. While the NCCD recommends adopting more objective biochemical terminology to describe the mechanisms of cell death, the metaphors in question retain an important function by highlighting the broader context within which cell death occurs. Scientific metaphors act as conceptual 'tools' which fulfill various roles, from highlighting a phenomenon as of particular interest, situating it in a particular context, or suggesting explanatory causal mechanisms. Copyright © 2014 Elsevier Ltd. All rights reserved.

Parthanatos, a messenger of death.

PubMed

David, Karen Kate; Andrabi, Shaida Ahmad; Dawson, Ted Murray; Dawson, Valina Lynn

2009-01-01

Poly-ADP-ribose polymerase-1 (PARP-1)'s roles in the cell span from maintaining life to inducing death. The processes PARP-1 is involved in include DNA repair, DNA transcription, mitosis, and cell death. Of PARP-1's different cellular functions, its role in cell death is of particular interest to designing therapies for diseases. Genetic deletion of PARP-1 revealed that PARP-1 overactivation underlies cell death in models of stroke, diabetes, inflammation and neurodegeneration. Since interfering with PARP-1 mediated cell death will be clinically beneficial, great effort has been invested into understanding mechanisms downstream of PARP-1 overactivation. Recent evidence shows that poly-ADP ribose (PAR) polymer itself can act as a cell death effector downstream of PARP-1. We coined the term parthanatos after Thanatos, the personification of death in Greek mythology, to refer to PAR-mediated cell death. In this review, we will present evidence and questions raised by these recent findings, and summarize the proposed mechanisms by which PARP-1 overactivation kills. It is evident that further understanding of parthanatos opens up new avenues for therapy in ameliorating diseases related to PARP-1 overactivation.

Catalytically active Yersinia outer protein P induces cleavage of RIP and caspase-8 at the level of the DISC independently of death receptors in dendritic cells.

PubMed

Gröbner, Sabine; Adkins, Irena; Schulz, Sebastian; Richter, Kathleen; Borgmann, Stefan; Wesselborg, Sebastian; Ruckdeschel, Klaus; Micheau, Olivier; Autenrieth, Ingo B

2007-10-01

Yersinia outer protein P (YopP) is injected by Y. enterocolitica into host cells thereby inducing apoptotic and necrosis-like cell death in dendritic cells (DC). Here we show the pathways involved in DC death caused by the catalytic activity of YopP. Infection with Yersinia enterocolitica, translocating catalytically active YopP into DC, triggered procaspase-8 cleavage and c-FLIPL degradation. YopP-dependent caspase-8 activation was, however, not mediated by tumor necrosis factor (TNF) receptor family members since the expression of both CD95/Fas/APO-1 and TRAIL-R2 on DC was low, and DC were resistant to apoptosis induced by agonistic anti-CD95 antibodies or TNF-related apoptosis-inducing ligand (TRAIL). Moreover, DC from TNF-Rp55-/- mice were not protected against YopP-induced cell death demonstrating that TNF-R1 is also not involved in this process. Activation of caspase-8 was further investigated by coimmunoprecitation of FADD from Yersinia-infected DC. We found that both cleaved caspase-8 and receptor interacting protein 1 (RIP1) were associated with the Fas-associated death domain (FADD) indicating the formation of an atypical death-inducing signaling complex (DISC). Furthermore, degradation of RIP mediated by the Hsp90 inhibitor geldanamycin significantly impaired YopP-induced cell death. Altogether our findings indicate that Yersinia-induced DC death is independent of death domain containing receptors, but mediated by RIP and caspase-8 at the level of DISC.

Nicotiana benthamiana MAPK-WRKY pathway confers resistance to a necrotrophic pathogen Botrytis cinerea.

PubMed

Adachi, Hiroaki; Ishihama, Nobuaki; Nakano, Takaaki; Yoshioka, Miki; Yoshioka, Hirofumi

2016-06-02

MEK2-SIPK/WIPK cascade, a Nicotiana benthamiana mitogen-activated protein kinase (MAPK) cascade, is an essential signaling pathway for plant immunity and involved in hypersensitive response (HR) accompanied by cell death. WRKY transcription factors as substrates of SIPK and WIPK have been isolated and implicated in HR cell death. Here, we show virus-induced gene silencing of WRKY genes compromised constitutively active MEK2-triggered cell death in N. benthamiana leaves. In general, HR cell death enhances susceptibility to necrotrophic pathogens such as Botrytis cinerea. However, the WRKY gene silencing elevated susceptibility to B. cinerea. These findings suggest that downstream WRKYs of MEK2-SIPK/WIPK cascade are required for cell death-dependent and -independent immunities in N. benthamiana.

Autophagy induced by baicalin involves downregulation of CD147 in SMMC-7721 cells in vitro

PubMed Central

ZHANG, XIANJIAO; TANG, XU; LIU, HANQIANG; LI, LIANXIANG; HOU, QIAN; GAO, JIANMIN

2012-01-01

Baicalin has been demonstrated to exert anticancer effects mainly through induction of tumor cell apoptosis and cell cycle arrest. However, the precise mechanisms underlying its anticancer role remain to be elucidated. In the present study, we investigated whether autophagy was involved in the anticancer activity of baicalin in the human hepatocellular carcinoma (HCC) cell line SMMC-7721 and the possible molecular mechanisms. Our data showed that the viability of SMMC-7721 cells was significantly inhibited by baicalin in a dose- and time-dependent manner. Alongside apoptosis, autophagy was also induced by baicalin dose- and time-dependently with the involvement of the autophagy-associated protein Beclin 1. Moreover, we demonstrated that cell death induced by baicalin was significantly inhibited by the apoptosis inhibitor z-DEVD-fmk or the autophagy inhibitor 3-MA, respectively. In addition, we found that CD147, a key molecule related both to apoptosis and autophagy, was markedly downregulated at the protein level in SMMC-7721 cells treated with baicalin. Collectively, this is the first study to suggest that baicalin induces autophagic cell death in SMMC-7721 cells, which involves the downregulation of CD147. Our study reveals a new mechanism for the anticancer effects of baicalin and puts forward a potential crucial role of CD147 in baicalin-induced cancer cell death. PMID:22200845

p53 death signal is mainly mediated by Nuc1(EndoG) in the yeast Saccharomyces cerevisiae.

PubMed

Palermo, Vanessa; Mangiapelo, Eleonora; Piloto, Cristina; Pieri, Luisa; Muscolini, Michela; Tuosto, Loretta; Mazzoni, Cristina

2013-11-01

The tumor suppressor p53 plays a central role in the regulation of cellular growth and apoptosis. In the yeast Saccharomyces cerevisiae, the overexpression of the human p53 leads to growth inhibition and apoptotic cell death on minimal medium. In the present work, we show that p53-expressing cells are more susceptible to cell death after an apoptotic stimulus such as H2O2. The analysis of mutants involved in yeast apoptosis-like death suggests that the observed cell death is Yca1 independent and mainly mediated through Nuc1p. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

NADE, a p75NTR-associated cell death executor, is involved in signal transduction mediated by the common neurotrophin receptor p75NTR.

PubMed

Mukai, J; Hachiya, T; Shoji-Hoshino, S; Kimura, M T; Nadano, D; Suvanto, P; Hanaoka, T; Li, Y; Irie, S; Greene, L A; Sato, T A

2000-06-09

The low affinity neurotrophin receptor p75NTR can mediate cell survival as well as cell death of neural cells by NGF and other neurotrophins. To elucidate p75NTR-mediated signal transduction, we screened p75NTR-associated proteins by a yeast two-hybrid system. We identified one positive clone and named NADE (p75NTR-associated cell death executor). Mouse NADE has marked homology to the human HGR74 protein. NADE specifically binds to the cell-death domain of p75NTR. Co-expression of NADE and p75NTR induced caspase-2 and caspase-3 activities and the fragmentation of nuclear DNA in 293T cells. However, in the absence of p75NTR, NADE failed to induce apoptosis, suggesting that NADE expression is necessary but insufficient for p75NTR-mediated apoptosis. Furthermore, p75NTR/NADE-induced cell death was dependent on NGF but not BDNF, NT-3, or NT-4/5, and the recruitment of NADE to p75NTR (intracellular domain) was dose-dependent. We obtained similar results from PC12 cells, nnr5 cells, and oligodendrocytes. Taken together, NADE is the first signaling adaptor molecule identified in the involvement of p75NTR-mediated apoptosis induced by NGF, and it may play an important role in the pathogenesis of neurogenetic diseases.

Involvement of mitochondrial proteins in calcium signaling and cell death induced by staurosporine in Neurospora crassa.

PubMed

Gonçalves, A Pedro; Cordeiro, J Miguel; Monteiro, João; Lucchi, Chiara; Correia-de-Sá, Paulo; Videira, Arnaldo

2015-10-01

Staurosporine-induced cell death in Neurospora crassa includes a well defined sequence of alterations in cytosolic calcium levels, comprising extracellular Ca(2+) influx and mobilization of Ca(2+) from internal stores. Here, we show that cells undergoing respiratory stress due to the lack of certain components of the mitochondrial complex I (like the 51kDa and 14kDa subunits) or the Ca(2+)-binding alternative NADPH dehydrogenase NDE-1 are hypersensitive to staurosporine and incapable of setting up a proper intracellular Ca(2+) response. Cells expressing mutant forms of NUO51 that mimic human metabolic diseases also presented Ca(2+) signaling deficiencies. Accumulation of reactive oxygen species is increased in cells lacking NDE-1 and seems to be required for Ca(2+) oscillations in response to staurosporine. Measurement of the mitochondrial levels of Ca(2+) further supported the involvement of these organelles in staurosporine-induced Ca(2+) signaling. In summary, our data indicate that staurosporine-induced fungal cell death involves a sophisticated response linking Ca(2+) dynamics and bioenergetics. Copyright © 2015 Elsevier B.V. All rights reserved.

UV-Induced cell death in plants.

PubMed

Nawkar, Ganesh M; Maibam, Punyakishore; Park, Jung Hoon; Sahi, Vaidurya Pratap; Lee, Sang Yeol; Kang, Chang Ho

2013-01-14

Plants are photosynthetic organisms that depend on sunlight for energy. Plants respond to light through different photoreceptors and show photomorphogenic development. Apart from Photosynthetically Active Radiation (PAR; 400-700 nm), plants are exposed to UV light, which is comprised of UV-C (below 280 nm), UV-B (280-320 nm) and UV-A (320-390 nm). The atmospheric ozone layer protects UV-C radiation from reaching earth while the UVR8 protein acts as a receptor for UV-B radiation. Low levels of UV-B exposure initiate signaling through UVR8 and induce secondary metabolite genes involved in protection against UV while higher dosages are very detrimental to plants. It has also been reported that genes involved in MAPK cascade help the plant in providing tolerance against UV radiation. The important targets of UV radiation in plant cells are DNA, lipids and proteins and also vital processes such as photosynthesis. Recent studies showed that, in response to UV radiation, mitochondria and chloroplasts produce a reactive oxygen species (ROS). Arabidopsis metacaspase-8 (AtMC8) is induced in response to oxidative stress caused by ROS, which acts downstream of the radical induced cell death (AtRCD1) gene making plants vulnerable to cell death. The studies on salicylic and jasmonic acid signaling mutants revealed that SA and JA regulate the ROS level and antagonize ROS mediated cell death. Recently, molecular studies have revealed genes involved in response to UV exposure, with respect to programmed cell death (PCD).

UV-Induced Cell Death in Plants

PubMed Central

Nawkar, Ganesh M.; Maibam, Punyakishore; Park, Jung Hoon; Sahi, Vaidurya Pratap; Lee, Sang Yeol; Kang, Chang Ho

2013-01-01

Plants are photosynthetic organisms that depend on sunlight for energy. Plants respond to light through different photoreceptors and show photomorphogenic development. Apart from Photosynthetically Active Radiation (PAR; 400–700 nm), plants are exposed to UV light, which is comprised of UV-C (below 280 nm), UV-B (280–320 nm) and UV-A (320–390 nm). The atmospheric ozone layer protects UV-C radiation from reaching earth while the UVR8 protein acts as a receptor for UV-B radiation. Low levels of UV-B exposure initiate signaling through UVR8 and induce secondary metabolite genes involved in protection against UV while higher dosages are very detrimental to plants. It has also been reported that genes involved in MAPK cascade help the plant in providing tolerance against UV radiation. The important targets of UV radiation in plant cells are DNA, lipids and proteins and also vital processes such as photosynthesis. Recent studies showed that, in response to UV radiation, mitochondria and chloroplasts produce a reactive oxygen species (ROS). Arabidopsis metacaspase-8 (AtMC8) is induced in response to oxidative stress caused by ROS, which acts downstream of the radical induced cell death (AtRCD1) gene making plants vulnerable to cell death. The studies on salicylic and jasmonic acid signaling mutants revealed that SA and JA regulate the ROS level and antagonize ROS mediated cell death. Recently, molecular studies have revealed genes involved in response to UV exposure, with respect to programmed cell death (PCD). PMID:23344059

Ophiobolin A, a sesterpenoid fungal phytotoxin, displays different mechanisms of cell death in mammalian cells depending upon the cancer cell origin.

PubMed

Morrison, Rachel; Lodge, Tiffany; Evidente, Antonio; Kiss, Robert; Townley, Helen

2017-03-01

Herein we have undertaken a systematic analysis of the effects of the fungal derivative ophiobolin A (OphA) on eight cancer cell lines from different tissue types. The LD50 for each cell line was determined and the change in cell size determined. Flow cytometric analysis and western blotting were used to assess the cell death markers for early apoptosis, late apoptosis and necrosis, and the involvement of the caspase signalling pathway. Alterations in calcium levels and reactive oxygen species were assessed due to their integral involvement in intracellular signalling. Subsequently, the endoplasmic reticulum (ER) and mitochondrial responses were investigated more closely. The extent of ER swelling, and the upregulation of proteins involved in the unfolded protein responses (UPR) were seen to vary according to cell line. The mitochondria were also shown to behave differently in response to the OphA in the different cell lines in terms of the change in membrane potential, the total area of mitochondria in the cell and the number of mitochondrial bifurcations. The data obtained in the present study indicate that the cancer cell lines tested are unable to successfully activate the ER stress/UPR responses, and that the mitochondria appear to be a central player in OphA-induced cancer cell death.

Autophagic cell death induced by reactive oxygen species is involved in hyperthermic sensitization to ionizing radiation in human hepatocellular carcinoma cells.

PubMed

Yuan, Guang-Jin; Deng, Jun-Jian; Cao, De-Dong; Shi, Lei; Chen, Xin; Lei, Jin-Ju; Xu, Xi-Ming

2017-08-14

To investigate whether autophagic cell death is involved in hyperthermic sensitization to ionizing radiation in human hepatocellular carcinoma cells, and to explore the underlying mechanism. Human hepatocellular carcinoma cells were treated with hyperthermia and ionizing radiation. MTT and clonogenic assays were performed to determine cell survival. Cell autophagy was detected using acridine orange staining and flow cytometric analysis, and the expression of autophagy-associated proteins, LC3 and p62, was determined by Western blot analysis. Intracellular reactive oxygen species (ROS) were quantified using the fluorescent probe DCFH-DA. Treatment with hyperthermia and ionizing radiation significantly decreased cell viability and surviving fraction as compared with hyperthermia or ionizing radiation alone. Cell autophagy was significantly increased after ionizing radiation combined with hyperthermia treatment, as evidenced by increased formation of acidic vesicular organelles, increased expression of LC3II and decreased expression of p62. Intracellular ROS were also increased after combined treatment with hyperthermia and ionizing radiation. Pretreatment with N-acetylcysteine, an ROS scavenger, markedly inhibited the cytotoxicity and cell autophagy induced by hyperthermia and ionizing radiation. Autophagic cell death is involved in hyperthermic sensitization of cancer cells to ionizing radiation, and its induction may be due to the increased intracellular ROS.

Autophagy promotes caspase-dependent cell death during Drosophila development.

PubMed

Mohseni, Nilufar; McMillan, Stephanie C; Chaudhary, Roopali; Mok, Jane; Reed, Bruce H

2009-04-01

The relationship between autophagic cell death and apoptosis is a poorly understood aspect of programmed cell death (PCD). We have examined this relationship by studying the elimination of an extra-embryonic tissue, known as the amnioserosa (AS), during Drosophila development. The AS becomes autophagic during the final stages of embryogenesis; ultimately, however, the elimination of the AS involves caspase-dependent nuclear fragmentation, tissue dissociation and engulfment by phagocytic macrophages. Mutants that are defective in the activation or execution of caspase-dependent PCD fail to degrade and eliminate the AS but show no abatement in AS autophagy. Sustained autophagy does not, therefore, necessarily result in cell death. Surprisingly, the downregulation of autophagy also results in a persistent AS phenotype and reduced cell death. Conversely, upregulation of autophagy results in caspase-dependent premature AS dissociation. These observations are consistent with the interpretation that autophagy is a prerequisite for caspase-dependent cell death in the AS.

Persistent random walk of cells involving anomalous effects and random death

NASA Astrophysics Data System (ADS)

Fedotov, Sergei; Tan, Abby; Zubarev, Andrey

2015-04-01

The purpose of this paper is to implement a random death process into a persistent random walk model which produces sub-ballistic superdiffusion (Lévy walk). We develop a stochastic two-velocity jump model of cell motility for which the switching rate depends upon the time which the cell has spent moving in one direction. It is assumed that the switching rate is a decreasing function of residence (running) time. This assumption leads to the power law for the velocity switching time distribution. This describes the anomalous persistence of cell motility: the longer the cell moves in one direction, the smaller the switching probability to another direction becomes. We derive master equations for the cell densities with the generalized switching terms involving the tempered fractional material derivatives. We show that the random death of cells has an important implication for the transport process through tempering of the superdiffusive process. In the long-time limit we write stationary master equations in terms of exponentially truncated fractional derivatives in which the rate of death plays the role of tempering of a Lévy jump distribution. We find the upper and lower bounds for the stationary profiles corresponding to the ballistic transport and diffusion with the death-rate-dependent diffusion coefficient. Monte Carlo simulations confirm these bounds.

Autophagy protein p62/SQSTM1 is involved in HAMLET-induced cell death by modulating apotosis in U87MG cells

PubMed Central

Zhang, Y-B; Gong, J-L; Xing, T-Y; Zheng, S-P; Ding, W

2013-01-01

HAMLET is a complex of oleic acids and decalcified α-lactalbumin that was discovered to selectively kill tumor cells both in vitro and in vivo. Autophagy is an important cellular process involved in drug-induced cell death of glioma cells. We treated U87MG human glioma cells with HAMLET and found that the cell viability was significantly decreased and accompanied with the activation of autophagy. Interestingly, we observed an increase in p62/SQSTM1, an important substrate of autophagosome enzymes, at the protein level upon HAMLET treatment for short periods. To better understand the functionality of autophagy and p62/SQSTM1 in HAMLET-induced cell death, we modulated the level of autophagy or p62/SQSTM1 with biochemical or genetic methods. The results showed that inhibition of autophagy aggravated HAMLET-induced cell death, whereas activation of authophagy attenuated this process. Meanwhile, we found that overexpression of wild-type p62/SQSTM1 was able to activate caspase-8, and then promote HAMLET-induced apoptosis, whereas knockdown of p62/SQSTM1 manifested the opposite effect. We further demonstrated that the function of p62/SQSTM1 following HAMLET treatment required its C-terminus UBA domain. Our results indicated that in addition to being a marker of autophagy activation in HAMLET-treated glioma cells, p62/SQSTM1 could also function as an important mediator for the activation of caspase-8-dependent cell death. PMID:23519119

Autophagy protein p62/SQSTM1 is involved in HAMLET-induced cell death by modulating apotosis in U87MG cells.

PubMed

Zhang, Y-B; Gong, J-L; Xing, T-Y; Zheng, S-P; Ding, W

2013-03-21

HAMLET is a complex of oleic acids and decalcified α-lactalbumin that was discovered to selectively kill tumor cells both in vitro and in vivo. Autophagy is an important cellular process involved in drug-induced cell death of glioma cells. We treated U87MG human glioma cells with HAMLET and found that the cell viability was significantly decreased and accompanied with the activation of autophagy. Interestingly, we observed an increase in p62/SQSTM1, an important substrate of autophagosome enzymes, at the protein level upon HAMLET treatment for short periods. To better understand the functionality of autophagy and p62/SQSTM1 in HAMLET-induced cell death, we modulated the level of autophagy or p62/SQSTM1 with biochemical or genetic methods. The results showed that inhibition of autophagy aggravated HAMLET-induced cell death, whereas activation of authophagy attenuated this process. Meanwhile, we found that overexpression of wild-type p62/SQSTM1 was able to activate caspase-8, and then promote HAMLET-induced apoptosis, whereas knockdown of p62/SQSTM1 manifested the opposite effect. We further demonstrated that the function of p62/SQSTM1 following HAMLET treatment required its C-terminus UBA domain. Our results indicated that in addition to being a marker of autophagy activation in HAMLET-treated glioma cells, p62/SQSTM1 could also function as an important mediator for the activation of caspase-8-dependent cell death.

Endoplasmic reticulum calcium release potentiates the ER stress and cell death caused by an oxidative stress in MCF-7 cells.

PubMed

Dejeans, Nicolas; Tajeddine, Nicolas; Beck, Raphaël; Verrax, Julien; Taper, Henryk; Gailly, Philippe; Calderon, Pedro Buc

2010-05-01

Increase in cytosolic calcium concentration ([Ca2+](c)), release of endoplasmic reticulum (ER) calcium ([Ca2+](er)) and ER stress have been proposed to be involved in oxidative toxicity. Nevertheless, their relative involvements in the processes leading to cell death are not well defined. In this study, we investigated whether oxidative stress generated during ascorbate-driven menadione redox cycling (Asc/Men) could trigger these three events, and, if so, whether they contributed to Asc/Men cytoxicity in MCF-7 cells. Using microspectrofluorimetry, we demonstrated that Asc/Men-generated oxidative stress was associated with a slow and moderate increase in [Ca2+](c), largely preceding permeation of propidium iodide, and thus cell death. Asc/Men treatment was shown to partially deplete ER calcium stores after 90 min (decrease by 45% compared to control). This event was associated with ER stress activation, as shown by analysis of eIF2 phosphorylation and expression of the molecular chaperone GRP94. Thapsigargin (TG) was then used to study the effect of complete [Ca2+](er) emptying during the oxidative stress generated by Asc/Men. Surprisingly, the combination of TG and Asc/Men increased ER stress to a level considerably higher than that observed for either treatment alone, suggesting that [Ca2+](er) release alone is not sufficient to explain ER stress activation during oxidative stress. Finally, TG-mediated [Ca2+](er) release largely potentiated ER stress, DNA fragmentation and cell death caused by Asc/Men, supporting a role of ER stress in the process of Asc/Men cytotoxicity. Taken together, our results highlight the involvement of ER stress and [Ca2+](er) decrease in the process of oxidative stress-induced cell death in MCF-7 cells. 2009 Elsevier Inc. All rights reserved.

Necroptosis, necrostatins and tissue injury

PubMed Central

Smith, Christopher CT; Yellon, Derek M

2011-01-01

Abstract Cell death is an integral part of the life of an organism being necessary for the maintenance of organs and tissues. If, however, cell death is allowed to proceed unrestricted, tissue damage and degenerative disease may ensue. Until recently, three morphologically distinct types of cell death were recognized, apoptosis (type I), autophagy (type II) and necrosis (type III). Apoptosis is a highly regulated, genetically determined mechanism designed to dismantle cells systematically (e.g. cells that are no longer functionally viable), via protease (caspase) action, and maintain homeostasis. Autophagy is responsible for the degradation of cytoplasmic material, e.g. proteins and organelles, through autophagosome formation and subsequent proteolytic degradation by lysosomes, and is normally considered in the context of survival although it is sometimes associated with cell death. Necrosis was formerly considered to be an accidental, unregulated form of cell death resulting from excessive stress, although it has been suggested that this is an over-simplistic view as necrosis may under certain circumstances involve the mobilization of specific transduction mechanisms. Indeed, recently, an alternative death pathway, termed necroptosis, was delineated and proposed as a form of ‘programmed necrosis’. Identified with the aid of specific inhibitors called necrostatins, necroptosis shares characteristics with both necrosis and apoptosis. Necroptosis involves Fas/tumour necrosis factor-α death domain receptor activation and inhibition of receptor-interacting protein I kinase, and it has been suggested that it may contribute to the development of neurological and myocardial diseases. Significantly, necrostatin-like drugs have been mooted as possible future therapeutic agents for the treatment of degenerative conditions. PMID:21564515

Host-Cell Survival and Death During Chlamydia Infection

PubMed Central

Ying, Songmin; Pettengill, Matthew; Ojcius, David M.; Häcker, Georg

2008-01-01

Different Chlamydia trachomatis strains are responsible for prevalent bacterial sexually-transmitted disease and represent the leading cause of preventable blindness worldwide. Factors that predispose individuals to disease and mechanisms by which chlamydiae cause inflammation and tissue damage remain unclear. Results from recent studies indicate that prolonged survival and subsequent death of infected cells and their effect on immune effector cells during chlamydial infection may be important in determining the outcome. Survival of infected cells is favored at early times of infection through inhibition of the mitochondrial pathway of apoptosis. Death at later times displays features of both apoptosis and necrosis, but pro-apoptotic caspases are not involved. Most studies on chlamydial modulation of host-cell death until now have been performed in cell lines. The consequences for pathogenesis and the immune response will require animal models of chlamydial infection, preferably mice with targeted deletions of genes that play a role in cell survival and death. PMID:18843378

"(Not) all (dead) things share the same breath": identification of cell death mechanisms in anticancer therapy.

PubMed

Rello-Varona, Santiago; Herrero-Martín, David; López-Alemany, Roser; Muñoz-Pinedo, Cristina; Tirado, Oscar M

2015-03-15

During the last decades, the knowledge of cell death mechanisms involved in anticancer therapy has grown exponentially. However, in many studies, cell death is still described in an incomplete manner. The frequent use of indirect proliferation assays, unspecific probes, or bulk analyses leads too often to misunderstandings regarding cell death events. There is a trend to focus on molecular or genetic regulations of cell demise without a proper characterization of the phenotype that is the object of this study. Sometimes, cancer researchers can feel overwhelmed or confused when faced with such a corpus of detailed insights, nomenclature rules, and debates about the accuracy of a particular probe or assay. On the basis of the information available, we propose a simple guide to distinguish forms of cell death in experimental settings using cancer cell lines. ©2015 American Association for Cancer Research.

NADPH Oxidase Activation Contributes to Heavy Ion Irradiation–Induced Cell Death

PubMed Central

Wang, Yupei; Liu, Qing; Zhao, Weiping; Zhou, Xin; Miao, Guoying; Sun, Chao

2017-01-01

Increased oxidative stress plays an important role in heavy ion radiation–induced cell death. The mechanism involved in the generation of elevated reactive oxygen species (ROS) is not fully illustrated. Here we show that NADPH oxidase activation is closely related to heavy ion radiation–induced cell death via excessive ROS generation. Cell death and cellular ROS can be greatly reduced in irradiated cancer cells with the preincubation of diphenyleneiodium, an inhibitor of NADPH oxidase. Most of the NADPH oxidase (NOX) family proteins (NOX1, NOX2, NOX3, NOX4, and NOX5) showed increased expression after heavy ion irradiation. Meanwhile, the cytoplasmic subunit p47phox was translocated to the cell membrane and localized with NOX2 to form reactive NADPH oxidase. Our data suggest for the first time that ROS generation, as mediated by NADPH oxidase activation, could be an important contributor to heavy ion irradiation–induced cell death. PMID:28473742

CDIP, a novel pro-apoptotic gene, regulates TNFalpha-mediated apoptosis in a p53-dependent manner.

PubMed

Brown, Lauren; Ongusaha, Pat P; Kim, Hyung-Gu; Nuti, Shanthy; Mandinova, Anna; Lee, Ji Won; Khosravi-Far, Roya; Aaronson, Stuart A; Lee, Sam W

2007-07-25

We have identified a novel pro-apoptotic p53 target gene named CDIP (Cell Death Involved p53-target). Inhibition of CDIP abrogates p53-mediated apoptotic responses, demonstrating that CDIP is an important p53 apoptotic effector. CDIP itself potently induces apoptosis that is associated with caspase-8 cleavage, implicating the extrinsic cell death pathway in apoptosis mediated by CDIP. siRNA-directed knockdown of caspase-8 results in a severe impairment of CDIP-dependent cell death. In investigating the potential involvement of extrinsic cell death pathway in CDIP-mediated apoptosis, we found that TNF-alpha expression tightly correlates with CDIP expression, and that inhibition of TNF-alpha signaling attenuates CDIP-dependent apoptosis. We also demonstrate that TNF-alpha is upregulated in response to p53 and p53 inducing genotoxic stress, in a CDIP-dependent manner. Consistently, knockdown of TNF-alpha impairs p53-mediated stress-induced apoptosis. Together, these findings support a novel p53 --> CDIP --> TNF-alpha apoptotic pathway that directs apoptosis after exposure of cells to genotoxic stress. Thus, CDIP provides a new link between p53-mediated intrinsic and death receptor-mediated extrinsic apoptotic signaling, providing a novel target for cancer therapeutics aimed at maximizing the p53 apoptotic response of cancer cells to drug therapy.

The MST/Hippo Pathway and Cell Death: A Non-Canonical Affair

PubMed Central

Fallahi, Emma; O’Driscoll, Niamh A.; Matallanas, David

2016-01-01

The MST/Hippo signalling pathway was first described over a decade ago in Drosophila melanogaster and the core of the pathway is evolutionary conserved in mammals. The mammalian MST/Hippo pathway regulates organ size, cell proliferation and cell death. In addition, it has been shown to play a central role in the regulation of cellular homeostasis and it is commonly deregulated in human tumours. The delineation of the canonical pathway resembles the behaviour of the Hippo pathway in the fly where the activation of the core kinases of the pathway prevents the proliferative signal mediated by the key effector of the pathway YAP. Nevertheless, several lines of evidence support the idea that the mammalian MST/Hippo pathway has acquired new features during evolution, including different regulators and effectors, crosstalk with other essential signalling pathways involved in cellular homeostasis and the ability to actively trigger cell death. Here we describe the current knowledge of the mechanisms that mediate MST/Hippo dependent cell death, especially apoptosis. We include evidence for the existence of complex signalling networks where the core proteins of the pathway play a central role in controlling the balance between survival and cell death. Finally, we discuss the possible involvement of these signalling networks in several human diseases such as cancer, diabetes and neurodegenerative disorders. PMID:27322327

Ceramide, sphingoid bases, and sphingoid base metabolites as lipid mediators in signaling pathways leading to cell death and disease

USDA-ARS?s Scientific Manuscript database

Increased ceramide generation de novo is known to be involved in the mechanism of action of many chemotherapeutic agents and conditions which disrupt cell cycle progression and induce cell death. Conversely, the metabolism of ceramide to sphingoid bases and and sphingoid base 1-phosphates has been i...

Sulfur mustard-induced apoptosis in hairless guinea pig skin.

PubMed

Kan, Robert K; Pleva, Christina M; Hamilton, Tracey A; Anderson, Dana R; Petrali, John P

2003-01-01

The present study was aimed to examine whether apoptosis is involved in the pathogenesis of sulfur mustard (SM)-induced basal cell death. Skin sites of the hairless guinea pig exposed to SM vapor for 8 minutes were harvested at 3, 6, 12, 24, and 48 hours postexposure. Immunohistochemical detection of basal cell apoptosis was performed using the ApopTag in situ apoptosis labeling kit. Only occasional apoptotic basal cells (BC)were observed in nonexposed and perilesional control sites. At lesional sites, apoptosis of BC was not detected at 3 hours postexposure. However, at 6 hours and 12 hours postexposure, 18% and 59% of BC were apoptotic, respectively. At 24 and 48 hours postexposure, individual apoptotic basal cells were not clearly recognizable due to necrosis. At the ultrastructural level, degenerating BC exhibited typical apoptotic morphology including nuclear condensation and chromatin margination. The results suggest that apoptotic cell death is a cytotoxic mechanism with the number of BC undergoing apoptosis significantly increasing from 6 to 12 hours postexposure. In addition, because necrosis is preferential at 24 hours postexposure, we believe that SM-induced cell death involves early apoptosis and late necrosis, which temporally overlap to produce a single cell death pathway along an apoptotic-necrotic continuum.

Novel function of STAT1beta in B cells: induction of cell death by a mechanism different from that of STAT1alpha.

PubMed

Najjar, Imen; Schischmanoff, Pierre Olivier; Baran-Marszak, Fanny; Deglesne, Pierre-Antoine; Youlyouz-Marfak, Ibtissam; Pampin, Mathieu; Feuillard, Jean; Bornkamm, Georg W; Chelbi-Alix, Mounira K; Fagard, Remi

2008-12-01

Alternate splicing of STAT1 produces two isoforms: alpha, known as the active form, and beta, previously shown to act as a dominant-negative factor. Most studies have dealt with STAT1alpha, showing its involvement in cell growth control and cell death. To examine the specific function of either isoform in cell death, a naturally STAT1-deficient human B cell line was transfected to express STAT1alpha or STAT1beta. STAT1alpha, expressed alone, enhanced cell death, potentiated the fludarabine-induced apoptosis, and enhanced the nuclear location, the phosphorylation, and the transcriptional activity of p53. Unexpectedly, STAT1beta, expressed alone, induced cell death through a mechanism that was independent of the nuclear function of p53. Indeed, in STAT1beta-expressing B cells, p53 was strictly cytoplasmic where it formed clusters, and there was no induction of the transcriptional activity of p53. These data reveal a novel role of STAT1beta in programmed cell death, which is independent of p53.

Nitric oxide released from JS-K induces cell death by mitotic catastrophe as part of necrosis in glioblastoma multiforme

PubMed Central

Günzle, Jessica; Osterberg, Nadja; Saavedra, Joseph E; Weyerbrock, Astrid

2016-01-01

The nitric oxide (NO) donor JS-K is specifically activated by glutathione S-transferases (GSTs) in GST-overexpressing cells. We have shown the induction of cell death in glioblastoma multiforme (GBM) cells at high JS-K doses but the mechanism remains unclear. The aim of this study was to determine whether NO-induced cell death is triggered by induction of apoptotic or necrotic pathways. For the first time, we demonstrate that NO induces cell death via mitotic catastrophe (MC) with non-apoptotic mechanisms in GBM cells. Moreover, the level of morphological changes indicating MC correlates with increased necrosis. Therefore, we conclude that MC is the main mechanism by which GBM cells undergo cell death after treatment with JS-K associated with necrosis rather than apoptosis. In addition, we show that PARP1 is not an exclusive marker for late apoptosis but is also involved in MC. Activating an alternative way of cell death can be useful for the multimodal cancer therapy of GBM known for its strong anti-apoptotic mechanisms and drug resistance. PMID:27584787

Nitric oxide released from JS-K induces cell death by mitotic catastrophe as part of necrosis in glioblastoma multiforme.

PubMed

Günzle, Jessica; Osterberg, Nadja; Saavedra, Joseph E; Weyerbrock, Astrid

2016-09-01

The nitric oxide (NO) donor JS-K is specifically activated by glutathione S-transferases (GSTs) in GST-overexpressing cells. We have shown the induction of cell death in glioblastoma multiforme (GBM) cells at high JS-K doses but the mechanism remains unclear. The aim of this study was to determine whether NO-induced cell death is triggered by induction of apoptotic or necrotic pathways. For the first time, we demonstrate that NO induces cell death via mitotic catastrophe (MC) with non-apoptotic mechanisms in GBM cells. Moreover, the level of morphological changes indicating MC correlates with increased necrosis. Therefore, we conclude that MC is the main mechanism by which GBM cells undergo cell death after treatment with JS-K associated with necrosis rather than apoptosis. In addition, we show that PARP1 is not an exclusive marker for late apoptosis but is also involved in MC. Activating an alternative way of cell death can be useful for the multimodal cancer therapy of GBM known for its strong anti-apoptotic mechanisms and drug resistance.

Glutathione Efflux and Cell Death

PubMed Central

2012-01-01

Abstract Significance: Glutathione (GSH) depletion is a central signaling event that regulates the activation of cell death pathways. GSH depletion is often taken as a marker of oxidative stress and thus, as a consequence of its antioxidant properties scavenging reactive species of both oxygen and nitrogen (ROS/RNS). Recent Advances: There is increasing evidence demonstrating that GSH loss is an active phenomenon regulating the redox signaling events modulating cell death activation and progression. Critical Issues: In this work, we review the role of GSH depletion by its efflux, as an important event regulating alterations in the cellular redox balance during cell death independent from oxidative stress and ROS/RNS formation. We discuss the mechanisms involved in GSH efflux during cell death progression and the redox signaling events by which GSH depletion regulates the activation of the cell death machinery. Future Directions: The evidence summarized here clearly places GSH transport as a central mechanism mediating redox signaling during cell death progression. Future studies should be directed toward identifying the molecular identity of GSH transporters mediating GSH extrusion during cell death, and addressing the lack of sensitive approaches to quantify GSH efflux. Antioxid. Redox Signal. 17, 1694–1713. PMID:22656858

TNF-induced necroptosis requires the plasma membrane localization of the MLKL protein | Center for Cancer Research

Cancer.gov

The cell signaling protein tumor necrosis factor (TNF), produced by white blood cells, promotes inflammation and immunity processes such as fever and is involved in tumorigenesis and apoptosis (programmed cell death). However, dysregulation of TNF can also lead to another form of programmed cell death called necroptosis, which is characterized by a rise in intracellular Ca2+,

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schock, Sarah C.; Edrissi, Hamidreza; Burger, Dylan

Highlights: • Microparticles are elevated in the plasma in a rodent model of chronic cerebral ischemia. • These microparticles initiate apoptosis in cultured cells. • Microparticles contain caspase 3 and they activate receptors for TNF-α and TRAIL. - Abstract: Circulating microparticles (MPs) are involved in many physiological processes and numbers are increased in a variety of cardiovascular disorders. The present aims were to characterize levels of MPs in a rodent model of chronic cerebral hypoperfusion (CCH) and to determine their signaling properties. MPs were isolated from the plasma of rats exposed to CCH and quantified by flow cytometry. When MPsmore » were added to cultured endothelial cells or normal rat kidney cells they induced cell death in a time and dose dependent manner. Analysis of pellets by electron microscopy indicates that cell death signals are carried by particles in the range of 400 nm in diameter or less. Cell death involved the activation of caspase 3 and was not a consequence of oxidative stress. Inhibition of the Fas/FasL signaling pathway also did not improve cell survival. MPs were found to contain caspase 3 and treating the MPs with a caspase 3 inhibitor significantly reduced cell death. A TNF-α receptor blocker and a TRAIL neutralizing antibody also significantly reduced cell death. Levels of circulating MPs are elevated in a rodent model of chronic cerebral ischemia. MPs with a diameter of 400 nm or less activate the TNF-α and TRAIL signaling pathways and may deliver caspase 3 to cultured cells.« less

A set of nutrient limitations trigger yeast cell death in a nitrogen-dependent manner during wine alcoholic fermentation

PubMed Central

Duc, Camille; Pradal, Martine; Sanchez, Isabelle; Noble, Jessica; Tesnière, Catherine

2017-01-01

Yeast cell death can occur during wine alcoholic fermentation. It is generally considered to result from ethanol stress that impacts membrane integrity. This cell death mainly occurs when grape musts processing reduces lipid availability, resulting in weaker membrane resistance to ethanol. However the mechanisms underlying cell death in these conditions remain unclear. We examined cell death occurrence considering yeast cells ability to elicit an appropriate response to a given nutrient limitation and thus survive starvation. We show here that a set of micronutrients (oleic acid, ergosterol, pantothenic acid and nicotinic acid) in low, growth-restricting concentrations trigger cell death in alcoholic fermentation when nitrogen level is high. We provide evidence that nitrogen signaling is involved in cell death and that either SCH9 deletion or Tor inhibition prevent cell death in several types of micronutrient limitation. Under such limitations, yeast cells fail to acquire any stress resistance and are unable to store glycogen. Unexpectedly, transcriptome analyses did not reveal any major changes in stress genes expression, suggesting that post-transcriptional events critical for stress response were not triggered by micronutrient starvation. Our data point to the fact that yeast cell death results from yeast inability to trigger an appropriate stress response under some conditions of nutrient limitations most likely not encountered by yeast in the wild. Our conclusions provide a novel frame for considering both cell death and the management of nutrients during alcoholic fermentation. PMID:28922393

Ophiobolin A, a sesterpenoid fungal phytotoxin, displays different mechanisms of cell death in mammalian cells depending upon the cancer cell origin

PubMed Central

Morrison, Rachel; Lodge, Tiffany; Evidente, Antonio; Kiss, Robert; Townley, Helen

2017-01-01

Herein we have undertaken a systematic analysis of the effects of the fungal derivative ophiobolin A (OphA) on eight cancer cell lines from different tissue types. The LD50 for each cell line was determined and the change in cell size determined. Flow cytometric analysis and western blotting were used to assess the cell death markers for early apoptosis, late apoptosis and necrosis, and the involvement of the caspase signalling pathway. Alterations in calcium levels and reactive oxygen species were assessed due to their integral involvement in intracellular signalling. Subsequently, the endoplasmic reticulum (ER) and mitochondrial responses were investigated more closely. The extent of ER swelling, and the upregulation of proteins involved in the unfolded protein responses (UPR) were seen to vary according to cell line. The mitochondria were also shown to behave differently in response to the OphA in the different cell lines in terms of the change in membrane potential, the total area of mitochondria in the cell and the number of mitochondrial bifurcations. The data obtained in the present study indicate that the cancer cell lines tested are unable to successfully activate the ER stress/UPR responses, and that the mitochondria appear to be a central player in OphA-induced cancer cell death. PMID:28112374

Tributyltin induces Yca1p-dependent cell death of yeast Saccharomyces cerevisiae.

PubMed

Chahomchuen, Thippayarat; Akiyama, Koichi; Sekito, Takayuki; Sugimoto, Naoko; Okabe, Masaaki; Nishimoto, Sogo; Sugahara, Takuya; Kakinuma, Yoshimi

2009-10-01

Tributyltin chloride (TBT), an environmental pollutant, is toxic to a variety of eukaryotic and prokaryotic organisms. Although it has been reported that TBT induces apoptotic cell death in mammalian, the action of TBT on eukaryotic microorganisms has not yet been fully investigated. In this study we examined the mechanism involved in cell death caused by TBT exposure in Saccharomyces cerevisiae. The median lethal concentration of TBT was 10 microM for the parent strain BY4741 and 3 microM for the pdr5Delta mutant defective in a major multidrug transporter, respectively. Fluorescence microscopic observations revealed nuclear condensation and chromatin fragmentation in cells treated with TBT indicating that cells underwent an apoptosis-like cell dearth. TBT-induced cell death was suppressed by deletion of the yca1 gene encoding a homologue of the mammalian caspase. In parallel, reactive oxygen species (ROS) were produced by TBT. These results suggest that TBT induces apoptosis-like cell death in yeast via an Yca1p-dependent pathway possibly downstream of the ROS production. This is the first report on TBT-induced apoptotic cell death in yeast.

Involvement of tumour necrosis factor-α-related apoptosis-inducing ligand in enhanced cytotoxicity of lipopolysaccharide-stimulated dendritic cells to activated T cells

PubMed Central

Yu, Yizhi; Liu, Shuxun; Wang, Wenya; Song, Wengang; Zhang, Minghui; Zhang, Weiping; Qin, Zhihai; Cao, Xuetao

2002-01-01

Dendritic cells (DC) are potent antigen-presenting cells (APC) specialized in T-cell mediated immune responses, and also play critical roles in the homeostasis of T cells for controlling immune responses. In the present study, we demonstrated that during mouse bone-marrow-derived DC activation of ovalbumin (OVA)-specific Ia-kb-restricted T hybridoma cells, MF2.2D9 and OVA257–264-specific H-2kb-restricted RF33.70 T cells, respectively, both hybridomas undergo cell death, partially mediated via apoptotic ligand–tumour necrosis factor-α (TNF-α)-related apoptosis-inducing ligand (TRAIL). Lipopolysaccharide enhanced the cytotoxic effect on the two activated T hybridoma cells, which was correlated with up-regulation of TRAIL-expression on DC to some extent. The activation of caspase-3 in activated T hybridoma cells cocultured with DC contributed to the programmed cell death pathway T cells underwent. Therefore, our results show that activation-induced cell death of T hybridoma cells can be influenced by DC, suggesting that DC may be involved in elimination of activated T cells at the end of primary immune responses. PMID:12100718

Involvement of tumour necrosis factor-alpha-related apoptosis-inducing ligand in enhanced cytotoxicity of lipopolysaccharide-stimulated dendritic cells to activated T cells.

PubMed

Yu, Yizhi; Liu, Shuxun; Wang, Wenya; Song, Wengang; Zhang, Minghui; Zhang, Weiping; Qin, Zhihai; Cao, Xuetao

2002-07-01

Dendritic cells (DC) are potent antigen-presenting cells (APC) specialized in T-cell mediated immune responses, and also play critical roles in the homeostasis of T cells for controlling immune responses. In the present study, we demonstrated that during mouse bone-marrow-derived DC activation of ovalbumin (OVA)-specific Ia-kb-restricted T hybridoma cells, MF2.2D9 and OVA257-264-specific H-2kb-restricted RF33.70 T cells, respectively, both hybridomas undergo cell death, partially mediated via apoptotic ligand-tumour necrosis factor-alpha (TNF-alpha)-related apoptosis-inducing ligand (TRAIL). Lipopolysaccharide enhanced the cytotoxic effect on the two activated T hybridoma cells, which was correlated with up-regulation of TRAIL-expression on DC to some extent. The activation of caspase-3 in activated T hybridoma cells cocultured with DC contributed to the programmed cell death pathway T cells underwent. Therefore, our results show that activation-induced cell death of T hybridoma cells can be influenced by DC, suggesting that DC may be involved in elimination of activated T cells at the end of primary immune responses.

Changes in the Antioxidant Systems as Part of the Signaling Pathway Responsible for the Programmed Cell Death Activated by Nitric Oxide and Reactive Oxygen Species in Tobacco Bright-Yellow 2 Cells1

PubMed Central

de Pinto, Maria Concetta; Tommasi, Franca; De Gara, Laura

2002-01-01

Nitric oxide (NO) has been postulated to be required, together with reactive oxygen species (ROS), for the activation of the hypersensitive reaction, a defense response induced in the noncompatible plant-pathogen interaction. However, its involvement in activating programmed cell death (PCD) in plant cells has been questioned. In this paper, the involvement of the cellular antioxidant metabolism in the signal transduction triggered by these bioactive molecules has been investigated. NO and ROS levels were singularly or simultaneously increased in tobacco (Nicotiana tabacum cv Bright-Yellow 2) cells by the addition to the culture medium of NO and/or ROS generators. The individual increase in NO or ROS had different effects on the studied parameters than the simultaneous increase in the two reactive species. NO generation did not cause an increase in phenylalanine ammonia-lyase (PAL) activity or induction of cellular death. It only induced minor changes in ascorbate (ASC) and glutathione (GSH) metabolisms. An increase in ROS induced oxidative stress in the cells, causing an oxidation of the ASC and GSH redox pairs; however, it had no effect on PAL activity and did not induce cell death when it was generated at low concentrations. In contrast, the simultaneous increase of NO and ROS activated a process of death with the typical cytological and biochemical features of hypersensitive PCD and a remarkable rise in PAL activity. Under the simultaneous generation of NO and ROS, the cellular antioxidant capabilities were also suppressed. The involvement of ASC and GSH as part of the transduction pathway leading to PCD is discussed. PMID:12376637

The serine protease inhibitor TLCK attenuates intrinsic death pathways in neurons upstream of mitochondrial demise.

PubMed

Reuther, C; Ganjam, G K; Dolga, A M; Culmsee, C

2014-11-01

It is well-established that activation of proteases, such as caspases, calpains and cathepsins are essential components in signaling pathways of programmed cell death (PCD). Although these proteases have also been linked to mechanisms of neuronal cell death, they are dispensable in paradigms of intrinsic death pathways, e.g. induced by oxidative stress. However, emerging evidence implicated a particular role for serine proteases in mechanisms of PCD in neurons. Here, we investigated the role of trypsin-like serine proteases in a model of glutamate toxicity in HT-22 cells. In these cells glutamate induces oxytosis, a form of caspase-independent cell death that involves activation of the pro-apoptotic protein BH3 interacting-domain death agonist (Bid), leading to mitochondrial demise and ensuing cell death. In this model system, the trypsin-like serine protease inhibitor Nα-tosyl-l-lysine chloromethyl ketone hydrochloride (TLCK) inhibited mitochondrial damage and cell death. Mitochondrial morphology alterations, the impairment of the mitochondrial membrane potential and ATP depletion were prevented and, moreover, lipid peroxidation induced by glutamate was completely abolished. Strikingly, truncated Bid-induced cell death was not affected by TLCK, suggesting a detrimental activity of serine proteases upstream of Bid activation and mitochondrial demise. In summary, this study demonstrates the protective effect of serine protease inhibition by TLCK against oxytosis-induced mitochondrial damage and cell death. These findings indicate that TLCK-sensitive serine proteases play a crucial role in cell death mechanisms upstream of mitochondrial demise and thus, may serve as therapeutic targets in diseases, where oxidative stress and intrinsic pathways of PCD mediate neuronal cell death.

Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis.

PubMed

Loor, Gabriel; Kondapalli, Jyothisri; Schriewer, Jacqueline M; Chandel, Navdeep S; Vanden Hoek, Terry L; Schumacker, Paul T

2010-12-15

Low levels of reactive oxygen species (ROS) can function as redox-active signaling messengers, whereas high levels of ROS induce cellular damage. Menadione generates ROS through redox cycling, and high concentrations trigger cell death. Previous work suggests that menadione triggers cytochrome c release from mitochondria, whereas other studies implicate the activation of the mitochondrial permeability transition pore as the mediator of cell death. We investigated menadione-induced cell death in genetically modified cells lacking specific death-associated proteins. In cardiomyocytes, oxidant stress was assessed using the redox sensor RoGFP, expressed in the cytosol or the mitochondrial matrix. Menadione elicited rapid oxidation in both compartments, whereas it decreased mitochondrial potential and triggered cytochrome c redistribution to the cytosol. Cell death was attenuated by N-acetylcysteine and exogenous glutathione or by overexpression of cytosolic or mitochondria-targeted catalase. By contrast, no protection was observed in cells overexpressing Cu,Zn-SOD or Mn-SOD. Overexpression of antiapoptotic Bcl-X(L) protected against staurosporine-induced cell death, but it failed to confer protection against menadione. Genetic deletion of Bax and Bak, cytochrome c, cyclophilin D, or caspase-9 conferred no protection against menadione-induced cell death. However, cells lacking PARP-1 showed a significant decrease in menadione-induced cell death. Thus, menadione induces cell death through the generation of oxidant stress in multiple subcellular compartments, yet cytochrome c, Bax/Bak, caspase-9, and cyclophilin D are dispensable for cell death in this model. These studies suggest that multiple redundant cell death pathways are activated by menadione, but that PARP plays an essential role in mediating each of them. Copyright © 2010 Elsevier Inc. All rights reserved.

Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis

PubMed Central

Loor, Gabriel; Kondapalli, Jyothisri; Schriewer, Jacqueline M.; Chandel, Navdeep S.; Vanden Hoek, Terry L.; Schumacker, Paul T.

2010-01-01

Low levels of reactive oxygen species (ROS) can function as redox-active signaling messengers, whereas high levels of ROS induce cellular damage. Menadione generates ROS through redox cycling, and high concentrations trigger cell death. Previous work suggests that menadione triggers cytochrome c release from mitochondria, while other studies implicate activation of the mitochondrial permeability transition poreas the mediator of cell death. We investigated menadione-induced cell death in genetically modified cells lacking specific death-associated proteins. In cardiomyocytes, oxidant stress was assessed using the redox sensor RoGFP, expressed in the cytosol or the mitochondrial matrix. Menadione elicited rapid oxidation in both compartments, while it decreased mitochondrial potential and triggered cytochrome c redistribution to the cytosol. Cell death was attenuated by N-acetyl cysteine and exogenous glutathione (GSH), or by over-expression of cytosolic or mitochondria-targeted catalase. By contrast, no protection was observed in cells over-expressing Cu, Zn-SOD or MnSOD. Over-expression of antiapoptotic Bcl-XLprotected against staurosporine-induced cell death, but it failed to confer protection against menadione. Genetic deletion of Bax and Bak, cytochrome c, cyclophilin D or caspase-9 conferred no protection against menadione-induced cell death. However, cells lacking PARP-1 showed a significant decrease in menadione-induced cell death. Thus, menadione induces cell death through the generation of oxidant stress in multiple subcellular compartments, yet cytochromec, Bax/Bak, caspase-9 and cyclophilin D are dispensable for cell death in this model. These studies suggest that multiple redundant cell death pathways are activated by menadione, but that PARP plays an essential role in mediating each of them. PMID:20937380

Lysosomal membrane permeabilization in cell death: new evidence and implications for health and disease.

PubMed

Serrano-Puebla, Ana; Boya, Patricia

2016-05-01

Recent studies have demonstrated that, in addition to their central role in cellular catabolic reactions, lysosomes are implicated in many cellular processes, including metabolism, membrane repair, and cell death. Lysosomal membrane permeabilization (LMP) has emerged as a pathway by which cell demise is regulated under physiological conditions and contributes to cell death in many pathological situations. Here, we review the latest evidence on LMP-mediated cell death, the upstream and downstream signals involved, and the role of LMP in the normal physiology of organisms. We also discuss the contributions of lysosomal damage and LMP to the pathogenic features of several disease states, such as lysosomal storage disorders and other neurodegenerative conditions. © 2015 New York Academy of Sciences.

The Netrin-4/ Neogenin-1 axis promotes neuroblastoma cell survival and migration

PubMed Central

Villanueva, Andrea A.; Falcón, Paulina; Espinoza, Natalie; Luis, Solano R.; Milla, Luis A.; Hernandez-SanMiguel, Esther; Torres, Vicente A.; Sanchez-Gomez, Pilar; Palma, Verónica

2017-01-01

Neogenin-1 (NEO1) is a transmembrane receptor involved in axonal guidance, angiogenesis, neuronal cell migration and cell death, during both embryonic development and adult homeostasis. It has been described as a dependence receptor, because it promotes cell death in the absence of its ligands (Netrin and Repulsive Guidance Molecule (RGM) families) and cell survival when they are present. Although NEO1 and its ligands are involved in tumor progression, their precise role in tumor cell survival and migration remain unclear. Public databases contain extensive information regarding the expression of NEO1 and its ligands Netrin-1 (NTN1) and Netrin-4 (NTN4) in primary neuroblastoma (NB) tumors. Analysis of this data revealed that patients with high expression levels of both NEO1 and NTN4 have a poor survival rate. Accordingly, our analyses in NB cell lines with different genetic backgrounds revealed that knocking-down NEO1 reduces cell migration, whereas silencing of endogenous NTN4 induced cell death. Conversely, overexpression of NEO1 resulted in higher cell migration in the presence of NTN4, and increased apoptosis in the absence of ligand. Increased apoptosis was prevented when utilizing physiological concentrations of exogenous Netrin-4. Likewise, cell death induced after NTN4 knock-down was rescued when NEO1 was transiently silenced, thus revealing an important role for NEO1 in NB cell survival. In vivo analysis, using the chicken embryo chorioallantoic membrane (CAM) model, showed that NEO1 and endogenous NTN4 are involved in tumor extravasation and metastasis. Our data collectively demonstrate that endogenous NTN4/NEO1 maintain NB growth via both pro-survival and pro-migratory molecular signaling. PMID:28038459

Oxygenated monoterpenes citral and carvacrol cause oxidative damage in Escherichia coli without the involvement of tricarboxylic acid cycle and Fenton reaction.

PubMed

Chueca, Beatriz; Pagán, Rafael; García-Gonzalo, Diego

2014-10-17

Oxygenated monoterpenes citral and carvacrol are common constituents of many essential oils (EOs) that have been extensively studied as antimicrobial agents but whose mechanisms of microbial inactivation have not been totally elucidated. A recent study described a mechanism of Escherichia coli death for (+)-limonene, a hydrocarbon monoterpene also frequently present in EOs, similar to the common mechanism proposed for bactericidal antibiotics. This mechanism involves the formation of Fenton-mediated hydroxyl radical, a reactive oxygen species (ROS), via tricarboxylic acid (TCA) cycle, which would ultimately inactivate cells. Our objective was to determine whether E. coli MG1655 inactivation by citral and carvacrol follows a similar mechanism of cell death. Challenging experiments with 300μL/L citral and 100μL/L carvacrol inactivated at least 2.5log10cycles of exponentially growing cells in 3h under aerobic conditions. The presence of thiourea (an ROS scavenger) reduced cell inactivation in 2log10cycles, demonstrating the role of ROS in cell death. Decreased resistance of a ΔrecA mutant (deficient in an enzyme involved in SOS response to DNA damage) indicated that citral and carvacrol caused oxidative damage to DNA. Although the mechanism of E. coli inactivation by carvacrol and citral was similarly mediated by ROS, their formation did not follow the same pathways described for (+)-limonene and bactericidal drugs because neither Fenton reaction nor NADH production via the TCA cycle was involved in cell death. Moreover, further experiments demonstrated antimicrobial activity of citral and carvacrol in anaerobic environments without the involvement of ROS. As a consequence, cell death by carvacrol and citral in anaerobiosis follows a different mechanism than that observed under aerobic conditions. These results demonstrated a different mechanism of inactivation by citral and carvacrol with regard to (+)-limonene and bactericidal antibiotics, indicating the complexity of the mechanisms of bacterial inactivation among EO constituents. Advancements in the description of these mechanisms will help in extending and improving the use of these compounds as natural antimicrobials. Copyright © 2014 Elsevier B.V. All rights reserved.

The phenoptosis problem: what is causing the death of an organism? Lessons from acute kidney injury.

PubMed

Zorov, D B; Plotnikov, E Y; Jankauskas, S S; Isaev, N K; Silachev, D N; Zorova, L D; Pevzner, I B; Pulkova, N V; Zorov, S D; Morosanova, M A

2012-07-01

Programmed execution of various cells and intracellular structures is hypothesized to be not the only example of elimination of biological systems - the general mechanism can also involve programmed execution of organs and organisms. Modern rating of programmed cell death mechanisms includes 13 mechanistic types. As for some types, the mechanism of actuation and manifestation of cell execution has been basically elucidated, while the causes and intermediate steps of the process of fatal failure of organs and organisms remain unknown. The analysis of deaths resulting from a sudden heart arrest or multiple organ failure and other acute and chronic pathologies leads to the conclusion of a special role of mitochondria and oxidative stress activating the immune system. Possible mechanisms of mitochondria-mediated induction of the signaling cascades involved in organ failure and death of the organism are discussed. These mechanisms include generation of reactive oxygen species and damage-associated molecular patterns in mitochondria. Some examples of renal failure-induced deaths are presented with mechanisms and settings determined by some hypothetical super system rather than by the kidneys themselves. This system plays the key role in the process of physiological senescence and termination of an organism. The facts presented suggest that it is the immune system involved in mitochondrial signaling that can act as the system responsible for the organism's death.

Telomerase Is Involved in IL-7-Mediated Differential Survival of Naive and Memory CD4+ T Cells1

PubMed Central

Yang, Yinhua; An, Jie; Weng, Nan-ping

2008-01-01

IL-7 plays an essential role in T cell maintenance and survival. The survival effect of IL-7 is thought to be mediated through regulation of Bcl2 family proteins. After a comparative analysis of IL-7-induced growth and cell death of human naive and memory CD4+ T cells, we observed that more memory CD4+ T cells underwent cell division and proceeded to apoptosis than naive cells in response to IL-7. However, IL-7-induced expressions of Bcl2 family members (Bcl2, Bcl-xL, Bax, and Bad) were similar between naive and memory cells. Instead, we found that IL-7 induced higher levels of telomerase activity in naive cells than in memory cells, and the levels of IL-7-induced telomerase activity had a significant inverse correlation with cell death in CD4+ T cells. Furthermore, we showed that reducing expression of telomerase reverse transcriptase and telomerase activity significantly increased cell death of IL-7-cultured CD4+ T cells. Together, these findings demonstrate that telomerase is involved in IL-7-mediated differential survival of naive and memory CD4+ T cells. PMID:18322183

High cell surface death receptor expression determines type I versus type II signaling.

PubMed

Meng, Xue Wei; Peterson, Kevin L; Dai, Haiming; Schneider, Paula; Lee, Sun-Hee; Zhang, Jin-San; Koenig, Alexander; Bronk, Steve; Billadeau, Daniel D; Gores, Gregory J; Kaufmann, Scott H

2011-10-14

Previous studies have suggested that there are two signaling pathways leading from ligation of the Fas receptor to induction of apoptosis. Type I signaling involves Fas ligand-induced recruitment of large amounts of FADD (FAS-associated death domain protein) and procaspase 8, leading to direct activation of caspase 3, whereas type II signaling involves Bid-mediated mitochondrial perturbation to amplify a more modest death receptor-initiated signal. The biochemical basis for this dichotomy has previously been unclear. Here we show that type I cells have a longer half-life for Fas message and express higher amounts of cell surface Fas, explaining the increased recruitment of FADD and subsequent signaling. Moreover, we demonstrate that cells with type II Fas signaling (Jurkat or HCT-15) can signal through a type I pathway upon forced receptor overexpression and that shRNA-mediated Fas down-regulation converts cells with type I signaling (A498) to type II signaling. Importantly, the same cells can exhibit type I signaling for Fas and type II signaling for TRAIL (TNF-α-related apoptosis-inducing ligand), indicating that the choice of signaling pathway is related to the specific receptor, not some other cellular feature. Additional experiments revealed that up-regulation of cell surface death receptor 5 levels by treatment with 7-ethyl-10-hydroxy-camptothecin converted TRAIL signaling in HCT116 cells from type II to type I. Collectively, these results suggest that the type I/type II dichotomy reflects differences in cell surface death receptor expression.

Impact of nutrient imbalance on wine alcoholic fermentations: nitrogen excess enhances yeast cell death in lipid-limited must.

PubMed

Tesnière, Catherine; Delobel, Pierre; Pradal, Martine; Blondin, Bruno

2013-01-01

We evaluated the consequences of nutritional imbalances, particularly lipid/nitrogen imbalances, on wine yeast survival during alcoholic fermentation. We report that lipid limitation (ergosterol limitation in our model) led to a rapid loss of viability during the stationary phase of fermentation and that the cell death rate is strongly modulated by nitrogen availability and nature. Yeast survival was reduced in the presence of excess nitrogen in lipid-limited fermentations. The rapidly dying yeast cells in fermentations in high nitrogen and lipid-limited conditions displayed a lower storage of the carbohydrates trehalose and glycogen than observed in nitrogen-limited cells. We studied the cell stress response using HSP12 promoter-driven GFP expression as a marker, and found that lipid limitation triggered a weaker stress response than nitrogen limitation. We used a SCH9-deleted strain to assess the involvement of nitrogen signalling pathways in the triggering of cell death. Deletion of SCH9 increased yeast viability in the presence of excess nitrogen, indicating that a signalling pathway acting through Sch9p is involved in this nitrogen-triggered cell death. We also show that various nitrogen sources, but not histidine or proline, provoked cell death. Our various findings indicate that lipid limitation does not elicit a transcriptional programme that leads to a stress response protecting yeast cells and that nitrogen excess triggers cell death by modulating this stress response, but not through HSP12. These results reveal a possibly negative role of nitrogen in fermentation, with reported effects referring to ergosterol limitation conditions. These effects should be taken into account in the management of alcoholic fermentations.

Impact of Nutrient Imbalance on Wine Alcoholic Fermentations: Nitrogen Excess Enhances Yeast Cell Death in Lipid-Limited Must

PubMed Central

Tesnière, Catherine; Delobel, Pierre; Pradal, Martine; Blondin, Bruno

2013-01-01

We evaluated the consequences of nutritional imbalances, particularly lipid/nitrogen imbalances, on wine yeast survival during alcoholic fermentation. We report that lipid limitation (ergosterol limitation in our model) led to a rapid loss of viability during the stationary phase of fermentation and that the cell death rate is strongly modulated by nitrogen availability and nature. Yeast survival was reduced in the presence of excess nitrogen in lipid-limited fermentations. The rapidly dying yeast cells in fermentations in high nitrogen and lipid-limited conditions displayed a lower storage of the carbohydrates trehalose and glycogen than observed in nitrogen-limited cells. We studied the cell stress response using HSP12 promoter-driven GFP expression as a marker, and found that lipid limitation triggered a weaker stress response than nitrogen limitation. We used a SCH9-deleted strain to assess the involvement of nitrogen signalling pathways in the triggering of cell death. Deletion of SCH9 increased yeast viability in the presence of excess nitrogen, indicating that a signalling pathway acting through Sch9p is involved in this nitrogen-triggered cell death. We also show that various nitrogen sources, but not histidine or proline, provoked cell death. Our various findings indicate that lipid limitation does not elicit a transcriptional programme that leads to a stress response protecting yeast cells and that nitrogen excess triggers cell death by modulating this stress response, but not through HSP12. These results reveal a possibly negative role of nitrogen in fermentation, with reported effects referring to ergosterol limitation conditions. These effects should be taken into account in the management of alcoholic fermentations. PMID:23658613

Glutathione in Cancer Cell Death

PubMed Central

Ortega, Angel L.; Mena, Salvador; Estrela, Jose M.

2011-01-01

Glutathione (L-γ-glutamyl-L-cysteinyl-glycine; GSH) in cancer cells is particularly relevant in the regulation of carcinogenic mechanisms; sensitivity against cytotoxic drugs, ionizing radiations, and some cytokines; DNA synthesis; and cell proliferation and death. The intracellular thiol redox state (controlled by GSH) is one of the endogenous effectors involved in regulating the mitochondrial permeability transition pore complex and, in consequence, thiol oxidation can be a causal factor in the mitochondrion-based mechanism that leads to cell death. Nevertheless GSH depletion is a common feature not only of apoptosis but also of other types of cell death. Indeed rates of GSH synthesis and fluxes regulate its levels in cellular compartments, and potentially influence switches among different mechanisms of death. How changes in gene expression, post-translational modifications of proteins, and signaling cascades are implicated will be discussed. Furthermore, this review will finally analyze whether GSH depletion may facilitate cancer cell death under in vivo conditions, and how this can be applied to cancer therapy. PMID:24212662

Sevoflurane post-conditioning protects primary rat cortical neurons against oxygen-glucose deprivation/resuscitation via down-regulation in mitochondrial apoptosis axis of Bid, Bim, Puma-Bax and Bak mediated by Erk1/2.

PubMed

Zhang, Li-Min; Zhao, Xiao-Chun; Sun, Wen-Bo; Li, Rui; Jiang, Xiao-Jing

2015-10-15

Temporal post-conditioning helps provide neuroprotection against brain injury secondary to ischemia-reperfusion and is considered an effective intervention, but the exact mechanism of sevoflurane post-conditioning is unclear. The essential axis involves activator Bid, Bim, Puma (BH3s), Bax, and Bak; activates the mitochondrial death program; and might be involved in a cell death signal. Extracellular signal-related kinases 1/2 (Erk1/2) play a pivotal role in cell growth and proliferation. We hypothesized that sevoflurane post-conditioning might inhibit Bid, Bim, Puma, Bax, and Bak expression and is activated by phosphor-Erk1/2 to decrease neuronal death. To test this hypothesis, we exposed primary cortical neuron cultures to oxygen-glucose deprivation for 1h, along with resuscitation for 24h (OGD/R). MTT assays, propidium iodide uptake (PI), JC-1 fluorescence, and Western blot indicated the following: decreased cell viability (P<0.05); increased cell death (P<0.05); decreased mitochondrial membrane potential (P<0.05); and decreased Bid, Bim, Puma, Bax, and Bak expression with OGD/R exposure. Inhibition of Erk1/2 phosphorylation could attenuate sevoflurane post-conditioning that mediated an increase in neuronal viability and mitochondrial membrane potential, as well as a decrease in cell death and Bid, Bim, Puma, Bax, and Bak expression after OGD/R treatment. The results demonstrated that sevoflurane post-conditioning caused a marked decrease in cortical neuronal death secondary to OGD/R exposure through the downregulation of the mitochondrial apoptosis axis involving Bid, Bim, Puma, Bax, and Bak that was mediated by the phosphorylation/activation of Erk1/2. Copyright © 2015 Elsevier B.V. All rights reserved.

Mechanisms of Action and Cell Death Associated with Clostridium perfringens Toxins.

PubMed

Navarro, Mauricio A; McClane, Bruce A; Uzal, Francisco A

2018-05-22

Clostridium perfringens uses its large arsenal of protein toxins to produce histotoxic, neurologic and intestinal infections in humans and animals. The major toxins involved in diseases are alpha (CPA), beta (CPB), epsilon (ETX), iota (ITX), enterotoxin (CPE), and necrotic B-like (NetB) toxins. CPA is the main virulence factor involved in gas gangrene in humans, whereas its role in animal diseases is limited and controversial. CPB is responsible for necrotizing enteritis and enterotoxemia, mostly in neonatal individuals of many animal species, including humans. ETX is the main toxin involved in enterotoxemia of sheep and goats. ITX has been implicated in cases of enteritis in rabbits and other animal species; however, its specific role in causing disease has not been proved. CPE is responsible for human food-poisoning and non-foodborne C. perfringens -mediated diarrhea. NetB is the cause of necrotic enteritis in chickens. In most cases, host⁻toxin interaction starts on the plasma membrane of target cells via specific receptors, resulting in the activation of intracellular pathways with a variety of effects, commonly including cell death. In general, the molecular mechanisms of cell death associated with C. perfringens toxins involve features of apoptosis, necrosis and/or necroptosis.

Mechanisms of Action and Cell Death Associated with Clostridium perfringens Toxins

PubMed Central

Navarro, Mauricio A.; Uzal, Francisco A.

2018-01-01

Clostridium perfringens uses its large arsenal of protein toxins to produce histotoxic, neurologic and intestinal infections in humans and animals. The major toxins involved in diseases are alpha (CPA), beta (CPB), epsilon (ETX), iota (ITX), enterotoxin (CPE), and necrotic B-like (NetB) toxins. CPA is the main virulence factor involved in gas gangrene in humans, whereas its role in animal diseases is limited and controversial. CPB is responsible for necrotizing enteritis and enterotoxemia, mostly in neonatal individuals of many animal species, including humans. ETX is the main toxin involved in enterotoxemia of sheep and goats. ITX has been implicated in cases of enteritis in rabbits and other animal species; however, its specific role in causing disease has not been proved. CPE is responsible for human food-poisoning and non-foodborne C. perfringens-mediated diarrhea. NetB is the cause of necrotic enteritis in chickens. In most cases, host–toxin interaction starts on the plasma membrane of target cells via specific receptors, resulting in the activation of intracellular pathways with a variety of effects, commonly including cell death. In general, the molecular mechanisms of cell death associated with C. perfringens toxins involve features of apoptosis, necrosis and/or necroptosis. PMID:29786671

Regulated Forms of Cell Death in Fungi

PubMed Central

Gonçalves, A. Pedro; Heller, Jens; Daskalov, Asen; Videira, Arnaldo; Glass, N. Louise

2017-01-01

Cell death occurs in all domains of life. While some cells die in an uncontrolled way due to exposure to external cues, other cells die in a regulated manner as part of a genetically encoded developmental program. Like other eukaryotic species, fungi undergo programmed cell death (PCD) in response to various triggers. For example, exposure to external stress conditions can activate PCD pathways in fungi. Calcium redistribution between the extracellular space, the cytoplasm and intracellular storage organelles appears to be pivotal for this kind of cell death. PCD is also part of the fungal life cycle, in which it occurs during sexual and asexual reproduction, aging, and as part of development associated with infection in phytopathogenic fungi. Additionally, a fungal non-self-recognition mechanism termed heterokaryon incompatibility (HI) also involves PCD. Some of the molecular players mediating PCD during HI show remarkable similarities to major constituents involved in innate immunity in metazoans and plants. In this review we discuss recent research on fungal PCD mechanisms in comparison to more characterized mechanisms in metazoans. We highlight the role of PCD in fungi in response to exogenic compounds, fungal development and non-self-recognition processes and discuss identified intracellular signaling pathways and molecules that regulate fungal PCD. PMID:28983298

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jeong Eun; Hanyang Biomedical Research Institute, Seoul; Park, Jae Hyeon

Oxidative stress can lead to expression of inflammatory transcription factors, which are important regulatory elements in the induction of inflammatory responses. One of the transcription factors, nuclear transcription factor kappa-B (NF-κB) plays a significant role in the inflammation regulatory process. Inflammatory cell death has been implicated in neuronal cell death in some neurodegenerative disorders such as Parkinson's disease (PD). In this study, we investigated the molecular mechanisms underlying apoptosis initiated by chlorpyrifos (CPF)-mediated oxidative stress. Based on the cytotoxic mechanism of CPF, we examined the neuroprotective effects of rosiglitazone (RGZ), a peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist, against CPF-induced neuronalmore » cell death. The treatment of SH-SY5Y cells with CPF induced oxidative stress. In addition, CPF activated the p38, JNK and ERK mitogen-activated protein kinases (MAPKs), and induced increases in the inflammatory genes such as COX-2 and TNF-α. CPF also induced nuclear translocation of NF-κB and inhibitors of NF-κB abolished the CPF-induced COX-2 expression. Pretreatment with RGZ significantly reduced ROS generation and enhanced HO-1 expression in CPF-exposed cells. RGZ blocked the activation of both p38 and JNK signaling, while ERK activation was strengthened. RGZ also attenuated CPF-induced cell death through the reduction of NF-κB-mediated proinflammatory factors. Results from this study suggest that RGZ may exert an anti-apoptotic effect against CPF-induced cytotoxicity by attenuation of oxidative stress as well as inhibition of the inflammatory cascade via inactivation of signaling by p38 and JNK, and NF-κB. - Highlights: • CPF induces apoptotic cell death in SH-SY5Y cells • ROS involved in CPF-mediated apoptotic cell death • Inflammation involved in CPF-mediated apoptotic cell death • Rosiglitazone modulates ROS and inflammatory response in CPF-treated cells.« less

Parthanatos, a messenger of death

PubMed Central

David, Karen Kate; Andrabi, Shaida Ahmad; Dawson, Ted Murray; Dawson, Valina Lynn

2015-01-01

Poly-ADP-ribose polymerase-1 (PARP-1)'s multiple roles in the cell span from maintaining life to inducing death. The processes PARP-1 is involved in include, but are not limited to DNA repair, DNA transcription, mitosis, and cell death. Of PARP-1's different cellular functions, its active role in cell death is of particular interest to designing therapies for diseases. Genetic deletion of PARP-1 revealed that PARP-1 over activation underlies cell death in experimental models of stroke, diabetes, inflammation and neurodegeneration. Since interfering with PARP-1 mediated cell death will be clinically beneficial, great effort has been invested into designing PARP-1 inhibitors and understanding mechanisms downstream of PARP-1 over activation. PARP-1 overactivation may kill by depleting cellular energy through nicotinamide adenine dinucleotide (NAD+) consumption, and by releasing the cell death effector apoptosis-inducing factor (AIF). Unexpectedly, recent evidence shows that poly-ADP ribose (PAR) polymer itself, and not the consumption of NAD+ is the source of cytotoxicity. Thus, PAR polymer acts as a cell death effector downstream of PARP-1-mediated cell death signaling. We coined the term parthanatos after Thanatos, the personification of death in Greek mythology, to refer to PAR-mediated cell death. In this review, we will summarize the proposed mechanisms by which PARP-1 overactivation kills. We will present evidence for parthanatos, and the questions raised by these recent findings. It is evident that further understanding of parthanatos opens up new avenues for therapy in ameliorating diseases related to PARP-1 over activation. PMID:19273119

Simultaneous induction of apoptotic, autophagic, and necrosis-like cell death by monoclonal antibodies recognizing chicken transferrin receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohno, Yoshiya; Yagi, Hideki; Nakamura, Masanori

Programmed cell death (PCD) is categorized as apoptotic, autophagic, or necrosis-like. Although the possibility that plural (two or three) death signals could be induced by a given stimulus has been reported, the precise mechanisms regulating PCD are not well understood. Recently, we have obtained two anti-chicken transferrin receptor (TfR) monoclonal antibodies (mAbs; D18 and D19) inducing a unique cell death. Although the cell death had several features of apoptosis, autophagic and necrosis-like morphological alterations were simultaneously observed in electron microphotographs. In addition to cells with condensed chromatin and an intact plasma membrane (apoptotic cells), cells having many vacuoles in themore » cytoplasm (autophagic cells), and enlarged cells with ruptured plasma membranes (necrosis-like cells) were observed in DT40 cells treated with the mAbs, however, the latter two types of dead cells were not detected upon treatment with staurosporine, a typical apoptosis inducer. In autophagic cells, numerous membrane-bound vesicles occupying most of the cytoplasmic space, which frequently contained electron-dense materials from cytoplasmic fragments and organelles, were observed. The simultaneous induction of multiple death signals from a stimulus via the TfR is of great interest to those researching cell death. In addition, activation of caspases was observed in DT40 cells treated with D19, however, the cell death was not inhibited with z-VAD-fmk, a pan-caspase inhibitor, suggesting that at least in part, a caspase-independent pathway is involved in the TfR-mediated cell death.« less

Harnessing tumor necrosis factor receptors to enhance antitumor activities of drugs.

PubMed

Muntané, Jordi

2011-10-17

Cancer is the second-leading cause of death in the U.S. behind heart disease and over stroke. The hallmarks of cancer comprise six biological capabilities acquired during the multistep development of human tumors. The inhibition of cell death pathways is one of these tumor characteristics which also include sustained proliferative signaling, evading growth suppressor signaling, replicative immortality, angiogenesis, and promotion of invasion and metastasis. Cell death is mediated through death receptor (DR) stimulation initiated by specific ligands that transmit signaling to the cell death machinery or through the participation of mitochondria. Cell death involving DR is mediated by the superfamily of tumor necrosis factor receptor (TNF-R) which includes TNF-R type I, CD95, DR3, TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 (TRAIL-R1) and -2 (TRAIL-R2), DR6, ectodysplasin A (EDA) receptor (EDAR), and the nerve growth factor (NGF) receptor (NGFR). The expression of these receptors in healthy and tumor cells induces treatment side effects that limit the systemic administration of cell death-inducing therapies. The present review is focused on the different therapeutic strategies such as targeted antibodies or small molecules addressed to selective stimulated DR-mediated apoptosis or reduce cell proliferation in cancer cells.

Ayanin diacetate-induced cell death is amplified by TRAIL in human leukemia cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marrero, Maria Teresa; Estevez, Sara; Negrin, Gledy

Highlights: Black-Right-Pointing-Pointer Ayanin diacetate as apoptotic inducer in leukemia cells. Black-Right-Pointing-Pointer Cell death was prevented by caspase inhibitors and by the overexpression of Bcl-x{sub L}. Black-Right-Pointing-Pointer The intrinsic and the extrinsic pathways are involved in the mechanism of action. Black-Right-Pointing-Pointer Death receptors are up-regulated and TRAIL enhances apoptotic cell death. -- Abstract: Here we demonstrate that the semi-synthetic flavonoid ayanin diacetate induces cell death selectively in leukemia cells without affecting the proliferation of normal lymphocytes. Incubation of human leukemia cells with ayanin diacetate induced G{sub 2}-M phase cell cycle arrest and apoptosis which was prevented by the non-specific caspase inhibitormore » z-VAD-fmk and reduced by the overexpression of Bcl-x{sub L}. Ayanin diacetate-induced cell death was found to be associated with: (i) loss of inner mitochondrial membrane potential, (ii) the release of cytochrome c, (iii) the activation of multiple caspases, (iv) cleavage of poly(ADP-ribose) polymerase and (v) the up-regulation of death receptors for TRAIL, DR4 and DR5. Moreover, the combined treatment with ayanin diacetate and TRAIL amplified cell death, compared to single treatments. These results provide a basis for further exploring the potential applications of this combination for the treatment of cancer.« less

Cell death proteomics database: consolidating proteomics data on cell death.

PubMed

Arntzen, Magnus Ø; Bull, Vibeke H; Thiede, Bernd

2013-05-03

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.

CDIP, a novel pro-apoptotic gene, regulates TNFα-mediated apoptosis in a p53-dependent manner

PubMed Central

Brown, Lauren; Ongusaha, Pat P; Kim, Hyung-Gu; Nuti, Shanthy; Mandinova, Anna; Lee, Ji Won; Khosravi-Far, Roya; Aaronson, Stuart A; Lee, Sam W

2007-01-01

We have identified a novel pro-apoptotic p53 target gene named CDIP (Cell Death Involved p53-target). Inhibition of CDIP abrogates p53-mediated apoptotic responses, demonstrating that CDIP is an important p53 apoptotic effector. CDIP itself potently induces apoptosis that is associated with caspase-8 cleavage, implicating the extrinsic cell death pathway in apoptosis mediated by CDIP. siRNA-directed knockdown of caspase-8 results in a severe impairment of CDIP-dependent cell death. In investigating the potential involvement of extrinsic cell death pathway in CDIP-mediated apoptosis, we found that TNF-α expression tightly correlates with CDIP expression, and that inhibition of TNF-α signaling attenuates CDIP-dependent apoptosis. We also demonstrate that TNF-α is upregulated in response to p53 and p53 inducing genotoxic stress, in a CDIP-dependent manner. Consistently, knockdown of TNF-α impairs p53-mediated stress-induced apoptosis. Together, these findings support a novel p53 → CDIP → TNF-α apoptotic pathway that directs apoptosis after exposure of cells to genotoxic stress. Thus, CDIP provides a new link between p53-mediated intrinsic and death receptor-mediated extrinsic apoptotic signaling, providing a novel target for cancer therapeutics aimed at maximizing the p53 apoptotic response of cancer cells to drug therapy. PMID:17599062

Eosinophilic Otitis Media: the Aftermath of Eosinophil Extracellular Trap Cell Death.

PubMed

Ueki, Shigeharu; Ohta, Nobuo; Takeda, Masahide; Konno, Yasunori; Hirokawa, Makoto

2017-05-01

Eosinophilic otitis media (EOM) is a refractory disease characterized by the accumulation of eosinophils in middle ear effusion and mucosa. We summarize current knowledge regarding the clinical characteristics and management of EOM. Although eosinophil activation in inflamed foci is involved in the pathogenesis of EOM, little is known about the fate of the eosinophils and aftermath of their cell death. We discuss the possibility that eosinophils undergo non-apoptotic cell death that worsens tissue damage and increases effusion viscosity. Unlike chronic otitis media, EOM is strongly associated with an allergic background. Corticosteroids are currently the only effective pharmacological treatment, and surgical intervention is often required. Mucosal eosinophils infiltrate extensively into the middle ear cavity where they are stimulated by locally produced activators including interleukin-5 and eotaxin. The eosinophils undergo cytolysis in the effusion, which represents a major fate of activated eosinophils in vivo. Recent data revealed cytolysis could be renamed as extracellular trap cell death (ETosis). ETosis represents suicidal cell death involving total cell degranulation and development of sticky chromatin structures (extracellular traps (ETs)). The characteristics of eosinophil- and neutrophil-derived ET polymers might contribute to the difference in viscosity of secretions between EOM and common chronic otitis media. The extracellular products remaining after eosinophil ETosis are an important aspect of EOM pathology. The concept of ETosis also has novel implications for potential therapeutic modalities in various eosinophilic disorders.

Hop/STI1 modulates retinal proliferation and cell death independent of PrP{sup C}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arruda-Carvalho, Maithe; Njaine, Brian; Silveira, Mariana S.

Hop/STI1 is a co-chaperone adaptor protein for Hsp70/Hsp90 complexes. Hop/STI1 is found extracellularly and modulates cell death and differentiation through interaction with the prion protein (PrP{sup C}). Here, we investigated the expression of hop/STI1 and its role upon cell proliferation and cell death in the developing retina. Hop/STI1 is more expressed in developing rat retina than in the mature tissue. Hop/STI1 blocks retinal cell death in the neuroblastic layer (NBL) in a PrP{sup C} dependent manner, but failed to protect ganglion cells against axotomy-induced cell death. An antibody raised against hop/STI1 ({alpha}-STI1) blocked both ganglion cell and NBL cell deathmore » independent of PrP{sup C}. cAMP/PKA, ERK, PI3K and PKC signaling pathways were not involved in these effects. Hop/STI1 treatment reduced proliferation, while {alpha}-STI1 increased proliferation in the developing retina, both independent of PrP{sup C}. We conclude that hop/STI1 can modulate both proliferation and cell death in the developing retina independent of PrP{sup C}.« less

ARTD1 (PARP1) activation and NAD+ in DNA repair and cell death

PubMed Central

Fouquerel, Elise; Sobol, Robert W.

2014-01-01

Nicotinamide adenine dinucleotide, NAD+, is a small metabolite coenzyme that is essential for the progress of crucial cellular pathways including glycolysis, the tricarboxylic acid cycle (TCA) and mitochondrial respiration. These processes consume and produce both oxidative and reduced forms of NAD (NAD+ and NADH). NAD+ is also important for ADP(ribosyl)ation reactions mediated by the ADP-ribosyltransferase enzymes (ARTDs) or deacetylation reactions catalysed by the sirtuins (SIRTs) which use NAD+ as a substrate. In this review, we highlight the significance of NAD+ catabolism in DNA repair and cell death through its utilization by ARTDs and SIRTs. We summarize the current findings on the involvement of ARTD1 activity in DNA repair and most specifically its involvement in the trigger of cell death mediated by energy depletion. By sharing the same substrate, the activities of ARTDs and SIRTs are tightly linked and dependent on each other and are thereby involved in the same cellular processes that play an important role in cancer biology, inflammatory diseases and ischemia/reperfusion. PMID:25283336

Remodelling of lace plant leaves: antioxidants and ROS are key regulators of programmed cell death.

PubMed

Dauphinee, Adrian N; Fletcher, Jacob I; Denbigh, Georgia L; Lacroix, Christian R; Gunawardena, Arunika H L A N

2017-07-01

Antioxidants and reactive oxygen species are integral for programmed cell death signaling during perforation formation in the lace plant ( Aponogeton madagascariensis ). The lace plant is an excellent model system for studying developmentally regulated programmed cell death (PCD). During early lace plant leaf development, PCD systematically deletes cells resulting in a perforated leaf morphology that is unique in planta. A distinct feature in young lace plant leaves is an abundance of anthocyanins, which have antioxidant properties. The first sign of PCD induction is the loss of anthocyanin pigmentation in cells that are targeted for destruction, which results in a visible gradient of cell death. The cellular dynamics and time course of lace plant PCD are well documented; however, the signals involved in the pathway remain elusive. This study investigates the roles of antioxidants and ROS in developmental PCD signaling during lace plant perforation formation. The involvement of antioxidants and ROS in the pathway was determined using a variety of techniques including pharmacological whole plant experimentation, long-term live cell imaging, the 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid anti-radical activity assay, and western blot analysis. Results indicate that antioxidants and ROS are key regulators of PCD during the remodelling of lace plant leaves.

Mitochondrial Ca2+ influx targets cardiolipin to disintegrate respiratory chain complex II for cell death induction

PubMed Central

Hwang, M-S; Schwall, C T; Pazarentzos, E; Datler, C; Alder, N N; Grimm, S

2014-01-01

Massive Ca2+ influx into mitochondria is critically involved in cell death induction but it is unknown how this activates the organelle for cell destruction. Using multiple approaches including subcellular fractionation, FRET in intact cells, and in vitro reconstitutions, we show that mitochondrial Ca2+ influx prompts complex II of the respiratory chain to disintegrate, thereby releasing an enzymatically competent sub-complex that generates excessive reactive oxygen species (ROS) for cell death induction. This Ca2+-dependent dissociation of complex II is also observed in model membrane systems, but not when cardiolipin is replaced with a lipid devoid of Ca2+ binding. Cardiolipin is known to associate with complex II and upon Ca2+ binding coalesces into separate homotypic clusters. When complex II is deprived of this lipid, it disintegrates for ROS formation and cell death. Our results reveal Ca2+ binding to cardiolipin for complex II disintegration as a pivotal step for oxidative stress and cell death induction. PMID:24948011

Apoptotic death in cerebral hemisphere cells is density dependent and modulated by transient oxygen and glucose deprivation.

PubMed

Yavin, E; Billia, D M

1997-03-01

Flow cytometry, light and fluorescence microscopy, and designated biochemical techniques were used to examine the type of death which occurs in cerebral cortex cells when grown under crowded vs. sparse conditions or after brief anoxia/hypoglycemia. A 4 hr episode of anoxia combined with glucose deprivation enhanced apoptotic cell death as assessed by 4',6-diamidino-2-phenylindole (DAPI) staining and reduced neutral red eye uptake. An additional form of cell death involving exclusion of the nucleus was recorded by time lapse cinematography and DAPI stain. The presence of the endonuclease inhibitor aurintricarboxylic acid (0.1 mM) reduced cell death by 56.6%, while the protein and RNA synthesis inhibitors actinomycin D and cycloheximide (each at 5 micrograms/ml) effectively decreased cell death by 83.3% and 90.6%, respectively. In contrast, 5 mM glutamate had no effect on cell death in accord with the immature state of the cells. Growth of cells under crowded conditions improved cell survival; after 2 h or 4 days in culture, cells seeded at high density (34 microgram cellular DNA/cm2) showed a nearly 3-fold decline in the amount of cell death in comparison to cells seeded at low density (5 micrograms cellular DNA/cm2). At high cell density, anoxic episodes enhanced cell death most likely by preventing a cell density-mediated rescue. Neutral red dye uptake, an index for cell viability, was enhanced with increasing cell density and in vitro maturation, but was reduced in dense cultures exposed to anoxic/hypoglycemic conditions. The data suggest that cell density may play a critical role in brain organogenesis and that anoxic stress is more deleterious in dense than sparse cell assemblies.

TNFα sensitizes neuroblastoma cells to FasL-, cisplatin- and etoposide-induced cell death by NF-κB-mediated expression of Fas.

PubMed

Galenkamp, Koen Mo; Carriba, Paulina; Urresti, Jorge; Planells-Ferrer, Laura; Coccia, Elena; Lopez-Soriano, Joaquín; Barneda-Zahonero, Bruna; Moubarak, Rana S; Segura, Miguel F; Comella, Joan X

2015-03-19

Patients with high-risk neuroblastoma (NBL) tumors have a high mortality rate. Consequently, there is an urgent need for the development of new treatments for this condition. Targeting death receptor signaling has been proposed as an alternative to standard chemo- and radio-therapies in various tumors. In NBL, this therapeutic strategy has been largely disregarded, possibly because ~50-70% of all human NBLs are characterized by caspase-8 silencing. However, the expression of caspase-8 is detected in a significant group of NBL patients, and they could therefore benefit from treatments that induce cell death through death receptor activation. Given that cytokines, such as TNFα, are able to upregulate Fas expression, we sought to address the therapeutic relevance of co-treatment with TNFα and FasL in NBL. For the purpose of the study we used a set of eight NBL cell lines. Here we explore the cell death induced by TNFα, FasL, cisplatin, and etoposide, or a combination thereof by Hoechst staining and calcein viability assay. Further assessment of the signaling pathways involved was performed by caspase activity assays and Western blot experiments. Characterization of Fas expression levels was achieved by qRT-PCR, cell surface biotinylation assays, and cytometry. We have found that TNFα is able to increase FasL-induced cell death by a mechanism that involves the NF-κB-mediated induction of the Fas receptor. Moreover, TNFα sensitized NBL cells to DNA-damaging agents (i.e. cisplatin and etoposide) that induce the expression of FasL. Priming to FasL-, cisplatin-, and etoposide-induced cell death could only be achieved in NBLs that display TNFα-induced upregulation of Fas. Further analysis denotes that the high degree of heterogeneity between NBLs is also manifested in Fas expression and modulation thereof by TNFα. In summary, our findings reveal that TNFα sensitizes NBL cells to FasL-induced cell death by NF-κB-mediated upregulation of Fas and unveil a new mechanism through which TNFα enhances the efficacy of currently used NBL treatments, cisplatin and etoposide.

Deciphering early events involved in hyperosmotic stress-induced programmed cell death in tobacco BY-2 cells.

PubMed

Monetti, Emanuela; Kadono, Takashi; Tran, Daniel; Azzarello, Elisa; Arbelet-Bonnin, Delphine; Biligui, Bernadette; Briand, Joël; Kawano, Tomonori; Mancuso, Stefano; Bouteau, François

2014-03-01

Hyperosmotic stresses represent one of the major constraints that adversely affect plants growth, development, and productivity. In this study, the focus was on early responses to hyperosmotic stress- (NaCl and sorbitol) induced reactive oxygen species (ROS) generation, cytosolic Ca(2+) concentration ([Ca(2+)]cyt) increase, ion fluxes, and mitochondrial potential variations, and on their links in pathways leading to programmed cell death (PCD). By using BY-2 tobacco cells, it was shown that both NaCl- and sorbitol-induced PCD seemed to be dependent on superoxide anion (O2·(-)) generation by NADPH-oxidase. In the case of NaCl, an early influx of sodium through non-selective cation channels participates in the development of PCD through mitochondrial dysfunction and NADPH-oxidase-dependent O2·(-) generation. This supports the hypothesis of different pathways in NaCl- and sorbitol-induced cell death. Surprisingly, other shared early responses, such as [Ca(2+)]cyt increase and singlet oxygen production, do not seem to be involved in PCD.

FasL-triggered death of Jurkat cells requires caspase 8-induced, ATP-dependent cross-talk between Fas and the purinergic receptor P2X(7).

PubMed

Aguirre, Adam; Shoji, Kenji F; Sáez, Juan C; Henríquez, Mauricio; Quest, Andrew F G

2013-02-01

Fas ligation via the ligand FasL activates the caspase-8/caspase-3-dependent extrinsic death pathway. In so-called type II cells, an additional mechanism involving tBid-mediated caspase-9 activation is required to efficiently trigger cell death. Other pathways linking FasL-Fas interaction to activation of the intrinsic cell death pathway remain unknown. However, ATP release and subsequent activation of purinergic P2X(7) receptors (P2X(7)Rs) favors cell death in some cells. Here, we evaluated the possibility that ATP release downstream of caspase-8 via pannexin1 hemichannels (Panx1 HCs) and subsequent activation of P2X(7)Rs participate in FasL-stimulated cell death. Indeed, upon FasL stimulation, ATP was released from Jurkat cells in a time- and caspase-8-dependent manner. Fas and Panx1 HCs colocalized and inhibition of the latter, but not connexin hemichannels, reduced FasL-induced ATP release. Extracellular apyrase, which hydrolyzes ATP, reduced FasL-induced death. Also, oxidized-ATP or Brilliant Blue G, two P2X(7)R blockers, reduced FasL-induced caspase-9 activation and cell death. These results represent the first evidence indicating that the two death receptors, Fas and P2X(7)R connect functionally via caspase-8 and Panx1 HC-mediated ATP release to promote caspase-9/caspase-3-dependent cell death in lymphoid cells. Thus, a hitherto unsuspected route was uncovered connecting the extrinsic to the intrinsic pathway to amplify death signals emanating from the Fas receptor in type II cells. Copyright © 2012 Wiley Periodicals, Inc.

Bad is not involved in DHA-induced apoptosis in human lung adenocarcinoma ASTC-a-1 cells

NASA Astrophysics Data System (ADS)

Yu, Huai-na; Lu, Ying-ying; Chen, Tong-sheng

2011-03-01

Dihydroartemisinin (DHA), a first-line anti-malarial drug with low toxicity, has been shown to possess promising anticancer activities and induce cancer cell death through apoptotic pathway, but the molecular mechanisms are not well understood. In this paper, we focus on whether Bad, a BH3-only pro-apoptotic protein, is involved in apoptotic cell death in DHA-treated human lung adenocarcinoma (ASTC-a-1) cells. Confocal fluorescence microscope imaging was used to monitor the temporal and spatial distribution of Bad in single living cells. Our results indicate that Bad is still located in cytoplasm and does not translocate to mitochondria after treatment with DHA for 24 h, while only a small proportion of Bad located in cytoplasm in the STS-treated cells for 6 h. These results show for the first time that Bad is not involved in DHA-induced apoptosis in ASTC-a-1 cells, which could give more evidence for the molecular mechanisms of apoptosis induced by DHA.

THAP5 is a DNA-binding transcriptional repressor that is regulated in melanoma cells during DNA damage-induced cell death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Balakrishnan, Meenakshi P.; Cilenti, Lucia; Ambivero, Camilla

2011-01-07

Research highlights: {yields} THAP5 is a DNA-binding protein and a transcriptional repressor. {yields} THAP5 is induced in melanoma cells upon exposure to UV or treatment with cisplatin. {yields} THAP5 induction correlates with the degree of apoptosis in melanoma cell population. {yields} THAP5 is a pro-apoptotic protein involved in melanoma cell death. -- Abstract: THAP5 was originally isolated as a specific interactor and substrate of the mitochondrial pro-apoptotic Omi/HtrA2 protease. It is a human zinc finger protein characterized by a restricted pattern of expression and the lack of orthologs in mouse and rat. The biological function of THAP5 is unknown butmore » our previous studies suggest it could regulate G2/M transition in kidney cells and could be involved in human cardiomyocyte cell death associated with coronary artery disease (CAD). In this report, we expanded our studies on the properties and function of THAP5 in human melanoma cells. THAP5 was expressed in primary human melanocytes as well as in all melanoma cell lines that were tested. THAP5 protein level was significantly induced by UV irradiation or cisplatin treatment, conditions known to cause DNA damage. The induction of THAP5 correlated with a significant increase in apoptotic cell death. In addition, we show that THAP5 is a nuclear protein that could recognize and bind a specific DNA motif. THAP5 could also repress the transcription of a reporter gene in a heterologous system. Our work suggests that THAP5 is a DNA-binding protein and a transcriptional repressor. Furthermore, THAP5 has a pro-apoptotic function and it was induced in melanoma cells under conditions that promoted cell death.« less

Ketamine-induced apoptosis in cultured rat cortical neurons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takadera, Tsuneo; Ishida, Akira; Ohyashiki, Takao

2006-01-15

Recent data suggest that anesthetic drugs cause neurodegeneration during development. Ketamine is frequently used in infants and toddlers for elective surgeries. The purpose of this study is to determine whether glycogen synthase kinase-3 (GSK-3) is involved in ketamine-induced apoptosis. Ketamine increased apoptotic cell death with morphological changes which were characterized by cell shrinkage, nuclear condensation or fragmentation. In addition, insulin growth factor-1 completely blocked the ketamine-induced apoptotic cell death. Ketamine decreased Akt phosphorylation. GSK-3 is known as a downstream target of Akt. The selective inhibitors of GSK-3 prevented the ketamine-induced apoptosis. Moreover, caspase-3 activation was accompanied by the ketamine-induced cellmore » death and inhibited by the GSK-3 inhibitors. These results suggest that activation of GSK-3 is involved in ketamine-induced apoptosis in rat cortical neurons.« less

DRAM Triggers Lysosomal Membrane Permeabilization and Cell Death in CD4+ T Cells Infected with HIV

PubMed Central

Laforge, Mireille; Limou, Sophie; Harper, Francis; Casartelli, Nicoletta; Rodrigues, Vasco; Silvestre, Ricardo; Haloui, Houda; Zagury, Jean-Francois; Senik, Anna; Estaquier, Jerome

2013-01-01

Productive HIV infection of CD4+ T cells leads to a caspase-independent cell death pathway associated with lysosomal membrane permeabilization (LMP) and cathepsin release, resulting in mitochondrial outer membrane permeabilization (MOMP). Herein, we demonstrate that HIV infection induces damage-regulated autophagy modulator (DRAM) expression in a p53-dependent manner. Knocking down the expression of DRAM and p53 genes with specific siRNAs inhibited autophagy and LMP. However, inhibition of Atg5 and Beclin genes that prevents autophagy had a minor effect on LMP and cell death. The knock down of DRAM gene inhibited cytochrome C release, MOMP and cell death. However, knocking down DRAM, we increased viral infection and production. Our study shows for the first time the involvement of DRAM in host-pathogen interactions, which may represent a mechanism of defense via the elimination of infected cells. PMID:23658518

Glucose Deprivation Induces ATF4-Mediated Apoptosis through TRAIL Death Receptors

PubMed Central

Iurlaro, Raffaella; Püschel, Franziska; León-Annicchiarico, Clara Lucía; O'Connor, Hazel; Martin, Seamus J.; Palou-Gramón, Daniel; Lucendo, Estefanía

2017-01-01

ABSTRACT Metabolic stress occurs frequently in tumors and in normal tissues undergoing transient ischemia. Nutrient deprivation triggers, among many potential cell death-inducing pathways, an endoplasmic reticulum (ER) stress response with the induction of the integrated stress response transcription factor ATF4. However, how this results in cell death remains unknown. Here we show that glucose deprivation triggered ER stress and induced the unfolded protein response transcription factors ATF4 and CHOP. This was associated with the nontranscriptional accumulation of TRAIL receptor 1 (TRAIL-R1) (DR4) and with the ATF4-mediated, CHOP-independent induction of TRAIL-R2 (DR5), suggesting that cell death in this context may involve death receptor signaling. Consistent with this, the ablation of TRAIL-R1, TRAIL-R2, FADD, Bid, and caspase-8 attenuated cell death, although the downregulation of TRAIL did not, suggesting ligand-independent activation of TRAIL receptors. These data indicate that stress triggered by glucose deprivation promotes the ATF4-dependent upregulation of TRAIL-R2/DR5 and TRAIL receptor-mediated cell death. PMID:28242652

3-Decylcatechol induces autophagy-mediated cell death through the IRE1α/JNK/p62 in hepatocellular carcinoma cells

PubMed Central

Kim, Jin-A; Jo, In-Hwa; Han, Yeon Soo; Jo, Yong Hun; Kim, Kwang-Youn; Seo, Young-Kyo; Moon, Jae-Hak; Jung, Chang Hwa; Jeon, Tae-Il

2017-01-01

The natural, phenolic lipid urushiol exhibits both antioxidant and anticancer activities; however, its biological activity on hepatocellular carcinoma (HCC) has not been previously investigated. Here, we demonstrate that an urushiol derivative, 3-decylcatechol (DC), induces human HCC Huh7 cell death by induction of autophagy. DC initiates the autophagic process by activation of the mammalian target of rapamycin signaling pathway via Unc-51-like autophagy activating kinase 1, leading to autophagosome formation. The autophagy inhibitor, chloroquine, suppressed autolysosome formation and cell death induction by DC, indicating an autophagic cell death. Interestingly, DC also activated the endoplasmic reticulum (ER) stress response that promotes autophagy via p62 transcriptional activation involving the inositol-requiring enzyme 1α/c-Jun N-terminal kinase/c-jun pathway. We also show that cytosolic calcium mobilization is necessary for the ER stress response and autophagy induction by DC. These findings reveal a novel mechanism by which this urushiol derivative induces autophagic cell death in HCC. PMID:28938597

Combined inhibition of autophagy and caspases fails to prevent developmental nurse cell death in the Drosophila melanogaster ovary.

PubMed

Peterson, Jeanne S; McCall, Kimberly

2013-01-01

During the final stages of Drosophila melanogaster oogenesis fifteen nurse cells, sister cells to the oocyte, degenerate as part of normal development. This process involves at least two cell death mechanisms, caspase-dependent cell death and autophagy, as indicated by apoptosis and autophagy markers. In addition, mutations affecting either caspases or autophagy partially reduce nurse cell removal, leaving behind end-stage egg chambers with persisting nurse cell nuclei. To determine whether apoptosis and autophagy work in parallel to degrade and remove these cells as is the case with salivary glands during pupariation, we generated mutants doubly affecting caspases and autophagy. We found no significant increase in either the number of late stage egg chambers containing persisting nuclei or in the number of persisting nuclei per egg chamber in the double mutants compared to single mutants. These findings suggest that there is another cell death mechanism functioning in the ovary to remove all nurse cell remnants from late stage egg chambers.

Combating rituximab resistance by inducing ceramide/lysosome-involved cell death through initiation of CD20-TNFR1 co-localization

PubMed Central

Zhang, Fan; Yang, Junlan; Li, Huafei; Liu, Moyan; Zhang, Jie; Zhao, Lichao; Wang, Lingxiong; LingHu, RuiXia; Feng, Fan; Gao, Xudong; Dong, Biqin; Liu, Xiaohan; Zi, Jian; Zhang, Weijing; Hu, Yi; Pan, Jingkun; Tian, Lei; Hu, Yazuo; Han, Zhitao; Zhang, Honghong; Wang, Xiaoning; Zhao, Lei

2016-01-01

ABSTRACT Despite the success of CD20 antibody rituximab in immunotherapy, acquired resistance is one of the prime obstacles for the successful treatment of B-cell malignancies. There is an urgent need to intensify efforts against resistance in cancer treatment. Growing evidence indicated that lysosomes may form an “Achilles heel” for cancer cells by sensitizing them to death pathways. Here, we uncover an important role of CD20 in initiation of ceramide/lysosomal membrane permeabilization (LMP)-mediated cell death, showing that colocalization of CD20-TNFR1 after type II CD20 antibody ligation can stimulate de novo ceramide synthesis by ceramide synthase and consequently induce remarkable lysosomal permeabilization (LMP) and lysosome-mediated cell death. Further studies show that the potent lysosome-mediated cell death induced by CD20 antibodies exhibits a profound killing effect against both rituximab-sensitive and -resistant (RR) lymphoma. Furthermore, engineering of rituximab by introducing a point mutation endows it with the ability to induce potent ceramide/LMP-mediated cell death in both RR lymphoma and primary B-cell malignancies from patients with rituximab-refractory, suggesting the potential clinical application to combat rituximab resistance. PMID:27467962

Silibinin, a natural flavonoid, induces autophagy via ROS-dependent mitochondrial dysfunction and loss of ATP involving BNIP3 in human MCF7 breast cancer cells.

PubMed

Jiang, Kai; Wang, Wei; Jin, Xin; Wang, Zhaoyang; Ji, Zhiwei; Meng, Guanmin

2015-06-01

Silibinin, derived from the milk thistle plant (Silybum marianum), has anticancer and chemopreventive properties. Silibinin has been reported to inhibit the growth of various types of cancer cells. However, the mechanisms by which silibinin exerts an anticancer effect are poorly defined. The present study aimed to investigate whether silibinin-induced cell death might be attributed to autophagy and the underlying mechanisms in human MCF7 breast cancer cells. Our results showed that silibinin-induced cell death was greatly abrogated by two specific autophagy inhibitors, 3-methyladenine (3-MA) and bafilomycin-A1 (Baf-A1). In addition, silibinin triggered the conversion of light chain 3 (LC3)-I to LC3-II, promoted the upregulation of Atg12-Atg5 formation, increased Beclin-1 expression, and decreased the Bcl-2 level. Moreover, we noted elevated reactive oxygen species (ROS) generation, concomitant with the dissipation of mitochondrial transmembrane potential (ΔΨm) and a drastic decline in ATP levels following silibinin treatment, which were effectively prevented by the antioxidants, N-acetylcysteine and ascorbic acid. Silibinin stimulated the expression of Bcl-2 adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), a pro-death Bcl-2 family member, and silencing of BNIP3 greatly inhibited silibinin-induced cell death, decreased ROS production, and sustained ΔΨm and ATP levels. Taken together, these findings revealed that silibinin induced autophagic cell death through ROS-dependent mitochondrial dysfunction and ATP depletion involving BNIP3 in MCF7 cells.

Peroxide accumulation and cell death in filamentous fungi induced by contact with a contestant.

PubMed

Silar, Philippe

2005-02-01

Podospora anserina and Coprinopsis cinerea (syn. Coprinus cinereus) are endowed with a defence system able to differentiate self vs. non-self and involving the generation of peroxide. Indeed, they produce peroxide when confronted with a filamentous fungus, only in non-self confrontations. Both species are not able to recognize yeasts and show a differential response to bacteria. The accumulation of peroxides in the ascomycete Podospora anserina requires an NADPH oxidase and a MAP kinase cascade, previously shown to be involved in fruit body formation, cell differentiation and cell degeneration. Confrontation is accompanied by the death of the contestant hyphae only in specific combinations of species. As in animals and plants, data suggest that peroxide is likely involved in signalling rather than playing a direct toxic role. Fungi display more complex behaviours than generally acknowledged, i.e. they are able to recognize potential contestants and built up defence reactions involving evolutionary conserved enzymes.

Involvement of the PI3K/Akt/GSK3β pathway in photodynamic injury of neurons and glial cells

NASA Astrophysics Data System (ADS)

Komandirov, M. A.; Knyazeva, E. A.; Fedorenko, Y. P.; Rudkovskii, M. V.; Stetsurin, D. A.; Uzdensky, A. B.

2010-10-01

Photodynamic treatment causes intense oxidative stress and kills cells. It is currently used in neurooncology. However, along with tumor it damages surrounding healthy neuronal and glial cells. In order to study the possible role of the phosphatidylinositol 3-kinase/protein kinase Akt/glycogen synthase kinase-3β signaling pathway in photodynamic damage to normal neurons and glia, we used isolated crayfish stretch receptor that consists only of a single neuron surrounded by glial cells. It was photosensitized with alumophthalocyanine Photosens (100 nM). The laser diode (670nm, 0.4W/cm2) was used as a light source. Application of specific inhibitors of the enzymes involved in this pathway showed that phosphatidylinositol 3-kinase did not participate in photoinduced death of neurons and glia. Protein kinase Akt was involved in photoinduced necrosis but not in apoptosis of neurons and glia. Glycogen synthase kinase-3β participated in photoinduced apoptosis of glial cells and in necrosis of neurons. Therefore, the phosphatidylinositol 3-kinase/protein kinase Akt/glycogen synthase kinase-3β pathway was not involved as a whole in photodynamic injury of crayfish neurons and glial cells but its components, protein kinase Akt and glycogen synthase kinase-3β, independently and cell-specifically regulated photoinduced death of neurons and glial cells. These data showed that in this system necrosis was not non-regulated and catastrophic mode of cell death. It was controlled by some signaling proteins. The obtained results may be used for search of pharmacological agents that selectively modulate injury of normal neurons and glial cells during photodynamic therapy of brain tumors.

Involvement of the PI3K/Akt/GSK3β pathway in photodynamic injury of neurons and glial cells

NASA Astrophysics Data System (ADS)

Komandirov, M. A.; Knyazeva, E. A.; Fedorenko, Y. P.; Rudkovskii, M. V.; Stetsurin, D. A.; Uzdensky, A. B.

2011-03-01

Photodynamic treatment causes intense oxidative stress and kills cells. It is currently used in neurooncology. However, along with tumor it damages surrounding healthy neuronal and glial cells. In order to study the possible role of the phosphatidylinositol 3-kinase/protein kinase Akt/glycogen synthase kinase-3β signaling pathway in photodynamic damage to normal neurons and glia, we used isolated crayfish stretch receptor that consists only of a single neuron surrounded by glial cells. It was photosensitized with alumophthalocyanine Photosens (100 nM). The laser diode (670nm, 0.4W/cm2) was used as a light source. Application of specific inhibitors of the enzymes involved in this pathway showed that phosphatidylinositol 3-kinase did not participate in photoinduced death of neurons and glia. Protein kinase Akt was involved in photoinduced necrosis but not in apoptosis of neurons and glia. Glycogen synthase kinase-3β participated in photoinduced apoptosis of glial cells and in necrosis of neurons. Therefore, the phosphatidylinositol 3-kinase/protein kinase Akt/glycogen synthase kinase-3β pathway was not involved as a whole in photodynamic injury of crayfish neurons and glial cells but its components, protein kinase Akt and glycogen synthase kinase-3β, independently and cell-specifically regulated photoinduced death of neurons and glial cells. These data showed that in this system necrosis was not non-regulated and catastrophic mode of cell death. It was controlled by some signaling proteins. The obtained results may be used for search of pharmacological agents that selectively modulate injury of normal neurons and glial cells during photodynamic therapy of brain tumors.

Autophagy Protects Against Aminochrome-Induced Cell Death in Substantia Nigra-Derived Cell Line

PubMed Central

Paris, Irmgard; Muñoz, Patricia; Huenchuguala, Sandro; Couve, Eduardo; Sanders, Laurie H.; Greenamyre, John Timothy; Caviedes, Pablo; Segura-Aguilar, Juan

2011-01-01

Aminochrome, the precursor of neuromelanin, has been proposed to be involved in the neurodegeneration neuromelanin-containing dopaminergic neurons in Parkinson’s disease. We aimed to study the mechanism of aminochrome-dependent cell death in a cell line derived from rat substantia nigra. We found that aminochrome (50μM), in the presence of NAD(P)H-quinone oxidoreductase, EC 1.6.99.2 (DT)-diaphorase inhibitor dicoumarol (DIC) (100μM), induces significant cell death (62 ± 3%; p < 0.01), increase in caspase-3 activation (p < 0.001), release of cytochrome C, disruption of mitochondrial membrane potential (p < 0.01), damage of mitochondrial DNA, damage of mitochondria determined with transmission electron microscopy, a dramatic morphological change characterized as cell shrinkage, and significant increase in number of autophagic vacuoles. To determine the role of autophagy on aminochrome-induced cell death, we incubated the cells in the presence of vinblastine and rapamycin. Interestingly, 10μM vinblastine induces a 5.9-fold (p < 0.001) and twofold (p < 0.01) significant increase in cell death when the cells were incubated with 30μM aminochrome in the absence and presence of DIC, respectively, whereas 10μM rapamycin preincubated 24 h before addition of 50μM aminochrome in the absence and the presence of 100μM DIC induces a significant decrease (p < 0.001) in cell death. In conclusion, autophagy seems to be an important protective mechanism against two different aminochrome-induced cell deaths that initially showed apoptotic features. The cell death induced by aminochrome when DT-diaphorase is inhibited requires activation of mitochondrial pathway, whereas the cell death induced by aminochrome alone requires inhibition of autophagy-dependent degrading of damaged organelles and recycling through lysosomes. PMID:21427056

Calcium and mitochondrial metabolism in ceramide-induced cardiomyocyte death

PubMed Central

Parra, Valentina; Moraga, Francisco; Kuzmicic, Jovan; López-Crisosto, Camila; Troncoso, Rodrigo; Torrealba, Natalia; Criollo, Alfredo; Díaz-Elizondo, Jessica; Rothermel, Beverly A.; Quest, Andrew F.G.; Lavandero, Sergio

2014-01-01

Ceramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulatenumerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca2+ overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca2+ levels, mitochondrial function and cardiomyocyte death. Our data show that C2-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca2+ influx, mitochondrial network fragmentation and loss of the mitochondrial Ca2+ buffer capacity. These biochemical events increase cytosolic Ca2+ levels and trigger cardiomyocyte death via the activation of calpains. PMID:23602992

Sodium fluoride induces apoptosis in mouse embryonic stem cells through ROS-dependent and caspase- and JNK-mediated pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nguyen Ngoc, Tam Dan; Son, Young-Ok; Lim, Shin-Saeng

2012-03-15

Sodium fluoride (NaF) is used as a source of fluoride ions in diverse applications. Fluoride salt is an effective prophylactic for dental caries and is an essential element required for bone health. However, fluoride is known to cause cytotoxicity in a concentration-dependent manner. Further, no information is available on the effects of NaF on mouse embryonic stem cells (mESCs). We investigated the mode of cell death induced by NaF and the mechanisms involved. NaF treatment greater than 1 mM reduced viability and DNA synthesis in mESCs and induced cell cycle arrest in the G{sub 2}/M phase. The addition of NaFmore » induced cell death mainly by apoptosis rather than necrosis. Catalase (CAT) treatment significantly inhibited the NaF-mediated cell death and also suppressed the NaF-mediated increase in phospho-c-Jun N-terminal kinase (p-JNK) levels. Pre-treatment with SP600125 or z-VAD-fmk significantly attenuated the NaF-mediated reduction in cell viability. In contrast, intracellular free calcium chelator, but not of sodium or calcium ion channel blockers, facilitated NaF-induced toxicity in the cells. A JNK specific inhibitor (SP600125) prevented the NaF-induced increase in growth arrest and the DNA damage-inducible protein 45α. Further, NaF-mediated loss of mitochondrial membrane potential was apparently inhibited by pifithrin-α or CAT inhibitor. These findings suggest that NaF affects viability of mESCs in a concentration-dependent manner, where more than 1 mM NaF causes apoptosis through hydroxyl radical-dependent and caspase- and JNK-mediated pathways. -- Highlights: ► The mode of NaF-induced cell death and the mechanisms involved were examined. ► NaF induced mainly apoptotic death of mouse embryonic stem cells (mESCs). ► NaF induced mitochondrial-mediated and caspase-dependent apoptosis. ► JNK- and p53-mediated pathways are involved in NaF-mediated apoptosis in the cells. ► ROS are the up-stream effector in NaF-mediated activation of JNK and p53 in mESCs.« less

Susceptibility of Mycobacterium tuberculosis-infected host cells to phospho-MLKL driven necroptosis is dependent on cell type and presence of TNFα.

PubMed

Butler, Rachel E; Krishnan, Nitya; Garcia-Jimenez, Waldo; Francis, Robert; Martyn, Abbe; Mendum, Tom; Felemban, Shaza; Locker, Nicolas; Salguero, Francisco J; Robertson, Brian; Stewart, Graham R

2017-11-17

An important feature of Mycobacterium tuberculosis pathogenesis is the ability to control cell death in infected host cells, including inhibition of apoptosis and stimulation of necrosis. Recently an alternative form of programmed cell death, necroptosis, has been described where necrotic cell death is induced by apoptotic stimuli under conditions where apoptotic execution is inhibited. We show for the first time that M. tuberculosis and TNFα synergise to induce necroptosis in murine fibroblasts via RIPK1-dependent mechanisms and characterized by phosphorylation of Ser345 of the MLKL necroptosis death effector. However, in murine macrophages M. tuberculosis and TNFα induce non-necroptotic cell death that is RIPK1-dependent but independent of MLKL phosphorylation. Instead, M. tuberculosis-infected macrophages undergo RIPK3-dependent cell death which occurs both in the presence and absence of TNFα and involves the production of mitochondrial ROS. Immunocytochemical staining for MLKL phosphorylation further demonstrated the occurrence of necroptosis in vivo in murine M. tuberculosis granulomas. Phosphorylated-MLKL immunoreactivity was observed associated with the cytoplasm and nucleus of fusiform cells in M. tuberculosis lesions but not in proximal macrophages. Thus whereas pMLKL-driven necroptosis does not appear to be a feature of M. tuberculosis-infected macrophage cell death, it may contribute to TNFα-induced cytotoxicity of the lung stroma and therefore contribute to necrotic cavitation and bacterial dissemination.

Alpha Particles Induce Autophagy in Multiple Myeloma Cells.

PubMed

Gorin, Jean-Baptiste; Gouard, Sébastien; Ménager, Jérémie; Morgenstern, Alfred; Bruchertseifer, Frank; Faivre-Chauvet, Alain; Guilloux, Yannick; Chérel, Michel; Davodeau, François; Gaschet, Joëlle

2015-01-01

Radiation emitted by the radionuclides in radioimmunotherapy (RIT) approaches induce direct killing of the targeted cells as well as indirect killing through the bystander effect. Our research group is dedicated to the development of α-RIT, i.e., RIT using α-particles especially for the treatment of multiple myeloma (MM). γ-irradiation and β-irradiation have been shown to trigger apoptosis in tumor cells. Cell death mode induced by (213)Bi α-irradiation appears more controversial. We therefore decided to investigate the effects of (213)Bi on MM cell radiobiology, notably cell death mechanisms as well as tumor cell immunogenicity after irradiation. Murine 5T33 and human LP-1 MM cell lines were used to study the effects of such α-particles. We first examined the effects of (213)Bi on proliferation rate, double-strand DNA breaks, cell cycle, and cell death. Then, we investigated autophagy after (213)Bi irradiation. Finally, a coculture of dendritic cells (DCs) with irradiated tumor cells or their culture media was performed to test whether it would induce DC activation. We showed that (213)Bi induces DNA double-strand breaks, cell cycle arrest, and autophagy in both cell lines, but we detected only slight levels of early apoptosis within the 120 h following irradiation in 5T33 and LP-1. Inhibition of autophagy prevented (213)Bi-induced inhibition of proliferation in LP-1 suggesting that this mechanism is involved in cell death after irradiation. We then assessed the immunogenicity of irradiated cells and found that irradiated LP-1 can activate DC through the secretion of soluble factor(s); however, no increase in membrane or extracellular expression of danger-associated molecular patterns was observed after irradiation. This study demonstrates that (213)Bi induces mainly necrosis in MM cells, low levels of apoptosis, and autophagy that might be involved in tumor cell death.

Molecular Characterization of Propolis-Induced Cell Death in Saccharomyces cerevisiae▿†

PubMed Central

de Castro, Patrícia Alves; Savoldi, Marcela; Bonatto, Diego; Barros, Mário Henrique; Goldman, Maria Helena S.; Berretta, Andresa A.; Goldman, Gustavo Henrique

2011-01-01

Propolis, a natural product of plant resins, is used by the bees to seal holes in their honeycombs and protect the hive entrance. However, propolis has also been used in folk medicine for centuries. Here, we apply the power of Saccharomyces cerevisiae as a model organism for studies of genetics, cell biology, and genomics to determine how propolis affects fungi at the cellular level. Propolis is able to induce an apoptosis cell death response. However, increased exposure to propolis provides a corresponding increase in the necrosis response. We showed that cytochrome c but not endonuclease G (Nuc1p) is involved in propolis-mediated cell death in S. cerevisiae. We also observed that the metacaspase YCA1 gene is important for propolis-mediated cell death. To elucidate the gene functions that may be required for propolis sensitivity in eukaryotes, the full collection of about 4,800 haploid S. cerevisiae deletion strains was screened for propolis sensitivity. We were able to identify 138 deletion strains that have different degrees of propolis sensitivity compared to the corresponding wild-type strains. Systems biology revealed enrichment for genes involved in the mitochondrial electron transport chain, vacuolar acidification, negative regulation of transcription from RNA polymerase II promoter, regulation of macroautophagy associated with protein targeting to vacuoles, and cellular response to starvation. Validation studies indicated that propolis sensitivity is dependent on the mitochondrial function and that vacuolar acidification and autophagy are important for yeast cell death caused by propolis. PMID:21193549

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

PubMed

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

2008-01-01

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

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

PubMed

Muñoz-Pinedo, Cristina

2012-01-01

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

The TIR domain of TIR-NB-LRR resistance proteins is a signaling domain involved in cell death induction.

PubMed

Swiderski, Michal R; Birker, Doris; Jones, Jonathan D G

2009-02-01

In plants, the TIR (toll interleukin 1 receptor) domain is found almost exclusively in nucleotide-binding (NB) leucine-rich repeat resistance proteins and their truncated homologs, and has been proposed to play a signaling role during resistance responses mediated by TIR containing R proteins. Transient expression in Nicotiana benthamiana leaves of "TIR + 80", the RPS4 truncation without the NB-ARC domain, leads to EDS1-, SGT1-, and HSP90-dependent cell death. Transgenic Arabidopsis plants expressing the RPS4 TIR+80 from either dexamethasone or estradiol-inducible promoters display inducer-dependent cell death. Cell death is also elicited by transient expression of similarly truncated constructs from two other R proteins, RPP1A and At4g19530, but is not elicited by similar constructs representing RPP2A and RPP2B proteins. Site-directed mutagenesis of the RPS4 TIR domain identified many loss-of-function mutations but also revealed several gain-of function substitutions. Lack of cell death induction by the E160A substitution suggests that amino acids outside of the TIR domain contribute to cell death signaling in addition to the TIR domain itself. This is consistent with previous observations that the TIR domain itself is insufficient to induce cell death upon transient expression.

miR-14 regulates autophagy during developmental cell death by targeting ip3-kinase 2.

PubMed

Nelson, Charles; Ambros, Victor; Baehrecke, Eric H

2014-11-06

Macroautophagy (autophagy) is a lysosome-dependent degradation process that has been implicated in age-associated diseases. Autophagy is involved in both cell survival and cell death, but little is known about the mechanisms that distinguish its use during these distinct cell fates. Here, we identify the microRNA miR-14 as being both necessary and sufficient for autophagy during developmentally regulated cell death in Drosophila. Loss of miR-14 prevented induction of autophagy during salivary gland cell death, but had no effect on starvation-induced autophagy in the fat body. Moreover, misexpression of miR-14 was sufficient to prematurely induce autophagy in salivary glands, but not in the fat body. Importantly, miR-14 regulates this context-specific autophagy through its target, inositol 1,4,5-trisphosphate kinase 2 (ip3k2), thereby affecting inositol 1,4,5-trisphosphate (IP3) signaling and calcium levels during salivary gland cell death. This study provides in vivo evidence of microRNA regulation of autophagy through modulation of IP3 signaling. Copyright © 2014 Elsevier Inc. All rights reserved.

Necroptosis in cancer: An angel or a demon?

PubMed

Wang, Tianzhen; Jin, Yinji; Yang, Weiwei; Zhang, Lei; Jin, Xiaoming; Liu, Xi; He, Yan; Li, Xiaobo

2017-06-01

In the past few decades, apoptosis has been regarded as the only form of programmed cell death. However, the traditional view has been challenged by the identification of several forms of regulated necrosis, including necroptosis. Necroptosis is typified by a necrotic cell death morphology and is controlled by RIP1, RIP3, and mixed lineage kinase domain-like protein. The physiological role of necroptosis is to serve as a "fail-safe" form of cell death for cells that fail to undergo apoptosis during embryonic development and disease defense. Currently, established studies have indicated that necroptosis is involved in cancer initiation and progression. Although elevated necroptosis contributes to cancer cell death, extensive cell death also increases the risk of proliferation and metastasis of the surviving cells by inducing the generation reactive oxygen species, activation of inflammation, and suppression of the immune response. Thus, questions regarding the overall impact of necroptosis on cancer remain open. In this review, we introduce the basic knowledge regarding necroptosis, summarize its dual effects on cancer progression, and analyze its advantages and disadvantages in clinical applications.

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

PubMed Central

Caprariello, Andrew V.; Henry, Tyler J.; Tsutsui, Shigeki; Chu, Tak H.; Schenk, Geert J.; Yong, V. Wee

2017-01-01

Cellular injury and death are ubiquitous features of disease, yet tools to detect them are limited and insensitive to subtle pathological changes. Acridine orange (AO), a nucleic acid dye with unique spectral properties, enables real-time measurement of RNA and DNA as proxies for cell viability during exposure to various noxious stimuli. This tool illuminates spectral signatures unique to various modes of cell death, such as cells undergoing apoptosis versus necrosis/necroptosis. This new approach also shows that cellular RNA decreases during necrotic, necroptotic, and apoptotic cell death caused by demyelinating, ischemic, and traumatic injuries, implying its involvement in a wide spectrum of tissue pathologies. Furthermore, cells with pathologically low levels of cytoplasmic RNA are detected earlier and in higher numbers than with standard markers including TdT-mediated dUTP biotin nick-end labeling and cleaved caspase 3 immunofluorescence. Our technique highlights AO-labeled cytoplasmic RNA as an important early marker of cellular injury and a sensitive indicator of various modes of cell death in a range of experimental models. PMID:28264914

Ultra-violet B (UVB)-induced skin cell death occurs through a cyclophilin D intrinsic signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ji, Chao; Yang, Bo; Yang, Zhi

Highlights: Black-Right-Pointing-Pointer UVB radiated skin keratinocytes show cyclophilin D (Cyp-D) upregulation. Black-Right-Pointing-Pointer NAC inhibits UVB induced Cyp-D expression, while H{sub 2}O{sub 2} facilitates it. Black-Right-Pointing-Pointer Cyp-D-deficient cells are significantly less susceptible to UVB induced cell death. Black-Right-Pointing-Pointer Over-expression of Cyp-D causes spontaneous keratinocytes cell death. -- Abstract: UVB-induced skin cell damage involves the opening of mitochondrial permeability transition pore (mPTP), which leads to both apoptotic and necrotic cell death. Cyclophilin D (Cyp-D) translocation to the inner membrane of mitochondrion acts as a key component to open the mPTP. Our Western-Blot results in primary cultured human skin keratinocytes and in HaCaTmore » cell line demonstrated that UVB radiation and hydrogen peroxide (H{sub 2}O{sub 2}) induced Cyp-D expression, which was inhibited by anti-oxidant N-acetyl cysteine (NAC). We created a stable Cyp-D deficiency skin keratinocytes by expressing Cyp-D-shRNA through lentiviral infection. Cyp-D-deficient cells were significantly less susceptible than their counterparts to UVB- or H{sub 2}O{sub 2}-induced cell death. Further, cyclosporine A (Cs-A), a Cyp-D inhibitor, inhibited UVB- or H{sub 2}O{sub 2}-induced keratinocytes cell death. Reversely, over-expression of Cyp-D in primary keratinocytes caused spontaneous keratinocytes cell death. These results suggest Cyp-D's critical role in UVB/oxidative stress-induced skin cell death.« less

Bcl-2 Family Members and Functional Electron Transport Chain Regulate Oxygen Deprivation-Induced Cell Death

PubMed Central

McClintock, David S.; Santore, Matthew T.; Lee, Vivian Y.; Brunelle, Joslyn; Budinger, G. R. Scott; Zong, Wei-Xing; Thompson, Craig B.; Hay, Nissim; Chandel, Navdeep S.

2002-01-01

The mechanisms underlying cell death during oxygen deprivation are unknown. We report here a model for oxygen deprivation-induced apoptosis. The death observed during oxygen deprivation involves a decrease in the mitochondrial membrane potential, followed by the release of cytochrome c and the activation of caspase-9. Bcl-XL prevented oxygen deprivation-induced cell death by inhibiting the release of cytochrome c and caspase-9 activation. The ability of Bcl-XL to prevent cell death was dependent on allowing the import of glycolytic ATP into the mitochondria to generate an inner mitochondrial membrane potential through the F1F0-ATP synthase. In contrast, although activated Akt has been shown to inhibit apoptosis induced by a variety of apoptotic stimuli, it did not prevent cell death during oxygen deprivation. In addition to Bcl-XL, cells devoid of mitochondrial DNA (ρ° cells) that lack a functional electron transport chain were resistant to oxygen deprivation. Further, murine embryonic fibroblasts from bax−/− bak−/− mice did not die in response to oxygen deprivation. These data suggest that when subjected to oxygen deprivation, cells die as a result of an inability to maintain a mitochondrial membrane potential through the import of glycolytic ATP. Proapoptotic Bcl-2 family members and a functional electron transport chain are required to initiate cell death in response to oxygen deprivation. PMID:11739725

Collapsing Aged Culture of the Cyanobacterium Synechococcus elongatus Produces Compound(s) Toxic to Photosynthetic Organisms

PubMed Central

Cohen, Assaf; Sendersky, Eleonora; Carmeli, Shmuel; Schwarz, Rakefet

2014-01-01

Phytoplankton mortality allows effective nutrient cycling, and thus plays a pivotal role in driving biogeochemical cycles. A growing body of literature demonstrates the involvement of regulated death programs in the abrupt collapse of phytoplankton populations, and particularly implicates processes that exhibit characteristics of metazoan programmed cell death. Here, we report that the cell-free, extracellular fluid (conditioned medium) of a collapsing aged culture of the cyanobacterium Synechococcus elongatus is toxic to exponentially growing cells of this cyanobacterium, as well as to a large variety of photosynthetic organisms, but not to eubacteria. The toxic effect, which is light-dependent, involves oxidative stress, as suggested by damage alleviation by antioxidants, and the very high sensitivity of a catalase-mutant to the conditioned medium. At relatively high cell densities, S. elongatus cells survived the deleterious effect of conditioned medium in a process that required de novo protein synthesis. Application of conditioned medium from a collapsing culture caused severe pigment bleaching not only in S. elongatus cells, but also resulted in bleaching of pigments in a cell free extract. The latter observation indicates that the elicited damage is a direct effect that does not require an intact cell, and therefore, is mechanistically different from the metazoan-like programmed cell death described for phytoplankton. We suggest that S. elongatus in aged cultures are triggered to produce a toxic compound, and thus, this process may be envisaged as a novel regulated death program. PMID:24959874

Necroptosis: Fifty shades of RIPKs.

PubMed

Ichim, Gabriel; Tait, Stephen Wg

2015-01-01

Apoptosis and necroptosis are 2 major, yet distinct, forms of regulated cell death. Whereas apoptosis requires caspase protease function, necroptosis requires activation of the receptor interacting protein kinases 1 (RIPK1) and RIPK3. Following activation, RIPK3 phosphorylates mixed-lineage kinase domain-like (MLKL), leading to cell death. Apoptosis and necroptosis are deeply intertwined such that a given death stimulus can often engage either form of cell death. Recent studies published in Cell Death and Differentiation by the Han, Oberst, and Vaux laboratories provide exciting new insights into necroptosis and how it interconnects with apoptosis. As we will discuss, their findings address key questions including: How does a cell choose between apoptosis or necroptosis? How can RIPK3 also induce apoptosis? What is the nature of the RIPK1-3 signaling cascade leading to necroptosis? Finally, data from the Oberst and Han groups strongly argue that RIPK1 is not only involved in executing necroptosis, but also protects against this process in some settings.

Histologic pattern of Merkel cell carcinoma sentinel lymph node metastasis improves stratification of Stage III patients

PubMed Central

Ko, Jennifer S; Prieto, Victor G; Elson, Paul; Vilain, Ricardo E; Pulitzer, Melissa; Scolyer, Richard A; Reynolds, Jordan P; Piliang, Melissa; Ernstoff, Marc S; Gastman, Brian; Billings, Steven D

2016-01-01

Sentinel lymph node biopsy is used to stage Merkel cell carcinoma, but its prognostic value has been questioned. Furthermore, predictors of outcome in sentinel lymph node positive Merkel cell carcinoma patients are poorly defined. In breast carcinoma, isolated immunohistochemically positive tumor cells have no impact, but in melanoma they are considered significant. The significance of sentinel lymph node metastasis tumor burden (including isolated tumor cells) and pattern of involvement in Merkel cell carcinoma are unknown. In this study, 64 Merkel cell carcinomas involving sentinel lymph nodes and corresponding immunohistochemical stains were reviewed and clinicopathologic predictors of outcome were sought. Five metastatic patterns were identified: 1, sheet-like (n=38, 59%); 2, non-solid parafollicular (n=4, 6%); 3, sinusoidal, (n=11, 17%); 4, perivascular hilar (n=1, 2%) and 5, rare scattered parenchymal cells (n=10, 16%). At the time of follow-up, 30/63 (48%) patients had died with 21(33%) attributable to Merkel cell carcinoma. Patients with pattern 1 metastases had poorer overall survival compared with patients with patterns 2–5 metastases (p=0.03), with 22/30 (73%) deaths occurring in pattern 1 patients. 3 (10%) deaths occurred in patients showing pattern 5, all of whom were immunosuppressed. 4 (13%) deaths occurred in pattern 3 patients and 1 (3%) death occurred in a pattern 2 patient. In multivariable analysis, the number of positive sentinel lymph node (1 or 2 versus >2, p<.0001), age (<70 versus ≥70, p=.01), sentinel lymph node metastasis pattern (patterns 2–5 versus 1, p=.02), and immune status (immunocompetent versus suppressed, p=.03) were independent predictors of outcome, and could be used to stratify Stage III patients into 3 groups with markedly different outcomes. In Merkel cell carcinoma, the pattern of sentinel lymph node involvement provides important prognostic information and utilizing this data with other clinicopathologic features facilitates risk stratification of Merkel cell carcinoma patients that may have management implications. PMID:26541273

Histological pattern of Merkel cell carcinoma sentinel lymph node metastasis improves stratification of Stage III patients.

PubMed

Ko, Jennifer S; Prieto, Victor G; Elson, Paul J; Vilain, Ricardo E; Pulitzer, Melissa P; Scolyer, Richard A; Reynolds, Jordan P; Piliang, Melissa P; Ernstoff, Marc S; Gastman, Brian R; Billings, Steven D

2016-02-01

Sentinel lymph node biopsy is used to stage Merkel cell carcinoma, but its prognostic value has been questioned. Furthermore, predictors of outcome in sentinel lymph node positive Merkel cell carcinoma patients are poorly defined. In breast carcinoma, isolated immunohistochemically positive tumor cells have no impact, but in melanoma they are considered significant. The significance of sentinel lymph node metastasis tumor burden (including isolated tumor cells) and pattern of involvement in Merkel cell carcinoma are unknown. In this study, 64 Merkel cell carcinomas involving sentinel lymph nodes and corresponding immunohistochemical stains were reviewed and clinicopathological predictors of outcome were sought. Five metastatic patterns were identified: (1) sheet-like (n=38, 59%); (2) non-solid parafollicular (n=4, 6%); (3) sinusoidal, (n=11, 17%); (4) perivascular hilar (n=1, 2%); and (5) rare scattered parenchymal cells (n=10, 16%). At the time of follow-up, 30/63 (48%) patients had died with 21 (33%) attributable to Merkel cell carcinoma. Patients with pattern 1 metastases had poorer overall survival compared with patients with patterns 2-5 metastases (P=0.03), with 22/30 (73%) deaths occurring in pattern 1 patients. Three (10%) deaths occurred in patients showing pattern 5, all of whom were immunosuppressed. Four (13%) deaths occurred in pattern 3 patients and 1 (3%) death occurred in a pattern 2 patient. In multivariable analysis, the number of positive sentinel lymph nodes (1 or 2 versus >2, P<0.0001), age (<70 versus ≥70, P=0.01), sentinel lymph node metastasis pattern (patterns 2-5 versus 1, P=0.02), and immune status (immunocompetent versus suppressed, P=0.03) were independent predictors of outcome, and could be used to stratify Stage III patients into three groups with markedly different outcomes. In Merkel cell carcinoma, the pattern of sentinel lymph node involvement provides important prognostic information and utilizing this data with other clinicopathological features facilitates risk stratification of Merkel cell carcinoma patients who may have management implications.

Kex1 protease is involved in yeast cell death induced by defective N-glycosylation, acetic acid, and chronological aging.

PubMed

Hauptmann, Peter; Lehle, Ludwig

2008-07-04

N-glycosylation in the endoplasmic reticulum is an essential protein modification and highly conserved in evolution from yeast to humans. The key step of this pathway is the transfer of the lipid-linked core oligosaccharide to the nascent polypeptide chain, catalyzed by the oligosaccharyltransferase complex. Temperature-sensitive oligosaccharyltransferase mutants of Saccharomyces cerevisiae at the restrictive temperature, such as wbp1-1, as well as wild-type cells in the presence of the N-glycosylation inhibitor tunicamycin display typical apoptotic phenotypes like nuclear condensation, DNA fragmentation, phosphatidylserine translocation, caspase-like activity, and reactive oxygen species accumulation. Since deletion of the yeast metacaspase YCA1 did not abrogate this death pathway, we postulated a different proteolytic process to be responsible. Here, we show that Kex1 protease is involved in the programmed cell death caused by defective N-glycosylation. Its disruption decreases caspase-like activity, production of reactive oxygen species, and fragmentation of mitochondria and, conversely, improves growth and survival of cells. Moreover, we demonstrate that Kex1 contributes also to the active cell death program induced by acetic acid stress or during chronological aging, suggesting that Kex1 plays a more general role in cellular suicide of yeast.

Activation of apoptosis signal-regulating kinase 1 is a key factor in paraquat-induced cell death: modulation by the Nrf2/Trx axis.

PubMed

Niso-Santano, Mireia; González-Polo, Rosa A; Bravo-San Pedro, José M; Gómez-Sánchez, Rubén; Lastres-Becker, Isabel; Ortiz-Ortiz, Miguel A; Soler, Germán; Morán, José M; Cuadrado, Antonio; Fuentes, José M

2010-05-15

Although oxidative stress is fundamental to the etiopathology of Parkinson disease, the signaling molecules involved in transduction after oxidant exposure to cell death are ill-defined, thus making it difficult to identify molecular targets of therapeutic relevance. We have addressed this question in human dopaminergic neuroblastoma SH-SY5Y cells exposed to the parkinsonian toxin paraquat (PQ). This toxin elicited a dose-dependent increase in reactive oxygen species and cell death that correlated with activation of ASK1 and the stress kinases p38 and JNK. The relevance of these kinases in channeling PQ neurotoxicity was demonstrated with the use of interference RNA for ASK1 and two well-established pharmaceutical inhibitors for JNK and p38. The toxic effect of PQ was substantially attenuated by preincubation with vitamin E, blocking ASK1 pathways and preventing oxidative stress and cell death. In a search for a physiological pathway that might counterbalance PQ-induced ASK1 activation, we analyzed the role of the transcription factor Nrf2, master regulator of redox homeostasis, and its target thioredoxin (Trx), which binds and inhibits ASK1. Trx levels were undetectable in Nrf2-deficient mouse embryo fibroblasts (MEFs), whereas they were constitutively high in Keap1-deficient MEFs as well as in SH-SY5Y cells treated with sulforaphane (SFN). Consistent with these data, Nrf2-deficient MEFs were more sensitive and Keap1-deficient MEFs and SH-SY5Y cells incubated with SFN were more resistant to PQ-induced cell death. This study identifies ASK1/JNK and ASK1/p38 as two critical pathways involved in the activation of cell death under oxidative stress conditions and identifies the Nrf2/Trx axis as a new target to block these pathways and protect from oxidant exposure such as that found in Parkinson and other neurodegenerative diseases. Copyright 2010 Elsevier Inc. All rights reserved.

Chronic inhibition of glycogen synthase kinase-3 protects against rotenone-induced cell death in human neuron-like cells by increasing BDNF secretion.

PubMed

Giménez-Cassina, Alfredo; Lim, Filip; Díaz-Nido, Javier

2012-12-07

Mitochondrial dysfunction is a common feature of many neurodegenerative disorders. Likewise, activation of glycogen synthase kinase-3 (GSK-3) has been proposed to play an important role in neurodegeneration. This multifunctional protein kinase is involved in a number of cellular functions and we previously showed that chronic inhibition of GSK-3 protects neuronal cells against mitochondrial dysfunction-elicited cell death, through a mechanism involving increased glucose metabolism and the translocation of hexokinase II (HKII) to mitochondria. Here, we sought to gain deeper insight into the molecular basis of this neuroprotection. We found that chronic inhibition of GSK-3, either genetically or pharmacologically, elicited a marked increase in brain-derived neurotrophic factor (BDNF) secretion, which in turn conferred resistance to mitochondrial dysfunction through subcellular re-distribution of HKII. These results define a molecular pathway through which chronic inhibition of GSK-3 may protect neuronal cells from death. Moreover, they highlight the potential benefits of enhanced neurotrophic factor secretion as a therapeutic approach to treat neurodegenerative diseases. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Deciphering early events involved in hyperosmotic stress-induced programmed cell death in tobacco BY-2 cells

PubMed Central

Monetti, Emanuela; Kadono, Takashi; Bouteau, François

2014-01-01

Hyperosmotic stresses represent one of the major constraints that adversely affect plants growth, development, and productivity. In this study, the focus was on early responses to hyperosmotic stress- (NaCl and sorbitol) induced reactive oxygen species (ROS) generation, cytosolic Ca2+ concentration ([Ca2+]cyt) increase, ion fluxes, and mitochondrial potential variations, and on their links in pathways leading to programmed cell death (PCD). By using BY-2 tobacco cells, it was shown that both NaCl- and sorbitol-induced PCD seemed to be dependent on superoxide anion (O2·–) generation by NADPH-oxidase. In the case of NaCl, an early influx of sodium through non-selective cation channels participates in the development of PCD through mitochondrial dysfunction and NADPH-oxidase-dependent O2·– generation. This supports the hypothesis of different pathways in NaCl- and sorbitol-induced cell death. Surprisingly, other shared early responses, such as [Ca2+]cyt increase and singlet oxygen production, do not seem to be involved in PCD. PMID:24420571

LAMP-2 mediates oxidative stress-dependent cell death in Zn2+-treated lung epithelium cells.

PubMed

Qin, Xia; Zhang, Jun; Wang, Bin; Xu, Ge; Zou, Zhen

2017-06-17

Zinc is an essential element for the biological system. However, excessive exogenous Zn 2+ would disrupt cellular Zn 2+ homeostasis and cause toxicity. In particular, Zinc salts or ZnO nanoparticles exposure could induce respiratory injury. Although previous studies have indicated that organelle damage (including mitochondria or lysosomes) and reactive oxygen species (ROS) production are involved in Zn 2+ -induced toxicity, the interplay between mitochondria/lysosomes damage and ROS production is obscure. Herein, we demonstrated that Zn 2+ could induce deglycosylation of lysosome-associated membrane protein 1 and 2 (LAMP-1 and LAMP-2), which primarily locate in late endosomes/lysosomes, in A549 lung epithelium cells. Intriguingly, LAMP-2 knockdown further aggravated Zn 2+ -mediated ROS production and cell death, indicating LAMP-2 (not LAMP-1) was involved in Zn 2+ -induced toxicity. Our results provide a new insight that LAMP-2 contributes to the ROS clearance and cell death induced by Zn 2+ treatment, which would help us to get a better understanding of Zn 2+ -induced toxicity in respiratory system. Copyright © 2017 Elsevier Inc. All rights reserved.

Endonuclease G mediates α-synuclein cytotoxicity during Parkinson's disease.

PubMed

Büttner, Sabrina; Habernig, Lukas; Broeskamp, Filomena; Ruli, Doris; Vögtle, F Nora; Vlachos, Manolis; Macchi, Francesca; Küttner, Victoria; Carmona-Gutierrez, Didac; Eisenberg, Tobias; Ring, Julia; Markaki, Maria; Taskin, Asli Aras; Benke, Stefan; Ruckenstuhl, Christoph; Braun, Ralf; Van den Haute, Chris; Bammens, Tine; van der Perren, Anke; Fröhlich, Kai-Uwe; Winderickx, Joris; Kroemer, Guido; Baekelandt, Veerle; Tavernarakis, Nektarios; Kovacs, Gabor G; Dengjel, Jörn; Meisinger, Chris; Sigrist, Stephan J; Madeo, Frank

2013-11-27

Malfunctioning of the protein α-synuclein is critically involved in the demise of dopaminergic neurons relevant to Parkinson's disease. Nonetheless, the precise mechanisms explaining this pathogenic neuronal cell death remain elusive. Endonuclease G (EndoG) is a mitochondrially localized nuclease that triggers DNA degradation and cell death upon translocation from mitochondria to the nucleus. Here, we show that EndoG displays cytotoxic nuclear localization in dopaminergic neurons of human Parkinson-diseased patients, while EndoG depletion largely reduces α-synuclein-induced cell death in human neuroblastoma cells. Xenogenic expression of human α-synuclein in yeast cells triggers mitochondria-nuclear translocation of EndoG and EndoG-mediated DNA degradation through a mechanism that requires a functional kynurenine pathway and the permeability transition pore. In nematodes and flies, EndoG is essential for the α-synuclein-driven degeneration of dopaminergic neurons. Moreover, the locomotion and survival of α-synuclein-expressing flies is compromised, but reinstalled by parallel depletion of EndoG. In sum, we unravel a phylogenetically conserved pathway that involves EndoG as a critical downstream executor of α-synuclein cytotoxicity.

Multimodal immunogenic cancer cell death as a consequence of anticancer cytotoxic treatments

PubMed Central

Inoue, H; Tani, K

2014-01-01

Apoptotic cell death generally characterized by a morphologically homogenous entity has been considered to be essentially non-immunogenic. However, apoptotic cancer cell death, also known as type 1 programmed cell death (PCD), was recently found to be immunogenic after treatment with several chemotherapeutic agents and oncolytic viruses through the emission of various danger-associated molecular patterns (DAMPs). Extensive studies have revealed that two different types of immunogenic cell death (ICD) inducers, recently classified by their distinct actions in endoplasmic reticulum (ER) stress, can reinitiate immune responses suppressed by the tumor microenvironment. Indeed, recent clinical studies have shown that several immunotherapeutic modalities including therapeutic cancer vaccines and oncolytic viruses, but not conventional chemotherapies, culminate in beneficial outcomes, probably because of their different mechanisms of ICD induction. Furthermore, interests in PCD of cancer cells have shifted from its classical form to novel forms involving autophagic cell death (ACD), programmed necrotic cell death (necroptosis), and pyroptosis, some of which entail immunogenicity after anticancer treatments. In this review, we provide a brief outline of the well-characterized DAMPs such as calreticulin (CRT) exposure, high-mobility group protein B1 (HMGB1), and adenosine triphosphate (ATP) release, which are induced by the morphologically distinct types of cell death. In the latter part, our review focuses on how emerging oncolytic viruses induce different forms of cell death and the combinations of oncolytic virotherapies with further immunomodulation by cyclophosphamide and other immunotherapeutic modalities foster dendritic cell (DC)-mediated induction of antitumor immunity. Accordingly, it is increasingly important to fully understand how and which ICD inducers cause multimodal ICD, which should aid the design of reasonably multifaceted anticancer modalities to maximize ICD-triggered antitumor immunity and eliminate residual or metastasized tumors while sparing autoimmune diseases. PMID:23832118

Physician Education: Apoptosis.

PubMed

Kataoka; Tsuruo

1996-01-01

We have come to understand apoptosis as not merely a single form of cell death, but as a fundamental theme in cell biology that has far-reaching implications in the fields of physiology and pathology. At the present time, however, the mechanism of apoptosis is not clearly understood, as research into apoptosis is still at the initial stages. Nevertheless, the links between apoptosis and a variety of pathological conditions are gradually becoming clearer. In this article, we will provide a simple explanation of apoptosis and its mechanism as a novel concept of cell death and discuss the way in which apoptosis has been linked to a variety of pathological conditions. WHAT IS APOPTOSIS?: In normal tissue, cells that are no longer needed are rapidly eliminated without affecting the overall function of the tissue. In this process cells undergo an active and spontaneous suicide called programmed cell death. In fact, the majority of physiological cell deaths take the form of apoptosis. The word apoptosis is used, in contrast to necrosis, to describe the situation in which a cell actively pursues a course toward death upon receiving certain stimuli [1]. The morphological changes of apoptosis found in most cell types first involve contraction in cell volume and condensation of the nucleus. When this happens the intracellular organelles such as the mitochondria retain their normal morphology. As apoptosis proceeds, blebbing of the plasma membrane occurs, and the nucleus becomes fragmented. Finally, the cell itself fragments to form apoptotic bodies that are engulfed by nearby phagocytes. With respect to biochemical changes, it is known that the chromosomes become fragmented into nucleosome units, and DNA forms characteristic ladder patterns when subjected to agarose gel electrophoresis. MECHANISM OF APOPTOSIS: It has been reported that apoptosis is induced in various cells by many kinds of irritations, but the precise mechanism is still unclear. Cell injuries that induce apoptosis include those that cause DNA damage such as radiation and anticancer drugs, those that are mediated by the TNF receptor and Fas receptor (the so-called "death signal receptors"), and the deprivation of cytokines that supply survival signals such as IL-3 and erythropoietin. The tumor suppressor gene p53 plays a very important role in apoptosis induced by damage to DNA. This has been demonstrated by studying resistance to apoptosis of cells derived from p53 knockout mice [2]. Other than the irritations that induce apoptosis, molecules that have been strongly implicated as major players in the drama of apoptosis include the Bcl-2 family proteins and the IL-1 converting enzyme (ICE) and its homolog proteases (caspase family). Both groups of proteins show homology with proteins that affect cell death in nematodes. It is believed that molecules that contribute to cell death have been well conserved in multicellular organisms all the way from the relatively primitive nematodes to mammals including humans. It was discovered that Bcl-2 suppressed apoptosis induced in IL-3 dependent cells by deprivation of IL-3 [3]. It has since become the gene around which apoptosis research revolves. Recently, it has become clear that cell death involving the Bcl-2 protein is under the control of similar proteins from the same family [4]. It is interesting that the phenomenon of cell death may be regulated by the balance of the molecules involved in it. APOPTOSIS ABNORMALITIES AND DISEASE: Physiological cell death plays a major role in the growth and permanent maintenance of the human body [5]. In the process of forming the nervous system, neurons that do not form proper connections die. Physiological cell death also accompanies the removal of virus-infected cells by cytotoxic T cells, the elimination of autoreactive immune cells, the formation of the gut, the reconstitution of cartilage and bone, etc. When physiological cell death that normally should occur is inhibited, inappropriate physiological cell death may occur that is harmful to the body and forms the basis of disease. For example, in patients with neural degenerative disorders such as Alzheimer's disease and Parkinson's disease, we can find premature cell death in a particular subset of neurons. The death of T cells in AIDS patients is also a form of physiological cell death. Inhibition of cell death in the immune system enables the survival of autoreactive B cells and T cells, and is therefore a cause of autoimmune disorders. Apoptosis has been particularly linked to cancer. Normal cells are programmed for death if they are subjected to many types of non-physiological stress such as anticancer drugs or radiation, if they become isolated from surrounding cells and are unable to receive their tissue-specific survival signals [6], or if oncogenes are expressed haphazardly [7]. On the other hand, it is believed that the ability to survive is enhanced in transformed cancer cells because they are more resistant to apoptosis, they exhibit resistance to anticancer drugs, they are no longer dependent on survival signals, and they can metastasize. Therefore, the cancer progresses as the cancer cells maintain the proliferative superiority they acquire from their oncogenes. In other words, when cancer cells become resistant to apoptosis, they become resistant to treatment, metastasize, and proliferate destructively. The concept that the malignancy of cancer is due to its resistance to apoptosis is a relatively new one and is worthy of further study.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Asare, Nana; Landvik, Nina E.; Lagadic-Gossmann, Dominique

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 thatmore » 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.« less

Calcium and mitochondrial metabolism in ceramide-induced cardiomyocyte death.

PubMed

Parra, Valentina; Moraga, Francisco; Kuzmicic, Jovan; López-Crisosto, Camila; Troncoso, Rodrigo; Torrealba, Natalia; Criollo, Alfredo; Díaz-Elizondo, Jessica; Rothermel, Beverly A; Quest, Andrew F G; Lavandero, Sergio

2013-08-01

Ceramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulate numerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca(2+) overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca(2+) levels, mitochondrial function and cardiomyocyte death. Our data show that C2-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca(2+) influx, mitochondrial network fragmentation and loss of the mitochondrial Ca(2+) buffer capacity. These biochemical events increase cytosolic Ca(2+) levels and trigger cardiomyocyte death via the activation of calpains. Copyright © 2013 Elsevier B.V. All rights reserved.

Constitutive Activation of PINK1 Protein Leads to Proteasome-mediated and Non-apoptotic Cell Death Independently of Mitochondrial Autophagy*

PubMed Central

Akabane, Shiori; Matsuzaki, Kohei; Yamashita, Shun-ichi; Arai, Kana; Okatsu, Kei; Kanki, Tomotake; Matsuda, Noriyuki; Oka, Toshihiko

2016-01-01

Phosphatase and tensin homolog-induced putative kinase 1 (PINK1), a Ser/Thr kinase, and PARKIN, a ubiquitin ligase, are causal genes for autosomal recessive early-onset parkinsonism. Multiple lines of evidence indicate that PINK1 and PARKIN cooperatively control the quality of the mitochondrial population via selective degradation of damaged mitochondria by autophagy. Here, we report that PINK1 and PARKIN induce cell death with a 12-h delay after mitochondrial depolarization, which differs from the time profile of selective autophagy of mitochondria. This type of cell death exhibited definite morphologic features such as plasma membrane rupture, was insensitive to a pan-caspase inhibitor, and did not involve mitochondrial permeability transition. Expression of a constitutively active form of PINK1 caused cell death in the presence of a pan-caspase inhibitor, irrespective of the mitochondrial membrane potential. PINK1-mediated cell death depended on the activities of PARKIN and proteasomes, but it was not affected by disruption of the genes required for autophagy. Furthermore, fluorescence and electron microscopic analyses revealed that mitochondria were still retained in the dead cells, indicating that PINK1-mediated cell death is not caused by mitochondrial loss. Our findings suggest that PINK1 and PARKIN play critical roles in selective cell death in which damaged mitochondria are retained, independent of mitochondrial autophagy. PMID:27302064

Inducible nitric oxide synthase in T cells regulates T cell death and immune memory

PubMed Central

Vig, Monika; Srivastava, Smita; Kandpal, Usha; Sade, Hadassah; Lewis, Virginia; Sarin, Apurva; George, Anna; Bal, Vineeta; Durdik, Jeannine M.; Rath, Satyajit

2004-01-01

The progeny of T lymphocytes responding to immunization mostly die rapidly, leaving a few long-lived survivors functioning as immune memory. Thus, control of this choice of death versus survival is critical for immune memory. There are indications that reactive radicals may be involved in this death pathway. We now show that, in mice lacking inducible nitric oxide synthase (iNOS), higher frequencies of both CD4 and CD8 memory T cells persist in response to immunization, even when iNOS+/+ APCs are used for immunization. Postactivation T cell death by neglect is reduced in iNOS–/– T cells, and levels of the antiapoptotic proteins Bcl-2 and Bcl-xL are increased. Inhibitors of the iNOS-peroxynitrite pathway also enhance memory responses and block postactivation death by neglect in both mouse and human T cells. However, early primary immune responses are not enhanced, which suggests that altered survival, rather than enhanced activation, is responsible for the persistent immunity observed. Thus, in primary immune responses, iNOS in activated T cells autocrinely controls their susceptibility to death by neglect to determine the level of persisting CD4 and CD8 T cell memory, and modulation of this pathway can enhance the persistence of immune memory in response to vaccination. PMID:15199408

Tributyltin-induced apoptosis requires glycolytic adenosine trisphosphate production.

PubMed

Stridh, H; Fava, E; Single, B; Nicotera, P; Orrenius, S; Leist, M

1999-10-01

The toxicity of tributyltin chloride (TBT) involves Ca(2+) overload, cytoskeletal damage, and mitochondrial failure leading to cell death by apoptosis or necrosis. Here, we examined whether the intracellular ATP level modulates the mode of cell death after exposure to TBT. When Jurkat cells were energized by the mitochondrial substrate, pyruvate, low concentrations of TBT (1-2 microM) triggered an immediate depletion of intracellular ATP followed by necrotic death. When ATP levels were maintained by the addition of glucose, the mode of cell death was typically apoptotic. Glycolytic ATP production was required for apoptosis at two distinct steps. First, maintenance of adequate ATP levels accelerated the decrease of mitochondrial membrane potential, and the release of the intermembrane proteins adenylate kinase and cytochrome c from mitochondria. A possible role of the adenine nucleotide exchanger in this first ATP-dependent step is suggested by experiments performed with the specific inhibitor, bongkrekic acid. This substance delayed cytochrome c release in a manner similar to that caused by ATP depletion. Second, caspase activation following cytochrome c release was only observed in ATP-containing cells. Bcl-2 had only a minor effect on TBT-triggered caspase activation or cell death. We conclude that intracellular ATP concentrations control the mode of cell death in TBT-treated Jurkat cells at both the mitochondrial and caspase activation levels.

Lysophosphatidylcholine-induced cytotoxicity in osteoblast-like MG-63 cells: involvement of transient receptor potential vanilloid 2 (TRPV2) channels.

PubMed

Fallah, Abdallah; Pierre, Rachel; Abed, Elie; Moreau, Robert

2013-01-01

Epidemiological studies indicate that patients suffering from atherosclerosis are predisposed to develop osteoporosis. Accordingly, atherogenic determinants such as oxidized low density lipoprotein (OxLDL) particles have been shown to alter bone cell functions. In this work, we investigated the cytotoxicity of lysophosphatidylcholine (lysoPC), a major phospholipid component generated upon LDL oxidation, on bone-forming MG-63 osteoblast-like cells. Cell viability was reduced by lysoPC in a concentration-dependent manner with a LC50 of 18.7±0.7 μM. LysoPC-induced cell death was attributed to induction of both apoptosis and necrosis. Since impairment of intracellular calcium homeostasis is often involved in mechanism of cell death, we determined the involvement of calcium in lysoPC-induced cytotoxicity. LysoPC promoted a rapid and transient increase in intracellular calcium attributed to mobilization from calcium stores, followed by a sustained influx. Intracellular calcium mobilization was associated to phospholipase C (PLC)-dependent mobilization of calcium from the endoplasmic reticulum since inhibition of PLC or calcium depletion of reticulum endoplasmic with thapsigargin prevented the calcium mobilization. The calcium influx induced by lysoPC was abolished by inhibition of transient receptor potential vanilloid (TRPV) channels with ruthenium red whereas gadolinium, which inhibits canonical TRP (TRPC) channels, was without effect. Accordingly, expression of TRPV2 and TRPV4 were shown in MG-63 cells. The addition of TRPV2 inhibitor Tranilast in the incubation medium prevent the calcium influx triggered by lysoPC and reduced lysoPC-induced cytotoxicity whereas TRPV4 inhibitor RN 1734 was without effect, which confirms the involvement of TRPV2 activation in lysoPC-induced cell death.

Different toxic effects of YTX in tumor K-562 and lymphoblastoid cell lines

PubMed Central

Fernández-Araujo, Andrea; Sánchez, Jon A.; Alfonso, Amparo; Vieytes, Mercedes R.; Botana, Luis M.

2015-01-01

Yessotoxin (YTX) modulates cellular phosphodiesterases (PDEs). In this regard, opposite effects had been described in the tumor model K-562 cell line and fresh human lymphocytes in terms of cell viability, cyclic adenosine 3',5'-cyclic monophosphate (cAMP) production and protein expression after YTX treatment. Studies in depth of the pathways activated by YTX in K-562 cell line, have demonstrated the activation of two different cell death types, apoptosis, and autophagy after 24 and 48 h of treatment, respectively. Furthermore, the key role of type 4A PDE (PDE4A) in both pathways activated by YTX was demonstrated. Therefore, taking into account the differences between cellular lines and fresh cells, a study of cell death pathways activated by YTX in a non-tumor cell line with mitotic activity, was performed. The cellular model used was the lymphoblastoid cell line that represents a non-tumor model with normal apoptotic and mitotic machinery. In this context, cell viability and cell proliferation, expression of proteins involved in cell death activated by YTX and mitochondrial mass, were studied after the incubation with the toxin. Opposite to the tumor model, no cell death activation was observed in lymphoblastoid cell line in the presence of YTX. In this sense, variations in apoptosis hallmarks were not detected in the lymphoblastoid cell line after YTX incubation, whereas this type I of programmed cell death was observed in K-562 cells. On the other hand, autophagy cell death was triggered in this cellular line, while other autophagic process is suggested in lymphoblastoid cells. These YTX effects are related to PDE4A in both cellular lines. In addition, while cell death is triggered in K-562 cells after YTX treatment, in lymphoblastoid cells the toxin stops cellular proliferation. These results point to YTX as a specific toxic compound of tumor cells, since in the non-tumor lymphoblastoid cell line, no cell death hallmarks are observed. PMID:26136685

The sirtuin 1/2 inhibitor tenovin-1 induces a nonlinear apoptosis-inducing factor-dependent cell death in a p53 null Ewing's sarcoma cell line.

PubMed

Marx, Christian; Marx-Blümel, Lisa; Lindig, Nora; Thierbach, René; Hoelzer, Doerte; Becker, Sabine; Wittig, Susan; Lehmann, Roland; Slevogt, Hortense; Heinzel, Thorsten; Wang, Zhao-Qi; Beck, James F; Sonnemann, Jürgen

2018-06-01

The sirtuin 1/2 inhibitor tenovin-1 activates p53 and may have potential in the management of cancer. Here, we investigated the responsiveness of Ewing's sarcoma cells to tenovin-1. We examined its effects in two Ewing's sarcoma cell lines with different p53 status, i.e. in p53 wild-type and p53 null cells. Effects were assessed by flow cytometric analyses of cell death, mitochondrial membrane depolarization and reactive oxygen species (ROS) generation, by caspase 3/7 activity measurement, by mRNA expression profiling and by immunoblotting. Tenovin-1 elicited caspase-mediated cell death in p53 wild-type cells, but caspase-independent cell death in p53 null cells. Remarkably, it induced a nonlinear concentration response in the latter: low concentrations of tenovin-1 were much more effective than were higher concentrations. Tenovin-1's effects in p53 null cells involved gene expression changes of Bcl-2 family members, mitochondrial membrane depolarization, nuclear translocation of apoptosis-inducing factor, ROS formation and DNA damage; all these effects followed a bell-shaped pattern. In conclusion, our results provide new insights into tenovin-1's mode of action by demonstrating that it can induce different pathways of cell death.

c-FLIP is involved in erythropoietin-mediated protection of erythroid-differentiated cells from TNF-alpha-induced apoptosis.

PubMed

Vittori, Daniela; Vota, Daiana; Callero, Mariana; Chamorro, María E; Nesse, Alcira

2010-05-04

The TNF-alpha (tumour necrosis factor) affects a wide range of biological activities, such as cell proliferation and apoptosis. Cell life or death responses to this cytokine might depend on cell conditions. This study focused on the modulation of factors that would affect the sensitivity of erythroid-differentiated cells to TNF-alpha. Hemin-differentiated K562 cells showed higher sensitivity to TNF-induced apoptosis than undifferentiated cells. At the same time, hemin-induced erythroid differentiation reduced c-FLIP (cellular FLICE-inhibitory protein) expression. However, this negative effect was prevented by prior treatment with Epo (erythropoietin), which allowed the cell line to maintain c-FLIP levels. On the other hand, erythroid-differentiated UT-7 cells - dependent on Epo for survival - showed resistance to TNF-alpha pro-apoptotic action. Only after the inhibition of PI3K (phosphatidylinositol-3 kinase)-mediated pathways, which was accompanied by negative c-FLIP modulation and increased erythroid differentiation, were UT-7 cells sensitive to TNF-alpha-triggered apoptosis. In summary, erythroid differentiation might deregulate the balance between growth promotion and death signals induced by TNF-alpha, depending on cell type and environmental conditions. The role of c-FLIP seemed to be critical in the protection of erythroid-differentiated cells from apoptosis or in the determination of their sensitivity to TNF-mediated programmed cell death. Epo, which for the first time was found to be involved in the prevention of c-FLIP down-regulation, proved to have an anti-apoptotic effect against the pro-inflammatory factor. The identification of signals related to cell life/death switching would have significant implications in the control of proliferative diseases and would contribute to the understanding of mechanisms underlying the anaemia associated with inflammatory processes.

Interferon-alpha and interferon-gamma modulate Fas-mediated apoptosis in mitomycin-C-resistant human Tenon's fibroblasts.

PubMed

Wang, Xiao Yang; Crowston, Jonathan G; White, Andrew J R; Zoellner, Hans; Healey, Paul R

2014-08-01

The aim of the study was to investigate, using a native mitomycin-C-resistant human Tenon's fibroblast cell line, the possibility that interferon-alpha and gamma could be used with Fas agonists as an alternative anti-fibrotic strategy to mitomycin-C in trabeculectomy. A clinically resistant and in vitro verified mitomycin-C-resistant human Tenon's fibroblast cell line was pretreated with interferon-alpha and interferon-gamma for 48 h before stimulation with an agonistic Fas antibody (CH11) for 2 days to induce cell death. Cell death assays were undertaken. Changes in apoptosis-related proteins were determined by flow cytometry and Western blot. Pretreatment with interferon-alpha or interferon-gamma for 48 h increased Fas, Fas-associated protein with death domain and caspase-8 expression. Protein expression was further increased by combined exposure to interferon-alpha and gamma. Pretreatment with cytokines had no effect on Fas-L and Bcl-2. Interferon-alpha alone did not change the rate of induced cell death. A combination of interferon-alpha and gamma synergistically increased the sensitivity of mitomycin-C-resistant human Tenon's fibroblast cell line to induced cell death. An antagonistic anti-Fas antibody (ZB4) completely blocked induced cell death. Broad caspase inhibitors specific for caspases-8 and -3 reduced induced deaths in interferon pretreated mitomycin-C-resistant human Tenon's fibroblast cell line in a dose-dependent manner. Interferon-alpha and interferon-gamma render mitomycin-C-resistant human Tenon's fibroblast cell line sensitive to Fas-mediated apoptosis. The mechanism involves increased death-inducing signalling complex formation by upregulation of Fas, Fas-associated protein with death domain and caspase-8 expression. © 2013 Royal Australian and New Zealand College of Ophthalmologists.

Fasting boosts sensitivity of human skin melanoma to cisplatin-induced cell death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Antunes, Fernanda; Corazzari, Marco; National Institute for Infectious Diseases IRCCS “Lazzaro Spallanzani”

Melanoma is one of leading cause of tumor death worldwide. Anti-cancer strategy includes combination of different chemo-therapeutic agents as well as radiation; however these treatments have limited efficacy and induce significant toxic effects on healthy cells. One of most promising novel therapeutic approach to cancer therapy is the combination of anti-cancer drugs with calorie restriction. Here we investigated the effect Cisplatin (CDDP), one of the most potent chemotherapeutic agent used to treat tumors, in association with fasting in wild type and mutated BRAF{sup V600E} melanoma cell lines. Here we show that nutrient deprivation can consistently enhance the sensitivity of tumormore » cells to cell death induction by CDDP, also of those malignancies particularly resistant to any treatment, such as oncogenic BRAF melanomas. Mechanistic studies revealed that the combined therapy induced cell death is characterized by ROS accumulation and ATF4 in the absence of ER-stress. In addition, we show that autophagy is not involved in the enhanced sensitivity of melanoma cells to combined CDDP/EBSS-induced apoptosis. While, the exposure to 2-DG further enhanced the apoptotic rate observed in SK Mel 28 cells upon treatment with both CDDP and EBSS. - Highlights: • Calorie restriction associated to chemo-therapeutic drugs enhance cell death induction in many resistant malignancies • Cisplatin in association with starvation significantly increases cell death also in those high resistant melanoma cells bearing BRAF mutations • Combined treatment also including 2-DG results in similar cell death levels in both wild type and mutated BRAF cells.« less

Patterns of cell death in the embryonic antenna of the grasshopper Schistocerca gregaria.

PubMed

Boyan, George; Graf, Philip; Ehrhardt, Erica

2018-03-01

We have investigated the pattern of apoptosis in the antennal epithelium during embryonic development of the grasshopper Schistocerca gregaria. The molecular labels lachesin and annulin reveal that the antennal epithelium becomes subdivided into segment-like meristal annuli within which sensory cell clusters later differentiate. To determine whether apoptosis is involved in the development of such sensory cell clusters, we examined the expression pattern of the cell death labels acridine orange and TUNEL in the epithelium. We found stereotypic, age-dependent, wave-like patterns of cell death in the antenna. Early in embryogenesis, apoptosis is restricted to the most basal meristal annuli but subsequently spreads to encompass almost the entire antenna. Cell death then declines in more basal annuli and is only found in the tip region later in embryogenesis. Apoptosis is restricted throughout to the midregion of a given annulus and away from its border with neighboring annuli, arguing against a causal role in annular formation. Double-labeling for cell death and sensory cell differentiation reveals apoptosis occurring within bands of differentiating sensory cell clusters, matching the meristal organization of the apical antenna. Examination of the individual epithelial lineages which generate sensory cells reveals that apoptosis begins peripherally within a lineage and with age expands to encompass the differentiated sensory cell at the base. We conclude that complete lineages can undergo apoptosis and that the youngest cells in these lineages appear to die first, with the sensory neuron dying last. Lineage-based death in combination with cell death patterns in different regions of the antenna may contribute to odor-mediated behaviors in the grasshopper.

Apoptosis inducing factor gene depletion inhibits zearalenone-induced cell death in a goat Leydig cell line.

PubMed

Yang, Diqi; Jiang, Tingting; Lin, Pengfei; Chen, Huatao; Wang, Lei; Wang, Nan; Zhao, Fan; Tang, Keqiong; Zhou, Dong; Wang, Aihua; Jin, Yaping

2017-01-01

Zearalenone (ZEA) is a contaminant of human food and animal feedstuffs that causes health hazards. However, the signal pathways underlying ZEA toxicity remain elusive. The aims of this study were to determine which pathways are involved in ZEA-induced cell death and investigate the effect of apoptosis inducing factor (AIF) on cell death during ZEA treatment in the immortalized goat Leydig cell line hTERT-GLC. This study showed that ZEA-induced cell death in hTERT-GLCs works via endoplasmic reticulum (ER) stress, the caspase-dependent pathway, the caspase-independent pathway and autophagy. Recombinant lentiviral vectors were constructed to silence AIF expression in hTERT-GLCs. Flow cytometry results showed that knockdown of AIF diminished ZEA-induced cell apoptosis in hTERT-GLCs. Furthermore, we found AIF depletion down-regulated phosphoIRE1α, GRP78, CHOP and promoted the switch of LC3-I to LC3-II. Therefore, ZEA induces cytotoxicity in hTERT-GLCs via different pathways, while AIF-mediated signaling plays a critical role in ZEA-induced cell death in hTERT-GLCs. Copyright © 2016 Elsevier Inc. All rights reserved.

Oxidative stress-induced necrotic cell death via mitochondira-dependent burst of reactive oxygen species.

PubMed

Choi, Kyungsun; Kim, Jinho; Kim, Gyung W; Choi, Chulhee

2009-11-01

Oxidative stress is deeply involved in various brain diseases, including neurodegenerative diseases, stroke, and ischemia/reperfusion injury. Mitochondria are thought to be the target and source of oxidative stress. We investigated the role of mitochondria in oxidative stress-induced necrotic neuronal cell death in a neuroblastoma cell line and a mouse model of middle cerebral artery occlusion. The exogenous administration of hydrogen peroxide was used to study the role of oxidative stress on neuronal cell survival and mitochondrial function in vitro. Hydrogen peroxide induced non-apoptotic neuronal cell death in a c-Jun N-terminal kinase- and poly(ADP-ribosyl) polymerase-dependent manner. Unexpectedly, hydrogen peroxide treatment induced transient hyperpolarization of the mitochondrial membrane potential and a subsequent delayed burst of endogenous reactive oxygen species (ROS). The inhibition of mitochondrial hyperpolarization by diphenylene iodonium or rotenone, potent inhibitors of mitochondrial respiratory chain complex I, resulted in reduced ROS production and subsequent neuronal cell death in vitro and in vivo. The inhibition of mitochondrial hyperpolarization can protect neuronal cells from oxidative stress-induced necrotic cell death, suggesting a novel method of therapeutic intervention in oxidative stress-induced neurological disease.

Involvement of Alveolar Epithelial Cell Necroptosis in IPF Pathogenesis.

PubMed

Lee, Ji-Min; Yoshida, Masahiro; Kim, Mi-So; Lee, June-Hyuk; Baek, Ae-Rin; Jang, An Soo; Kim, Do Jin; Minagawa, Shunsuke; Chin, Su Sie; Park, Choon-Sik; Araya, Jun; Kuwano, Kazuyoshi; Park, Sung Woo

2018-02-14

Alveolar epithelial cell (AEC) injury leading to cell death is involved in the process of fibrosis development during idiopathic pulmonary fibrosis (IPF). Among regulated/programmed cell death, the excessive apoptosis of AECs has been widely implicated in IPF pathogenesis. Necroptosis is a type of regulated/programmed necrosis. A multiprotein complex composed of receptor-interacting protein kinase-1 and -3 (RIPK1/3) plays a key regulatory role in initiating necroptosis. Although necroptosis participates in disease pathogeneses through the release of damage-associated molecular patterns (DAMPs), its association with IPF progression remains elusive. In this study, we attempted to illuminate the involvement of RIPK3-regulated necroptosis in IPF pathogenesis. IPF lung tissues were used to detect necroptosis, and the role of RIPK3 was determined using cell culturing models of AECs. Lung fibrosis models of bleomycin (BLM) treatment were also used. RIPK3 expression levels were increased in IPF lungs and both apoptosis and necroptosis were detected mainly in AECs. Necrostatin-1 and RIPK3 knockdown experiments in AECs revealed the participation of necroptosis in BLM and hydrogen peroxide-induced cell death. BLM treatment induced RIPK3 expression in AECs and increased High Mobility Group Box 1 (HMGB1) and interleukin 1β (IL-1β) levels in mouse lungs. The efficient attenuation of BLM-induced lung inflammation and fibrosis was determined in RIPK3 knockout mice and by necrostatin-1 with a concomitant reduction in HMGB1 and IL-1β. RIPK3-regulated necroptosis in AECs is involved in the mechanism of lung fibrosis development through the release of DAMPs as part of the pathogenic sequence of IPF.

Tritrichomonas foetus Induces Apoptotic Cell Death in Bovine Vaginal Epithelial Cells

PubMed Central

Singh, B. N.; Lucas, J. J.; Hayes, G. R.; Kumar, Ish; Beach, D. H.; Frajblat, Marcel; Gilbert, R. O.; Sommer, U.; Costello, C. E.

2004-01-01

Tritrichomonas foetus is a serious veterinary pathogen, causing bovine trichomoniasis, a sexually transmitted disease leading to infertility and abortion. T. foetus infects the mucosal surfaces of the reproductive tract. Infection with T. foetus leads to apoptotic cell death of bovine vaginal epithelial cells (BVECs) in culture. An affinity-purified cysteine protease (CP) fraction yielding on sodium dodecyl sulfate-polyacrylamide gel electrophoresis a single band with an apparent molecular mass of 30 kDa (CP30) also induces BVEC apoptosis. Treatment of CP30 with the protease inhibitors TLCK (Nα-p-tosyl-l-lysine chloromethyl ketone) and E-64 [l-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane] greatly reduces induction of BVEC apoptosis. Matrix-assisted laser desorption ionization-time-of-flight MALDI-TOF mass spectrometry analysis of CP30 reveals a single peak with a molecular mass of 23.7 kDa. Mass spectral peptide sequence analysis of proteolytically digested CP30 reveals homologies to a previously reported cDNA clone, CP8 (D. J. Mallinson, J. Livingstone, K. M. Appleton, S. J. Lees, G. H. Coombs, and M. J. North, Microbiology 141:3077-3085, 1995). Induction of apoptosis is highly species specific, since the related human parasite Trichomonas vaginalis and associated purified CPs did not induce BVEC death. Fluorescence microscopy along with the Cell Death Detection ELISAPLUS assay and flow cytometry analyses were used to detect apoptotic nuclear condensation, DNA fragmentation, and changes in plasma membrane asymmetry in host cells undergoing apoptosis in response to T. foetus infection or incubation with CP30. Additionally, the activation of caspase-3 and inhibition of cell death by caspase inhibitors indicates that caspases are involved in BVEC apoptosis. These results imply that apoptosis is involved in the pathogenesis of T. foetus infection in vivo, which may have important implications for therapeutic interference with host cell death that could alter the course of the pathology in vivo. PMID:15213160

Towards a unifying, systems biology understanding of large-scale cellular death and destruction caused by poorly liganded iron: Parkinson’s, Huntington’s, Alzheimer’s, prions, bactericides, chemical toxicology and others as examples

PubMed Central

2010-01-01

Exposure to a variety of toxins and/or infectious agents leads to disease, degeneration and death, often characterised by circumstances in which cells or tissues do not merely die and cease to function but may be more or less entirely obliterated. It is then legitimate to ask the question as to whether, despite the many kinds of agent involved, there may be at least some unifying mechanisms of such cell death and destruction. I summarise the evidence that in a great many cases, one underlying mechanism, providing major stresses of this type, entails continuing and autocatalytic production (based on positive feedback mechanisms) of hydroxyl radicals via Fenton chemistry involving poorly liganded iron, leading to cell death via apoptosis (probably including via pathways induced by changes in the NF-κB system). While every pathway is in some sense connected to every other one, I highlight the literature evidence suggesting that the degenerative effects of many diseases and toxicological insults converge on iron dysregulation. This highlights specifically the role of iron metabolism, and the detailed speciation of iron, in chemical and other toxicology, and has significant implications for the use of iron chelating substances (probably in partnership with appropriate anti-oxidants) as nutritional or therapeutic agents in inhibiting both the progression of these mainly degenerative diseases and the sequelae of both chronic and acute toxin exposure. The complexity of biochemical networks, especially those involving autocatalytic behaviour and positive feedbacks, means that multiple interventions (e.g. of iron chelators plus antioxidants) are likely to prove most effective. A variety of systems biology approaches, that I summarise, can predict both the mechanisms involved in these cell death pathways and the optimal sites of action for nutritional or pharmacological interventions. PMID:20967426

bis-Dehydroxy-Curcumin triggers mitochondrial-associated cell death in human colon cancer cells through ER-stress induced autophagy.

PubMed

Basile, Valentina; Belluti, Silvia; Ferrari, Erika; Gozzoli, Chiara; Ganassi, Sonia; Quaglino, Daniela; Saladini, Monica; Imbriano, Carol

2013-01-01

The activation of autophagy has been extensively described as a pro-survival strategy, which helps to keep cells alive following deprivation of nutrients/growth factors and other stressful cellular conditions. In addition to cytoprotective effects, autophagy can accompany cell death. Autophagic vacuoles can be observed before or during cell death, but the role of autophagy in the death process is still controversial. A complex interplay between autophagy and apoptosis has come to light, taking into account that numerous genes, such as p53 and Bcl-2 family members, are shared between these two pathways. In this study we showed a potent and irreversible cytotoxic activity of the stable Curcumin derivative bis-DeHydroxyCurcumin (bDHC) on human colon cancer cells, but not on human normal cells. Autophagy is elicited by bDHC before cell death as demonstrated by increased autophagosome formation -measured by electron microscopy, fluorescent LC3 puncta and LC3 lipidation- and autophagic flux -measured by interfering LC3-II turnover. The accumulation of poly-ubiquitinated proteins and ER-stress occurred upstream of autophagy induction and resulted in cell death. Cell cycle and Western blot analyses highlighted the activation of a mitochondrial-dependent apoptosis, which involves caspase 7, 8, 9 and Cytochrome C release. Using pharmacological inhibitions and RNAi experiments, we showed that ER-stress induced autophagy has a major role in triggering bDHC-cell death. Our findings describe the mechanism through which bDHC promotes tumor selective inhibition of proliferation, providing unequivocal evidence of the role of autophagy in contrasting the proliferation of colon cancer cells.

Oxaliplatin triggers necrosis as well as apoptosis in gastric cancer SGC-7901 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Ping; Zhu, Xueping; Jin, Wei

Intrinsic apoptotic pathway is considered to be responsible for cell death induced by platinum anticancer drugs. While in this study, we found that, necrosis is an indispensable pathway besides apoptosis in oxaliplatin-treated gastric cancer SGC-7901 cells. Upon exposure to oxaliplatin, both apoptotic and necrotic features were observed. The majority of dead cells were double positive for Annexin V and propidium iodide (PI). Moreover, mitochondrial membrane potential collapsed and caspase cascades were activated. However, ultrastructural changes under transmission electron microscope, coupled with the release of cellular contents, demonstrated the rupture of the plasma membrane. Oxaliplatin administration did not stimulate reactive oxygenmore » species (ROS) production and autophagy, but elevated the protein level of Bmf. In addition, receptor interacting protein 1 (RIP1), but not receptor interacting protein 3 (RIP3) and its downstream components participated in this death process. Necrostatin-1 (Nec-1) blocked oxaliplatin-induced cell death nearly completely, whereas z-VAD-fmk could partially suppress cell death. Oxaliplatin treatment resulted in poly(ADP-ribose) polymerase-1 (PARP-1) overactivation, as indicated by the increase of poly(ADP-ribose) (PAR), which led to NAD{sup +} and ATP depletion. PARP-1 inhibitor, olaparib, could significantly block oxaliplatin-induced cell death, thus confirming that PARP-1 activation is mainly responsible for the cytotoxicity of oxaliplatin. Phosphorylation of H2AX at Ser139 and translocalization of apoptosis-inducing factor (AIF) are critical for this death process. Taken together, these results indicate that oxaliplatin can bypass canonical cell death pathways to kill gastric cancer cells, which may be of therapeutic advantage in the treatment of gastric cancer. - Highlights: • Oxaliplatin induces apoptotic and necrotic cell death. • Nec-1 can inhibit oxaliplatin-induced cell death nearly completely. • RIP3 and its downstream components are not involved in this process. • PARP-1 overactivation-mediated energy depletion, H2AX phosphorylation and AIF translocation are crucial for this cell death.« less

Sugar suppresses cell death caused by disruption of fumarylacetoacetate hydrolase in Arabidopsis.

PubMed

Zhi, Tiantian; Zhou, Zhou; Huang, Yi; Han, Chengyun; Liu, Yan; Zhu, Qi; Ren, Chunmei

2016-09-01

Sugar negatively regulates cell death resulting from the loss of fumarylacetoacetate hydrolase that catalyzes the last step in the Tyr degradation pathway in Arabidopsis . Fumarylacetoacetate hydrolase (FAH) hydrolyzes fumarylacetoacetate to fumarate and acetoacetate, the final step in the tyrosine (Tyr) degradation pathway that is essential to animals. Previously, we first found that the Tyr degradation pathway plays an important role in plants. Mutation of the SSCD1 gene encoding FAH in Arabidopsis leads to spontaneous cell death under short-day conditions. In this study, we presented that the lethal phenotype of the short-day sensitive cell death1 (sscd1) seedlings was suppressed by sugars including sucrose, glucose, fructose, and maltose in a dose-dependent manner. Real-time quantitative PCR (RT-qPCR) analysis showed the expression of Tyr degradation pathway genes homogentisate dioxygenase and maleylacetoacetate isomerase, and sucrose-processing genes cell-wall invertase 1 and alkaline/neutral invertase G, was up-regulated in the sscd1 mutant, however, this up-regulation could be repressed by sugar. In addition, a high concentration of sugar attenuated cell death of Arabidopsis wild-type seedlings caused by treatment with exogenous succinylacetone, an abnormal metabolite resulting from the loss of FAH in the Tyr degradation pathway. These results indicated that (1) sugar could suppress cell death in sscd1, which might be because sugar supply enhances the resistance of Arabidopsis seedlings to toxic effects of succinylacetone and reduces the accumulation of Tyr degradation intermediates, resulting in suppression of cell death; and (2) sucrose-processing genes cell-wall invertase 1 and alkaline/neutral invertase G might be involved in the cell death in sscd1. Our work provides insights into the relationship between sugar and sscd1-mediated cell death, and contributes to elucidation of the regulation of cell death resulting from the loss of FAH in plants.

Experimental Proof for the Role of Nonlinear Photoionization in Plasmonic Phototherapy.

PubMed

Minai, Limor; Zeidan, Adel; Yeheskely-Hayon, Daniella; Yudovich, Shimon; Kviatkovsky, Inna; Yelin, Dvir

2016-07-13

Targeting individual cells within a heterogeneous tissue is a key challenge in cancer therapy, encouraging new approaches for cancer treatment that complement the shortcomings of conventional therapies. The highly localized interactions triggered by focused laser beams promise great potential for targeting single cells or small cell clusters; however, most laser-tissue interactions often involve macroscopic processes that may harm healthy nearby tissue and reduce specificity. Specific targeting of living cells using femtosecond pulses and nanoparticles has been demonstrated promising for various potential therapeutic applications including drug delivery via optoporation, drug release, and selective cell death. Here, using an intense resonant femtosecond pulse and cell-specific gold nanorods, we show that at certain irradiation parameters cell death is triggered by nonlinear plasmonic photoionization and not by thermally driven processes. The experimental results are supported by a physical model for the pulse-particle-medium interactions. A good correlation is found between the calculated total number and energy of the generated free electrons and the observed cell death, suggesting that femtosecond photoionization plays the dominant role in cell death.

Nuclear DAMP complex-mediated RAGE-dependent macrophage cell death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Ruochan; Department of Infectious Diseases and State Key Lab of Viral Hepatitis, Xiangya Hospital, Central South University, Changsha, Hunan 410008; Fu, Sha

High mobility group box 1 (HMGB1), histone, and DNA are essential nuclear components involved in the regulation of chromosome structure and function. In addition to their nuclear function, these molecules act as damage-associated molecular patterns (DAMPs) alone or together when released extracellularly. The synergistic effect of these nuclear DNA-HMGB1-histone complexes as DAMP complexes (nDCs) on immune cells remains largely unexplored. Here, we demonstrate that nDCs limit survival of macrophages (e.g., RAW264.7 and peritoneal macrophages) but not cancer cells (e.g., HCT116, HepG2 and Hepa1-6). nDCs promote production of inflammatory tumor necrosis factor α (TNFα) release, triggering reactive oxygen species-dependent apoptosis andmore » necrosis. Moreover, the receptor for advanced glycation end products (RAGE), but not toll-like receptor (TLR)-4 and TLR-2, was required for Akt-dependent TNFα release and subsequent cell death following treatment with nDCs. Genetic depletion of RAGE by RNAi, antioxidant N-Acetyl-L-cysteine, and TNFα neutralizing antibody significantly attenuated nDC-induced cell death. These findings provide evidence supporting novel signaling mechanisms linking nDCs and inflammation in macrophage cell death. - Highlights: • Nuclear DAMP complexes (nDCs) selectively induce cell death in macrophages, but not cancer cells. • TNFα-mediated oxidative stress is required for nDC-induced death. • RAGE-mediated Akt activation is required for nDC-induced TNFα release. • Blocking RAGE and TNFα inhibits nDC-induced macrophage cell death.« less

Transcriptomic Analyses Reveal Differential Gene Expression of Immune and Cell Death Pathways in the Brains of Mice Infected with West Nile Virus and Chikungunya Virus

PubMed Central

Lim, Stephanie M.; van den Ham, Henk-Jan; Oduber, Minoushka; Martina, Eurydice; Zaaraoui-Boutahar, Fatiha; Roose, Jeroen M.; van IJcken, Wilfred F. J.; Osterhaus, Albert D. M. E.; Andeweg, Arno C.; Koraka, Penelope; Martina, Byron E. E.

2017-01-01

West Nile virus (WNV) and chikungunya virus (CHIKV) are arboviruses that are constantly (re-)emerging and expanding their territory. Both viruses often cause a mild form of disease, but severe forms of the disease can consist of neurological symptoms, most often observed in the elderly and young children, respectively, for which the mechanisms are poorly understood. To further elucidate the mechanisms responsible for end-stage WNV and CHIKV neuroinvasive disease, we used transcriptomics to compare the induction of effector pathways in the brain during the early and late stage of disease in young mice. In addition to the more commonly described cell death pathways such as apoptosis and autophagy, we also found evidence for the differential expression of pyroptosis and necroptosis cell death markers during both WNV and CHIKV neuroinvasive disease. In contrast, no evidence of cell dysfunction was observed, indicating that cell death may be the most important mechanism of disease. Interestingly, there was overlap when comparing immune markers involved in neuroinvasive disease to those seen in neurodegenerative diseases. Nonetheless, further validation studies are needed to determine the activation and involvement of these effector pathways at the end stage of disease. Furthermore, evidence for a strong inflammatory response was found in mice infected with WNV and CHIKV. The transcriptomics profile measured in mice with WNV and CHIKV neuroinvasive disease in our study showed strong overlap with the mRNA profile described in the literature for other viral neuroinvasive diseases. More studies are warranted to decipher the role of cell inflammation and cell death in viral neuroinvasive disease and whether common mechanisms are active in both neurodegenerative and brain infectious diseases. PMID:28861067

Transcriptomic Analyses Reveal Differential Gene Expression of Immune and Cell Death Pathways in the Brains of Mice Infected with West Nile Virus and Chikungunya Virus.

PubMed

Lim, Stephanie M; van den Ham, Henk-Jan; Oduber, Minoushka; Martina, Eurydice; Zaaraoui-Boutahar, Fatiha; Roose, Jeroen M; van IJcken, Wilfred F J; Osterhaus, Albert D M E; Andeweg, Arno C; Koraka, Penelope; Martina, Byron E E

2017-01-01

West Nile virus (WNV) and chikungunya virus (CHIKV) are arboviruses that are constantly (re-)emerging and expanding their territory. Both viruses often cause a mild form of disease, but severe forms of the disease can consist of neurological symptoms, most often observed in the elderly and young children, respectively, for which the mechanisms are poorly understood. To further elucidate the mechanisms responsible for end-stage WNV and CHIKV neuroinvasive disease, we used transcriptomics to compare the induction of effector pathways in the brain during the early and late stage of disease in young mice. In addition to the more commonly described cell death pathways such as apoptosis and autophagy, we also found evidence for the differential expression of pyroptosis and necroptosis cell death markers during both WNV and CHIKV neuroinvasive disease. In contrast, no evidence of cell dysfunction was observed, indicating that cell death may be the most important mechanism of disease. Interestingly, there was overlap when comparing immune markers involved in neuroinvasive disease to those seen in neurodegenerative diseases. Nonetheless, further validation studies are needed to determine the activation and involvement of these effector pathways at the end stage of disease. Furthermore, evidence for a strong inflammatory response was found in mice infected with WNV and CHIKV. The transcriptomics profile measured in mice with WNV and CHIKV neuroinvasive disease in our study showed strong overlap with the mRNA profile described in the literature for other viral neuroinvasive diseases. More studies are warranted to decipher the role of cell inflammation and cell death in viral neuroinvasive disease and whether common mechanisms are active in both neurodegenerative and brain infectious diseases.

PMCA2 silencing potentiates MDA-MB-231 breast cancer cell death initiated with the Bcl-2 inhibitor ABT-263.

PubMed

Curry, Merril; Roberts-Thomson, Sarah J; Monteith, Gregory R

2016-09-30

PMCA2 overexpression in some breast cancers suggests that this calcium pump isoform may play a role in breast pathophysiology. To investigate PMCA2 as a potential drug target for breast cancer therapy, we assessed the functional consequence of PMCA2 silencing on cell death pathways and calcium signals in the basal-like MDA-MB-231 breast cancer cell line. Silencing PMCA2 expression alone has no effect on MDA-MB-231 cell viability, however, PMCA2 silencing promotes calcium-induced cell death initiated with the calcium ionophore ionomycin. Assessment of cytoplasmic calcium responses generated with various agents including ionomycin demonstrates that in MDA-MB-231 cells, PMCA2 does not play a major role in shaping global calcium signals. We also examined the ability of PMCA2 silencing to modulate caspase-dependent cell death triggered by a Bcl-2 inhibitor that is in clinical development for the treatment of various cancers, ABT-263 (Navitoclax). Despite the lack of effect on global calcium responses, PMCA2 silencing augmented Bcl-2 inhibitor (ABT-263)-mediated MDA-MB-231 breast cancer cell death. These studies provide evidence that PMCA2 inhibitors could sensitize PMCA2-positive breast cancers to cell death initiators that work through mechanisms involving the Bcl-2 survival pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

Is necroptosis a death pathway in aluminum-induced neuroblastoma cell demise?

PubMed

Zhang, Q L; Niu, Q; Ji, X L; Conti, P; Boscolo, P

2008-01-01

Besides being an aggravating factor secondary to major physiological alterations in degenerative diseases, aluminum has also been considered as a risk factor in the etiology. Although many in vivo and in vitro data are in favor of apoptosis and necrosis being involved in Al induced neurodegenerative processes, there is considerable evidence that very complex events may contribute to neural cell death. Necroptosis, a novel cell death pathway, was recently reported to contribute to ischemia brain injury. It is different from, but associated with, apoptosis and necrosis, the two common major pathways of cell demise. In the present study, SH-SY5Y cells were put under stress by Al, a potential degenerative cell death inducer. Nec-1, a specific inhibitor, was used to identify necroptosis. The characteristics observed in Nec-1 and Al treated SH-SY5Y cells showed that necrotic morphological changes were reduced, and a sharp decrease of necrotic rate was detected. Besides, there were Al-induced mitochondria membrane potential decreasing, reactive oxygen species remaining, and autophagosomes declining. The mechanism of Nec-1s effect on cell death may be related to caspases pathways. To our best knowledge, this is the pioneer report on necroptosis in mixed human neural cell death pathways, which might offer a novel therapeutic target for neurodegenerative diseases, and an extended window for neuroprotection.

The binding protein BiP attenuates stress-induced cell death in soybean via modulation of the N-rich protein-mediated signaling pathway.

PubMed

Reis, Pedro A A; Rosado, Gustavo L; Silva, Lucas A C; Oliveira, Luciana C; Oliveira, Lucas B; Costa, Maximiller D L; Alvim, Fátima C; Fontes, Elizabeth P B

2011-12-01

The molecular chaperone binding protein (BiP) participates in the constitutive function of the endoplasmic reticulum (ER) and protects the cell against stresses. In this study, we investigated the underlying mechanism by which BiP protects plant cells from stress-induced cell death. We found that enhanced expression of BiP in soybean (Glycine max) attenuated ER stress- and osmotic stress-mediated cell death. Ectopic expression of BiP in transgenic lines attenuated the leaf necrotic lesions that are caused by the ER stress inducer tunicamycin and also maintained shoot turgidity upon polyethylene glycol-induced dehydration. BiP-mediated attenuation of stress-induced cell death was confirmed by the decreased percentage of dead cell, the reduced induction of the senescence-associated marker gene GmCystP, and reduced DNA fragmentation in BiP-overexpressing lines. These phenotypes were accompanied by a delay in the induction of the cell death marker genes N-RICH PROTEIN-A (NRP-A), NRP-B, and GmNAC6, which are involved in transducing a cell death signal generated by ER stress and osmotic stress through the NRP-mediated signaling pathway. The prosurvival effect of BiP was associated with modulation of the ER stress- and osmotic stress-induced NRP-mediated cell death signaling, as determined in transgenic tobacco (Nicotiana tabacum) lines with enhanced (sense) and suppressed (antisense) BiP levels. Enhanced expression of BiP prevented NRP- and NAC6-mediated chlorosis and the appearance of senescence-associated markers, whereas silencing of endogenous BiP accelerated the onset of leaf senescence mediated by NRPs and GmNAC6. Collectively, these results implicate BiP as a negative regulator of the stress-induced NRP-mediated cell death response.

The Binding Protein BiP Attenuates Stress-Induced Cell Death in Soybean via Modulation of the N-Rich Protein-Mediated Signaling Pathway1[C][W][OA

PubMed Central

Reis, Pedro A.A.; Rosado, Gustavo L.; Silva, Lucas A.C.; Oliveira, Luciana C.; Oliveira, Lucas B.; Costa, Maximiller D.L.; Alvim, Fátima C.; Fontes, Elizabeth P.B.

2011-01-01

The molecular chaperone binding protein (BiP) participates in the constitutive function of the endoplasmic reticulum (ER) and protects the cell against stresses. In this study, we investigated the underlying mechanism by which BiP protects plant cells from stress-induced cell death. We found that enhanced expression of BiP in soybean (Glycine max) attenuated ER stress- and osmotic stress-mediated cell death. Ectopic expression of BiP in transgenic lines attenuated the leaf necrotic lesions that are caused by the ER stress inducer tunicamycin and also maintained shoot turgidity upon polyethylene glycol-induced dehydration. BiP-mediated attenuation of stress-induced cell death was confirmed by the decreased percentage of dead cell, the reduced induction of the senescence-associated marker gene GmCystP, and reduced DNA fragmentation in BiP-overexpressing lines. These phenotypes were accompanied by a delay in the induction of the cell death marker genes N-RICH PROTEIN-A (NRP-A), NRP-B, and GmNAC6, which are involved in transducing a cell death signal generated by ER stress and osmotic stress through the NRP-mediated signaling pathway. The prosurvival effect of BiP was associated with modulation of the ER stress- and osmotic stress-induced NRP-mediated cell death signaling, as determined in transgenic tobacco (Nicotiana tabacum) lines with enhanced (sense) and suppressed (antisense) BiP levels. Enhanced expression of BiP prevented NRP- and NAC6-mediated chlorosis and the appearance of senescence-associated markers, whereas silencing of endogenous BiP accelerated the onset of leaf senescence mediated by NRPs and GmNAC6. Collectively, these results implicate BiP as a negative regulator of the stress-induced NRP-mediated cell death response. PMID:22007022

Release mechanism of high mobility group nucleosome binding domain 1 from lipopolysaccharide-stimulated macrophages.

PubMed

Murakami, Taisuke; Hu, Zhongshuang; Tamura, Hiroshi; Nagaoka, Isao

2016-04-01

Alarmins are identified as endogenous mediators that have potent immune-activating abilities. High mobility group nucleosome binding domain 1 (HMGN1), a highly conserved, non-histone chromosomal protein, which binds to the inner side of the nucleosomal DNA, regulates chromatin dynamics and transcription in cells. Furthermore, HMGN1 acts as a cytokine in the extracellular milieu by inducing the recruitment and maturation of antigen-presenting cells (dendritic cells) to enhance Th1-type antigen-specific immune responses. Thus, HMGN1 is expected to act as an alarmin, when released into the extracellular milieu. The present study investigated the release mechanism of HMGN1 from macrophages using mouse macrophage‑like RAW264.7 cells. The results indicated that HMGN1 was released from lipopolysaccharide (LPS)‑stimulated RAW264.7 cells, accompanied by cell death as assessed by the release of lactate dehydrogenase (LDH). Subsequently, the patterns of cell death involved in HMGN1 release from LPS‑stimulated RAW264.7 cells were determined using a caspase‑1 inhibitor, YVAD, and a necroptosis inhibitor, Nec‑1. YVAD and Nec‑1 did not alter LPS‑induced HMGN1 and LDH release, suggesting that pyroptosis (caspase‑1‑activated cell death) and necroptosis are not involved in the release of HMGN1 from LPS‑stimulated RAW264.7 cells. In addition, flow cytometric analysis indicated that LPS stimulation did not induce apoptosis but substantially augmented necrosis, as evidenced by staining with annexin V/propidium iodide. Together these findings suggest that HMGN1 is extracellularly released from LPS‑stimulated RAW264.7 macrophage‑like cells, accompanied by unprogrammed necrotic cell death but not pyroptosis, necroptosis or apoptosis.

[Novel Anticancer Strategy Targeting Switch Mechanisms in Two Types of Cell Death: Necrosis and Apoptosis].

PubMed

Sato, Akira

2017-01-01

 Two types of cell death, necrosis and apoptosis, are defined in terms of cell death morphological features. We have been studying the mechanisms by which cell death processes are switched during the treatment of mouse tumor FM3A with anticancer, 5-fluoro-2'-deoxyuridine (FUdR): it induces original clone F28-7 to necrosis, but its sub-clone F28-7-A to apoptosis. We identified several such switch regulators of cell death: heat shock protein 90 (HSP90), lamin-B1, cytokeratin-19, and activating transcription factor 3 (ATF3), by using transcriptomic, proteomic analyses and siRNA screening. For example, the inhibition of HSP90 by its inhibitor geldanamycin in F28-7 caused a shift from necrosis to apoptosis. We also observed that the knockdown of lamin-B1, cytokeratin-19, or ATF3 expression in F28-7 resulted in a shift from necrosis to apoptosis. Recently, we used microRNA (miRNA, miR) microarray analyses to investigate the miRNA expression profiles in these sister cells. The miR-351 and miR-743a were expressed at higher levels in F28-7-A than in F28-7. Higher expression of miR-351 or miR-743a in F28-7, induced by transfecting the miR mimics, resulted in a switch of cell death mode: necrosis to apoptosis. Furthermore, transfection of an miR-351 inhibitor into F28-7-A resulted in morphological changes, and mode of cell death from apoptosis to necrosis. These findings suggest that the identified cell death regulators may have key roles in switching cell death mode. Possible mechanisms involving cell death regulators in the switch of necrosis or apoptosis are discussed. We propose a novel anticancer strategy targeting the switch regulators of necrosis or apoptosis.

UPREGULATION OF BNIP3 AND TRANSLOCATION TO MITOCHONDRIA MEDIATES CYANIDE-INDUCED APOPTOSIS IN CORTICAL CELLS

PubMed Central

Prabhakaran, K.; Li, L.; Zhang, L.; Borowitz, J.L.; Isom, G.E.

2008-01-01

BNIP3, a BH3 domain only Bcl-2 protein, has been identified as a mitochrondrial mediator of hypoxia-induced cell death. Since cyanide produces histotoxic anoxia (chemical hypoxia), the present study was undertaken in primary cortical cells to determine involvement of the BNIP3 signaling pathway in cyanide-induced death. Over a 20 h exposure KCN increased BNIP3 expression, followed by a concentration-related apoptotic death. To determine if BNIP3 plays a role in the cell death, expression was either overexpressed with BNIP3 cDNA (BNIP3+) or knocked down with small interfering RNA (RNAi). In BNIP3+ cells, cyanide-induced apoptotic death was markedly enhanced and preceded by reduction of mitochondrial membrane potential (Δψm), release of cytochrome c from mitochondria and elevated caspase 3 and 7 activity. Pretreatment with the pan caspase inhibitor zVAD-fmk suppressed BNIP3+-mediated cell death, thus confirming a caspase-dependent apoptosis. On the other hand, BNIP3 knock down by RNAi or antagonism of BNIP3 by a transmembrane-deleted dominant-negative mutant (BNIP3ΔTM) markedly reduced cell death. Immunohistochemical imaging showed that cyanide stimulated translocation of BNIP3 from cytosol to mitochondria and displacement studies with BNIP3ΔTM showed that integration of BNIP3 into the mitochondrial outer membrane was necessary for the cell death. In BNIP3+ cells, cyclosporin-A, an inhibitor of mitochondrial pore transition, blocked the cyanide-induced reduction of Δψm and decreased the apoptotic death. These results demonstrate in cortical cells that cyanide induces a rapid upregulation of BNIP3 expression, followed by translocation to the mitochondrial outer membrane to reduceΔψm This was followed by mitochondrial release of cytochrome c to execute a caspase-dependent cell death. PMID:17980495

Bax and Bid act in synergy to bring about T11TS-mediated glioma apoptosis via the release of mitochondrial cytochrome c and subsequent caspase activation.

PubMed

Bhattacharjee, M; Acharya, S; Ghosh, A; Sarkar, P; Chatterjee, S; Kumar, P; Chaudhuri, S

2008-12-01

The specific apoptotic role of T11TS has been well established in glioma animal models. T11TS specifically induces the glioma cells to die an apoptotic death via immune cross-talk with the two intracranial immune competent cells-microglia and the brain-infiltrating lymphocytes. To unearth the molecular cascades operative within the glioma cells and to some extent in the two interacting immunocytes, we had initiated studies where preliminary findings not only had indicated the involvement of death receptors but had also hinted to the involvement of other apoptotic regulators. Hence, to identify the molecular pathway of apoptosis involving other apoptotic regulators in the three cell types, the cells were studied for the intrinsic apoptotic death regulators that were engaged to maintain the mitochondrial membrane integrity. The proteins that were selected could be divided into three broad classes-the Bcl-2 family of proteins-Bid, Bax and Bcl-2; the guardian of the genome p53 and the proteins downstream of mitochondria-Apaf-1, cytochrome c, caspase-9 and caspase-3. Activated Bid as well as maximal p53 expression was observed in the first dose of T11TS thus dually activating the pro-apoptotic Bax in the first and second dose in the glioma cells. Concurrently, the pro-survival protein Bcl-2's expression level was very much down-regulated in the same two doses favoring the internal microenvironment to proceed for apoptosis. High expression of cytochrome c and Apaf-1 and the presence of active caspase-9 and active caspase-3 in all the T11TS-treated tumor-bearing groups further adjudicated apoptosis of the glioma cells with clear involvement of mitochondrial death pathway in the T11TS-treated animals. Even though expression of the apoptotic regulators remained more or less the same indicating the involvement of mitochondria in the two interacting immunocytes, the intensity of expression of these proteins was much lower than the tumor cells. The present work focuses on the mechanistic approach of how T11TS mediates apoptosis and hence is the first approach of its kind in the field of immunology where the immunotherapeutic molecule's mode of action has been worked out.

The involvement of protein kinase C-ε in isoflurane induced preconditioning of human embryonic stem cell--derived Nkx2.5(+) cardiac progenitor cells.

PubMed

Song, In-Ae; Oh, Ah-Young; Kim, Jin-Hee; Choi, Young-Min; Jeon, Young-Tae; Ryu, Jung-Hee; Hwang, Jung-Won

2016-02-20

Anesthetic preconditioning can improve survival of cardiac progenitor cells exposed to oxidative stress. We investigated the role of protein kinase C and isoform protein kinase C-ε in isoflurane-induced preconditioning of cardiac progenitor cells exposed to oxidative stress. Cardiac progenitor cells were obtained from undifferentiated human embryonic stem cells. Immunostaining with anti-Nkx2.5 was used to confirm the differentiated cardiac progenitor cells. Oxidative stress was induced by H2O2 and FeSO4. For anesthetic preconditioning, cardiac progenitor cells were exposed to 0.25, 0.5, and 1.0 mM of isoflurane. PMA and chelerythrine were used for protein kinase C activation and inhibition, while εψRACK and εV1-2 were used for protein kinase C -ε activation and inhibition, respectively. Isoflurane-preconditioning decreased the death rate of Cardiac progenitor cells exposed to oxidative stress (death rates isoflurane 0.5 mM 12.7 ± 9.3%, 1.0 mM 12.0 ± 7.7% vs. control 31.4 ± 10.2%). Inhibitors of both protein kinase C and protein kinase C -ε abolished the preconditioning effect of isoflurane 0.5 mM (death rates 27.6 ± 13.5% and 25.9 ± 8.7% respectively), and activators of both protein kinase C and protein kinase C - ε had protective effects from oxidative stress (death rates 16.0 ± 3.2% and 10.6 ± 3.8% respectively). Both PKC and PKC-ε are involved in isoflurane-induced preconditioning of human embryonic stem cells -derived Nkx2.5(+) Cardiac progenitor cells under oxidative stress.

Cationic polystyrene nanospheres induce autophagic cell death through the induction of endoplasmic reticulum stress

NASA Astrophysics Data System (ADS)

Chiu, Hui-Wen; Xia, Tian; Lee, Yu-Hsuan; Chen, Chun-Wan; Tsai, Jui-Chen; Wang, Ying-Jan

2014-12-01

Nanoparticles (NPs) have been used to produce a wide range of products that have applications in imaging and drug delivery in medicine. Due to their chemical stability, well-controlled sizes and surface charges, polystyrene (PS) NPs have been developed as biosensors and drug delivery carriers. However, the possible adverse biological effects and underlying mechanisms are still unclear. Recently, autophagy has been implicated in the regulation of cell death. In this study, we evaluated a library of PS NPs with different surface charges. We found that NH2-labeled polystyrene (NH2-PS) nanospheres were highly toxic with enhanced uptake in macrophage (RAW 264.7) and lung epithelial (BEAS-2B) cells. Furthermore, NH2-PS could induce autophagic cell death. NH2-PS increased autophagic flux due to reactive oxygen species (ROS) generation and endoplasmic reticulum (ER) stress caused by misfolded protein aggregation. The inhibition of ER stress decreased cytotoxicity and autophagy in the NH2-PS-treated cells. In addition, the Akt/mTOR and AMPK signaling pathways were involved in the regulation of NH2-PS-triggered autophagic cell death. These results suggest an important role of autophagy in cationic NP-induced cell death and provide mechanistic insights into the inhibition of the toxicity and safe material design.Nanoparticles (NPs) have been used to produce a wide range of products that have applications in imaging and drug delivery in medicine. Due to their chemical stability, well-controlled sizes and surface charges, polystyrene (PS) NPs have been developed as biosensors and drug delivery carriers. However, the possible adverse biological effects and underlying mechanisms are still unclear. Recently, autophagy has been implicated in the regulation of cell death. In this study, we evaluated a library of PS NPs with different surface charges. We found that NH2-labeled polystyrene (NH2-PS) nanospheres were highly toxic with enhanced uptake in macrophage (RAW 264.7) and lung epithelial (BEAS-2B) cells. Furthermore, NH2-PS could induce autophagic cell death. NH2-PS increased autophagic flux due to reactive oxygen species (ROS) generation and endoplasmic reticulum (ER) stress caused by misfolded protein aggregation. The inhibition of ER stress decreased cytotoxicity and autophagy in the NH2-PS-treated cells. In addition, the Akt/mTOR and AMPK signaling pathways were involved in the regulation of NH2-PS-triggered autophagic cell death. These results suggest an important role of autophagy in cationic NP-induced cell death and provide mechanistic insights into the inhibition of the toxicity and safe material design. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr05509h

Large-Scale Identification and Characterization of Heterodera avenae Putative Effectors Suppressing or Inducing Cell Death in Nicotiana benthamiana

PubMed Central

Chen, Changlong; Chen, Yongpan; Jian, Heng; Yang, Dan; Dai, Yiran; Pan, Lingling; Shi, Fengwei; Yang, Shanshan; Liu, Qian

2018-01-01

Heterodera avenae is one of the most important plant pathogens and causes vast losses in cereal crops. As a sedentary endoparasitic nematode, H. avenae secretes effectors that modify plant defenses and promote its biotrophic infection of its hosts. However, the number of effectors involved in the interaction between H. avenae and host defenses remains unclear. Here, we report the identification of putative effectors in H. avenae that regulate plant defenses on a large scale. Our results showed that 78 of the 95 putative effectors suppressed programmed cell death (PCD) triggered by BAX and that 7 of the putative effectors themselves caused cell death in Nicotiana benthamiana. Among the cell-death-inducing effectors, three were found to be dependent on their specific domains to trigger cell death and to be expressed in esophageal gland cells by in situ hybridization. Ten candidate effectors that suppressed BAX-triggered PCD also suppressed PCD triggered by the elicitor PsojNIP and at least one R-protein/cognate effector pair, suggesting that they are active in suppressing both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Notably, with the exception of isotig16060, these putative effectors could also suppress PCD triggered by cell-death-inducing effectors from H. avenae, indicating that those effectors may cooperate to promote nematode parasitism. Collectively, our results indicate that the majority of the tested effectors of H. avenae may play important roles in suppressing cell death induced by different elicitors in N. benthamiana. PMID:29379510

Mis-specified cells die by an active gene-directed process, and inhibition of this death results in cell fate transformation in Drosophila

PubMed Central

Werz, Christian; Lee, Tom V.; Lee, Peter L.; Lackey, Melinda; Bolduc, Clare; Stein, David S.; Bergmann, Andreas

2009-01-01

Summary Incorrectly specified or mis-specified cells often undergo cell death or are transformed to adopt a different cell fate during development. The underlying cause for this distinction is largely unknown. In many developmental mutants in Drosophila, large numbers of mis-specified cells die synchronously, providing a convenient model for analysis of this phenomenon. The maternal mutant bicoid is particularly useful model with which to address this issue because its mutant phenotype is a combination of both transformation of tissue (acron to telson) and cell death in the presumptive head and thorax regions. We show that a subset of these mis-specified cells die through an active gene-directed process involving transcriptional upregulation of the cell death inducer hid. Upregulation of hid also occurs in oskar mutants and other segmentation mutants. In hid bicoid double mutants, mis-specified cells in the presumptive head and thorax survive and continue to develop, but they are transformed to adopt a different cell fate. We provide evidence that the terminal torso signaling pathway protects the mis-specified telson tissue in bicoid mutants from hid-induced cell death, whereas mis-specified cells in the head and thorax die, presumably because equivalent survival signals are lacking. These data support a model whereby mis-specification can be tolerated if a survival pathway is provided, resulting in cellular transformation. PMID:16280349

MicroRNA-155 Reinforces HIV Latency*

PubMed Central

Ruelas, Debbie S.; Chan, Jonathan K.; Oh, Eugene; Heidersbach, Amy J.; Hebbeler, Andrew M.; Chavez, Leonard; Verdin, Eric; Rape, Michael; Greene, Warner C.

2015-01-01

The presence of a small number of infected but transcriptionally dormant cells currently thwarts a cure for the more than 35 million individuals infected with HIV. Reactivation of these latently infected cells may result in three fates: 1) cell death due to a viral cytopathic effect, 2) cell death due to immune clearance, or 3) a retreat into latency. Uncovering the dynamics of HIV gene expression and silencing in the latent reservoir will be crucial for developing an HIV-1 cure. Here we identify and characterize an intracellular circuit involving TRIM32, an HIV activator, and miR-155, a microRNA that may promote a return to latency in these transiently activated reservoir cells. Notably, we demonstrate that TRIM32, an E3 ubiquitin ligase, promotes reactivation from latency by directly modifying IκBα, leading to a novel mechanism of NF-κB induction not involving IκB kinase activation. PMID:25873391

Gastrointestinal stromal tumors (GIST): Facing cell death between autophagy and apoptosis.

PubMed

Ravegnini, Gloria; Sammarini, Giulia; Nannini, Margherita; Pantaleo, Maria A; Biasco, Guido; Hrelia, Patrizia; Angelini, Sabrina

2017-03-04

Autophagy and apoptosis are 2 fundamental biological mechanisms that may cooperate or be antagonistic, although both are involved in deciding the fate of cells in physiological or pathological conditions. These 2 mechanisms coexist simultaneously in cells and share common upstream signals and stimuli. Autophagy and apoptosis play pivotal roles in cancer development. Autophagy plays a key function in maintaining tumor cell survival by providing energy during unfavorable metabolic conditions through its recycling mechanism, and supporting the high energy requirement for metabolism and growth. This review focuses on gastrointestinal stromal tumors and cell death through autophagy and apoptosis, taking into account the involvement of both of these processes in tumor development and growth and as mechanisms of drug resistance. We also focus on the crosstalk between autophagy and apoptosis as an emerging field with major implications for the development of novel therapeutic options.

Involvement of the MAPK and PI3K pathways in chitinase 3-like 1-regulated hyperoxia-induced airway epithelial cell death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Mi Na; Lee, Kyung Eun; Hong, Jung Yeon

Highlights: Black-Right-Pointing-Pointer Hyperoxia induces apoptosis and chitinase 3-like 1 expression in human airway epithelial cells. Black-Right-Pointing-Pointer Presence of chitinase 3-like 1 affects airway epithelial cell death after hyperoxic exposure. Black-Right-Pointing-Pointer Silencing chitinase 3-like 1 manipulate the phosphorylation of ERK, p38 and Akt. -- Abstract: Background: Exposure to 100% oxygen causes hyperoxic acute lung injury characterized by cell death and injury of alveolar epithelial cells. Recently, the role of chitinase 3-like 1 (CHI3L1), a member of the glycosyl hydrolase 18 family that lacks chitinase activity, in oxidative stress was demonstrated in murine models. High levels of serum CHI3L1 have been associatedmore » with various diseases of the lung, such as asthma, chronic obstructive pulmonary disease, and cancer. However, the role of CHI3L1 in human airway epithelial cells undergoing oxidative stress remains unknown. In addition, the signaling pathways associated with CHI3L1 in this process are poorly understood. Purpose: In this study, we demonstrate the role of CHI3L1, along with the MAPK and PI3K signaling pathways, in hyperoxia-exposed airway epithelial cells. Method: The human airway epithelial cell line, BEAS-2B, was exposed to >95% oxygen (hyperoxia) for up to 72 h. Hyperoxia-induced cell death was determined by assessing cell viability, Annexin-V FITC staining, caspase-3 and -7 expression, and electron microscopy. CHI3L1 knockdown and overexpression studies were conducted in BEAS-2B cells to examine the role of CHI3L1 in hyperoxia-induced apoptosis. Activation of the MAPK and PI3K pathways was also investigated to determine the role of these signaling cascades in this process. Results: Hyperoxia exposure increased CHI3L1 expression and apoptosis in a time-dependent manner. CHI3L1 knockdown protected cells from hyperoxia-induced apoptosis. In contrast, CHI3L1 overexpression promoted cell death after hyperoxia exposure. Finally, phosphorylation of ERK1/2, p38, and Akt were affected by CHI3L1 knockdown. Conclusion: This study indicates that CHI3L1 is involved in hyperoxia-induced cell death, suggesting that CHI3L1 may be one of several cell death regulators influencing the MAPK and PI3K pathways during oxidative stress in human airway epithelial cells.« less

Sigma-2 ligands and PARP inhibitors synergistically trigger cell death in breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

McDonald, Elizabeth S.; Mankoff, Julia; Makvandi, Mehran

The sigma-2 receptor is overexpressed in proliferating cells compared to quiescent cells and has been used as a target for imaging solid tumors by positron emission tomography. Recent work has suggested that the sigma-2 receptor may also be an effective therapeutic target for cancer therapy. Poly (ADP-ribose) polymerase (PARP) is a family of enzymes involved in DNA damage response. In this study, we looked for potential synergy of cytotoxicity between PARP inhibitors and sigma-2 receptor ligands in breast cancer cell lines. We showed that the PARP inhibitor, YUN3-6, sensitized mouse breast cancer cell line, EMT6, to sigma-2 receptor ligand (SV119,more » WC-26, and RHM-138) induced cell death determined by cell viability assay and colony forming assay. The PARP inhibitor, olaparib, sensitized tumor cells to a different sigma-2 receptor ligand SW43-induced apoptosis and cell death in human triple negative cell line, MDA-MB-231. Olaparib inhibited PARP activity and cell proliferation, and arrested cells in G2/M phase of the cell cycle in MDA-MB-231 cells. Subsequently cells became sensitized to SW43 induced cell death. In conclusion, the combination of sigma-2 receptor ligands and PARP inhibitors appears to hold promise for synergistically triggering cell death in certain types of breast cancer cells and merits further investigation. - Highlights: • PARPi, YUN3-6 and olaparib, and σ2 ligands, SV119 and SW43, were evaluated. • Mouse and human breast cancer cells, EMT6 and MDA-MB-231 respectively, were used. • YUN3-6 and SV119 synergistically triggered cell death in EMT6 cells. • Olaparib and SW43 additively triggered cell death in MDA-MB-231 cells. • Olaparib arrested cells in G2/M in MDA-MB-231 cells.« less

Respective effects of oxygen and energy substrate deprivation on beta cell viability.

PubMed

Lablanche, Sandrine; Cottet-Rousselle, Cécile; Argaud, Laurent; Laporte, Camille; Lamarche, Frédéric; Richard, Marie-Jeanne; Berney, Thierry; Benhamou, Pierre-Yves; Fontaine, Eric

2015-01-01

Deficit in oxygen and energetic substrates delivery is a key factor in islet loss during islet transplantation. Permeability transition pore (PTP) is a mitochondrial channel involved in cell death. We have studied the respective effects of oxygen and energy substrate deprivation on beta cell viability as well as the involvement of oxidative stress and PTP opening. Energy substrate deprivation for 1h followed by incubation in normal conditions led to a cyclosporin A (CsA)-sensitive-PTP-opening in INS-1 cells and human islets. Such a procedure dramatically decreased INS-1 cells viability except when transient removal of energy substrates was performed in anoxia, in the presence of antioxidant N-acetylcysteine (NAC) or when CsA or metformin inhibited PTP opening. Superoxide production increased during removal of energy substrates and increased again when normal energy substrates were restored. NAC, anoxia or metformin prevented the two phases of oxidative stress while CsA prevented the second one only. Hypoxia or anoxia alone did not induce oxidative stress, PTP opening or cell death. In conclusion, energy substrate deprivation leads to an oxidative stress followed by PTP opening, triggering beta cell death. Pharmacological prevention of PTP opening during islet transplantation may be a suitable option to improve islet survival and graft success. Copyright © 2015 Elsevier B.V. All rights reserved.

Novel Mechanisms of Target Cell Death and Survival and of Therapeutic Action of IVIg in Pemphigus

PubMed Central

Arredondo, Juan; Chernyavsky, Alexander I.; Karaouni, Ali; Grando, Sergei A.

2005-01-01

Pemphigus vulgaris (PV) is a potentially lethal mucocutaneous blistering disease characterized by cell-cell detachment within the stratified epithelium (acantholysis) caused by IgG autoantibodies. Intravenous immunoglobulin (IVIg) therapy effectively treats PV, but the mechanism is not fully understood. To further understand acantholysis and the efficacy of IVIg, we measured effects of IgG fractions from PV patients on keratinocyte death processes. Using IgGs from representative PV patients who improved with IVIg, we identified apoptotic and oncotic signaling pathways in in vitro and in vivo PV models. We identified two groups of PV patients, each producing autoantibodies activating predominantly either apoptotic or oncotic cell death pathway. Experimental treatments with caspase 3 or calpain inhibitors demonstrated that PV IgGs induced acantholysis through both pathways. Upstream, the apoptotic signaling involved activation of caspases 8 and 3 and up-regulation of Fas ligand mRNA, whereas calpain-mediated cell death depended on elevated intracellular free Ca2+. IVIg reduced PV IgG-mediated acantholysis and cell death and up-regulated the caspase inhibitor FLIP and the calpain inhibitor calpastatin. These results indicate that in different PV patients, IgG-induced acantholysis proceeds predominantly via distinct, yet complementary, pathways of programmed cell death differentially mediated by apoptosis and oncosis effectors, with IVIg protecting target cells by up-regulating endogenous caspase and calpain inhibitors. PMID:16314468

Changes in metabolic markers in insulin-producing β-cells during hypoxia-induced cell death as studied by NMR metabolomics.

PubMed

Tian, Lianji; Kim, Hoe Suk; Kim, Heyonjin; Jin, Xing; Jung, Hye Seung; Park, Kyong Soo; Cho, Kyoung Won; Park, Sunghyouk; Moon, Woo Kyung

2013-08-02

This study was designed to investigate changes in the metabolites in the intracellular fluid of the pancreatic β-cell line INS-1 to identify potential early and late biomarkers for predicting hypoxia-induced cell death. INS-1 cells were incubated under normoxic conditions (95% air, 5% CO₂) or hypoxic conditions (1% O₂, 5% CO₂, 95% N₂) for 2, 4, 6, 12, or 24 h. The biological changes indicating the process of cell death were analyzed using the MTT assay, flow cytometry, Western blotting, and immunostaining. Changes in the metabolic profiles from cell lysates were identified using ¹H nuclear magnetic resonance (¹H NMR) spectroscopy, and the spectra were analyzed by the multivariate model Orthogonal Projections to Latent Structure-Discriminant Analysis. Cell viability decreased approximately 40% after 12-24 h of hypoxia, coincident with a high level of cleaved caspase-3. A high level of HIF-1α was detected in the 12-24 h hypoxic conditions. The metabolite profiles were altered according to the degree of exposure to hypoxia. A spectral analysis showed significant differences in creatine-containing compounds at the early stage (2-6 h) and taurine-containing compounds at the late stage (12-24 h), with the detection of HIF-1α and cleaved caspase-3 in cells exposed to hypoxia compared to normoxia. Glycerophosphocholine decreased during the early stage hypoxia. The change in taurine- and creatine-containing compounds and choline species could be involved in the β-cell death process as inhibitors or activators of cell death. Our results imply that assessment by ¹H NMR spectroscopy would be a useful tool to predict the cell death process and to identify molecules regulating hypoxia-induced cell death mechanisms.

VOSalophen: a vanadium complex with a stilbene derivative-induction of apoptosis, autophagy, and efficiency in experimental cutaneous leishmaniasis.

PubMed

Machado, Patrícia de A; Morais, Jessica O F; Carvalho, Gustavo S G; Lima, Wallace P; Macedo, Gilson C; Britta, Elizandra A; Nakamura, Celso V; da Silva, Adilson D; Cuin, Alexandre; Coimbra, Elaine S

2017-08-01

In our previous work, we demonstrated the promising in vitro effect of VOSalophen, a vanadium complex with a stilbene derivative, against Leishmania amazonensis. Its antileishmanial activity has been associated with oxidative stress in L. amazonensis promastigotes and L. amazonensis-infected macrophages. In the present study, the mechanism involved in the death of parasites after treatment with VOSalophen, as well as in vivo effect in the murine model cutaneous leishmaniasis, has been investigated. Promastigotes of L. amazonensis treated with VOSalophen presented apoptotic cells features, such as cell volume decrease, phosphatidylserine externalization, and DNA fragmentation. An increase in autophagic vacuoles formation in treated promastigotes was also observed, showing that autophagy also may be involved in the death of these parasites. In intracellular amastigotes, DNA fragmentation was observed after treatment with VOSalophen, but this effect was not observed in host cells, highlighting the selective effect of this vanadium complex. In addition, VOSalophen showed activity in the murine model of cutaneous leishmaniasis, without hepatic and renal damages. The outcome described here points out that VOSalophen had promising antileishmanial properties and these data also contribute to the understanding of the mechanisms involved in the death of protozoa induced by metal complexes.

Necrostatin-1 attenuates early brain injury after subarachnoid hemorrhage in rats by inhibiting necroptosis.

PubMed

Chen, Fuxiang; Su, Xingfen; Lin, Zhangya; Lin, Yuanxiang; Yu, Lianghong; Cai, Jiawei; Kang, Dezhi; Hu, Liwen

2017-01-01

Necroptosis is programmed cell death that has been recently proposed and reported to be involved in several neurologic diseases. However, the role of necroptosis in early brain injury after subarachnoid hemorrhage (SAH) is still unknown. The purpose of this study was to investigate whether necroptosis was involved in SAH-induced early brain injury, and to assess the possible neuroprotective effect of necrostatin-1 using an endovascular perforation rat model of SAH. Our results showed that the expression levels of necroptosis-related proteins including RIP1, RIP3 and MLKL in the basal cortex all increased at 3 hours after SAH ( P <0.05) and peaked at 48 hours after SAH ( P <0.05). However, they were greatly reduced after treatment with necrostatin-1 ( P <0.05). Concurrently, neurologic outcomes were significantly improved after necrostatin-1 treatment ( P <0.05). Furthermore, brain edema, blood-brain barrier disruption, necrotic cell death and neuroinflammation were also greatly inhibited after necrostatin-1 treatment. These results indicate that necroptosis is an important mechanism of cell death involved in the early brain injury after experimental SAH. Necrostatin-1 perhaps can serve as a promising neuroprotective agent for SAH treatment.

Circadian Stress Regimes Affect the Circadian Clock and Cause Jasmonic Acid-Dependent Cell Death in Cytokinin-Deficient Arabidopsis Plants[OPEN

PubMed Central

Nitschke, Silvia; Cortleven, Anne; Iven, Tim; Havaux, Michel; Schmülling, Thomas

2016-01-01

The circadian clock helps plants measure daylength and adapt to changes in the day-night rhythm. We found that changes in the light-dark regime triggered stress responses, eventually leading to cell death, in leaves of Arabidopsis thaliana plants with reduced cytokinin levels or defective cytokinin signaling. Prolonged light treatment followed by a dark period induced stress and cell death marker genes while reducing photosynthetic efficiency. This response, called circadian stress, is also characterized by altered expression of clock and clock output genes. In particular, this treatment strongly reduced the expression of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). Intriguingly, similar changes in gene expression and cell death were observed in clock mutants lacking proper CCA1 and LHY function. Circadian stress caused strong changes in reactive oxygen species- and jasmonic acid (JA)-related gene expression. The activation of the JA pathway, involving the accumulation of JA metabolites, was crucial for the induction of cell death, since the cell death phenotype was strongly reduced in the jasmonate resistant1 mutant background. We propose that adaptation to circadian stress regimes requires a normal cytokinin status which, acting primarily through the AHK3 receptor, supports circadian clock function to guard against the detrimental effects of circadian stress. PMID:27354555

Lysosome-mediated Cell Death and Autophagy-Dependent Multidrug Resistance in Breast Cancer

DTIC Science & Technology

2008-10-01

gene links mitochondria and cell death, the data suggests that Bcl2 may be involved in autophagic cell death and AD-MDR. GeneGo analysis also...GSK3 beta GSK3 beta E2A p53 p21 p21 E2F1 PPAR -gamma JNK1(MA PK8) JNK1(M APK8) ESR1 (nuclear) RARalpha Androgen receptor Androge n receptor p53...RelA (p65 NF-kB subunit) Erk (MAPK1/3 ) Erk (MAPK1/ 3) PPAR - gamma SOX9 Bcl-2 Bcl-2 RARalpha SP1 EGFR EGFR RelA (p65 NF- kB subunit) RARalpha RelA

Docetaxel-induced prostate cancer cell death involves concomitant activation of caspase and lysosomal pathways and is attenuated by LEDGF/p75

PubMed Central

Mediavilla-Varela, Melanie; Pacheco, Fabio J; Almaguel, Frankis; Perez, Jossymar; Sahakian, Eva; Daniels, Tracy R; Leoh, Lai Sum; Padilla, Amelia; Wall, Nathan R; Lilly, Michael B; De Leon, Marino; Casiano, Carlos A

2009-01-01

Background Hormone-refractory prostate cancer (HRPC) is characterized by poor response to chemotherapy and high mortality, particularly among African American men when compared to other racial/ethnic groups. It is generally accepted that docetaxel, the standard of care for chemotherapy of HRPC, primarily exerts tumor cell death by inducing mitotic catastrophe and caspase-dependent apoptosis following inhibition of microtubule depolymerization. However, there is a gap in our knowledge of mechanistic events underlying docetaxel-induced caspase-independent cell death, and the genes that antagonize this process. This knowledge is important for circumventing HRPC chemoresistance and reducing disparities in prostate cancer mortality. Results We investigated mechanistic events associated with docetaxel-induced death in HRPC cell lines using various approaches that distinguish caspase-dependent from caspase-independent cell death. Docetaxel induced both mitotic catastrophe and caspase-dependent apoptosis at various concentrations. However, caspase activity was not essential for docetaxel-induced cytotoxicity since cell death associated with lysosomal membrane permeabilization still occurred in the presence of caspase inhibitors. Partial inhibition of docetaxel-induced cytotoxicity was observed after inhibition of cathepsin B, but not inhibition of cathepsins D and L, suggesting that docetaxel induces caspase-independent, lysosomal cell death. Simultaneous inhibition of caspases and cathepsin B dramatically reduced docetaxel-induced cell death. Ectopic expression of lens epithelium-derived growth factor p75 (LEDGF/p75), a stress survival autoantigen and transcription co-activator, attenuated docetaxel-induced lysosomal destabilization and cell death. Interestingly, LEDGF/p75 overexpression did not protect cells against DTX-induced mitotic catastrophe, and against apoptosis induced by tumor necrosis factor related apoptosis inducing ligand (TRAIL), suggesting selectivity in its pro-survival activity. Conclusion These results underscore the ability of docetaxel to induce concomitantly caspase-dependent and independent death pathways in prostate cancer cells. The results also point to LEDGF/p75 as a potential contributor to cellular resistance to docetaxel-induced lysosomal destabilization and cell death, and an attractive candidate for molecular targeting in HRPC. PMID:19715609

Crystalline structure of pulverized dental calculus induces cell death in oral epithelial cells.

PubMed

Ziauddin, S M; Yoshimura, A; Montenegro Raudales, J L; Ozaki, Y; Higuchi, K; Ukai, T; Kaneko, T; Miyazaki, T; Latz, E; Hara, Y

2018-06-01

Dental calculus is a mineralized deposit attached to the tooth surface. We have shown that cellular uptake of dental calculus triggers nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation, leading to the processing of the interleukin-1β precursor into its mature form in mouse and human phagocytes. The activation of the NLRP3 inflammasome also induced a lytic form of programmed cell death, pyroptosis, in these cells. However, the effects of dental calculus on other cell types in periodontal tissue have not been investigated. The aim of this study was to determine whether dental calculus can induce cell death in oral epithelial cells. HSC-2 human oral squamous carcinoma cells, HOMK107 human primary oral epithelial cells and immortalized mouse macrophages were exposed to dental calculus or 1 of its components, hydroxyapatite crystals. For inhibition assays, the cells were exposed to dental calculus in the presence or absence of cytochalasin D (endocytosis inhibitor), z-YVAD-fmk (caspase-1 inhibitor) or glyburide (NLRP3 inflammasome inhibitor). Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release and staining with propidium iodide. Tumor necrosis factor-α production was quantified by enzyme-linked immunosorbent assay. Oral epithelial barrier function was examined by permeability assay. Dental calculus induced cell death in HSC-2 cells, as judged by LDH release and propidium iodide staining. Dental calculus also induced LDH release from HOMK107 cells. Following heat treatment, dental calculus lost its capacity to induce tumor necrosis factor-α in mouse macrophages, but could induce LDH release in HSC-2 cells, indicating a major role of inorganic components in cell death. Hydroxyapatite crystals also induced cell death in both HSC-2 and HOMK107 cells, as judged by LDH release, indicating the capacity of crystal particles to induce cell death. Cell death induced by dental calculus was significantly inhibited by cytochalasin D, z-YVAD-fmk and glyburide, indicating NLRP3 inflammasome involvement. In permeability assays, dental calculus attenuated the barrier function of HSC-2 cell monolayers. Dental calculus induces pyroptotic cell death in human oral epithelial cells and the crystalline structure plays a major role in this process. Oral epithelial cell death induced by dental calculus might be important for the etiology of periodontitis. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Differential activation of cell death and autophagy results in an increased cytotoxic potential for trifluorothymidine compared to 5-fluorouracil in colon cancer cells.

PubMed

Bijnsdorp, Irene V; Peters, Godefridus J; Temmink, Olaf H; Fukushima, Masakazu; Kruyt, Frank A

2010-05-15

Trifluorothymidine (TFT) is part of the oral drug formulation TAS-102. Both 5-fluorouracil (5-FU) and TFT can inhibit thymidylate synthase and be incorporated into DNA. TFT shows only moderate cross-resistance to 5-FU. Therefore, we examined whether mechanistic differences in cell death could underlie their different modes of action in colorectal cancer cell lines (WiDR, Lovo92 and Colo320). Drug cytotoxicity was determined by SRB- and clonogenic assays, cell death by flow cytometry (PI and annexin V), caspase cleavage by Western blotting and activity assays and in vivo activity in the hollow fiber assay. The IC(50) values of TFT were 1-6 fold lower than for 5-FU, and clonogenic survival was less than 0.9% at 3 muM TFT, while 2-20% of the cells still survived after 20 muM 5-FU. In general, TFT was a more potent inducer of apoptosis than 5-FU, although the contribution of caspases varied between the used cell lines and necrosis-like cell death was detected. Accordingly, both drugs induced caspase (Z-VAD) independent cell death and lysosomal cathepsin B was involved. Activation of autophagy recovery mechanisms was only triggered by 5-FU, but not by TFT as determined by LC3B expression and cleavage. Inhibition of autophagy by 3-MA in 5-FU exposed cells reduced cell survival. Also, in vivo TFT (as TAS-102) caused more cell death than a 5-FU formulation. We conclude that TFT and 5-FU induce cell death via both caspase-dependent and independent mechanisms. The TFT was more potent than 5-FU, because it induces higher levels of cell death and does not elicit an autophagic survival response in the cancer cell lines. This provides a strong molecular basis for further application of TFT in cancer therapy.

In vivo inhibition of the mitochondrial H+-ATP synthase in neurons promotes metabolic preconditioning.

PubMed

Formentini, Laura; Pereira, Marta P; Sánchez-Cenizo, Laura; Santacatterina, Fulvio; Lucas, José J; Navarro, Carmen; Martínez-Serrano, Alberto; Cuezva, José M

2014-04-01

A key transducer in energy conservation and signaling cell death is the mitochondrial H(+)-ATP synthase. The expression of the ATPase inhibitory factor 1 (IF1) is a strategy used by cancer cells to inhibit the activity of the H(+)-ATP synthase to generate a ROS signal that switches on cellular programs of survival. We have generated a mouse model expressing a mutant of human IF1 in brain neurons to assess the role of the H(+)-ATP synthase in cell death in vivo. The expression of hIF1 inhibits the activity of oxidative phosphorylation and mediates the shift of neurons to an enhanced aerobic glycolysis. Metabolic reprogramming induces brain preconditioning affording protection against quinolinic acid-induced excitotoxicity. Mechanistically, preconditioning involves the activation of the Akt/p70S6K and PARP repair pathways and Bcl-xL protection from cell death. Overall, our findings provide the first in vivo evidence highlighting the H(+)-ATP synthase as a target to prevent neuronal cell death.

Life and death of female gametes during oogenesis and folliculogenesis.

PubMed

Krysko, Dmitri V; Diez-Fraile, Araceli; Criel, Godelieve; Svistunov, Andrei A; Vandenabeele, Peter; D'Herde, Katharina

2008-09-01

The vertebrate ovary is an extremely dynamic organ in which excessive or defective follicles are rapidly and effectively eliminated early in ontogeny and thereafter continuously throughout reproductive life. More than 99% of follicles disappear, primarily due to apoptosis of granulosa cells, and only a minute fraction of the surviving follicles successfully complete the path to ovulation. The balance between signals for cell death and survival determines the destiny of the follicles. An abnormally high rate of cell death followed by atresia can negatively affect fertility and eventually lead irreversibly to premature ovarian failure. In this review we provide a short overview of the role of programmed cell death in prenatal differentiation of the primordial germ cells and in postnatal folliculogenesis. We also discuss the issue of neo-oogenesis. Next, we highlight molecules involved in regulation of granulosa cell apoptosis. We further discuss the potential use of scores for apoptosis in granulosa cells and characteristics of follicular fluid as prognostic markers for predicting the outcome of assisted reproduction. Potential therapeutic strategies for combating premature ovarian failure are also addressed.

SN56 neuronal cell death after 24 h and 14 days chlorpyrifos exposure through glutamate transmission dysfunction, increase of GSK-3β enzyme, β-amyloid and tau protein levels.

PubMed

Moyano, Paula; Frejo, María Teresa; Anadon, María José; García, José Manuel; Díaz, María Jesús; Lobo, Margarita; Sola, Emma; García, Jimena; Del Pino, Javier

2018-06-01

Chlorpyrifos (CPF) is an organophosphate insecticide described to induce cognitive disorders, both after acute and repeated administration. However, the mechanisms through which it induces these effects are unknown. CPF has been reported to produce basal forebrain cholinergic neuronal cell death, involved on learning and memory regulation, which could be the cause of such cognitive disorders. Neuronal cell death was partially mediated by oxidative stress generation, P75 NTR and α 7 -nAChRs gene expression alteration triggered through acetylcholinesterase (AChE) variants disruption, suggesting other mechanisms are involved. In this regard, CPF induces Aβ and tau proteins production and activation of GSK3β enzyme and alters glutamatergic transmission, which have been related with basal forebrain cholinergic neuronal cell death and development of cognitive disorders. According to these data, we hypothesized that CPF induces basal forebrain cholinergic neuronal cell death through induction of Aβ and tau proteins production, activation of GSK-3β enzyme and disruption of glutamatergic transmission. We evaluated this hypothesis in septal SN56 basal forebrain cholinergic neurons, after 24 h and 14 days CPF exposure. This study shows that CPF increases glutamate levels, upregulates GSK-3β gene expression, and increases the production of Aβ and phosphorylated tau proteins and all these effects reduced cell viability. CPF increases glutaminase activity and upregulates the VGLUT1 gene expression, which could mediate the disruption of glutamatergic transmission. Our present results provide new understanding of the mechanisms contributing to the harmful effects of CPF, and its possible relevance in the pathogenesis of neurodegenerative diseases. Copyright © 2018 Elsevier B.V. All rights reserved.

Crosstalk of ROS/RNS and autophagy in silibinin-induced apoptosis of MCF-7 human breast cancer cells in vitro.

PubMed

Zheng, Nan; Liu, Lu; Liu, Wei-Wei; Li, Fei; Hayashi, Toshihiko; Tashiro, Shin-Ichi; Onodera, Satoshi; Ikejima, Takashi

2017-02-01

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) play important roles in regulating cell survival and death. Silibinin is a natural polyphenolic flavonoid isolated from milk thistle with anti-tumor activities, but it was found to induce cytoprotective ROS/RNS in human breast cancer MCF-7 cells. Furthermore, treatment with silibinin down-regulates ERα expression in MCF-7 cells, and inducing both autophagy and apoptosis. In this study we explored the relationship between ER-associated pathways and RNS/ROS in MCF-7 cells. We also investigated the molecular mechanisms underlying the reciprocal regulation between ROS/RNS levels and autophagy in the death signaling pathways in silibinin-treated MCF-7 cells. Silibinin (100-300 μmol/L) dose-dependently increased ROS/RNS generation in MCF-7 cells (with high expression of ERα and low expression of ERβ) and MDA-MB-231 cells (with low expression of ERα and high expression of ERβ). Scavenging ROS/RNS significantly enhanced silibinin-induced death of MCF-7 cells, but not MDA-MB231 cells. Pharmacological activation or blockade of ERα in MCF-7 cells significantly enhanced or decreased, respectively, silibinin-induced ROS/RNS generation, whereas activation or block of ERβ had no effect. In silibinin-treated MCF-7 cells, exposure to the ROS/RNS donators decreased the autophagic levels, whereas inhibition of autophagy with 3-MA significantly increased ROS/RNS levels. We further showed that increases in ROS/RNS generation, ERα activation or autophagy down-regulation had protective roles in silibinin-treated MCF-7 cells. Under a condition of ERα activation, scavenging ROS/RNS or stimulating autophagy enhanced the cytotoxicity of silibinin. These results demonstrate the existence of two conflicting pathways in silibinin-induced death of MCF-7 cells: one involves the down-regulation of ERα and thereby augmenting the pro-apoptotic autophagy downstream, leading to cell death; the other involves the up-regulation of pro-survival ROS/RNS; and that the generation of ROS/RNS and autophagy form a negative feedback loop whose balance is regulated by ERα.

Crosstalk of ROS/RNS and autophagy in silibinin-induced apoptosis of MCF-7 human breast cancer cells in vitro

PubMed Central

Zheng, Nan; Liu, Lu; Liu, Wei-wei; Li, Fei; Hayashi, Toshihiko; Tashiro, Shin-ichi; Onodera, Satoshi; Ikejima, Takashi

2017-01-01

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) play important roles in regulating cell survival and death. Silibinin is a natural polyphenolic flavonoid isolated from milk thistle with anti-tumor activities, but it was found to induce cytoprotective ROS/RNS in human breast cancer MCF-7 cells. Furthermore, treatment with silibinin down-regulates ERα expression in MCF-7 cells, and inducing both autophagy and apoptosis. In this study we explored the relationship between ER-associated pathways and RNS/ROS in MCF-7 cells. We also investigated the molecular mechanisms underlying the reciprocal regulation between ROS/RNS levels and autophagy in the death signaling pathways in silibinin-treated MCF-7 cells. Silibinin (100–300 μmol/L) dose-dependently increased ROS/RNS generation in MCF-7 cells (with high expression of ERα and low expression of ERβ) and MDA-MB-231 cells (with low expression of ERα and high expression of ERβ). Scavenging ROS/RNS significantly enhanced silibinin-induced death of MCF-7 cells, but not MDA-MB231 cells. Pharmacological activation or blockade of ERα in MCF-7 cells significantly enhanced or decreased, respectively, silibinin-induced ROS/RNS generation, whereas activation or block of ERβ had no effect. In silibinin-treated MCF-7 cells, exposure to the ROS/RNS donators decreased the autophagic levels, whereas inhibition of autophagy with 3-MA significantly increased ROS/RNS levels. We further showed that increases in ROS/RNS generation, ERα activation or autophagy down-regulation had protective roles in silibinin-treated MCF-7 cells. Under a condition of ERα activation, scavenging ROS/RNS or stimulating autophagy enhanced the cytotoxicity of silibinin. These results demonstrate the existence of two conflicting pathways in silibinin-induced death of MCF-7 cells: one involves the down-regulation of ERα and thereby augmenting the pro-apoptotic autophagy downstream, leading to cell death; the other involves the up-regulation of pro-survival ROS/RNS; and that the generation of ROS/RNS and autophagy form a negative feedback loop whose balance is regulated by ERα. PMID:27867187

In vitro cell injury by oxidized low density lipoprotein involves lipid hydroperoxide-induced formation of alkoxyl, lipid, and peroxyl radicals.

PubMed Central

Coffey, M D; Cole, R A; Colles, S M; Chisolm, G M

1995-01-01

Mounting evidence supports current theories linking lipoprotein oxidation to atherosclerosis. We sought the cellular biochemical mechanism by which oxidized LDL inflicts cell injury. Inhibitors of candidate pathways of cell death were used to treat human fibroblast target cells exposed to oxidized LDL.. Ebselen, which degrades lipid hydroperoxides, inhibited oxidized LDL toxicity, consistent with our recent report that 7 beta-hydroperoxycholesterol (7 beta-OOH chol) is the major cytotoxin of oxidized LDL. Intracellular chelation of metal ions inhibited, while preloading cells with iron enhanced, toxicity, Inhibition of oxidized LDL and 7 beta-OOH chol toxicity by 2-keto-4-thiolmethyl butyric acid, a putative alkoxyl radical scavenger and by vitamin E, probucol and diphenylphenylenediamine, putative scavengers of peroxyl radicals was consistent with the involvement of these radicals in the lethal sequence. Cell death was thus postulated to occur due to lipid peroxidation via a sequence involving lipid hydroperoxide-induced, iron-mediated formation of alkoxyl, lipid, and peroxyl radicals. Pathways involving other reactive oxygen species, new protein synthesis, or altered cholesterol metabolism were considered less likely, since putative inhibitors failed to lessen toxicity. Understanding the mechanism of cell injury by oxidized LDL and its toxic moiety, 7 beta-OOH chol, may indicate specific interventions in the cell injury believed to accompany vascular lesion development. PMID:7560078

Cell Death in the Epithelia of the Intestine and Hepatopancreas in Neocaridina heteropoda (Crustacea, Malacostraca)

PubMed Central

Sonakowska, Lidia; Włodarczyk, Agnieszka; Wilczek, Grażyna; Wilczek, Piotr; Student, Sebastian; Rost-Roszkowska, Magdalena Maria

2016-01-01

The endodermal region of the digestive system in the freshwater shrimp Neocaridina heteropoda (Crustacea, Malacostraca) consists of a tube-shaped intestine and large hepatopancreas, which is formed by numerous blind-ended tubules. The precise structure and ultrastructure of these regions were presented in our previous studies, while here we focused on the cell death processes and their effect on the functioning of the midgut. We used transmission electron microscopy, light and confocal microscopes to describe and detect cell death, while a quantitative assessment of cells with depolarized mitochondria helped us to establish whether there is the relationship between cell death and the inactivation of mitochondria. Three types of the cell death were observed in the intestine and hepatopancreas–apoptosis, necrosis and autophagy. No differences were observed in the course of these processes in males and females and or in the intestine and hepatopancreas of the shrimp that were examined. Our studies revealed that apoptosis, necrosis and autophagy only involves the fully developed cells of the midgut epithelium that have contact with the midgut lumen–D-cells in the intestine and B- and F-cells in hepatopancreas, while E-cells (midgut stem cells) did not die. A distinct correlation between the accumulation of E-cells and the activation of apoptosis was detected in the anterior region of the intestine, while necrosis was an accidental process. Degenerating organelles, mainly mitochondria were neutralized and eventually, the activation of cell death was prevented in the entire epithelium due to autophagy. Therefore, we state that autophagy plays a role of the survival factor. PMID:26844766

Small heat shock proteins HSP27 (HspB1), αB-crystallin (HspB5) and HSP22 (HspB8) as regulators of cell death.

PubMed

Acunzo, Julie; Katsogiannou, Maria; Rocchi, Palma

2012-10-01

Hsp27, αB-crystallin and HSP22 are ubiquitous small heat shock proteins (sHsp) whose expression is induced in response to a wide variety of unfavorable physiological and environmental conditions. These sHsp protect cells from otherwise lethal conditions mainly by their involvement in cell death pathways such as necrosis, apoptosis or autophagy. At a molecular level, the mechanisms accounting for sHsp functions in cell death are (1) prevention of denatured proteins aggregation, (2) regulation of caspase activity, (3) regulation of the intracellular redox state, (4) function in actin polymerization and cytoskeleton integrity and (5) proteasome-mediated degradation of selected proteins. In cancer cells, these sHsp are often overexpressed and associated with increased tumorigenicity, cancer cells metastatic potential and resistance to chemotherapy. Altogether, these properties suggest that Hsp27, αB-crystallin and Hsp22 are appropriate targets for modulating cell death pathways. In the present, we briefly review recent reports showing molecular evidence of cell death regulation by these sHsp and co-chaperones. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

PubMed

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

2009-01-01

Programmed cell death is necessary for homeostasis in multicellular organisms and it is also widely recognized to occur in unicellular organisms. However, the mechanisms through which it occurs in unicells, and the enzymes involved within the final response is still the subject of heated debate. It is shown here that exposure of the unicellular microalga Dunaliella viridis to several environmental stresses, induced different cell death morphotypes, depending on the stimulus received. Senescent cells demonstrated classical and unambiguous apoptotic-like characteristics such as chromatin condensation, DNA fragmentation, intact organelles, and blebbing of the cell membrane. Acute heat shock caused general swelling and altered plasma membrane, but the presence of chromatin clusters and DNA strand breaks suggested a necrotic-like event. UV irradiated cells presented changes typical for necrosis, together with apoptotic characteristics resembling an intermediate cell-death phenotype termed aponecrosis-like. Cells subjected to hyperosmotic shock revealed chromatin spotting without DNA fragmentation, and extensive cytoplasmic swelling and vacuolization, comparable to a paraptotic-like cell death phenotype. Nitrogen-starved cells showed pyknosis, blebbing, and cytoplasmic consumption, indicating a similarity to autophagic/vacuolar-like cell death. The caspase-like activity DEVDase was measured by using the fluorescent substrate Ac-DEVD-AMC and antibodies against the human caspase-3 active enzyme cross-reacted with bands, the intensity of which paralleled the activity. All the environmental stresses tested produced a substantial increase in both DEVDase activity and protein levels. The irreversible caspase-3 inhibitor Z-DEVD-FMK completely inhibited the enzymatic activity whereas serine and aspartyl proteases inhibitors did not. These results show that cell death in D. viridis does not conform to a single pattern and that environmental stimuli may produce different types of cell death depending on the type and intensity of the stimulus, all of which help to understand the cell death-dependent and cell death-independent functions of caspase-like proteins. Hence, these data support the theory that alternative, non-apoptotic programmed cell death (PCDs), exist either in parallel or in an independent manner with apoptosis and were already present in single-celled organisms that evolved some 1.2-1.6 billion years ago.

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

PubMed Central

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

2009-01-01

Programmed cell death is necessary for homeostasis in multicellular organisms and it is also widely recognized to occur in unicellular organisms. However, the mechanisms through which it occurs in unicells, and the enzymes involved within the final response is still the subject of heated debate. It is shown here that exposure of the unicellular microalga Dunaliella viridis to several environmental stresses, induced different cell death morphotypes, depending on the stimulus received. Senescent cells demonstrated classical and unambiguous apoptotic-like characteristics such as chromatin condensation, DNA fragmentation, intact organelles, and blebbing of the cell membrane. Acute heat shock caused general swelling and altered plasma membrane, but the presence of chromatin clusters and DNA strand breaks suggested a necrotic-like event. UV irradiated cells presented changes typical for necrosis, together with apoptotic characteristics resembling an intermediate cell-death phenotype termed aponecrosis-like. Cells subjected to hyperosmotic shock revealed chromatin spotting without DNA fragmentation, and extensive cytoplasmic swelling and vacuolization, comparable to a paraptotic-like cell death phenotype. Nitrogen-starved cells showed pyknosis, blebbing, and cytoplasmic consumption, indicating a similarity to autophagic/vacuolar-like cell death. The caspase-like activity DEVDase was measured by using the fluorescent substrate Ac-DEVD-AMC and antibodies against the human caspase-3 active enzyme cross-reacted with bands, the intensity of which paralleled the activity. All the environmental stresses tested produced a substantial increase in both DEVDase activity and protein levels. The irreversible caspase-3 inhibitor Z-DEVD-FMK completely inhibited the enzymatic activity whereas serine and aspartyl proteases inhibitors did not. These results show that cell death in D. viridis does not conform to a single pattern and that environmental stimuli may produce different types of cell death depending on the type and intensity of the stimulus, all of which help to understand the cell death-dependent and cell death-independent functions of caspase-like proteins. Hence, these data support the theory that alternative, non-apoptotic programmed cell death (PCDs), exist either in parallel or in an independent manner with apoptosis and were already present in single-celled organisms that evolved some 1.2-1.6 billion years ago. PMID:19251986

Detecting Anastasis In Vivo by CaspaseTracker Biosensor.

PubMed

Tang, Ho Man; Fung, Ming Chiu; Tang, Ho Lam

2018-02-01

Anastasis (Greek for "rising to life") is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Influence on cell death of high frequency motion of magnetic nanoparticles during magnetic hyperthermia experiments

NASA Astrophysics Data System (ADS)

Hallali, N.; Clerc, P.; Fourmy, D.; Gigoux, V.; Carrey, J.

2016-07-01

Studies with transplanted tumors in animals and clinical trials have provided the proof-of-concept of magnetic hyperthermia (MH) therapy of cancers using iron oxide nanoparticles. Interestingly, in several studies, the application of an alternating magnetic field (AMF) to tumor cells having internalized and accumulated magnetic nanoparticles (MNPs) into their lysosomes can induce cell death without detectable temperature increase. To explain these results, among other hypotheses, it was proposed that cell death could be due to the high-frequency translational motion of MNPs under the influence of the AMF gradient generated involuntarily by most inductors. Such mechanical actions of MNPs might cause cellular damages and participate in the induction of cell death under MH conditions. To test this hypothesis, we developed a setup maximizing this effect. It is composed of an anti-Helmholtz coil and two permanent magnets, which produce an AMF gradient and a superimposed static MF. We have measured the MNP heating power and treated tumor cells by a standard AMF and by an AMF gradient, on which was added or not a static magnetic field. We showed that the presence of a static magnetic field prevents MNP heating and cell death in standard MH conditions. The heating power of MNPs in an AMF gradient is weak, position-dependent, and related to the presence of a non-zero AMF. Under an AMF gradient and a static field, no MNP heating and cell death were measured. Consequently, the hypothesis that translational motions could be involved in cell death during MH experiments is ruled out by our experiments.

Hydrogen Suppresses Hypoxia/Reoxygenation-Induced Cell Death in Hippocampal Neurons Through Reducing Oxidative Stress.

PubMed

Wei, Rong; Zhang, Rufang; Xie, Yewei; Shen, Li; Chen, Fang

2015-01-01

Deep hypothermic circulatory arrest (DHCA) is a cerebral protection technique that has been used in the operations involving the aortic arch and brain aneurysm for decades. We previous showed that DHCA treated rats developed a significant oxidative stress and apoptosis in neurons. We here intend to investigate the protective the effect of hydrogen against oxidative stress-induced cell injury and the involved mechanisms using an in vitro experimental model of hypoxia/reoxygenation (H/R) on HT-22 cells. The model of H/R was established using an airtight culture container and the anaeropack. Measurement of mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) production was used H2DCFDA and JC-1 staining. Western blot was used for the quantification of Akt, p-Akt, Bcl-2, Bax and cleaved caspase-3 proteins. The microRNA (miRNA) profile in hippocampal neurons from rat model of DHCA was determined by miRNA deep sequencing. The elevation of ROS and reduction of MMP were significantly induced by the treatment with hypoxia for 18 h followed by reoxygenation for 6 h. Hydrogen treatment significantly reduced H/R-caused cell death. The levels of p-Akt (Ser 473) and Bcl-2 were significantly increased while Bax and cleaved caspase-3 were decreased by hydrogen treatment on the model of H/R. The expression of miR-200 family was significantly elevated in model of DHCA and H/R. Hydrogen administration inhibited the H/R-induced expression of miR-200 family in HT-22 cells. In addition, inhibition of miR-200 family suppressed H/R-caused cell death through reducing ROS production. These results suggest that H/R causes oxidative stress-induced cell death and that the hydrogen protects against H/R-induced cell death in HT22 cells, in part, due to reducing expression of miR-200 family. © 2015 S. Karger AG, Basel.

Hemoglobins, programmed cell death and somatic embryogenesis.

PubMed

Hill, Robert D; Huang, Shuanglong; Stasolla, Claudio

2013-10-01

Programmed cell death (PCD) is a universal process in all multicellular organisms. It is a critical component in a diverse number of processes ranging from growth and differentiation to response to stress. Somatic embryogenesis is one such process where PCD is significantly involved. Nitric oxide is increasingly being recognized as playing a significant role in regulating PCD in both mammalian and plant systems. Plant hemoglobins scavenge NO, and evidence is accumulating that events that modify NO levels in plants also affect hemoglobin expression. Here, we review the process of PCD, describing the involvement of NO and plant hemoglobins in the process. NO is an effector of cell death in both plants and vertebrates, triggering the cascade of events leading to targeted cell death that is a part of an organism's response to stress or to tissue differentiation and development. Expression of specific hemoglobins can alter this response in plants by scavenging the NO, thus, interrupting the death process. Somatic embryogenesis is used as a model system to demonstrate how cell-specific expression of different classes of hemoglobins can alter the embryogenic process, affecting hormone synthesis, cell metabolite levels and genes associated with PCD and embryogenic competence. We propose that plant hemoglobins influence somatic embryogenesis and PCD through cell-specific expression of a distinct plant hemoglobin. It is based on the premise that both embryogenic competence and PCD are strongly influenced by cellular NO levels. Increases in cellular NO levels result in elevated Zn(2+) and reactive-oxygen species associated with PCD, but they also result in decreased expression of MYC2, a transcription factor that is a negative effector of indoleacetic acid synthesis, a hormone that positively influences embryogenic competence. Cell-specific hemoglobin expression reduces NO levels as a result of NO scavenging, resulting in cell survival. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Tumor suppressor Spred2 interaction with LC3 promotes autophagosome maturation and induces autophagy-dependent cell death.

PubMed

Jiang, Ke; Liu, Min; Lin, Guibin; Mao, Beibei; Cheng, Wei; Liu, Han; Gal, Jozsef; Zhu, Haining; Yuan, Zengqiang; Deng, Wuguo; Liu, Quentin; Gong, Peng; Bi, Xiaolin; Meng, Songshu

2016-05-03

The tumor suppressor Spred2 (Sprouty-related EVH1 domain-2) induces cell death in a variety of cancers. However, the underlying mechanism remains to be elucidated. Here we show that Spred2 induces caspase-independent but autophagy-dependent cell death in human cervical carcinoma HeLa and lung cancer A549 cells. We demonstrate that ectopic Spred2 increased both the conversion of microtubule-associated protein 1 light chain 3 (LC3), GFP-LC3 puncta formation and p62/SQSTM1 degradation in A549 and HeLa cells. Conversely, knockdown of Spred2 in tumor cells inhibited upregulation of autophagosome maturation induced by the autophagy inducer Rapamycin, which could be reversed by the rescue Spred2. These data suggest that Spred2 promotes autophagy in tumor cells. Mechanistically, Spred2 co-localized and interacted with LC3 via the LC3-interacting region (LIR) motifs in its SPR domain. Mutations in the LIR motifs or deletion of the SPR domain impaired Spred2-mediated autophagosome maturation and tumor cell death, indicating that functional LIR is required for Spred2 to trigger tumor cell death. Additionally, Spred2 interacted and co-localized with p62/SQSTM1 through its SPR domain. Furthermore, the co-localization of Spred2, p62 and LAMP2 in HeLa cells indicates that p62 may be involved in Spred2-mediated autophagosome maturation. Inhibition of autophagy using the lysosomal inhibitor chloroquine, reduced Spred2-mediated HeLa cell death. Silencing the expression of autophagy-related genes ATG5, LC3 or p62 in HeLa and A549 cells gave similar results, suggesting that autophagy is required for Spred2-induced tumor cell death. Collectively, these data indicate that Spred2 induces tumor cell death in an autophagy-dependent manner.

Tumor suppressor Spred2 interaction with LC3 promotes autophagosome maturation and induces autophagy-dependent cell death

PubMed Central

Lin, Guibin; Mao, Beibei; Cheng, Wei; Liu, Han; Gal, Jozsef; Zhu, Haining; Yuan, Zengqiang; Deng, Wuguo; Liu, Quentin; Gong, Peng; Bi, Xiaolin; Meng, Songshu

2016-01-01

The tumor suppressor Spred2 (Sprouty-related EVH1 domain-2) induces cell death in a variety of cancers. However, the underlying mechanism remains to be elucidated. Here we show that Spred2 induces caspase-independent but autophagy-dependent cell death in human cervical carcinoma HeLa and lung cancer A549 cells. We demonstrate that ectopic Spred2 increased both the conversion of microtubule-associated protein 1 light chain 3 (LC3), GFP-LC3 puncta formation and p62/SQSTM1 degradation in A549 and HeLa cells. Conversely, knockdown of Spred2 in tumor cells inhibited upregulation of autophagosome maturation induced by the autophagy inducer Rapamycin, which could be reversed by the rescue Spred2. These data suggest that Spred2 promotes autophagy in tumor cells. Mechanistically, Spred2 co-localized and interacted with LC3 via the LC3-interacting region (LIR) motifs in its SPR domain. Mutations in the LIR motifs or deletion of the SPR domain impaired Spred2-mediated autophagosome maturation and tumor cell death, indicating that functional LIR is required for Spred2 to trigger tumor cell death. Additionally, Spred2 interacted and co-localized with p62/SQSTM1 through its SPR domain. Furthermore, the co-localization of Spred2, p62 and LAMP2 in HeLa cells indicates that p62 may be involved in Spred2-mediated autophagosome maturation. Inhibition of autophagy using the lysosomal inhibitor chloroquine, reduced Spred2-mediated HeLa cell death. Silencing the expression of autophagy-related genes ATG5, LC3 or p62 in HeLa and A549 cells gave similar results, suggesting that autophagy is required for Spred2-induced tumor cell death. Collectively, these data indicate that Spred2 induces tumor cell death in an autophagy-dependent manner. PMID:27028858

Therapeutic approaches to preventing cell death in Huntington disease

PubMed Central

Kaplan, Anna; Stockwell, Brent R.

2012-01-01

Neurodegenerative diseases affect the lives of millions of patients and their families. Due to the complexity of these diseases and our limited understanding of their pathogenesis, the design of therapeutic agents that can effectively treat these diseases has been challenging. Huntington disease (HD) is one of several neurological disorders with few therapeutic options. HD, like numerous other neurodegenerative diseases, involves extensive neuronal cell loss. One potential strategy to combat HD and other neurodegenerative disorders is to intervene in the execution of neuronal cell death. Inhibiting neuronal cell death pathways may slow the development of neurodegeneration. However, discovering small molecule inhibitors of neuronal cell death remains a significant challenge. Here, we review candidate therapeutic targets controlling cell death mechanisms that have been the focus of research in HD, as well as an emerging strategy that has been applied to developing small molecule inhibitors—fragment-based drug discovery (FBDD). FBDD has been successfully used in both industry and academia to identify selective and potent small molecule inhibitors, with a focus on challenging proteins that are not amenable to traditional high-throughput screening approaches. FBDD has been used to generate potent leads, pre-clinical candidates, and has led to the development of an FDA approved drug. This approach can be valuable for identifying modulators of cell-death-regulating proteins; such compounds may prove to be the key to halting the progression of HD and other neurodegenerative disorders. PMID:22967354

Image Analysis Algorithms for Immunohistochemical Assessment of Cell Death Events and Fibrosis in Tissue Sections

PubMed Central

Krajewska, Maryla; Smith, Layton H.; Rong, Juan; Huang, Xianshu; Hyer, Marc L.; Zeps, Nikolajs; Iacopetta, Barry; Linke, Steven P.; Olson, Allen H.; Reed, John C.; Krajewski, Stan

2009-01-01

Cell death is of broad physiological and pathological importance, making quantification of biochemical events associated with cell demise a high priority for experimental pathology. Fibrosis is a common consequence of tissue injury involving necrotic cell death. Using tissue specimens from experimental mouse models of traumatic brain injury, cardiac fibrosis, and cancer, as well as human tumor specimens assembled in tissue microarray (TMA) format, we undertook computer-assisted quantification of specific immunohistochemical and histological parameters that characterize processes associated with cell death. In this study, we demonstrated the utility of image analysis algorithms for color deconvolution, colocalization, and nuclear morphometry to characterize cell death events in tissue specimens: (a) subjected to immunostaining for detecting cleaved caspase-3, cleaved poly(ADP-ribose)-polymerase, cleaved lamin-A, phosphorylated histone H2AX, and Bcl-2; (b) analyzed by terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling assay to detect DNA fragmentation; and (c) evaluated with Masson's trichrome staining. We developed novel algorithm-based scoring methods and validated them using TMAs as a high-throughput format. The proposed computer-assisted scoring methods for digital images by brightfield microscopy permit linear quantification of immunohistochemical and histochemical stainings. Examples are provided of digital image analysis performed in automated or semiautomated fashion for successful quantification of molecular events associated with cell death in tissue sections. (J Histochem Cytochem 57:649–663, 2009) PMID:19289554

Inhibition of paraquat-induced autophagy accelerates the apoptotic cell death in neuroblastoma SH-SY5Y cells.

PubMed

González-Polo, Rosa A; Niso-Santano, Mireia; Ortíz-Ortíz, Miguel A; Gómez-Martín, Ana; Morán, José M; García-Rubio, Lourdes; Francisco-Morcillo, Javier; Zaragoza, Concepción; Soler, Germán; Fuentes, José M

2007-06-01

Autophagy is a degradative mechanism involved in the recycling and turnover of cytoplasmic constituents from eukaryotic cells. This phenomenon of autophagy has been observed in neurons from patients with Parkinson's disease (PD), suggesting a functional role for autophagy in neuronal cell death. On the other hand, it has been demonstrated that exposure to pesticides can be a risk factor in the incidence of PD. In this sense, paraquat (PQ) (1,1'-dimethyl-4,4'-bipyridinium dichloride), a widely used herbicide that is structurally similar to the known dopaminergic neurotoxicant MPP(+) (1-methyl-4-phenyl-pyridine), has been suggested as a potential etiologic factor for the development of PD. The current study shows, for the first time, that low concentrations of PQ induce several characteristics of autophagy in human neuroblastoma SH-SY5Y cells. In this way, PQ induced the accumulation of autophagic vacuoles (AVs) in the cytoplasm and the recruitment of a LC3-GFP fusion protein to AVs. Furthermore, the cells treated with PQ showed an increase of the long-lived protein degradation which is blocked in the presence of the autophagy inhibitor 3-methyladenine and regulated by the mammalian target of rapamycin (mTOR) signaling. Finally, the cells succumbed to cell death with hallmarks of apoptosis such as phosphatidylserine exposure, caspase activation, and chromatin condensation. While caspase inhibition retarded cell death, autophagy inhibition accelerated the apoptotic cell death induced by PQ. Altogether, these findings show the relationship between autophagy and apoptotic cell death in human neuroblastoma cells treated with PQ.

Melatonin partially protects 661W cells from H2O2-induced death by inhibiting Fas/FasL-caspase-3.

PubMed

Sánchez-Bretaño, Aída; Baba, Kenkichi; Janjua, Uzair; Piano, Ilaria; Gargini, Claudia; Tosini, Gianluca

2017-01-01

Previous studies have shown that melatonin (MEL) signaling is involved in the modulation of photoreceptor viability during aging. Recent work by our laboratory suggested that MEL may protect cones by modulating the Fas/FasL-caspase-3 pathway. In this study, we first investigated the presence of MEL receptors (MT 1 and MT 2 ) in 661W cells, then whether MEL can prevent H 2 O 2 -induced cell death, and last, through which pathway MEL confers protection. The mRNA and proteins of the MEL receptors were detected with quantitative PCR (q-PCR) and immunocytochemistry, respectively. To test the protective effect of MEL, 661W cells were treated with H 2 O 2 for 2 h in the presence or absence of MEL, a MEL agonist, and an antagonist. To study the pathways involved in H 2 O 2 -mediated cell death, a Fas/FasL antagonist was used before the exposure to H 2 O 2 . Finally, Fas/FasL and caspase-3 mRNA was analyzed with q-PCR and immunocytochemistry in cells treated with H 2 O 2 and/or MEL. Cell viability was analyzed by using Trypan Blue. Both MEL receptors (MT 1 and MT 2 ) were detected at the mRNA and protein levels in 661W cells. MEL partially prevented H 2 O 2 -mediated cell death (20-25%). This effect was replicated with IIK7 (a melatonin receptor agonist) when used at a concentration of 1 µM. Preincubation with luzindole (a melatonin receptor antagonist) blocked MEL protection. Kp7-6, an antagonist of Fas/FasL, blocked cell death caused by H 2 O 2 similarly to what was observed for MEL. Fas, FasL, and caspase-3 expression was increased in cells treated with H 2 O 2 , and this effect was prevented by MEL. Finally, MEL treatment partially prevented the activation of caspase-3 caused by H 2 O 2 . The results demonstrate that MEL receptors are present and functional in 661W cells. MEL can prevent photoreceptor cell death induced by H 2 O 2 via the inhibition of the proapoptotic pathway Fas/FasL-caspase-3.

Improving Accuracy in Arrhenius Models of Cell Death: Adding a Temperature-Dependent Time Delay.

PubMed

Pearce, John A

2015-12-01

The Arrhenius formulation for single-step irreversible unimolecular reactions has been used for many decades to describe the thermal damage and cell death processes. Arrhenius predictions are acceptably accurate for structural proteins, for some cell death assays, and for cell death at higher temperatures in most cell lines, above about 55 °C. However, in many cases--and particularly at hyperthermic temperatures, between about 43 and 55 °C--the particular intrinsic cell death or damage process under study exhibits a significant "shoulder" region that constant-rate Arrhenius models are unable to represent with acceptable accuracy. The primary limitation is that Arrhenius calculations always overestimate the cell death fraction, which leads to severely overoptimistic predictions of heating effectiveness in tumor treatment. Several more sophisticated mathematical model approaches have been suggested and show much-improved performance. But simpler models that have adequate accuracy would provide useful and practical alternatives to intricate biochemical analyses. Typical transient intrinsic cell death processes at hyperthermic temperatures consist of a slowly developing shoulder region followed by an essentially constant-rate region. The shoulder regions have been demonstrated to arise chiefly from complex functional protein signaling cascades that generate delays in the onset of the constant-rate region, but may involve heat shock protein activity as well. This paper shows that acceptably accurate and much-improved predictions in the simpler Arrhenius models can be obtained by adding a temperature-dependent time delay. Kinetic coefficients and the appropriate time delay are obtained from the constant-rate regions of the measured survival curves. The resulting predictions are seen to provide acceptably accurate results while not overestimating cell death. The method can be relatively easily incorporated into numerical models. Additionally, evidence is presented to support the application of compensation law behavior to the cell death processes--that is, the strong correlation between the kinetic coefficients, ln{A} and E(a), is confirmed.

Ergosterol Peroxide Isolated from Ganoderma lucidum Abolishes MicroRNA miR-378-Mediated Tumor Cells on Chemoresistance

PubMed Central

Li, Xiang-Min; Yang, Weining; Jiao, Chun-Wei; Fang, Ling; Li, Sen-Zhu; Pan, Hong-Hui; Yee, Albert J.; Lee, Daniel Y.; Li, Chong; Zhang, Zhi; Guo, Jun; Yang, Burton B.

2012-01-01

Due to an altered expression of oncogenic factors and tumor suppressors, aggressive cancer cells have an intrinsic or acquired resistance to chemotherapeutic agents. This typically contributes to cancer recurrence after chemotherapy. microRNAs are short non-coding RNAs that are involved in both cell self-renewal and cancer development. Here we report that tumor cells transfected with miR-378 acquired properties of aggressive cancer cells. Overexpression of miR-378 enhanced both cell survival and colony formation, and contributed to multiple drug resistance. Higher concentrations of chemotherapeutic drugs were needed to induce death of miR-378-transfected cells than to induce death of control cells. We found that the biologically active component isolated from Ganoderma lucidum could overcome the drug-resistance conferred by miR-378. We purified and identified the biologically active component of Ganoderma lucidum as ergosterol peroxide. We demonstrated that ergosterol peroxide produced greater activity in inducing death of miR-378 cells than the GFP cells. Lower concentrations of ergosterol peroxide were needed to induce death of the miR-378-transfected cells than in the control cells. With further clinical development, ergosterol peroxide represents a promising new reagent that can overcome the drug-resistance of tumor cells. PMID:22952996

Tumor Necrosis Factor-α (TNFα)-induced Ceramide Generation via Ceramide Synthases Regulates Loss of Focal Adhesion Kinase (FAK) and Programmed Cell Death.

PubMed

Hernández-Corbacho, María José; Canals, Daniel; Adada, Mohamad M; Liu, Mengling; Senkal, Can E; Yi, Jae Kyo; Mao, Cungui; Luberto, Chiara; Hannun, Yusuf A; Obeid, Lina M

2015-10-16

Ceramide synthases (CerS1-CerS6), which catalyze the N-acylation of the (dihydro)sphingosine backbone to produce (dihydro)ceramide in both the de novo and the salvage or recycling pathway of ceramide generation, have been implicated in the control of programmed cell death. However, the regulation of the de novo pathway compared with the salvage pathway is not fully understood. In the current study, we have found that late accumulation of multiple ceramide and dihydroceramide species in MCF-7 cells treated with TNFα occurred by up-regulation of both pathways of ceramide synthesis. Nevertheless, fumonisin B1 but not myriocin was able to protect from TNFα-induced cell death, suggesting that ceramide synthase activity is crucial for the progression of cell death and that the pool of ceramide involved derives from the salvage pathway rather than de novo biosynthesis. Furthermore, compared with control cells, TNFα-treated cells exhibited reduced focal adhesion kinase and subsequent plasma membrane permeabilization, which was blocked exclusively by fumonisin B1. In addition, exogenously added C6-ceramide mimicked the effects of TNFα that lead to cell death, which were inhibited by fumonisin B1. Knockdown of individual ceramide synthases identified CerS6 and its product C16-ceramide as the ceramide synthase isoform essential for the regulation of cell death. In summary, our data suggest a novel role for CerS6/C16-ceramide as an upstream effector of the loss of focal adhesion protein and plasma membrane permeabilization, via the activation of caspase-7, and identify the salvage pathway as the critical mechanism of ceramide generation that controls cell death. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Tanshinone IIA protects H9c2 cells from oxidative stress-induced cell death via microRNA-133 upregulation and Akt activation.

PubMed

Gu, Yunfei; Liang, Zhuo; Wang, Haijun; Jin, Jun; Zhang, Shouyan; Xue, Shufeng; Chen, Jianfeng; He, Huijuan; Duan, Kadan; Wang, Jing; Chang, Xuewei; Qiu, Chunguang

2016-08-01

The aim of the present study was to investigate the cardioprotective effect of tanshinone IIA and the underlying molecular mechanisms. An in vitro model of oxidative stress injury was established in cardiac H9c2 cells, and the effects of tanshinone IIa were investigated using cell viability, reverse transcription-quantitative polymerase chain reaction and western blotting assays. The results demonstrated that tanshinone IIA protects H9c2 cells from H 2 O 2 -induced cell death in a concentration-dependent manner, via a mechanism involving microRNA-133 (miR-133), and that treatment with TIIA alone exerted no cytotoxic effects on H9c2. In order to further elucidate the mechanisms underlying the actions of TIIA, reverse transcription-quantitative polymease chain reaction and western blot analysis were performed. Reductions in miR-133 expression levels induced by increasing concentrations of H 2 O 2 were reversed by treatment with tanshinone IIA. In addition, the inhibition of miR-133 by transfection with an miR-133 inhibitor abolished the cardioprotective effects of tanshinone IIA against H 2 O 2 -induced cell death. Furthermore, western blot analysis demonstrated that tanshinone IIA activated Akt kinase via the phosphorylation of serine 473. Inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway by pretreatment with the PI3K specific inhibitors wortmannin and LY294002 also eliminated the cardioprotective effects of tanshinone IIA against H 2 O 2 -induced cell death. Western blot analysis demonstrated that H 2 O 2 -induced reductions in B cell lymphoma 2 (Bcl-2) expression levels were reversed by tanshinone IIA. In addition, the effect of tanshinone IIA on Bcl-2 protein expression level in an oxidative environment was suppressed by a PI3K inhibitor, wortmannin, indicating that tanshinone IIA exerts cardioprotective effects against H 2 O 2 -induced cell death via the activation of the PI3K/Akt signal transduction pathway and the consequent upregulation of Bcl-2. In conclusion, the present study demonstrates that TIIA is able to protcet H9c2 cells from oxidative stress-induced cell death through signalling pathways involving miR-133 and Akt, and that tanshinone IIA is a promising natural cardioprotective agent.

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

PubMed

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

2009-11-30

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

Modulating cell-to-cell variability and sensitivity to death ligands by co-drugging

NASA Astrophysics Data System (ADS)

Flusberg, Deborah A.; Sorger, Peter K.

2013-06-01

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) holds promise as an anti-cancer therapeutic but efficiently induces apoptosis in only a subset of tumor cell lines. Moreover, even in clonal populations of responsive lines, only a fraction of cells dies in response to TRAIL and individual cells exhibit cell-to-cell variability in the timing of cell death. Fractional killing in these cell populations appears to arise not from genetic differences among cells but rather from differences in gene expression states, fluctuations in protein levels and the extent to which TRAIL-induced death or survival pathways become activated. In this study, we ask how cell-to-cell variability manifests in cell types with different sensitivities to TRAIL, as well as how it changes when cells are exposed to combinations of drugs. We show that individual cells that survive treatment with TRAIL can regenerate the sensitivity and death-time distribution of the parental population, demonstrating that fractional killing is a stable property of cell populations. We also show that cell-to-cell variability in the timing and probability of apoptosis in response to treatment can be tuned using combinations of drugs that together increase apoptotic sensitivity compared to treatment with one drug alone. In the case of TRAIL, modulation of cell-to-cell variability by co-drugging appears to involve a reduction in the threshold for mitochondrial outer membrane permeabilization.

Bax-mediated mitochondrial outer membrane permeabilization (MOMP), distinct from the mitochondrial permeability transition, is a key mechanism in diclofenac-induced hepatocyte injury: Multiple protective roles of cyclosporin A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Siu, W.P.; Pun, Pamela Boon Li; Latchoumycandane, Calivarathan

2008-03-15

Diclofenac, a widely used nonsteroidal anti-inflammatory drug, has been associated with rare but severe cases of clinical hepatotoxicity. Diclofenac causes concentration-dependent cell death in human hepatocytes (after 24-48 h) by mitochondrial permeabilization via poorly defined mechanisms. To explore whether the cyclophilin D (CyD)-dependent mitochondrial permeability transition (mPT) and/or the mitochondrial outer membrane permeabilization (MOMP) was primarily involved in mediating cell death, we exposed immortalized human hepatocytes (HC-04) to apoptogenic concentrations of diclofenac (> 500 {mu}M) in the presence or absence of inhibitors of upstream mediators. The CyD inhibitor, cyclosporin A (CsA, 2 {mu}M) fully inhibited diclofenac-induced cell injury, suggesting thatmore » mPT was involved. However, CyD gene silencing using siRNA left the cells susceptible to diclofenac toxicity, and CsA still protected the CyD-negative cells from lethal injury. Diclofenac induced early (9 h) activation of Bax and Bak and caused mitochondrial translocation of Bax, indicating that MOMP was involved in cell death. Inhibition of Bax protein expression by using siRNA significantly protected HC-04 from diclofenac-induced cell injury. Diclofenac also induced early Bid activation (tBid formation, 6 h), which is an upstream mechanism that initiates Bax activation and mitochondrial translocation. Bid activation was sensitive to the Ca{sup 2+} chelator, BAPTA. In conclusion, we found that Bax/Bak-mediated MOMP is a key mechanism of diclofenac-induced lethal cell injury in human hepatocytes, and that CsA can prevent MOMP through inhibition of Bax activation. These data support our concept that the Ca{sup 2+}-Bid-Bax-MOMP axis is a critical pathway in diclofenac (metabolite)-induced hepatocyte injury.« less

Glucose deprivation activates a metabolic and signaling amplification loop leading to cell death

PubMed Central

Graham, Nicholas A; Tahmasian, Martik; Kohli, Bitika; Komisopoulou, Evangelia; Zhu, Maggie; Vivanco, Igor; Teitell, Michael A; Wu, Hong; Ribas, Antoni; Lo, Roger S; Mellinghoff, Ingo K; Mischel, Paul S; Graeber, Thomas G

2012-01-01

The altered metabolism of cancer can render cells dependent on the availability of metabolic substrates for viability. Investigating the signaling mechanisms underlying cell death in cells dependent upon glucose for survival, we demonstrate that glucose withdrawal rapidly induces supra-physiological levels of phospho-tyrosine signaling, even in cells expressing constitutively active tyrosine kinases. Using unbiased mass spectrometry-based phospho-proteomics, we show that glucose withdrawal initiates a unique signature of phospho-tyrosine activation that is associated with focal adhesions. Building upon this observation, we demonstrate that glucose withdrawal activates a positive feedback loop involving generation of reactive oxygen species (ROS) by NADPH oxidase and mitochondria, inhibition of protein tyrosine phosphatases by oxidation, and increased tyrosine kinase signaling. In cells dependent on glucose for survival, glucose withdrawal-induced ROS generation and tyrosine kinase signaling synergize to amplify ROS levels, ultimately resulting in ROS-mediated cell death. Taken together, these findings illustrate the systems-level cross-talk between metabolism and signaling in the maintenance of cancer cell homeostasis. PMID:22735335

Trimethyltin-induced apoptosis is associated with upregulation of inducible nitric oxide synthase and Bax in a hippocampal cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, L.; Li, L.; Prabhakaran, K.

2006-10-01

Trimethyltin (TMT) produces selective neuronal degeneration in the central nervous system (CNS), in which the hippocampus is the most sensitive area. Since previous studies have been conducted in either non-neural cells or mixed primary cultures, an immortalized hippocampal neuronal cell line (HT-22 cell) was used to assess the mechanism and mode of death produced by TMT. The compound produced a time- and concentration-dependent apoptotic death that was caspase-mediated. Excessive generation of reactive oxygen species (ROS) and subsequent reduction of mitochondrial membrane potential ({delta}{psi}{sub m}) were involved in the cytotoxicity{sub .} Scavenging of ROS by a free radical trapping agent ormore » inhibition of the mitochondrial permeability transition (MPT) pore significantly reduced cell death. Additionally, TMT increased expression of inducible nitric oxide synthase (iNOS) by activation of the redox-sensitive transcription factor NF{kappa}B. Pharmacologic inhibition studies showed that the iNOS-mediated NO generation increased expression of Bax and then mitochondrial-mediated apoptosis. It was concluded that excessive ROS generation initiated the apoptotic cell death by upregulating iNOS followed by increased Bax expression which then led to loss of {delta}{psi}{sub m} and caspase-executed cell death. This study is the first to report in a neuronal cell model that TMT stimulates induction of iNOS, which then increases cellular levels of reactive nitrogen species (RNS) to initiate apoptotic death.« less

Modulation of notch signaling pathway to prevent H2O2/menadione-induced SK-N-MC cells death by EUK134.

PubMed

Kamarehei, Maryam; Yazdanparast, Razieh

2014-10-01

The brain in Alzheimer's disease is under increased oxidative stress, and this may have a role in the pathogenesis and neural death in this disorder. It has been verified that numerous signaling pathways involved in neurodegenerative disorders are activated in response to reactive oxygen species (ROS). EUK134, a synthetic salen-manganese antioxidant complex, has been found to possess many interesting pharmacological activities awaiting exploration. The present study is to characterize the role of Notch signaling in apoptotic cell death of SK-N-MC cells. The cells were treated with hydrogen peroxide (H2O2) or menadione to induce oxidative stress. The free-radical scavenging capabilities of EUK134 were studied through the MTT assay, glutathione peroxidase (GPx) enzyme activity assay, and glutathione (GSH) Levels. The extents of lipid peroxidation, protein carbonyl formation, and intracellular ROS levels, as markers of oxidative stress, were also studied. Our results showed that H2O2/menadione reduced GSH levels and GPx activity. However, EUK134 protected cells against ROS-induced cell death by down-regulation of lipid peroxidation and protein carbonyl formation as well as restoration of antioxidant enzymes activity. ROS induced apoptosis and increased NICD and HES1 expression. Inhibition of NICD production proved that Notch signaling is involved in apoptosis through p53 activation. Moreover, H2O2/menadione led to Numb protein down-regulation which upon EUK134 pretreatment, its level increased and subsequently prevented Notch pathway activation. We indicated that EUK134 can be a promising candidate in designing natural-based drugs for ROS-induced neurodegenerative diseases. Collectively, ROS activated Notch signaling in SK-N-MC cells leading to cell apoptosis.

Proteomics and Functional Analyses of Pepper Abscisic Acid–Responsive 1 (ABR1), Which Is Involved in Cell Death and Defense Signaling[C][W

PubMed Central

Choi, Du Seok; Hwang, Byung Kook

2011-01-01

Abscisic acid (ABA) is a key regulator of plant growth and development, as well as plant defense responses. A high-throughput in planta proteome screen identified the pepper (Capsicum annuum) GRAM (for glucosyltransferases, Rab-like GTPase activators, and myotubularins) domain-containing ABA-RESPONSIVE1 (ABR1), which is highly induced by infection with avirulent Xanthomonas campestris pv vesicatoria and also by treatment with ABA. The GRAM domain is essential for the cell death response and for the nuclear localization of ABR1. ABR1 is required for priming cell death and reactive oxygen species production, as well as ABA-salicylic acid (SA) antagonism. Silencing of ABR1 significantly compromised the hypersensitive response but enhanced bacterial pathogen growth and ABA levels in pepper. High levels of ABA in ABR1-silenced plants antagonized the SA levels induced by pathogen infection. Heterologous transgenic expression of ABR1 in Arabidopsis thaliana conferred enhanced resistance to Pseudomonas syringae pv tomato and Hyaloperonospora arabidopsidis infection. The susceptibility of the Arabidopsis ABR1 putative ortholog mutant, abr1, to these pathogens also supports the involvement of ABR1 in disease resistance. Together, these results reveal ABR1 as a novel negative regulator of ABA signaling and suggest that the nuclear ABR1 pool is essential for the cell death induction associated with ABA-SA antagonism. PMID:21335377

TRAIL enhances paracetamol-induced liver sinusoidal endothelial cell death in a Bim- and Bid-dependent manner

PubMed Central

Badmann, A; Langsch, S; Keogh, A; Brunner, T; Kaufmann, T; Corazza, N

2012-01-01

Paracetamol (acetaminophen, APAP) is a universally used analgesic and antipyretic agent. Considered safe at therapeutic doses, overdoses cause acute liver damage characterized by centrilobular hepatic necrosis. One of the major clinical problems of paracetamol-induced liver disease is the development of hemorrhagic alterations. Although hepatocytes represent the main target of the cytotoxic effect of paracetamol overdose, perturbations within the endothelium involving morphological changes of liver sinusoidal endothelial cells (LSECs) have also been described in paracetamol-induced liver disease. Recently, we have shown that paracetamol-induced liver damage is synergistically enhanced by the TRAIL signaling pathway. As LSECs are constantly exposed to activated immune cells expressing death ligands, including TRAIL, we investigated the effect of TRAIL on paracetamol-induced LSEC death. We here demonstrate for the first time that TRAIL strongly enhances paracetamol-mediated LSEC death with typical features of apoptosis. Inhibition of caspases using specific inhibitors resulted in a strong reduction of cell death. TRAIL appears to enhance paracetamol-induced LSEC death via the activation of the pro-apoptotic BH3-only proteins Bid and Bim, which initiate the mitochondrial apoptotic pathway. Taken together this study shows that the liver endothelial layer, mainly LSECs, represent a direct target of the cytotoxic effect of paracetamol and that activation of TRAIL receptor synergistically enhances paracetamol-induced LSEC death via the mitochondrial apoptotic pathway. TRAIL-mediated acceleration of paracetamol-induced cell death may thus contribute to the pathogenesis of paracetamol-induced liver damage. PMID:23254290

Neurons other than motor neurons in motor neuron disease.

PubMed

Ruffoli, Riccardo; Biagioni, Francesca; Busceti, Carla L; Gaglione, Anderson; Ryskalin, Larisa; Gambardella, Stefano; Frati, Alessandro; Fornai, Francesco

2017-11-01

Amyotrophic lateral sclerosis (ALS) is typically defined by a loss of motor neurons in the central nervous system. Accordingly, morphological analysis for decades considered motor neurons (in the cortex, brainstem and spinal cord) as the neuronal population selectively involved in ALS. Similarly, this was considered the pathological marker to score disease severity ex vivo both in patients and experimental models. However, the concept of non-autonomous motor neuron death was used recently to indicate the need for additional cell types to produce motor neuron death in ALS. This means that motor neuron loss occurs only when they are connected with other cell types. This concept originally emphasized the need for resident glia as well as non-resident inflammatory cells. Nowadays, the additional role of neurons other than motor neurons emerged in the scenario to induce non-autonomous motor neuron death. In fact, in ALS neurons diverse from motor neurons are involved. These cells play multiple roles in ALS: (i) they participate in the chain of events to produce motor neuron loss; (ii) they may even degenerate more than and before motor neurons. In the present manuscript evidence about multi-neuronal involvement in ALS patients and experimental models is discussed. Specific sub-classes of neurons in the whole spinal cord are reported either to degenerate or to trigger neuronal degeneration, thus portraying ALS as a whole spinal cord disorder rather than a disease affecting motor neurons solely. This is associated with a novel concept in motor neuron disease which recruits abnormal mechanisms of cell to cell communication.

Stathmin Mediates Hepatocyte Resistance to Death from Oxidative Stress by down Regulating JNK

PubMed Central

Zhao, Enpeng; Amir, Muhammad; Lin, Yu; Czaja, Mark J.

2014-01-01

Stathmin 1 performs a critical function in cell proliferation by regulating microtubule polymerization. This proliferative function is thought to explain the frequent overexpression of stathmin in human cancer and its correlation with a bad prognosis. Whether stathmin also functions in cell death pathways is unclear. Stathmin regulates microtubules in part by binding free tubulin, a process inhibited by stathmin phosphorylation from kinases including c-Jun N-terminal kinase (JNK). The involvement of JNK activation both in stathmin phosphorylation, and in hepatocellular resistance to oxidative stress, led to an examination of the role of stathmin/JNK crosstalk in oxidant-induced hepatocyte death. Oxidative stress from menadione-generated superoxide induced JNK-dependent stathmin phosphorylation at Ser-16, Ser-25 and Ser-38 in hepatocytes. A stathmin knockdown sensitized hepatocytes to both apoptotic and necrotic cell death from menadione without altering levels of oxidant generation. The absence of stathmin during oxidative stress led to JNK overactivation that was the mechanism of cell death as a concomitant knockdown of JNK1 or JNK2 blocked death. Hepatocyte death from JNK overactivation was mediated by the effects of JNK on mitochondria. Mitochondrial outer membrane permeabilization occurred in stathmin knockdown cells at low concentrations of menadione that triggered apoptosis, whereas mitochondrial β-oxidation and ATP homeostasis were compromised at higher, necrotic menadione concentrations. Stathmin therefore mediates hepatocyte resistance to death from oxidative stress by down regulating JNK and maintaining mitochondrial integrity. These findings demonstrate a new mechanism by which stathmin promotes cell survival and potentially tumor growth. PMID:25285524

Recommendations from the INHAND Apoptosis/Necrosis Working Group.

PubMed

Elmore, Susan A; Dixon, Darlene; Hailey, James R; Harada, Takanori; Herbert, Ronald A; Maronpot, Robert R; Nolte, Thomas; Rehg, Jerold E; Rittinghausen, Susanne; Rosol, Thomas J; Satoh, Hiroshi; Vidal, Justin D; Willard-Mack, Cynthia L; Creasy, Dianne M

2016-02-01

Historically, there has been confusion relating to the diagnostic nomenclature for individual cell death. Toxicologic pathologists have generally used the terms "single cell necrosis" and "apoptosis" interchangeably. Increased research on the mechanisms of cell death in recent years has led to the understanding that apoptosis and necrosis involve different cellular pathways and that these differences can have important implications when considering overall mechanisms of toxicity, and, for these reasons, the separate terms of apoptosis and necrosis should be used whenever differentiation is possible. However, it is also recognized that differentiation of the precise pathway of cell death may not be important, necessary, or possible in routine toxicity studies and so a more general term to indicate cell death is warranted in these situations. Morphological distinction between these two forms of cell death can sometimes be straightforward but can also be challenging. This article provides a brief discussion of the cellular mechanisms and morphological features of apoptosis and necrosis as well as guidance on when the pathologist should use these terms. It provides recommended nomenclature along with diagnostic criteria (in hematoxylin and eosin [H&E]-stained sections) for the most common forms of cell death (apoptosis and necrosis). This document is intended to serve as current guidance for the nomenclature of cell death for the International Harmonization of Nomenclature and Diagnostic Criteria Organ Working Groups and the toxicologic pathology community at large. The specific recommendations are:Use necrosis and apoptosis as separate diagnostic terms.Use modifiers to denote the distribution of necrosis (e.g., necrosis, single cell; necrosis, focal; necrosis, diffuse; etc.).Use the combined term apoptosis/single cell necrosis whenThere is no requirement or need to split the processes, orWhen the nature of cell death cannot be determined with certainty, orWhen both processes are present together. The diagnosis should be based primarily on the morphological features in H&E-stained sections. When needed, additional, special techniques to identify and characterize apoptosis can also be used. © The Author(s) 2016.

Circadian Stress Regimes Affect the Circadian Clock and Cause Jasmonic Acid-Dependent Cell Death in Cytokinin-Deficient Arabidopsis Plants.

PubMed

Nitschke, Silvia; Cortleven, Anne; Iven, Tim; Feussner, Ivo; Havaux, Michel; Riefler, Michael; Schmülling, Thomas

2016-07-01

The circadian clock helps plants measure daylength and adapt to changes in the day-night rhythm. We found that changes in the light-dark regime triggered stress responses, eventually leading to cell death, in leaves of Arabidopsis thaliana plants with reduced cytokinin levels or defective cytokinin signaling. Prolonged light treatment followed by a dark period induced stress and cell death marker genes while reducing photosynthetic efficiency. This response, called circadian stress, is also characterized by altered expression of clock and clock output genes. In particular, this treatment strongly reduced the expression of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). Intriguingly, similar changes in gene expression and cell death were observed in clock mutants lacking proper CCA1 and LHY function. Circadian stress caused strong changes in reactive oxygen species- and jasmonic acid (JA)-related gene expression. The activation of the JA pathway, involving the accumulation of JA metabolites, was crucial for the induction of cell death, since the cell death phenotype was strongly reduced in the jasmonate resistant1 mutant background. We propose that adaptation to circadian stress regimes requires a normal cytokinin status which, acting primarily through the AHK3 receptor, supports circadian clock function to guard against the detrimental effects of circadian stress. © 2016 American Society of Plant Biologists. All rights reserved.

Over-expression of Trxo1 increases the viability of tobacco BY-2 cells under H2O2 treatment

PubMed Central

Ortiz-Espín, Ana; Locato, Vittoria; Camejo, Daymi; Schiermeyer, Andreas; De Gara, Laura; Sevilla, Francisca; Jiménez, Ana

2015-01-01

Background and Aims Reactive oxygen species (ROS), especially hydrogen peroxide, play a critical role in the regulation of plant development and in the induction of plant defence responses during stress adaptation, as well as in plant cell death. The antioxidant system is responsible for controlling ROS levels in these processes but redox homeostasis is also a key factor in plant cell metabolism under normal and stress situations. Thioredoxins (Trxs) are ubiquitous small proteins found in different cell compartments, including mitochondria and nuclei (Trxo1), and are involved in the regulation of target proteins through reduction of disulphide bonds, although their role under oxidative stress has been less well studied. This study describes over-expression of a Trxo1 for the first time, using a cell-culture model subjected to an oxidative treatment provoked by H2O2. Methods Control and over-expressing PsTrxo1 tobacco (Nicotiana tabacum) BY-2 cells were treated with 35 mm H2O2 and the effects were analysed by studying the growth dynamics of the cultures together with oxidative stress parameters, as well as several components of the antioxidant systems involved in the metabolism of H2O2. Analysis of different hallmarks of programmed cell death was also carried out. Key Results Over-expression of PsTrxo1 caused significant differences in the response of TBY-2 cells to high concentrations of H2O2, namely higher and maintained viability in over-expressing cells, whilst the control line presented a severe decrease in viability and marked indications of oxidative stress, with generalized cell death after 3 d of treatment. In over-expressing cells, an increase in catalase activity, decreases in H2O2 and nitric oxide contents and maintenance of the glutathione redox state were observed. Conclusions A decreased content of endogenous H2O2 may be responsible in part for the delayed cell death found in over-expressing cells, in which changes in oxidative parameters and antioxidants were less extended after the oxidative treatment. It is concluded that PsTrxo1 transformation protects TBY-2 cells from exogenous H2O2, thus increasing their viability via a process in which not only antioxidants but also Trxo1 seem to be involved. PMID:26041732

PI3K and Inhibitor of Apoptosis Proteins Modulate Gentamicin- Induced Hair Cell Death in the Zebrafish Lateral Line

PubMed Central

Wiedenhoft, Heather; Hayashi, Lauren; Coffin, Allison B.

2017-01-01

Inner ear hair cell death leads to sensorineural hearing loss and can be a direct consequence of aminoglycoside antibiotic treatment. Aminoglycosides such as gentamicin are effective therapy for serious Gram-negative bacterial infections such as some forms of meningitis, pneumonia, and sepsis. Aminoglycosides enter hair cells through mechanotransduction channels at the apical end of hair bundles and initiate intrinsic cell death cascades, but the precise cell signaling that leads to hair cell death is incompletely understood. Here, we examine the cell death pathways involved in aminoglycoside damage using the zebrafish (Danio rerio). The zebrafish lateral line contains hair cell-bearing organs called neuromasts that are homologous to hair cells of the mammalian inner ear and represents an excellent model to study ototoxicity. Based on previous research demonstrating a role for p53, Bcl2 signaling, autophagy, and proteasomal degradation in aminoglycoside-damaged hair cells, we used the Cytoscape GeneMANIA Database to identify additional proteins that might play a role in neomycin or gentamicin ototoxicity. Our bioinformatics analysis identified the pro-survival proteins phosphoinositide-dependent kinase-1 (PDK1) and X-linked inhibitor of apoptosis protein (Xiap) as potential mediators of gentamicin-induced hair cell damage. Pharmacological inhibition of PDK1 or its downstream mediator protein kinase C facilitated gentamicin toxicity, as did Xiap mutation, suggesting that both PI3K and endogenous Xiap confer protection. Surprisingly, aminoglycoside-induced hair cell death was highly attenuated in wild type Tupfel long-fin (TL fish; the background strain for the Xiap mutant line) compared to wild type ∗AB zebrafish. Pharmacologic manipulation of p53 suggested that the strain difference might result from decreased p53 in TL hair cells, allowing for increased hair cell survival. Overall, our studies identified additional steps in the cell death cascade triggered by aminoglycoside damage, suggesting possible drug targets to combat hearing loss resulting from aminoglycoside exposure. PMID:29093665

RIPK1/RIPK3/MLKL-mediated necroptosis contributes to compression-induced rat nucleus pulposus cells death.

PubMed

Chen, Songfeng; Lv, Xiao; Hu, Binwu; Shao, Zengwu; Wang, Baichuan; Ma, Kaige; Lin, Hui; Cui, Min

2017-05-01

The aim of this study was to systematically investigate the role of necroptosis in compression-induced rat nucleus pulposus (NP) cells death, as well as explore the underlying mechanisms involved. Rat NP cells underwent various periods of exposure to 1.0 MPa pressure. Cell viability and cell death were quantified by using cell counting kit-8 (CCK-8), and Calcein-AM/propidium iodine (PI) staining respectively. Necroptosis-associated target molecules receptor-interacing protein kinase 1 (RIPK1), phosphorylated RIPK1 (pRIPK1), receptor-interacing protein kinase 3 (RIPK3), phosphorylated RIPK3 (pRIPK3) and mixed lineage kinase domain-like (MLKL) were analyzed by Western-blot and RT-PCR. NP cells were also examined for morphological and ultrastructural changes, which can indicate necroptosis. To indirectly establish the presence of necroptosis, the RIPK1 specific inhibitor necrostatin-1 (Nec-1), RIPK3 inhibitor GSK'872, MLKL inhibitor necrosulfonamide (NSA) and small interfering RNA (siRNA) were utilized. The results established necroptosis was taking place in NP cells. The level of necroptosis increased in a time-dependent manner, and this effect was reduced by Nec-1 in vitro. Additionally, NP cells death were significantly attenuated following treatment with Nec-1, GSK'872 or NSA. SiRNA-induced knockdown of RIPK3 or MLKL increased cell survival rate, while knockdown of RIPK1 resulted in a decreased cell survival rate. In summary, RIPK1/RIPK3/MLKL-mediated necroptosis may play an important role in NP cells death induced by continuous mechanical stress. Treatment strategies which aim to regulate necroptosis may prove beneficial, by both reducing NP cells death and slowing IVD degeneration.

GILZ overexpression attenuates endoplasmic reticulum stress-mediated cell death via the activation of mitochondrial oxidative phosphorylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

André, Fanny; Corazao-Rozas, Paola; Idziorek, Thierry

The Glucocorticoïd-induced leucine zipper (GILZ) protein has profound anti-inflammatory activities in haematopoietic cells. GILZ regulates numerous signal transduction pathways involved in proliferation and survival of normal and neoplastic cells. Here, we have demonstrated the potential of GILZ in alleviating apoptosis induced by ER stress inducers. Whereas the glucocorticoid, dexamethasone, protects from tunicamycin-induced cell death, silencing endogeneous GILZ in dexamethasone-treated cancer cells alter the capacity of glucocorticoids to protect from tunicamycin-mediated apoptosis. Under ER stress conditions, overexpression of GILZ significantly reduced activation of mitochondrial pathway of apoptosis by maintaining Bcl-xl level. GILZ protein affects the UPR signaling shifting the balance towardsmore » pro-survival signals as judged by down-regulation of CHOP, ATF4, XBP1s mRNA and increase in GRP78 protein level. Interestingly, GILZ sustains high mitochondrial OXPHOS during ER stress and cytoprotection mediated by GILZ is abolished in cells depleted of mitochondrial DNA, which are OXPHOS-deficient. These findings reveal a new role of GILZ, which acts as a cytoprotector against ER stress through a pathway involving mitochondrial OXPHOS. - Highlights: • GILZ attenuates apoptotic cell death induced by ER stress conditions. • GILZ promotes pro-survival signaling of the UPR. • GILZ overexpression sustains high mitochondrial activity under ER stress. • Mitochondrial OXPHOX is required for GILZ protective effects against ER stress-mediated apoptosis.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woo, Sang Hyeok; Seo, Sung-Keum; An, Sungkwan

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

Programmed cell death during development of cowpea (Vigna unguiculata (L.) Walp.) seed coat.

PubMed

Lima, Nathália Bastos; Trindade, Fernanda Gomes; da Cunha, Maura; Oliveira, Antônia Elenir Amâncio; Topping, Jennifer; Lindsey, Keith; Fernandes, Kátia Valevski Sales

2015-04-01

The seed coat develops primarily from maternal tissues and comprises multiple cell layers at maturity, providing a metabolically dynamic interface between the developing embryo and the environment during embryogenesis, dormancy and germination of seeds. Seed coat development involves dramatic cellular changes, and the aim of this research was to investigate the role of programmed cell death (PCD) events during the development of seed coats of cowpea [Vigna unguiculata (L.) Walp.]. We demonstrate that cells of the developing cowpea seed coats undergo a programme of autolytic cell death, detected as cellular morphological changes in nuclei, mitochondria, chloroplasts and vacuoles, DNA fragmentation and oligonucleosome accumulation in the cytoplasm, and loss of membrane viability. We show for the first time that classes 6 and 8 caspase-like enzymes are active during seed coat development, and that these activities may be compartmentalized by translocation between vacuoles and cytoplasm during PCD events. © 2014 John Wiley & Sons Ltd.

Metal-accelerated oxidation in plant cell death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Czuba, M.

1993-05-01

Cadmium and mercury toxicity is further enhanced by external oxidizing conditions O[sub 3] or inherent plant processes. Lepidium sativum L, Lycopersicon esculentum Mill., or Phaseolus vulgaris L, were grown inpeat-lite to maturity under continuous cadmium exposure followed by one oxidant (O[sub 3]-6 hr. 30 pphm) exposure, with or without foliar calcium pretreatments. In comparison, Daucus carota, L and other species grown in a 71-V suspension, with or without 2,4-D were exposed continuously to low levels of methylmercury during exponential growth and analyzed in aggregates of distinct populations. Proteins were extracted and analyzed. Mechanisms of toxicity and eventual cell death aremore » Ca-mediated and involve chloroplast, stomatal-water relations and changes in oxidant-anti-oxidant components in cells. Whether the metal-accelerated oxidative damage proceeds to cell death, depends on the species and its differential biotransformation system and cell association component.« less

Reduction of conventional dendritic cells during Plasmodium infection is dependent on activation induced cell death by type I and II interferons.

PubMed

Tamura, Takahiko; Kimura, Kazumi; Yui, Katsuyuki; Yoshida, Shigeto

2015-12-01

Dendritic cells (DCs) play critical roles in innate and adaptive immunity and in pathogenesis during the blood stage of malaria infection. The mechanisms underlying DC homeostasis during malaria infection are not well understood. In this study, the numbers of conventional DCs (cDCs) and plasmacytoid DCs (pDCs) in the spleens after lethal rodent malaria infection were examined, and were found to be significantly reduced. Concomitant with up-regulation of maturation-associated molecules, activation of caspase-3 was significantly increased, suggesting induction of cell death. Studies using neutralizing antibody and gene-deficient mice showed that type I and II interferons were critically involved in activation induced cell death of cDCs during malaria infection. These results demonstrate that DCs rapidly disappeared following IFN-mediated DC activation, and that homeostasis of DCs was significantly impaired during malaria infection. Copyright © 2015 Elsevier Inc. All rights reserved.

In-depth proteomic profiling of left ventricular tissues in human end-stage dilated cardiomyopathy.

PubMed

Liu, Shanshan; Xia, Yan; Liu, Xiaohui; Wang, Yi; Chen, Zhangwei; Xie, Juanjuan; Qian, Juying; Shen, Huali; Yang, Pengyuan

2017-07-18

Dilated cardiomyopathy (DCM) is caused by reduced left ventricular (LV) myocardial function, which is one of the most common causes of heart failure (HF). We performed iTRAQ-coupled 2D-LC-MS/MS to profile the cardiac proteome of LV tissues from healthy controls and patients with end-stage DCM. We identified 4263 proteins, of which 125 were differentially expressed in DCM tissues compared to LV controls. The majority of these were membrane proteins related to cellular junctions and neuronal metabolism. In addition, these proteins were involved in membrane organization, mitochondrial organization, translation, protein transport, and cell death process. Four key proteins involved in the cell death process were also detected by western blotting, indicated that cell death was activated in DCM tissues. Furthermore, S100A1 and eEF2 were enriched in the "cellular assembly and organization" and "cell cycle" networks, respectively. We verified decreases in these two proteins in end-stage DCM LV samples through multiple reaction monitoring (MRM). These observations demonstrate that our understanding of the mechanisms underlying DCM can be deepened through comparison of the proteomes of normal LV tissues with that from end-stage DCM in humans.

Mechanism of Cisplatin-Induced Cytotoxicity Is Correlated to Impaired Metabolism Due to Mitochondrial ROS Generation.

PubMed

Choi, Yong-Min; Kim, Han-Kyul; Shim, Wooyoung; Anwar, Muhammad Ayaz; Kwon, Ji-Woong; Kwon, Hyuk-Kwon; Kim, Hyung Joong; Jeong, Hyobin; Kim, Hwan Myung; Hwang, Daehee; Kim, Hyung Sik; Choi, Sangdun

2015-01-01

The chemotherapeutic use of cisplatin is limited by its severe side effects. In this study, by conducting different omics data analyses, we demonstrated that cisplatin induces cell death in a proximal tubular cell line by suppressing glycolysis- and tricarboxylic acid (TCA)/mitochondria-related genes. Furthermore, analysis of the urine from cisplatin-treated rats revealed the lower expression levels of enzymes involved in glycolysis, TCA cycle, and genes related to mitochondrial stability and confirmed the cisplatin-related metabolic abnormalities. Additionally, an increase in the level of p53, which directly inhibits glycolysis, has been observed. Inhibition of p53 restored glycolysis and significantly reduced the rate of cell death at 24 h and 48 h due to p53 inhibition. The foremost reason of cisplatin-related cytotoxicity has been correlated to the generation of mitochondrial reactive oxygen species (ROS) that influence multiple pathways. Abnormalities in these pathways resulted in the collapse of mitochondrial energy production, which in turn sensitized the cells to death. The quenching of ROS led to the amelioration of the affected pathways. Considering these observations, it can be concluded that there is a significant correlation between cisplatin and metabolic dysfunctions involving mROS as the major player.

Gasdermin: A new player to the inflammasome game.

PubMed

Ramos-Junior, Erivan S; Morandini, Ana Carolina

2017-12-01

Pyroptosis is a lytic type of programmed cell death that was traditionally associated with the involvement of inflammatory caspases, such as caspase-1. These inflammatory caspases are activated within multi-protein complexes called inflammasomes that are assembled in response to invading pathogens and/or danger signals. Pyroptotic cell death was suggested to evolve via the formation of pores in the plasma membrane, but the exact mechanism underlying the formation of these pores remained unclear. Recently, gasdermin D, a member of the gasdermin protein family was identified as a caspase substrate and essential effector of pyroptosis, being identified as the protagonist of membrane pore formation. Gasdermins have emerged as a family of new class of cell death inducers, but many questions remain unanswered. Here, we present an overview of recent work being done in the area of programmed cell death and the latest evidence regarding the role and participation of gasdermin D as an effector of pyroptosis. Copyright © 2017 Chang Gung University. Published by Elsevier B.V. All rights reserved.

Anti-Yo antibody uptake and interaction with its intracellular target antigen causes Purkinje cell death in rat cerebellar slice cultures: a possible mechanism for paraneoplastic cerebellar degeneration in humans with gynecological or breast cancers.

PubMed

Greenlee, John E; Clawson, Susan A; Hill, Kenneth E; Wood, Blair; Clardy, Stacey L; Tsunoda, Ikuo; Carlson, Noel G

2015-01-01

Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat cerebellar slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat cerebellar slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not initiated by brain mononuclear cells. Purkinje cell death was not simply due to intraneuronal antibody accumulation.

Anti-Yo Antibody Uptake and Interaction with Its Intracellular Target Antigen Causes Purkinje Cell Death in Rat Cerebellar Slice Cultures: A Possible Mechanism for Paraneoplastic Cerebellar Degeneration in Humans with Gynecological or Breast Cancers

PubMed Central

Greenlee, John E.; Clawson, Susan A.; Hill, Kenneth E.; Wood, Blair; Clardy, Stacey L.; Tsunoda, Ikuo; Carlson, Noel G.

2015-01-01

Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat cerebellar slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat cerebellar slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not initiated by brain mononuclear cells. Purkinje cell death was not simply due to intraneuronal antibody accumulation. PMID:25885452

Ursodeoxycholic acid effectively kills drug-resistant gastric cancer cells through induction of autophagic death.

PubMed

Lim, Sung-Chul; Han, Song Iy

2015-09-01

Carcinoma cells that have acquired drug resistance often exhibit cross-resistance to various other cytotoxic stimuli. Here, we investigated the effects of ursodeoxycholic acid (UDCA), a gastrointestinal tumor-suppressor, on a cisplatin‑resistant SNU601 gastric cancer subline (SNU601/R). While other anticancer drugs, including L-OHP, etoposide, and death ligand TRAIL, had minimal effects on the viability of these resistant cells, they were sensitive to UDCA. The UDCA‑induced reduction in the viability of the SNU601/R cells was accomplished through autophagy while the primary means of cell death in the parental SNU601 cells (SNU601/WT) was apoptosis. Previously, we demonstrated that the UDCA-triggered apoptosis of gastric cancer cells was regulated by a cell surface death receptor, TRAIL-R2/DR5, which was upregulated and re-distributed on lipid rafts. The UDCA stimulation of TRAIL-R2/DR5 also occurred in the SNU601/R cells despite the lack of apoptosis. In the present study, we found that CD95/Fas, another cell surface death receptor, was also translocated into lipid rafts in response to UDCA although it was not involved in the decrease in cell viability. Specifically, raft relocalization of CD95/Fas was triggered by UDCA in the SNU601/WT cells in which apoptosis occurred, but not in the SNU601/R cells where autophagic death occurred. Notably, UDCA reduced ATG5 levels, an essential component of autophagy, in the SNU601/WT, but not in the SNU601/R cell line. Moreover, in CD95/Fas-silenced SNU601/WT cells, UDCA did not decrease ATG5 levels and induced autophagic cell death rather than apoptosis. These results imply that raft‑distributed CD95/Fas may support UDCA-induced apoptosis via downregulation of ATG5 levels, preventing the autophagic pathway. Taken together, these results suggest that UDCA induces both apoptotic and autophagic cell death depending on the intracellular signaling environment, thereby conferring the advantage to overcome drug resistance through apoptotic defects.

Hypoglycemic neuronal death is triggered by glucose reperfusion and activation of neuronal NADPH oxidase

PubMed Central

Suh, Sang Won; Gum, Elizabeth T.; Hamby, Aaron M.; Chan, Pak H.; Swanson, Raymond A.

2007-01-01

Hypoglycemic coma and brain injury are potential complications of insulin therapy. Certain neurons in the hippocampus and cerebral cortex are uniquely vulnerable to hypoglycemic cell death, and oxidative stress is a key event in this cell death process. Here we show that hypoglycemia-induced oxidative stress and neuronal death are attributable primarily to the activation of neuronal NADPH oxidase during glucose reperfusion. Superoxide production and neuronal death were blocked by the NADPH oxidase inhibitor apocynin in both cell culture and in vivo models of insulin-induced hypoglycemia. Superoxide production and neuronal death were also blocked in studies using mice or cultured neurons deficient in the p47phox subunit of NADPH oxidase. Chelation of zinc with calcium disodium EDTA blocked both the assembly of the neuronal NADPH oxidase complex and superoxide production. Inhibition of the hexose monophosphate shunt, which utilizes glucose to regenerate NADPH, also prevented superoxide formation and neuronal death, suggesting a mechanism linking glucose reperfusion to superoxide formation. Moreover, the degree of superoxide production and neuronal death increased with increasing glucose concentrations during the reperfusion period. These results suggest that high blood glucose concentrations following hypoglycemic coma can initiate neuronal death by a mechanism involving extracellular zinc release and activation of neuronal NADPH oxidase. PMID:17404617

Methadone induces CAD degradation and AIF-mediated necrotic-like cell death in neuroblastoma cells.

PubMed

Perez-Alvarez, Sergio; Iglesias-Guimarais, Victoria; Solesio, María E; Melero-Fernandez de Mera, Raquel María; Yuste, Víctor J; Galindo, María F; Jordán, Joaquín

2011-04-01

Methadone (d,l-methadone hydrochloride) is a full-opioid agonist, originally developed as a substitution for heroin or other opiates abusers. Nowadays methadone is also being applied as long-lasting analgesics in cancer, and it is proposed as a promising agent for leukemia therapy. Previously, we have demonstrated that high concentrations of methadone (0.5mM) induced necrotic-like cell death in SH-SY5Y cells. The pathway involved is caspase-independent but involves impairment of mitochondrial ATP synthesis and mitochondrial cytochrome c release. However, the downstream mitochondrial pathways remained unclear. Here, we studied the participation of apoptosis inducing factor (AIF) in methadone-induced cell death. Methadone resulted in a translocation of AIF from mitochondria to the nucleus. Translocation was inhibited by cyclosporine A, but not by lack of Bax protein. Therefore the effect seems mediated by the formation of the mitochondrial transition pore, but is apparently independent of Bax. Furthermore, methadone-treated SH-SY5Y nuclei show characteristics that are typical for stage I nuclear condensation. Methadone did not induce degradation of DNA into oligonucleosomal fragments or into high molecular weight DNA fragments. Absence of DNA fragmentation coincided with a considerable decrease in the levels of the caspase-actived endonuclase DNase and its chaperone-inhibitor ICAD. In conclusion, our results provide mechanistic insights into the molecular mechanisms that underlie methadone-induced cell death. This knowledge may prove useful to develop novel strategies to prevent toxic side-effects of methadone thereby sustaining its use as therapeutical agent against tumors. Copyright © 2010 Elsevier Ltd. All rights reserved.

Cocaine-Mediated Autophagy in Astrocytes Involves Sigma 1 Receptor, PI3K, mTOR, Atg5/7, Beclin-1 and Induces Type II Programed Cell Death.

PubMed

Cao, Lu; Walker, Mary P; Vaidya, Naveen K; Fu, Mingui; Kumar, Santosh; Kumar, Anil

2016-09-01

Cocaine, a commonly used drug of abuse, has been shown to cause neuropathological dysfunction and damage in the human brain. However, the role of autophagy in this process is not defined. Autophagy, generally protective in nature, can also be destructive leading to autophagic cell death. This study was designed to investigate whether cocaine induces autophagy in the cells of CNS origin. We employed astrocyte, the most abundant cell in the CNS, to define the effects of cocaine on autophagy. We measured levels of the autophagic marker protein LC3II in SVGA astrocytes after exposure with cocaine. The results showed that cocaine caused an increase in LC3II level in a dose- and time-dependent manner, with the peak observed at 1 mM cocaine after 6-h exposure. This result was also confirmed by detecting LC3II in SVGA astrocytes using confocal microscopy and transmission electron microscopy. Next, we sought to explore the mechanism by which cocaine induces the autophagic response. We found that cocaine-induced autophagy was mediated by sigma 1 receptor, and autophagy signaling proteins p-mTOR, Atg5, Atg7, and p-Bcl-2/Beclin-1 were also involved, and this was confirmed by using selective inhibitors and small interfering RNAs (siRNAs). In addition, we found that chronic treatment with cocaine resulted in cell death, which is caspase-3 independent and can be ameliorated by autophagy inhibitor. Therefore, this study demonstrated that cocaine induces autophagy in astrocytes and is associated with autophagic cell death.

Cocaine-mediated autophagy in astrocytes involves sigma 1 receptor, PI3K, m-TOR, Atg5/7, Beclin-1 and induces Type II programed cell death

PubMed Central

Cao, Lu; Walker, Mary P; Vaidya, Naveen K; Fu, Mingui; Kumar, Santosh; Kumar, Anil

2015-01-01

Cocaine, a commonly used drug of abuse, has been shown to cause neuropathological dysfunction and damage in the human brain. However, the role of autophagy in this process is not defined. Autophagy generally protective in nature, can also be destructive leading to autophagic cell death. This study was designed to investigate whether cocaine induces autophagy in the cells of CNS origin. We employed astrocyte, the most abundant cell in the CNS, to define the effects of cocaine on autophagy. We measured levels of the autophagic marker protein LC3II in SVGA astrocytes after exposure with cocaine. The results showed that cocaine caused an increase in LC3II level in a dose- and time-dependent manner, with the peak observed at 1 mM cocaine after 6 hours exposure. This result was also confirmed by detecting LC3II in SVGA astrocytes using confocal microscopy and transmission electron microscopy. Next, we sought to explore the mechanism by which cocaine induces the autophagic response. We found that cocaine-induced autophagy was mediated by sigma 1 receptor, and autophagy signaling proteins p-mTOR, Atg5, Atg7 and p-Bcl-2/Beclin-1 were also involved and this was confirmed by using selective inhibitors and siRNAs. In addition, we found that chronic treatment with cocaine resulted in cell death, which is caspase-3 independent, and can be ameliorated by autophagy inhibitor. Therefore, this study demonstrated that cocaine induces autophagy in astrocytes and is associated with autophagic cell death. PMID:26243186

Involvement of Programmed Cell Death in Neurotoxicity of Metallic Nanoparticles: Recent Advances and Future Perspectives

NASA Astrophysics Data System (ADS)

Song, Bin; Zhou, Ting; Liu, Jia; Shao, LongQuan

2016-11-01

The widespread application of metallic nanoparticles (NPs) or NP-based products has increased the risk of exposure to NPs in humans. The brain is an important organ that is more susceptible to exogenous stimuli. Moreover, any impairment to the brain is irreversible. Recently, several in vivo studies have found that metallic NPs can be absorbed into the animal body and then translocated into the brain, mainly through the blood-brain barrier and olfactory pathway after systemic administration. Furthermore, metallic NPs can cross the placental barrier to accumulate in the fetal brain, causing developmental neurotoxicity on exposure during pregnancy. Therefore, metallic NPs become a big threat to the brain. However, the mechanisms underlying the neurotoxicity of metallic NPs remain unclear. Programmed cell death (PCD), which is different from necrosis, is defined as active cell death and is regulated by certain genes. PCD can be mainly classified into apoptosis, autophagy, necroptosis, and pyroptosis. It is involved in brain development, neurodegenerative disorders, psychiatric disorders, and brain injury. Given the pivotal role of PCD in neurological functions, we reviewed relevant articles and tried to summarize the recent advances and future perspectives of PCD involvement in the neurotoxicity of metallic NPs, with the purpose of comprehensively understanding the neurotoxic mechanisms of NPs.

Role of the immune modulator programmed cell death-1 during development and apoptosis of mouse retinal ganglion cells

PubMed Central

Chen, Ling; Sham, Caroline W.; Chan, Ann M.; Francisco, Loise M.; Wu, Yin; Mareninov, Sergey; Sharpe, Arlene H.; Freeman, Gordon J.; Yang, Xian-Jie; Braun, Jonathan; Gordon, Lynn K.

2011-01-01

PURPOSE Mammalian programmed cell death-1 (PD-1) is a membrane-associated receptor regulating the balance between T cell activation, tolerance and immunopathology, however its role in neurons has not yet been defined. We investigate the hypothesis that PD-1 signaling actively promotes retinal ganglion cell (RGC) death within the developing mouse retina. METHODS Mature retinal cell types expressing PD-1 were identified by immunofluorescence staining of vertical retina sections; developmental expression was localized by immunostaining and quantified by Western analysis. PD-1 involvement in developmental RGC survival was assessed in vitro using retina explants and in vivo using PD-1 knockout mice. PD-1 ligand gene expression was detected by RT-PCR. RESULTS PD-1 is expressed in most adult RGCs, and undergoes dynamic upregulation during the early postnatal window of retinal cell maturation and physiological programmed cell death (PCD). In vitro blockade of PD-1 signaling during this time selectively increases survival of RGCs. Furthermore, PD-1 deficient mice show a selective increase in RGC number in the neonatal retina at the peak of developmental RGC death. Lastly, throughout postnatal retina maturation, we find gene expression of both immune PD-1 ligand genes, PD-L1 and PD-L2. CONCLUSIONS These findings collectively support a novel role for a PD-1-mediated signaling pathway in developmental PCD during postnatal RGC maturation. PMID:19420345

Serratia marcescens Induces Apoptotic Cell Death in Host Immune Cells via a Lipopolysaccharide- and Flagella-dependent Mechanism*

PubMed Central

Ishii, Kenichi; Adachi, Tatsuo; Imamura, Katsutoshi; Takano, Shinya; Usui, Kimihito; Suzuki, Kazushi; Hamamoto, Hiroshi; Watanabe, Takeshi; Sekimizu, Kazuhisa

2012-01-01

Injection of Serratia marcescens into the blood (hemolymph) of the silkworm, Bombyx mori, induced the activation of c-Jun NH2-terminal kinase (JNK), followed by caspase activation and apoptosis of blood cells (hemocytes). This process impaired the innate immune response in which pathogen cell wall components, such as glucan, stimulate hemocytes, leading to the activation of insect cytokine paralytic peptide. S. marcescens induced apoptotic cell death of silkworm hemocytes and mouse peritoneal macrophages in vitro. We searched for S. marcescens transposon mutants with attenuated ability to induce apoptosis of silkworm hemocytes. Among the genes identified, disruption mutants of wecA (a gene involved in lipopolysaccharide O-antigen synthesis), and flhD and fliR (essential genes in flagella synthesis) showed reduced motility and impaired induction of mouse macrophage cell death. These findings suggest that S. marcescens induces apoptosis of host immune cells via lipopolysaccharide- and flagella-dependent motility, leading to the suppression of host innate immunity. PMID:22859304

Effect of hypothermia on doxorubicin-induced cardiac myoblast signaling and cell death.

PubMed

L'Ecuyer, Thomas J; Aggarwal, Sanjeev; Zhang, Jiang Ping; Van der Heide, Richard S

2012-01-01

Anthracyclines (AC) are useful chemotherapeutic agents whose principal limitation is cardiac toxicity, which may progress to heart failure, transplantation or even death. We have shown that this toxicity involves oxidative stress-induced activation of the DNA damage pathway. Hypothermia has been shown to be protective against other diseases involving oxidative stress but has not been studied in models of AC toxicity. In the current experiments, H9C2 cardiac myoblasts were treated with varying concentrations of the AC doxorubicin (DOX) during normothermia (37°C) or mild hypothermia (35°C). Total cell death was assayed using trypan blue exclusion and apoptosis by terminal deoxynucleotidyl transferase-mediated deoxyuridine-biotin nick end labeling (TUNEL) staining. Oxidative stress was assayed using the fluorescent indicator 2'7'-dichlorofluorescein diacetate. DNA damage pathway activation was assayed by immunostaining for H2AX and p53. Mitochondrial membrane potential was assayed by JC-1 staining. At all concentrations of DOX examined (1, 2.5 and 5 μM), hypothermia reduced oxidative stress, activation of H2AX and p53, loss of mitochondrial membrane potential and total and apoptotic cell death (P=.001-.03 for each observation). The reduction of oxidative stress-induced activation of the DNA damage pathway and consequent cell death by mild hypothermia supports a possible protective role to reduce the clinical impact of DOX-induced cardiac toxicity. Such an approach may allow expanded use of these effective chemotherapeutic agents to increase cancer cure rates. Copyright © 2012 Elsevier Inc. All rights reserved.

Modes of Cell Death Induced by Photodynamic Therapy Using Zinc Phthalocyanine in Lung Cancer Cells Grown as a Monolayer and Three-Dimensional Multicellular Spheroids.

PubMed

Manoto, Sello L; Houreld, Nicolette; Hodgkinson, Natasha; Abrahamse, Heidi

2017-05-16

Photodynamic therapy (PDT) involves interaction of a photosensitizer, light, and molecular oxygen which produces singlet oxygen and subsequent tumour eradication. The development of second generation photosensitizers, such as phthalocyanines, has improved this technology. Customary monolayer cell culture techniques are, unfortunately, too simple to replicate treatment effects in vivo. Multicellular tumour spheroids may provide a better alternative since they mimic aspects of the human tumour environment. This study aimed to profile 84 genes involved in apoptosis following treatment with PDT on lung cancer cells (A549) grown in a monolayer versus three-dimensional multicellular tumour spheroids (250 and 500 μm). Gene expression profiling was performed 24 h post irradiation (680 nm; 5 J/cm²) with zinc sulfophthalocyanine (ZnPcS mix ) to determine the genes involved in apoptotic cell death. In the monolayer cells, eight pro-apoptotic genes were upregulated, and two were downregulated. In the multicellular tumour spheroids (250 µm) there was upregulation of only 1 gene while there was downregulation of 56 genes. Apoptosis in the monolayer cultured cells was induced via both the intrinsic and extrinsic apoptotic pathways. However, in the multicellular tumour spheroids (250 and 500 µm) the apoptotic pathway that was followed was not conclusive.

Experimental results using 3-bromopyruvate in mesothelioma: in vitro and in vivo studies.

PubMed

Icard, Philippe; Philippe, Icard; Zhang, Xiao-Dong; Xiao-Dong, Zhang; Lemoisson, Edwige; Edwige, Lemoisson; Louis, Marie-Hélène; Marie-Hélène, Louis; Allouche, Stéphane; Stéphane, Allouche; Lincet, Hubert; Hubert, Lincet; Poulain, Laurent; Laurent, Poulain

2012-02-01

Over many years we have taken advantage of the special metabolism of cancer cells involving an increased consumption of glucose associated with lactic acid production even in the presence of oxygen, a phenomenon referred to as the "Warburg effect", to counteract cancer cell growth. We have tested 3-bromopyruvate (3-BrPA), an inhibitor of pyruvate-associated reactions. Firstly, we tested this agent, in vitro, in two mesothelioma cell lines. Cellular response would appear to depend on the mode of administration (immediately or 24 h after seeding). Depending on the line, 3-BrPA induced a cytostatic or cytotoxic effect. This effect was accompanied by cell death induction even in cells highly refractory to cisplatin. Mitochondrial apoptotic death appeared to involve both lines; however, a different death pathway such as necrosis cannot be excluded. Interestingly, 3-BrPA leads to a diminution of the expression of the anti-apotptoic protein Mcl-1. We then tested 3-BrPA in vivo. Survival of nude mice bearing human mesothelioma was significantly prolonged (p < 0.0001). Toxicity and clinical studies should be performed to test 3- BrPA as local therapy for patients suffering from pleural or peritoneal mesothelioma. Association with cisplatin should be particularly considered.

Lysozyme amyloid oligomers and fibrils induce cellular death via different apoptotic/necrotic pathways.

PubMed

Gharibyan, Anna L; Zamotin, Vladimir; Yanamandra, Kiran; Moskaleva, Olesya S; Margulis, Boris A; Kostanyan, Irina A; Morozova-Roche, Ludmilla A

2007-02-02

Among the newly discovered amyloid properties, its cytotoxicity plays a key role. Lysozyme is a ubiquitous protein involved in systemic amyloidoses in vivo and forming amyloid under destabilising conditions in vitro. We characterized both oligomers and fibrils of hen lysozyme by atomic force microscopy and demonstrated their dose (5-50 microM) and time-dependent (6-48 h) effect on neuroblastoma SH-SY5Y cell viability. We revealed that fibrils induce a decrease of cell viability after 6 h due to membrane damage shown by inhibition of WST-1 reduction, early lactate dehydrogenase release, and propidium iodide intake; by contrast, oligomers activate caspases after 6 h but cause the cell viability to decline only after 48 h, as shown by fluorescent-labelled annexin V binding to externalized phosphatidylserine, propidium iodide DNA staining, lactate dehydrogenase release, and by typical apoptotic shrinking of cells. We conclude that oligomers induce apoptosis-like cell death, while the fibrils lead to necrosis-like death. As polymorphism is a common property of an amyloid, we demonstrated that it is not a single uniform species but rather a continuum of cross-beta-sheet-containing amyloids that are cytotoxic. An abundance of lysozyme highlights a universal feature of this phenomenon, indicating that amyloid toxicity should be assessed in all clinical applications involving proteinaceous materials.

Sensory hair cell death and regeneration in fishes

PubMed Central

Monroe, Jerry D.; Rajadinakaran, Gopinath; Smith, Michael E.

2015-01-01

Sensory hair cells are specialized mechanotransductive receptors required for hearing and vestibular function. Loss of hair cells in humans and other mammals is permanent and causes reduced hearing and balance. In the early 1980’s, it was shown that hair cells continue to be added to the inner ear sensory epithelia in cartilaginous and bony fishes. Soon thereafter, hair cell regeneration was documented in the chick cochlea following acoustic trauma. Since then, research using chick and other avian models has led to great insights into hair cell death and regeneration. However, with the rise of the zebrafish as a model organism for studying disease and developmental processes, there has been an increased interest in studying sensory hair cell death and regeneration in its lateral line and inner ears. Advances derived from studies in zebrafish and other fish species include understanding the effect of ototoxins on hair cells and finding otoprotectants to mitigate ototoxin damage, the role of cellular proliferation vs. direct transdifferentiation during hair cell regeneration, and elucidating cellular pathways involved in the regeneration process. This review will summarize research on hair cell death and regeneration using fish models, indicate the potential strengths and weaknesses of these models, and discuss several emerging areas of future studies. PMID:25954154

BAX Inhibitor-1, an ancient cell death suppressor in animals and plants with prokaryotic relatives.

PubMed

Hückelhoven, R

2004-05-01

BAX Inhibitor-1 (BI-1) was originally described as testis enhanced gene transcript in mammals. Functional screening in yeast for human proteins that can inhibit the cell death provoking function of BAX, a proapoptotic Bcl-2 family member, led to functional characterisation and renaming of BI-1. The identification of functional homologues of BI-1 in plants and yeast widened the understanding of BI-1 function as an ancient suppressor of programmed cell death. BI-1 is one of the few cell death suppressors conserved in animals and plants. Computer predictions and experimental data together suggest that BI-1 is a membrane spanning protein with 6 to 7 transmembrane domains and a cytoplasmic C-terminus sticking in the endoplasmatic reticulum and nuclear envelope. Proteins similar to BI-1 are present in other eukaryotes, bacteria, and even viruses encode BI-1 like proteins. BI-1 is involved in development, response to biotic and abiotic stress and probably represents an indispensable cell protectant. BI-1 appears to suppress cell death induced by mitochondrial dysfunction, reactive oxygen species or elevated cytosolic Ca(2+) levels. This review focuses on the present understanding about BI-1 and suggests potential directions for further analyses of this increasingly noticed protein.

GPR30 Activation Contributes to the Puerarin-Mediated Neuroprotection in MPP+-Induced SH-SY5Y Cell Death.

PubMed

Cheng, Yue-Fa; Zhu, Guoqi; Wu, Qing-Wen; Xie, Yue-Sheng; Jiang, Yan; Guo, Lan; Guan, Ya-Li; Liu, Ying-Shuo; Zhang, Jun

2017-02-01

The neuroprotective action of puerarin in Parkinson's disease (PD) models has been well investigated. However, the mechanisms involved in protection have not been completely understood. G protein-coupled receptor 30 (GPR30) is a G protein-coupled estrogen receptor and considered a potential target in the neuroprotection against PD. In this study, we investigated whether puerarin prevented against 1-methyl-4-phenylpyridinium (MPP + )-induced cell death via GPR30. Our results showed that the GPR30 agonist, G1, exhibited puerarin-mediated neuroprotection against MPP + -induced cell death of SH-SY5Y cells. This protective action was reversed by the GPR30 antagonist. Moreover, a time- and concentration-dependent effect of puerarin on GPR30 expression was verified at the protein level but not at the mRNA level. Further, we showed that an mTor-dependent new GPR30 synthesis contributed to the protection conferred by puerarin. Finally, glial cell line-derived neurotrophic factor (GDNF) levels were enhanced by puerarin and G1 in both control and MPP + -lesioned cells via GPR30. Taken together, our data strongly suggest that puerarin prevents MPP + -induced cell death via facilitating GPR30 expression and GDNF release.

The relationship between neurotrophic factors and CaMKII in the death and survival of retinal ganglion cells.

PubMed

Cooper, N G F; Laabich, A; Fan, W; Wang, X

2008-01-01

The scientific discourse relating to the causes and treatments for glaucoma are becoming reflective of the need to protect and preserve retinal neurons from degenerative changes, which result from the injurious environment associated with this disease. Knowledge, in particular, of the signal transduction pathways which affect death and survival of the retinal ganglion cells is critical to this discourse and to the development of a suitable neurotherapeutic strategy for this disease. The goal of this chapter is to review what is known of the chief suspects involved in initiating the cell death/survival pathways in these cells, and what still remains to be uncovered. The least controversial aspect of the subject relates to the potential role of neurotrophic factors in the protection of the retinal ganglion cells. On the other hand, the postulated triggers for signaling cell death in glaucoma remain controversial. Certainly, the restricted flow of neurotrophic factors has been cited as one possible trigger. However, the connections between glaucoma and other factors present in the retina, such as glutamate, long held to be a prospective culprit in retinal ganglion cell death are still being questioned. Whatever the outcome of this particular debate, it is clear that the downstream intersections between the cell death and survival pathways should provide important foci for future studies whose goal is to protect retinal neurons, situated as they are, in the stressful environment of a cell destroying disease. The evidence for CaMKII being one of these intersecting points is discussed.

Therapeutic approaches to preventing cell death in Huntington disease.

PubMed

Kaplan, Anna; Stockwell, Brent R

2012-12-01

Neurodegenerative diseases affect the lives of millions of patients and their families. Due to the complexity of these diseases and our limited understanding of their pathogenesis, the design of therapeutic agents that can effectively treat these diseases has been challenging. Huntington disease (HD) is one of several neurological disorders with few therapeutic options. HD, like numerous other neurodegenerative diseases, involves extensive neuronal cell loss. One potential strategy to combat HD and other neurodegenerative disorders is to intervene in the execution of neuronal cell death. Inhibiting neuronal cell death pathways may slow the development of neurodegeneration. However, discovering small molecule inhibitors of neuronal cell death remains a significant challenge. Here, we review candidate therapeutic targets controlling cell death mechanisms that have been the focus of research in HD, as well as an emerging strategy that has been applied to developing small molecule inhibitors-fragment-based drug discovery (FBDD). FBDD has been successfully used in both industry and academia to identify selective and potent small molecule inhibitors, with a focus on challenging proteins that are not amenable to traditional high-throughput screening approaches. FBDD has been used to generate potent leads, pre-clinical candidates, and has led to the development of an FDA approved drug. This approach can be valuable for identifying modulators of cell-death-regulating proteins; such compounds may prove to be the key to halting the progression of HD and other neurodegenerative disorders. Copyright © 2012 Elsevier Ltd. All rights reserved.

Cell death pathways associated with PDT

NASA Astrophysics Data System (ADS)

Kessel, David; Reiners, John J., Jr.

2006-02-01

Photodynamic therapy leads to both direct and indirect tumor cell death. The latter also involves the consequences of vascular shut-down and immunologic effects. While these factors are a major factor in tumor eradication, there is usually an element of direct cell killing that can reduce the cell population by as much as 2-3 logs. Necrosis was initially believed to represent the predominant PDT death mechanism. An apoptotic response to PDT was first reported by Oleinick in 1991, using a sensitizer that targets the anti-apoptotic protein Bcl-2. Apoptosis leads to fragmentation of DNA and of cells into apoptotic bodies that are removed by phagocytosis. Inflammatory effects are minimized, and the auto- catalytic elements of the process can amplify the death signal. In this study, we examined consequences of Bcl-2 photodamage by a porphycene sensitizer that targets the ER and causes photodamage to the anti-apoptotic protein Bcl-2. Death patterns after Bcl-2 inactivation by a small-molecular antagonist were also assessed. In addition to apoptosis, we also characterized a hitherto undescribed PDT effect, the initiation of autophagy. Autophagy was initially identified as a cell survival pathway, allowing the recycling of components as nutrients become scarce. We propose that autophagy can also represent both a potential survival pathway after PDT damage to cellular organelles, as well as a cell-death pathway. Recent literature reports indicate that autophagy, as well as apoptosis, can be evoked after down-regulation of Bcl-2, a result consistent with results reported here.

Insights into the mechanism of human papillomavirus E2-induced procaspase-8 activation and cell death

NASA Astrophysics Data System (ADS)

Singh, Nitu; Senapati, Sanjib; Bose, Kakoli

2016-02-01

High-risk human papillomavirus (HR-HPV) E2 protein, the master regulator of viral life cycle, induces apoptosis of host cell that is independent of its virus-associated regulatory functions. E2 protein of HR-HPV18 has been found to be involved in novel FADD-independent activation of caspase-8, however, the molecular basis of this unique non-death-fold E2-mediated apoptosis is poorly understood. Here, with an interdisciplinary approach that involves in silico, mutational, biochemical and biophysical probes, we dissected and characterized the E2-procasapse-8 binding interface. Our data demonstrate direct non-homotypic interaction of HPV18 E2 transactivation domain (TAD) with α2/α5 helices of procaspase-8 death effector domain-B (DED-B). The observed interaction mimics the homotypic DED-DED complexes, wherein the conserved hydrophobic motif of procaspase-8 DED-B (F122/L123) occupies a groove between α2/α3 helices of E2 TAD. This interaction possibly drives DED oligomerization leading to caspase-8 activation and subsequent cell death. Furthermore, our data establish a model for E2-induced apoptosis in HR-HPV types and provide important clues for designing E2 analogs that might modulate procaspase-8 activation and hence apoptosis.

TRPV2 activation induces apoptotic cell death in human T24 bladder cancer cells: a potential therapeutic target for bladder cancer.

PubMed

Yamada, Takahiro; Ueda, Takashi; Shibata, Yasuhiro; Ikegami, Yosuke; Saito, Masaki; Ishida, Yusuke; Ugawa, Shinya; Kohri, Kenjiro; Shimada, Shoichi

2010-08-01

To investigate the functional expression of the transient receptor potential vanilloid 2 (TRPV2) channel protein in human urothelial carcinoma (UC) cells and to determine whether calcium influx into UC cells through TRPV2 is involved in apoptotic cell death. The expression of TRPV2 mRNA in bladder cancer cell lines (T24, a poorly differentiated UC cell line and RT4, a well-differentiated UC cell line) was analyzed using reverse transcriptase-polymerase chain reaction. The calcium permeability of TRPV2 channels in T24 cells was investigated using a calcium imaging assay that used cannabidiol (CBD), a relatively selective TRPV2 agonist, and ruthenium red (RuR), a nonselective TRPV channel antagonist. The death of T24 or RT4 cells in the presence of CBD was evaluated using a cellular viability assay. Apoptosis of T24 cells caused by CBD was confirmed using an annexin-V assay and small interfering RNA (siRNA) silencing of TRPV2. TRPV2 mRNA was abundantly expressed in T24 cells. The expression level in UC cells was correlated with high-grade disease. The administration of CBD increased intracellular calcium concentrations in T24 cells. In addition, the viability of T24 cells progressively decreased with increasing concentrations of CBD, whereas RT4 cells were mostly unaffected. Cell death occurred via apoptosis caused by continuous influx of calcium through TRPV2. TRPV2 channels in UC cells are calcium-permeable and the regulation of calcium influx through these channels leads directly to the death of UC cells. TRPV2 channels in UC cells may be a potential new therapeutic target, especially in higher-grade UC cells. Copyright 2010 Elsevier Inc. All rights reserved.

HIV gp120- and methamphetamine-mediated oxidative stress induces astrocyte apoptosis via cytochrome P450 2E1

PubMed Central

Shah, A; Kumar, S; Simon, S D; Singh, D P; Kumar, A

2013-01-01

HIV-1 glycoprotein 120 (gp120) is known to cause neurotoxicity via several mechanisms including production of proinflammatory cytokines/chemokines and oxidative stress. Likewise, drug abuse is thought to have a direct impact on the pathology of HIV-associated neuroinflammation through the induction of proinflammatory cytokines/chemokines and oxidative stress. In the present study, we demonstrate that gp120 and methamphetamine (MA) causes apoptotic cell death by inducing oxidative stress through the cytochrome P450 (CYP) and NADPH oxidase (NOX) pathways. The results showed that both MA and gp120 induced reactive oxygen species (ROS) production in concentration- and time-dependent manners. The combination of gp120 and MA also induced CYP2E1 expression at both mRNA (1.7±0.2- and 2.8±0.3-fold in SVGA and primary astrocytes, respectively) and protein (1.3±0.1-fold in SVGA and 1.4±0.03-fold in primary astrocytes) levels, suggesting the involvement of CYP2E1 in ROS production. This was further confirmed by using a selective inhibitor of CYP2E1, diallylsulfide (DAS), and CYP2E1 knockdown using siRNA, which significantly reduced ROS production (30–60%). As the CYP pathway is known to be coupled with the NOX pathway, including Fenton–Weiss–Haber (FWH) reaction, we examined whether the NOX pathway is also involved in ROS production induced by either gp120 or MA. Our results showed that selective inhibitors of NOX, diphenyleneiodonium (DPI), and FWH reaction, deferoxamine (DFO), also significantly reduced ROS production. These findings were further confirmed using specific siRNAs against NOX2 and NOX4 (NADPH oxidase family). We then showed that gp120 and MA both induced apoptosis (caspase-3 activity and DNA lesion using TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling) assay) and cell death. Furthermore, we showed that DAS, DPI, and DFO completely abolished apoptosis and cell death, suggesting the involvement of CYP and NOX pathways in ROS-mediated apoptotic cell death. In conclusion, this is the first report on the involvement of CYP and NOX pathways in gp120/MA-induced oxidative stress and apoptotic cell death in astrocytes, which has clinical implications in neurodegenerative diseases, including neuroAIDS. PMID:24113184

Voltage-Dependent Anion Channel 1 As an Emerging Drug Target for Novel Anti-Cancer Therapeutics

PubMed Central

Shoshan-Barmatz, Varda; Krelin, Yakov; Shteinfer-Kuzmine, Anna; Arif, Tasleem

2017-01-01

Cancer cells share several properties, high proliferation potential, reprogramed metabolism, and resistance to apoptotic cues. Acquiring these hallmarks involves changes in key oncogenes and non-oncogenes essential for cancer cell survival and prosperity, and is accompanied by the increased energy requirements of proliferating cells. Mitochondria occupy a central position in cell life and death with mitochondrial bioenergetics, biosynthesis, and signaling are critical for tumorigenesis. Voltage-dependent anion channel 1 (VDAC1) is situated in the outer mitochondrial membrane (OMM) and serving as a mitochondrial gatekeeper. VDAC1 allowing the transfer of metabolites, fatty acid ions, Ca2+, reactive oxygen species, and cholesterol across the OMM and is a key player in mitochondrial-mediate apoptosis. Moreover, VDAC1 serves as a hub protein, interacting with diverse sets of proteins from the cytosol, endoplasmic reticulum, and mitochondria that together regulate metabolic and signaling pathways. The observation that VDAC1 is over-expressed in many cancers suggests that the protein may play a pivotal role in cancer cell survival. However, VDAC1 is also important in mitochondria-mediated apoptosis, mediating release of apoptotic proteins and interacting with anti-apoptotic proteins, such as B-cell lymphoma 2 (Bcl-2), Bcl-xL, and hexokinase (HK), which are also highly expressed in many cancers. Strategically located in a “bottleneck” position, controlling metabolic homeostasis and apoptosis, VDAC1 thus represents an emerging target for anti-cancer drugs. This review presents an overview on the multi-functional mitochondrial protein VDAC1 performing several functions and interacting with distinct sets of partners to regulate both cell life and death, and highlights the importance of the protein for cancer cell survival. We address recent results related to the mechanisms of VDAC1-mediated apoptosis and the potential of associated proteins to modulate of VDAC1 activity, with the aim of developing VDAC1-based approaches. The first strategy involves modification of cell metabolism using VDAC1-specific small interfering RNA leading to inhibition of cancer cell and tumor growth and reversed oncogenic properties. The second strategy involves activation of cancer cell death using VDAC1-based peptides that prevent cell death induction by anti-apoptotic proteins. Finally, we discuss the potential therapeutic benefits of treatments and drugs leading to enhanced VDAC1 expression or targeting VDAC1 to induce apoptosis. PMID:28824871

BAG3 promoted starvation-induced apoptosis of thyroid cancer cells via attenuation of autophagy.

PubMed

Li, Si; Zhang, Hai-Yan; Wang, Tian; Meng, Xin; Zong, Zhi-Hong; Kong, De-Hui; Wang, Hua-Qin; Du, Zhen-Xian

2014-11-01

BAG3 plays a regulatory role in a number of cellular processes. Recent studies have attracted much attention on its role in activation of selective autophagy. In addition, we have very recently reported that BAG3 is implicated in a BECN1-independent autophagy, namely noncanonical autophagy. The current study aimed to investigate the potential involvement of BAG3 in canonical autophagy triggered by Earle's Balanced Salt Solution (EBSS) starvation. Replacement of complete medium with EBSS was used to trigger canonical autophagy. BAG3 expression was measured using real-time RT-PCR and Western blot. Autophagy was monitored using LC3-II transition and p62/SQSTM1 accumulation by Western blot, as well as punctate distribution of LC3 by immunofluorescence staining. Cell growth and apoptotic cell death was investigated using real-time cell analyzer and flowcytometry, respectively. BAG3 expression was potently reduced by EBSS starvation. Forced expression of BAG3 suppressed autophagy and promoted apoptotic cell death of thyroid cancer cells elicited by starvation. In addition, in the presence of autophagy inhibitor, the enhancing effect of BAG3 on apoptotic cell death was attenuated. These results suggest that BAG3 promotes apoptotic cell death in starved thyroid cancer cells, at least in part by autophagy attenuation.

The role of necroptosis in the treatment of diseases.

PubMed

Cho, Young Sik

2018-04-11

Necroptosis is an emerging form of programmed cell death occurring via active and well-regulated necrosis, distinct from apoptosis morphologically, and biochemically. Necroptosis is mainly unmasked when apoptosis is compromised in response to cell stress. Unlike apoptotic cells, which are cleared by macrophages or neighboring cells, necrotic cells release danger signals, triggering inflammation, and exacerbating tissue damage. Evidence increasingly suggests that programmed necrosis is not only associated with pathophysiology of disease, but also induces innate immune response to viral infection. Therefore, necroptotic cell death plays both physiological and pathological roles. Physiologically, necroptosis induce an innate immune response as well as premature assembly of viral particles in cells infected with virus that abrogates host apoptotic machinery. On the other hand, necroptosis per se is detrimental, causing various diseases such as sepsis, neurodegenerative diseases and ischemic reperfusion injury. This review discusses the signaling pathways leading to necroptosis, associated necroptotic proteins with target-specific inhibitors and diseases involved. Several studies currently focus on protective approaches to inhibit necroptotic cell death. In cancer biology, however, anticancer drug resistance severely hampers the efficacy of chemotherapy based on apoptosis. Pharmacological switch of cell death finds therapeutic application in drug-resistant cancers. Therefore, the possible clinical role of necroptosis in cancer control will be discussed in brief.

The proinflammatory cytokine interleukin-18 alters multiple signaling pathways to inhibit natural killer cell death

USGS Publications Warehouse

Hodge, D.L.; Subleski, J.J.; Reynolds, D.A.; Buschman, M.D.; Schill, W.B.; Burkett, M.W.; Malyguine, A.M.; Young, H.A.

2006-01-01

The proinflammatory cytokine, interleukin-18 (IL-18), is a natural killer (NK) cell activator that induces NK cell cytotoxicity and interferon-?? (IFN-??) expression. In this report, we define a novel role for IL-18 as an NK cell protective agent. Specifically, IL-18 prevents NK cell death initiated by different and distinct stress mechanisms. IL-18 reduces NK cell self-destruction during NK-targeted cell killing, and in the presence of staurosporin, a potent apoptotic inducer, IL-18 reduces caspase-3 activity. The critical regulatory step in this process is downstream of the mitochondrion and involves reduced cleavage and activation of caspase-9 and caspase-3. The ability of IL-18 to regulate cell survival is not limited to a caspase death pathway in that IL-18 augments tumor necrosis factor (TNF) signaling, resulting in increased and prolonged mRNA expression of c-apoptosis inhibitor 2 (cIAP2), a prosurvival factor and caspase-3 inhibitor, and TNF receptor-associated factor 1 (TRAF1), a prosurvival protein. The cumulative effects of IL-18 define a novel role for this cytokine as a molecular survival switch that functions to both decrease cell death through inhibition of the mitochondrial apoptotic pathway and enhance TNF induction of prosurvival factors. ?? Mary Ann Liebert, Inc.

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

PubMed Central

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

2017-01-01

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

Unexpected cross-reactivity of anti-cathepsin B antibodies leads to uncertainties regarding the mechanism of action of anti-CD20 monoclonal antibody GA101.

PubMed

Chien, Wei Wen; Niogret, Charlène; Jugé, Romain; Lionnard, Loïc; Cornut-Thibaut, Aurélie; Kucharczak, Jérôme; Savina, Ariel; Salles, Gilles; Aouacheria, Abdel

2017-04-01

GA101, also known as obinutuzumab or Gazyva (Gazyvaro), is a glycoengineered type II humanized antibody that targets the CD20 antigen expressed at the surface of B-cells. This novel anti-CD20 antibody is currently assessed in clinical trials with promising results as a single agent or as part of therapeutic combinations for the treatment of B-cell malignancies. Detailed understanding of the mechanisms of GA101-induced cell death is needed to get insight into possible resistance mechanisms occurring in patients. Although multiple in vitro and in vivo mechanisms have been suggested to describe the effects of GA101 on B-cells, currently available data are ambiguous. The aim of our study was to clarify the cellular mechanisms involved in GA101-induced cell death in vitro, and more particularly the respective roles played by lysosomal and mitochondrial membrane permeabilization. Our results confirm previous reports suggesting that GA101 triggers homotypic adhesion and caspase-independent cell death, two processes that are dependent on actin remodeling and involve the production of reactive oxygen species. With respect to lysosomal membrane permeabilization (LMP), our data suggest that lack of specificity of available antibodies directed against cathepsin B may have confounded previously published results, possibly challenging current LMP-driven model of GA101 action mode. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evidence for the activation of pyroptotic and apoptotic pathways in RPE cells associated with NLRP3 inflammasome in the rodent eye.

PubMed

Gao, Jiangyuan; Cui, Jing Z; To, Eleanor; Cao, Sijia; Matsubara, Joanne A

2018-01-12

Age-related macular degeneration (AMD) is a devastating eye disease causing irreversible vision loss in the elderly. Retinal pigment epithelium (RPE), the primary cell type that is afflicted in AMD, undergoes programmed cell death in the late stages of the disease. However, the exact mechanisms for RPE degeneration in AMD are still unresolved. The prevailing theories consider that each cell death pathway works independently and without regulation of each other. Building upon our previous work in which we induced a short burst of inflammasome activity in vivo, we now investigate the effects of prolonged inflammasome activity on RPE cell death mechanisms in rats. Long-Evans rats received three intravitreal injections of amyloid beta (Aβ), once every 4 days, and were sacrificed at day 14. The vitreous samples were collected to assess the levels of secreted cytokines. The inflammasome activity was evaluated by both immunohistochemistry and western blot. The types of RPE cell death mechanisms were determined using specific cell death markers and morphological characterizations. We found robust inflammasome activation evident by enhanced caspase-1 immunoreactivity, augmented NF-κB nuclear translocalization, increased IL-1β vitreal secretion, and IL-18 protein levels. Moreover, we observed elevated proteolytic cleavage of caspase-3 and gasdermin D, markers for apoptosis and pyroptosis, respectively, in RPE-choroid tissues. There was also a significant reduction in the anti-apoptotic factor, X-linked inhibitor of apoptosis protein, consistent with the overall changes of RPE cells. Morphological analysis showed phenotypic characteristics of pyroptosis including RPE cell swelling. Our data suggest that two cell death pathways, pyroptosis and apoptosis, were activated in RPE cells after exposure to prolonged inflammasome activation, induced by a drusen component, Aβ. The involvement of two distinct cell death pathways in RPE sheds light on the potential interplay between these pathways and provides insights on the future development of therapeutic strategies for AMD.

Cytotoxicity of citral against melanoma cells: The involvement of oxidative stress generation and cell growth protein reduction.

PubMed

Sanches, Larissa Juliani; Marinello, Poliana Camila; Panis, Carolina; Fagundes, Tatiane Renata; Morgado-Díaz, José Andrés; de-Freitas-Junior, Julio Cesar Madureira; Cecchini, Rubens; Cecchini, Alessandra Lourenço; Luiz, Rodrigo Cabral

2017-03-01

Citral is a natural compound that has shown cytotoxic and antiproliferative effects on breast and hematopoietic cancer cells; however, there are few studies on melanoma cells. Oxidative stress is known to be involved in all stages of melanoma development and is able to modulate intracellular pathways related to cellular proliferation and death. In this study, we hypothesize that citral exerts its cytotoxic effect on melanoma cells by the modulation of cellular oxidative status and/or intracellular signaling. To test this hypothesis, we investigated the antiproliferative and cytotoxic effects of citral on B16F10 murine melanoma cells evaluating its effects on cellular oxidative stress, DNA damage, cell death, and important signaling pathways, as these pathways, namely, extracellular signal-regulated kinases 1/2 (ERK1/2), AKT, and phosphatidylinositol-3 kinase, are involved in cell proliferation and differentiation. The p53 and nuclear factor kappa B were also investigated due to their ability to respond to intracellular stress. We observed that citral exerted antiproliferative and cytotoxic effects in B16F10; induced oxidative stress, DNA lesions, and p53 nuclear translocation; and reduced nitric oxide levels and nuclear factor kappa B, ERK1/2, and AKT. To investigate citral specificity, we used non-neoplastic human and murine cells, HaCaT (human skin keratinocytes) and NIH-3T3 cells (murine fibroblasts), and observed that although citral effects were not specific for cancer cells, non-neoplastic cells were more resistant to citral than B16F10. These findings highlight the potential clinical utility of citral in melanoma, with a mechanism of action involving the oxidative stress generation, nitric oxide depletion, and interference in signaling pathways related to cell proliferation.

A Disease-associated Mutant of NLRC4 Shows Enhanced Interaction with SUG1 Leading to Constitutive FADD-dependent Caspase-8 Activation and Cell Death.

PubMed

Raghawan, Akhouri Kishore; Sripada, Anand; Gopinath, Gayathri; Pushpanjali, Pendyala; Kumar, Yatender; Radha, Vegesna; Swarup, Ghanshyam

2017-01-27

Nod-like receptor family card containing 4 (NLRC4)/Ipaf is involved in recognition of pathogen-associated molecular patterns leading to caspase-1 activation and cytokine release, which mediate protective innate immune response. Point mutations in NLRC4 cause autoinflammatory syndromes. Although all the mutations result in constitutive caspase-1 activation, their phenotypic presentations are different, implying that these mutations cause different alterations in properties of NLRC4. NLRC4 interacts with SUG1 and induces caspase-8-mediated cell death. Here, we show that one of the autoinflammatory syndrome-causing mutants of NLRC4, H443P, but not T337A and V341A, constitutively activates caspase-8 and induces apoptotic cell death in human lung epithelial cells. Compared with wild type NLRC4, the H443P mutant shows stronger interaction with SUG1 and with ubiquitinated cellular proteins. Phosphorylation of NLRC4 at Ser 533 plays a crucial role in caspase-8 activation and cell death. However, H443P mutant does not require Ser 533 phosphorylation for caspase-8 activation and cell death. Caspase-8 activation by NLRC4 and its H443P mutant are dependent on the adaptor protein FADD. A phosphomimicking mutant of NLRC4, S533D does not require SUG1 activity for inducing cell death. Ubiquitin-tagged NLRC4 could induce cell death and activate caspase-8 independent of Ser 533 phosphorylation. Our work suggests that SUG1-mediated signaling results in enhanced ubiquitination and regulates FADD-dependent caspase-8 activation by NLRC4. We show that the autoinflammation-associated H443P mutant is altered in interaction with SUG1 and ubiquitinated proteins, triggering constitutive caspase-8-mediated cell death dependent on FADD but independent of Ser 533 phosphorylation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Detection of Necroptosis in Ligand-Mediated and Hypoxia-Induced Injury of Hepatocytes Using a Novel Optic Probe Detecting Receptor-Interacting Protein (RIP)1/RIP3 Binding.

PubMed

Haga, Sanae; Kanno, Akira; Ozawa, Takeaki; Morita, Naoki; Asano, Mami; Ozaki, Michitaka

2017-07-21

Liver injury is often observed in various pathological conditions including posthepatectomy state and cancer chemotherapy. It occurs mainly as a consequence of the combined necrotic and apoptotic types of cell death. In order to study liver/hepatocyte injury by necrotic type of cell death, we studied signal-regulated necrosis (necroptosis) by newly developing an optic probe detecting receptor-interacting protein (RIP)1/RIP3 binding, an essential process for necroptosis induction. In the mouse hepatocyte cell line, TIB-73 cells, TNF-a/cycloheximide (T/C) induced RIP1/3 binding only when caspase activity was suppressed by z-VAD-fmk (zVAD), a caspase-specific inhibitor. T/C/zVADinduced RIP1/3-binding was inhibited by necrostatin-1 (Nec-1), an allosteric inhibitor of RIP1. The reduced cell survival by T/C/zVAD was improved by Nec-1. These facts indicate that T/C induces necroptosis of hepatocytes when apoptotic pathway is inhibited/unavailable. FasL also induced cell death which was only partially inhibited by zVAD, indicating the possible involvement of necroptosis other than apoptosis. FasL activated caspase-3 and, similarly, induced RIP1/3-binding when caspases were inactivated. Interestingly, FasL-induced RIP1/3 binding was significantly suppressed by the antioxidants, Trolox and N-acetyl cysteine (NAC), suggesting the involvement of reactive oxygen species (ROS) in FasL-induced necroptotic cellular processes. H₂O₂, by itself, induced RIP1/3 binding that was suppressed by Nec-1, but not by zVAD. Hypoxia induced RIP1/3 binding after reoxygenation, which was suppressed by Nec-1 or by the antioxidants. Cell death induced by hypoxia/reoxygenation (H/R) was also improved by Nec-1. Similar to H₂O₂, H/R did not require caspase inhibition for RIP1/3 binding, suggesting the involvement of a caspase-independent mechanism for non-ligand induced and/or redox-mediated necroptosis. These data indicate that ROS induce necroptosis, and mediate the FasL- and hypoxia-induced necroptosis via a molecular mechanism that differs from a conventional caspase-dependent pathway. In conclusion, necroptosis is potentially involved in liver/hepatocyte injury induced by oxidative stress and FasL, other than apoptosis.

Oxidative Stress Facilitates IFN-γ-Induced Mimic Extracellular Trap Cell Death in A549 Lung Epithelial Cancer Cells.

PubMed

Lin, Chiou-Feng; Chen, Chia-Ling; Chien, Shun-Yi; Tseng, Po-Chun; Wang, Yu-Chih; Tsai, Tsung-Ting

2016-01-01

We previously demonstrated that IFN-γ induces an autophagy-regulated mimic extracellular trap cell death (ETosis) in A549 human lung cancer cells. Regarding reactive oxygen species (ROS) are involved in ETosis, this study investigated the role of oxidative stress. After IFN-γ stimulation, a necrosis-like cell death mimic ETosis occurred accompanied by the inhibition of cell growth, aberrant nuclear staining, and nucleosome release. ROS were generated in a time-dependent manner with an increase in NADPH oxidase component protein expression. STAT1-mediated IFN regulatory factor-1 activation was essential for upregulating ROS production. By genetically silencing p47phox, IFN-γ-induced ROS and mimic ETosis were significantly attenuated. This mechanistic study indicated that ROS may mediate DNA damage followed by histone H3 citrullination. Furthermore, ROS promoted IFN-γ-induced mimic ETosis in cooperation with autophagy. These findings further demonstrate that ROS regulates IFN-γ-induced mimic ETosis in lung epithelial malignancy.

Oxidative Stress Facilitates IFN-γ-Induced Mimic Extracellular Trap Cell Death in A549 Lung Epithelial Cancer Cells

PubMed Central

Lin, Chiou-Feng; Chen, Chia-Ling; Chien, Shun-Yi; Tseng, Po-Chun; Wang, Yu-Chih; Tsai, Tsung-Ting

2016-01-01

We previously demonstrated that IFN-γ induces an autophagy-regulated mimic extracellular trap cell death (ETosis) in A549 human lung cancer cells. Regarding reactive oxygen species (ROS) are involved in ETosis, this study investigated the role of oxidative stress. After IFN-γ stimulation, a necrosis-like cell death mimic ETosis occurred accompanied by the inhibition of cell growth, aberrant nuclear staining, and nucleosome release. ROS were generated in a time-dependent manner with an increase in NADPH oxidase component protein expression. STAT1-mediated IFN regulatory factor-1 activation was essential for upregulating ROS production. By genetically silencing p47phox, IFN-γ-induced ROS and mimic ETosis were significantly attenuated. This mechanistic study indicated that ROS may mediate DNA damage followed by histone H3 citrullination. Furthermore, ROS promoted IFN-γ-induced mimic ETosis in cooperation with autophagy. These findings further demonstrate that ROS regulates IFN-γ-induced mimic ETosis in lung epithelial malignancy. PMID:27575372

Dissecting mitochondrial apoptosis pathways by gain-of-function cell culture screens.

PubMed

Grimm, Stefan

2013-05-01

While more primitive organism such as Caenorhabditis elegans and Drosophila melanogaster feature a limited, and by now probably mostly known, array of basic cell death factors, the mammalian cell is replete with additional regulators of the cell's demise. This abundance of apoptosis mediators has made it imperative to set up a systematic inventory of mammalian cell death genes. Genetic screens in this biological system have recently uncovered the rich diversity of cell death signalling and have in particular highlighted mitochondria as an organelle loaded with apoptosis regulators. Many of the screens that have addressed this utilised the novel technique of RNA interference but some also looked at gain-of-functions with transfected cDNAs. Here we give an overview of the rationale for the latter approach, present the genes discovered by this strategy and in particular describe the involvement of mitochondria and their signalling pathways defined by those genes. Copyright © 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Cyanide-induced death of dopaminergic cells is mediated by uncoupling protein-2 up-regulation and reduced Bcl-2 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, X.; Li, L.; Zhang, L.

Cyanide is a potent inhibitor of mitochondrial oxidative metabolism and produces mitochondria-mediated death of dopaminergic neurons and sublethal intoxications that are associated with a Parkinson-like syndrome. Cyanide toxicity is enhanced when mitochondrial uncoupling is stimulated following up-regulation of uncoupling protein-2 (UCP-2). In this study, the role of a pro-survival protein, Bcl-2, in cyanide-mediated cell death was determined in a rat dopaminergic immortalized mesencephalic cell line (N27 cells). Following pharmacological up-regulation of UCP-2 by treatment with Wy14,643, cyanide reduced cellular Bcl-2 expression by increasing proteasomal degradation of the protein. The increased turnover of Bcl-2 was mediated by an increase of oxidativemore » stress following UCP-2 up-regulation. The oxidative stress involved depletion of mitochondrial glutathione (mtGSH) and increased H{sub 2}O{sub 2} generation. Repletion of mtGSH by loading cells with glutathione ethyl ester reduced H{sub 2}O{sub 2} generation and in turn blocked the cyanide-induced decrease of Bcl-2. To determine if UCP-2 mediated the response, RNAi knock down was conducted. The RNAi decreased cyanide-induced depletion of mtGSH, reduced H{sub 2}O{sub 2} accumulation, and inhibited down-regulation of Bcl-2, thus blocking cell death. To confirm the role of Bcl-2 down-regulation in the cell death, it was shown that over-expression of Bcl-2 by cDNA transfection attenuated the enhancement of cyanide toxicity after UCP-2 up-regulation. It was concluded that UCP-2 up-regulation sensitizes cells to cyanide by increasing cellular oxidative stress, leading to an increase of Bcl-2 degradation. Then the reduced Bcl-2 levels sensitize the cells to cyanide-mediated cell death.« less

Caspase inhibition augmented oridonin-induced cell death in murine fibrosarcoma l929 by enhancing reactive oxygen species generation.

PubMed

Wu, Jin-Nan; Huang, Jian; Yang, Jia; Tashiro, Shin-Ichi; Onodera, Satoshi; Ikejima, Takashi

2008-09-01

Oridonin, a diterpenoid isolated from Rabdosia rubescences, has been reported to have antitumor effects. In this study, the growth-inhibitory activity of oridonin for L929 cells was exerted in a time-and dose-dependent manner. After treatment with oridonin for 24 h, L929 cells underwent both apoptosis and necrosis as measured by an lactate dehydrogenase (LDH) activity-based assay. A rapid generation of reactive oxygen species (ROS) was triggered by oridonin, and subsequently up-regulation of phospho-p53 (ser 15) expression and an increased expression ratio of Bax/Bcl-2 was observed. Furthermore, there was a significant fall in mitochondrial membrane potential (MMP) and increase in caspase-3 activity after exposure to oridonin for 24 h. Surprisingly, the pan-caspase inhibitor z-VAD-fmk and caspase3 inhibitor z-DEVD-fmk rendered L929 cells more sensitive to oridonin, rather than preventing oridonin-induced cell death. Oridonin and z-VAD-fmk co-treatment not only resulted in an even higher ROS production, but also made a more significant reduction in the MMP. Pretreatment of ROS scavenger N-acetylcysteine (NAC) led to a complete inhibition of oridonin-induced cell death, intracellular ROS generation, and MMP collapse. NAC treatment also reversed the potentiation of cell death by the pan-caspase inhibitor z-VAD-fmk. Taken together, these observations showed that oridonin-induced cell death in L929 cells involved intracellular ROS generation, activation of phospho-p53 (ser 15), and up-regulation of the Bax/Bcl-2 ratio; and the augmented cell death by z-VAD-fmk was dependent on an increased ROS production.

Inhibition of MPP+-induced mitochondrial damage and cell death by trifluoperazine and W-7 in PC12 cells.

PubMed

Lee, Chung Soo; Park, Se Young; Ko, Hyun Hee; Song, Jin Ho; Shin, Yong Kyoo; Han, Eun Sook

2005-01-01

Opening of the mitochondrial permeability transition pore has been recognized to be involved in cell death. The present study investigated the effect of trifluoperazine and W-7 on the MPP+-induced mitochondrial damage and cell death in undifferentiated PC12 cells. Calmodulin antagonists (trifluoperazine, W-7 and calmidazolium) at 0.5-1 microM significantly reduced the loss of cell viability in PC12 cells treated with 500 microM MPP+. Trifluoperazine and W-7 (0.5-1 microM) inhibited the nuclear damage, the loss of the mitochondrial transmembrane potential followed by cytochrome c release, and the elevation of intracellular Ca2+ levels due to MPP+ in PC12 cells and attenuated the formation of reactive oxygen species and the depletion of GSH. Calmodulin antagonists at 5-10 microM exhibited a cytotoxic effect on PC12 cells, and compounds at 10 microM did not attenuate cytotoxicity of MPP+. Calmodulin antagonists (0.5-1 microM) significantly reduced rotenone-induced mitochondrial damage and cell death, whereas they did not attenuate cell death and elevation of intracellular Ca2+ levels due to H2O2 or ionomycin. The results show that trifluoperazine and W-7 exhibit a differential inhibitory effect against cytotoxicity of MPP+ depending on concentration. Both compounds at the concentrations less than 5 microM may attenuate the MPP+-induced viability loss in PC12 cells by suppressing change in the mitochondrial membrane permeability and by lowering the intracellular Ca2+ levels.

Withaferin A Induces Cell Death Selectively in Androgen-Independent Prostate Cancer Cells but Not in Normal Fibroblast Cells

PubMed Central

Nishikawa, Yukihiro; Okuzaki, Daisuke; Fukushima, Kohshiro; Mukai, Satomi; Ohno, Shouichi; Ozaki, Yuki; Yabuta, Norikazu; Nojima, Hiroshi

2015-01-01

Withaferin A (WA), a major bioactive component of the Indian herb Withania somnifera, induces cell death (apoptosis/necrosis) in multiple types of tumor cells, but the molecular mechanism underlying this cytotoxicity remains elusive. We report here that 2 μM WA induced cell death selectively in androgen-insensitive PC-3 and DU-145 prostate adenocarcinoma cells, whereas its toxicity was less severe in androgen-sensitive LNCaP prostate adenocarcinoma cells and normal human fibroblasts (TIG-1 and KD). WA also killed PC-3 cells in spheroid-forming medium. DNA microarray analysis revealed that WA significantly increased mRNA levels of c-Fos and 11 heat-shock proteins (HSPs) in PC-3 and DU-145, but not in LNCaP and TIG-1. Western analysis revealed increased expression of c-Fos and reduced expression of the anti-apoptotic protein c-FLIP(L). Expression of HSPs such as HSPA6 and Hsp70 was conspicuously elevated; however, because siRNA-mediated depletion of HSF-1, an HSP-inducing transcription factor, reduced PC-3 cell viability, it is likely that these heat-shock genes were involved in protecting against cell death. Moreover, WA induced generation of reactive oxygen species (ROS) in PC-3 and DU-145, but not in normal fibroblasts. Immunocytochemistry and immuno-electron microscopy revealed that WA disrupted the vimentin cytoskeleton, possibly inducing the ROS generation, c-Fos expression and c-FLIP(L) suppression. These observations suggest that multiple events followed by disruption of the vimentin cytoskeleton play pivotal roles in WA-mediated cell death. PMID:26230090

Ecdysone-dependent and ecdysone-independent programmed cell death in the developing optic lobe of Drosophila.

PubMed

Hara, Yusuke; Hirai, Keiichiro; Togane, Yu; Akagawa, Hiromi; Iwabuchi, Kikuo; Tsujimura, Hidenobu

2013-02-01

The adult optic lobe of Drosophila develops from the primordium during metamorphosis from mid-3rd larval stage to adult. Many cells die during development of the optic lobe with a peak of the number of dying cells at 24 h after puparium formation (h APF). Dying cells were observed in spatio-temporal specific clusters. Here, we analyzed the function of a component of the insect steroid hormone receptor, EcR, in this cell death. We examined expression patterns of two EcR isoforms, EcR-A and EcR-B1, in the optic lobe. Expression of each isoform altered during development in isoform-specific manner. EcR-B1 was not expressed in optic lobe neurons from 0 to 6h APF, but was expressed between 9 and 48 h APF and then disappeared by 60 h APF. In each cortex, its expression was stronger in older glia-ensheathed neurons than in younger ones. EcR-B1 was also expressed in some types of glia. EcR-A was expressed in optic lobe neurons and many types of glia from 0 to 60 h APF in a different pattern from EcR-B1. Then, we genetically analyzed EcR function in the optic lobe cell death. At 0 h APF, the optic lobe cell death was independent of any EcR isoforms. In contrast, EcR-B1 was required for most optic lobe cell death after 24 h APF. It was suggested that cell death cell-autonomously required EcR-B1 expressed after puparium formation. βFTZ-F1 was also involved in cell death in many dying-cell clusters, but not in some of them at 24 h APF. Altogether, the optic lobe cell death occurred in ecdysone-independent manner at prepupal stage and ecdysone-dependent manner after 24 h APF. The acquisition of ecdysone-dependence was not directly correlated with the initiation or increase of EcR-B1 expression. Copyright © 2012 Elsevier Inc. All rights reserved.

Constitutive expression of the maltoporin LamB in the absence of OmpR damages the cell envelope.

PubMed

Reimann, Sylvia A; Wolfe, Alan J

2011-02-01

Cells experience multiple environmental stimuli simultaneously. To survive, they must respond accordingly. Unfortunately, the proper response to one stress easily could make the cell more susceptible to a second coexistent stress. To deal with such a problem, a cell must possess a mechanism that balances the need to respond simultaneously to both stresses. Our recent studies of ompR malT(Con) double mutants show that elevated expression of LamB, the outer membrane porin responsible for maltose uptake, causes cell death when the osmoregulator OmpR is disabled. To obtain insight into the nature of the death experienced by ompR malT(Con) mutants, we described the death process. On the basis of microscopic and biochemical approaches, we conclude that death results from a loss of membrane integrity. On the basis of an unbiased genome-wide search for suppressor mutations, we conclude that this loss of membrane integrity results from a LamB-induced envelope stress that the cells do not sufficiently perceive and thus do not adequately accommodate. Finally, we conclude that this envelope stress involves an imbalance in the lipopolysaccharide/porin composition of the outer membrane and an increased requirement for inorganic phosphate.

Involvement of PPAR-γ in the neuroprotective and anti-inflammatory effects of angiotensin type 1 receptor inhibition: effects of the receptor antagonist telmisartan and receptor deletion in a mouse MPTP model of Parkinson's disease.

PubMed

Garrido-Gil, Pablo; Joglar, Belen; Rodriguez-Perez, Ana I; Guerra, Maria J; Labandeira-Garcia, Jose L

2012-02-22

Several recent studies have shown that angiotensin type 1 receptor (AT1) antagonists such as candesartan inhibit the microglial inflammatory response and dopaminergic cell loss in animal models of Parkinson's disease. However, the mechanisms involved in the neuroprotective and anti-inflammatory effects of AT1 blockers in the brain have not been clarified. A number of studies have reported that AT1 blockers activate peroxisome proliferator-activated receptor gamma (PPAR γ). PPAR-γ activation inhibits inflammation, and may be responsible for neuroprotective effects, independently of AT1 blocking actions. We have investigated whether oral treatment with telmisartan (the most potent PPAR-γ activator among AT1 blockers) provides neuroprotection against dopaminergic cell death and neuroinflammation, and the possible role of PPAR-γ activation in any such neuroprotection. We used a mouse model of parkinsonism induced by the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and co-administration of the PPAR-γ antagonist GW9662 to study the role of PPAR-γ activation. In addition, we used AT1a-null mice lesioned with MPTP to study whether deletion of AT1 in the absence of any pharmacological effect of AT1 blockers provides neuroprotection, and investigated whether PPAR-γ activation may also be involved in any such effect of AT1 deletion by co-administration of the PPAR-γ antagonist GW9662. We observed that telmisartan protects mouse dopaminergic neurons and inhibits the microglial response induced by administration of MPTP. The protective effects of telmisartan on dopaminergic cell death and microglial activation were inhibited by co-administration of GW9662. Dopaminergic cell death and microglial activation were significantly lower in AT1a-null mice treated with MPTP than in mice not subjected to AT1a deletion. Interestingly, the protective effects of AT1 deletion were also inhibited by co-administration of GW9662. The results suggest that telmisartan provides effective neuroprotection against dopaminergic cell death and that the neuroprotective effect is mediated by PPAR-γ activation. However, the results in AT1-deficient mice show that blockage of AT1, unrelated to the pharmacological properties of AT1 blockers, also protects against dopaminergic cell death and neuroinflammation. Furthermore, the results show that PPAR-γ activation is involved in the anti-inflammatory and neuroprotective effects of AT1 deletion.

The Roles of 4β-Hydroxywithanolide E from Physalis peruviana on the Nrf2-Anti-Oxidant System and the Cell Cycle in Breast Cancer Cells.

PubMed

Peng, Chieh Yu; You, Bang Jau; Lee, Chia Lin; Wu, Yang Chang; Lin, Wen Hsin; Lu, Te Ling; Chang, Fei-Ching; Lee, Hong Zin

2016-01-01

4[Formula: see text]-Hydroxywithanolide E is an active component of the extract of Physalis peruviana that has been reported to exhibit antitumor effects. Although the involvement of reactive oxygen species (ROS) production and the ataxia-telangiectasia mutated protein (ATM)-dependent DNA damage signaling pathway in 4[Formula: see text]-hydroxywithanolide E-induced apoptosis of breast cancer MCF-7 cells was demonstrated in our previous study, the relationship between ROS production and the cellular defense system response in 4[Formula: see text]-hydroxywithanolide E-induced cell death requires further verification. The present study suggests that ROS play an important role in 4[Formula: see text]-hydroxywithanolide E-induced MCF-7 cell death in which anti-oxidants, such as glutathione or N-acetylcysteine, can resist the 4[Formula: see text]-hydroxywithanolide E-induced accumulation of ROS and cell death. Furthermore, N-acetylcysteine or glutathione can reverse the 4[Formula: see text]-hydroxywithanolide E-induced changes in the cell cycle distribution and the expression of cell cycle regulators. We found that the 4[Formula: see text]-hydroxywithanolide E-induced ROS accumulation was correlated with the upregulation of Nrf2 and Nrf2-downstream genes, such as antioxidative defense enzymes. In general, the activity of Nrf2 is regulated by the Ras signalling pathway. However, we demonstrated that Nrf2 was activated during 4[Formula: see text]-hydroxywithanolide E-induced MCF-7 cell death in spite of the 4[Formula: see text]-hydroxywithanolide E-induced inhibition of the Ras/Raf/ERK pathway. The activity and protein expression of superoxide dismutase and catalase were involved in the 4[Formula: see text]-hydroxywithanolide E-induced ROS production in MCF-7 cells. Furthermore, 4[Formula: see text]-hydroxywithanolide E was demonstrated to significantly reduce the sizes of the tumor nodules in the human breast cancer MDA-MB231 xenograft tumor model.

Ongoing cell death and immune influences on regeneration in the vestibular sensory organs

NASA Technical Reports Server (NTRS)

Warchol, M. E.; Matsui, J. I.; Simkus, E. L.; Ogilive, J. M.

2001-01-01

Hair cells in the vestibular organs of birds have a relatively short life span. Mature hair cells appear to die spontaneously and are then quickly replaced by new hair cells that arise from the division of epithelial supporting cells. A similar regenerative mechanism also results in hair cell replacement after ototoxic damage. The cellular basis of hair cell turnover in the avian ear is not understood. We are investigating the signaling pathways that lead to hair cell death and the relationship between ongoing cell death and cell production. In addition, work from our lab and others has demonstrated that the avian inner ear contains a resident population of macrophages and that enhanced numbers of macrophages are recruited to sites of hair cells lesions. Those observations suggest that macrophages and their secretory products (cytokines) may be involved in hair cell regeneration. Consistent with that suggestion, we have found that treatment with the anti-inflammatory drug dexamethasone reduces regenerative cell proliferation in the avian ear, and that certain macrophage-secreted cytokines can influence the proliferation of vestibular supporting cells and the survival of statoacoustic neurons. Those results suggest a role for the immune system in the process of sensory regeneration in the inner ear.

Very Long Chain Fatty Acids Are Functionally Involved in Necroptosis.

PubMed

Parisi, Laura R; Li, Nasi; Atilla-Gokcumen, G Ekin

2017-12-21

Necroptosis is a form of regulated cell death that is linked to various human diseases. Distinct membrane-related, thus lipid-dependent, alterations take place during necroptosis. However, little is known about the roles of specific lipids in this process. We used an untargeted LC-MS-based approach to reveal that distinct lipid species are regulated at the molecular level during necroptosis. We found that ceramides and very long chain fatty acids accumulate during this process. Intrigued by the specificity of very long chain fatty acid accumulation, we focused on characterizing their involvement during necroptosis. Biochemical characterizations suggested that activated fatty acid biosynthesis and elongation could be responsible for these accumulations. We further showed that inhibition of fatty acid biosynthesis and depletion of very long chain fatty acids prevented loss of plasma membrane integrity and cell death, strongly suggesting that very long chain fatty acids are functionally involved in necroptosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

The role of the Fas/FasL signaling pathway in environmental toxicant-induced testicular cell apoptosis: An update.

PubMed

Wang, Mei; Su, Ping

2018-04-01

The Fas/FasL signaling pathway is one of the major pathways that regulate apoptosis. Increasing studies have shown that the activation of the Fas/FasL signaling pathway is closely associated with testicular cell apoptosis. However, the mechanism involved is still unclear. We discuss recent findings regarding the molecular mechanisms by which environmental toxicants induce testicular pathology via Fas/FasL signaling. These findings suggest that Fas/FasL signaling is employed to impact the sensitivity (a response to external factors) of germ cells, disrupt steroidogenic hormone and cytokine metabolism mediated by Sertoli cells, and elicit the activation of NFAT (nuclear factor of activated T-cells) in Leydig cell apoptosis. Consequently, degeneration of testicular somatic (Sertoli and Leydig) and spermatogenic cells, leads to decreased numbers of mature sperm and subsequently translates into infertility issues. Collectively, these findings illustrate that it is beneficial to develop potential targets for a new generation of new pharmaceutical therapies that would alleviate testicular dysfunctions. BTB: blood-testis barrier; DD: death domains; DR3: death receptor 3; DR4: death receptor 4; DR5: death receptor 5; DED: death effector domain; DISC: death-inducing signaling complex; ERα: estrogen receptor alpha; FADD: Fas-associated death domain; FSH: follicle- stimulating hormone; IL-1β: interleukin 1 beta; LH: luteinizing hormone; LPS: lipopolysaccharide; mFas: membrane Fas; MMP2: matrix metalloproteinase-2; MTA1: metastasis-associated protein 1; NAC: N-acetylcysteine; NCCD: the Nomenclature Committee on Cell Death; NFAT: nuclear factor of activated T-cells; NF-kB: nuclear transcription factor-kappaB; NO: nitric oxide; NP: 4-nonylphenol; PCD: programmed cell death; PP1/PP2A: protein phosphatase 1 and 2A; ROS: reactive oxygen species; sFas: soluble Fas; T: testosterone; TGF-β: transforming growth factor-beta; THD: TNF homology domain; TIMP-2: tissue inhibitor of metalloproteinase-2; TNF: tumor necrosis factor; TNF-α: tumor necrosis factor-alpha; TNF-R1: Tumor necrosis factor receptor 1; TNFRSF1A: TNF receptor superfamily member 1A.

Endotoxin-activated microglia injure brain derived endothelial cells via NF-κB, JAK-STAT and JNK stress kinase pathways

PubMed Central

2011-01-01

Background We previously showed that microglia damage blood brain barrier (BBB) components following ischemic brain insults, but the underlying mechanism(s) is/are not well known. Recent work has established the contribution of toll-like receptor 4 (TLR4) activation to several brain pathologies including ischemia, neurodegeneration and sepsis. The present study established the requirement of microglia for lipopolysaccharide (LPS) mediated endothelial cell death, and explored pathways involved in this toxicity. LPS is a classic TLR4 agonist, and is used here to model aspects of brain conditions where TLR4 stimulation occurs. Methods/Results In monocultures, LPS induced death in microglia, but not brain derived endothelial cells (EC). However, LPS increased EC death when cocultured with microglia. LPS led to nitric oxide (NO) and inducible NO synthase (iNOS) induction in microglia, but not in EC. Inhibiting microglial activation by blocking iNOS and other generators of NO or blocking reactive oxygen species (ROS) also prevented injury in these cocultures. To assess the signaling pathway(s) involved, inhibitors of several downstream TLR-4 activated pathways were studied. Inhibitors of NF-κB, JAK-STAT and JNK/SAPK decreased microglial activation and prevented cell death, although the effect of blocking JNK/SAPK was rather modest. Inhibitors of PI3K, ERK, and p38 MAPK had no effect. Conclusions We show that LPS-activated microglia promote BBB disruption through injury to endothelial cells, and the specific blockade of JAK-STAT, NF-κB may prove to be especially useful anti-inflammatory strategies to confer cerebrovascular protection. PMID:21385378

Targeting Metastasis with Snake Toxins: Molecular Mechanisms

PubMed Central

Urra, Félix A.

2017-01-01

Metastasis involves the migration of cancer cells from a primary tumor to invade and establish secondary tumors in distant organs, and it is the main cause for cancer-related deaths. Currently, the conventional cytostatic drugs target the proliferation of malignant cells, being ineffective in metastatic disease. This highlights the need to find new anti-metastatic drugs. Toxins isolated from snake venoms are a natural source of potentially useful molecular scaffolds to obtain agents with anti-migratory and anti-invasive effects in cancer cells. While there is greater evidence concerning the mechanisms of cell death induction of several snake toxin classes on cancer cells; only a reduced number of toxin classes have been reported (i.e., disintegrins/disintegrin-like proteins, C-type lectin-like proteins, C-type lectins, serinproteases, cardiotoxins, snake venom cystatins) as inhibitors of adhesion, migration, and invasion of cancer cells. Here, we discuss the anti-metastatic mechanisms of snake toxins, distinguishing three targets, which involve (1) inhibition of extracellular matrix components-dependent adhesion and migration, (2) inhibition of epithelial-mesenchymal transition, and (3) inhibition of migration by alterations in the actin/cytoskeleton network. PMID:29189742

Relevance of Endoplasmic Reticulum Stress Cell Signaling in Liver Cold Ischemia Reperfusion Injury

PubMed Central

Folch-Puy, Emma; Panisello, Arnau; Oliva, Joan; Lopez, Alexandre; Castro Benítez, Carlos; Adam, René; Roselló-Catafau, Joan

2016-01-01

The endoplasmic reticulum (ER) is involved in calcium homeostasis, protein folding and lipid biosynthesis. Perturbations in its normal functions lead to a condition called endoplasmic reticulum stress (ERS). This can be triggered by many physiopathological conditions such as alcoholic steatohepatitis, insulin resistance or ischemia-reperfusion injury. The cell reacts to ERS by initiating a defensive process known as the unfolded protein response (UPR), which comprises cellular mechanisms for adaptation and the safeguarding of cell survival or, in cases of excessively severe stress, for the initiation of the cell death program. Recent experimental data suggest the involvement of ERS in ischemia/reperfusion injury (IRI) of the liver graft, which has been considered as one of major problems influencing outcome after liver transplantation. The purpose of this review is to summarize updated data on the molecular mechanisms of ERS/UPR and the consequences of this pathology, focusing specifically on solid organ preservation and liver transplantation models. We will also discuss the potential role of ERS, beyond the simple adaptive response and the regulation of cell death, in the modification of cell functional properties and phenotypic changes. PMID:27231901

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

PubMed

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

2006-09-01

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

The RING-Type E3 Ligase XBAT35.2 Is Involved in Cell Death Induction and Pathogen Response1[OPEN

PubMed Central

Liu, Hongxia; Ravichandran, Sridhar; McVey, Sarah; Lilley, Carly; Teresinski, Howard J.; Gonzalez-Ferrer, Carmen; Prithiviraj, Balakrishnan

2017-01-01

XBAT35 belongs to a subfamily of Arabidopsis (Arabidopsis thaliana) RING-type E3s that are similar in domain architecture to the rice (Oryza sativa) XA21 Binding Protein3, a defense protein. The XBAT35 transcript undergoes alternative splicing to produce two protein isoforms, XBAT35.1 and XBAT35.2. Here, we demonstrate that XBAT35.2 localizes predominantly to the Golgi and is involved in cell death induction and pathogen response. XBAT35.2, but not XBAT35.1, was found to trigger cell death when overexpressed in tobacco (Nicotiana benthamiana) leaves and does so in a manner that requires its RING domain. Loss of XBAT35 gene function disrupts the plant’s ability to defend against pathogen attack, whereas overexpression of XBAT35.2 enhances resistance to pathogens. XBAT35.2 was found to be unstable and promotes its own degradation, suggesting self-regulation. Inoculation with virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae pv tomato DC3000 results in a drastic reduction in the levels of ubiquitinated XBAT35.2 and an increase in the abundance of the E3. This implies that pathogen infection prohibits XBAT35.2 self-regulation and stabilizes the E3. In agreement with a role in defending against pathogens, XBAT35.2 interacts with defense-related Accelerated Cell Death11 (ACD11) in planta and promotes the proteasome-dependent turnover of ACD11 in cell-free degradation assays. In accordance with regulation by a stabilized XBAT35.2, the levels of ubiquitinated ACD11 increased considerably, and the abundance of ACD11 was reduced following pathogen infection. In addition, treatment of transgenic seedlings with a proteasome inhibitor results in the accumulation of ACD11, confirming proteasome-dependent degradation. Collectively, these results highlight a novel role for XBAT35.2 in cell death induction and defense against pathogens. PMID:28951488

Mitofusin 1 degradation is induced by a disruptor of mitochondrial calcium homeostasis, CGP37157: a role in apoptosis in prostate cancer cells.

PubMed

Choudhary, Vivek; Kaddour-Djebbar, Ismail; Alaisami, Rabei; Kumar, M Vijay; Bollag, Wendy B

2014-05-01

Mitochondria constantly divide (mitochondrial fission) and fuse (mitochondrial fusion) in a normal cell. Disturbances in the balance between these two physiological processes may lead to cell dysfunction or to cell death. Induction of cell death is the prime goal of prostate cancer chemotherapy. Our previous study demonstrated that androgens increase the expression of a mitochondrial protein involved in fission and facilitate an apoptotic response to CGP37157 (CGP), an inhibitor of mitochondrial calcium efflux, in prostate cancer cells. However, the regulation and role of mitochondrial fusion proteins in the death of these cells have not been examined. Therefore, our objective was to investigate the effect of CGP on a key mitochondrial fusion protein, mitofusin 1 (Mfn1), and the role of Mfn1 in prostate cancer cell apoptosis. We used various prostate cancer cell lines and western blot analysis, qRT-PCR, siRNA, M30 apoptosis assay and immunoprecipitation techniques to determine mechanisms regulating Mfn1. Treatment of prostate cancer cells with CGP resulted in selective degradation of Mfn1. Mfn1 ubiquitination was detected following immunoprecipitation of overexpressed Myc-tagged Mfn1 protein from CGP-treated cells, and treatment with the proteasomal inhibitor lactacystin, as well as siRNA-mediated knockdown of the E3 ubiquitin ligase March5, protected Mfn1 from CGP-induced degradation. These data indicate the involvement of the ubiquitin-proteasome pathway in CGP-induced degradation of Mfn1. We also demonstrated that downregulation of Mfn1 by siRNA enhanced the apoptotic response of LNCaP cells to CGP, suggesting a likely pro-survival role for Mfn1 in these cells. Our results suggest that manipulation of mitofusins may provide a novel therapeutic advantage in treating prostate cancer.

Differential expression of virulence genes in Legionella pneumophila growing in Acanthamoeba and human monocytes.

PubMed

Mou, Qianqian; Leung, Polly H M

2018-01-01

Legionella pneumophila, the causative agent of Legionnaires' disease, is widely distributed throughout natural and artificial water systems and can replicate in macrophages and amoebae. Amoebae are the natural hosts of L. pneumophila, whereas macrophages are incidentally infected. The life cycle of L. pneumophila comprises a replicative phase within the Legionella-containing vacuole (LCV) and a transmissive phase during which bacterial cells become motile and are released via killing of the host. Although the host death mechanisms induced by L. pneumophila have been studied, the expression patterns of related L. pneumophila genes have not been reported. The present study compared the expression patterns of host cell death-associated genes in L. pneumophila grown in the human monocytic cell line THP-1 and Acanthamoeba castellanii. Notably, when L. pneumophila was grown in THP-1, expression of the gene flaA, which is involved in the induction of pyroptosis, was downregulated during the course of infection. In contrast, sdhA associated indirectly with host death, was upregulated. Expression of the genes vipD and sidF, which are involved in the induction and suppression of apoptosis, changed by less than 2-fold. Notably, a lower percentage of pyroptotic cells was observed among infected THP-1 cells relative to uninfected cells, and the latter exhibited stronger expression of caspase-1. A different pattern was observed when L. pneumophila was grown in A. castellanii: flaA and vipD were activated, whereas sdhA and sidF were downregulated during the later stage of replication. The percentage of non-viable (annexin-V + PI + or annexin-V + PI - ) A. castellanii organisms increased with Legionella infection, and the expression of metacaspase-1, which is involved in encystation was up-regulated at late infection time. In summary, L. pneumophila can multiply intracellularly in both amoebae and macrophages to induce cell death and secondary infection, and this characteristic is essential for its survival in water and the lungs. The gene expression profiles observed in this study indicated the increased cytotoxicity of L. pneumophila in A. castellanii, suggesting an increased adaptation of Legionella to this host.

The Alarmin Properties of DNA and DNA-associated Nuclear Proteins.

PubMed

Magna, Melinda; Pisetsky, David S

2016-05-01

The communication of cell injury and death is a critical element in host defense. Although immune cells can serve this function by elaborating cytokines and chemokines, somatic cells can repurpose nuclear macromolecules to function as damage-associated molecular patterns (DAMPs) or alarmins to exert similar activity. Among these molecules, DNA, high-mobility group box-1, and histone proteins can all act as DAMPs once they are in an extracellular location. This review describes current information on the role of the nuclear DAMPs, their translocation to the outside of cells, and pathways of activation after uptake into the inside of immune cells. MEDLINE and PubMed databases were searched for citations (1990-2016) in English related to the following terms: DAMPs, high-mobility group box-1, DNA, histones, cell death, danger, and immune activation. Selected articles with the most relevant studies were included for a more detailed consideration. Although nuclear molecules have important structural and genetic regulatory roles inside the cell nucleus, when released into the extracellular space during cell death, these molecules can acquire immune activity and serve as alarmins or DAMPs. Although apoptosis is generally considered the source of extracellular nuclear material, other cell death pathways such as necroptosis, NETosis, and pyroptosis can contribute to the release of nuclear molecules. Importantly, the release of nuclear DAMPs occurs with both soluble and particulate forms of these molecules. The activity of nuclear molecules may depend on posttranslational modifications, redox changes, and the binding of other molecules. Once in an extracellular location, nuclear DAMPs can engage the same pattern recognition receptors as do pathogen-associated molecular patterns. These interactions can activate immune cells and lead to cytokine and chemokine production. Among these receptors, internal receptors for DNA are key to the response to this molecule; the likely function of these internal sensors is the recognition of DNA from intracellular infection by bacteria or viruses. Activation of these receptors requires translocation of extracellular DNA into specialized compartments. In addition to nuclear DNA, mitochondrial DNA can also serve as a DAMP. The communication of cell injury and death is a critical element in host defense and involves the repurposing of nuclear molecules as immune triggers. As such, the presence of extracellular nuclear material can serve as novel biomarkers for conditions involving cell injury and death. Targeting of these molecules may also represent an important new approach to therapy. Published by Elsevier Inc.

TGFbeta type II receptor signaling controls Schwann cell death and proliferation in developing nerves.

PubMed

D'Antonio, Maurizio; Droggiti, Anna; Feltri, M Laura; Roes, Jürgen; Wrabetz, Lawrence; Mirsky, Rhona; Jessen, Kristján R

2006-08-16

During development, Schwann cell numbers are precisely adjusted to match the number of axons. It is essentially unknown which growth factors or receptors carry out this important control in vivo. Here, we tested whether the type II transforming growth factor (TGF) beta receptor has a role in this process. We generated a conditional knock-out mouse in which the type II TGFbeta receptor is specifically ablated only in Schwann cells. Inactivation of the receptor, evident at least from embryonic day 18, resulted in suppressed Schwann cell death in normally developing and injured nerves. Notably, the mutants also showed a strong reduction in Schwann cell proliferation. Consequently, Schwann cell numbers in wild-type and mutant nerves remained similar. Lack of TGFbeta signaling did not appear to affect other processes in which TGFbeta had been implicated previously, including myelination and response of adult nerves to injury. This is the first in vivo evidence for a growth factor receptor involved in promoting Schwann cell division during development and the first genetic evidence for a receptor that controls normal developmental Schwann cell death.

Spinosad induces programmed cell death involves mitochondrial dysfunction and cytochrome C release in Spodoptera frugiperda Sf9 cells.

PubMed

Yang, Mingjun; Wang, Bo; Gao, Jufang; Zhang, Yang; Xu, Wenping; Tao, Liming

2017-02-01

Spinosad, a reduced-risk insecticide, acts on the nicotinic acetylcholine receptors and the gamma-aminobutyric acid receptor in the nervous system of target insects. However, its mechanism of action in non-neural insect cells is unclear. This study aimed to evaluate mitochondrial functional changes associated with spinosad in Spodoptera frugiperda (Sf9) insect cells. Our results indicate that in Sf9 cells, spinosad induces programmed cell death and mitochondrial dysfunction through enhanced reactive oxygen species production, mitochondrial permeability transition pore (mPTP) opening, and mitochondrial membrane potential collapse, eventually leading to cytochrome C release and apoptosis. The cytochrome C release induced by spinosad treatment was partly inhibited by the mPTP inhibitors cyclosporin A and bongkrekic acid. Subsequently, we found that spinosad downregulated Bcl-2 expression and upregulated p53 and Bax expressions, activated caspase-9 and caspase-3, and triggered PARP cleavage in Sf9 cells. These findings suggested that spinosad-induced programmed cell death was modulated by mitochondrial dysfunction and cytochrome C release. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Btk-dependent PIP5K1γ lipid kinase activation by Fas counteracts FasL-induced cell death.

PubMed

Rossin, Aurélie; Lounnas, Nadia; Durivault, Jérôme; Miloro, Giorgia; Gagnoux-Palacios, Laurent; Hueber, Anne-Odile

2017-11-01

The Fas/FasL system plays a critical role in death by apoptosis and immune escape of cancer cells. The Fas receptor being ubiquitously expressed in tissues, its apoptotic-inducing function, initiated upon FasL binding, is tightly regulated by several negative regulatory mechanisms to prevent inappropriate cell death. One of them, involving the non-receptor tyrosine kinase Btk, was reported mainly in B cells and only poorly described. We report here that Btk negatively regulates, through its tyrosine kinase activity, the FasL-mediated cell death in epithelial cell lines from colon cancer origin. More importantly, we show that Btk interacts not only with Fas but also with the phosphatidylinositol-4-phosphate 5-kinase, PIP5K1γ, which, upon stimulation by Fas ligand, is responsible of a rapid and transient synthesis of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ). This production requires both the presence and the tyrosine kinase activity of Btk, and participates in the negative regulation of FasL-mediated cell death since knocking down PIP5K1γ expression significantly strengthens the apoptotic signal upon FasL engagement. Altogether, our data demonstrate the cooperative role of Btk and PIP5K1γ in a FasL-induced PI(4,5)P 2 production, both proteins participating to the threshold setting of FasL-induced apoptotic commitment in colorectal cell lines.

The NF-κB regulator Bcl-3 and the BH3-only proteins Bim and Puma control the death of activated T cells

PubMed Central

Bauer, Anette; Villunger, Andreas; Labi, Verena; Fischer, Silke F.; Strasser, Andreas; Wagner, Hermann; Schmid, Roland M.; Häcker, Georg

2006-01-01

Apoptosis of activated T cells is critical for the termination of immune responses. Here we show that adjuvant-stimulated dendritic cells secrete cytokines that prime activated T cells for survival and analyze the roles of the NF-κB regulator Bcl-3 and the proapoptotic Bcl-2 family members Bim and Puma. Bcl-3 overexpression increased survival, and activated bcl-3−/− T cells died abnormally rapidly. Cytokines from adjuvant-stimulated dendritic cells induced Bcl-3, but survival through cytokine priming was Bcl-3-independent. Apoptosis inhibition by Bcl-3 involved blockade of Bim activation, because Bim was overactivated in Bcl-3-deficient cells, and Bcl-3 failed to increase survival of bim−/− T cells. However, adjuvants increased survival also in Bim-deficient T cells. This Bim-independent death pathway is at least in part regulated by Puma, as shown by analysis of puma−/− and noxa−/− T cells. IL-1, IL-7, and IL-15 primed T cells for survival even in the absence of Bim or Puma. Our data define interrelations and a Bim-independent pathway to activated T cell death. PMID:16832056

Methylglyoxal-bis(guanylhydrazone), a polyamine analogue, sensitized γ-radiation-induced cell death in HL-60 leukemia cells Sensitizing effect of MGBG on γ-radiation-induced cell death.

PubMed

Kim, Jin Sik; Lee, Jin; Chung, Hai Won; Choi, Han; Paik, Sang Gi; Kim, In Gyu

2006-09-01

Methylglyoxal-bis(guanylhydrazone) (MGBG), a polyamine analogue, has been known to inhibit the biosynthesis of polyamines, which are important in cell proliferation. We showed that MGBG treatment significantly affected γ-radiation-induced cell cycle transition (G(1)/G(0)→S→G(2)/M) and thus γ-radiation-induced cell death. As determined by micronuclei and comet assay, we showed that it sensitized the cytotoxic effect induced by γ-radiation. One of the reasons is that polyamine depletion by MGBG treatment did not effectively protect against the chemical (OH) or physical damage to DNA caused by γ-radiation. Through in vitro experiment, we confirmed that DNA strand breaks induced by γ-radiation was prevented more effectively in the presence of polyamines (spermine and spermidine) than in the absence of polyamines. MGBG also blocks the cell cycle transition caused by γ-radiation (G(2) arrest), which helps protect cells by allowing time for DNA repair before entry into mitosis or apoptosis, via the down regulation of cyclin D1, which mediates the transition from G(1) to S phase of cell cycle, and ataxia telangiectasia mutated, which is involved in the DNA sensing, repair and cell cycle check point. Therefore, the abrogation of G(2) arrest sensitizes cells to the effect of γ-radiation. As a result, γ-radiation-induced cell death increased by about 2.5-3.0-fold in cells treated with MGBG. However, exogenous spermidine supplement partially relieved this γ-radiation-induced cytotoxicity and cell death. These findings suggest a potentially therapeutic strategy for increasing the cytotoxic efficacy of γ-radiation.

Biological pathways involved in the development of inflammatory bowel disease.

PubMed

Zemljic, Mateja; Pejkovic, Bozena; Krajnc, Ivan; Lipovsek, Saska

2014-10-01

Apoptosis, autophagy and necrosis are three distinct functional types of the mammalian cell death network. All of them are characterized by a number of cell's morphological changes. The inappropriate induction of cell death is involved in the pathogenesis of a number of diseases.Pathogenesis of inflammatory bowel diseases (ulcerative colitis, Crohn's disease) includes an abnormal immunological response to disturbed intestinal microflora. One of the most important reason in pathogenesis of chronic inflammatory disease and subsequent multiple organ pathology is a barrier function of the gut, regulating cellular viability. Recent findings have begun to explain the mechanisms by which intestinal epithelial cells are able to survive in such an environment and how loss of normal regulatory processes may lead to inflammatory bowel disease (IBD).This review focuses on the regulation of biological pathways in development and homeostasis in IBD. Better understanding of the physiological functions of biological pathways and their influence on inflammation, immunity, and barrier function will simplify our expertice of homeostasis in the gastrointestinal tract and in upgrading diagnosis and treatment.

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

PubMed

Twumasi, Peter; Iakimova, Elena T; Qian, Tian; van Ieperen, Wim; Schel, Jan H N; Emons, Anne Mie C; van Kooten, Olaf; Woltering, Ernst J

2010-08-06

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

Mammalian follicular development and atresia: role of apoptosis.

PubMed

Asselin, E; Xiao, C W; Wang, Y F; Tsang, B K

2000-01-01

The regulation of follicular development and atresia is a complex process and involves interactions between endocrine factors (gonadotropins) and intraovarian regulators (sex steroids, growth factors and cytokines) in the control of follicular cell fate (i.e. proliferation, differentiation and programmed cell death). Granulosa and theca cells are key players in this fascinating process. As atresia is the fate of most follicles, understanding of how these physiological regulators participate in determining the destiny of the follicle (to degenerate or to ovulate) at cellular and subcellular levels is fundamental. This short review summarizes the role of intraovarian modulators of programmed cell death in the induction of atresia during follicular development. Copyright 2000 S. Karger AG, Basel

Chrysophanol-induced cell death (necrosis) in human lung cancer A549 cells is mediated through increasing reactive oxygen species and decreasing the level of mitochondrial membrane potential.

PubMed

Ni, Chien-Hang; Yu, Chun-Shu; Lu, Hsu-Feng; Yang, Jai-Sing; Huang, Hui-Ying; Chen, Po-Yuan; Wu, Shin-Hwar; Ip, Siu-Wan; Chiang, Su-Yin; Lin, Jaung-Geng; Chung, Jing-Gung

2014-05-01

Chrysophanol (1,8-dihydroxy-3-methylanthraquinone) is one of the anthraquinone compounds, and it has been shown to induce cell death in different types of cancer cells. The effects of chrysophanol on human lung cancer cell death have not been well studied. The purpose of this study is to examine chrysophanol-induced cytotoxic effects and also to investigate such influences that involved apoptosis or necrosis in A549 human lung cancer cells in vitro. Our results indicated that chrysophanol decreased the viable A549 cells in a dose- and time-dependent manner. Chrysophanol also promoted the release of reactive oxygen species (ROS) and Ca(2+) and decreased the levels of mitochondria membrane potential (ΔΨm ) and adenosine triphosphate in A549 cells. Furthermore, chrysophanol triggered DNA damage by using Comet assay and DAPI staining. Importantly, chrysophanol only stimulated the cytocheome c release, but it did not activate other apoptosis-associated protein levels including caspase-3, caspase-8, Apaf-1, and AIF. In conclusion, human lung cancer A549 cells treated with chrysophanol exhibited a cellular pattern associated with necrotic cell death and not apoptosis in vitro. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 740-749, 2014. Copyright © 2012 Wiley Periodicals, Inc., a Wiley company.

Novel Genome-Wide Screening Method Identifies Genes Important to Breast Cancer Metastasis | Center for Cancer Research

Cancer.gov

For patients with solid tumors, the primary cause of illness and death is metastasis, a complex process involving multiple steps and cooperation between cancerous and normal cells. Many genes must be involved, but few have been found and characterized.

AMPA receptor-mediated toxicity in oligodendrocyte progenitors involves free radical generation and activation of JNK, calpain and caspase 3.

PubMed

Liu, Hsueh-Ning; Giasson, Benoit I; Mushynski, Walter E; Almazan, Guillermina

2002-07-01

The molecular mechanisms underlying AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) receptor-mediated excitotoxicity were characterized in rat oligodendrocyte progenitor cultures. Activation of AMPA receptors, in the presence of cyclothiazide to selectively block desensitization, produced a massive Ca(2+) influx and cytotoxicity which were blocked by the antagonists CNQX and GYKI 52466. A role for free radical generation in oligodendrocyte progenitor cell death was deduced from three observations: (i) treatment with AMPA agonists decreased intracellular glutathione; (ii) depletion of intracellular glutathione with buthionine sulfoximine potentiated cell death; and (iii) the antioxidant N -acetylcysteine replenished intracellular glutathione and protected cultures from AMPA receptor-mediated toxicity. Cell death displayed some characteristics of apoptosis, including DNA fragmentation, chromatin condensation and activation of caspase-3 and c-Jun N-terminal kinase (JNK). A substrate of calpain and caspase-3, alpha-spectrin, was cleaved into characteristic products following treatment with AMPA agonists. In contrast, inhibition of either caspase-3 by DEVD-CHO or calpain by PD 150606 protected cells from excitotoxicity. Our results indicate that overactivation of AMPA receptors causes apoptosis in oligodendrocyte progenitors through mechanisms involving Ca(2+) influx, depletion of glutathione, and activation of JNK, calpain, and caspase-3.

Mechanism of Cisplatin-Induced Cytotoxicity Is Correlated to Impaired Metabolism Due to Mitochondrial ROS Generation

PubMed Central

Shim, Wooyoung; Anwar, Muhammad Ayaz; Kwon, Ji-Woong; Kwon, Hyuk-Kwon; Kim, Hyung Joong; Jeong, Hyobin; Kim, Hwan Myung; Hwang, Daehee; Kim, Hyung Sik; Choi, Sangdun

2015-01-01

The chemotherapeutic use of cisplatin is limited by its severe side effects. In this study, by conducting different omics data analyses, we demonstrated that cisplatin induces cell death in a proximal tubular cell line by suppressing glycolysis- and tricarboxylic acid (TCA)/mitochondria-related genes. Furthermore, analysis of the urine from cisplatin-treated rats revealed the lower expression levels of enzymes involved in glycolysis, TCA cycle, and genes related to mitochondrial stability and confirmed the cisplatin-related metabolic abnormalities. Additionally, an increase in the level of p53, which directly inhibits glycolysis, has been observed. Inhibition of p53 restored glycolysis and significantly reduced the rate of cell death at 24 h and 48 h due to p53 inhibition. The foremost reason of cisplatin-related cytotoxicity has been correlated to the generation of mitochondrial reactive oxygen species (ROS) that influence multiple pathways. Abnormalities in these pathways resulted in the collapse of mitochondrial energy production, which in turn sensitized the cells to death. The quenching of ROS led to the amelioration of the affected pathways. Considering these observations, it can be concluded that there is a significant correlation between cisplatin and metabolic dysfunctions involving mROS as the major player. PMID:26247588

A mechanistic study on the effect of dexamethasone in moderating cell death in Chinese Hamster Ovary cell cultures.

PubMed

Jing, Ying; Qian, Yueming; Ghandi, Mahmoud; He, Aiqing; Borys, Michael C; Pan, Shih-Hsie; Li, Zheng Jian

2012-01-01

Dexamethasone (DEX) was previously shown (Jing et al., Biotechnol Bioeng. 2010;107:488-496) to play a dual role in increasing sialylation of recombinant glycoproteins produced by Chinese Hamster Ovary (CHO) cells. DEX addition increased sialic acid levels of a recombinant fusion protein through increased expression of α2,3-sialyltransferase and β1,4-galactosyltransferase, but also decreased the sialidase-mediated, extracellular degradation of sialic acid through slowing cell death at the end of the culture period. This study examines the underlying mechanism for this cytoprotective action by studying the transcriptional response of the CHO cell genome upon DEX treatment using DNA microarrays and gene ontology term analysis. Many of those genes showing a significant transcriptional response were associated with the regulation of programmed cell death. The gene with the highest change in expression level, as validated by Quantitative PCR assays with TaqMan® probes and confirmed by Western Blot analysis, was the antiapoptotic gene Tsc22d3, also referred to as GILZ (glucocorticoid-induced leucine zipper). The pathway by which DEX suppressed cell death towards the end of the culture period was also confirmed by showing involvement of glucocorticoid receptors and GILZ through studies using the glucocorticoid antagonist mifepristone (RU-486). These findings advance the understanding of the mechanism by which DEX suppresses cell death in CHO cells and provide a rationale for the application of glucocorticoids in CHO cell culture processes. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

Cullin-4 regulates Wingless and JNK signaling-mediated cell death in the Drosophila eye

PubMed Central

Tare, Meghana; Sarkar, Ankita; Bedi, Shimpi; Kango-Singh, Madhuri; Singh, Amit

2016-01-01

In all multicellular organisms, the fundamental processes of cell proliferation and cell death are crucial for growth regulation during organogenesis. Strict regulation of cell death is important to maintain tissue homeostasis by affecting processes like regulation of cell number, and elimination of unwanted/unfit cells. The developing Drosophila eye is a versatile model to study patterning and growth, where complex signaling pathways regulate growth and cell survival. However, the molecular mechanisms underlying regulation of these processes is not fully understood. In a gain-of-function screen, we found that misexpression of cullin-4 (cul-4), an ubiquitin ligase, can rescue reduced eye mutant phenotypes. Previously, cul-4 has been shown to regulate chromatin remodeling, cell cycle and cell division. Genetic characterization of cul-4 in the developing eye revealed that loss-of-function of cul-4 exhibits a reduced eye phenotype. Analysis of twin-spots showed that in comparison with their wild-type counterparts, the cul-4 loss-of-function clones fail to survive. Here we show that cul-4 clones are eliminated by induction of cell death due to activation of caspases. Aberrant activation of signaling pathways is known to trigger cell death in the developing eye. We found that Wingless (Wg) and c-Jun-amino-terminal-(NH2)-Kinase (JNK) signaling are ectopically induced in cul-4 mutant clones, and these signals co-localize with the dying cells. Modulating levels of Wg and JNK signaling by using agonists and antagonists of these pathways demonstrated that activation of Wg and JNK signaling enhances cul-4 mutant phenotype, whereas downregulation of Wg and JNK signaling rescues the cul-4 mutant phenotypes of reduced eye. Here we present evidences to demonstrate that cul-4 is involved in restricting Wg signaling and downregulation of JNK signaling-mediated cell death during early eye development. Overall, our studies provide insights into a novel role of cul-4 in promoting cell survival in the developing Drosophila eye. PMID:28032862

The Hippo pathway: regulators and regulations

PubMed Central

Yu, Fa-Xing; Guan, Kun-Liang

2013-01-01

Control of cell number is crucial in animal development and tissue homeostasis, and its dysregulation may result in tumor formation or organ degeneration. The Hippo pathway in both Drosophila and mammals regulates cell number by modulating cell proliferation, cell death, and cell differentiation. Recently, numerous upstream components involved in the Hippo pathway have been identified, such as cell polarity, mechanotransduction, and G-protein-coupled receptor (GPCR) signaling. Actin cytoskeleton or cellular tension appears to be the master mediator that integrates and transmits upstream signals to the core Hippo signaling cascade. Here, we review regulatory mechanisms of the Hippo pathway and discuss potential implications involved in different physiological and pathological conditions. PMID:23431053

Epigallocatechin-3-Gallate Suppresses Human Herpesvirus 8 Replication and Induces ROS Leading to Apoptosis and Autophagy in Primary Effusion Lymphoma Cells

PubMed Central

Tsai, Ching-Yi; Chen, Chang-Yu; Chiou, Yee-Hsuan; Shyu, Huey-Wen; Lin, Kuan-Hua; Chou, Miao-Chen; Huang, Mei-Han; Wang, Yi-Fen

2017-01-01

Epigallocatechin-3-gallate (EGCG), the major constituent of green tea, has been shown to induce cell death in cancer cells. Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by human herpesvirus 8 (HHV8). In this study, we examined the role of EGCG on PEL cells in cell death and HHV8 replication. We performed trypan blue exclusion assay to assess the cell viability of PEL cells, flow cytometry analysis to examine the cell cycle distribution and reactive oxygen species (ROS) generation, caspase-3 activity to assay apoptosis, acridine orange staining to determine autophagy, and immunoblotting to detect the protein levels involved in apoptosis and autophagy as well as mitogen activated protein kinases (MAPKs) activation upon EGCG treatment. The expression of the HHV8 lytic gene was determined by luciferase reporter assay and reverse transcription-PCR, and viral progeny production was determined by PCR. Results revealed that EGCG induced cell death and ROS generation in PEL cells in a dose-dependent manner. N-acetylcysteine (NAC) inhibited the EGCG-induced ROS and rescued the cell from EGCG-induced cell death. Even though EGCG induced ROS generation in PEL cells, it reduced the production of progeny virus from PEL cells without causing HHV8 reactivation. These results suggest that EGCG may represent a novel strategy for the treatment of HHV8 infection and HHV8-associated lymphomas. PMID:29267216

Seizure-like activity leads to the release of BAD from 14-3-3 protein and cell death in hippocampal neurons in vitro.

PubMed

Meller, R; Schindler, C K; Chu, X P; Xiong, Z G; Cameron, J A; Simon, R P; Henshall, D C

2003-05-01

Seizure-induced neuronal death may involve engagement of the BCL-2 family of apoptosis-regulating proteins. In the present study we examined the activation of proapoptotic BAD in cultured hippocampal neurons following seizures induced by removal of chronic glutamatergic transmission blockade. Kynurenic acid withdrawal elicited an increase in seizure-like electrical activity, which was inhibited by blockers of AMPA (CNQX) and NMDA (MK801 and AP5) receptor function. However, only NMDA receptor antagonists inhibited calcium entry as assessed by fura-2, and cell death of hippocampal neurons. Seizures increased proteolysis of caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) of cells. Seizure-like activity induced dephosphorylation of BAD and the disruption of its constitutive interaction with 14-3-3 proteins. In turn, BAD dimerized with antiapoptotic BCL-Xl after seizures. However, the absence of neuroprotective effects of pathway intervention suggests that BAD may perform a reinforcement rather than instigator role in cell death following seizures in vitro.

Pore-forming toxin-mediated ion dysregulation leads to death receptor-independent necroptosis of lung epithelial cells during bacterial pneumonia

PubMed Central

González-Juarbe, Norberto; Bradley, Kelley Margaret; Shenoy, Anukul Taranath; Gilley, Ryan Paul; Reyes, Luis Felipe; Hinojosa, Cecilia Anahí; Restrepo, Marcos Ignacio; Dube, Peter Herman; Bergman, Molly Ann; Orihuela, Carlos Javier

2017-01-01

We report that pore-forming toxins (PFTs) induce respiratory epithelial cell necroptosis independently of death receptor signaling during bacterial pneumonia. Instead, necroptosis was activated as a result of ion dysregulation arising from membrane permeabilization. PFT-induced necroptosis required RIP1, RIP3 and MLKL, and could be induced in the absence or inhibition of TNFR1, TNFR2 and TLR4 signaling. We detected activated MLKL in the lungs from mice and nonhuman primates experiencing Serratia marcescens and Streptococcus pneumoniae pneumonia, respectively. We subsequently identified calcium influx and potassium efflux as the key initiating signals responsible for necroptosis; also that mitochondrial damage was not required for necroptosis activation but was exacerbated by MLKL activation. PFT-induced necroptosis in respiratory epithelial cells did not involve CamKII or reactive oxygen species. KO mice deficient in MLKL or RIP3 had increased survival and reduced pulmonary injury during S. marcescens pneumonia. Our results establish necroptosis as a major cell death pathway active during bacterial pneumonia and that necroptosis can occur without death receptor signaling. PMID:28387756

Pore-forming toxin-mediated ion dysregulation leads to death receptor-independent necroptosis of lung epithelial cells during bacterial pneumonia.

PubMed

González-Juarbe, Norberto; Bradley, Kelley Margaret; Shenoy, Anukul Taranath; Gilley, Ryan Paul; Reyes, Luis Felipe; Hinojosa, Cecilia Anahí; Restrepo, Marcos Ignacio; Dube, Peter Herman; Bergman, Molly Ann; Orihuela, Carlos Javier

2017-05-01

We report that pore-forming toxins (PFTs) induce respiratory epithelial cell necroptosis independently of death receptor signaling during bacterial pneumonia. Instead, necroptosis was activated as a result of ion dysregulation arising from membrane permeabilization. PFT-induced necroptosis required RIP1, RIP3 and MLKL, and could be induced in the absence or inhibition of TNFR1, TNFR2 and TLR4 signaling. We detected activated MLKL in the lungs from mice and nonhuman primates experiencing Serratia marcescens and Streptococcus pneumoniae pneumonia, respectively. We subsequently identified calcium influx and potassium efflux as the key initiating signals responsible for necroptosis; also that mitochondrial damage was not required for necroptosis activation but was exacerbated by MLKL activation. PFT-induced necroptosis in respiratory epithelial cells did not involve CamKII or reactive oxygen species. KO mice deficient in MLKL or RIP3 had increased survival and reduced pulmonary injury during S. marcescens pneumonia. Our results establish necroptosis as a major cell death pathway active during bacterial pneumonia and that necroptosis can occur without death receptor signaling.

Involvement of the Electrophilic Isothiocyanate Sulforaphane in Arabidopsis Local Defense Responses1

PubMed Central

Andersson, Mats X.; Nilsson, Anders K.; Johansson, Oskar N.; Boztaş, Gülin; Adolfsson, Lisa E.; Pinosa, Francesco; Petit, Christel Garcia; Aronsson, Henrik; Mackey, David; Tör, Mahmut; Hamberg, Mats; Ellerström, Mats

2015-01-01

Plants defend themselves against microbial pathogens through a range of highly sophisticated and integrated molecular systems. Recognition of pathogen-secreted effector proteins often triggers the hypersensitive response (HR), a complex multicellular defense reaction where programmed cell death of cells surrounding the primary site of infection is a prominent feature. Even though the HR was described almost a century ago, cell-to-cell factors acting at the local level generating the full defense reaction have remained obscure. In this study, we sought to identify diffusible molecules produced during the HR that could induce cell death in naive tissue. We found that 4-methylsulfinylbutyl isothiocyanate (sulforaphane) is released by Arabidopsis (Arabidopsis thaliana) leaf tissue undergoing the HR and that this compound induces cell death as well as primes defense in naive tissue. Two different mutants impaired in the pathogen-induced accumulation of sulforaphane displayed attenuated programmed cell death upon bacterial and oomycete effector recognition as well as decreased resistance to several isolates of the plant pathogen Hyaloperonospora arabidopsidis. Treatment with sulforaphane provided protection against a virulent H. arabidopsidis isolate. Glucosinolate breakdown products are recognized as antifeeding compounds toward insects and recently also as intracellular signaling and bacteriostatic molecules in Arabidopsis. The data presented here indicate that these compounds also trigger local defense responses in Arabidopsis tissue. PMID:25371552

Critical Involvement of Extracellular ATP Acting on P2RX7 Purinergic Receptors in Photoreceptor Cell Death

PubMed Central

Notomi, Shoji; Hisatomi, Toshio; Kanemaru, Takaaki; Takeda, Atsunobu; Ikeda, Yasuhiro; Enaida, Hiroshi; Kroemer, Guido; Ishibashi, Tatsuro

2011-01-01

Stressed cells release ATP, which participates in neurodegenerative processes through the specific ligation of P2RX7 purinergic receptors. Here, we demonstrate that extracellular ATP and the more specific P2RX7 agonist, 2′- and 3′-O-(4-benzoylbenzoyl)-ATP, both induce photoreceptor cell death when added to primary retinal cell cultures or when injected into the eyes from wild-type mice, but not into the eyes from P2RX7−/− mice. Photoreceptor cell death was accompanied by the activation of caspase-8 and -9, translocation of apoptosis-inducing factor from mitochondria to nuclei, and TUNEL-detectable chromatin fragmentation. All hallmarks of photoreceptor apoptosis were prevented by premedication or co-application of Brilliant Blue G, a selective P2RX7 antagonist that is already approved for the staining of internal limiting membranes during ocular surgery. ATP release is up-regulated by nutrient starvation in primary retinal cell cultures and seems to be an initializing event that triggers primary and/or secondary cell death via the positive feedback loop on P2RX7. Our results encourage the potential application of Brilliant Blue G as a novel neuroprotective agent in retinal diseases or similar neurodegenerative pathologies linked to excessive extracellular ATP. PMID:21983632

Cisplatin resistance induced in germ cell tumour cells is due to reduced susceptibility towards cell death but not to altered DNA damage induction or repair.

PubMed

Fenske, Annabelle E; Glaesener, Stephanie; Bokemeyer, Carsten; Thomale, Juergen; Dahm-Daphi, Jochen; Honecker, Friedemann; Dartsch, Dorothee C

2012-11-28

To identify factors involved in cisplatin (CDDP) resistance of germ cell tumours (GCTs), we exposed NTERA-2 cells, and the platinum-adapted subline NTERA-2R to CDDP and compared their response. While both cell lines showed comparable proliferation, NTERA-2R cells were clearly more resistant to the drug than the parental NTERA-2 cell line. Interestingly, the two lines showed identical extent of DNA adduct formation and elimination, indicating that neither changes in CDDP uptake, nor altered drug efflux, DNA binding, or repair caused the difference in resistance. Similarly, no difference occurred in the time-course of γH2AX formation, which was not linked to 53BP1 accumulation. In contrast, NTERA-2R cells showed a more pronounced dose-dependent S phase delay, a transient G(2)/M-block, and subsequent release into immediate cell death. We thus conclude that the enhanced resistance against CDDP is linked to reduced susceptibility to cell death rather than to an altered DNA adduct formation or adduct removal. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Characterization of Puma-Dependent and Puma-Independent Neuronal Cell Death Pathways following Prolonged Proteasomal Inhibition▿

PubMed Central

Tuffy, Liam P.; Concannon, Caoimhín G.; D'Orsi, Beatrice; King, Matthew A.; Woods, Ina; Huber, Heinrich J.; Ward, Manus W.; Prehn, Jochen H. M.

2010-01-01

Proteasomal stress and the accumulation of polyubiquitinated proteins are key features of numerous neurodegenerative disorders. Previously we demonstrated that stabilization of p53 and activation of its target gene, puma (p53-upregulated mediator of apoptosis), mediated proteasome inhibitor-induced apoptosis in cancer cells. Here we demonstrated that Puma also contributed to proteasome inhibitor-induced apoptosis in mouse neocortical neurons. Although protection afforded by puma gene deletion was incomplete, we found little evidence indicating contributions from other proapoptotic BH3-only proteins. Attenuation of bax expression did not further reduce Puma-independent apoptosis, suggesting that pathways other than the mitochondrial apoptosis pathway were activated. Real-time imaging experiments in wild-type and puma-deficient neurons using a fluorescence resonance energy transfer (FRET)-based caspase sensor confirmed the involvement of a second cell death pathway characterized by caspase activation prior to mitochondrial permeabilization and, more prominently, a third, caspase-independent and Puma-independent pathway characterized by rapid cell shrinkage and nuclear condensation. This pathway involved lysosomal permeabilization in the absence of autophagy activation and was sensitive to cathepsin but not autophagy inhibition. Our data demonstrate that proteasomal stress activates distinct cell death pathways in neurons, leading to both caspase-dependent and caspase-independent apoptosis, and demonstrate independent roles for Puma and lysosomal permeabilization in this model. PMID:20921277

ER Stress-Mediated Signaling: Action Potential and Ca(2+) as Key Players.

PubMed

Bahar, Entaz; Kim, Hyongsuk; Yoon, Hyonok

2016-09-15

The proper functioning of the endoplasmic reticulum (ER) is crucial for multiple cellular activities and survival. Disturbances in the normal ER functions lead to the accumulation and aggregation of unfolded proteins, which initiates an adaptive response, the unfolded protein response (UPR), in order to regain normal ER functions. Failure to activate the adaptive response initiates the process of programmed cell death or apoptosis. Apoptosis plays an important role in cell elimination, which is essential for embryogenesis, development, and tissue homeostasis. Impaired apoptosis can lead to the development of various pathological conditions, such as neurodegenerative and autoimmune diseases, cancer, or acquired immune deficiency syndrome (AIDS). Calcium (Ca(2+)) is one of the key regulators of cell survival and it can induce ER stress-mediated apoptosis in response to various conditions. Ca(2+) regulates cell death both at the early and late stages of apoptosis. Severe Ca(2+) dysregulation can promote cell death through apoptosis. Action potential, an electrical signal transmitted along the neurons and muscle fibers, is important for conveying information to, from, and within the brain. Upon the initiation of the action potential, increased levels of cytosolic Ca(2+) (depolarization) lead to the activation of the ER stress response involved in the initiation of apoptosis. In this review, we discuss the involvement of Ca(2+) and action potential in ER stress-mediated apoptosis.

Over-expression of Trxo1 increases the viability of tobacco BY-2 cells under H2O2 treatment.

PubMed

Ortiz-Espín, Ana; Locato, Vittoria; Camejo, Daymi; Schiermeyer, Andreas; De Gara, Laura; Sevilla, Francisca; Jiménez, Ana

2015-09-01

Reactive oxygen species (ROS), especially hydrogen peroxide, play a critical role in the regulation of plant development and in the induction of plant defence responses during stress adaptation, as well as in plant cell death. The antioxidant system is responsible for controlling ROS levels in these processes but redox homeostasis is also a key factor in plant cell metabolism under normal and stress situations. Thioredoxins (Trxs) are ubiquitous small proteins found in different cell compartments, including mitochondria and nuclei (Trxo1), and are involved in the regulation of target proteins through reduction of disulphide bonds, although their role under oxidative stress has been less well studied. This study describes over-expression of a Trxo1 for the first time, using a cell-culture model subjected to an oxidative treatment provoked by H2O2. Control and over-expressing PsTrxo1 tobacco (Nicotiana tabacum) BY-2 cells were treated with 35 mm H2O2 and the effects were analysed by studying the growth dynamics of the cultures together with oxidative stress parameters, as well as several components of the antioxidant systems involved in the metabolism of H2O2. Analysis of different hallmarks of programmed cell death was also carried out. Over-expression of PsTrxo1 caused significant differences in the response of TBY-2 cells to high concentrations of H2O2, namely higher and maintained viability in over-expressing cells, whilst the control line presented a severe decrease in viability and marked indications of oxidative stress, with generalized cell death after 3 d of treatment. In over-expressing cells, an increase in catalase activity, decreases in H2O2 and nitric oxide contents and maintenance of the glutathione redox state were observed. A decreased content of endogenous H2O2 may be responsible in part for the delayed cell death found in over-expressing cells, in which changes in oxidative parameters and antioxidants were less extended after the oxidative treatment. It is concluded that PsTrxo1 transformation protects TBY-2 cells from exogenous H2O2, thus increasing their viability via a process in which not only antioxidants but also Trxo1 seem to be involved. © The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Altered Cytochrome c Display Precedes Apoptotic Cell Death in Drosophila

PubMed Central

Varkey, Johnson; Chen, Po; Jemmerson, Ronald; Abrams, John M.

1999-01-01

Drosophila affords a genetically well-defined system to study apoptosis in vivo. It offers a powerful extension to in vitro models that have implicated a requirement for cytochrome c in caspase activation and apoptosis. We found that an overt alteration in cytochrome c anticipates programmed cell death (PCD) in Drosophila tissues, occurring at a time that considerably precedes other known indicators of apoptosis. The altered configuration is manifested by display of an otherwise hidden epitope and occurs without release of the protein into the cytosol. Conditional expression of the Drosophila death activators, reaper or grim, provoked apoptogenic cytochrome c display and, surprisingly, caspase activity was necessary and sufficient to induce this alteration. In cell-free studies, cytosolic caspase activation was triggered by mitochondria from apoptotic cells but identical preparations from healthy cells were inactive. Our observations provide compelling validation of an early role for altered cytochrome c in PCD and suggest propagation of apoptotic physiology through reciprocal, feed-forward amplification involving cytochrome c and caspases. PMID:10037791

Human replication protein Cdc6 is selectively cleaved by caspase 3 during apoptosis

PubMed Central

Pelizon, Cristina; d’Adda di Fagagna, Fabrizio; Farrace, Lorena; Laskey, Ronald A.

2002-01-01

In eukaryotes, the initiation of DNA replication involves the ordered assembly on chromatin of pre-replicative complexes (pre-RCs), including the origin recognition complex (ORC), Cdc6, Cdt1 and the minichromosome maintenance proteins (MCMs). In light of its indispensable role in the formation of pre-RCs, Cdc6 binding to chromatin represents a key step in the regulation of DNA replication and cell proliferation. Here, we study the human Cdc6 (HuCdc6) protein during programmed cell death (apoptosis). We find that HuCdc6, but not HuOrc2 (a member of the ORC) or HuMcm5 (one of the MCMs), is specifically cleaved in several human cell lines induced to undergo apoptosis by a variety of stimuli. Expression of caspase-uncleavable mutant HuCdc6 attenuates apoptosis, delaying cell death. Therefore, an important function for cleavage of HuCdc6 is to prevent a wounded cell from replicating and to facilitate death. PMID:12151338

Histone deacetylase inhibitors promote the tumoricidal effect of HAMLET.

PubMed

Brest, Patrick; Gustafsson, Mattias; Mossberg, Ann-Kristin; Gustafsson, Lotta; Duringer, Caroline; Hamiche, Ali; Svanborg, Catharina

2007-12-01

Histone deacetylase inhibitors (HDIs) and HAMLET (human alpha-lactalbumin made lethal to tumor cells) interact with histones, modify the structure of chromatin, and trigger tumor cell death. This study investigated how the combination of HDIs and HAMLET influences cell viability, histone acetylation, and DNA integrity. The pretreatment of tumor cells with HDIs was shown to enhance the lethal effect of HAMLET and the histone hyperacetylation response to HDIs increased even further after HAMLET treatment. HDIs and HAMLET were shown to target different histone domains as HAMLET bound tailless core histones, whereas HDIs modify the acetylation of the histone tail. DNA damage in response to HAMLET was increased by HDIs. The DNA repair response (p21WAFI expression) was induced by both agonists but abolished when the two agonists were combined. The results suggest that the synergy of HDIs and HAMLET is based on different but converging death pathways, both involving chromatin alterations. We speculate that HAMLET and HDIs might be combined to promote tumor cell death in vivo.

Bone marrow stromal-B cell interactions in polycyclic aromatic hydrocarbon-induced pro/pre-B cell apoptosis.

PubMed

Allan, Lenka L; Mann, Koren K; Matulka, Raymond A; Ryu, Heui-Young; Schlezinger, Jennifer J; Sherr, David H

2003-12-01

Environmental polycyclic aromatic hydrocarbons (PAH) and related halogenated hydrocarbons are immunotoxic in a variety of systems. In a model system of B lymphopoiesis, PAH exposure rapidly induces apoptosis in CD43- pre-B and CD43+ pro/pre-B cells. Apoptosis induction by 7,12-dimethylbenzo[a]anthracene (DMBA) is dependent upon AhR+ bone marrow stromal cells and likely involves DMBA metabolism within the stromal cell. However, it is not known if PAH-treated stromal cells release free metabolites or soluble factors that may directly induce B cell death or if the effector death signal is delivered by stromal cell-B cell contact. Here, we demonstrate that supernatants from DMBA-treated bone marrow stromal cells contain an activity capable of inducing apoptosis in pro/pre-B cells cocultured with stromal cells. This activity (1) is not produced when stromal cells are cotreated with DMBA and alpha-naphthoflavone (alpha-NF), an aryl hydrocarbon receptor (AhR) and cytochrome P-450 inhibitor, (2) is > or = 50 kDa, (3) is trypsin and heat sensitive, and (4) is dependent on AhR+ stromal cells, which in turn deliver the effector death signal to pro/pre-B cells. The results (1) argue against a role for a soluble, stromal cell-derived cytokine as the effector of PAH-induced pro/pre-B cell death, (2) exclude the possibility of a free metabolite acting directly on AhR- pro/pre-B cell targets, and (3) suggest the elaboration by stromal cells of a relatively stable, DMBA metabolite-protein complex capable of acting on other stromal cells at some distance. Collectively, these studies suggest that, while stromal cell products, e.g., metabolite-protein complexes, may affect the function of distant stromal cells, the effector death signal delivered by stromal cells to bone marrow B cells is mediated by cell-cell contact.

Artesunate induces AIF-dependent apoptosis in A549 cells

NASA Astrophysics Data System (ADS)

Zhou, Chen-juan; Chen, Tong-Sheng

2012-03-01

Artesunate (ART), a semi-synthetic derivative of the sesquiterpene artemisinin extracted from the Chinese herb Artemisia annua, exerts a broad spectrum of clinical activity against human cancers. It has been shown that ART induces cancer cells death through apoptosis pathway. This study investigated whether ART treatment induced reactive oxygen species (ROS)-dependent cell death in the apoptosis fashion in human lung adenocarconoma A549 cell line and the proapoptotic protein apoptosis inducing factor (AIF) is involved in ART-induced apoptosis. Cells treated with ART exhibited typical apoptotic morphology as chromatin condensation, margination and shrunken nucleus. ART treatment also induced a loss of mitochondrial membrane potential and AIF release from mitochondria. Silencing AIF can remarkable attenuated ART-induced apoptosis. Collectively, ART induces apoptosis by caspase-independent intrinsic pathway in A549 cells.

Involvement of the CD200 receptor complex in microglia activation in experimental glaucoma

PubMed Central

Taylor, Sarah; Calder, Claudia J.; Albon, Julie; Erichsen, Jonathan T.; Boulton, Micheal E.; Morgan, James E.

2013-01-01

The interaction of the myeloid restricted molecule CD200R with its widely expressed ligand CD200 is involved in the down-regulation of microglia activation. In the present study, we examined the involvement of CD200R in microglia activation in experimental ocular hypertension to determine the role of microglia activation in retinal ganglion cell (RGC) death, the key pathological event in glaucoma. Experimental glaucoma was induced in adult Brown Norway rats by sclerosis of the episcleral veins with the injection of hypertonic saline. Immunohistochemical methods were used to determine the involvement of microglia using GFAP, CD45, OX42 and OX41 and the involvement of CD200 and CD200R in the optic nerve head. Our data demonstrate the increased presence of microglia within the optic nerve head during ocular hypertension, identified by positive staining with OX42 and OX41. The peak of microglia correlates with peak in RGC death at days 20–27 (T3) post OHT induction. In addition, CD200 and CD200R positive cells were increased in ocular hypertensive eyes. Increased expression of CD200 was detected in the early phase (days 1–7; T1) of OHT and decreased over time, whilst the expression of CD200R was detected in the middle phase (days 20–27; T3) of OHT, correlating with the increase in microglia markers. Changes in the expression of CD200R/CD200 occur early in experimental glaucoma and precede the peak in microglia infiltration and RGC death, suggesting that CD200R-positive microglia play an important role in the initiation of RGC death during OHT, indicating a potential area for therapeutic intervention in treating glaucoma. PMID:21296076

Cytokinin-induced cell death is associated with elevated expression of alternative oxidase in tobacco BY-2 cells.

PubMed

Mlejnek, Petr

2013-10-01

N(6)-benzyladenine (BA) and N(6)-benzyladenosine ([9R]BA) induce massive production of reactive oxygen species (ROS) that is eventually followed by a loss of cell viability in tobacco BY-2 cells (Mlejnek et al. Plant Cell Environ 26:1723-1735, 2003, Plant Sci 168:389-395, 2005). Results presented in this work suggest that the main sources of ROS are likely mitochondria and that the maintenance of the mitochondrial transmembrane potential is crucial for ROS production in cytokinin-treaded BY-2 cells. Therefore, the possible involvement of alternative oxidase (AOX) in cell death process induced by BA and [9R]BA was studied. About three- to fourfold increase in mRNA levels of AOX1 was observed a few hours after the BA and [9R]BA addition into the growth medium. The elevated expression of AOX1 mRNA could be prevented by adding adenine and adenosine which simultaneously reduced the cytotoxic effects of BA and [9R]BA, respectively. N(6)-benzyladenine 7-β-D-glucoside ([7G]BA) which is a common non-toxic metabolite of BA and [9R]BA did not affect the AOX1 mRNA expression. Although AOX1 seemed to be involved in protection of BY-2 cells against the abiotic stress induced by BA and [9R]BA, the results do not support the idea that it protects cells from death exclusively by scavenging of reactive oxygen species. Indeed, N-propyl gallate, an inhibitor of AOX, decreased cell survival despite it concomitantly decreased the ROS production. This finding is in contrast to the effect of salicylhydroxamic acid, another well-known inhibitor of AOX, which also increased the number of dying cells while it increased the ROS production.

Rotenone-induced death of RGC-5 cells is caspase independent, involves the JNK and p38 pathways and is attenuated by specific green tea flavonoids.

PubMed

Kamalden, T A; Ji, D; Osborne, N N

2012-05-01

The aim of the present studies was to characterise cell death following inhibition of mitochondrial complex I with rotenone in a transformed cell line (RGC-5 cells) and to examine the neuroprotective properties of the flavonoids genistein, epigallocatechin gallate (EGCG), epicatechin (EC) and baicalin. Rotenone-induced cell death of RGC-5 cells results in a generation of reactive oxygen species, a breakdown of DNA, the translocation of membrane phosphatidylserine, an up-regulation of haemoxygenase-1 and is unaffected by necrostatin-1 (inhibitor of necroptosis), z-VAD-fmk (pan caspase inhibitor) or NU1025 (PARP inhibitor) but attenuated with SP600125 (JNK inhibitor). Rotenone-induced toxicity of RGC-5 cells also caused an activation of mitogen-activated kinases indicated by an up-regulation and translocation into mitochondria of p-c-Jun, pJNK and pp38. Exposure of RGC-5 cells to rotenone does not affect apoptosis inducing factor or significantly stimulate caspase-3 activity. EGCG and EC both significantly blunt rotenone toxicity of RGC-5 cells at concentrations of 50 μM while genistein and baicalin were without effect. Significantly, genistein is approximately 20 times less efficacious than EGCG (IC(50) 2.5 μM) and EC (IC(50) 1.5 μM) at inhibiting sodium nitroprusside-induced lipid peroxidation. These studies show that rotenone toxicity of RGC-5 cells is neither necroptosis nor caspase-dependent apoptosis but involves the activation of mitogen-activated kinases and is inhibited by a JNK inhibitor, EGCG and EC. Genistein attenuates lipid peroxidation less efficaciously than EC and EGCG and does not affect rotenone toxicity of RGC-5 cells.

Experimental methods for testing the effects of neurotrophic peptide, ADNF-9, against alcohol-induced apoptosis during pregnancy in c57bl/6 mice.

PubMed

Sari, Youssef

2013-04-24

Experimental designs for investigating the effects of prenatal alcohol exposure during early embryonic stages in fetal brain growth are challenging. This is mostly due to the difficulty of microdissection of fetal brains and their sectioning for determination of apoptotic cells caused by prenatal exposure to alcohol. The experiments described here provide visualized techniques from mice breeding to the identification of cell death in fetal brain tissue. This study used C57BL/6 mice as the animal model for studying fetal alcohol exposure and the role of trophic peptide against alcohol-induced apoptosis. The breeding consists of a 2-hr matting window to determine the exact stage of embryonic age. An established fetal alcohol exposure model has been used in this study to determine the effects of prenatal alcohol exposure in fetal brains. This involves free access to alcohol or pair-fed liquid diets as the sole source of nutrients for the pregnant mice. The techniques involving dissection of fetuses and microdissection of fetal brains are described carefully, since the latter can be challenging. Microdissection requires a stereomicroscope and ultra-fine forceps. Step-by-step procedures for dissecting the fetal brains are provided visually. The fetal brains are dissected from the base of the primordium olfactory bulb to the base of the metencephalon. For investigating apoptosis, fetal brains are first embedded in gelatin using a peel-away mold to facilitate their sectioning with a vibratome apparatus. Fetal brains embedded and fixed in paraformaldehyde are easily sectioned, and the free floating sections can be mounted in superfrost plus slides for determination of apoptosis or cell death. TUNEL (TdT-mediated dUTP Nick End Labeling; TdT: terminal deoxynucleotidyl transferase) assay has been used to identify cell death or apoptotic cells. It is noteworthy that apoptosis and cell-mediated cytotoxicity are characterized by DNA fragmentation. Thus, the visualized TUNEL-positive cells are indicative of cell death or apoptotic cells. The experimental designs here provide information about the use of an established liquid diet for studying the effects of alcohol and the role of neurotrophic peptides during pregnancy in fetal brains. This involves breeding and feeding pregnant mice, microdissecting fetal brains, and determining apoptosis. Together, these visual and textual techniques might be a source for investigating prenatal exposure of harmful agents in fetal brains.

Role of astrocytes in reproduction and neuroprotection.

PubMed

Mahesh, Virendra B; Dhandapani, Krishnan M; Brann, Darrell W

2006-02-26

Hypothalamic astrocytes secrete TGF-beta and 3 alpha,5 alpha-tetrahydro progesterone (3 alpha,5 alpha-THP) in culture. When the astrocyte-conditioned medium (ACM) was incubated with the hypothalamic cell line GT1-7, it resulted in the secretion of GnRH. Immunoneutralization with TGF-beta antibody or ultra-filteration with a 10 kDa cut off filter resulted in attenuation of the GnRH releasing ability of ACM, indicating that TGF-beta was a major factor involved with GnRH release. Treatment with estrogens increases TGF-beta secretion. These observations indicate a significant role of astrocytes in GnRH secretion. Serum-deprivation results in the death of GT1-7 neurons in culture and addition of ACM or TGF-beta to the culture, attenuates cell death. The mechanism of protection from cell death appears to involve phosphorylation of MKK4, JNK, c-Jun(Ser63), and enhancement of AP-1 binding. Co-administration of JNK inhibitors, but not MEK inhibitors attenuated ACM or TGF-beta-induced c-Jun(Ser63) phosphorylation and their neuroprotective effects. These studies suggest that astrocytes can protect neurons, at least in part, by the release of TGF-beta and activation of a c-Jun/AP-1 protective pathway.

Human Cytomegalovirus Protein pUL38 Prevents Premature Cell Death by Binding to Ubiquitin-Specific Protease 24 and Regulating Iron Metabolism.

PubMed

Sun, Yamei; Bao, Qunchao; Xuan, Baoqin; Xu, Wenjia; Pan, Deng; Li, Qi; Qian, Zhikang

2018-07-01

Human cytomegalovirus (HCMV) protein pUL38 has been shown to prevent premature cell death by antagonizing cellular stress responses; however, the underlying mechanism remains unknown. In this study, we identified the host protein ubiquitin-specific protease 24 (USP24) as an interaction partner of pUL38. Mutagenesis analysis of pUL38 revealed that amino acids TFV at positions 227 to 230 were critical for its interaction with USP24. Mutant pUL38 TFV/AAA protein did not bind to USP24 and failed to prevent cell death induced by pUL38-deficient HCMV infection. Knockdown of USP24 suppressed the cell death during pUL38-deficient HCMV infection, suggesting that pUL38 achieved its function by antagonizing the function of USP24. We investigated the cellular pathways regulated by USP24 that might be involved in the cell death phenotype by testing several small-molecule compounds known to have a protective effect during stress-induced cell death. The iron chelators ciclopirox olamine and Tiron specifically protected cells from pUL38-deficient HCMV infection-induced cell death, thus identifying deregulated iron homeostasis as a potential mechanism. Protein levels of nuclear receptor coactivator 4 (NCOA4) and lysosomal ferritin degradation, a process called ferritinophagy, were also regulated by pUL38 and USP24 during HCMV infection. Knockdown of USP24 decreased NCOA4 protein stability and ferritin heavy chain degradation in lysosomes. Blockage of ferritinophagy by genetic inhibition of NCOA4 or Atg5/Atg7 prevented pUL38-deficient HCMV infection-induced cell death. Overall, these results support the hypothesis that pUL38 binds to USP24 to reduce ferritinophagy, which may then protect cells from lysosome dysfunction-induced cell death. IMPORTANCE Premature cell death is considered a first line of defense against various pathogens. Human cytomegalovirus (HCMV) is a slow-replicating virus that encodes several cell death inhibitors, such as pUL36 and pUL37x1, which allow it to overcome both extrinsic and intrinsic mitochondrion-mediated apoptosis. We previously identified HCMV protein pUL38 as another virus-encoded cell death inhibitor. In this study, we demonstrated that pUL38 achieved its activity by interacting with and antagonizing the function of the host protein ubiquitin-specific protease 24 (USP24). pUL38 blocked USP24-mediated ferritin degradation in lysosomes, which could otherwise be detrimental to the lysosome and initiate cell death. These novel findings suggest that iron metabolism is finely tuned during HCMV infection to avoid cellular toxicity. The results also provide a solid basis for further investigations of the role of USP24 in regulating iron metabolism during infection and other diseases. Copyright © 2018 American Society for Microbiology.

A new lactoferrin- and iron-dependent lysosomal death pathway is induced by benzo[a]pyrene in hepatic epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gorria, Morgane; Tekpli, Xavier; Rissel, Mary

2008-04-15

While lysosomal disruption seems to be a late step of necrosis, a moderate lysosomal destabilization has been suggested to participate early in the apoptotic cascade. The origin of lysosomal dysfunction and its precise role in apoptosis or apoptosis-like process still needs to be clarified, especially upon carcinogen exposure. In this study, we focused on the implication of lysosomes in cell death induced by the prototype carcinogen benzo[a]pyrene (B[a]P; 50 nM) in rat hepatic epithelial F258 cells. We first demonstrated that B[a]P affected lysosomal morphology (increase in size) and pH (alkalinization), and that these changes were involved in caspase-3 activation andmore » cell death. Subsequently, we showed that lysosomal modifications were partly dependent on mitochondrial dysfunction, and that lysosomes together with mitochondria participate in B[a]P-induced oxidative stress. Using two iron chelators (desferrioxamine and deferiprone) and siRNA targeting the lysosomal iron-binding protease lactoferrin, we further demonstrated that both lysosomal iron content and lactoferrin were required for caspase-3 activation and apoptosis-like cell death.« less

Oxidative stress and cell death in the cerebral cortex as a long-term consequence of neonatal hypoglycemia.

PubMed

Anju, T R; Akhilraj, P R; Paulose, C S

2016-09-01

Neonatal hypoglycemia limits glucose supply to cells leading to long-term consequences in brain function. The present study evaluated antioxidant and cell death factors' alterations in cerebral cortex of 1-month-old rats exposed to neonatal hypoglycemia. Gene expression studies by real-time PCR were carried out using gene-specific TaqMan probes. Fluorescent dyes were used for immunohistochemistry and nuclear staining and imaged by confocal microscope. Total antioxidant level and expression of antioxidant enzymes - superoxide dismutase (SOD) and gluthathione peroxide (GPx) - mRNA was significantly reduced along with high peroxide level in the cerebral cortex of 1-month-old rats exposed to neonatal hypoglycemia. Real-time PCR analysis showed an upregulation of Bax, caspase 3, and caspase 8 gene expression. Confocal imaging with TOPRO-3 staining and immunohistochemistry with caspase 3 antibody indicated cell death activation. The reduced free radical scavenging capability coupled with the expression of key factors involved in cell death pathway points to the possibility of oxidative stress in the cortex of 1-month-old rats exposed to neonatal hypoglycemia. The observed results indicate the effects of neonatal hypoglycemia in determining the antioxidant capability of cerebral cortex in a later stage of life.

Involvement of apoptotic cell death and cell cycle perturbation in retinoic acid-induced cleft palate in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okano, Junko; Suzuki, Shigehiko; Shiota, Kohei

2007-05-15

Retinoic acid (RA), a metabolite of vitamin A, plays a key role in a variety of biological processes and is essential for normal embryonic development. On the other hand, exogenous RA could cause cleft palate in offspring when it is given to pregnant animals at either the early or late phases of palatogenesis, but the pathogenetic mechanism of cleft palate caused by excess RA remains not fully elucidated. The aim of the present study was to investigate the effects of excess of RA on early palatogenesis in mouse fetuses and analyze the teratogenic mechanism, especially at the stage prior tomore » palatal shelf elevation. We gave all-trans RA (100 mg/kg) orally to E11.5 ICR pregnant mice and observed the changes occurring in the palatal shelves of their fetuses. It was found that apoptotic cell death increased not only in the epithelium of the palatal shelves but also in the tongue primordium, which might affect tongue withdrawal movement during palatogenesis and impair the horizontal elevation of palatal shelves. In addition, RA was found to prevent the G{sub 1}/S progression of palatal mesenchymal cells through upregulation of p21 {sup Cip1}, leading to Rb hypophospholylation. Thus, RA appears to cause G{sub 1} arrest in palatal mesenchymal cells in a similar manner as in various cancer and embryonic cells. It is likely that apoptotic cell death and cell cycle disruption are involved in cleft palate formation induced by RA.« less

Neem oil limonoids induces p53-independent apoptosis and autophagy

PubMed Central

Chandra, Dhyan

2012-01-01

Azadirachta indica, commonly known as neem, has a wide range of medicinal properties. Neem extracts and its purified products have been examined for induction of apoptosis in multiple cancer cell types; however, its underlying mechanisms remain undefined. We show that neem oil (i.e., neem), which contains majority of neem limonoids including azadirachtin, induced apoptotic and autophagic cell death. Gene silencing demonstrated that caspase cascade was initiated by the activation of caspase-9, whereas caspase-8 was also activated late during neem-induced apoptosis. Pretreatment of cancer cells with pan caspase inhibitor, z-VAD inhibited activities of both initiator caspases (e.g., caspase-8 and -9) and executioner caspase-3. Neem induced the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, suggesting the involvement of both caspase-dependent and AIF-mediated apoptosis. p21 deficiency caused an increase in caspase activities at lower doses of neem, whereas p53 deficiency did not modulate neem-induced caspase activation. Additionally, neem treatment resulted in the accumulation of LC3-II in cancer cells, suggesting the involvement of autophagy in neem-induced cancer cell death. Low doses of autophagy inhibitors (i.e., 3-methyladenine and LY294002) did not prevent accumulation of neem-induced LC3-II in cancer cells. Silencing of ATG5 or Beclin-1 further enhanced neem-induced cell death. Phosphoinositide 3-kinase (PI3K) or autophagy inhibitors increased neem-induced caspase-3 activation and inhibition of caspases enhanced neem-induced autophagy. Together, for the first time, we demonstrate that neem induces caspase-dependent and AIF-mediated apoptosis, and autophagy in cancer cells. PMID:22915764

Neem oil limonoids induces p53-independent apoptosis and autophagy.

PubMed

Srivastava, Pragya; Yadav, Neelu; Lella, Ravi; Schneider, Andrea; Jones, Anthony; Marlowe, Timothy; Lovett, Gabrielle; O'Loughlin, Kieran; Minderman, Hans; Gogada, Raghu; Chandra, Dhyan

2012-11-01

Azadirachta indica, commonly known as neem, has a wide range of medicinal properties. Neem extracts and its purified products have been examined for induction of apoptosis in multiple cancer cell types; however, its underlying mechanisms remain undefined. We show that neem oil (i.e., neem), which contains majority of neem limonoids including azadirachtin, induced apoptotic and autophagic cell death. Gene silencing demonstrated that caspase cascade was initiated by the activation of caspase-9, whereas caspase-8 was also activated late during neem-induced apoptosis. Pretreatment of cancer cells with pan caspase inhibitor, z-VAD inhibited activities of both initiator caspases (e.g., caspase-8 and -9) and executioner caspase-3. Neem induced the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, suggesting the involvement of both caspase-dependent and AIF-mediated apoptosis. p21 deficiency caused an increase in caspase activities at lower doses of neem, whereas p53 deficiency did not modulate neem-induced caspase activation. Additionally, neem treatment resulted in the accumulation of LC3-II in cancer cells, suggesting the involvement of autophagy in neem-induced cancer cell death. Low doses of autophagy inhibitors (i.e., 3-methyladenine and LY294002) did not prevent accumulation of neem-induced LC3-II in cancer cells. Silencing of ATG5 or Beclin-1 further enhanced neem-induced cell death. Phosphoinositide 3-kinase (PI3K) or autophagy inhibitors increased neem-induced caspase-3 activation and inhibition of caspases enhanced neem-induced autophagy. Together, for the first time, we demonstrate that neem induces caspase-dependent and AIF-mediated apoptosis, and autophagy in cancer cells.

Zinc oxide nanoparticles decrease the expression and activity of plasma membrane calcium ATPase, disrupt the intracellular calcium homeostasis in rat retinal ganglion cells.

PubMed

Guo, Dadong; Bi, Hongsheng; Wang, Daoguang; Wu, Qiuxin

2013-08-01

Zinc oxide nanoparticle is one of the most important materials with diverse applications. However, it has been reported that zinc oxide nanoparticles are toxic to organisms, and that oxidative stress is often hypothesized to be an important factor in cytotoxicity mediated by zinc oxide nanoparticles. Nevertheless, the mechanism of toxicity of zinc oxide nanoparticles has not been completely understood. In this study, we investigated the cytotoxic effect of zinc oxide nanoparticles and the possible molecular mechanism involved in calcium homeostasis mediated by plasma membrane calcium ATPase in rat retinal ganglion cells. Real-time cell electronic sensing assay showed that zinc oxide nanoparticles could exert cytotoxic effect on rat retinal ganglion cells in a concentration-dependent manner; flow cytometric analysis indicated that zinc oxide nanoparticles could lead to cell damage by inducing the overproduction of reactive oxygen species. Furthermore, zinc oxide nanoparticles could also apparently decrease the expression level and their activity of plasma membrane calcium ATPase, which finally disrupt the intracellular calcium homeostasis and result in cell death. Taken together, zinc oxide nanoparticles could apparently decrease the plasma membrane calcium ATPase expression, inhibit their activity, cause the elevated intracellular calcium ion level and disrupt the intracellular calcium homeostasis. Further, the disrupted calcium homeostasis will trigger mitochondrial dysfunction, generate excessive reactive oxygen species, and finally initiate cell death. Thus, the disrupted calcium homeostasis is involved in the zinc oxide nanoparticle-induced rat retinal ganglion cell death. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

PubMed

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

2018-01-24

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

Enniatin B-induced cell death and inflammatory responses in RAW 267.4 murine macrophages

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gammelsrud, A.; Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo; Solhaug, A.

2012-05-15

The mycotoxin enniatin B (EnnB) is predominantly produced by species of the Fusarium genera, and often found in grain. The cytotoxic effect of EnnB has been suggested to be related to its ability to form ionophores in cell membranes. The present study examines the effects of EnnB on cell death, differentiation, proliferation and pro-inflammatory responses in the murine monocyte–macrophage cell line RAW 264.7. Exposure to EnnB for 24 h caused an accumulation of cells in the G0/G1-phase with a corresponding decrease in cyclin D1. This cell cycle-arrest was possibly also linked to the reduced cellular ability to capture and internalizemore » receptors as illustrated by the lipid marker ganglioside GM1. EnnB also increased the number of apoptotic, early apoptotic and necrotic cells, as well as cells with elongated spindle-like morphology. The Neutral Red assay indicated that EnnB induced lysosomal damage; supported by transmission electron microscopy (TEM) showing accumulation of lipids inside the lysosomes forming lamellar structures/myelin bodies. Enhanced levels of activated caspase-1 were observed after EnnB exposure and the caspase-1 specific inhibitor ZYVAD-FMK reduced EnnB-induced apoptosis. Moreover, EnnB increased the release of interleukin-1beta (IL-1β) in cells primed with lipopolysaccharide (LPS), and this response was reduced by both ZYVAD-FMK and the cathepsin B inhibitor CA-074Me. In conclusion, EnnB was found to induce cell cycle arrest, cell death and inflammation. Caspase-1 appeared to be involved in the apoptosis and release of IL-1β and possibly activation of the inflammasome through lysosomal damage and leakage of cathepsin B. -- Highlights: ► The mycotoxin EnnB induced cell cycle arrest, cell death and inflammation. ► The G0/G1-arrest was linked to a reduced ability to internalize receptors. ► EnnB caused lysosomal damage, leakage of cathepsin B and caspase-1 cleavage. ► Caspase-1 was partly involved in both apoptosis and release of IL-1β. ► There was a synergistic action between EnnB and bacterial LPS.« less

Effect of Immortalization-Upregulated Protein-2 (IMUP-2) on Cell Death of Trophoblast

PubMed Central

Jung, Ran; Choi, Jong Ho; Lee, Hyun Jung; Kim, Jin Kyeoung; Kim, Gi Jin

2013-01-01

Trophoblasts, in the placenta, play a role for placental development as well as implantation in the early pregnancy. The characteristics and functions of trophoblast are identified by their localization and potency for proliferation, differentiation, and invasion. Thus, inadequate trophoblast cell death induces trophoblast dysfunction resulting in abnormal placental development and several gynecological diseases. Recently, it was reported that increased immortalization-upregulated protein-2 (IMUP-2) by hypoxia influences trophoblast apoptosis. However, IMUP-2 function on autophagy, which is type II programmed cell death remains unclear. In this study, we analyzed IMUP-2 expression in trophoblast cells (HTR8-SVneo) and compared IMUP-2 effects on cell death including apoptosis and autophagy in trophoblast regardless of IMUP-2 expression. Increased IMUP-2 in trophoblast by IMUP-2 gene transfection induces cell death, especially, apoptosis increases more than autophagy (p<0.05). However, the decreased IMUP-2 in trophoblasts after siRNA treatment decreased apoptosis with the decreased activities of caspase 3 and 7. The expressions of LC3 and MDC as an autophagosome makers and phosphorylated mTOR, which is a negative regulator for autophagy, increased. In addition, the S phase of cell cycle increased in trophoblasts when IMUP-2 expression decreased. Taken together, the alteration of IMUP-2 can control the balance between apoptosis and autophagy of trophoblasts resulting in functional involvement in placental development and in gynecological diseases by regulating the function of trophoblasts. PMID:25949126

A Unifying Mechanism for Cancer Cell Death through Ion Channel Activation by HAMLET

PubMed Central

Storm, Petter; Kjaer Klausen, Thomas; Trulsson, Maria; Ho CS, James; Dosnon, Marion; Westergren, Tomas; Chao, Yinxia; Rydström, Anna; Yang, Henry; Pedersen, Stine Falsig; Svanborg, Catharina

2013-01-01

Ion channels and ion fluxes control many aspects of tissue homeostasis. During oncogenic transformation, critical ion channel functions may be perturbed but conserved tumor specific ion fluxes remain to be defined. Here we used the tumoricidal protein-lipid complex HAMLET as a probe to identify ion fluxes involved in tumor cell death. We show that HAMLET activates a non-selective cation current, which reached a magnitude of 2.74±0.88 nA within 1.43±0.13 min from HAMLET application. Rapid ion fluxes were essential for HAMLET-induced carcinoma cell death as inhibitors (amiloride, BaCl2), preventing the changes in free cellular Na+ and K+ concentrations also prevented essential steps accompanying carcinoma cell death, including changes in morphology, uptake, global transcription, and MAP kinase activation. Through global transcriptional analysis and phosphorylation arrays, a strong ion flux dependent p38 MAPK response was detected and inhibition of p38 signaling delayed HAMLET-induced death. Healthy, differentiated cells were resistant to HAMLET challenge, which was accompanied by innate immunity rather than p38-activation. The results suggest, for the first time, a unifying mechanism for the initiation of HAMLET’s broad and rapid lethal effect on tumor cells. These findings are particularly significant in view of HAMLET’s documented therapeutic efficacy in human studies and animal models. The results also suggest that HAMLET offers a two-tiered therapeutic approach, killing cancer cells while stimulating an innate immune response in surrounding healthy tissues. PMID:23505537

A unifying mechanism for cancer cell death through ion channel activation by HAMLET.

PubMed

Storm, Petter; Klausen, Thomas Kjaer; Trulsson, Maria; Ho C S, James; Dosnon, Marion; Westergren, Tomas; Chao, Yinxia; Rydström, Anna; Yang, Henry; Pedersen, Stine Falsig; Svanborg, Catharina

2013-01-01

Ion channels and ion fluxes control many aspects of tissue homeostasis. During oncogenic transformation, critical ion channel functions may be perturbed but conserved tumor specific ion fluxes remain to be defined. Here we used the tumoricidal protein-lipid complex HAMLET as a probe to identify ion fluxes involved in tumor cell death. We show that HAMLET activates a non-selective cation current, which reached a magnitude of 2.74±0.88 nA within 1.43±0.13 min from HAMLET application. Rapid ion fluxes were essential for HAMLET-induced carcinoma cell death as inhibitors (amiloride, BaCl2), preventing the changes in free cellular Na(+) and K(+) concentrations also prevented essential steps accompanying carcinoma cell death, including changes in morphology, uptake, global transcription, and MAP kinase activation. Through global transcriptional analysis and phosphorylation arrays, a strong ion flux dependent p38 MAPK response was detected and inhibition of p38 signaling delayed HAMLET-induced death. Healthy, differentiated cells were resistant to HAMLET challenge, which was accompanied by innate immunity rather than p38-activation. The results suggest, for the first time, a unifying mechanism for the initiation of HAMLET's broad and rapid lethal effect on tumor cells. These findings are particularly significant in view of HAMLET's documented therapeutic efficacy in human studies and animal models. The results also suggest that HAMLET offers a two-tiered therapeutic approach, killing cancer cells while stimulating an innate immune response in surrounding healthy tissues.

Cell Surface Translocation of Annexin A2 Facilitates Glutamate-induced Extracellular Proteolysis*

PubMed Central

Valapala, Mallika; Maji, Sayantan; Borejdo, Julian; Vishwanatha, Jamboor K.

2014-01-01

Glutamate-induced elevation in intracellular Ca2+ has been implicated in excitotoxic cell death. Neurons respond to increased glutamate levels by activating an extracellular proteolytic cascade involving the components of the plasmin-plasminogen system. AnxA2 is a Ca2+-dependent phospholipid binding protein and serves as an extracellular proteolytic center by recruiting the tissue plasminogen activator and plasminogen and mediating the localized generation of plasmin. Ratiometric Ca2+ imaging and time-lapse confocal microscopy demonstrated glutamate-induced Ca2+ influx. We showed that glutamate translocated both endogenous and AnxA2-GFP to the cell surface in a process dependent on the activity of the NMDA receptor. Glutamate-induced translocation of AnxA2 is dependent on the phosphorylation of tyrosine 23 at the N terminus, and mutation of tyrosine 23 to a non-phosphomimetic variant inhibits the translocation process. The cell surface-translocated AnxA2 forms an active plasmin-generating complex, and this activity can be neutralized by a hexapeptide directed against the N terminus. These results suggest an involvement of AnxA2 in potentiating glutamate-induced cell death processes. PMID:24742684

Role of estrogens in anterior pituitary gland remodeling during the estrous cycle.

PubMed

Zárate, S; Zaldivar, V; Jaita, G; Magri, L; Radl, D; Pisera, D; Seilicovich, A

2010-01-01

In this review, we analyze the action of estrogens leading to the remodeling of the anterior pituitary gland, especially during the estrous cycle. Proliferation and death of anterior pituitary cells and especially lactotropes is regulated by estrogens, which act by sensitizing these cells to both mitotic and apoptotic stimuli such as TNF-alpha, FasL and dopamine. During the estrous cycle, the changing pattern of gonadal steroids is thought to modulate both cell proliferation and death in the anterior pituitary gland, estrogens being key players in cell turnover. The mechanisms involved in estrogen-modulated cell renewal in the anterior pituitary gland during the estrous cycle could include an increase in the expression of proapoptotic cytokines as well as the increase in the Bax/Bcl-2 ratio at proestrus, when estrogen levels are highest and a peak of apoptosis, in particular of lactotropes, is evident in this gland. Estrogens exert rapid antimitogenic and proapoptotic actions in the anterior pituitary through membrane-associated estrogen receptors, a mechanism that might also be involved in remodeling of this gland during the estrous cycle. Copyright (c) 2010 S. Karger AG, Basel.

Brucella abortus Induces the Premature Death of Human Neutrophils through the Action of Its Lipopolysaccharide

PubMed Central

Barquero-Calvo, Elías; Mora-Cartín, Ricardo; Arce-Gorvel, Vilma; de Diego, Juana L.; Chacón-Díaz, Carlos; Chaves-Olarte, Esteban; Guzmán-Verri, Caterina; Buret, Andre G.; Gorvel, Jean-Pierre; Moreno, Edgardo

2015-01-01

Most bacterial infections induce the activation of polymorphonuclear neutrophils (PMNs), enhance their microbicidal function, and promote the survival of these leukocytes for protracted periods of time. Brucella abortus is a stealthy pathogen that evades innate immunity, barely activates PMNs, and resists the killing mechanisms of these phagocytes. Intriguing clinical signs observed during brucellosis are the low numbers of Brucella infected PMNs in the target organs and neutropenia in a proportion of the patients; features that deserve further attention. Here we demonstrate that B. abortus prematurely kills human PMNs in a dose-dependent and cell-specific manner. Death of PMNs is concomitant with the intracellular Brucella lipopolysaccharide (Br-LPS) release within vacuoles. This molecule and its lipid A reproduce the premature cell death of PMNs, a phenomenon associated to the low production of proinflammatory cytokines. Blocking of CD14 but not TLR4 prevents the Br-LPS-induced cell death. The PMNs cell death departs from necrosis, NETosis and classical apoptosis. The mechanism of PMN cell death is linked to the activation of NADPH-oxidase and a modest but steadily increase of ROS mediators. These effectors generate DNA damage, recruitments of check point kinase 1, caspases 5 and to minor extent of caspase 4, RIP1 and Ca++ release. The production of IL-1β by PMNs was barely stimulated by B. abortus infection or Br-LPS treatment. Likewise, inhibition of caspase 1 did not hamper the Br-LPS induced PMN cell death, suggesting that the inflammasome pathway was not involved. Although activation of caspases 8 and 9 was observed, they did not seem to participate in the initial triggering mechanisms, since inhibition of these caspases scarcely blocked PMN cell death. These findings suggest a mechanism for neutropenia in chronic brucellosis and reveal a novel Brucella-host cross-talk through which B. abortus is able to hinder the innate function of PMN. PMID:25946018

Investigating the Mechanisms Underlying Neuronal Death in Ischemia Using In Vitro Oxygen-Glucose Deprivation: Potential Involvement of Protein SUMOylation

PubMed Central

CIMAROSTI, HELENA; HENLEY, JEREMY M.

2012-01-01

It is well established that brain ischemia can cause neuronal death via different signaling cascades. The relative importance and interrelationships between these pathways, however, remain poorly understood. Here is presented an overview of studies using oxygen-glucose deprivation of organotypic hippocampal slice cultures to investigate the molecular mechanisms involved in ischemia. The culturing techniques, setup of the oxygen-glucose deprivation model, and analytical tools are reviewed. The authors focus on SUMOylation, a posttranslational protein modification that has recently been implicated in ischemia from whole animal studies as an example of how these powerful tools can be applied and could be of interest to investigate the molecular pathways underlying ischemic cell death. PMID:19029060

Fas-Fas Ligand: Checkpoint of T Cell Functions in Multiple Sclerosis.

PubMed

Volpe, Elisabetta; Sambucci, Manolo; Battistini, Luca; Borsellino, Giovanna

2016-01-01

Fas and Fas Ligand (FasL) are two molecules involved in the regulation of cell death. Their interaction leads to apoptosis of thymocytes that fail to rearrange correctly their T cell receptor (TCR) genes and of those that recognize self-antigens, a process called negative selection; moreover, Fas-FasL interaction leads to activation-induced cell death, a form of apoptosis induced by repeated TCR stimulation, responsible for the peripheral deletion of activated T cells. Both control mechanisms are particularly relevant in the context of autoimmune diseases, such as multiple sclerosis (MS), where T cells exert an immune response against self-antigens. This concept is well demonstrated by the development of autoimmune diseases in mice and humans with defects in Fas or FasL. In recent years, several new aspects of T cell functions in MS have been elucidated, such as the pathogenic role of T helper (Th) 17 cells and the protective role of T regulatory (Treg) cells. Thus, in this review, we summarize the role of the Fas-FasL pathway, with particular focus on its involvement in MS. We then discuss recent advances concerning the role of Fas-FasL in regulating Th17 and Treg cells' functions, in the context of MS.

Inflammatory caspases are critical for enhanced cell death in the target tissue of Sjögren’s syndrome prior to disease onset

PubMed Central

Bulosan, Marievic; Pauley, Kaleb; Yo, Kyumee; Chan, Edward K.; Katz, Joseph; Peck, Ammon B.; Cha, Seunghee

2015-01-01

To date, little is known why exocrine glands are subject to immune cell infiltrations in Sjögren’s syndrome (SjS). Studies with SjS-prone-C57BL/6.NOD-Aec1Aec2 mice showed altered glandular homeostasis in the submandibular glands (SMX) at 8 weeks prior to disease onset and suggested potential involvement of inflammatory caspases (caspases-11 and -1). To determine if inflammatory caspases are critical for the increased epithelial cell death prior to SjS-like disease, we investigated molecular events involving caspase-11/caspase-1 axis. Our results revealed concurrent up-regulation of caspase-11 in macrophages, STAT-1 activity, caspase-1 activity, and apoptotic epithelial cells in the SMX of C57BL/6.NOD-Aec1Aec2 at 8 weeks. Caspase-1, a critical factor for IL-1β and IL-18 secretion, resulted in elevated level of IL-18 in saliva. Interestingly, TUNEL-positive cells in the SMX of C57BL/6.NOD-Aec1Aec2 were not co-localized with caspase-11, indicating that caspase-11 functions in a non-cell autonomous manner. Increased apoptosis of a human salivary gland (HSG) cell line occurred only in the presence of LPS-and IFN-γ-stimulated human monocytic THP-1 cells, which was reversed when caspase-1 in THP-1 cells was targeted by siRNA. Taken together, our study discovered that inflammatory caspases are essential in promoting pro-inflammatory microenvironment and influencing increased epithelial cell death in the target tissues of SjS before disease onset. PMID:18936772

Osteopontin splice variants expression is involved on docetaxel resistance in PC3 prostate cancer cells.

PubMed

Nakamura, K D M; Tilli, T M; Wanderley, J L; Palumbo, A; Mattos, R M; Ferreira, A C; Klumb, C E; Nasciutti, L E; Gimba, E R

2016-02-01

Osteopontin (OPN) is a phosphoprotein that activates several aspects of tumor progression. Alternative splicing of the OPN primary transcript generates three splicing isoforms, OPNa, OPNb and OPNc. In this report, we investigated some cellular mechanisms by which OPN splice variants could mediate PC3 prostate cancer (PCa) cell survival and growth in response to docetaxel (DXT)-induced cell death. Cell survival before and after DXT treatment was analyzed by phase-contrast microscopy and crystal-violet staining assays. Quantitative real-time PCR and immunocytochemical staining assays were used to evaluate the putative involvement of epithelial-mesenchymal transition (EMT) and OPN isoforms on mediating PC3 cell survival. Upon DXT treatment, PC3 cells overexpressing OPNb or OPNc isoforms showed higher cell densities, compared to cells overexpressing OPNa and controls. Notably, cells overexpressing OPNb or OPNc isoforms showed a downregulated pattern of EMT epithelial cell markers, while mesenchymal markers were mostly upregulated in these experimental conditions. We concluded that OPNc or OPNb overexpression in PC3 cells can mediate resistance and cell survival features in response to DXT-induced cell death. Our data also provide evidence the EMT program could be one of the molecular mechanisms mediating survival in OPNb- or OPNc-overexpressing cells in response to DXT treatment. These data could further contribute to a better understanding of the mechanisms by which PCa cells acquire resistance to DXT treatment.

Control of mitochondrial physiology and cell death by the Bcl-2 family proteins Bax and Bok.

PubMed

D'Orsi, Beatrice; Mateyka, Julia; Prehn, Jochen H M

2017-10-01

Neuronal cell death is often triggered by events that involve intracellular increases in Ca 2+ . Under resting conditions, the intracellular Ca 2+ concentration is tightly controlled by a number of extrusion and sequestering mechanisms involving the plasma membrane, mitochondria, and ER. These mechanisms act to prevent a disruption of neuronal ion homeostasis. As these processes require ATP, excessive Ca 2+ overloading may cause energy depletion, mitochondrial dysfunction, and may eventually lead to Ca 2+ -dependent cell death. Excessive Ca 2+ entry though glutamate receptors (excitotoxicity) has been implicated in several neurologic and chronic neurodegenerative diseases, including ischemic stroke, epilepsy, and Alzheimer's disease. Recent evidence has revealed that excitotoxic cell death is regulated by the B-cell lymphoma-2 (Bcl-2) family of proteins. Bcl-2 proteins, comprising of both pro-apoptotic and anti-apoptotic members, have been shown to not only mediate the intrinsic apoptosis pathway by controlling mitochondrial outer membrane (MOM) integrity, but to also control neuronal Ca 2+ homeostasis and energetics. In this review, the role of Bcl-2 family proteins in the regulation of apoptosis, their expression in the central nervous system and how they control Ca 2+ -dependent neuronal injury are summarized. We review the current knowledge on Bcl-2 family proteins in the regulation of mitochondrial function and bioenergetics, including the fusion and fission machinery, and their role in Ca 2+ homeostasis regulation at the mitochondria and ER. Specifically, we discuss how the 'pro-apoptotic' Bcl-2 family proteins, Bax and Bok, physiologically expressed in the nervous system, regulate such 'non-apoptotic/daytime' functions. Copyright © 2017 Elsevier Ltd. All rights reserved.

M1 muscarinic receptor activation mediates cell death in M1-HEK293 cells.

PubMed

Graham, E Scott; Woo, Kerhan K; Aalderink, Miranda; Fry, Sandie; Greenwood, Jeffrey M; Glass, Michelle; Dragunow, Mike

2013-01-01

HEK293 cells have been used extensively to generate stable cell lines to study G protein-coupled receptors, such as muscarinic acetylcholine receptors (mAChRs). The activation of M1 mAChRs in various cell types in vitro has been shown to be protective. To further investigate M1 mAChR-mediated cell survival, we generated stable HEK293 cell-lines expressing the human M1 mAChR. M1 mAChRs were efficiently expressed at the cell surface and efficiently internalised within 1 h by carbachol. Carbachol also induced early signalling cascades similar to previous reports. Thus, ectopically expressed M1 receptors behaved in a similar fashion to the native receptor over short time periods of analysis. However, substantial cell death was observed in HEK293-M1 cells within 24 h after carbachol application. Death was only observed in HEK cells expressing M1 receptors and fully blocked by M1 antagonists. M1 mAChR-stimulation mediated prolonged activation of the MEK-ERK pathway and resulted in prolonged induction of the transcription factor EGR-1 (>24 h). Blockade of ERK signalling with U0126 did not reduce M1 mAChR-mediated cell-death significantly but inhibited the acute induction of EGR-1. We investigated the time-course of cell death using time-lapse microscopy and xCELLigence technology. Both revealed the M1 mAChR cytotoxicity occurs within several hours of M1 activation. The xCELLigence assay also confirmed that the ERK pathway was not involved in cell-death. Interestingly, the MEK blocker did reduce carbachol-mediated cleaved caspase 3 expression in HEK293-M1 cells. The HEK293 cell line is a widely used pharmacological tool for studying G-protein coupled receptors, including mAChRs. Our results highlight the importance of investigating the longer term fate of these cells in short term signalling studies. Identifying how and why activation of the M1 mAChR signals apoptosis in these cells may lead to a better understanding of how mAChRs regulate cell-fate decisions.

Molecular mechanism of cardol, isolated from Trigona incisa stingless bee propolis, induced apoptosis in the SW620 human colorectal cancer cell line.

PubMed

Kustiawan, Paula Mariana; Lirdprapamongkol, Kriengsak; Palaga, Tanapat; Puthong, Songchan; Phuwapraisirisan, Preecha; Svasti, Jisnuson; Chanchao, Chanpen

2017-05-04

Cardol is a major bioactive constituent in the Trigona incisa propolis from Indonesia, with a strong in vitro antiproliferative activity against the SW620 colorectal adenocarcinoma cell line (IC 50 of 4.51 ± 0.76 μg/mL). Cardol induced G 0 /G 1 cell cycle arrest and apoptotic cell death. The present study was designed to reveal the mechanism of cardol's antiproliferative effect and induction of apoptosis. Changes in cell morphology were observed by light microscopy. To determine whether the mitochondrial apoptotic pathway was involved in cell death, caspase-3 and caspase-9 activities, western blot analysis, mitochondrial membrane potential, and intracellular reactive oxygen species (ROS) levels were assayed. Changes in the cell morphology and the significantly increased caspase-3 and caspase-9 activities, plus the cleavage of pro-caspase-3, pro-caspase-9 and PARP, supported that cardol caused apoptosis in SW620 cells within 2 h after treatment by cardol. In addition, cardol decreased the mitochondrial membrane potential while increasing the intracellular ROS levels in a time- and dose-dependent manner. Antioxidant treatment supported that the cardol-induced cell death was dependent on ROS production. Cardol induced cell death in SW620 cells was mediated by oxidative stress elevation and the mitochondrial apoptotic pathway, and these could be the potential molecular mechanism for the antiproliferative effect of cardol.

Cellular Stress and p53-Associated Apoptosis by Juniperus communis L. Berry Extract Treatment in the Human SH-SY5Y Neuroblastoma Cells.

PubMed

Lantto, Tiina A; Laakso, Into; Dorman, H J Damien; Mauriala, Timo; Hiltunen, Raimo; Kõks, Sulev; Raasmaja, Atso

2016-07-13

Plant phenolics have shown to activate apoptotic cell death in different tumourigenic cell lines. In this study, we evaluated the effects of juniper berry extract (Juniperus communis L.) on p53 protein, gene expression and DNA fragmentation in human neuroblastoma SH-SY5Y cells. In addition, we analyzed the phenolic composition of the extract. We found that juniper berry extract activated cellular relocalization of p53 and DNA fragmentation-dependent cell death. Differentially expressed genes between treated and non-treated cells were evaluated with the cDNA-RDA (representational difference analysis) method at the early time point of apoptotic process when p53 started to be activated and no caspase activity was detected. Twenty one overexpressed genes related to cellular stress, protein synthesis, cell survival and death were detected. Interestingly, they included endoplasmic reticulum (ER) stress inducer and sensor HSPA5 and other ER stress-related genes CALM2 and YKT6 indicating that ER stress response was involved in juniper berry extract mediated cell death. In composition analysis, we identified and quantified low concentrations of fifteen phenolic compounds. The main groups of them were flavones, flavonols, phenolic acids, flavanol and biflavonoid including glycosides of quercetin, apigenin, isoscutellarein and hypolaetin. It is suggested that juniper berry extract induced the p53-associated apoptosis through the potentiation and synergism by several phenolic compounds.

Black rice extract protected HepG2 cells from oxidative stress-induced cell death via ERK1/2 and Akt activation

PubMed Central

Yoon, Jaemin; Ham, Hyeonmi; Sung, Jeehye; Kim, Younghwa; Choi, Youngmin; Lee, Jeom-Sig; Jeong, Heon-Sang; Lee, Junsoo

2014-01-01

BACKGROUND/OBJECTIVES The objective of this study was to evaluate the protective effect of black rice extract (BRE) on tert-butyl hydroperoxide (TBHP)-induced oxidative injury in HepG2 cells. MATERIALS/METHODS Methanolic extract from black rice was evaluated for the protective effect on TBHP-induced oxidative injury in HepG2 cells. Several biomarkers that modulate cell survival and death including reactive oxygen species (ROS), caspase-3 activity, and related cellular kinases were determined. RESULTS TBHP induced cell death and apoptosis by a rapid increase in ROS generation and caspase-3 activity. Moreover, TBHP-induced oxidative stress resulted in a transient ERK1/2 activation and a sustained increase of JNK1/2 activation. While, BRE pretreatment protects the cells against oxidative stress by reducing cell death, caspase-3 activity, and ROS generation and also by preventing ERKs deactivation and the prolonged JNKs activation. Moreover, pretreatment of BRE increased the activation of ERKs and Akt which are pro-survival signal proteins. However, this effect was blunted in the presence of ERKs and Akt inhibitors. CONCLUSIONS These results suggest that activation of ERKs and Akt pathway might be involved in the cytoprotective effect of BRE against oxidative stress. Our findings provide new insights into the cytoprotective effects and its possible mechanism of black rice against oxidative stress. PMID:24741394

Hemeoxygenase-1 Mediates an Adaptive Response to Spermidine-Induced Cell Death in Human Endothelial Cells

PubMed Central

Yang, Hana; Lee, Seung Eun; Kim, Gun-Dong; Park, Hye Rim; Park, Yong Seek

2013-01-01

Spermidine (SPD) is a ubiquitous polycation that is commonly distributed in living organisms. Intracellular levels of SPD are tightly regulated, and SPD controls cell proliferation and death. However, SPD undergoes oxidation in the presence of serum, producing aldehydes, hydrogen peroxide, and ammonia, which exert cytotoxic effect on cells. Hemeoxygenase-1 (HO-1) is thought to have a protective effect against oxidative stress. Upregulation of HO-1 in endothelial cells is considered to be beneficial in the cardiovascular disease. In the present study, we demonstrate that the ubiquitous polyamine, SPD, induces HO-1 in human umbilical vein endothelial cells (HUVECs). SPD-induced HO-1 expression was examined by Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Involvement of reactive oxygen species, serum amine oxidase, PI3K/Akt signaling pathway, and transcription factor Nrf2 in the induction of HO-1 by SPD was also investigated. Furthermore, small interfering RNA knockdown of Nrf2 or HO-1 and treatment with the specific HO-1 inhibitor ZnPP exhibited a noteworthy increase of death of SPD-stimulated HUVECs. In conclusion, these results suggest that SPD induces PI3K/Akt-Nrf2-mediated HO-1 expression in human endothelial cells, which may have a role in cytoprotection of the cells against oxidative stress-induced death. PMID:23983896

Constitutive Expression of the Maltoporin LamB in the Absence of OmpR Damages the Cell Envelope▿ †

PubMed Central

Reimann, Sylvia A.; Wolfe, Alan J.

2011-01-01

Cells experience multiple environmental stimuli simultaneously. To survive, they must respond accordingly. Unfortunately, the proper response to one stress easily could make the cell more susceptible to a second coexistent stress. To deal with such a problem, a cell must possess a mechanism that balances the need to respond simultaneously to both stresses. Our recent studies of ompR malT(Con) double mutants show that elevated expression of LamB, the outer membrane porin responsible for maltose uptake, causes cell death when the osmoregulator OmpR is disabled. To obtain insight into the nature of the death experienced by ompR malT(Con) mutants, we described the death process. On the basis of microscopic and biochemical approaches, we conclude that death results from a loss of membrane integrity. On the basis of an unbiased genome-wide search for suppressor mutations, we conclude that this loss of membrane integrity results from a LamB-induced envelope stress that the cells do not sufficiently perceive and thus do not adequately accommodate. Finally, we conclude that this envelope stress involves an imbalance in the lipopolysaccharide/porin composition of the outer membrane and an increased requirement for inorganic phosphate. PMID:21131484

Protective effect of T-type calcium channel blocker flunarizine on cisplatin-induced death of auditory cells.

PubMed

So, Hong-Seob; Park, Channy; Kim, Hyung-Jin; Lee, Jung-Han; Park, Sung-Yeol; Lee, Jai-Hyung; Lee, Zee-Won; Kim, Hyung-Min; Kalinec, Federico; Lim, David J; Park, Raekil

2005-06-01

Changes in intracellular Ca2+ level are involved in a number of intracellular events, including triggering of apoptosis. The role of intracellular calcium mobilization in cisplatin-induced hair cell death, however, is still unknown. In this study, the effect of calcium channel blocker flunarizine (Sibelium), which is used to prescribe for vertigo and tinnitus, on cisplatin-induced hair cell death was investigated in a cochlear organ of Corti-derived cell line, HEI-OC1, and the neonatal (P2) rat organ of Corti explant. Cisplatin induced apoptotic cell death showing nuclear fragmentation, DNA ladder, and TUNEL positive in both HEI-OC1 and primary organ of Corti explant. Flunarizine significantly inhibited the cisplatin-induced apoptosis. Unexpectedly, flunarizine increased the intracellular calcium ([Ca2+]i) levels of HEI-OC1. However, the protective effect of flunarizine against cisplatin was not mediated by modulation of intracellular calcium level. Treatment of cisplatin resulted in ROS generation and lipid peroxidation in HEI-OC1. Flunarizine did not attenuate ROS production but inhibited lipid peroxidation and mitochondrial permeability transition in cisplatin-treated cells. This result suggests that the protective mechanism of flunarizine on cisplatin-induced cytotoxicity is associated with direct inhibition of lipid peroxidation and mitochondrial permeability transition.

Identification of the key pathway of oxazolinoanthracyclines mechanism of action in cells derived from human solid tumors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Denel-Bobrowska, Marta, E-mail: mdenel@biol.uni.lo

Oxazolinodoxorubicin (O-DOX) and oxazolinodaunorubicin (O-DAU) are novel anthracycline derivatives with a modified daunosamine moiety. In the present study, we evaluated the cytotoxicities, genotoxicities and abilities of O-DOX and O-DAU to induce apoptosis in cancer cell lines (SKOV-3; A549; HepG2), and compared the results with their parent drugs. We assessed antiproliferative activity by MTT assay. We evaluated apoptosis-inducing ability by double-staining with fluorescent probes (Hoechst 33258/propidium iodide), and by determining expression levels of genes involved in programmed cell death by reverse transcription-polymerase chain reaction. Genotoxicities of the compounds were tested by comet assays. Oxazolinoanthracyclines demonstrated high anti-tumor activity. O-DOX had significantlymore » higher cytotoxicity, apoptosis-inducing ability, and genotoxicity compared with parental doxorubicin (DOX) in all tested conditions, while O-DAU activity differed among cell lines. The mechanism of oxazoline analog action appeared to involve the mitochondrial pathway of programmed cell death. These results provide further information about oxazoline derivatives of commonly used anthracycline chemotherapy agents. O-DOX and O-DAU have the ability to induce apoptosis in tumor cells. - Highlights: • Substituted amino group increased the anticancer activity of anthracyclines. • Mitochondrial apoptotic pathway seems to be involved in the mechanism of action. • Favorable biological properties of oxazoline derivatives were confirmed.« less

Prodigiosin activates endoplasmic reticulum stress cell death pathway in human breast carcinoma cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pan, Mu-Yun; Shen, Yuh-Chiang; National Research Institute of Chinese Medicine, Taipei, Taiwan

Prodigiosin is a bacterial tripyrrole pigment with potent cytotoxicity against diverse human cancer cell lines. Endoplasmic reticulum (ER) stress is initiated by accumulation of unfolded or misfolded proteins in the ER lumen and may induce cell death when irremediable. In this study, the role of ER stress in prodigiosin-induced cytotoxicity was elucidated for the first time. Comparable to the ER stress inducer thapsigargin, prodigiosin up-regulated signature ER stress markers GRP78 and CHOP in addition to activating the IRE1, PERK and ATF6 branches of the unfolded protein response (UPR) in multiple human breast carcinoma cell lines, confirming prodigiosin as an ERmore » stress inducer. Prodigiosin transcriptionally up-regulated CHOP, as evidenced by its promoting effect on the CHOP promoter activity. Of note, knockdown of CHOP effectively lowered prodigiosin's capacity to evoke PARP cleavage, reduce cell viability and suppress colony formation, highlighting an essential role of CHOP in prodigiosin-induced cytotoxic ER stress response. In addition, prodigiosin down-regulated BCL2 in a CHOP-dependent manner. Importantly, restoration of BCL2 expression blocked prodigiosin-induced PARP cleavage and greatly enhanced the survival of prodigiosin-treated cells, suggesting that CHOP-dependent BCL2 suppression mediates prodigiosin-elicited cell death. Moreover, pharmacological inhibition of JNK by SP600125 or dominant-negative blockade of PERK-mediated eIF2α phosphorylation impaired prodigiosin-induced CHOP up-regulation and PARP cleavage. Collectively, these results identified ER stress-mediated cell death as a mode-of-action of prodigiosin's tumoricidal effect. Mechanistically, prodigiosin engages the IRE1–JNK and PERK–eIF2α branches of the UPR signaling to up-regulate CHOP, which in turn mediates BCL2 suppression to induce cell death. Highlights: ► Prodigiosin is a bacterial tripyrrole pigment with potent anticancer effect. ► Prodigiosin is herein identified as an endoplasmic reticulum (ER) stress inducer. ► Prodigiosin-induced cytotoxicity involves ER stress-mediated cell death. ► Prodigiosin transcriptionally induces CHOP to suppress BCL2 for evoking cell death. ► Prodigiosin engages the IRE1–JNK and PERK–eIF2α pathways to up-regulate CHOP.« less

CD47-ligation induced cell death in T-acute lymphoblastic leukemia.

PubMed

Leclair, Pascal; Liu, Chi-Chao; Monajemi, Mahdis; Reid, Gregor S; Sly, Laura M; Lim, Chinten James

2018-05-10

CD47 is a cell-surface marker well recognized for its anti-phagocytic functions. As such, an emerging avenue for targeted cancer therapies involves neutralizing the anti-phagocytic function using monoclonal antibodies (mAbs) to enhance tumour cell immunogenicity. A lesser known consequence of CD47 receptor ligation is the direct induction of tumour cell death. While several mAbs and their derivatives with this property have been studied, the best characterized is the commercially available mAb B6H12, which requires immobilization for induction of cell death. Here, we describe a commercially available mAb, CC2C6, which induces T-cell acute lymphoblastic leukemia (ALL) cell death in soluble form. Soluble CC2C6 induces CD47-dependent cell death in a manner consistent with immobilized B6H12, which is characterized by mitochondrial deficiencies but is independent of caspase activation. Titration studies indicated that CC2C6 shares a common CD47-epitope with B6H12. Importantly, CC2C6 retains the anti-phagocytic neutralizing function, thus possessing dual anti-tumour properties. Although CD47-ligation induced cell death occurs in a caspase-independent manner, CC2C6 was found to stimulate increases in Mcl-1 and NOXA levels, two Bcl-2 family proteins that govern the intrinsic apoptosis pathway. Further analysis revealed that the ratio of Mcl-1:NOXA were minimally altered for cells treated with CC2C6, in comparison to cells treated with agents that induced caspase-dependent apoptosis which alter this ratio in favour of NOXA. Finally, we found that CC2C6 can synergize with low dose chemotherapeutic agents that induce classical apoptosis, giving rise to the possibility of an effective combination treatment with reduced long-term sequelae associated with high-dose chemotherapies in childhood ALL.

The Progression of Cell Death Affects the Rejection of Allogeneic Tumors in Immune-Competent Mice – Implications for Cancer Therapy

PubMed Central

Chaurio, Ricardo A.; Muñoz, Luis E.; Maueröder, Christian; Janko, Christina; Harrer, Thomas; Fürnrohr, Barbara G.; Niederweis, Michael; Bilyy, Rostyslav; Schett, Georg; Herrmann, Martin; Berens, Christian

2014-01-01

Large amounts of dead and dying cells are produced during cancer therapy and allograft rejection. Depending on the death pathway and stimuli involved, dying cells exhibit diverse features, resulting in defined physiological consequences for the host. It is not fully understood how dying and dead cells modulate the immune response of the host. To address this problem, different death stimuli were studied in B16F10 melanoma cells by regulated inducible transgene expression of the pro-apoptotic active forms of caspase-3 (revCasp-3), Bid (tBid), and the Mycobacterium tuberculosis-necrosis inducing toxin (CpnTCTD). The immune outcome elicited for each death stimulus was assessed by evaluating the allograft rejection of melanoma tumors implanted subcutaneously in BALB/c mice immunized with dying cells. Expression of all proteins efficiently killed cells in vitro (>90%) and displayed distinctive morphological and physiological features as assessed by multiparametric flow cytometry analysis. BALB/c mice immunized with allogeneic dying melanoma cells expressing revCasp-3 or CpnTCTD showed strong rejection of the allogeneic challenge. In contrast, mice immunized with cells dying either after expression of tBid or irradiation with UVB did not, suggesting an immunologically silent cell death. Surprisingly, immunogenic cell death induced by expression of revCasp-3 or CpnTCTD correlated with elevated intracellular reactive oxygen species (ROS) levels at the time point of immunization. Conversely, early mitochondrial dysfunction induced by tBid expression or UVB irradiation accounted for the absence of intracellular ROS accumulation at the time point of immunization. Although ROS inhibition in vitro was not sufficient to abrogate the immunogenicity in our allo-immunization model, we suggest that the point of ROS generation and its intracellular accumulation may be an important factor for its role as damage associated molecular pattern in the development of allogeneic responses. PMID:25426116

Involvement of miR17 pathway in glucocorticoid-induced cell death in pediatric acute lymphoblastic leukemia.

PubMed

Harada, Masako; Pokrovskaja-Tamm, Katja; Söderhäll, Stefan; Heyman, Mats; Grander, Dan; Corcoran, Martin

2012-10-01

Analysis of the microRNA transcriptome following dexa- methasone treatment of the acute lymphocytic leukemia (ALL) cell line RS4;11 showed a global down-regulation of microRNA levels. MIR17HG was rapidly down-regulated following treatment, with chromatin immunoprecipitation (ChIP) analysis demonstrating the promoter to be a direct target of glucocorticoid (GC)-transcriptional repression and revealing the miR17-92 cluster as a prime target for dexamethasone-induced repression. The loss of miR17 family expression and concomitant increases in the miR17 target Bim occurred in an additional ALL cell line SUP-B15 but not in the dexamethasone-resistant REH. Alteration of miR17 levels through up-regulation or inhibition resulted in an decrease and increase, respectively, in Bim protein levels and dexamethasone-induced cell death. Primary ex vivo ALL cells that underwent apoptosis induced by dexamethasone also down-regulated miR17 levels. Thus, down-regulation of miR17 plays an important role in glucocorticoid-induced cell death suggesting that targeting miR17 may improve the current ALL combination therapy.

Role of Bax in death of uninfected retinal cells during murine cytomegalovirus retinitis.

PubMed

Mo, Juan; Marshall, Brendan; Covar, Jason; Zhang, Nancy Y; Smith, Sylvia B; Atherton, Sally S; Zhang, Ming

2014-10-08

Extensive death of uninfected bystander neuronal cells is an important component of the pathogenesis of cytomegalovirus retinitis. Our previous results have shown that caspase 3-dependent and -independent pathways are involved in death of uninfected bystander cells during murine cytomegalovirus (MCMV) retinitis and also that Bcl-2, an important inhibitor of apoptosis via the Bax-mediated mitochondrial pathway, is downregulated during this process. The purpose of this study was to determine whether Bax-mediated mitochondrial damage has a significant role in the death of uninfected retinal cells. BALB/c mice, Bax(-/-) mice, or Bax(+/+) mice were immunosuppressed with methylprednisolone and infected with 5 × 10(3) plaque-forming units (PFU) of the K181 strain of MCMV via the supraciliary route. Injected eyes were analyzed by plaque assay, electron microscopy, hematoxylin and eosin (H&E) staining, TUNEL assay, Western blot (for caspase 3, caspase 12, Bax, receptor interacting protein-1 [RIP1] and receptor interacting protein-3 [RIP3]), as well as immunohistochemical staining for MCMV early antigen and cleaved caspase 3. Significantly more Bax was detected in mitochondrial fractions of MCMV-infected eyes than in mitochondrial fractions of mock-infected control eyes. Furthermore, the level of cleaved caspase 3 was significantly lower in MCMV-infected Bax(-/-) eyes than in MCMV-infected Bax(+/+) eyes. However, more caspase 3-independent cell death of uninfected bystander retinal cells and more cleaved RIP1 were observed in Bax(-/-) than in Bax(+/+) eyes. During MCMV retinitis, Bax is activated and has an important role in death of uninfected bystander retinal cells by caspase 3-dependent apoptosis. Although the exact mechanism remains to be deciphered, active Bax might also prevent death of some types of uninfected retinal cells by a caspase 3-independent pathway. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

Role of Bax in Death of Uninfected Retinal Cells During Murine Cytomegalovirus Retinitis

PubMed Central

Mo, Juan; Marshall, Brendan; Covar, Jason; Zhang, Nancy Y.; Smith, Sylvia B.; Atherton, Sally S.; Zhang, Ming

2014-01-01

Purpose. Extensive death of uninfected bystander neuronal cells is an important component of the pathogenesis of cytomegalovirus retinitis. Our previous results have shown that caspase 3–dependent and –independent pathways are involved in death of uninfected bystander cells during murine cytomegalovirus (MCMV) retinitis and also that Bcl-2, an important inhibitor of apoptosis via the Bax-mediated mitochondrial pathway, is downregulated during this process. The purpose of this study was to determine whether Bax-mediated mitochondrial damage has a significant role in the death of uninfected retinal cells. Methods. BALB/c mice, Bax−/− mice, or Bax+/+ mice were immunosuppressed with methylprednisolone and infected with 5 × 103 plaque-forming units (PFU) of the K181 strain of MCMV via the supraciliary route. Injected eyes were analyzed by plaque assay, electron microscopy, hematoxylin and eosin (H&E) staining, TUNEL assay, Western blot (for caspase 3, caspase 12, Bax, receptor interacting protein-1 [RIP1] and receptor interacting protein-3 [RIP3]), as well as immunohistochemical staining for MCMV early antigen and cleaved caspase 3. Results. Significantly more Bax was detected in mitochondrial fractions of MCMV-infected eyes than in mitochondrial fractions of mock-infected control eyes. Furthermore, the level of cleaved caspase 3 was significantly lower in MCMV-infected Bax−/− eyes than in MCMV-infected Bax+/+ eyes. However, more caspase 3–independent cell death of uninfected bystander retinal cells and more cleaved RIP1 were observed in Bax−/− than in Bax+/+ eyes. Conclusions. During MCMV retinitis, Bax is activated and has an important role in death of uninfected bystander retinal cells by caspase 3–dependent apoptosis. Although the exact mechanism remains to be deciphered, active Bax might also prevent death of some types of uninfected retinal cells by a caspase 3–independent pathway. PMID:25298417

Caspases in retinal ganglion cell death and axon regeneration

PubMed Central

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

2017-01-01

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

Natural Killer Cell Memory

PubMed Central

O’Sullivan, Timothy E.; Sun, Joseph C.; Lanier, Lewis L.

2015-01-01

Natural killer (NK) cells have historically been considered short-lived cytolytic cells that can rapidly respond against pathogens and tumors in an antigen-independent manner, and then undergo cell death. Recently, however, NK cells have been shown to possess traits of adaptive immunity, and can acquire immunological memory in a similar manner to T and B cells. In this review, we discuss evidence for NK cell memory and the mechanisms involved in the generation and survival of these innate lymphocytes. PMID:26488815

Multicolor Fluorescence Imaging of Traumatic Brain Injury in a Cryolesion Mouse Model

PubMed Central

2012-01-01

Traumatic brain injury is characterized by initial tissue damage, which then can lead to secondary processes such as cell death and blood-brain-barrier disruption. Clinical and preclinical studies of traumatic brain injury typically employ anatomical imaging techniques and there is a need for new molecular imaging methods that provide complementary biochemical information. Here, we assess the ability of a targeted, near-infrared fluorescent probe, named PSS-794, to detect cell death in a brain cryolesion mouse model that replicates certain features of traumatic brain injury. In short, the model involves brief contact of a cold rod to the head of a living, anesthetized mouse. Using noninvasive whole-body fluorescence imaging, PSS-794 permitted visualization of the cryolesion in the living animal. Ex vivo imaging and histological analysis confirmed PSS-794 localization to site of brain cell death. The nontargeted, deep-red Tracer-653 was validated as a tracer dye for monitoring blood-brain-barrier disruption, and a binary mixture of PSS-794 and Tracer-653 was employed for multicolor imaging of cell death and blood-brain-barrier permeability in a single animal. The imaging data indicates that at 3 days after brain cryoinjury the amount of cell death had decreased significantly, but the integrity of the blood-brain-barrier was still impaired; at 7 days, the blood-brain-barrier was still three times more permeable than before cryoinjury. PMID:22860222

Regulation of necrotic cell death p53, PARP1 and Cyclophilin D -overlapping pathways of regulated necrosis?

PubMed Central

Ying, Yuan; Padanilam, Babu J.

2017-01-01

In contrast to apoptosis and autophagy, necrotic cell death was considered to be a random, passive cell death without definable mediators. However, this dogma has been challenged by recent developments suggesting that necrotic cell death can also be a regulated process. Regulated necrosis includes multiple cell death modalities such as necroptosis, parthanatos, ferroptosis, pyroptosis, and mitochondrial permeability transition pore (MPTP)-mediated necrosis. Several distinctive executive molecules, particularly residing on the mitochondrial inner and outer membrane, amalgamating to form the MPTP have been defined. The c-subunit of the F1F0ATP synthase on the inner membrane and Bax/Bak on the outer membrane are considered to be the long sought components that form the MPTP. Opening of the MPTP results in loss of mitochondrial inner membrane potential, disruption of ATP production, increased ROS production, organelle swelling, mitochondrial dysfunction and consequent necrosis. Cyclophilin D, along with adenine nucleotide translocator (ANT) and the phosphate carrier (PiC) are considered to be important regulators involved in the opening of MPTP. Increased production of ROS can further trigger other necrotic pathways mediated through molecules such as PARP1, leading to irreversible cell damage. This review examines the roles of PARP1 and cyclophilin D in necrotic cell death. The hierarchical role of p53 in regulation and integration of key components of signaling pathway to elicit MPTP-mediated necrosis and ferroptosis is explored. In the context of recent insights, the indistinct role of necroptosis signaling in tubular necrosis after ischemic kidney injury is scrutinized. We conclude by discussing the participation of p53, PARP1 and cyclophilin D and their overlapping pathways to elicit MPTP-mediated necrosis and ferroptosis in acute kidney injury. PMID:27048819

Regulation of necrotic cell death: p53, PARP1 and cyclophilin D-overlapping pathways of regulated necrosis?

PubMed

Ying, Yuan; Padanilam, Babu J

2016-06-01

In contrast to apoptosis and autophagy, necrotic cell death was considered to be a random, passive cell death without definable mediators. However, this dogma has been challenged by recent developments suggesting that necrotic cell death can also be a regulated process. Regulated necrosis includes multiple cell death modalities such as necroptosis, parthanatos, ferroptosis, pyroptosis, and mitochondrial permeability transition pore (MPTP)-mediated necrosis. Several distinctive executive molecules, particularly residing on the mitochondrial inner and outer membrane, amalgamating to form the MPTP have been defined. The c-subunit of the F1F0ATP synthase on the inner membrane and Bax/Bak on the outer membrane are considered to be the long sought components that form the MPTP. Opening of the MPTP results in loss of mitochondrial inner membrane potential, disruption of ATP production, increased ROS production, organelle swelling, mitochondrial dysfunction and consequent necrosis. Cyclophilin D, along with adenine nucleotide translocator and the phosphate carrier are considered to be important regulators involved in the opening of MPTP. Increased production of ROS can further trigger other necrotic pathways mediated through molecules such as PARP1, leading to irreversible cell damage. This review examines the roles of PARP1 and cyclophilin D in necrotic cell death. The hierarchical role of p53 in regulation and integration of key components of signaling pathway to elicit MPTP-mediated necrosis and ferroptosis is explored. In the context of recent insights, the indistinct role of necroptosis signaling in tubular necrosis after ischemic kidney injury is scrutinized. We conclude by discussing the participation of p53, PARP1 and cyclophilin D and their overlapping pathways to elicit MPTP-mediated necrosis and ferroptosis in acute kidney injury.

Fas–Fas Ligand: Checkpoint of T Cell Functions in Multiple Sclerosis

PubMed Central

Volpe, Elisabetta; Sambucci, Manolo; Battistini, Luca; Borsellino, Giovanna

2016-01-01

Fas and Fas Ligand (FasL) are two molecules involved in the regulation of cell death. Their interaction leads to apoptosis of thymocytes that fail to rearrange correctly their T cell receptor (TCR) genes and of those that recognize self-antigens, a process called negative selection; moreover, Fas–FasL interaction leads to activation-induced cell death, a form of apoptosis induced by repeated TCR stimulation, responsible for the peripheral deletion of activated T cells. Both control mechanisms are particularly relevant in the context of autoimmune diseases, such as multiple sclerosis (MS), where T cells exert an immune response against self-antigens. This concept is well demonstrated by the development of autoimmune diseases in mice and humans with defects in Fas or FasL. In recent years, several new aspects of T cell functions in MS have been elucidated, such as the pathogenic role of T helper (Th) 17 cells and the protective role of T regulatory (Treg) cells. Thus, in this review, we summarize the role of the Fas–FasL pathway, with particular focus on its involvement in MS. We then discuss recent advances concerning the role of Fas–FasL in regulating Th17 and Treg cells’ functions, in the context of MS. PMID:27729910

DNA damage response in nephrotoxic and ischemic kidney injury

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Mingjuan; Tang, Chengyuan

DNA damage activates specific cell signaling cascades for DNA repair, cell cycle arrest, senescence, and/or cell death. Recent studies have demonstrated DNA damage response (DDR) in experimental models of acute kidney injury (AKI). In cisplatin-induced AKI or nephrotoxicity, the DDR pathway of ATR/Chk2/p53 is activated and contributes to renal tubular cell apoptosis. In ischemic AKI, DDR seems more complex and involves at least the ataxia telangiectasia mutated (ATM), a member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, and p53; however, while ATM may promote DNA repair, p53 may trigger cell death. Targeting DDR for kidney protection in AKI therefore reliesmore » on a thorough elucidation of the DDR pathways in various forms of AKI.« less

Molecular Processes that Drive Cigarette Smoke–Induced Epithelial Cell Fate of the Lung

PubMed Central

Nyunoya, Toru; Mebratu, Yohannes; Contreras, Amelia; Delgado, Monica; Chand, Hitendra S.

2014-01-01

Cigarette smoke contains numerous chemical compounds, including abundant reactive oxygen/nitrogen species and aldehydes, and many other carcinogens. Long-term cigarette smoking significantly increases the risk of various lung diseases, including chronic obstructive pulmonary disease and lung cancer, and contributes to premature death. Many in vitro and in vivo studies have elucidated mechanisms involved in cigarette smoke–induced inflammation, DNA damage, and autophagy, and the subsequent cell fates, including cell death, cellular senescence, and transformation. In this Translational Review, we summarize the known pathways underlying these processes in airway epithelial cells to help reveal future challenges and describe possible directions of research that could lead to better management and treatment of these diseases. PMID:24111585

TNF-induced necroptosis requires the plasma membrane localization of the MLKL protein | Center for Cancer Research

Cancer.gov

The cell signaling protein tumor necrosis factor (TNF), produced by white blood cells, promotes inflammation and immunity processes such as fever and is involved in tumorigenesis and apoptosis (programmed cell death). However, dysregulation of TNF can also lead to another form of programmed cell death called necroptosis, which is characterized by a rise in intracellular Ca2+, generation of reactive oxygen species (ROS), intracellular acidity, depletion of ATP, and, eventually, plasma membrane rupture. TNF-induced necroptosis has been associated with a wide variety of diseases including neurodegenerative diseases, major depression, rheumatoid arthritis, and cancer. Whereas the signaling mechanisms underlying TNF-induced apoptosis have largely been determined, the events precipitating in TNF-initiated necroptosis are still unknown.

Pivotal Role of Receptor-Interacting Protein Kinase 1 and Mixed Lineage Kinase Domain-Like in Neuronal Cell Death Induced by the Human Neuroinvasive Coronavirus OC43

PubMed Central

Meessen-Pinard, Mathieu; Le Coupanec, Alain

2016-01-01

ABSTRACT Human coronaviruses (HCoV) are respiratory pathogens with neuroinvasive, neurotropic, and neurovirulent properties, highlighting the importance of studying the potential implication of these viruses in neurological diseases. The OC43 strain (HCoV-OC43) was reported to induce neuronal cell death, which may participate in neuropathogenesis. Here, we show that HCoV-OC43 harboring two point mutations in the spike glycoprotein (rOC/Us183–241) was more neurovirulent than the wild-type HCoV-OC43 (rOC/ATCC) in mice and induced more cell death in murine and human neuronal cells. To evaluate the role of regulated cell death (RCD) in HCoV-OC43-mediated neural pathogenesis, we determined if knockdown of Bax, a key regulator of apoptosis, or RIP1, a key regulator of necroptosis, altered the percentage of neuronal cell death following HCoV-OC43 infection. We found that Bax-dependent apoptosis did not play a significant role in RCD following infection, as inhibition of Bax expression mediated by RNA interference did not confer cellular protection against the cell death process. On the other hand, we demonstrated that RIP1 and MLKL were involved in neuronal cell death, as RIP1 knockdown and chemical inhibition of MLKL significantly increased cell survival after infection. Taken together, these results indicate that RIP1 and MLKL contribute to necroptotic cell death after HCoV-OC43 infection to limit viral replication. However, this RCD could lead to neuronal loss in the mouse CNS and accentuate the neuroinflammation process, reflecting the severity of neuropathogenesis. IMPORTANCE Because they are naturally neuroinvasive and neurotropic, human coronaviruses are suspected to participate in the development of neurological diseases. Given that the strain OC43 is neurovirulent in mice and induces neuronal cell death, we explored the neuronal response to infection by characterizing the activation of RCD. Our results revealed that classical apoptosis associated with the Bax protein does not play a significant role in HCoV-OC43-induced neuronal cell death and that RIP1 and MLKL, two cellular proteins usually associated with necroptosis (an RCD back-up system when apoptosis is not adequately induced), both play a pivotal role in the process. As necroptosis disrupts cellular membranes and allows the release of damage-associated molecular patterns (DAMP) and possibly induces the production of proinflammatory cytokines, it may represent a proinflammatory cell death mechanism that contributes to excessive neuroinflammation and neurodegeneration and eventually to neurological disorders after a coronavirus infection. PMID:27795420

Protective Effects of Fisetin Against 6-OHDA-Induced Apoptosis by Activation of PI3K-Akt Signaling in Human Neuroblastoma SH-SY5Y Cells.

PubMed

Watanabe, Ryoko; Kurose, Takumi; Morishige, Yuta; Fujimori, Ko

2018-02-01

6-Hydroxydopamine (6-OHDA) induces the production of reactive oxygen species (ROS) that are associated with various neurodegenerative diseases such as Parkinson's disease. 3,3',4',7-Tetrahydroxyflavone (fisetin), a plant flavonoid has a variety of physiological effects such as antioxidant activity. In this study, we investigated the molecular mechanism of the neuroprotective effects of fisetin against 6-OHDA-induced cell death in human neuroblastoma SH-SY5Y cells. 6-OHDA-mediated cell toxicity was reduced in a fisetin concentration-dependent manner. 6-OHDA-mediated elevation of the expression of the oxidative stress-related genes such as hemeoxygenase-1, NAD(P)H dehydrogenase quinone 1, NF-E2-related factor 2, and γ-glutamate-cysteine ligase modifier was suppressed by fisetin. Fisetin also lowered the ratio of the proapoptotic Bax protein and the antiapoptotic Bcl-2 protein in SH-SY5Y cells. Moreover, fisetin effectively suppressed 6-OHDA-mediated activation of caspase-3 and caspase-9, which leads to the cell death, while, 6-OHDA-induced caspase-3/7 activity was lowered. Furthermore, fisetin activated the PI3K-Akt signaling, which inhibits the caspase cascade, and fisetin-mediated inhibition of 6-OHDA-induced cell death was negated by the co-treatment with an Akt inhibitor. These results indicate that fisetin protects 6-OHDA-induced cell death by activating PI3K-Akt signaling in human neuronal SH-SY5Y cells. This is the first report that the PI3K-Akt signaling is involved in the fisetin-protected ROS-mediated neuronal cell death.

atRA-induced apoptosis of mouse embryonic palate mesenchymal cells involves activation of MAPK pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu Zengli; Xing Ying

2006-08-15

Our previous studies have shown that atRA treatment resulted in cell-cycle block and growth inhibition in mouse embryonic palatal mesenchymal (MEPM). In the current study, gestation day (GD) 13 MEPM cells were used to test the hypothesis that the growth inhibition by atRA is due to apoptosis. The effects of atRA on apoptosis were assessed by performing MTT assay, Cell Death Detection ELISA and flow cytometry, respectively. Data analysis confirmed that atRA treatment induced apoptosis-like cell death, as shown by decreased cell viability and increased fragmented DNA and sub-G1 fraction. atRA-induced apoptosis was associated with upregulation of bcl-2, translocation ofmore » bax protein to the mitochondria from the cytosol, activation of caspase-3 and cytochrome c release into cytosol. atRA-induced apoptosis was abrogated by z-DEVD-fmk, a caspase-3 specific inhibitor, and z-VAD-fmk, a general caspase inhibitor, suggesting that the atRA-induced cell death of MEPM cells occurs through the cytochrome c- and caspase-3-dependent pathways. In addition, atRA treatment caused a strong and sustained activation of c-Jun N-terminal kinase (JNK) and p38 kinase (p38), as well as an early but transient activation of extracellular signal-regulated kinase (ERK). Importantly, atRA-induced DNA fragmentation and capase-3 activation were prevented by pretreatment with the JNK inhibitor (SP600125) and the p38 MAPK inhibitor (SB202190), but not by pretreatment with MEK inhibitor (U0126). From these results, we suggest that mitogen-activated protein kinase-dependent pathways is involved in the atRA-induced apoptosis of MEPM cells.« less

Role of glycolysis inhibition and poly(ADP-ribose) polymerase activation in necrotic-like cell death caused by ascorbate/menadione-induced oxidative stress in K562 human chronic myelogenous leukemic cells.

PubMed

Verrax, Julien; Vanbever, Stéphanie; Stockis, Julie; Taper, Henryk; Calderon, Pedro Buc

2007-03-15

Among different features of cancer cells, two of them have retained our interest: their nearly universal glycolytic phenotype and their sensitivity towards an oxidative stress. Therefore, we took advantage of these features to develop an experimental approach by selectively exposing cancer cells to an oxidant insult induced by the combination of menadione (vitamin K(3)) and ascorbate (vitamin C). Ascorbate enhances the menadione redox cycling, increases the formation of reactive oxygen species and kills K562 cells as shown by more than 65% of LDH leakage after 24 hr of incubation. Since both lactate formation and ATP content are depressed by about 80% following ascorbate/menadione exposure, we suggest that the major intracellular event involved in such a cytotoxicity is related to the impairment of glycolysis. Indeed, NAD(+) is rapidly and severely depleted, a fact most probably related to a strong Poly(ADP-ribose) polymerase (PARP) activation, as shown by the high amount of poly-ADP-ribosylated proteins. The addition of N-acetylcysteine (NAC) restores most of the ATP content and the production of lactate as well. The PARP inhibitor dihydroxyisoquinoline (DiQ) was able to partially restore both parameters as well as cell death induced by ascorbate/menadione. These results suggest that the PARP activation induced by the oxidative stress is a major but not the only intracellular event involved in cell death by ascorbate/menadione. Due to the high energetic dependence of cancer cells on glycolysis, the impairment of such an essential pathway may explain the effectiveness of this combination to kill cancer cells. (c) 2006 Wiley-Liss, Inc.

Milestones and recent discoveries on cell death mediated by mitochondria and their interactions with biologically active amines.

PubMed

Grancara, Silvia; Ohkubo, Shinji; Artico, Marco; Ciccariello, Mauro; Manente, Sabrina; Bragadin, Marcantonio; Toninello, Antonio; Agostinelli, Enzo

2016-10-01

Mitochondria represent cell "powerhouses," being involved in energy transduction from the electrochemical gradient to ATP synthesis. The morphology of their cell types may change, according to various metabolic processes or osmotic pressure. A new morphology of the inner membrane and mitochondrial cristae, significantly different from the previous one, has been proposed for the inner membrane and mitochondrial cristae, based on the technique of electron tomography. Mitochondrial Ca(2+) transport (the transporter has been isolated) generates reactive oxygen species and induces the mitochondrial permeability transition of both inner and outer mitochondrial membranes, leading to induction of necrosis and apoptosis. In the mitochondria of several cell types (liver, kidney, and heart), mitochondrial oxidative stress is an essential step in the induction of cell death, although not in brain, in which the phenomenon is caused by a different mechanism. Mitochondrial permeability transition drives both apoptosis and necrosis, whereas mitochondrial outer membrane permeability is characteristic of apoptosis. Adenine nucleotide translocase remains the most important component involved in membrane permeability, with the opening of the transition pore, although other proteins, such as ATP synthase or phosphate carriers, have been proposed. Intrinsic cell death is triggered by the release from mitochondria of proteic factors, such as cytochrome c, apoptosis inducing factor, and Smac/DIABLO, with the activation of caspases upon mitochondrial permeability transition or mitochondrial outer membrane permeability induction. Mitochondrial permeability transition induces the permeability of the inner membrane in sites in contact with the outer membrane; mitochondrial outer membrane permeability forms channels on the outer membrane by means of various stimuli involving Bcl-2 family proteins. The biologically active amines, spermine, and agmatine, have specific functions on mitochondria which distinguish them from other amines. Enzymatic oxidative deamination of spermine by amine oxidases in tumor cells may produce reactive oxygen species, leading to transition pore opening and apoptosis. This process could be exploited as a new therapeutic strategy to combat cancer.

E4F1 deficiency results in oxidative stress–mediated cell death of leukemic cells

PubMed Central

Hatchi, Elodie; Rodier, Genevieve; Lacroix, Matthieu; Caramel, Julie; Kirsh, Olivier; Jacquet, Chantal; Schrepfer, Emilie; Lagarrigue, Sylviane; Linares, Laetitia Karine; Lledo, Gwendaline; Tondeur, Sylvie; Dubus, Pierre

2011-01-01

The multifunctional E4F1 protein was originally discovered as a target of the E1A viral oncoprotein. Growing evidence indicates that E4F1 is involved in key signaling pathways commonly deregulated during cell transformation. In this study, we investigate the influence of E4F1 on tumorigenesis. Wild-type mice injected with fetal liver cells from mice lacking CDKN2A, the gene encoding Ink4a/Arf, developed histiocytic sarcomas (HSs), a tumor originating from the monocytic/macrophagic lineage. Cre-mediated deletion of E4F1 resulted in the death of HS cells and tumor regression in vivo and extended the lifespan of recipient animals. In murine and human HS cell lines, E4F1 inactivation resulted in mitochondrial defects and increased production of reactive oxygen species (ROS) that triggered massive cell death. Notably, these defects of E4F1 depletion were observed in HS cells but not healthy primary macrophages. Short hairpin RNA–mediated depletion of E4F1 induced mitochondrial defects and ROS-mediated death in several human myeloid leukemia cell lines. E4F1 protein is overexpressed in a large subset of human acute myeloid leukemia samples. Together, these data reveal a role for E4F1 in the survival of myeloid leukemic cells and support the notion that targeting E4F1 activities might have therapeutic interest. PMID:21708927

Potentiation of lead-induced cell death in PC12 cells by glutamate: Protection by N-acetylcysteine amide (NACA), a novel thiol antioxidant

DOE Office of Scientific and Technical Information (OSTI.GOV)

Penugonda, Suman; Mare, Suneetha; Lutz, P.

2006-10-15

Oxidative stress has been implicated as an important factor in many neurological diseases. Oxidative toxicity in a number of these conditions is induced by excessive glutamate release and subsequent glutamatergic neuronal stimulation. This, in turn, causes increased generation of reactive oxygen species (ROS), oxidative stress, excitotoxicity, and neuronal damage. Recent studies indicate that the glutamatergic neurotransmitter system is involved in lead-induced neurotoxicity. Therefore, this study aimed to (1) investigate the potential effects of glutamate on lead-induced PC12 cell death and (2) elucidate whether the novel thiol antioxidant N-acetylcysteine amide (NACA) had any protective abilities against such cytotoxicity. Our results suggestmore » that glutamate (1 mM) potentiates lead-induced cytotoxicity by increased generation of ROS, decreased proliferation (MTS), decreased glutathione (GSH) levels, and depletion of cellular adenosine-triphosphate (ATP). Consistent with its ability to decrease ATP levels and induce cell death, lead also increased caspase-3 activity, an effect potentiated by glutamate. Exposure to glutamate and lead elevated the cellular malondialdehyde (MDA) levels and phospholipase-A{sub 2} (PLA{sub 2}) activity and diminished the glutamine synthetase (GS) activity. NACA protected PC12 cells from the cytotoxic effects of glutamate plus lead, as evaluated by MTS assay. NACA reduced the decrease in the cellular ATP levels and restored the intracellular GSH levels. The increased levels of ROS and MDA in glutamate-lead treated cells were significantly decreased by NACA. In conclusion, our data showed that glutamate potentiated the effects of lead-induced PC12 cell death by a mechanism involving mitochondrial dysfunction (ATP depletion) and oxidative stress. NACA had a protective role against the combined toxic effects of glutamate and lead by inhibiting lipid peroxidation and scavenging ROS, thus preserving intracellular GSH.« less

Apoptotic actions of p53 require transcriptional activation of PUMA and do not involve a direct mitochondrial/cytoplasmic site of action in postnatal cortical neurons.

PubMed

Uo, Takuma; Kinoshita, Yoshito; Morrison, Richard S

2007-11-07

Recent studies in non-neuronal cells have shown that the tumor suppressor p53 can promote cell death through a transcription-independent mechanism involving its direct action with a subset of Bcl-2 family member proteins in the cytosol and at the mitochondria. In cultured cortical neurons, however, we could not find evidence supporting a significant contribution of the cytosolic/mitochondrial p53 pathway, and available evidence instead corroborated the requirement for the transcriptional activity of p53. When directly targeted to the cytosol/mitochondria, wild-type p53 lost its apoptosis-inducing activity in neurons but not in non-neuronal cells. The N-terminal p53 fragment (transactivation and proline-rich domains), which induces apoptosis in non-neuronal cells via the cytosolic/mitochondrial pathway, displayed no apoptogenic activity in neurons. In neuronal apoptosis induced by camptothecin or an MDM2 (murine double minute 2) inhibitor, nutlin-3, endogenous p53 protein did not accumulate in the cytosol/mitochondria, and transcriptional inhibition after p53 induction effectively blocked cell death. In addition, overexpression of a dominant-negative form of p53 (R273H) completely suppressed induction of proapoptotic p53 target genes and cell death. PUMA (p53-upregulated modulator of apoptosis) was one such gene induced by camptothecin, and its overexpression was sufficient to induce Bax (Bcl-2-associated X protein)-dependent neuronal death, whereas Noxa was not apoptogenic. These results collectively demonstrate that, in contrast to non-neuronal cells, the apoptotic activity of p53 in postnatal cortical neurons does not rely on its direct action at the cytosol/mitochondria but is exclusively mediated through its transcription-dependent functions. The uniqueness of p53-mediated apoptotic signaling in postnatal cortical neurons was further illustrated by the dispensable function of the proline-rich domain of p53.

Akt phosphorylation and NFkappaB activation are counterregulated under conditions of oxidative stress.

PubMed

Taylor, Juliet M; Crack, Peter J; Gould, Jodee A; Ali, Uğur; Hertzog, Paul J; Iannello, Rocco C

2004-11-01

This study was designed to elucidate the mechanisms involved in elevated cell death arising from an altered endogenous oxidant state. Increased levels of cell death were detected in cells lacking Gpx1 following the addition of exogenous H2O2. This increased apoptosis correlated with a down-regulation in the activation of the PI(3)K-Akt survival pathway. The importance of this pathway in protecting against H2O2-induced cell death was highlighted by the increased susceptibility of wild-type cells to apoptosis when treated with the PI(3)K inhibitor, LY294002. Activation of the oxidative stress sensitive transcription factor, NFkappaB, was elevated in the Gpx1-/- cells. Significantly, NFkappaB activation could be increased in wild-type cells through the addition of dominant-negative Akt. Therefore, our results suggest that the increased susceptibility of Gpx1-/- cells to H2O2-induced apoptosis can be attributed in part to diminished activation of Akt despite an up-regulation in the activation of the prosurvival NFkappaB. Thus, the PI(3)K-Akt and NFkappaB pathways can act independently of each other in an endogenous model of oxidative stress.

Vesicles between plasma membrane and cell wall prior to visible senescence of Iris and Dendrobium flowers.

PubMed

Kamdee, Channatika; Kirasak, Kanjana; Ketsa, Saichol; van Doorn, Wouter G

2015-09-01

Cut Iris flowers (Iris x hollandica, cv. Blue Magic) show visible senescence about two days after full opening. Epidermal cells of the outer tepals collapse due to programmed cell death (PCD). Transmission electron microscopy (TEM) showed irregular swelling of the cell walls, starting prior to cell collapse. Compared to cells in flowers that had just opened, wall thickness increased up to tenfold prior to cell death. Fibrils were visible in the swollen walls. After cell death very little of the cell wall remained. Prior to and during visible wall swelling, vesicles (paramural bodies) were observed between the plasma membrane and the cell walls. The vesicles were also found in groups and were accompanied by amorphous substance. They usually showed a single membrane, and had a variety of diameters and electron densities. Cut Dendrobium hybrid cv. Lucky Duan flowers exhibited visible senescence about 14 days after full flower opening. Paramural bodies were also found in Dendrobium tepal epidermis and mesophyll cells, related to wall swelling and degradation. Although alternative explanations are well possible, it is hypothesized that paramural bodies carry enzymes involved in cell wall breakdown. The literature has not yet reported such bodies in association with senescence/PCD. Copyright © 2015 Elsevier GmbH. All rights reserved.

Signal transducers and activators of transcription: STATs-mediated mitochondrial neuroprotection.

PubMed

Lin, Hung Wen; Thompson, John W; Morris, Kahlilia C; Perez-Pinzon, Miguel A

2011-05-15

Cerebral ischemia is defined as little or no blood flow in cerebral circulation, characterized by low tissue oxygen and glucose levels, which promotes neuronal mitochondria dysfunction leading to cell death. A strategy to counteract cerebral ischemia-induced neuronal cell death is ischemic preconditioning (IPC). IPC results in neuroprotection, which is conferred by a mild ischemic challenge prior to a normally lethal ischemic insult. Although many IPC-induced mechanisms have been described, many cellular and subcellular mechanisms remain undefined. Some reports have suggested key signal transduction pathways of IPC, such as activation of protein kinase C epsilon, mitogen-activated protein kinase, and hypoxia-inducible factors, that are likely involved in IPC-induced mitochondria mediated-neuroprotection. Moreover, recent findings suggest that signal transducers and activators of transcription (STATs), a family of transcription factors involved in many cellular activities, may be intimately involved in IPC-induced ischemic tolerance. In this review, we explore current signal transduction pathways involved in IPC-induced mitochondria mediated-neuroprotection, STAT activation in the mitochondria as it relates to IPC, and functional significance of STATs in cerebral ischemia.

Signal Transducers and Activators of Transcription: STATs-Mediated Mitochondrial Neuroprotection

PubMed Central

Lin, Hung Wen; Thompson, John W.; Morris, Kahlilia C.

2011-01-01

Abstract Cerebral ischemia is defined as little or no blood flow in cerebral circulation, characterized by low tissue oxygen and glucose levels, which promotes neuronal mitochondria dysfunction leading to cell death. A strategy to counteract cerebral ischemia-induced neuronal cell death is ischemic preconditioning (IPC). IPC results in neuroprotection, which is conferred by a mild ischemic challenge prior to a normally lethal ischemic insult. Although many IPC-induced mechanisms have been described, many cellular and subcellular mechanisms remain undefined. Some reports have suggested key signal transduction pathways of IPC, such as activation of protein kinase C epsilon, mitogen-activated protein kinase, and hypoxia-inducible factors, that are likely involved in IPC-induced mitochondria mediated-neuroprotection. Moreover, recent findings suggest that signal transducers and activators of transcription (STATs), a family of transcription factors involved in many cellular activities, may be intimately involved in IPC-induced ischemic tolerance. In this review, we explore current signal transduction pathways involved in IPC-induced mitochondria mediated-neuroprotection, STAT activation in the mitochondria as it relates to IPC, and functional significance of STATs in cerebral ischemia. Antioxid. Redox Signal. 14, 1853–1861. PMID:20712401

Cytotoxicity of Manganese (III) Complex in Human Breast Adenocarcinoma Cell Line Is Mediated by the Generation of Reactive Oxygen Species Followed by Mitochondrial Damage.

PubMed

Al-Anbaky, Qudes; Al-Karakooly, Zeiyad; Kilaparty, Surya P; Agrawal, Megha; Albkuri, Yahya M; RanguMagar, Ambar B; Ghosh, Anindya; Ali, Nawab

2016-11-01

Manganese (Mn) complexes are widely studied because of their important catalytic properties in synthetic and biochemical reactions. A Mn (III) complex of an amidoamine ligand was synthesized using a tetradentate amidoamine ligand. In this study, the Mn (III) complex was evaluated for its biological activity by measuring its cytotoxicity in human breast adenocarcinoma cell line (MCF-7). Cytotoxic effects of the Mn (III) complex were determined using established biomarkers in an attempt to delineate the mechanism of action and the utility of the complex as a potential anticancer drug. The Mn (III) complex induces cell death in a dose- and time-dependent manner as shown by microculture tetrazolium assay, a measure of cytotoxic cell death. Our results demonstrated that cytotoxic effects were significantly increased at higher concentrations of Mn (III) complex and with longer time of treatment. The IC 50 (Inhibitor concentration that results in 50% cell death) value of Mn (III) complex in MCF-7 cells was determined to be 2.5 mmol/L for 24 hours of treatment. In additional experiments, we determined the Mn (III) complex-mediated cell death was due to both apoptotic and nonspecific necrotic cell death mechanisms. This was assessed by ethidium bromide/acridine orange staining and flow cytometry techniques. The Mn (III) complex produced reactive oxygen species (ROS) triggering the expression of manganese superoxide dismutase 1 and ultimately damaging the mitochondrial function as is evident by a decline in mitochondrial membrane potential. Treatment of the cells with free radical scavenger, N, N-dimethylthiourea decreased Mn (III) complex-mediated generation of ROS and attenuated apoptosis. Together, these results suggest that the Mn (III) complex-mediated MCF-7 cell death utilizes combined mechanism involving apoptosis and necrosis perhaps due to the generation of ROS. © The Author(s) 2016.

AgHalo: A Facile Fluorogenic Sensor to Detect Drug-Induced Proteome Stress.

PubMed

Liu, Yu; Fares, Matthew; Dunham, Noah P; Gao, Zi; Miao, Kun; Jiang, Xueyuan; Bollinger, Samuel S; Boal, Amie K; Zhang, Xin

2017-07-17

Drug-induced proteome stress that involves protein aggregation may cause adverse effects and undermine the safety profile of a drug. Safety of drugs is regularly evaluated using cytotoxicity assays that measure cell death. However, these assays provide limited insights into the presence of proteome stress in live cells. A fluorogenic protein sensor is reported to detect drug-induced proteome stress prior to cell death. An aggregation prone Halo-tag mutant (AgHalo) was evolved to sense proteome stress through its aggregation. Detection of such conformational changes was enabled by a fluorogenic ligand that fluoresces upon AgHalo forming soluble aggregates. Using 5 common anticancer drugs, we exemplified detection of differential proteome stress before any cell death was observed. Thus, this sensor can be used to evaluate drug safety in a regime that the current cytotoxicity assays cannot cover and be generally applied to detect proteome stress induced by other toxins. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Manipulating the mitochondria activity in human hepatic cell line Huh7 by low-power laser irradiation

PubMed Central

Lynnyk, Anna; Lunova, Mariia; Jirsa, Milan; Egorova, Daria; Kulikov, Andrei; Kubinová, Šárka; Lunov, Oleg; Dejneka, Alexandr

2018-01-01

Low-power laser irradiation of red light has been recognized as a promising tool across a vast variety of biomedical applications. However, deep understanding of the molecular mechanisms behind laser-induced cellular effects remains a significant challenge. Here, we investigated mechanisms involved in the death process in human hepatic cell line Huh7 at a laser irradiation. We decoupled distinct cell death pathways targeted by laser irradiations of different powers. Our data demonstrate that high dose laser irradiation exhibited the highest levels of total reactive oxygen species production, leading to cyclophilin D-related necrosis via the mitochondrial permeability transition. On the contrary, low dose laser irradiation resulted in the nuclear accumulation of superoxide and apoptosis execution. Our findings offer a novel insight into laser-induced cellular responses, and reveal distinct cell death pathways triggered by laser irradiation. The observed link between mitochondria depolarization and triggering ROS could be a fundamental phenomenon in laser-induced cellular responses. PMID:29541521

Neuroprotective effect against axonal damage-induced retinal ganglion cell death in apolipoprotein E-deficient mice through the suppression of kainate receptor signaling.

PubMed

Omodaka, Kazuko; Nishiguchi, Koji M; Yasuda, Masayuki; Tanaka, Yuji; Sato, Kota; Nakamura, Orie; Maruyama, Kazuichi; Nakazawa, Toru

2014-10-24

Apolipoprotein E (ApoE) plays important roles in the body, including a carrier of cholesterols, an anti-oxidant, and a ligand for the low-density lipoprotein receptors. In the nervous system, the presence of ApoE4 isoforms is associated with Alzheimer's disease. ApoE gene polymorphisms are also associated with glaucoma, but the function of ApoE in the retina remains unclear. In this study, we investigated the role of ApoE in axonal damage-induced RGC death. ApoE was detected in the astrocytes and Müller cells in the wild-type (WT) retina. RGC damage was induced in adult ApoE-deficient mice (male, 10-12 weeks old) through ocular hypertension (OH), optic nerve crush (NC), or by administering kainic acid (KA) intravitreally. The WT mice were treated with a glutamate receptor antagonist (MK801 or CNQX) 30 min before performing NC or left untreated. Seven days later, the retinas were flat mounted and Fluorogold-labeled RGCs were counted. We found that the RGCs in the ApoE-deficient mice were resistant to OH-induced RGC death and optic nerve degeneration 4 weeks after induction. In WT mice, NC effectively induced RGC death (control: 4085±331 cells/mm(2), NC: 1728±170 cells/mm(2)). CNQX, an inhibitor of KA receptors, suppressed this RGC death (3031±246 cells/mm(2)), but MK801, an inhibitor of NMDA receptors, did not (1769±212 cells/mm(2)). This indicated the involvement of KA receptor signaling in NC-induced RGC death. We found that NC- or KA-induced RGC death was significantly less in the ApoE-deficient mice than in the WT mice. These data suggest that the ApoE deficiency had a neuroprotective effect against axonal damage-induced RGC death by suppressing the KA receptor signaling. Copyright © 2014 Elsevier B.V. All rights reserved.

The contribution of apoptosis and necrosis in freezing injury of sea urchin embryonic cells.

PubMed

Boroda, Andrey V; Kipryushina, Yulia O; Yakovlev, Konstantin V; Odintsova, Nelly A

2016-08-01

Sea urchins have recently been reported to be a promising tool for investigations of oxidative stress, UV light perturbations and senescence. However, few available data describe the pathway of cell death that occurs in sea urchin embryonic cells after cryopreservation. Our study is focused on the morphological and functional alterations that occur in cells of these animals during the induction of different cell death pathways in response to cold injury. To estimate the effect of cryopreservation on sea urchin cell cultures and identify the involved cell death pathways, we analyzed cell viability (via trypan blue exclusion test, MTT assay and DAPI staining), caspase activity (via flow cytometry and spectrophotometry), the level of apoptosis (via annexin V-FITC staining), and cell ultrastructure alterations (via transmission electron microscopy). Using general caspase detection, we found that the level of caspase activity was low in unfrozen control cells, whereas the number of apoptotic cells with activated caspases rose after freezing-thawing depending on cryoprotectants used, also as the number of dead cells and cells in a late apoptosis. The data using annexin V-binding assay revealed a very high apoptosis level in all tested samples, even in unfrozen cells (about 66%). Thus, annexin V assay appears to be unsuitable for sea urchin embryonic cells. Typical necrotic cells with damaged mitochondria were not detected after freezing in sea urchin cell cultures. Our results assume that physical cell disruption but not freezing-induced apoptosis or necrosis is the predominant reason of cell death in sea urchin cultures after freezing-thawing with any cryoprotectant combination. Copyright © 2016 Elsevier Inc. All rights reserved.

Leukemia cell proliferation and death in chronic lymphocytic leukemia patients on therapy with the BTK inhibitor ibrutinib.

PubMed

Burger, Jan A; Li, Kelvin W; Keating, Michael J; Sivina, Mariela; Amer, Ahmed M; Garg, Naveen; Ferrajoli, Alessandra; Huang, Xuelin; Kantarjian, Hagop; Wierda, William G; O'Brien, Susan; Hellerstein, Marc K; Turner, Scott M; Emson, Claire L; Chen, Shih-Shih; Yan, Xiao-Jie; Wodarz, Dominik; Chiorazzi, Nicholas

2017-01-26

BACKGROUND. Ibrutinib is an effective targeted therapy for patients with chronic lymphocytic leukemia (CLL) that inhibits Bruton's tyrosine kinase (BTK), a kinase involved in B cell receptor signaling. METHODS. We used stable isotopic labeling with deuterated water ( 2 H 2 O) to measure directly the effects of ibrutinib on leukemia cell proliferation and death in 30 patients with CLL. RESULTS. The measured average CLL cell proliferation ("birth") rate before ibrutinib therapy was 0.39% of the clone per day (range 0.17%-1.04%); this decreased to 0.05% per day (range 0%-0.36%) with treatment. Death rates of blood CLL cells increased from 0.18% per day (average, range 0%-0.7%) prior to treatment to 1.5% per day (range 0%-3.0%) during ibrutinib therapy, and they were even higher in tissue compartments. CONCLUSIONS. This study provides the first direct in vivo measurements to our knowledge of ibrutinib's antileukemia actions, demonstrating profound and immediate inhibition of CLL cell proliferation and promotion of high rates of CLL cell death. TRIAL REGISTRATION. This trial was registered at clinicaltrials.gov (NCT01752426). FUNDING. This study was supported by a Cancer Center Support Grant (National Cancer Institute grant P30 CA016672), an NIH grant (CA081554) from the National Cancer Institute, MD Anderson's Moon Shots Program in CLL, and Pharmacyclics, an AbbVie company.

Critical involvement of extracellular ATP acting on P2RX7 purinergic receptors in photoreceptor cell death.

PubMed

Notomi, Shoji; Hisatomi, Toshio; Kanemaru, Takaaki; Takeda, Atsunobu; Ikeda, Yasuhiro; Enaida, Hiroshi; Kroemer, Guido; Ishibashi, Tatsuro

2011-12-01

Stressed cells release ATP, which participates in neurodegenerative processes through the specific ligation of P2RX7 purinergic receptors. Here, we demonstrate that extracellular ATP and the more specific P2RX7 agonist, 2'- and 3'-O-(4-benzoylbenzoyl)-ATP, both induce photoreceptor cell death when added to primary retinal cell cultures or when injected into the eyes from wild-type mice, but not into the eyes from P2RX7(-/-) mice. Photoreceptor cell death was accompanied by the activation of caspase-8 and -9, translocation of apoptosis-inducing factor from mitochondria to nuclei, and TUNEL-detectable chromatin fragmentation. All hallmarks of photoreceptor apoptosis were prevented by premedication or co-application of Brilliant Blue G, a selective P2RX7 antagonist that is already approved for the staining of internal limiting membranes during ocular surgery. ATP release is up-regulated by nutrient starvation in primary retinal cell cultures and seems to be an initializing event that triggers primary and/or secondary cell death via the positive feedback loop on P2RX7. Our results encourage the potential application of Brilliant Blue G as a novel neuroprotective agent in retinal diseases or similar neurodegenerative pathologies linked to excessive extracellular ATP. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

PsANT, the adenine nucleotide translocase of Puccinia striiformis, promotes cell death and fungal growth

PubMed Central

Tang, Chunlei; Wei, Jinping; Han, Qingmei; Liu, Rui; Duan, Xiaoyuan; Fu, Yanping; Huang, Xueling; Wang, Xiaojie; Kang, Zhensheng

2015-01-01

Adenine nucleotide translocase (ANT) is a constitutive mitochondrial component that is involved in ADP/ATP exchange and mitochondrion-mediated apoptosis in yeast and mammals. However, little is known about the function of ANT in pathogenic fungi. In this study, we identified an ANT gene of Puccinia striiformis f. sp. tritici (Pst), designated PsANT. The PsANT protein contains three typical conserved mitochondrion-carrier-protein (mito-carr) domains and shares more than 70% identity with its orthologs from other fungi, suggesting that ANT is conserved in fungi. Immuno-cytochemical localization confirmed the mitochondrial localization of PsANT in normal Pst hyphal cells or collapsed cells. Over-expression of PsANT indicated that PsANT promotes cell death in tobacco, wheat and fission yeast cells. Further study showed that the three mito-carr domains are all needed to induce cell death. qRT-PCR analyses revealed an in-planta induced expression of PsANT during infection. Knockdown of PsANT using a host-induced gene silencing system (HIGS) attenuated the growth and development of virulent Pst at the early infection stage but not enough to alter its pathogenicity. These results provide new insight into the function of PsANT in fungal cell death and growth and might be useful in the search for and design of novel disease control strategies. PMID:26058921

Up-regulated Ectonucleotidases in Fas-Associated Death Domain Protein- and Receptor-Interacting Protein Kinase 1-Deficient Jurkat Leukemia Cells Counteract Extracellular ATP/AMP Accumulation via Pannexin-1 Channels during Chemotherapeutic Drug-Induced Apoptosis.

PubMed

Boyd-Tressler, Andrea M; Lane, Graham S; Dubyak, George R

2017-07-01

Pannexin-1 (Panx1) channels mediate the efflux of ATP and AMP from cancer cells in response to induction of extrinsic apoptosis by death receptors or intrinsic apoptosis by chemotherapeutic agents. We previously described the accumulation of extracellular ATP /AMP during chemotherapy-induced apoptosis in Jurkat human leukemia cells. In this study, we compared how different signaling pathways determine extracellular nucleotide pools in control Jurkat cells versus Jurkat lines that lack the Fas-associated death domain (FADD) or receptor-interacting protein kinase 1 (RIP1) cell death regulatory proteins. Tumor necrosis factor- α induced extrinsic apoptosis in control Jurkat cells and necroptosis in FADD-deficient cells; treatment of both lines with chemotherapeutic drugs elicited similar intrinsic apoptosis. Robust extracellular ATP/AMP accumulation was observed in the FADD-deficient cells during necroptosis, but not during apoptotic activation of Panx1 channels. Accumulation of extracellular ATP/AMP was similarly absent in RIP1-deficient Jurkat cells during apoptotic responses to chemotherapeutic agents. Apoptotic activation triggered equivalent proteolytic gating of Panx1 channels in all three Jurkat cell lines. The differences in extracellular ATP/AMP accumulation correlated with cell-line-specific expression of ectonucleotidases that metabolized the released ATP/AMP. CD73 mRNA, and α β -methylene-ADP-inhibitable ecto-AMPase activity were elevated in the FADD-deficient cells. In contrast, the RIP1-deficient cells were defined by increased expression of tartrate-sensitive prostatic acid phosphatase as a broadly acting ectonucleotidase. Thus, extracellular nucleotide accumulation during regulated tumor cell death involves interplay between ATP/AMP efflux pathways and different cell-autonomous ectonucleotidases. Differential expression of particular ectonucleotidases in tumor cell variants will determine whether chemotherapy-induced activation of Panx1 channels drives accumulation of immunostimulatory ATP versus immunosuppressive adenosine within the tumor microenvironment. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

7-Ketocholesterol Incorporation into Sphingolipid/Cholesterol-enriched (Lipid Raft) Domains Is Impaired by Vitamin E

PubMed Central

Royer, Marie-Charlotte; Lemaire-Ewing, Stéphanie; Desrumaux, Catherine; Monier, Serge; Pais de Barros, Jean-Paul; Athias, Anne; Néel, Dominique; Lagrost, Laurent

2009-01-01

Cholesterol oxides, in particular 7-ketocholesterol, are proatherogenic compounds that induce cell death in the vascular wall when localized in lipid raft domains of the cell membrane. Deleterious effects of 7-ketocholesterol can be prevented by vitamin E, but the molecular mechanism involved is unclear. In this study, unlike γ-tocopherol, the α-tocopherol vitamin E form was found to prevent 7-ketocholesterol-mediated apoptosis of A7R5 smooth muscle cells. To be operative, α-tocopherol needed to be added to the cells before 7-ketocholesterol, and its anti-apoptotic effect was reduced and even suppressed when added together or after 7-ketocholesterol, respectively. Both pre- and co-treatment of the cells with α-tocopherol resulted in the redistribution of 7-ketocholesterol out of the sphingolipid/cholesterol-enriched (lipid raft) domains. In turn, fewer amounts of α-tocopherol associated with lipid rafts on 7-ketocholesterol-pretreated cells compared with untreated cells, with no prevention of cell death in this case. In further support of the implication of lipid raft domains, the dephosphorylation/inactivation of Akt-PKB was involved in the 7-ketocholesterol-induced apoptosis. Akt-PKB dephosphorylation was prevented by α-tocopherol, but not γ-tocopherol pretreatment. PMID:19351882

Arabinogalactan Proteins Are Involved in Salt-Adaptation and Vesicle Trafficking in Tobacco by-2 Cell Cultures

PubMed Central

Olmos, Enrique; García De La Garma, Jesús; Gomez-Jimenez, Maria C.; Fernandez-Garcia, Nieves

2017-01-01

Arabinogalactan proteins (AGPs) are a highly diverse family of glycoproteins that are commonly found in most plant species. However, little is known about the physiological and molecular mechanisms of their function. AGPs are involved in different biological processes such as cell differentiation, cell expansion, tissue development and somatic embryogenesis. AGPs are also involved in abiotic stress response such as salinity modulating cell wall expansion. In this study, we describe how salt-adaptation in tobacco BY-2 cell cultures induces important changes in arabinogalactan proteins distribution and contents. Using the immuno-dot blot technique with different anti-AGP antibodies (JIM13, JIM15, and others), we observed that AGPs were highly accumulated in the culture medium of salt-adapted tobacco cells, probably due to the action of phospholipases. We located these AGP epitopes using immunogold labeling in the cytoplasm associated to the endoplasmic reticulum, the golgi apparatus, and vesicles, plasma membrane and tonoplast. Our results show that salt-adaptation induced a significant reduction of the cytoplasm, plasma membrane and tonoplast content of these epitopes. Yariv reagent was added to the control and salt-adapted tobacco cell cultures, leading to cell death induction in control cells but not in salt-adapted cells. Ultrastructural and immunogold labeling revealed that cell death induced by Yariv reagent in control cells was due to the interaction of Yariv reagent with the AGPs linked to the plasma membranes. Finally, we propose a new function of AGPs as a possible sodium carrier through the mechanism of vesicle trafficking from the apoplast to the vacuoles in salt-adapted tobacco BY-2 cells. This mechanism may contribute to sodium homeostasis during salt-adaptation to high saline concentrations. PMID:28676820

Arabinogalactan Proteins Are Involved in Salt-Adaptation and Vesicle Trafficking in Tobacco by-2 Cell Cultures.

PubMed

Olmos, Enrique; García De La Garma, Jesús; Gomez-Jimenez, Maria C; Fernandez-Garcia, Nieves

2017-01-01

Arabinogalactan proteins (AGPs) are a highly diverse family of glycoproteins that are commonly found in most plant species. However, little is known about the physiological and molecular mechanisms of their function. AGPs are involved in different biological processes such as cell differentiation, cell expansion, tissue development and somatic embryogenesis. AGPs are also involved in abiotic stress response such as salinity modulating cell wall expansion. In this study, we describe how salt-adaptation in tobacco BY-2 cell cultures induces important changes in arabinogalactan proteins distribution and contents. Using the immuno-dot blot technique with different anti-AGP antibodies (JIM13, JIM15, and others), we observed that AGPs were highly accumulated in the culture medium of salt-adapted tobacco cells, probably due to the action of phospholipases. We located these AGP epitopes using immunogold labeling in the cytoplasm associated to the endoplasmic reticulum, the golgi apparatus, and vesicles, plasma membrane and tonoplast. Our results show that salt-adaptation induced a significant reduction of the cytoplasm, plasma membrane and tonoplast content of these epitopes. Yariv reagent was added to the control and salt-adapted tobacco cell cultures, leading to cell death induction in control cells but not in salt-adapted cells. Ultrastructural and immunogold labeling revealed that cell death induced by Yariv reagent in control cells was due to the interaction of Yariv reagent with the AGPs linked to the plasma membranes. Finally, we propose a new function of AGPs as a possible sodium carrier through the mechanism of vesicle trafficking from the apoplast to the vacuoles in salt-adapted tobacco BY-2 cells. This mechanism may contribute to sodium homeostasis during salt-adaptation to high saline concentrations.

Phospholipase C delta 4 (PLCδ4) is a nuclear protein involved in cell proliferation and senescence in mesenchymal stromal stem cells.

PubMed

Kunrath-Lima, Marianna; de Miranda, Marcelo Coutinho; Ferreira, Andrea da Fonseca; Faraco, Camila Cristina Fraga; de Melo, Mariane Izabella Abreu; Goes, Alfredo Miranda; Rodrigues, Michele Angela; Faria, Jerusa Araújo Quintão Arantes; Gomes, Dawidson Assis

2018-06-01

Ca 2+ is an important second messenger, and it is involved in many cellular processes such as cell death and proliferation. The rise in intracellular Ca 2+ levels can be due to the generation of inositol 1,4,5-trisphosphate (InsP 3 ), which is a product of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) hydrolysis by phospholipases C (PLCs), that leads to Ca 2+ release from endoplasmic reticulum by InsP 3 receptors (InsP 3 R). Ca 2+ signaling patterns can vary in different regions of the cell and increases in nuclear Ca 2+ levels have specific biological effects that differ from those of Ca 2+ increase in the cytoplasm. There are PLCs in the cytoplasm and nucleus, but little is known about the functions of nuclear PLCs. This work aimed to characterize phenotypically the human PLCδ4 (hPLCδ4) in mesenchymal stem cells. This nuclear isoform of PLC is present in different cell types and has a possible role in proliferative processes. In this work, hPLCδ4 was found to be mainly nuclear in human adipose-derived mesenchymal stem cells (hASC). PLCδ4 knockdown demonstrated that it is essential for hASC proliferation, without inducing cell death. An increase of cells in G1, and a reduction of cells on interphase and G2/M in knockdown cells were seen. Furthermore, PLCδ4 knockdown increased the percentage of senescent cells, p16 INK4A+ and p21 Cip1 mRNAs expression, which could explain the impaired cell proliferation. The results show that hPLCδ4 is in involved in cellular proliferation and senescence in hASC. Copyright © 2018 Elsevier Inc. All rights reserved.

Recent advances in inflammasome biology.

PubMed

Place, David E; Kanneganti, Thirumala-Devi

2018-02-01

The inflammasome is a complex of proteins that through the activity of caspase-1 and the downstream substrates gasdermin D, IL-1β, and IL-18 execute an inflammatory form of cell death termed pyroptosis. Activation of this complex often involves the adaptor protein ASC and upstream sensors including NLRP1, NLRP3, NLRC4, AIM2, and pyrin, which are activated by different stimuli including infectious agents and changes in cell homeostasis. Here we discuss new regulatory mechanisms that have been identified for the canonical inflammasomes, the most recently identified NLRP9b inflammasome, and the new gasdermin family of proteins that mediate pyroptosis and other forms of regulated cell death. Copyright © 2017 Elsevier Ltd. All rights reserved.

Calnexin Is Essential for Survival under Nitrogen Starvation and Stationary Phase in Schizosaccharomyces pombe

PubMed Central

Rokeach, Luis A.

2015-01-01

Cell fate is determined by the balance of conserved molecular mechanisms regulating death (apoptosis) and survival (autophagy). Autophagy is a process by which cells recycle their organelles and macromolecules through degradation within the vacuole in yeast and plants, and lysosome in metazoa. In the yeast Schizosaccharomyces pombe, autophagy is strongly induced under nitrogen starvation and in aging cells. Previously, we demonstrated that calnexin (Cnx1p), a highly conserved transmembrane chaperone of the endoplasmic reticulum (ER), regulates apoptosis under ER stress or inositol starvation. Moreover, we showed that in stationary phase, Cnx1p is cleaved into two moieties, L_Cnx1p and S_Cnx1p. Here, we show that the processing of Cnx1p is regulated by autophagy, induced by nitrogen starvation or cell aging. The cleavage of Cnx1p involves two vacuolar proteases: Isp6, which is essential for autophagy, and its paralogue Psp3. Blocking autophagy through the knockout of autophagy-related genes (atg) results in inhibition of both, the cleavage and the trafficking of Cnx1p from the ER to the vacuole. We demonstrate that Cnx1p is required for cell survival under nitrogen-starvation and in chronological aging cultures. The death of the mini_cnx1 mutant (overlapping S_cnx1p) cells is accompanied by accumulation of high levels of reactive-oxygen species (ROS), a slowdown in endocytosis and severe cell-wall defects. Moreover, mutant cells expressing only S_Cnx1p showed cell wall defects. Co-expressing mutant overlapping the L_Cnx1p and S_Cnx1p cleavage products reverses the death, ROS phenotype and cell wall defect to wild-type levels. As it is involved in both apoptosis and autophagy, Cnx1p could be a nexus for the crosstalk between these pro-death and pro-survival mechanisms. Ours, and observations in mammalian systems, suggest that the multiple roles of calnexin depend on its sub-cellular localization and on its cleavage. The use of S. pombe should assist in further shedding light on the multiple roles of calnexin. PMID:25803873

Modeling activity and target-dependent developmental cell death of mouse retinal ganglion cells ex vivo.

PubMed

Voyatzis, Sylvie; Muzerelle, Aude; Gaspar, Patricia; Nicol, Xavier

2012-01-01

Programmed cell death is widespread during the development of the central nervous system and serves multiple purposes including the establishment of neural connections. In the mouse retina a substantial reduction of retinal ganglion cells (RGCs) occurs during the first postnatal week, coinciding with the formation of retinotopic maps in the superior colliculus (SC). We previously established a retino-collicular culture preparation which recapitulates the progressive topographic ordering of RGC projections during early post-natal life. Here, we questioned whether this model could also be suitable to examine the mechanisms underlying developmental cell death of RGCs. Brn3a was used as a marker of the RGCs. A developmental decline in the number of Brn3a-immunolabelled neurons was found in the retinal explant with a timing that paralleled that observed in vivo. In contrast, the density of photoreceptors or of starburst amacrine cells increased, mimicking the evolution of these cell populations in vivo. Blockade of neural activity with tetrodotoxin increased the number of surviving Brn3a-labelled neurons in the retinal explant, as did the increase in target availability when one retinal explant was confronted with 2 or 4 collicular slices. Thus, this ex vivo model reproduces the developmental reduction of RGCs and recapitulates its regulation by neural activity and target availability. It therefore offers a simple way to analyze developmental cell death in this classic system. Using this model, we show that ephrin-A signaling does not participate to the regulation of the Brn3a population size in the retina, indicating that eprhin-A-mediated elimination of exuberant projections does not involve developmental cell death.

Novel role of TRPV2 in promoting the cytotoxicity of H2O2-mediated oxidative stress in human hepatoma cells.

PubMed

Ma, Wenbo; Li, Caiyue; Yin, Shikui; Liu, Jingxin; Gao, Chao; Lin, Zuoxian; Huang, Rongqi; Huang, Jufang; Li, Zhiyuan

2015-12-01

Oxidative stress is important for the initiation and progression of cancers, which confers the cells with a survival advantage by inducing oxidative adaption and drug resistance. Therefore, developing strategies to promote oxidative stress-induced cytotoxicity could be important for cancer therapy. Herein, we found that H2O2-mediated oxidative stress increases TRPV2 expression in human hepatoma (HepG2 and Huh-7) cells. This occurred at the mRNA and protein levels in a dose-dependent manner. The significance of TRPV2 in promoting H2O2-induced cell death was demonstrated in gain and loss of function studies with overexpression and knockdown of TRPV2, respectively. Mechanistically, H2O2-induced cell death involves inhibition of pro-survival signaling proteins (Akt, Nrf2) and activation of pro-death signaling proteins (p38, JNK1). Overexpression of TRPV2 in H2O2-treated hepatoma cells aggravates the inhibition of Akt and Nrf2, while it enhances the activation of p38 and JNK1 at the early stage of cell death. Interestingly, increased expression of TRPV2 in HepG2 cells improved the efficacy of stress-associated chemicals to induce cell death. Our findings suggest that TRPV2 acts as an important enhancer for H2O2-induced cytotoxicity. This process occurred by the inhibition of Akt and Nrf2 as well as the early activation of p38 and JNK1. These findings have important implications for inhibition of oxidative adaption and drug resistance. Copyright © 2015 Elsevier Inc. All rights reserved.

The antioxidant N-acetyl cysteine suppresses lidocaine-induced intracellular reactive oxygen species production and cell death in neuronal SH-SY5Y cells.

PubMed

Okamoto, Akihisa; Tanaka, Masahiro; Sumi, Chisato; Oku, Kanako; Kusunoki, Munenori; Nishi, Kenichiro; Matsuo, Yoshiyuki; Takenaga, Keizo; Shingu, Koh; Hirota, Kiichi

2016-10-24

The local anesthetic lidocaine can affect intra- and extra-cellular signaling pathways in both neuronal and non-neuronal cells, resulting in long-term modulation of biological functions, including cell growth and death. Indeed, lidocaine was shown to induce necrosis and apoptosis in vitro. While several studies have suggested that lidocaine-induced apoptosis is mitochondrial pathway-dependent, it remains unclear whether reactive oxygen species (ROS) are involved in this process and whether the observed cell death can be prevented by antioxidant treatment. The effects of lidocaine and antioxidants on cell viability and death were evaluated using SH-SY5Y cells, HeLa cells, and HeLa cell derivatives. Cell viability was examined via MTS/PES ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]/phenazine ethosulfate) assay. Meanwhile, cell apoptosis and necrosis were evaluated using a cell death detection assay with Annexin V-FITC and PI staining, as well as by assaying for caspase-3/7 and caspase-9 activity, and by measuring the release of lactate dehydrogenase, respectively. Mitochondrial transmembrane potential (ΔΨm) was assessed using the fluorescent probe tetramethylrhodamine ethyl ester. Lidocaine treatment resulted in suppression of the mitochondrial electron transport chain and subsequent attenuation of mitochondrial membrane potential, as well as enhanced ROS production, activation of caspase-3/7 and caspase-9, and induction of apoptosis and necrosis in SH-SY5Y cells in a dose- and time-dependent manner. Likewise, the anesthetics mepivacaine and bupivacaine also induced apoptosis in SH-SY5Y cells. Notably, the antioxidants N-acetyl cysteine (NAC) and Trolox successfully scavenged the mitochondria-derived ROS and suppressed local lidocaine-induced cell death. Our findings demonstrate that the local anesthetics lidocaine, mepivacaine, and bupivacaine inhibited the activity of mitochondria and induced apoptosis and necrosis in a dose-dependent manner. Furthermore, they demonstrate that treatment with the antioxidants NAC, Trolox, and GGA resulted in preservation of mitochondrial voltage and inhibition of apoptosis via suppression of caspase activation.

Transfer hydrogenation catalysis in cells as a new approach to anticancer drug design

PubMed Central

Soldevila-Barreda, Joan J.; Romero-Canelón, Isolda; Habtemariam, Abraha; Sadler, Peter J.

2015-01-01

Organometallic complexes are effective hydrogenation catalysts for organic reactions. For example, Noyori-type ruthenium complexes catalyse reduction of ketones by transfer of hydride from formate. Here we show that such catalytic reactions can be achieved in cancer cells, offering a new strategy for the design of safe metal-based anticancer drugs. The activity of ruthenium(II) sulfonamido ethyleneamine complexes towards human ovarian cancer cells is enhanced by up to 50 × in the presence of low non-toxic doses of formate. The extent of conversion of coenzyme NAD+ to NADH in cells is dependent on formate concentration. This novel reductive stress mechanism of cell death does not involve apoptosis or perturbation of mitochondrial membrane potentials. In contrast, iridium cyclopentadienyl catalysts cause cancer cell death by oxidative stress. Organometallic complexes therefore have an extraordinary ability to modulate the redox status of cancer cells. PMID:25791197

Bcl-2△21 and Ac-DEVD-CHO Inhibit Death of Wheat Microspores

PubMed Central

Sinha, Rakesh K.; Pospíšil, Pavel; Maheshwari, Priti; Eudes, François

2016-01-01

Microspore cell death and low green plant production efficiency are an integral obstacle in the development of doubled haploid production in wheat. The aim of the current study was to determine the effect of anti-apoptotic recombinant human B-cell lymphoma-2 (Bcl-2△21) and caspase-3-inhibitor (Ac-DEVD-CHO) in microspore cell death in bread wheat cultivars AC Fielder and AC Andrew. Induction medium containing Bcl-2△21 and Ac-DEVD-CHO yielded a significantly higher number of viable microspores, embryo-like structures and total green plants in wheat cultivars AC Fielder and AC Andrew. Total peroxidase activity was lower in Bcl-2△21 treated microspore cultures at 96 h of treatment compared to control and Ac-DEVD-CHO. Electron paramagnetic resonance study of total microspore protein showed a different scavenging activity for Bcl-2△21 and Ac-DEVD-CHO. Bcl-2△21 scavenged approximately 50% hydroxyl radical (HO•) formed, whereas Ac-DEVD-CHO scavenged approximately 20% of HO•. Conversely, reduced caspase-3-like activities were detected in the presence of Bcl-2△21 and Ac-DEVD-CHO, supporting the involvement of Bcl-2△21 and Ac-DEVD-CHO in increasing microspore viability by reducing oxidative stress and caspase-3-like activity. Our results indicate that Bcl-2△21 and Ac-DEVD-CHO protects cells from cell death following different pathways. Bcl-2△21 prevents cell damage by detoxifying HO• and suppressing caspase-3-like activity, while Ac-DEVD-CHO inhibits the cell death pathways by modulating caspase-like activity. PMID:28082995

Up-Regulation of PAI-1 and Down-Regulation of uPA Are Involved in Suppression of Invasiveness and Motility of Hepatocellular Carcinoma Cells by a Natural Compound Berberine.

PubMed

Wang, Xuanbin; Wang, Ning; Li, Hongliang; Liu, Ming; Cao, Fengjun; Yu, Xianjun; Zhang, Jingxuan; Tan, Yan; Xiang, Longchao; Feng, Yibin

2016-04-16

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death and its prognosis remains poor due to the high risk of tumor recurrence and metastasis. Berberine (BBR) is a natural compound derived from some medicinal plants, and accumulating evidence has shown its potent anti-tumor activity with diverse action on tumor cells, including inducing cancer cell death and blocking cell cycle and migration. Molecular targets of berberine involved in its inhibitory effect on the invasiveness remains not yet clear. In this study, we identified that berberine exhibits a potent inhibition on the invasion and migration of HCC cells. This was accompanied by a dose-dependent down-regulation of expression of Cyclooxygenase-2 (COX-2), nuclear factor kappa B (NF-κB), urokinase-type plasminogen activator (uPA) and matrix metalloproteinase (MMP)-9 in berberine-treated HCC cells. Furthermore, berberine inactivated p38 and Erk1/2 signaling pathway in HCC cells. Primarily, this may be attributed to the up-regulation of plasminogen activator inhibitor-1 (PAI-1), a tumor suppressor that can antagonize uPA receptor and down-regulation of uPA. Blockade of uPA receptor-associated pathways leads to reduced invasiveness and motility of berberine-treated HCC cells. In conclusion, our findings identified for the first time that inactivation of uPA receptor by up-regulation of PAI-1 and down-regulation of uPA is involved in the inhibitory effect of berberine on HCC cell invasion and migration.

The hnRNP-Htt axis regulates necrotic cell death induced by transcriptional repression through impaired RNA splicing.

PubMed

Mao, Y; Tamura, T; Yuki, Y; Abe, D; Tamada, Y; Imoto, S; Tanaka, H; Homma, H; Tagawa, K; Miyano, S; Okazawa, H

2016-04-28

In this study, we identify signaling network of necrotic cell death induced by transcriptional repression (TRIAD) by α-amanitin (AMA), the selective RNA polymerase II inhibitor, as a model of neurodegenerative cell death. We performed genetic screen of a knockdown (KD) fly library by measuring the ratio of transformation from pupa to larva (PL ratio) under TRIAD, and selected the cell death-promoting genes. Systems biology analysis of the positive genes mapped on protein-protein interaction databases predicted the signaling network of TRIAD and the core pathway including heterogeneous nuclear ribonucleoproteins (hnRNPs) and huntingtin (Htt). RNA sequencing revealed that AMA impaired transcription and RNA splicing of Htt, which is known as an endoplasmic reticulum (ER)-stabilizing molecule. The impairment in RNA splicing and PL ratio was rescued by overexpresion of hnRNP that had been also affected by transcriptional repression. Fly genetics with suppressor or expresser of Htt and hnRNP worsened or ameliorated the decreased PL ratio by AMA, respectively. Collectively, these results suggested involvement of RNA splicing and a regulatory role of the hnRNP-Htt axis in the process of the transcriptional repression-induced necrosis.

Calcein+/PI- as an early apoptotic feature in Leishmania.

PubMed

Basmaciyan, Louise; Azas, Nadine; Casanova, Magali

2017-01-01

Although leishmaniases are responsible for high morbidity and mortality all over the world, no really satisfying treatment exists. Furthermore, the corresponding parasite Leishmania undergoes a very characteristic form of programmed cell death. Indeed, different stimuli can induce morphological and biochemical apoptotic-like features. However, the key proteins involved in mammal apoptosis, such as caspases and death receptors, are not encoded in the genome of this parasite. Currently, little is known about Leishmania apoptosis, notably owing to the lack of specific tools for programmed cell death analysis in these parasites. Furthermore, there is a need for a better understanding of Leishmania programmed cell death in order (i) to better understand the role of apoptosis in unicellular organisms, (ii) to better understand apoptosis in general through the study of an ancestral eukaryote, and (iii) to identify new therapeutic targets against leishmaniases. To advance understanding of apoptosis in Leishmania, in this study we developed a new tool based on the quantification of calcein and propidium iodide by flow cytometry. This double labeling can be employed to distinguish early apoptosis, late apoptosis and necrosis in Leishmania live cells with a very simple and rapid assay. This paper should, therefore, be of interest for people working on Leishmania and related parasites.

Vacuolar processing enzyme in plant programmed cell death

PubMed Central

Hatsugai, Noriyuki; Yamada, Kenji; Goto-Yamada, Shino; Hara-Nishimura, Ikuko

2015-01-01

Vacuolar processing enzyme (VPE) is a cysteine proteinase originally identified as the proteinase responsible for the maturation and activation of vacuolar proteins in plants, and it is known to be an ortholog of animal asparaginyl endopeptidase (AEP/VPE/legumain). VPE has been shown to exhibit enzymatic properties similar to that of caspase 1, which is a cysteine protease that mediates the programmed cell death (PCD) pathway in animals. Although there is limited sequence identity between VPE and caspase 1, their predicted three-dimensional structures revealed that the essential amino-acid residues for these enzymes form similar pockets for the substrate peptide YVAD. In contrast to the cytosolic localization of caspases, VPE is localized in vacuoles. VPE provokes vacuolar rupture, initiating the proteolytic cascade leading to PCD in the plant immune response. It has become apparent that the VPE-dependent PCD pathway is involved not only in the immune response, but also in the responses to a variety of stress inducers and in the development of various tissues. This review summarizes the current knowledge on the contribution of VPE to plant PCD and its role in vacuole-mediated cell death, and it also compares VPE with the animal cell death executor caspase 1. PMID:25914711

Roles of zinc and metallothionein-3 in oxidative stress-induced lysosomal dysfunction, cell death, and autophagy in neurons and astrocytes.

PubMed

Lee, Sook-Jeong; Koh, Jae-Young

2010-10-26

Zinc dyshomeostasis has been recognized as an important mechanism for cell death in acute brain injury. An increase in the level of free or histochemically reactive zinc in astrocytes and neurons is considered one of the major causes of death of these cells in ischemia and trauma. Although zinc dyshomeostasis can lead to cell death via diverse routes, the major pathway appears to involve oxidative stress.Recently, we found that a rise of zinc in autophagic vacuoles, including autolysosomes, is a prerequisite for lysosomal membrane permeabilization and cell death in cultured brain cells exposed to oxidative stress conditions. The source of zinc in this process is likely redox-sensitive zinc-binding proteins such as metallothioneins, which release zinc under oxidative conditions. Of the metallothioneins, metallothionein-3 is especially enriched in the central nervous system, but its physiologic role in this tissue is not well established. Like other metallothioneins, metallothionein-3 may function as metal detoxicant, but is also known to inhibit neurite outgrowth and, sometimes, promote neuronal death, likely by serving as a source of toxic zinc release. In addition, metallothionein-3 regulates lysosomal functions. In the absence of metallothionein-3, there are changes in lysosome-associated membrane protein-1 and -2, and reductions in certain lysosomal enzymes that result in decreased autophagic flux. This may have dual effects on cell survival. In acute oxidative injury, zinc dyshomeostasis and lysosomal membrane permeabilization are diminished in metallothionein-3 null cells, resulting in less cell death. But over the longer term, diminished lysosomal function may lead to the accumulation of abnormal proteins and cause cytotoxicity.The roles of zinc and metallothionein-3 in autophagy and/or lysosomal function have just begun to be investigated. In light of evidence that autophagy and lysosomes may play significant roles in the pathogenesis of various neurological diseases, further insight into the contribution of zinc dynamics and metallothionein-3 function may help provide ways to effectively regulate these processes in brain cells.

Association between histological alterations in the thymus and sudden infant death syndrome.

PubMed

Varga, Ivan; Bódi, Ildikó; Mešťanová, Veronika; Kováč, Martin; Klein, Martin

2018-04-01

Sudden infant death syndrome (SIDS) involves the death of an infant during the first year of life and it is among the leading causes of infant mortality worldwide. One hypothesis regarding the pathogenesis of SIDS is that it results from a combination of three independent factors: endogenous vulnerability, a critical time window during postnatal development, and exogenous stressors. This hypothesis is known as the "triple-risk model". In this study, we used an immunohistological approach to compare the cellular microenvironments of thymuses from 19 infants whose sudden death was classified as SIDS and a control group, which consisted of thymuses from age-matched children undergoing surgery for various congenital heart defects. We hypothesized that morphological signs of stress-related thymic involution would be present. Based on our observations, we found evidence that the proliferation and maturation of T-lymphocytes in the thymuses of infants with SIDS were suppressed. We observed enhanced macrophage activity, suggesting an increase in the apoptosis of lymphocytes and decrease in number of thymic dendritic cells and myoid cells. Significant apoptosis of thymic lymphocytes without cell regeneration typically leads to atrophy of the thymus. All cellular events we observed resemble the initial stage of stress-related thymic involution. These results support the "triple-risk model," suggesting that certain exogenous stressors might be involved in the pathogenesis of SIDS. This was probably not recognized during the autopsies of infants who died suddenly. Copyright © 2018 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Capsaicin sensitizes TRAIL-induced apoptosis through Sp1-mediated DR5 up-regulation: Involvement of Ca{sup 2+} influx

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moon, Dong-Oh; Kang, Chang-Hee; Kang, Sang-Hyuck

2012-02-15

Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various malignant cells, several cancers including human hepatocellular carcinoma (HCC) exhibit potent resistance to TRAIL-induced cell death. The aim of this study is to evaluate the anti-cancer potential of capsaicin in TRAIL-induced cancer cell death. As indicated by assays that measure phosphatidylserine exposure, mitochondrial activity and activation of caspases, capsaicin potentiated TRAIL-resistant cells to lead to cell death. In addition, we found that capsaicin induces the cell surface expression of TRAIL receptor DR5, but not DR4 through the activation Sp1 on its promoter region. Furthermore, we investigated that capsaicin-induced DR5more » expression and apoptosis are inhibited by calcium chelator or inhibitors for calmodulin-dependent protein kinase. Taken together, our data suggest that capsaicin sensitizes TRAIL-mediated HCC cell apoptosis by DR5 up-regulation via calcium influx-dependent Sp1 activation. Highlights: ► Capsaicin sensitizes TRAIL-induced apoptosis through activation of caspases. ► Capsaicin induces expression of DR5 through Sp1 activation. ► Capsaicin activates calcium signaling pathway.« less

Pharmacological inhibition of NADPH oxidase protects against cisplatin induced nephrotoxicity in mice by two step mechanism.

PubMed

Wang, Yimin; Luo, Xiao; Pan, Hao; Huang, Wei; Wang, Xueping; Wen, Huali; Shen, Kezhen; Jin, Baiye

2015-09-01

Cisplatin induced nephrotoxicity is primarily caused by ROS (Reactive Oxygen Species) induced proximal tubular cell death. NADPH oxidase is major source of ROS production by cisplatin. Here, we reported that pharmacological inhibition of NADPH oxidase by acetovanillone (obtained from medicinal herb Picrorhiza kurroa) led to reduced cisplatin nephrotoxicity in mice. In this study we used various molecular biology and biochemistry methods a clinically relevant model of nephropathy, induced by an important chemotherapeutic drug cisplatin. Cisplatin-induced nephrotoxicity was evident by histological damage from loss of the tubular structure. The damage was also marked by the increase in blood urea nitrogen, creatinine, protein nitration as well as cell death markers such as caspase 3/7 activity and DNA fragmentation. Tubular cell death by cisplatin led to pro-inflammatory response by production of TNFα and IL1β followed by leukocyte/neutrophil infiltration which resulted in new wave of ROS involving more NADPH oxidases. Cisplatin-induced markers of kidney damage such as oxidative stress, cell death, inflammatory cytokine production and nephrotoxicity were attenuated by acetovanillone. In addition to that, acetovanillone enhanced cancer cell killing efficacy of cisplatin. Thus, pharmacological inhibition of NADPH oxidase can be protective for cisplatin-induced nephrotoxicity in mice. Copyright © 2015. Published by Elsevier Ltd.

Death induction by recombinant native TRAIL and its prevention by a caspase 9 inhibitor in primary human esophageal epithelial cells.

PubMed

Kim, Seok-Hyun; Kim, Kunhong; Kwagh, Jae G; Dicker, David T; Herlyn, Meenhard; Rustgi, Anil K; Chen, Youhai; El-Deiry, Wafik S

2004-09-17

The cytotoxic death ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a tumor-specific agent under development as a novel anticancer therapeutic agent. However, some reports have demonstrated toxicity of certain TRAIL preparations toward human hepatocytes and keratinocytes through a caspase-dependent mechanism that involves activation of the extrinsic death pathway and Type II signaling through the mitochondria. We have isolated and purified both His-tagged protein and three versions of native recombinant human TRAIL protein from Escherichia coli. We found that 5 mm dithiothreitol in the purification process enhanced oligomerization of TRAIL and resulted in the formation of hyper-oligomerized TRAILs, including hexamers and nonomers with an extremely high potency in apoptosis induction. Although death-inducing signaling complex formation was much more efficient in cells treated with hyper-oligomerized TRAILs, this did not convert TRAIL-sensitive Type II HCT116 colon tumor cells to a Type I death pattern as judged by their continued sensitivity to a caspase 9 inhibitor. Moreover, TRAIL-resistant Type II Bax-null colon carcinoma cells were not converted to a TRAIL-sensitive Type I state by hyper-oligomerized TRAIL. Primary human esophageal epithelial 2 cells were found to be sensitive to all TRAIL preparations used, including trimer TRAIL. TRAIL-induced death in esophageal epithelial 2 cells was prevented by caspase 9 inhibition for up to 4 h after TRAIL exposure. This result suggests a possible therapeutic application of caspase 9 inhibition as a strategy to reverse TRAIL toxicity. Hyper-oligomerized TRAIL may be considered as an alternative agent for testing in clinical trials.

The role of altered [Ca2+]i regulation in apoptosis, oncosis, and necrosis.

PubMed

Trump, B F; Berezesky, I K

1996-10-11

Understanding the processes and events that occur when a cell undergoes a prelethal injury or that lead the cell to death following a lethal injury has been the aim of our research for a number of years. Throughout this period much has been learned, recently at rapid rates, not only by us but by many other investigators as well. Based on the data gathered, we proposed a working hypothesis over a decade ago and have since continually updated it as new experimentation is performed. Our laboratory has focused particularly on the role of cytoplasmic ionized calcium ([Ca2+]i) and the effects of its deregulation on prelethal events, including oncosis and apoptosis, and lethal events (necrosis) following cell death. [Ca2+]i appears to be a major link and signalling event. Understanding the mechanisms involved by using a variety of in vivo and in vitro models, coupled with state-of-the-art methodologies, should now allow us to prevent cell death by killing cells when necessary through gene therapy and cancer chemotherapy.

Microtubule antagonists activate programmed cell death (apoptosis) in cultured rat hepatocytes.

PubMed Central

Tsukidate, K.; Yamamoto, K.; Snyder, J. W.; Farber, J. L.

1993-01-01

We investigated the mechanism of lethal injury following the disruption of microtubules in cultured hepatocytes treated with vinblastine (VBL) or colchicine (COL). These agents kill hepatocytes by a process readily distinguished from two well-known pathways that lead to a loss of viability, namely, oxidative stress and inhibition of mitochondrial electron transport. Cell killing with VBL and COL was accompanied by fragmentation of DNA. Both the loss of viability and the fragmentation of DNA were prevented by the inhibition of protein synthesis within 6 hours following exposure to VBL or COL. Cell death and the fragmentation of DNA were also prevented when Ca2+ was removed from the culture medium. By contrast, the inhibition of protein kinase C prevented cell killing by VBL or COL, but did not alter the extent of DNA fragmentation. The requirements here for protein synthesis, extracellular Ca2+, and protein kinase C activity define a model of apoptosis, or programmed cell death, that seems to involve mechanisms that can be dissociated from the fragmentation of DNA. Images Figure 2 PMID:8362985

Giant cell phlebitis: a potentially lethal clinical entity.

PubMed

Kunieda, Takeshige; Murayama, Masanori; Ikeda, Tsuneko; Yamakita, Noriyoshi

2012-08-01

An 83-year-old woman presented to us with a 4-week history of general malaise, subjective fever and lower abdominal pain. Despite the intravenous infusion of antibiotics, her blood results and physical condition worsened, resulting in her sudden death. Autopsy study revealed that the medium-sized veins of the mesentery were infiltrated by eosinophil granulocytes, lymphocytes, macrophages and multinucleated giant cells; however, the arteries were not involved. Microscopically, venous giant cell infiltration was observed in the gastrointestinal tract, bladder, retroperitoneal tissues and myocardium. The final diagnosis was giant cell phlebitis, a rare disease of unknown aetiology. This case demonstrates for the first time that giant cell phlebitis involving extra-abdominal organs, including hearts, can cause serious morbidity.

The importance of serum serotonin levels in the measurement of radiation-induced bystander cell death in HaCaT cells.

PubMed

Lyng, Fiona M; Desplanques, Maxime; Jella, Kishore Kumar; Garcia, Amaya; McClean, Brendan

2012-10-01

The aim of this study was to investigate the importance of serum serotonin levels in the measurement of bystander cell death. The study was undertaken as part of an intercomparison exercise involving seven European laboratories funded under the European Union Sixth Framework Programme (FP6) Non-Targeted Effects (NOTE) integrated project. Three batches of foetal bovine serum were tested; serum with high and low serotonin content from the intercomparison exercise as well as serum from the home laboratory. Three sets of human keratinocytes (HaCaT cell line) were cultured in DMEM:F12 medium supplemented with serum with high or low serotonin content or serum from the home laboratory and both donor and recipient HaCaT cells were plated. The donor HaCaT cells were irradiated (0.5 Gy) using a cobalt 60 teletherapy unit, the medium was harvested 1 hour post irradiation and transferred to the recipient HaCaT cells. Bystander induced cell death was measured by the clonogenic survival assay and the Alamar blue viability assay. A significant reduction in cell survival, as measured by the clonogenic assay, and in cell viability, as measured by the Alamar blue assay, was observed in the recipient HaCaT cells treated with medium from irradiated cells compared to the cells treated with medium from unirradiated cells. No significant difference was found between the three batches of serum. The data suggest that in our cell system and with our endpoints (clonogenic assay and Alamar blue assay), serum serotonin levels do not play a role in bystander-induced cell death.

In vitro and in vivo anti-cancer activity of silymarin on oral cancer.

PubMed

Won, Dong-Hoon; Kim, Lee-Han; Jang, Boonsil; Yang, In-Hyoung; Kwon, Hye-Jeong; Jin, Bohwan; Oh, Seung Hyun; Kang, Ju-Hee; Hong, Seong-Doo; Shin, Ji-Ae; Cho, Sung-Dae

2018-05-01

Silymarin, a standardized extract from milk thistle fruits has been found to exhibit anti-cancer effects against various cancers. Here, we explored the anti-cancer activity of silymarin and its molecular target in human oral cancer in vitro and in vivo. Silymarin dose-dependently inhibited the proliferation of HSC-4 oral cancer cells and promoted caspase-dependent apoptosis. A human apoptosis protein array kit showed that death receptor 5 may be involved in silymarin-induced apoptosis, which was also shown through western blotting, immunocytochemistry, and reverse transcription-polymerase chain reaction. Silymarin increased cleaved caspase-8 and truncated Bid, leading to accumulation of cytochrome c. In addition, silymarin activated death receptor 5/caspase-8 to induce apoptotic cell death in two other oral cancer cell lines (YD15 and Ca9.22). Silymarin also suppressed tumor growth and volume without any hepatic or renal toxicity in vivo. Taken together, these results provide in vitro and in vivo evidence supporting the anti-cancer effect of silymarin and death receptor 5, and caspase-8 may be essential players in silymarin-mediated apoptosis in oral cancer.

Distinct cytoprotective roles of pyruvate and ATP by glucose metabolism on epithelial necroptosis and crypt proliferation in ischaemic gut

PubMed Central

Huang, Ching‐Ying; Kuo, Wei‐Ting; Huang, Chung‐Yen; Lee, Tsung‐Chun; Chen, Chin‐Tin; Peng, Wei‐Hao; Lu, Kuo‐Shyan; Yang, Chung‐Yi

2016-01-01

Key points Intestinal ischaemia causes epithelial death and crypt dysfunction, leading to barrier defects and gut bacteria‐derived septic complications.Enteral glucose protects against ischaemic injury; however, the roles played by glucose metabolites such as pyruvate and ATP on epithelial death and crypt dysfunction remain elusive.A novel form of necrotic death that involves the assembly and phosphorylation of receptor interacting protein kinase 1/3 complex was found in ischaemic enterocytes.Pyruvate suppressed epithelial cell death in an ATP‐independent manner and failed to maintain crypt function. Conversely, replenishment of ATP partly restored crypt proliferation but had no effect on epithelial necroptosis in ischaemic gut.Our data argue against the traditional view of ATP as the main cytoprotective factor by glucose metabolism, and indicate a novel anti‐necroptotic role of glycolytic pyruvate under ischaemic stress. Abstract Mesenteric ischaemia/reperfusion induces epithelial death in both forms of apoptosis and necrosis, leading to villus denudation and gut barrier damage. It remains unclear whether programmed cell necrosis [i.e. receptor‐interacting protein kinase (RIP)‐dependent necroptosis] is involved in ischaemic injury. Previous studies have demonstrated that enteral glucose uptake by sodium‐glucose transporter 1 ameliorated ischaemia/reperfusion‐induced epithelial injury, partly via anti‐apoptotic signalling and maintenance of crypt proliferation. Glucose metabolism is generally assumed to be cytoprotective; however, the roles played by glucose metabolites (e.g. pyruvate and ATP) on epithelial cell death and crypt dysfunction remain elusive. The present study aimed to investigate the cytoprotective effects exerted by distinct glycolytic metabolites in ischaemic gut. Wistar rats subjected to mesenteric ischaemia were enterally instilled glucose, pyruvate or liposomal ATP. The results showed that intestinal ischaemia caused RIP1‐dependent epithelial necroptosis and villus destruction accompanied by a reduction in crypt proliferation. Enteral glucose uptake decreased epithelial cell death and increased crypt proliferation, and ameliorated mucosal histological damage. Instillation of cell‐permeable pyruvate suppressed epithelial cell death in an ATP‐independent manner and improved the villus morphology but failed to maintain crypt function. Conversely, the administration of liposomal ATP partly restored crypt proliferation but did not reduce epithelial necroptosis and histopathological injury. Lastly, glucose and pyruvate attenuated mucosal‐to‐serosal macromolecular flux and prevented enteric bacterial translocation upon blood reperfusion. In conclusion, glucose metabolites protect against ischaemic injury through distinct modes and sites, including inhibition of epithelial necroptosis by pyruvate and the promotion of crypt proliferation by ATP. PMID:27121603

Lack of association of programmed cell death 1 gene (PDCD1) polymorphisms with susceptibility to chronic urticaria in patients with positive autologous serum skin test.

PubMed

Brzoza, Z; Grzeszczak, W; Trautsolt, W; Moczulski, D

2012-01-01

Autoimmune mechanisms play an important role in the pathophysiology of chronic urticaria (CU), and the autologous serum skin test (ASST) helps to identify patients with autoreactive CU. One of the factors involved in autoreactive mechanisms is the cell surface receptor programmed death-1 which is encoded by the programmed cell death 1 gene (PDCD1). To investigate whether PDCD1 polymorphisms influence susceptibility to CU. We enrolled 93 ASST-positive patients with CU and a control group consisting of 105 healthy volunteers. In all individuals, PD1.3 (7146 A/G; rs 11568821) and PD1.5 (7785 C/T; rs 2227981) polymorphisms were analyzed. No statistically significant differences were found between CU patients and controls for allele or genotype distribution. We also did not observe any association between PDCD1 genotypes and severity of urticaria or age of disease onset. PD1.3 and PD1.5 polymorphisms were not proven to be implicated in susceptibility to ASST-positive CU in the Polish population. A more comprehensive analysis of the 2q33-2q37 genomic region might reveal whether variants of 1 or more of the genes in this region are involved in susceptibility to CU.

A common biological mechanism in cancer and Alzheimer’s disease?

PubMed Central

Behrens, Maria I; Lendon, Corinne; Roe, Catherine M.

2009-01-01

Cancer and Alzheimer’s disease (AD) are two common disorders for which the final pathophysiological mechanism is not yet clearly defined. In a prospective longitudinal study we have previously shown an inverse association between AD and cancer, such that the rate of developing cancer in general with time was significantly slower in participants with AD, while participants with a history of cancer had a slower rate of developing AD. In cancer, cell regulation mechanisms are disrupted with augmentation of cell survival and/or proliferation, whereas conversely, AD is associated with increased neuronal death, either caused by, or concomitant with, beta amyloid (Aβ) and tau deposition. The possibility that perturbations of mechanisms involved in cell survival/death regulation could be involved in both disorders is discussed. Genetic polymorphisms, DNA methylation or other mechanisms that induce changes in activity of molecules with key roles in determining the decision to “repair and live”- or “die” could be involved in the pathogenesis of the two disorders. As examples, the role of p53, Pin1 and the Wnt signaling pathway are discussed as potential candidates that, speculatively, may explain inverse associations between AD and cancer. PMID:19519301

Differential effect of grape seed extract against human non-small-cell lung cancer cells: the role of reactive oxygen species and apoptosis induction.

PubMed

Tyagi, Alpna; Raina, Komal; Gangar, Subhash; Kaur, Manjinder; Agarwal, Rajesh; Agarwal, Chapla

2013-01-01

The present study examines grape seed extract (GSE) efficacy against a series of non-small-cell lung cancer (NSCLC) cell lines that differ in their Kras and p53 status to establish GSE potential as a cytotoxic agent against a wide range of lung cancer cells. GSE suppressed growth and induced apoptotic death in NSCLC cells irrespective of their k-Ras status, with more sensitivity toward H460 and H322 (wt k-Ras) than A549 and H1299 cells (mutated k-Ras). Mechanistic studies in A549 and H460 cells, selected, based on comparative efficacy of GSE at higher and lower doses, respectively, showed that apoptotic death involves cytochrome c release associated caspases 9 and 3 activation, and poly (ADP-ribosyl) polymerase cleavage, strong phosphorylation of ERK1/2 and JNK1/2, downregulation of cell survival proteins, and upregulated proapoptotic Bak expression. Importantly, GSE treatment caused a strong superoxide radical-associated oxidative stress, significantly decreased intracellular reduced glutathione levels, suggesting, for the first time, the involvement of GSE-caused oxidative stress in its apoptotic inducing activity in these cells. Because GSE is a widely-consumed dietary agent with no known untoward effects, our results support future studies to establish GSE efficacy and usefulness against NSCLC control.

Metabolic effects of photodynamically induced apoptosis in an erythroleukemic cell line. A (31)P NMR spectroscopic study of Victoria-Blue-BO-sensitized TF-1 cells.

PubMed

Viola, A; Lutz, N W; Maroc, C; Chabannon, C; Julliard, M; Cozzone, P J

2000-03-01

Victoria Blue BO (VB BO) is a new and promising photosensitizer currently being evaluated for photodynamic therapy (PDT). Its photochemical processes are mediated by oxygen radicals, but do not involve singlet oxygen. We used (31)P NMR spectroscopy of VB-BO sensitized TF-1 leukemic cells to gain further insight into the biochemical mechanisms underlying PDT-induced cell death. Sham-treatment experiments were performed to evaluate the effects of this photosensitizer in the absence of light irradiation. Significant metabolic differences were detected for TF-1 cells incubated with VB BO but not exposed to light, as compared with native cells (controls). These changes include reductions in phosphocreatine, UDP-hexose and phosphodiester levels (as percentage of total phosphate) and slightly reduced intracellular pH. Complete phosphocreatine depletion, significant acidification and concomitant inorganic-phosphate accumulation were observed for TF-1 cells irradiated after incubation with VB BO. Moreover, significant changes in phospholipid metabolites, i.e., accumulation of cytidine 5'-diphosphate choline and a decrease in phosphodiester levels, were observed for PDT-treated vs. sham-treated cells. Perturbations of phospholipid metabolism may be involved in programmed cell death, and the detection of a characteristic DNA ladder pattern by gel electrophoresis confirmed the existence of apoptosis in PDT-treated TF-1 cells. Copyright 2000 Wiley-Liss, Inc.

HYAL-2–WWOX–SMAD4 Signaling in Cell Death and Anticancer Response

PubMed Central

Hsu, Li-Jin; Chiang, Ming-Fu; Sze, Chun-I; Su, Wan-Pei; Yap, Ye Vone; Lee, I-Ting; Kuo, Hsiang-Ling; Chang, Nan-Shan

2016-01-01

Hyaluronidase HYAL-2 is a membrane-anchored protein and also localizes, in part, in the lysosome. Recent study from animal models revealed that both HYAL-1 and HYAL-2 are essential for the metabolism of hyaluronan (HA). Hyal-2 deficiency is associated with chronic thrombotic microangiopathy with hemolytic anemia in mice due to over accumulation of high molecular size HA. HYAL-2 is essential for platelet generation. Membrane HYAL-2 degrades HA bound by co-receptor CD44. Also, in a non-canonical signal pathway, HYAL-2 serves as a receptor for transforming growth factor beta (TGF-β) to signal with downstream tumor suppressors WWOX and SMAD4 to control gene transcription. When SMAD4 responsive element is overly driven by the HYAL-2–WWOX–SMAD4 signaling complex, cell death occurs. When rats are subjected to traumatic brain injury, over accumulation of a HYAL-2–WWOX complex occurs in the nucleus to cause neuronal death. HA induces the signaling of HYAL-2–WWOX–SMAD4 and relocation of the signaling complex to the nucleus. If the signaling complex is overexpressed, bubbling cell death occurs in WWOX-expressing cells. In addition, a small synthetic peptide Zfra (zinc finger-like protein that regulates apoptosis) binds membrane HYAL-2 of non-T/non-B spleen HYAL-2+ CD3− CD19− Z lymphocytes and activates the cells to generate memory anticancer response against many types of cancer cells in vivo. Whether the HYAL-2–WWOX–SMAD4 signaling complex is involved is discussed. In this review and opinion article, we have updated the current knowledge of HA, HYAL-2 and WWOX, HYAL-2–WWOX–SMAD4 signaling, bubbling cell death, and Z cell activation for memory anticancer response. PMID:27999774

Role of Hsp-70 in triptolide-mediated cell death of neuroblastoma.

PubMed

Antonoff, Mara B; Chugh, Rohit; Skube, Steven J; Dudeja, Vikas; Borja-Cacho, Daniel; Clawson, Kimberly A; Vickers, Selwyn M; Saluja, Ashok K

2010-09-01

Our recent work demonstrated that treatment of neuroblastoma with triptolide causes apoptotic cell death in vitro and decreases tumor size in vivo. Triptolide therapy has been associated with reduced expression of Hsp-70, suggesting a mechanism of cell killing involving Hsp-70 inhibition. The principal objective of this study was to investigate the role of Hsp-70 in triptolide-mediated cell death in neuroblastoma. Neuroblastoma cells were transfected with Hsp-70-specific siRNA. Viability, caspase activity, and phosphatidylserine externalization were subsequently measured. An orthotopic, syngeneic murine tumor model was developed, and randomized mice received daily injections of triptolide or vehicle. At 21 d, mice were sacrificed. Immunohistochemisty was used to characterize Hsp-70 levels in residual tumors, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed to identify cells undergoing apoptosis. Targeted silencing of Hsp-70 with siRNA significantly decreased cellular viability, augmented caspase-3 activity, and resulted in increased annexin-V staining. These effects parallel those findings obtained following treatment with triptolide. Residual tumors from triptolide-treated mice showed minimal staining with Hsp-70 immunohistochemistry, while control tumors stained prominently. Tumors from treated mice demonstrated marked staining with the TUNEL assay, while control tumors showed no evidence of apoptosis. Use of siRNA to suppress Hsp-70 expression in neuroblastoma resulted in apoptotic cell death, similar to the effects of triptolide. Residual tumors from triptolide-treated mice expressed decreased levels of Hsp-70 and demonstrated significant apoptosis. These findings support the hypothesis that Hsp-70 inhibition plays a significant role in triptolide-mediated neuroblastoma cell death. Copyright 2010 Elsevier Inc. All rights reserved.

Cellular Stress and p53-Associated Apoptosis by Juniperus communis L. Berry Extract Treatment in the Human SH-SY5Y Neuroblastoma Cells

PubMed Central

Lantto, Tiina A.; Laakso, Into; Dorman, H. J. Damien; Mauriala, Timo; Hiltunen, Raimo; Kõks, Sulev; Raasmaja, Atso

2016-01-01

Plant phenolics have shown to activate apoptotic cell death in different tumourigenic cell lines. In this study, we evaluated the effects of juniper berry extract (Juniperus communis L.) on p53 protein, gene expression and DNA fragmentation in human neuroblastoma SH-SY5Y cells. In addition, we analyzed the phenolic composition of the extract. We found that juniper berry extract activated cellular relocalization of p53 and DNA fragmentation-dependent cell death. Differentially expressed genes between treated and non-treated cells were evaluated with the cDNA-RDA (representational difference analysis) method at the early time point of apoptotic process when p53 started to be activated and no caspase activity was detected. Twenty one overexpressed genes related to cellular stress, protein synthesis, cell survival and death were detected. Interestingly, they included endoplasmic reticulum (ER) stress inducer and sensor HSPA5 and other ER stress-related genes CALM2 and YKT6 indicating that ER stress response was involved in juniper berry extract mediated cell death. In composition analysis, we identified and quantified low concentrations of fifteen phenolic compounds. The main groups of them were flavones, flavonols, phenolic acids, flavanol and biflavonoid including glycosides of quercetin, apigenin, isoscutellarein and hypolaetin. It is suggested that juniper berry extract induced the p53-associated apoptosis through the potentiation and synergism by several phenolic compounds. PMID:27420050

Targeting Cancer Cells with Reactive Oxygen and Nitrogen Species Generated by Atmospheric-Pressure Air Plasma

PubMed Central

Hoan, Nguyen Ngoc; Kim, Churl Ho; Moon, Eunpyo; Choi, Kyeong Sook; Yang, Sang Sik; Lee, Jong-Soo

2014-01-01

The plasma jet has been proposed as a novel therapeutic method for cancer. Anticancer activity of plasma has been reported to involve mitochondrial dysfunction. However, what constituents generated by plasma is linked to this anticancer process and its mechanism of action remain unclear. Here, we report that the therapeutic effects of air plasma result from generation of reactive oxygen/nitrogen species (ROS/RNS) including H2O2, Ox, OH−, •O2, NOx, leading to depolarization of mitochondrial membrane potential and mitochondrial ROS accumulation. Simultaneously, ROS/RNS activate c-Jun NH2-terminal kinase (JNK) and p38 kinase. As a consequence, treatment with air plasma jets induces apoptotic death in human cervical cancer HeLa cells. Pretreatment of the cells with antioxidants, JNK and p38 inhibitors, or JNK and p38 siRNA abrogates the depolarization of mitochondrial membrane potential and impairs the air plasma-induced apoptotic cell death, suggesting that the ROS/RNS generated by plasma trigger signaling pathways involving JNK and p38 and promote mitochondrial perturbation, leading to apoptosis. Therefore, administration of air plasma may be a feasible strategy to eliminate cancer cells. PMID:24465942

Increased oxidative stress and apoptosis in the hypothalamus of diabetic male mice in the insulin receptor substrate-2 knockout model

PubMed Central

Canelles, Sandra; Argente, Jesús; Barrios, Vicente

2016-01-01

ABSTRACT Insulin receptor substrate-2-deficient (IRS2−/−) mice are considered a good model to study the development of diabetes because IRS proteins mediate the pleiotropic effects of insulin-like growth factor-I (IGF-I) and insulin on metabolism, mitogenesis and cell survival. The hypothalamus might play a key role in the early onset of diabetes, owing to its involvement in the control of glucose homeostasis and energy balance. Because some inflammatory markers are elevated in the hypothalamus of diabetic IRS2−/− mice, our aim was to analyze whether the diabetes associated with the absence of IRS2 results in hypothalamic injury and to analyze the intracellular mechanisms involved. Only diabetic IRS2−/− mice showed increased cell death and activation of caspase-8 and -3 in the hypothalamus. Regulators of apoptosis such as FADD, Bcl-2, Bcl-xL and p53 were also increased, whereas p-IκB and c-FLIPL were decreased. This was accompanied by increased levels of Nox-4 and catalase, enzymes involved in oxidative stress. In summary, the hypothalamus of diabetic IRS2−/− mice showed an increase in oxidative stress and inflammatory markers that finally resulted in cell death via substantial activation of the extrinsic apoptotic pathway. Conversely, non-diabetic IRS2−/− mice did not show cell death in the hypothalamus, possibly owing to an increase in the levels of circulating IGF-I and in the enhanced hypothalamic IGF-IR phosphorylation that would lead to the stimulation of survival pathways. In conclusion, diabetes in IRS2-deficient male mice is associated with increased oxidative stress and apoptosis in the hypothalamus. PMID:27013528

Increased oxidative stress and apoptosis in the hypothalamus of diabetic male mice in the insulin receptor substrate-2 knockout model.

PubMed

Baquedano, Eva; Burgos-Ramos, Emma; Canelles, Sandra; González-Rodríguez, Agueda; Chowen, Julie A; Argente, Jesús; Barrios, Vicente; Valverde, Angela M; Frago, Laura M

2016-05-01

Insulin receptor substrate-2-deficient (IRS2(-/-)) mice are considered a good model to study the development of diabetes because IRS proteins mediate the pleiotropic effects of insulin-like growth factor-I (IGF-I) and insulin on metabolism, mitogenesis and cell survival. The hypothalamus might play a key role in the early onset of diabetes, owing to its involvement in the control of glucose homeostasis and energy balance. Because some inflammatory markers are elevated in the hypothalamus of diabetic IRS2(-/-) mice, our aim was to analyze whether the diabetes associated with the absence of IRS2 results in hypothalamic injury and to analyze the intracellular mechanisms involved. Only diabetic IRS2(-/-) mice showed increased cell death and activation of caspase-8 and -3 in the hypothalamus. Regulators of apoptosis such as FADD, Bcl-2, Bcl-xL and p53 were also increased, whereas p-IκB and c-FLIPL were decreased. This was accompanied by increased levels of Nox-4 and catalase, enzymes involved in oxidative stress. In summary, the hypothalamus of diabetic IRS2(-/-) mice showed an increase in oxidative stress and inflammatory markers that finally resulted in cell death via substantial activation of the extrinsic apoptotic pathway. Conversely, non-diabetic IRS2(-/-) mice did not show cell death in the hypothalamus, possibly owing to an increase in the levels of circulating IGF-I and in the enhanced hypothalamic IGF-IR phosphorylation that would lead to the stimulation of survival pathways. In conclusion, diabetes in IRS2-deficient male mice is associated with increased oxidative stress and apoptosis in the hypothalamus. © 2016. Published by The Company of Biologists Ltd.

Glucose Metabolism and AMPK Signaling Regulate Dopaminergic Cell Death Induced by Gene (α-Synuclein)-Environment (Paraquat) Interactions.

PubMed

Anandhan, Annadurai; Lei, Shulei; Levytskyy, Roman; Pappa, Aglaia; Panayiotidis, Mihalis I; Cerny, Ronald L; Khalimonchuk, Oleh; Powers, Robert; Franco, Rodrigo

2017-07-01

While environmental exposures are not the single cause of Parkinson's disease (PD), their interaction with genetic alterations is thought to contribute to neuronal dopaminergic degeneration. However, the mechanisms involved in dopaminergic cell death induced by gene-environment interactions remain unclear. In this work, we have revealed for the first time the role of central carbon metabolism and metabolic dysfunction in dopaminergic cell death induced by the paraquat (PQ)-α-synuclein interaction. The toxicity of PQ in dopaminergic N27 cells was significantly reduced by glucose deprivation, inhibition of hexokinase with 2-deoxy-D-glucose (2-DG), or equimolar substitution of glucose with galactose, which evidenced the contribution of glucose metabolism to PQ-induced cell death. PQ also stimulated an increase in glucose uptake, and in the levels of glucose transporter type 4 (GLUT4) and Na + -glucose transporters isoform 1 (SGLT1) proteins, but only inhibition of GLUT-like transport with STF-31 or ascorbic acid reduced PQ-induced cell death. Importantly, while autophagy protein 5 (ATG5)/unc-51 like autophagy activating kinase 1 (ULK1)-dependent autophagy protected against PQ toxicity, the inhibitory effect of glucose deprivation on cell death progression was largely independent of autophagy or mammalian target of rapamycin (mTOR) signaling. PQ selectively induced metabolomic alterations and adenosine monophosphate-activated protein kinase (AMPK) activation in the midbrain and striatum of mice chronically treated with PQ. Inhibition of AMPK signaling led to metabolic dysfunction and an enhanced sensitivity of dopaminergic cells to PQ. In addition, activation of AMPK by PQ was prevented by inhibition of the inducible nitric oxide syntase (iNOS) with 1400W, but PQ had no effect on iNOS levels. Overexpression of wild type or A53T mutant α-synuclein stimulated glucose accumulation and PQ toxicity, and this toxic synergism was reduced by inhibition of glucose metabolism/transport and the pentose phosphate pathway (6-aminonicotinamide). These results demonstrate that glucose metabolism and AMPK regulate dopaminergic cell death induced by gene (α-synuclein)-environment (PQ) interactions.

Cellular recovery from exposure to sub-optimal concentrations of AB toxins that inhibit protein synthesis.

PubMed

Cherubin, Patrick; Quiñones, Beatriz; Teter, Ken

2018-02-06

Ricin, Shiga toxin, exotoxin A, and diphtheria toxin are AB-type protein toxins that act within the host cytosol and kill the host cell through pathways involving the inhibition of protein synthesis. It is thought that a single molecule of cytosolic toxin is sufficient to kill the host cell. Intoxication is therefore viewed as an irreversible process. Using flow cytometry and a fluorescent reporter system to monitor protein synthesis, we show a single molecule of cytosolic toxin is not sufficient for complete inhibition of protein synthesis or cell death. Furthermore, cells can recover from intoxication: cells with a partial loss of protein synthesis will, upon removal of the toxin, increase the level of protein production and survive the toxin challenge. Thus, in contrast to the prevailing model, ongoing toxin delivery to the cytosol appears to be required for the death of cells exposed to sub-optimal toxin concentrations.

Analysis of epothilone B-induced cell death in normal ovarian cells.

PubMed

Rogalska, Aneta; Gajek, Arkadiusz; Marczak, Agnieszka

2013-12-01

We have investigated the mode of cell death induced by a new microtubule-stabilizing agent, epothilone B (EpoB, patupilone), and a clinically used medicine, paclitaxel (PTX), in normal ovarian cells. Using fluorescence microscopy, polyacrylamide gel electrophoresis preceding Western blot analysis, as well as spectrofluorimetric and colorimetric detection, we demonstrate that, compared to EpoB, PTX induced high time-dependent morphological and biochemical changes typical of apoptosis. Induction of apoptosis followed an early increase in p53 levels. Apoptosis reached its maximum at 24-48 h. At the same time, there was a significant increase in caspase-9 and -3 activity and PARP fragmentation, which suggests that an intrinsic path was involved. Apoptosis in MM14 cells was increased more by PTX than EpoB, and also induced more necrosis responsible for inflammation (1.4-fold) than EpoB. © 2013 International Federation for Cell Biology.

Bluetongue virus infection alters the impedance of monolayers of bovine endothelial cells as a result of cell death.

PubMed

Drew, Clifton P; Gardner, Ian A; Mayo, Christie E; Matsuo, Eiko; Roy, Polly; MacLachlan, N James

2010-07-01

Bluetongue virus (BTV) is the cause of bluetongue, an emerging, arthropod-transmitted disease of ungulates. Bluetongue is characterized by vascular injury with hemorrhage, tissue infarction and widespread edema, lesions that are consistent with those of the so-called viral hemorrhagic fevers. To further investigate the pathogenesis of vascular injury in bluetongue, we utilized an electrical impedance assay and immunofluorescence staining to compare the effects of BTV infection on cultured bovine endothelial cells (bPAEC) with those of inducers of cell death (Triton X-100) and interendothelial gap formation (tissue necrosis factor [TNF]). The data confirm that the adherens junctions of BTV-infected bPAECs remained intact until 24h post-infection, and that loss of monolayer impedance precisely coincided with onset of virus-induced cell death. In contrast, recombinant bovine TNF-alpha caused rapid loss of bPAEC monolayer impedance that was associated with interendothelial gap formation and redistribution of VE-cadherin, but without early cell death. The data from these in vitro studies are consistent with a pathogenesis of bluetongue that involves virus-induced vascular injury leading to thrombosis, hemorrhage and tissue necrosis. However, the contribution of cytokine-induced interendothelial gap formation with subsequent edema and hypovolemic shock contributes to the pathogenesis of bluetongue remains to be fully characterized. Copyright 2010 Elsevier B.V. All rights reserved.

Fluoxetine induces autophagic cell death via eEF2K-AMPK-mTOR-ULK complex axis in triple negative breast cancer.

PubMed

Sun, Dejuan; Zhu, Lingjuan; Zhao, Yuqian; Jiang, Yingnan; Chen, Lixia; Yu, Yang; Ouyang, Liang

2018-04-01

Triple negative breast cancer (TNBC) is a complex and intrinsically aggressive tumour with poor prognosis, and the discovery of targeted small-molecule drugs for TNBC treatment still remains in its infancy. In this study, we aimed to discover a small-molecule agent for TNBC treatment and illuminate its potential mechanisms. Cell viability was detected by using methylthiazoltetrazolium (MTT) assay. Electron microscopy, GFP-LC3 transfection, monodansylcadaverine staining and apoptosis assay were performed to determine Fluoxetine-induced autophagy and apoptosis. Western blotting and siRNA transfection were carried out to investigate the mechanisms of Fluoxetine-induced autophagy. iTRAQ-based proteomics analysis was used to explore the underlying mechanisms. We have demonstrated that Fluoxetine had remarkable anti-proliferative activities and induced autophagic cell death in MDA-MB-231 and MDA-MB-436 cells. The mechanism for Fluoxetine-induced autophagic cell death was associated with inhibition of eEF2K and activation of AMPK-mTOR-ULK complex axis. Further iTRAQ-based proteomics and network analyses revealed that Fluoxetine-induced mechanism was involved in BIRC6, BNIP1, SNAP29 and Bif-1. These results demonstrate that Fluoxetine induces apoptosis and autophagic cell death in TNBC, which will hold a promise for the future TNBC therapy. © 2017 John Wiley & Sons Ltd.

The Enemy within: Innate Surveillance-Mediated Cell Death, the Common Mechanism of Neurodegenerative Disease

PubMed Central

Richards, Robert I.; Robertson, Sarah A.; O'Keefe, Louise V.; Fornarino, Dani; Scott, Andrew; Lardelli, Michael; Baune, Bernhard T.

2016-01-01

Neurodegenerative diseases comprise an array of progressive neurological disorders all characterized by the selective death of neurons in the central nervous system. Although, rare (familial) and common (sporadic) forms can occur for the same disease, it is unclear whether this reflects several distinct pathogenic pathways or the convergence of different causes into a common form of nerve cell death. Remarkably, neurodegenerative diseases are increasingly found to be accompanied by activation of the innate immune surveillance system normally associated with pathogen recognition and response. Innate surveillance is the cell's quality control system for the purpose of detecting such danger signals and responding in an appropriate manner. Innate surveillance is an “intelligent system,” in that the manner of response is relevant to the magnitude and duration of the threat. If possible, the threat is dealt with within the cell in which it is detected, by degrading the danger signal(s) and restoring homeostasis. If this is not successful then an inflammatory response is instigated that is aimed at restricting the spread of the threat by elevating degradative pathways, sensitizing neighboring cells, and recruiting specialized cell types to the site. If the danger signal persists, then the ultimate response can include not only the programmed cell death of the original cell, but the contents of this dead cell can also bring about the death of adjacent sensitized cells. These responses are clearly aimed at destroying the ability of the detected pathogen to propagate and spread. Innate surveillance comprises intracellular, extracellular, non-cell autonomous and systemic processes. Recent studies have revealed how multiple steps in these processes involve proteins that, through their mutation, have been linked to many familial forms of neurodegenerative disease. This suggests that individuals harboring these mutations may have an amplified response to innate-mediated damage in neural tissues, and renders innate surveillance mediated cell death a plausible common pathogenic pathway responsible for neurodegenerative diseases, in both familial and sporadic forms. Here we have assembled evidence in favor of the hypothesis that neurodegenerative disease is the cumulative result of chronic activation of the innate surveillance pathway, triggered by endogenous or environmental danger or damage associated molecular patterns in a progressively expanding cascade of inflammation, tissue damage and cell death. PMID:27242399

Substance P Receptor Signaling Mediates Doxorubicin-Induced Cardiomyocyte Apoptosis and Triple-Negative Breast Cancer Chemoresistance

PubMed Central

Robinson, Prema; Kasembeli, Moses; Bharadwaj, Uddalak; Engineer, Nikita; Eckols, Kris T.; Tweardy, David J.

2016-01-01

Doxorubicin (DOX), an anthracycline, is broadly considered the most active single agent available for treating breast cancer but has been known to induce cardiotoxicity. Although DOX is highly effective in treating triple-negative breast cancer (TNBC), DOX can have poor outcomes owing to induction of chemoresistance. There is an urgent need to develop new therapies for TNBC aimed at improving DOX outcome and DOX-induced cardiotoxicity. Substance P (SP), a neuropeptide involved in pain transmission is known to stimulate production of reactive oxygen species (ROS). Elevated cardiac ROS is linked with heart injury and failure. We investigated the role of SP in chemotherapy-associated death of cardiomyocytes and chemoresistance. We showed that pretreating a cardiomyocyte cell line (H9C2) and a TNBC cell line (MDA-MB 231) with aprepitant, a SP receptor antagonist that is routinely used to treat chemotherapy-associated associated nausea, decreased DOX-induced reduction of cell viability, apoptotic cell death, and ROS production in cardiomyocytes and increased DOX-induced reduction of cell viability, apoptotic cell death, and ROS production in TNBC cells compared with cells treated with DOX alone. Our findings demonstrate the ability of aprepitant to decrease DOX-induced killing of cardiomyocytes and to increase cancer cell sensitivity to DOX, which has tremendous clinical significance. PMID:26981525

Genome-wide analysis of autophagy-related genes in banana highlights MaATG8s in cell death and autophagy in immune response to Fusarium wilt.

PubMed

Wei, Yunxie; Liu, Wen; Hu, Wei; Liu, Guoyin; Wu, Chunjie; Liu, Wei; Zeng, Hongqiu; He, Chaozu; Shi, Haitao

2017-08-01

MaATG8s play important roles in hypersensitive-like cell death and immune response, and autophagy is essential for disease resistance against Foc in banana. Autophagy is responsible for the degradation of damaged cytoplasmic constituents in the lysosomes or vacuoles. Although the effects of autophagy have been extensively revealed in model plants, the possible roles of autophagy-related gene in banana remain unknown. In this study, 32 MaATGs were identified in the draft genome, and the profiles of several MaATGs in response to fungal pathogen Fusarium oxysporum f. sp. cubense (Foc) were also revealled. We found that seven MaATG8s were commonly regulated by Foc. Through transient expression in Nicotiana benthamiana leaves, we highlight the novel roles of MaATG8s in conferring hypersensitive-like cell death, and MaATG8s-mediated hypersensitive response-like cell death is dependent on autophagy. Notablly, autophagy inhibitor 3-methyladenine (3-MA) treatment resulted in decreased disease resistance in response to Foc4, and the effect of 3-MA treatment could be rescued by exogenous salicylic acid, jasmonic acid and ethylene, indicating the involvement of autophagy-mediated plant hormones in banana resistance to Fusarium wilt. Taken together, this study may extend our understanding the putative role of MaATG8s in hypersensitive-like cell death and the essential role of autophagy in immune response against Foc in banana.

An S-Type Anion Channel SLAC1 Is Involved in Cryptogein-Induced Ion Fluxes and Modulates Hypersensitive Responses in Tobacco BY-2 Cells

PubMed Central

Horikoshi, Sonoko; Hanamata, Shigeru; Negi, Juntaro; Yagi, Chikako; Kitahata, Nobutaka; Iba, Koh; Kuchitsu, Kazuyuki

2013-01-01

Pharmacological evidence suggests that anion channel-mediated plasma membrane anion effluxes are crucial in early defense signaling to induce immune responses and hypersensitive cell death in plants. However, their molecular bases and regulation remain largely unknown. We overexpressed Arabidopsis SLAC1, an S-type anion channel involved in stomatal closure, in cultured tobacco BY-2 cells and analyzed the effect on cryptogein-induced defense responses including fluxes of Cl− and other ions, production of reactive oxygen species (ROS), gene expression and hypersensitive responses. The SLAC1-GFP fusion protein was localized at the plasma membrane in BY-2 cells. Overexpression of SLAC1 enhanced cryptogein-induced Cl− efflux and extracellular alkalinization as well as rapid/transient and slow/prolonged phases of NADPH oxidase-mediated ROS production, which was suppressed by an anion channel inhibitor, DIDS. The overexpressor also showed enhanced sensitivity to cryptogein to induce downstream immune responses, including the induction of defense marker genes and the hypersensitive cell death. These results suggest that SLAC1 expressed in BY-2 cells mediates cryptogein-induced plasma membrane Cl− efflux to positively modulate the elicitor-triggered activation of other ion fluxes, ROS as well as a wide range of defense signaling pathways. These findings shed light on the possible involvement of the SLAC/SLAH family anion channels in cryptogein signaling to trigger the plasma membrane ion channel cascade in the plant defense signal transduction network. PMID:23950973

An S-type anion channel SLAC1 is involved in cryptogein-induced ion fluxes and modulates hypersensitive responses in tobacco BY-2 cells.

PubMed

Kurusu, Takamitsu; Saito, Katsunori; Horikoshi, Sonoko; Hanamata, Shigeru; Negi, Juntaro; Yagi, Chikako; Kitahata, Nobutaka; Iba, Koh; Kuchitsu, Kazuyuki

2013-01-01

Pharmacological evidence suggests that anion channel-mediated plasma membrane anion effluxes are crucial in early defense signaling to induce immune responses and hypersensitive cell death in plants. However, their molecular bases and regulation remain largely unknown. We overexpressed Arabidopsis SLAC1, an S-type anion channel involved in stomatal closure, in cultured tobacco BY-2 cells and analyzed the effect on cryptogein-induced defense responses including fluxes of Cl(-) and other ions, production of reactive oxygen species (ROS), gene expression and hypersensitive responses. The SLAC1-GFP fusion protein was localized at the plasma membrane in BY-2 cells. Overexpression of SLAC1 enhanced cryptogein-induced Cl(-) efflux and extracellular alkalinization as well as rapid/transient and slow/prolonged phases of NADPH oxidase-mediated ROS production, which was suppressed by an anion channel inhibitor, DIDS. The overexpressor also showed enhanced sensitivity to cryptogein to induce downstream immune responses, including the induction of defense marker genes and the hypersensitive cell death. These results suggest that SLAC1 expressed in BY-2 cells mediates cryptogein-induced plasma membrane Cl(-) efflux to positively modulate the elicitor-triggered activation of other ion fluxes, ROS as well as a wide range of defense signaling pathways. These findings shed light on the possible involvement of the SLAC/SLAH family anion channels in cryptogein signaling to trigger the plasma membrane ion channel cascade in the plant defense signal transduction network.

Neuroprotection by GH against excitotoxic-induced cell death in retinal ganglion cells.

PubMed

Martínez-Moreno, Carlos G; Ávila-Mendoza, José; Wu, Yilun; Arellanes-Licea, Elvira Del Carmen; Louie, Marcela; Luna, Maricela; Arámburo, Carlos; Harvey, Steve

2016-08-01

Retinal growth hormone (GH) has been shown to promote cell survival in retinal ganglion cells (RGCs) during developmental waves of apoptosis during chicken embryonic development. The possibility that it might also against excitotoxicity-induced cell death was therefore examined in the present study, which utilized quail-derived QNR/D cells as an in vitro RGC model. QNR/D cell death was induced by glutamate in the presence of BSO (buthionine sulfoxamide) (an enhancer of oxidative stress), but this was significantly reduced (P<0.01) in the presence of exogenous recombinant chicken GH (rcGH). Similarly, QNR/D cells that had been prior transfected with a GH plasmid to overexpress secreted and non-secreted GH. This treatment reduced the number of TUNEL-labeled cells and blocked their release of lactate dehydrogenase (LDH). In a further experiment with dissected neuroretinal explants from ED (embryonic day) 10 embryos, rcGH treatment of the explants also reduced (P<0.01) the number of glutamate-BSO-induced apoptotic cells and blocked the explant release of LDH. This neuroprotective action was likely mediated by increased STAT5 phosphorylation and increased bcl-2 production, as induced by exogenous rcGH treatment and the media from GH-overexpressing QNR/D cells. As rcGH treatment and GH-overexpression cells also increased the content of IGF-1 and IGF-1 mRNA this neuroprotective action of GH is likely to be mediated, at least partially, through an IGF-1 mechanism. This possibility is supported by the fact that the siRNA knockdown of GH or IGF-1 significantly reduced QNR/D cell viability, as did the immunoneutralization of IGF-1. GH is therefore neuroprotective against excitotoxicity-induced RGC cell death by anti-apoptotic actions involving IGF-1 stimulation. Copyright © 2016 Elsevier Inc. All rights reserved.

Tocotrienols promote apoptosis in human breast cancer cells by inducing poly(ADP-ribose) polymerase cleavage and inhibiting nuclear factor kappa-B activity.

PubMed

Loganathan, R; Selvaduray, K R; Nesaretnam, K; Radhakrishnan, A K

2013-04-01

Tocotrienols and tocopherols are members of the vitamin E family, with similar structures; however, only tocotrienols have been reported to achieve potent anti-cancer effects. The study described here has evaluated anti-cancer activity of vitamin E to elucidate mechanisms of cell death, using human breast cancer cells. Anti-cancer activity of a tocotrienol-rich fraction (TRF) and a tocotrienol-enriched fraction (TEF) isolated from palm oil, as well as pure vitamin E analogues (α-tocopherol, α-, δ- and γ-tocotrienols) were studied using highly aggressive triple negative MDA-MB-231 cells and oestrogen-dependent MCF-7 cells, both of human breast cancer cell lines. Cell population growth was evaluated using a Coulter particle counter. Cell death mechanism, poly(ADP-ribose) polymerase cleavage and levels of NF-κB were determined using commercial ELISA kits. Tocotrienols exerted potent anti-proliferative effects on both types of cell by inducing apoptosis, the underlying mechanism of cell death being ascertained using respective IC50 concentrations of all test compounds. There was marked induction of apoptosis in both cell lines by tocotrienols compared to treatment with Paclitaxel, which was used as positive control. This activity was found to be associated with cleavage of poly(ADP-ribose) polymerase (a DNA repair protein), demonstrating involvement of the apoptotic cell death signalling pathway. Tocotrienols also inhibited expression of nuclear factor kappa-B (NF-κB), which in turn can increase sensitivity of cancer cells to apoptosis. Tocotrienols induced anti-proliferative and apoptotic effects in association with DNA fragmentation, poly(ADP-ribose) polymerase cleavage and NF-κB inhibition in the two human breast cancer cell lines. © 2013 Blackwell Publishing Ltd.

The flavonoid tangeretin activates the unfolded protein response and synergizes with imatinib in the erythroleukemia cell line K562.

PubMed

Lust, Sofie; Vanhoecke, Barbara; Van Gele, Mireille; Philippé, Jan; Bracke, Marc; Offner, Fritz

2010-06-01

We explored the mechanism of cell death of the polymethoxyflavone tangeretin (TAN) in K562 breakpoint cluster region-abelson murine leukemia (Bcr-Abl+) cells. Flow cytometric analysis showed that TAN arrested the cells in the G(2)/M phase and stimulated an accumulation of the cells in the sub-G(0) phase. TAN-induced cell death was evidenced by poly(ADP)-ribose polymerase cleavage, DNA laddering fragmentation, activation of the caspase cascade and downregulation of the antiapoptotic proteins Mcl-1 and Bcl-x(L). Pretreatment with the pancaspase inhibitor Z-VAD-FMK_blocked caspase activation and cell cycle arrest but did not inhibit apoptosis which suggest that other cell killing mechanisms like endoplasmic reticulum (ER)-associated cell death pathways could be involved. We demonstrated that TAN-induced apoptosis was preceded by a rapid activation of the proapoptotic arm of the unfolded protein response, namely PKR-like ER kinase. This was accompanied by enhanced levels of glucose-regulated protein of 78 kDa and of spliced X-box binding protein 1. Furthermore, TAN sensitized K562 cells to the cell killing effects of imatinib via an apoptotic mechanism. In conclusion, our results suggest that TAN is able to induce apoptosis in Bcr-Abl+ cells via cell cycle arrest and the induction of the unfolded protein response, and has synergistic cytotoxicity with imatinib.

In vivo contributions of BH3-only proteins to neuronal death following seizures, ischemia, and traumatic brain injury

PubMed Central

Engel, Tobias; Plesnila, Nikolaus; Prehn, Jochen H M; Henshall, David C

2011-01-01

The Bcl-2 homology (BH) domain 3-only proteins are a proapoptotic subgroup of the Bcl-2 gene family, which regulate cell death via effects on mitochondria. The BH3-only proteins react to various cell stressors and promote cell death by binding and inactivating antiapoptotic Bcl-2 family members and direct activation of proapoptotic multi-BH domain proteins such as Bax. Here, we review the in vivo evidence for their involvement in the pathophysiology of status epilepticus and contrast it to ischemia and traumatic brain injury. Seizures in rodents activate three potent proapoptotic BH3-only proteins: Bid, Bim, and Puma. Analysis of damage after seizures in mice singly deficient for each BH3-only protein supports a causal role for Puma and to a lesser extent Bim but, surprisingly, not Bid. In ischemia and trauma, where core aspects of the pathophysiology of cell death overlap, multiple BH3-only proteins are also activated and Bid has been shown to be required for neuronal death. The findings suggest that while each neurologic insult activates multiple BH3-only proteins, there may be specificity in their functional contribution. Future challenges include evaluating the remaining BH3-only proteins, explaining different causal contributions, and, if possible, exploring neurologic outcomes in mouse models deficient for multiple BH3-only proteins. PMID:21364604

Cell death in response to antimetabolites directed at thymidylate synthase.

PubMed

Barbour, Karen W; Berger, Franklin G

2008-02-01

Thymidylate synthase (TS) is an indispensable enzyme in the de novo biosynthesis of TMP during DNA replication and cell growth, and has, therefore, been an important target for several classes of antimetabolites used in cancer chemotherapy. While most investigations of the action of TS-directed agents have focused on apoptosis as the primary means of cell death, little is known regarding the role, if any, of non-apoptotic mechanisms. In the present study, we have examined the mode of cell death induced by several TS inhibitors. Apoptosis and necrosis in response to TS inhibitors was assessed. The roles of caspases and the transcriptional regulator nuclear factor kappa B (NFkappaB) in drug-induced cell death were analyzed. Finally, drug-mediated changes in expression of several proteins involved in regulation of apoptosis were analyzed. Though human colon tumor cells exposed to TS inhibitors undergo classical apoptosis, it is not the predominant mechanism of response; rather, a necrosis-like mechanism prevails. The apoptotic response to TS inhibitors is caspase-dependent, and is promoted by NFkappaB. In contrast, the necrosis-like response is independent of both caspases and NFkappaB. Exposure to TS inhibitors induces PARP cleavage, but does not alter expression of the pro or activated forms of caspases-3 or caspases-8, Fas, or FasL. Treatment with the death-inducing cytokine TNFalpha, like TS inhibitors, results in a limited extent of apoptosis that is both caspase- and NFkappaB-dependent; however, unlike TS inhibitors, the cytokine does not induce necrosis. Classical apoptosis occurs to a limited extent in human colon tumor cells exposed to TS inhibitors, with caspase-independent necrosis being the prinicipal mechanism of cell death. We suggest that the role of necrosis and necrosis-like mechanisms should be considered in future studies of the action of TS-directed antimetabolites, as well as other chemotherapeutic agents.

Excessive L-cysteine induces vacuole-like cell death by activating endoplasmic reticulum stress and mitogen-activated protein kinase signaling in intestinal porcine epithelial cells.

PubMed

Ji, Yun; Wu, Zhenlong; Dai, Zhaolai; Sun, Kaiji; Zhang, Qing; Wu, Guoyao

2016-01-01

High intake of dietary cysteine is extremely toxic to animals and the underlying mechanism remains largely unknown. This study was conducted to test the hypothesis that excessive L-cysteine induces cell death by activating endoplasmic reticulum (ER) stress and mitogen-activated protein kinase (MAPK) signaling in intestinal porcine epithelial cells. Jejunal enterocytes were cultured in the presence of 0-10 mmol/L L-cysteine. Cell viability, morphologic alterations, mRNA levels for genes involved in ER stress, protein abundances for glucose-regulated protein 78, C/EBP homologous protein (CHOP), alpha subunit of eukaryotic initiation factor-2 (eIF2α), extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal protein kinase (JNK1/2) were determined. The results showed that L-cysteine (5-10 mmol/L) reduced cell viability (P < 0.05) and led to vacuole-like cell death in intestinal porcine epithelial cells. These adverse effects of L-cysteine were not affected by the autophagy inhibitor 3-methyladenine. The protein abundances for CHOP, phosphorylated (p)-eIF2α, p-JNK1/2, p-p38 MAPK, and the spliced form of XBP-1 mRNA were enhanced (P < 0.05), whereas those for p-ERK1/2 were reduced (P < 0.05). Collectively, excessive L-cysteine induces vacuole-like cell death via the activation of ER stress and MAPK signaling in small intestinal epithelial cells. These signaling pathways may be potential targets for developing effective strategies to prevent the toxicity of dietary cysteine.

Activation of the endoplasmic reticulum stress response by the amyloid-beta 1-40 peptide in brain endothelial cells.

PubMed

Fonseca, Ana Catarina R G; Ferreiro, Elisabete; Oliveira, Catarina R; Cardoso, Sandra M; Pereira, Cláudia F

2013-12-01

Neurovascular dysfunction arising from endothelial cell damage is an early pathogenic event that contributes to the neurodegenerative process occurring in Alzheimer's disease (AD). Since the mechanisms underlying endothelial dysfunction are not fully elucidated, this study was aimed to explore the hypothesis that brain endothelial cell death is induced upon the sustained activation of the endoplasmic reticulum (ER) stress response by amyloid-beta (Aβ) peptide, which deposits in the cerebral vessels in many AD patients and transgenic mice. Incubation of rat brain endothelial cells (RBE4 cell line) with Aβ1-40 increased the levels of several markers of ER stress-induced unfolded protein response (UPR), in a time-dependent manner, and affected the Ca(2+) homeostasis due to the release of Ca(2+) from this intracellular store. Finally, Aβ1-40 was shown to activate both mitochondria-dependent and -independent apoptotic cell death pathways. Enhanced release of cytochrome c from mitochondria and activation of the downstream caspase-9 were observed in cells treated with Aβ1-40 concomitantly with caspase-12 activation. Furthermore, Aβ1-40 activated the apoptosis effectors' caspase-3 and promoted the translocation of apoptosis-inducing factor (AIF) to the nucleus demonstrating the involvement of caspase-dependent and -independent mechanisms during Aβ-induced endothelial cell death. In conclusion, our data demonstrate that ER stress plays a significant role in Aβ1-40-induced apoptotic cell death in brain endothelial cells suggesting that ER stress-targeted therapeutic strategies might be useful in AD to counteract vascular defects and ultimately neurodegeneration. © 2013.

Clostridium perfringens beta-toxin induces necrostatin-inhibitable, calpain-dependent necrosis in primary porcine endothelial cells.

PubMed

Autheman, Delphine; Wyder, Marianne; Popoff, Michel; D'Herde, Katharina; Christen, Stephan; Posthaus, Horst

2013-01-01

Clostridium perfringens β-toxin (CPB) is a β-barrel pore-forming toxin and an essential virulence factor of C. perfringens type C strains, which cause fatal hemorrhagic enteritis in animals and humans. We have previously shown that CPB is bound to endothelial cells within the intestine of affected pigs and humans, and that CPB is highly toxic to primary porcine endothelial cells (pEC) in vitro. The objective of the present study was to investigate the type of cell death induced by CPB in these cells, and to study potential host cell mechanisms involved in this process. CPB rapidly induced lactate dehydrogenase (LDH) release, propidium iodide uptake, ATP depletion, potassium efflux, a marked rise in intracellular calcium [Ca(2+)]i, release of high-mobility group protein B1 (HMGB1), and caused ultrastructural changes characteristic of necrotic cell death. Despite a certain level of caspase-3 activation, no appreciable DNA fragmentation was detected. CPB-induced LDH release and propidium iodide uptake were inhibited by necrostatin-1 and the two dissimilar calpain inhibitors PD150606 and calpeptin. Likewise, inhibition of potassium efflux, chelation of intracellular calcium and treatment of pEC with cyclosporin A also significantly inhibited CPB-induced LDH release. Our results demonstrate that rCPB primarily induces necrotic cell death in pEC, and that necrotic cell death is not merely a passive event caused by toxin-induced membrane disruption, but is propagated by host cell-dependent biochemical pathways activated by the rise in intracellular calcium and inhibitable by necrostatin-1, consistent with the emerging concept of programmed necrosis ("necroptosis").

The antidepressant fluoxetine induces necrosis by energy depletion and mitochondrial calcium overload.

PubMed

Charles, Emilie; Hammadi, Mehdi; Kischel, Philippe; Delcroix, Vanessa; Demaurex, Nicolas; Castelbou, Cyril; Vacher, Anne-Marie; Devin, Anne; Ducret, Thomas; Nunes, Paula; Vacher, Pierre

2017-01-10

Selective Serotonin Reuptake Inhibitor antidepressants, such as fluoxetine (Prozac), have been shown to induce cell death in cancer cells, paving the way for their potential use as cancer therapy. These compounds are able to increase cytosolic calcium concentration ([Ca2+]cyt), but the involved mechanisms and their physiological consequences are still not well understood. Here, we show that fluoxetine induces an increase in [Ca2+]cyt by emptying the endoplasmic reticulum (ER) through the translocon, an ER Ca2+ leakage structure. Our data also show that fluoxetine inhibits oxygen consumption and lowers mitochondrial ATP. This latter is essential for Ca2+ reuptake into the ER, and we postulated therefore that the fluoxetine-induced decrease in mitochondrial ATP production results in the emptying of the ER, leading to capacitative calcium entry. Furthermore, Ca2+ quickly accumulated in the mitochondria, leading to mitochondrial Ca2+ overload and cell death. We found that fluoxetine could induce an early necrosis in human peripheral blood lymphocytes and Jurkat cells, and could also induce late apoptosis, especially in the tumor cell line. These results shed light on fluoxetine-induced cell death and its potential use in cancer treatment.

The antidepressant fluoxetine induces necrosis by energy depletion and mitochondrial calcium overload

PubMed Central

Kischel, Philippe; Delcroix, Vanessa; Demaurex, Nicolas; Castelbou, Cyril; Vacher, Anne-Marie; Devin, Anne; Ducret, Thomas; Nunes, Paula; Vacher, Pierre

2017-01-01

Selective Serotonin Reuptake Inhibitor antidepressants, such as fluoxetine (Prozac), have been shown to induce cell death in cancer cells, paving the way for their potential use as cancer therapy. These compounds are able to increase cytosolic calcium concentration ([Ca2+]cyt), but the involved mechanisms and their physiological consequences are still not well understood. Here, we show that fluoxetine induces an increase in [Ca2+]cyt by emptying the endoplasmic reticulum (ER) through the translocon, an ER Ca2+ leakage structure. Our data also show that fluoxetine inhibits oxygen consumption and lowers mitochondrial ATP. This latter is essential for Ca2+ reuptake into the ER, and we postulated therefore that the fluoxetine-induced decrease in mitochondrial ATP production results in the emptying of the ER, leading to capacitative calcium entry. Furthermore, Ca2+ quickly accumulated in the mitochondria, leading to mitochondrial Ca2+ overload and cell death. We found that fluoxetine could induce an early necrosis in human peripheral blood lymphocytes and Jurkat cells, and could also induce late apoptosis, especially in the tumor cell line. These results shed light on fluoxetine-induced cell death and its potential use in cancer treatment. PMID:27911858

Calcium-mediated apoptosis in a plant hypersensitive disease resistance response.

PubMed

Levine, A; Pennell, R I; Alvarez, M E; Palmer, R; Lamb, C

1996-04-01

Avirulent pathogens elicit a battery of plant defenses, often accompanied by collapse of the challenged cells. In soybean cells, sustained accumulation of H2O2 from an oxidative burst cues localized host cell death. Such hypersensitive cell death appears to be an active process, but little is known about the mechanisms underlying cellular collapse. We show that H2O2 stimulates a rapid influx of Ca2+ into soybean cells, which activates a physiological cell death program resulting in the generation of large (approximately 50 kb) DNA fragments and cell corpse morphology--including cell shrinkage, plasma membrane blebbing and nuclear condensation--characteristic of apoptosis. In contrast, H2O2 induction of the cellular protectant gene glutathione S-transferase is Ca(2+)-independent. Apoptosis in soybean cells and leaf tissue was induced by avirulent Pseudomonas syringae pv. glycinea but was not observed at comparable stages of the compatible interaction with the isogenic virulent strain, which fails to elicit a hypersensitive response. Apoptosis was also observed at the onset of the hypersensitive response in Arabidopsis leaves inoculated with avirulent P. syringae pv. tomato and in tobacco cells treated with the fungal peptide cryptogein, which is involved in the induction of non-host resistance to Phytophthora cryptogea. These observations establish a signal function for Ca2+ downstream of the oxidative burst in the activation of a physiological cell death program in soybean cells that is similar to apoptosis in animals. That the characteristic cell corpse morphology is also induced in Arabidopsis and tobacco by different avirulence signals suggests that apoptosis may prove to be a common, but not necessarily ubiquitous, feature of incompatible plant-pathogen interactions. Emerging similarities between facets of hypersensitive disease resistance and the mammalian native immune system indicate that apoptosis is a widespread defence mechanism in eukaryotes.

CD147 is increased in HCC cells under starvation and reduces cell death through upregulating p-mTOR in vitro.

PubMed

Gou, Xingchun; Tang, Xu; Kong, Derek Kai; He, Xinying; Gao, Xingchun; Guo, Na; Hu, Zhifang; Zhao, Zhaohua; Chen, Yanke

2016-01-01

Transarterial chemoembolization (TACE) is the standard of care for treatment of intermediate hepatocellular carcinoma (HCC), however, key molecules involved in HCC cell survival and tumor metastasis post-TACE remain unclear. CD147 is a member of the immunoglobulin superfamily that is overexpressed on the surface of HCC cells and is associated with malignant potential and poor prognosis in HCC patients. In this study, using an Earle's Balanced Salt Solution medium culture model that mimics nutrient deprivation induced by TACE, we investigated the regulation of CD147 expression on HCC cells under starvation conditions and its functional effects on HCC cell death. During early stages of starvation, the expression of CD147 was considerably upregulated in SMMC7721, HepG2 and HCC9204 hepatoma cell lines at the protein levels. Downregulation of CD147 by specific small interfering RNA (siRNA) significantly promoted starvation-induced cell death. In addition, CD147 siRNA-transfected SMMC7721 cells demonstrated significantly increased levels of both apoptosis and autophagy as compared to cells transfected with control siRNA under starvation conditions, whereas no difference was observed between the two treatment groups under normal culture conditions. Furthermore, silencing of CD147 resulted in a remarkable downregulation of phosphorylated mammalian target of rapamycin (p-mTOR) in starved SMMC7721 cells. Finally, the combined treatment of starvation and anti-CD147 monoclonal antibody exhibited a synergistic HCC cell killing effect. Our study suggests that upregulation of CD147 under starvation may reduce hepatoma cell death by modulating both apoptosis and autophagy through mTOR signaling, and that CD147 may be a novel potential molecular target to improve the efficacy of TACE.

Crosstalk between p38, Hsp25 and Akt in spinal motor neurons after sciatic nerve injury

NASA Technical Reports Server (NTRS)

Murashov, A. K.; Ul Haq, I.; Hill, C.; Park, E.; Smith, M.; Wang, X.; Wang, X.; Goldberg, D. J.; Wolgemuth, D. J.

2001-01-01

The p38 stress-activated protein kinase pathway is involved in regulation of phosphorylation of Hsp25, which in turn regulates actin filament dynamic in non-neuronal cells. We report that p38, Hsp25 and Akt signaling pathways were specifically activated in spinal motor neurons after sciatic nerve axotomy. The activation of the p38 kinase was required for induction of Hsp25 expression. Furthermore, Hsp25 formed a complex with Akt, a member of PI-3 kinase pathway that prevents neuronal cell death. Together, our observations implicate Hsp25 as a central player in a complex system of signaling that may both promote regeneration of nerve fibers and prevent neuronal cell death in the injured spinal cord.

Nitrogen Mustard-Induced Corneal Injury Involves DNA Damage and Pathways Related to Inflammation, Epithelial-Stromal Separation, and Neovascularization.

PubMed

Goswami, Dinesh G; Tewari-Singh, Neera; Dhar, Deepanshi; Kumar, Dileep; Agarwal, Chapla; Ammar, David A; Kant, Rama; Enzenauer, Robert W; Petrash, J Mark; Agarwal, Rajesh

2016-02-01

To evaluate the toxic effects and associated mechanisms in corneal tissue exposed to the vesicating agent, nitrogen mustard (NM), a bifunctional alkylating analog of the chemical warfare agent sulfur mustard. Toxic effects and associated mechanisms were examined in maximally affected corneal tissue using corneal cultures and human corneal epithelial (HCE) cells exposed to NM. Analysis of ex vivo rabbit corneas showed that NM exposure increased apoptotic cell death, epithelial thickness, epithelial-stromal separation, and levels of vascular endothelial growth factor, cyclooxygenase 2, and matrix metalloproteinase-9. In HCE cells, NM exposure resulted in a dose-dependent decrease in cell viability and proliferation, which was associated with DNA damage in terms of an increase in p53 ser15, total p53, and H2A.X ser139 levels. NM exposure also induced caspase-3 and poly ADP ribose polymerase cleavage, suggesting their involvement in NM-induced apoptotic death in the rabbit cornea and HCE cells. Similar to rabbit cornea, NM exposure caused an increase in cyclooxygenase 2, matrix metalloproteinase-9, and vascular endothelial growth factor levels in HCE cells, indicating a role of these molecules and related pathways in NM-induced corneal inflammation, epithelial-stromal separation, and neovascularization. NM exposure also induced activation of activator protein 1 transcription factor proteins and upstream signaling pathways including mitogen-activated protein kinases and Akt protein kinase, suggesting that these could be key factors involved in NM-induced corneal injury. Results from this study provide insight into the molecular targets and pathways that could be involved in NM-induced corneal injuries laying the background for further investigation of these pathways in vesicant-induced ocular injuries, which could be helpful in the development of targeted therapies.

Nitrogen mustard-induced corneal injury involves DNA damage and pathways related to inflammation, epithelial-stromal separation and neovascularization

PubMed Central

Goswami, Dinesh G; Tewari-Singh, Neera; Dhar, Deepanshi; Kumar, Dileep; Agarwal, Chapla; Ammar, David A; Kant, Rama; Enzenauer, Robert W; Petrash, J Mark; Agarwal, Rajesh

2015-01-01

Purpose To evaluate the toxic effects and associated mechanisms in corneal tissue exposed to vesicating agent, nitrogen mustard (NM), a bi-functional alkylating analog of chemical warfare agent sulfur mustard (SM). Methods Toxic effects and associated mechanisms were examined in maximal affected corneal tissue employing corneal cultures and human corneal epithelial (HCE) cells exposed to nitrogen mustard (NM). Results Analysis of ex vivo rabbit corneas showed that NM exposure increased apoptotic cell death, epithelial thickness, epithelial-stromal separation and levels of VEGF, COX-2 and MMP-9. In HCE cells, NM exposure resulted in a dose-dependent decrease in cell viability and proliferation, which was associated with DNA damage in terms of an increase in p53 ser15, total p53 and H2A.X ser139 levels. NM exposure also induced caspase-3 and PARP cleavage, suggesting their involvement in NM-induced apoptotic death in rabbit cornea and HCE cells. Similar to rabbit cornea, NM exposure caused an increase in COX-2, MMP-9 and VEGF levels in HCE cells, indicating a role of these molecules and related pathways in NM-induced corneal inflammation, epithelial-stromal separation and neovascularization. NM exposure also induced activation of AP-1 transcription factor proteins and upstream signaling pathways including MAPKs and Akt, suggesting that these could be key factors involved in NM-induced corneal injury. Conclusion Results from this study provide insight into the molecular targets and pathways that could be involved in NM-induced corneal injuries laying the background for further investigation of these pathways in vesicant–induced ocular injuries, which could be helpful in the development of targeted therapies. PMID:26555588

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

Evaluation of the Effects of Airborne Particulate Matter on Bone Marrow-Mesenchymal Stem Cells (BM-MSCs): Cellular, Molecular and Systems Biological Approaches

PubMed Central

Abu-Elmagd, Muhammad; Alghamdi, Mansour A.; Shamy, Magdy; Khoder, Mamdouh I.; Costa, Max; Assidi, Mourad; Kadam, Roaa; Alsehli, Haneen; Gari, Mamdooh; Pushparaj, Peter Natesan; Kalamegam, Gauthaman; Al-Qahtani, Mohammed H.

2017-01-01

Particulate matter (PM) contains heavy metals that affect various cellular functions and gene expression associated with a range of acute and chronic diseases in humans. However, the specific effects they exert on the stem cells remain unclear. Here, we report the effects of PM collected from the city of Jeddah on proliferation, cell death, related gene expression and systems of biological analysis in bone marrow mesenchymal stem cells (BM-MSCs), with the aim of understanding the underlying mechanisms. PM2.5 and PM10 were tested in vitro at various concentrations (15 to 300 µg/mL) and durations (24 to 72 h). PMs induced cellular stress including membrane damage, shrinkage and death. Lower concentrations of PM2.5 increased proliferation of BM-MSCs, while higher concentrations served to decrease it. PM10 decreased BM-MSCs proliferation in a concentration-dependent manner. The X-ray fluorescence spectrometric analysis showed that PM contains high levels of heavy metals. Ingenuity Pathway Analysis (IPA) and hierarchical clustering analyses demonstrated that heavy metals were associated with signaling pathways involving cell stress/death, cancer and chronic diseases. qRT-PCR results showed differential expression of the apoptosis genes (BCL2, BAX); inflammation associated genes (TNF-α and IL-6) and the cell cycle regulation gene (p53). We conclude that PM causes inflammation and cell death, and thereby predisposes to chronic debilitating diseases. PMID:28425934

Toxicity of chemically generated nitric oxide towards pancreatic islet cells can be prevented by nicotinamide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kallmann, B.; Burkart, V.; Kolb, H.

1992-01-01

Previous studies have indicated that nitric oxide is involved in the lysis of pancreatic islet cells by inflammatory macrophages. Here the authors show that the incubation of islet cells with chemical NO-donors leads to cell lysis in a concentration and time dependent way. Islet cell death could be prevented by nicotinamide and 3-aminobenzamide, which are known to inhibit ADP-ribosylation, while several scavengers of oxygen radicals, N-acetylcysteine, dihydrolipoic acid, dimethylthiourea and citiolone, provided no protection.

ERK1/2-dependent bestrophin-3 expression prevents ER-stress-induced cell death in renal epithelial cells by reducing CHOP.

PubMed

Lee, Wing-Kee; Chakraborty, Prabir K; Roussa, Eleni; Wolff, Natascha A; Thévenod, Frank

2012-10-01

Upon endoplasmic reticulum (ER) stress induction, cells endeavor to survive by engaging the adaptive stress response known as the unfolded protein response or by removing aggregated proteins via autophagy. Chronic ER stress culminates in apoptotic cell death, which involves induction of pro-apoptotic CHOP. Here, we show that bestrophin-3 (Best-3), a protein previously associated with Ca(2+)-activated Cl(-) channel activity, is upregulated by the ER stressors, thapsigargin (TG), tunicamycin (TUN) and the toxic metal Cd(2+). In cultured rat kidney proximal tubule cells, ER stress, CHOP and cell death were induced after 6h by Cd(2+) (25μM), TG (3μM) and TUN (6μM), were associated with increased cytosolic Ca(2+) and downstream formation of reactive oxygen species and attenuated by the Ca(2+) chelator BAPTA-AM (10μM), the antioxidant α-tocopherol (100μM), or overexpression of catalase (CAT). Immunofluorescence staining showed Best-3 expression in the plasma membrane, nuclei and intracellular compartments, though not in the ER, in cultured cells and rat kidney cortex sections. Best-3 mRNA was augmented by ER stress and signaled through increased Ca(2+), oxidative stress and ERK1/2 phosphorylation, because it was attenuated by α-tocopherol, CAT expression, BAPTA-AM, calmodulin kinase inhibitor calmidazolium (40μM), ERK1/2 inhibitor U0126 (10μM), and ERK1/2 RNAi. Knockdown of Best-3 resulted in decreased cell number consequentially of cell death, as determined by nuclear staining and PARP-1 cleavage. Furthermore, reduced ER stress-cell death by Best-3 overexpression is attributed to diminished CHOP. Since Best-3 overexpression did not affect upstream signaling pathways, we hypothesize that Best-3 possibly interferes with CHOP transcription. From our novel observations, we conclude that ERK1/2-dependent Best-3 activation regulates cell fate decisions during ER stress by suppressing CHOP induction and death. Copyright © 2012 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Fen; Sun, Hong; Kluz, Thomas

Hexavalent chromium [Cr(VI)] is a human carcinogen that results in the generation of reactive oxygen species (ROS) and a variety of DNA lesions leading to cell death. Epigallocatechin-3-gallate (EGCG), the major polyphenol present in green tea, possesses potent antioxidative activity capable of protecting normal cells from various stimuli-induced oxidative stress and cell death. Here we demonstrated that co-treatment with EGCG protected human normal bronchial epithelial BEAS-2B cells from Cr(VI)-induced cell death in a dose-dependent manner. Cr(VI) induces apoptosis as the primary mode of cell death. Co-treatment of BEAS-2B cells with EGCG dose-dependently suppressed Cr(VI)-induced apoptosis. Fluorescence microscopic analyses and quantitativemore » measurement revealed that EGCG significantly decreased intracellular levels of ROS induced by Cr(VI) exposure. Using a well-established K{sup +}/SDS precipitation assay, we further showed that EGCG was able to dose-dependently reduce DNA–protein cross-links (DPC), lesions that could be partially attributed to Cr(VI)-induced oxidative stress. Finally, analyses of Affymetrix microarray containing 28,869 well-annotated genes revealed that, among the 3412 genes changed more than 1.5-fold by Cr(VI) treatment, changes of 2404 genes (70%) were inhibited by pretreatment of EGCG. Real-time PCR confirmed the induction of 3 genes involved in cell death and apoptosis by Cr(VI), which was eliminated by EGCG. In contrast, Cr(VI) reduced the expression of 3 genes related to cellular defense, and this reduction was inhibited by EGCG. Our results indicate that EGCG protects BEAS-2B cells from Cr(VI)-induced cytotoxicity presumably by scavenging ROS and modulating a subset of genes. EGCG, therefore, might serve as a potential chemopreventive agent against Cr(VI) carcinogenesis. -- Highlights: ► EGCG protected human normal bronchial epithelial BEAS-2B cells from Cr(VI)-induced cell death and apoptosis. ► EGCG significantly decreased intracellular levels of ROS induced by Cr(VI) exposure. ► EGCG reduced DNA-protein cross-links, lesions that could be partially attributed to Cr(VI)-induced oxidative stress. ► EGCG modulated 70% of the gene expression changes induced by Cr(VI) exposure.« less

Iron modulates cell survival in a Ras- and MAPK-dependent manner in ovarian cells

PubMed Central

Bauckman, K A; Haller, E; Flores, I; Nanjundan, M

2013-01-01

Ovarian cancer is a leading cause of cancer death in women in the United States. While the majority of ovarian cancers are serous, some rarer subtypes (i.e. clear cell) are often associated with endometriosis, a benign gynecological disease. Iron is rich in the cyst fluid of endometriosis-associated ovarian cancers and induces persistent oxidative stress. The role of iron, an essential nutrient involved in multiple cellular functions, in normal ovarian cell survival and ovarian cancer remains unclear. Iron, presented as ferric ammonium citrate (FAC), dramatically inhibits cell survival in ovarian cancer cell types associated with Ras mutations, while it is without effect in immortalized normal ovarian surface epithelial (T80) and endometriotic epithelial cells (lacking Ras mutations). Interestingly, FAC induced changes in cytoplasmic vacuolation concurrently with increases in LC3-II levels (an autophagy marker); these changes occurred in an ATG5/ATG7-dependent, beclin-1/hVps34-independent, and Ras-independent manner. Knockdown of autophagy mediators in HEY ovarian cancer cells reversed FAC-induced LC3-II levels, but there was little effect on reversing the cell death response. Intriguingly, transmission electron microscopy of FAC-treated T80 cells demonstrated abundant lysosomes (confirmed using Lysotracker) rich in iron particles, which occurred in a Ras-independent manner. Although the mitogen-activated protein kinase (MAPK) inhibitor, U0126, reversed FAC-induced LC3-II/autophagic punctae and lysosomes in a Ras-independent manner, it was remarkable that U0126 reversed cell death in malignant ovarian cells associated with Ras mutations. Moreover, FAC increased heme oxygenase-1 expression in H-Ras-overexpressing T80 cells, which was associated with increased cell death when overexpressed in T80 cells. Disruption of intracellular iron levels, via chelation of intracellular iron (deferoxamine), was also detrimental to malignant ovarian cell survival; thus, homeostatic intracellular iron levels are essential for cell survival. Collectively, our results implicate iron in modulating cell death in a Ras- and MAPK-dependent manner in ovarian cancer cells. PMID:23598404

Evaluation of a 99mTc-labeled AnnexinA5 variant for non-invasive SPECT imaging of cell death in liver, spleen and prostate.

PubMed

Greupink, Rick; Sio, Charles F; Ederveen, Antwan; Orsel, Joke

2009-12-01

We investigate radio-labeling and pharmacokinetics of a new AnnexinA5 variant (HYNIC-cys-AnxA5) and then assess its utility for the non-invasive detection of cell death in liver, spleen and prostate. AnnexinA5 binds to phosphatidylserine expressed on the surface of apoptotic and necrotic cells. Contrary to other AnnexinA5 variants, the new cys-AnxA5 allows for site-specific conjugation of a hydrazinonicotinamide-maleimide moiety and subsequent radio-labeling with (99m)Tc at a position not involved in the AnxA5-phosphatidylserine interaction. Distribution of (99m)Tc-HYNIC-cys-AnxA5 was studied in rats, both invasively and via SPECT/CT. Cycloheximide was used to induce cell death in liver and spleen, whereas apoptosis in the prostate was induced by castration. HYNIC-cys-AnxA5 was efficiently and reproducibly labeled with (99m)Tc. Blood clearance of radioactivity after iv-injection was adequately described by a two-compartment model, the renal cortex representing the main site of accumulation. Cycloheximide treatment resulted in increased accumulation of intravenous-injected (99m)Tc-HYNIC-cys-AnxA5 in liver and spleen over controls, which correlated well with TUNEL staining for cell death in corresponding tissue sections. However, the increase in TUNEL-positive prostate epithelial cells observed following castration was not paralleled by greater (99m)Tc-HYNIC-cys-AnxA5 accumulation. (99m)Tc-HYNIC-cys-AnxA5 appears a suitable tracer for assessment of cell death in liver and spleen, but not prostate.

Silver nanoparticles of different sizes induce a mixed type of programmed cell death in human pancreatic ductal adenocarcinoma

PubMed Central

Zielinska, Ewelina; Zauszkiewicz-Pawlak, Agata; Wojcik, Michal; Inkielewicz-Stepniak, Iwona

2018-01-01

Pancreatic ductal adenocarcinoma, with the high resistance to chemotherapeutic agents, remains the fourth leading cause of cancer-death in the world. Due to the wide range of biological activity and unique properties, silver nanoparticles (AgNPs) are indicated as agents with potential to overcome barriers involved in chemotherapy failure. Therefore, in our study we decided to assess the ability of AgNPs to kill pancreatic cancer cells, and then to identify the molecular mechanism underlying this effect. Moreover, we evaluated the cytotoxicity of AgNPs against non-tumor cell of the same tissue (hTERT-HPNE cells) for comparison. Our results indicated that AgNPs with size of 2.6 and 18 nm decreased viability, proliferation and caused death of pancreatic cancer cells in a size- and concentration-dependent manner. Ultrastructural analysis identified that cellular uptake of AgNPs resulted in apoptosis, autophagy, necroptosis and mitotic catastrophe. These alterations were associated with increased pro-apoptotic protein Bax and decreased level of anti-apoptotic protein Bcl-2. Moreover, AgNPs significantly elevated the level of tumor suppressor p53 protein as well as necroptosis- and autophagy-related proteins: RIP-1, RIP-3, MLKL and LC3-II, respectively. In addition, we found that PANC-1 cells were more vulnerable to AgNPs-induced cytotoxicity compared to pancreatic non-tumor cells. In conclusion, AgNPs by inducing mixed type of programmed cell death in PANC-1 cells, could provide a new therapeutic strategy to overcome chemoresistance in one of the deadliest human cancer. PMID:29435134

Cranberry proanthocyanidins inhibit esophageal adenocarcinoma in vitro and in vivo through pleiotropic cell death induction and PI3K/AKT/mTOR inactivation

PubMed Central

Kresty, Laura A.; Weh, Katherine M.; Zeyzus-Johns, Bree; Perez, Laura N.; Howell, Amy B.

2015-01-01

Cranberries are rich in bioactive constituents known to improve urinary tract health and more recent evidence supports cranberries possess cancer inhibitory properties. However, mechanisms of cancer inhibition by cranberries remain to be elucidated, particularly in vivo. Properties of a purified cranberry-derived proanthocyanidin extract (C-PAC) were investigated utilizing acid-sensitive and acid-resistant human esophageal adenocarcinoma (EAC) cell lines and esophageal tumor xenografts in athymic NU/NU mice. C-PAC induced caspase-independent cell death mainly via autophagy and low levels of apoptosis in acid-sensitive JHAD1 and OE33 cells, but resulted in cellular necrosis in acid-resistant OE19 cells. Similarly, C-PAC induced necrosis in JHAD1 cells pushed to acid-resistance via repeated exposures to an acidified bile cocktail. C-PAC associated cell death involved PI3K/AKT/mTOR inactivation, pro-apoptotic protein induction (BAX, BAK1, deamidated BCL-xL, Cytochrome C, PARP), modulation of MAPKs (P-P38/P-JNK) and G2-M cell cycle arrest in vitro. Importantly, oral delivery of C-PAC significantly inhibited OE19 tumor xenograft growth via modulation of AKT/mTOR/MAPK signaling and induction of the autophagic form of LC3B supporting in vivo efficacy against EAC for the first time. C-PAC is a potent inducer of EAC cell death and is efficacious in vivo at non-toxic behaviorally achievable concentrations, holding promise for preventive or therapeutic interventions in cohorts at increased risk for EAC, a rapidly rising and extremely deadly malignancy. PMID:26378019

X-ray-induced bystander responses reduce spontaneous mutations in V79 cells

PubMed Central

Maeda, Munetoshi; Kobayashi, Katsumi; Matsumoto, Hideki; Usami, Noriko; Tomita, Masanori

2013-01-01

The potential for carcinogenic risks is increased by radiation-induced bystander responses; these responses are the biological effects in unirradiated cells that receive signals from the neighboring irradiated cells. Bystander responses have attracted attention in modern radiobiology because they are characterized by non-linear responses to low-dose radiation. We used a synchrotron X-ray microbeam irradiation system developed at the Photon Factory, High Energy Accelerator Research Organization, KEK, and showed that nitric oxide (NO)-mediated bystander cell death increased biphasically in a dose-dependent manner. Here, we irradiated five cell nuclei using 10 × 10 µm2 5.35 keV X-ray beams and then measured the mutation frequency at the hypoxanthine-guanosine phosphoribosyl transferase (HPRT) locus in bystander cells. The mutation frequency with the null radiation dose was 2.6 × 10–5 (background level), and the frequency decreased to 5.3 × 10–6 with a dose of approximately 1 Gy (absorbed dose in the nucleus of irradiated cells). At high doses, the mutation frequency returned to the background level. A similar biphasic dose-response effect was observed for bystander cell death. Furthermore, we found that incubation with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), a specific scavenger of NO, suppressed not only the biphasic increase in bystander cell death but also the biphasic reduction in mutation frequency of bystander cells. These results indicate that the increase in bystander cell death involves mechanisms that suppress mutagenesis. This study has thus shown that radiation-induced bystander responses could affect processes that protect the cell against naturally occurring alterations such as mutations. PMID:23660275

Treatment with polyamine oxidase inhibitor reduces microglial activation and limits vascular injury in ischemic retinopathy

PubMed Central

Patel, C.; Xu, Z.; Shosha, E.; Xing, J.; Lucas, R.; Caldwell, R.W.; Caldwell, R.B.; Narayanan, S.P.

2016-01-01

Retinal vascular injury is a major cause of vision impairment in ischemic retinopathies. Insults such as hyperoxia, oxidative stress and inflammation contribute to this pathology. Previously, we showed that hyperoxia-induced retinal neurodegeneration is associated with increased polyamine oxidation. Here, we are studying the involvement of polyamine oxidases in hyperoxia-induced injury and death of retinal vascular endothelial cells. Newborn C57BL6/J mice were exposed to hyperoxia (70% O2) from postnatal day (P) 7 to 12 and were treated with the polyamine oxidase inhibitor MDL 72527 or vehicle starting at P6. Mice were sacrificed after different durations of hyperoxia and their retinas were analyzed to determine the effects on vascular injury, microglial cell activation, and inflammatory cytokine profiling. The results of this analysis showed that MDL 72527 treatment significantly reduced hyperoxia-induced retinal vascular injury and enhanced vascular sprouting as compared with the vehicle controls. These protective effects were correlated with significant decreases in microglial activation as well as levels of inflammatory cytokines and chemokines. In order to model the effects of polyamine oxidation in causing microglial activation in vitro, studies were performed using rat brain microvascular endothelial cells treated with conditioned-medium from rat retinal microglia stimulated with hydrogen peroxide. Conditioned-medium from activated microglial cultures induced cell stress signals and cell death in microvascular endothelial cells. These studies demonstrate the involvement of polyamine oxidases in hyperoxia-induced retinal vascular injury and retinal inflammation in ischemic retinopathy, through mechanisms involving cross-talk between endothelial cells and resident retinal microglia. PMID:27239699

Differences and Similarities in TRAIL- and Tumor Necrosis Factor-Mediated Necroptotic Signaling in Cancer Cells

PubMed Central

Philipp, Stephan; Fuchslocher Chico, Johaiber; Saggau, Carina; Fritsch, Jürgen; Föll, Alexandra; Plenge, Johannes; Arenz, Christoph; Pinkert, Thomas; Kalthoff, Holger; Trauzold, Anna; Schmitz, Ingo; Schütze, Stefan; Adam, Dieter

2016-01-01

Recently, a type of regulated necrosis (RN) called necroptosis was identified to be involved in many pathophysiological processes and emerged as an alternative method to eliminate cancer cells. However, only a few studies have elucidated components of TRAIL-mediated necroptosis useful for anticancer therapy. Therefore, we have compared this type of cell death to tumor necrosis factor (TNF)-mediated necroptosis and found similar signaling through acid and neutral sphingomyelinases, the mitochondrial serine protease HtrA2/Omi, Atg5, and vacuolar H+-ATPase. Notably, executive mechanisms of both TRAIL- and TNF-mediated necroptosis are independent of poly(ADP-ribose) polymerase 1 (PARP-1), and depletion of p38α increases the levels of both types of cell death. Moreover, we found differences in signaling between TNF- and TRAIL-mediated necroptosis, e.g., a lack of involvement of ubiquitin carboxyl hydrolase L1 (UCH-L1) and Atg16L1 in executive mechanisms of TRAIL-mediated necroptosis. Furthermore, we discovered indications of an altered involvement of mitochondrial components, since overexpression of the mitochondrial protein Bcl-2 protected Jurkat cells from TRAIL- and TNF-mediated necroptosis, and overexpression of Bcl-XL diminished only TRAIL-induced necroptosis in Colo357 cells. Furthermore, TRAIL does not require receptor internalization and endosome-lysosome acidification to mediate necroptosis. Taken together, pathways described for TRAIL-mediated necroptosis and differences from those for TNF-mediated necroptosis might be unique targets to increase or modify necroptotic signaling and eliminate tumor cells more specifically in future anticancer approaches. PMID:27528614

Disturbance of DKK1 level is partly involved in survival of lung cancer cells via regulation of ROMO1 and γ-radiation sensitivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, In Gyu, E-mail: igkim@kaeri.re.kr; Department of Radiation Biotechnology and Applied Radioisotope, University of Science and Technology; Kim, Seo Yoen

2014-01-03

Highlights: •DKK1 was expressed differently among non-small-cell lung cancer cell lines. •DKK1 negatively regulated ROMO1 gene expression. •Disturbance of DKK1 level induced the imbalance of cellular ROS. •DKK1/ROMO1-induced ROS imbalance is involved in cell survival in NSCLC. -- Abstract: Dickkopf1 (DKK1), a secreted protein involved in embryonic development, is a potent inhibitor of the Wnt signaling pathway and has been postulated to be a tumor suppressor or tumor promoter depending on the tumor type. In this study, we showed that DKK1 was expressed differently among non-small-cell lung cancer cell lines. The DKK1 expression level was much higher in A549 cellsmore » than in H460 cells. We revealed that blockage of DKK1 expression by silencing RNA in A549 cells caused up-regulation of intracellular reactive oxygen species (ROS) modulator (ROMO1) protein, followed by partial cell death, cell growth inhibition, and loss of epithelial–mesenchymal transition property caused by ROS, and it also increased γ-radiation sensitivity. DKK1 overexpression in H460 significantly inhibited cell survival with the decrease of ROMO1 level, which induced the decrease of cellular ROS. Thereafter, exogenous N-acetylcysteine, an antioxidant, or hydrogen peroxide, a pro-oxidant, partially rescued cells from death and growth inhibition. In each cell line, both overexpression and blockage of DKK1 not only elevated p-RB activation, which led to cell growth arrest, but also inactivated AKT/NF-kB, which increased radiation sensitivity and inhibited cell growth. This study is the first to demonstrate that strict modulation of DKK1 expression in different cell types partially maintains cell survival via tight regulation of the ROS-producing ROMO1 and radiation resistance.« less

Novel type 1 photosensitizers: viability of leukemia cells exposed to reactive intermediates generated in situ by in vitro photofragmentation

NASA Astrophysics Data System (ADS)

Rajagopalan, Raghavan; Karwa, Amol; Lusiak, Przemyslaw M.; Srivastava, Kripa; Poreddy, Amruta R.; Pandurangi, Raghootama S.; Galen, Karen P.; Neumann, William L.; Cantrell, Gary E.; Dorshow, Richard B.

2009-06-01

Photodynamic therapy of tumors involving Type 2 photosenstizers has been conspicuously successful, but the Type 1 process, in contrast, has not received much attention despite its considerable potential. Accordingly, several classes of molecules containing fragile bonds such as azido (-N=N=N), azo (-N=N-), sulfenato (-S-O-) and oxaza (-N-O-) functional groups that produce reactive intermediates such as radicals and nitrenes upon photoexcitation were prepared and tested for cell viability using U397 leukemia cell line. The azido photosensitizer was conjugated to leukemia cell binding peptide, SFFWRLS, for targeted cell viability study. The cells were incubated with the photosensitizer at various concentrations, and were illuminated for 5, 10, and 20 minutes. The results show that all the photosensitizers caused cell death compared to the controls when exposed to both the photosensitizers and light. Most importantly, selective cell death was observed with the azido peptide conjugate 6, which clearly demonstrates that these Type 1 sensitizers are useful for phototherapeutic applications.

Necroptosis in tumorigenesis, activation of anti-tumor immunity, and cancer therapy

PubMed Central

Wu, Zhi-Qiang; Shi, Yang-Yang; Zaorsky, Nicholas G.; Deng, Lei; Yuan, Zhi-Yong; Lu, You; Wang, Ping

2016-01-01

While the mechanisms underlying apoptosis and autophagy have been well characterized over recent decades, another regulated cell death event, necroptosis, remains poorly understood. Elucidating the signaling networks involved in the regulation of necroptosis may allow this form of regulated cell death to be exploited for diagnosis and treatment of cancer, and will contribute to the understanding of the complex tumor microenvironment. In this review, we have summarized the mechanisms and regulation of necroptosis, the converging and diverging features of necroptosis in tumorigenesis, activation of anti-tumor immunity, and cancer therapy, as well as attempts to exploit this newly gained knowledge to provide therapeutics for cancer. PMID:27429198

Molecular mechanisms involved in cochlear implantation trauma and the protection of hearing and auditory sensory cells by inhibition of c-Jun-N-terminal kinase signaling.

PubMed

Eshraghi, Adrien A; Gupta, Chhavi; Van De Water, Thomas R; Bohorquez, Jorge E; Garnham, Carolyn; Bas, Esperanza; Talamo, Victoria Maria

2013-03-01

To investigate the molecular mechanisms involved in electrode insertion trauma (EIT) and to test the otoprotective effect of locally delivered AM-111. An animal model of cochlear implantation. Guinea pigs' hearing thresholds were measured by auditory brainstem response (ABR) before and after cochlear implantation in four groups: EIT; pretreated with hyaluronate gel 30 minutes before EIT (EIT+Gel); pretreated with hyaluronate gel/AM-111 30 minutes before EIT (EIT+AM-111); and unoperated contralateral ears as controls. Neurofilament, synapsin, and fluorescein isothiocyanate (FITC)-phalloidin staining for hair cell counts were performed at 90 days post-EIT. Immunostaining for 4-hydroxy-2-nonenal (HNE), activated caspase-3, CellROX, and phospho-c-Jun were performed at 24 hours post-EIT. ABR thresholds increased post-EIT in the cochleae of EIT only and EIT+Gel treated animals. There was no significant increase in hearing thresholds in cochleae from either EIT+AM-111 treated or unoperated control ears. AM-111 protection of organ of Corti sensory elements (i.e., hair cells [HCs], supporting cells [SCs], nerve fibers, and synapses) was documented at 3 months post-EIT. Immunostaining of 24-hour post-EIT specimens demonstrated increased levels of HNE in HCs and SCs; increased levels of CellROX and activation of caspase-3 was observed only in SCs, and phosphorylation of c-Jun occurred only in HCs of the EIT-only and EIT+Gel specimens. There was no immunostaining for either HNE, CellROX, caspase-3, or phospho-c-Jun in the organ of Corti specimens from AM-111 treated cochleae. Molecular mechanisms involved in programmed cell death of HCs are different than the ones involved in programmed cell death of SCs. Local delivery of AM-111 provided a significant level of protection against EIT-induced hearing losses, HC losses, and damage to neural elements. Copyright © 2012 The American Laryngological, Rhinological, and Otological Society, Inc.

Apoptosis-like death was involved in freeze-drying-preserved fungus Mucor rouxii and can be inhibited by L-proline.

PubMed

Wang, Xiaoyun; Wang, Youzhi

2016-02-01

Freeze-drying is one of the most effective methods to preserve fungi for an extended period. However, it is associated with a loss of viability and shortened storage time in some fungi. This study evaluated the stresses that led to the death of freeze-dried Mucor rouxii by using cell apoptotic methods. The results showed there were apoptosis-inducing stresses, such as the generation of obvious intracellular reactive oxygen species (ROS) and metacaspase activation. Moreover, nuclear condensation and a delayed cell death peak were determined after rehydration and 24 h incubation in freeze-dried M. rouxii via a propidium iodide (PI) assay, which is similar to the phenomenon of cryopreservation-induced delayed-onset cell death (CIDOCD). Then, several protective agents were tested to decrease the apoptosis-inducing stresses and to improve the viability. Finally, it was found that 1.6 mM L-proline can effectively decrease the nuclear condensation rate and increase the survival rate in freeze-dried M. rouxii. (1) apoptosis-inducing factors occur in freeze-dried M. rouxii. (2) ROS and activated metacaspases lead to death in freeze-dried M. rouxii. (3)L-proline increases the survival rate of freeze-dried M. rouxii. Copyright © 2015 Elsevier Inc. All rights reserved.

Giant cell phlebitis: a potentially lethal clinical entity

PubMed Central

Kunieda, Takeshige; Murayama, Masanori; Ikeda, Tsuneko; Yamakita, Noriyoshi

2012-01-01

An 83-year-old woman presented to us with a 4-week history of general malaise, subjective fever and lower abdominal pain. Despite the intravenous infusion of antibiotics, her blood results and physical condition worsened, resulting in her sudden death. Autopsy study revealed that the medium-sized veins of the mesentery were infiltrated by eosinophil granulocytes, lymphocytes, macrophages and multinucleated giant cells; however, the arteries were not involved. Microscopically, venous giant cell infiltration was observed in the gastrointestinal tract, bladder, retroperitoneal tissues and myocardium. The final diagnosis was giant cell phlebitis, a rare disease of unknown aetiology. This case demonstrates for the first time that giant cell phlebitis involving extra-abdominal organs, including hearts, can cause serious morbidity. PMID:22859384

Hyperthermia: an effective strategy to induce apoptosis in cancer cells.

PubMed

Ahmed, Kanwal; Tabuchi, Yoshiaki; Kondo, Takashi

2015-11-01

Heat has been used as a medicinal and healing modality throughout human history. The combination of hyperthermia (HT) with radiation and anticancer agents has been used clinically and has shown positive results to a certain extent. However, the clinical results of HT treatment alone have been only partially satisfactory. Cell death following HT treatment is a function of both temperature and treatment duration. HT induces cancer cell death through apoptosis; the degree of apoptosis and the apoptotic pathway vary in different cancer cell types. HT-induced reactive oxygen species production are responsible for apoptosis in various cell types. However, the underlying mechanism of signal transduction and the genes related to this process still need to be elucidated. In this review, we summarize the molecular mechanism of apoptosis induced by HT, enhancement of heat-induced apoptosis, and the genetic network involved in HT-induced apoptosis.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Noda, Chiseko; Department of Genomic Science, Kobe University Graduate School of Medicine, Kobe, Hyogo 650-0017; He, Jinsong

(-)-Epigallocatechin-3-gallate (EGCG), a major constituent of green tea polyphenols, has been shown to suppress cancer cell proliferation and induce apoptosis. In this study we investigated its efficacy and the mechanism underlying its effect using human B lymphoblastoid cell line Ramos, and effect of co-treatment with EGCG and a chemotherapeutic agent on apoptotic cell death. EGCG induced dose- and time-dependent apoptotic cell death accompanied by loss of mitochondrial transmembrane potential, release of cytochrome c into the cytosol, and cleavage of pro-caspase-9 to its active form. EGCG also enhanced production of intracellular reactive oxygen species (ROS). Pretreatment with diphenylene iodonium chloride, anmore » inhibitor of NAD(P)H oxidase and an antioxidant, partially suppressed both EGCG-induced apoptosis and production of ROS, implying that oxidative stress is involved in the apoptotic response. Furthermore, we showed that combined-treatment with EGCG and a chemotherapeutic agent, etoposide, synergistically induced apoptosis in Ramos cells.« less

Akt mediated ROS-dependent selective targeting of mutant KRAS tumors.

PubMed

Iskandar, Kartini; Rezlan, Majidah; Pervaiz, Shazib

2014-10-01

Reactive oxygen species (ROS) play a critical role in a variety of cellular processes, ranging from cell survival and proliferation to cell death. Previously, we reported the ability of a small molecule compound, C1, to induce ROS dependent autophagy associated apoptosis in human cancer cell lines and primary tumor cells (Wong C. et al. 2010). Our ongoing investigations have unraveled a hitherto undefined novel signaling network involving hyper-phosphorylation of Akt and Akt-mediated ROS production in cancer cell lines. Interestingly, drug-induced Akt activation is selectively seen in cell lines that carry mutant KRAS; HCT116 cells that carry the V13D KRAS mutation respond favorably to C1 while HT29 cells expressing wild type KRAS are relatively resistant. Of note, not only does the compound target mutant KRAS expressing cells but also induces RAS activation as evidenced by the PAK pull down assay. Corroborating this, pharmacological inhibition as well as siRNA mediated silencing of KRAS or Akt, blocked C1-induced ROS production and rescued tumor colony forming ability in HCT116 cells. To further confirm the involvement of KRAS, we made use of mutant KRAS transformed RWPE-1 prostate epithelial cells. Notably, drug-induced ROS generation and death sensitivity was significantly higher in RWPE-1-KRAS cells than the RWPE-1-vector cells, thus confirming the results obtained with mutant KRAS colorectal carcinoma cell line. Lastly, we made use of HCT116 mutant KRAS knockout cells (KO) where the mutant KRAS allele had been deleted, thus expressing a single wild-type KRAS allele. Exposure of the KO cells to C1 failed to induce Akt activation and mitochondrial ROS production. Taken together, results show the involvement of activated Akt in ROS-mediated selective targeting of mutant KRAS expressing tumors, which could have therapeutic implications given the paucity of chemotherapeutic strategies specifically targeting KRAS mutant cancers. Copyright © 2014. Published by Elsevier Inc.

Cell Death, Neuronal Plasticity and Functional Loading in the Development of the Central Nervous System

NASA Technical Reports Server (NTRS)

Keefe, J. R.

1985-01-01

Research on the precise timing and regulation of neuron production and maturation in the vestibular and visual systems of Wistar rats and several inbred strains of mice (C57B16 and Pallid mutant) concentrated upon establishing a timing baseline for mitotic development of the neurons of the vestibular nuclei and the peripheral vestibular sensory structures (maculae, cristae). This involved studies of the timing and site of neuronal cell birth and preliminary studies of neuronal cell death in both central and peripheral elements of the mammalian vestibular system. Studies on neuronal generation and maturation in the retina were recently added to provide a mechanism for more properly defining the in utero' developmental age of the individual fetal subject and to closely monitor potential transplacental effects of environmentally stressed maternal systems. Information is given on current efforts concentrating upon the (1) perinatal period of development (E18 thru P14) and (2) the role of cell death in response to variation in the functional loading of the vestibular and proprioreceptive systems in developing mammalian organisms.

A Systems Model of Parkinson's Disease Using Biochemical Systems Theory.

PubMed

Sasidharakurup, Hemalatha; Melethadathil, Nidheesh; Nair, Bipin; Diwakar, Shyam

2017-08-01

Parkinson's disease (PD), a neurodegenerative disorder, affects millions of people and has gained attention because of its clinical roles affecting behaviors related to motor and nonmotor symptoms. Although studies on PD from various aspects are becoming popular, few rely on predictive systems modeling approaches. Using Biochemical Systems Theory (BST), this article attempts to model and characterize dopaminergic cell death and understand pathophysiology of progression of PD. PD pathways were modeled using stochastic differential equations incorporating law of mass action, and initial concentrations for the modeled proteins were obtained from literature. Simulations suggest that dopamine levels were reduced significantly due to an increase in dopaminergic quinones and 3,4-dihydroxyphenylacetaldehyde (DOPAL) relating to imbalances compared to control during PD progression. Associating to clinically observed PD-related cell death, simulations show abnormal parkin and reactive oxygen species levels with an increase in neurofibrillary tangles. While relating molecular mechanistic roles, the BST modeling helps predicting dopaminergic cell death processes involved in the progression of PD and provides a predictive understanding of neuronal dysfunction for translational neuroscience.

Can a single "powerless" mitochondrion in the malaria parasite contribute to parasite programmed cell death in the asexual stages?

PubMed

Ch'ng, Jun-Hong; Yeo, Su-Ping; Shyong-Wei Tan, Kevin

2013-05-01

The protozoan pathogens responsible for malaria are from the Plasmodium genus, with Plasmodium falciparum and Plasmodium vivax accounting for almost all clinical infections. With recent estimates of mortality exceeding 800,000 annually, malaria continues to take a terrible toll on lives and the early promises of medicine to eradicate the disease have yet to approach realization, in part due to the spread of drug resistant parasites. Recent reports of artemisinin-resistance have prompted renewed efforts to identify novel therapeutic options, and one such pathway being considered for antimalarial exploit is the parasite's programmed cell death (PCD) pathway. In this mini-review, we will discuss the roles of the plasmodium mitochondria in cell death and as a target of antimalarial compounds, taking into account recent data suggesting that PCD pathways involving the mitochondria may be attractive antimalarial targets. Copyright © 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

TRPM2, calcium and neurodegenerative diseases

PubMed Central

Xie, Yu-Feng; MacDonald, John F; Jackson, Michael F

2010-01-01

NMDA receptor overactivation triggers intracellular Ca2+ dysregulation, which has long been thought to be critical for initiating excitotoxic cell death cascades associated with stroke and neurodegenerative disease. The inability of NMDA receptor antagonists to afford neuroprotection in clinical stroke trials has led to a re-evaluation of excitotoxic models of cell death and has focused research efforts towards identifying additional Ca2+ influx pathways. Recent studies indicate that TRPM2, a member of the TRPM subfamily of Ca2+-permeant, non-selective cation channel, plays an important role in mediating cellular responses to a wide range of stimuli that, under certain situations, can induce cell death. These include reactive oxygen and nitrogen species, tumour necrosis factor as well as soluble oli-gomers of amyloid beta. However, the molecular basis of TRPM2 channel involvement in these processes is not fully understood. In this review, we summarize recent studies about the regulation of TRPM2, its interaction with calcium and the possible implications for neurodegenerative diseases. PMID:21383889

DICER1/Alu RNA dysmetabolism induces Caspase-8–mediated cell death in age-related macular degeneration

PubMed Central

Kim, Younghee; Tarallo, Valeria; Kerur, Nagaraj; Yasuma, Tetsuhiro; Gelfand, Bradley D.; Bastos-Carvalho, Ana; Hirano, Yoshio; Yasuma, Reo; Mizutani, Takeshi; Fowler, Benjamin J.; Li, Shengjian; Kaneko, Hiroki; Bogdanovich, Sasha; Ambati, Balamurali K.; Hinton, David R.; Hauswirth, William W.; Hakem, Razqallah; Wright, Charles; Ambati, Jayakrishna

2014-01-01

Geographic atrophy, an advanced form of age-related macular degeneration (AMD) characterized by death of the retinal pigmented epithelium (RPE), causes untreatable blindness in millions worldwide. The RPE of human eyes with geographic atrophy accumulates toxic Alu RNA in response to a deficit in the enzyme DICER1, which in turn leads to activation of the NLRP3 inflammasome and elaboration of IL-18. Despite these recent insights, it is still unclear how RPE cells die during the course of the disease. In this study, we implicate the involvement of Caspase-8 as a critical mediator of RPE degeneration. Here we show that DICER1 deficiency, Alu RNA accumulation, and IL-18 up-regulation lead to RPE cell death via activation of Caspase-8 through a Fas ligand-dependent mechanism. Coupled with our observation of increased Caspase-8 expression in the RPE of human eyes with geographic atrophy, our findings provide a rationale for targeting this apoptotic pathway in this disease. PMID:25349431

Dynamic metabolic exchange governs a marine algal-bacterial interaction.

PubMed

Segev, Einat; Wyche, Thomas P; Kim, Ki Hyun; Petersen, Jörn; Ellebrandt, Claire; Vlamakis, Hera; Barteneva, Natasha; Paulson, Joseph N; Chai, Liraz; Clardy, Jon; Kolter, Roberto

2016-11-18

Emiliania huxleyi is a model coccolithophore micro-alga that generates vast blooms in the ocean. Bacteria are not considered among the major factors influencing coccolithophore physiology. Here we show through a laboratory model system that the bacterium Phaeobacter inhibens , a well-studied member of the Roseobacter group, intimately interacts with E. huxleyi. While attached to the algal cell, bacteria initially promote algal growth but ultimately kill their algal host. Both algal growth enhancement and algal death are driven by the bacterially-produced phytohormone indole-3-acetic acid. Bacterial production of indole-3-acetic acid and attachment to algae are significantly increased by tryptophan, which is exuded from the algal cell. Algal death triggered by bacteria involves activation of pathways unique to oxidative stress response and programmed cell death. Our observations suggest that bacteria greatly influence the physiology and metabolism of E. huxleyi. Coccolithophore-bacteria interactions should be further studied in the environment to determine whether they impact micro-algal population dynamics on a global scale.

Evolution of apoptosis-like programmed cell death in unicellular protozoan parasites.

PubMed

Kaczanowski, Szymon; Sajid, Mohammed; Reece, Sarah E

2011-03-25

Apoptosis-like programmed cell death (PCD) has recently been described in multiple taxa of unicellular protists, including the protozoan parasites Plasmodium, Trypanosoma and Leishmania. Apoptosis-like PCD in protozoan parasites shares a number of morphological features with programmed cell death in multicellular organisms. However, both the evolutionary explanations and mechanisms involved in parasite PCD are poorly understood. Explaining why unicellular organisms appear to undergo 'suicide' is a challenge for evolutionary biology and uncovering death executors and pathways is a challenge for molecular and cell biology. Bioinformatics has the potential to integrate these approaches by revealing homologies in the PCD machinery of diverse taxa and evaluating their evolutionary trajectories. As the molecular mechanisms of apoptosis in model organisms are well characterised, and recent data suggest similar mechanisms operate in protozoan parasites, key questions can now be addressed. These questions include: which elements of apoptosis machinery appear to be shared between protozoan parasites and multicellular taxa and, have these mechanisms arisen through convergent or divergent evolution? We use bioinformatics to address these questions and our analyses suggest that apoptosis mechanisms in protozoan parasites and other taxa have diverged during their evolution, that some apoptosis factors are shared across taxa whilst others have been replaced by proteins with similar biochemical activities.

Evolution of apoptosis-like programmed cell death in unicellular protozoan parasites

PubMed Central

2011-01-01

Apoptosis-like programmed cell death (PCD) has recently been described in multiple taxa of unicellular protists, including the protozoan parasites Plasmodium, Trypanosoma and Leishmania. Apoptosis-like PCD in protozoan parasites shares a number of morphological features with programmed cell death in multicellular organisms. However, both the evolutionary explanations and mechanisms involved in parasite PCD are poorly understood. Explaining why unicellular organisms appear to undergo 'suicide' is a challenge for evolutionary biology and uncovering death executors and pathways is a challenge for molecular and cell biology. Bioinformatics has the potential to integrate these approaches by revealing homologies in the PCD machinery of diverse taxa and evaluating their evolutionary trajectories. As the molecular mechanisms of apoptosis in model organisms are well characterised, and recent data suggest similar mechanisms operate in protozoan parasites, key questions can now be addressed. These questions include: which elements of apoptosis machinery appear to be shared between protozoan parasites and multicellular taxa and, have these mechanisms arisen through convergent or divergent evolution? We use bioinformatics to address these questions and our analyses suggest that apoptosis mechanisms in protozoan parasites and other taxa have diverged during their evolution, that some apoptosis factors are shared across taxa whilst others have been replaced by proteins with similar biochemical activities. PMID:21439063

Differential effects of lesion mimic mutants in barley on disease development by facultative pathogens

PubMed Central

McGrann, Graham R. D.; Steed, , Andrew; Burt, Christopher; Nicholson, Paul; Brown, James K. M.

2015-01-01

Lesion mimic mutants display spontaneous necrotic spots and chlorotic leaves as a result of mis-regulated cell death programmes. Typically these mutants have increased resistance to biotrophic pathogens but their response to facultative fungi that cause necrotrophic diseases is less well studied. The effect of altered cell death regulation on the development of disease caused by Ramularia collo-cygni, Fusarium culmorum and Oculimacula yallundae was explored using a collection of barley necrotic (nec) lesion mimic mutants. nec8 mutants displayed lower levels of all three diseases compared to nec9 mutants, which had increased R. collo-cygni but decreased F. culmorum disease symptoms. nec1 mutants reduced disease development caused by both R. collo-cygni and F. culmorum. The severity of the nec1-induced lesion mimic phenotype and F. culmorum symptom development was reduced by mutation of the negative cell death regulator MLO. The significant reduction in R. collo-cygni symptoms caused by nec1 was completely abolished in the presence of the mlo-5 allele and both symptoms and fungal biomass were greater than in the wild-type. These results indicate that physiological pathways involved in regulation of cell death interact with one another in their effects on different fungal pathogens. PMID:25873675

Impairment of Fas-ligand-caveolin-1 interaction inhibits Fas-ligand translocation to rafts and Fas-ligand-induced cell death.

PubMed

Glukhova, Xenia A; Trizna, Julia A; Proussakova, Olga V; Gogvadze, Vladimir; Beletsky, Igor P

2018-01-22

Fas-ligand/CD178 belongs to the TNF family proteins and can induce apoptosis through death receptor Fas/CD95. The important requirement for Fas-ligand-dependent cell death induction is its localization to rafts, cholesterol- and sphingolipid-enriched micro-domains of membrane, involved in regulation of different signaling complexes. Here, we demonstrate that Fas-ligand physically associates with caveolin-1, the main protein component of rafts. Experiments with cells overexpressing Fas-ligand revealed a FasL N-terminal pre-prolin-rich region, which is essential for the association with caveolin-1. We found that the N-terminal domain of Fas-ligand bears two caveolin-binding sites. The first caveolin-binding site binds the N-terminal domain of caveolin-1, whereas the second one appears to interact with the C-terminal domain of caveolin-1. The deletion of both caveolin-binding sites in Fas-ligand impairs its distribution between cellular membranes, and attenuates a Fas-ligand-induced cytotoxicity. These results demonstrate that the interaction of Fas-ligand and caveolin-1 represents a molecular basis for Fas-ligand translocation to rafts, and the subsequent induction of Fas-ligand-dependent cell death. A possibility of a similar association between other TNF family members and caveolin-1 is discussed.

Anti-Cancerous Effect of Inonotus taiwanensis Polysaccharide Extract on Human Acute Monocytic Leukemia Cells through ROS-Independent Intrinsic Mitochondrial Pathway.

PubMed

Chao, Tsai-Ling; Wang, Ting-Yin; Lee, Chin-Huei; Yiin, Shuenn-Jiun; Ho, Chun-Te; Wu, Sheng-Hua; You, Huey-Ling; Chern, Chi-Liang

2018-01-29

Acute leukemia is one of the commonly diagnosed neoplasms and causes human death. However, the treatment for acute leukemia is not yet satisfactory. Studies have shown that mushroom-derived polysaccharides display low toxicity and have been used clinically for cancer therapy. Therefore, we set out to evaluate the anti-cancerous efficacy of a water-soluble polysaccharide extract from Inonotus taiwanensis (WSPIS) on human acute monocytic leukemia THP-1 and U937 cell lines in vitro. Under our experimental conditions, WSPIS elicited dose-dependent growth retardation and induced apoptotic cell death. Further analysis showed that WSPIS-induced apoptosis was associated with a mitochondrial apoptotic pathway, such as the disruption of mitochondrial membrane potential (MMP), followed by the activation of caspase-9, caspase-3, and PARP (poly(ADP-ribose) polymerase) cleavage. However, a broad caspase inhibitor, Z-VAD.fmk, could not prevent WSPIS-induced apoptosis. These data imply that mechanism(s) other than caspase might be involved. Thus, the involvement of endonuclease G (endoG), a mediator arbitrating caspase-independent oligonucleosomal DNA fragmentation, was examined. Western blotting demonstrated that WSPIS could elicit nuclear translocation of endoG. MMP disruption after WSPIS treatment was accompanied by intracellular reactive oxygen species (ROS) generation. However, pretreatment with N -acetyl-l-cysteine (NAC) could not attenuate WSPIS-induced apoptosis. In addition, our data also show that WSPIS could inhibit autophagy. Activation of autophagy by rapamycin decreased WSPIS-induced apoptosis and cell death. Taken together, our findings suggest that cell cycle arrest, endonuclease G-mediated apoptosis, and autophagy inhibition contribute to the anti-cancerous effect of WSPIS on human acute monocytic leukemia cells.

Absence of correlation between oxysterol accumulation in lipid raft microdomains, calcium increase, and apoptosis induction on 158N murine oligodendrocytes.

PubMed

Ragot, Kévin; Mackrill, John J; Zarrouk, Amira; Nury, Thomas; Aires, Virginie; Jacquin, Agnès; Athias, Anne; Pais de Barros, Jean-Paul; Véjux, Anne; Riedinger, Jean-Marc; Delmas, Dominique; Lizard, Gérard

2013-07-01

There is some evidence that oxidized derivatives of cholesterol, 7-ketocholesterol (7KC) and 7β-hydroxycholesterol (7βOHC), are increased in the plasma of patients with neurodegenerative diseases associated with demyelinization of the central nervous system (CNS). It was therefore of interest to investigate the effects of these oxysterols on oligodendrocytes, the myelin-forming cells in the CNS. To this end, 158N murine oligodendrocytes were treated with 7KC or 7βOHC inducing an apoptotic mode of cell death characterized by condensation/fragmentation of the nuclei, dephosphorylation of Akt and GSK3, mitochondrial depolarization involving Mcl-1, and caspase-3 activation. In contrast, under treatment with 27-hydroxycholesterol (27OHC), no cell death was observed. When the cells were stained with Fura-2, no significant Ca(2+) rise was found with the different oxysterols, whereas strong signals were detected with ionomycin used as positive control. At concentrations which induced apoptosis, 7KC but not 7βOHC accumulated in lipid rafts. Although not cytotoxic, 27OHC was mainly detected in lipid rafts. It is noteworthy that α-tocopherol (but not ellagic acid and resveratrol) was able to counteract 7KC- and 7βOHC-induced apoptosis and to decrease the accumulation of 7KC and 27OHC in lipid rafts. Thus, in 158N cells, the ability of oxysterols to trigger a mode of cell death by apoptosis involving GSK-3 and caspase-3 activation is independent of the increase in the Ca(2+) level and of their accumulation in lipid raft microdomains. Copyright © 2013 Elsevier Inc. All rights reserved.

Photosensitized rose Bengal-induced phototoxicity on human melanoma cell line under natural sunlight exposure.

PubMed

Srivastav, Ajeet K; Mujtaba, Syed Faiz; Dwivedi, Ashish; Amar, Saroj K; Goyal, Shruti; Verma, Ankit; Kushwaha, Hari N; Chaturvedi, Rajnish K; Ray, Ratan Singh

2016-03-01

Rose Bengal (RB) is an anionic water-soluble xanthene dye, which used for many years to assess eye cornea and conjunctiva damage. RB showed strong absorption maxima (λmax) under visible light followed by UV-B and UV-A. RB under sunlight exposure showed a time-dependent photodegradation. Our results show that photosensitized RB generates (1)O2 via Type-II photodynamic pathway and induced DNA damage under sunlight/UV-R exposure. 2'dGuO degradation, micronuclei formation, and single- and double-strand breakage were the outcome of photogenotoxicity caused by RB. Quenching studies with NaN3 advocate the involvement of (1)O2 in RB photogenotoxicity. RB induced linoleic acid photoperoxidation, which was parallel to (1)O2-mediated DNA damage. Oxidative stress in A375 cell line (human melanoma cell line) was detected through DCF-DA assay. Photosensitized RB decreased maximum cellular viability under sunlight followed by UV-B and UV-A exposures. Apoptosis was detected as a pattern of cell death through the increased of caspase-3 activity, decreased mitochondrial membrane potential, and PS translocation through inner to outer plasma membrane. Increased cytosolic levels of Bax also advocate the apoptotic cell death. We propose a p53-mediated apoptosis via increased expression of Bax gene and protein. Thus, the exact mechanism behind RB phototoxicity was the involvement of (1)O2, which induced oxidative stress-mediated DNA and membrane damage, finally apoptotic cell death under natural sunlight exposure. The study suggests that after the use of RB, sunlight exposure may avoid to prevent from its harmful effects. Copyright © 2015. Published by Elsevier B.V.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang Bo; Huang Bo; School of Public Health, University of South China, Hengyang, Hunan 421001

Mitotic catastrophe, a form of cell death resulting from abnormal mitosis, is a cytotoxic death pathway as well as an appealing mechanistic strategy for the development of anti-cancer drugs. In this study, 6-bromine-5-hydroxy-4-methoxybenzaldehyde was demonstrated to induce DNA double-strand break, multipolar spindles, sustain mitotic arrest and generate multinucleated cells, all of which indicate mitotic catastrophe, in human hepatoma HepG2 cells. We used proteomic profiling to identify the differentially expressed proteins underlying mitotic catastrophe. A total of 137 differentially expressed proteins (76 upregulated and 61 downregulated proteins) were identified. Some of the changed proteins have previously been associated with mitotic catastrophe,more » such as DNA-PKcs, FoxM1, RCC1, cyclin E, PLK1-pT210, 14-3-3{sigma} and HSP70. Multiple isoforms of 14-3-3, heat-shock proteins and tubulin were upregulated. Analysis of functional significance revealed that the 14-3-3-mediated signaling network was the most significantly enriched for the differentially expressed proteins. The modulated proteins were found to be involved in macromolecule complex assembly, cell death, cell cycle, chromatin remodeling and DNA repair, tubulin and cytoskeletal organization. These findings revealed the overall molecular events and functional signaling networks associated with spindle disruption and mitotic catastrophe. - Graphical abstract: Display Omitted Research highlights: > 6-bromoisovanillin induced spindle disruption and sustained mitotic arrest, consequently resulted in mitotic catastrophe. > Proteomic profiling identified 137 differentially expressed proteins associated mitotic catastrophe. > The 14-3-3-mediated signaling network was the most significantly enriched for the altered proteins. > The macromolecule complex assembly, cell cycle, chromatin remodeling and DNA repair, tubulin organization were also shown involved in mitotic catastrophe.« less

RIP3 attenuates the pancreatic damage induced by deletion of ATG7.

PubMed

Zhou, Xiaodong; Xie, Li; Xia, Leizhou; Bergmann, Frank; Büchler, Markus W; Kroemer, Guido; Hackert, Thilo; Fortunato, Franco

2017-07-13

Invalidation of pancreatic autophagy entails pancreatic atrophy, endocrine and exocrine insufficiency and pancreatitis. The aim of this study was to investigate whether depletion of Rip3, which is involved in necroptotic signaling, may attenuate the pancreatic atrophy and pancreatitis resulting from autophagy inhibition. Autophagy and necroptosis signaling were evaluated in mice lacking expression of Rip3 in all organs and Atg7 in the pancreas. Acinar cell death, inflammation and fibrosis were evaluated by using of a compendium of immunofluorescence methods and immunoblots. Mice deficient for pancreatic Atg7 developed acute pancreatitis, which progressed to chronic pancreatitis. This phenotype reduces autophagy, increase apoptosis and necroptosis, inflammation and fibrosis, as well as premature death of the animals. Knockout of Rip3 exacerbated the apoptotic death of acinar cells, increased tissue damage, reduced macrophage infiltration and further accelerated the death of the mice with Atg7-deficient pancreas. The pancreatic degeneration induced by autophagy inhibition was exacerbated by Rip3 deletion.

PUMA amplifies necroptosis signaling by activating cytosolic DNA sensors

PubMed Central

Tong, Jingshan; Yang, Liheng; Wei, Liang; Stolz, Donna B.; Yu, Jian; Zhang, Jianke; Zhang, Lin

2018-01-01

Necroptosis, a form of regulated necrotic cell death, is governed by RIP1/RIP3-mediated activation of MLKL. However, the signaling process leading to necroptotic death remains to be elucidated. In this study, we found that PUMA, a proapoptotic BH3-only Bcl-2 family member, is transcriptionally activated in an RIP3/MLKL-dependent manner following induction of necroptosis. The induction of PUMA, which is mediated by autocrine TNF-α and enhanced NF-κB activity, contributes to necroptotic death in RIP3-expressing cells with caspases inhibited. On induction, PUMA promotes the cytosolic release of mitochondrial DNA and activation of the DNA sensors DAI/Zbp1 and STING, leading to enhanced RIP3 and MLKL phosphorylation in a positive feedback loop. Furthermore, deletion of PUMA partially rescues necroptosis-mediated developmental defects in FADD-deficient embryos. Collectively, our results reveal a signal amplification mechanism mediated by PUMA and cytosolic DNA sensors that is involved in TNF-driven necroptotic death in vitro and in vivo. PMID:29581256

A platycoside-rich fraction from the root of Platycodon grandiflorum enhances cell death in A549 human lung carcinoma cells via mainly AMPK/mTOR/AKT signal-mediated autophagy induction.

PubMed

Yim, Nam-Hui; Hwang, Youn-Hwan; Liang, Chun; Ma, Jin Yeul

2016-12-24

The root of Platycodon grandiflorum (PG), commonly known as Kilkyong in Korea, Jiegeng in China, and Kikyo in Japan, has been extensively used as a traditional anti-inflammatory medicine in Asia for the treatment of respiratory conditions, such as bronchitis, asthma, and tonsillitis. Platycosides isolated from PG are especially well-known for their anti-cancer effects. We investigated the involvement of autophagic cell death and other potential molecular mechanisms induced by the platycoside-containing butanol fraction of PG (PGB) in human lung carcinoma cells. PGB-induced growth inhibition and cell death were measured using a 5-diphenyl-tetrazolium bromide (MTT) assay. The effects of PGB on autophagy were determined by observing microtubule-associated protein 1 light chain 3 (LC3) redistribution with confocal microscopy. The PGB-mediated regulation of autophagy-associated proteins was investigated using Western blotting analysis. Furthermore, the anti-cancer mechanism of PGB was confirmed using chemical inhibitors. A high-performance liquid chromatography (HPLC)-DAD system was used to analyze the platycosides in PGB. In A549 cells, PGB induced significant autophagic cell death. Specifically, PGB upregulated LC3-II in a time- and dose-dependent manner, and it redistributed LC3 via autophagosome formation in the cytoplasm. PGB treatment increased the phosphorylation of AMP-activated protein kinase (AMPK) and subsequently suppressed the AKT/mammalian target of the rapamycin (mTOR) pathway. Furthermore, PGB inhibited cell proliferation by regulating the mitogen-activated protein kinase (MAPK) pathways. In this study, six types of platycosides were identified in the PGB using HPLC. PGB efficiently induced cancer cell death via autophagy and the modulation of the AMPK/mTOR/AKT and MAPK signaling pathways in A549 cells. Therefore, PGB may be an efficacious herbal anti-cancer therapy. Copyright © 2016. Published by Elsevier Ireland Ltd.